
Proceedings of the AACR

Volume 57 | April 2016

**Part** **A: Abstracts 1-2696**

TABLE OF CONTENTS

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Altered Cellular Signaling and Cancer Metabolomics

Altered Glucose Metabolism in Cancer

Functional Genomics and Genomics of Model Systems

Genomic Analysis of Cancers

Genomic Profiling of Cancers

Intratumor Heterogeneity and Resistance

Kinases and Phosphatases

Mitochondria, Autophagy, and Metabolic Vulnerabilities

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Cellular Processes and Responses to Therapy

Combination Chemotherapy

Mechanisms of Drug Resistance 1

Novel Antitumor Agents

Novel Assays

PI3K/AKT Inhibitors

CLINICAL RESEARCH:

Biomarkers

Biomarkers for Genitourinary and Gynecological Cancers

Biomarkers for Melanoma and Uncommon Cancers

Circulating Biomarkers 1

Radiation Oncology

IMMUNOLOGY:

Genetic Determinants and Regulators of Cancer Immunity

Immune Modulating Agents 1

Therapeutic Antibodies

TUMOR BIOLOGY:

Drug Testing in Cell Lines and 3D Models

Human in Mouse Models

Mechanisms of Tumorigenesis in Animal Models of Cancer 1

Molecular Regulation of Tumor Invasion

Pro-Tumorigenic Microenvironment

Targeting the Microenvironment

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Systems Biology

EPIDEMIOLOGY:

Genes and Function and Risk

PREVENTION RESEARCH:

Models and Mechanisms in Cancer Prevention

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Novel and Integrative Analyses of Cancer Genome Data

CLINICAL RESEARCH:

Biomarkers to Direct Cancer Therapy

ENDOCRINOLOGY:

Molecular Pharmacology of Hormone-dependent Malignancies

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Antibody-targeted Therapy

Approaches to Elucidating and Overcoming Drug Resistance

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Disordered Gene Regulation and Chromatin State in Malignant Transformation

Oncogene and Tumor Suppressor Function and Targeting

PREVENTION RESEARCH:

Highlights in Cancer Prevention Advances

TUMOR BIOLOGY:

Immunomodulation in Cancer

The Relevance of Stemness Properties in Cancer

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell-Cell Interaction and Tumor Microenvironment

Circulating RNAs, Epigenetics, and Novel Non-coding RNAs in Cancer

LncRNAs and Mechanisms in Cancer

Metabolic Reprogramming and Autophagy

Metabolic Reprogramming in Cancer

MicroRNAs as Biomarkers and Therapeutics

MicroRNAs as Oncogenes and Tumor Suppressors

Oncogene Function, Regulation, and Targeting

Oncogenes and Tumor Suppressor Genes and Pathways

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Differentiation Therapy

Growth Factor Receptors and Surface Antigens as Therapeutic Targets

Novel Antitumor Agents and Epigenetics

Novel Molecular Targets

Regulation of Anticancer Drug Effects

CANCER CHEMISTRY:

Drug Delivery 1

Drug Delivery 2

CLINICAL RESEARCH:

Clinical Assay Development

Immune Response Monitoring: Clinical

Special Populations, Supportive Care, and Survivorship Research

IMMUNOLOGY:

Immune Microenvironment and Antitumor Immunity

Immune Modulating Agents and Therapeutic Antibodies

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Applications of Bioinformatic Tools to Analyze Cancer Data

TUMOR BIOLOGY:

Biomarkers and Profiling of Metastasis

Cellular and Molecular Mediators of Metastasis

Epithelial-Mesenchymal Transition in Metastasis

Metastasis-Promoting and -Suppressing Genes

Radiation Science

Regulators of Epithelial-Mesenchymal Transition and Metastasis

Stemness Properties of Intestinal, Pancreatic, and Hepatic Cancer

EPIDEMIOLOGY:

Diet, Tobacco, Smoking, and Other Lifestyle Factors in Cancer Epidemiology

Race/Ethnicity and Disparities in Diagnosis, Treatment, and Outcomes

PREVENTION RESEARCH:

Prevention Clinical Research

ENDOCRINOLOGY:

Molecular Endocrinology of Hormone-dependent Malignancies

REGULATORY SCIENCE AND POLICY:

Regulatory Science and Policy

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Assessment of Gene Regulation in the Malignant Context

GTPase, RAF, and Growth Factor Pathways

MicroRNAs in Metastasis and Cancer

Non-coding RNAs and Mechanisms in Cancer

Profiling MicroRNA Expression in Cancer

Regulation of Chromatin State and Gene Expression

Transcriptional Regulation and Gene Expression

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Clinical Pharmacology and Pharmacogenomics

Drug Delivery

Experimental Therapeutics

Mechanisms of Drug Resistance 2

New Drugs, Therapeutic Targets, and Treatment Approaches

CANCER CHEMISTRY:

High Throughput Screening and Natural Products

Nanotechnology and Drug Delivery

CLINICAL RESEARCH:

Adoptive Cell Therapy, Immune Checkpoints, and Vaccines

Biomarkers for Lung Cancer

Mechanisms of Response to Targeted Agents and Potential New Targets

IMMUNOLOGY:

Adoptive Cell Therapy

Immune Checkpoints 1

Therapeutic Antibodies and Vaccines

TUMOR BIOLOGY:

Clonal Heterogeneity and Evolution

Intratumor Heterogeneity and Treatment Responses

Pediatric Cancer Genomics, Genetics, and Epigenetics

Pediatric Cancer Molecular Pathways

Pediatric Cancer Targets, Models, Therapies, and Resistance

Stemness Properties and Therapeutic Targeting in Solid Tumors

Stemness Properties of Neuronal and Pediatric Tumors and New Approaches

EPIDEMIOLOGY:

Genetic Contributions to Cancer Epidemiology: Familial Cancers and GWAS

Risk Prediction, Screening, and Comparative Effectiveness Research

PREVENTION RESEARCH:

Targets, Markers, and Agents in Cancer Prevention

CLINICAL RESEARCH:

Genomic Landscapes

EPIDEMIOLOGY:

Biomarkers and Other Epidemiologic Factors in Cancer Prognosis

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Hitting the Target Harder: Preclinical Development of Potent and Selective Inhibitors

IMMUNOLOGY:

Immunotherapy Trial Results and Correlates

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Epigenetic Alterations in Cancer

LncRNAs, MicroRNAs, and Non-coding RNAs in Cancer

Mechanisms and Vulnerabilities of Metabolic Reprogramming

TUMOR BIOLOGY:

Mechanism and Dynamics of Cancer Metastasis

Mechanisms of Tumorigenesis in Mouse Models of Human Cancer

New Molecular Advances in Pediatric Cancer

Advocates Poster Session 1

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# Sunday, April 17, 2016

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Altered Cellular Signaling and Cancer Metabolomics

#2

Metabolomics analysis reveals distinct profiles of non-muscle invasive and muscle-invasive bladder cancer.

Divya Sahu,1 Yair Lotan,2 Bryan Wittmann,3 Bruce Neri,3 Donna Hansel1. 1 _UC San Diego, San Diego, CA;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX;_ 3 _Metabolon Inc., Durham, NC_.

Background: Bladder cancer affects >70,000 patients annually in the United States. Despite its high incidence, therapeutic options are limited in early or late stage. We wanted to identify key metabolic pathways that were altered in bladder cancer development and progression.

Experimental Design: We performed global metabolomics profiling of benign urothelium, high-grade non-muscle invasive bladder cancer and advanced, muscle-invasive bladder cancer using GC-MS and LC-MS platforms. This analysis was coupled with publicly available data on transcriptomics of key enzymes, to determine pathways that may be suitable for future therapeutics development.

Results: Categorical pathways globally dysregulated in cancer relative to benign urothelium included glucose, TCA cycle, lipid, amino acid and nucleotide pathways. Bladder cancers demonstrated Warburg metabolism, with elevated glucose utilization to

drive glycolysis and sorbitol pathway intermediates. Elevated late TCA cycle intermediates, coupled with higher levels of amino acids and dipeptides, suggest the possibility of anaplerotic activity in bladder cancer as a mechanism to sustain energy production. Medium and long chain fatty acids were produced at the expense of dicarboxylic fatty acids. Muscle-invasive bladder cancers showed enhanced use of COX and LOX metabolomics pathways and a possible role for inflammation in regulating NAD+ synthesis in muscle-invasive bladder cancer. Transcriptomic profiling validated that the majority of metabolomics pathway alterations corresponded to gene expression changes of enzymes responsible for metabolite production.

Conclusions: This study identifies multiple parallel metabolomics changes unique to non-muscle invasive and muscle-invasive bladder cancer that can be used to justify testing novel therapeutics targeting metabolic pathways in bladder cancer.

#3

Amino acid profiles indicate dependence on different metabolic pathways between leukemia subtypes.

Shannon R. Sweeney, Enrique Sentandreu, Stefano Tiziani. _The University of Texas at Austin, Austin, TX_.

Despite the growing body of evidence that the tumor microenvironment protects leukemia cells from chemotherapeutic stresses (1-3), the effect of many extracellular metabolites remains largely unknown. To explore the influence of extracellular metabolites on different leukemia subtypes, cells were treated for 24 hours in vitro with either a supplemental amino acid or amino acid derivative. From this initial screening, a subset of metabolites were chosen for metabolomics analysis. Mass spectrometry (UPLC-MS/MS) was performed on intracellular fractions to identify metabolic differences that resulted from supplementation. Metabolite profiles were also compared between leukemia cell types, namely AML, pre-B cell ALL, and T cell ALL. Of the metabolites tested, lysine and 4-hydroxyphenylpyruvate, an intermediate of tyrosine and phenylalanine metabolism, had the greatest impact on global amino acid profiles. In AML and T cell ALL cell lines, intracellular glutamate, glutamine, proline, and aspartate were increased relative to their respective controls. These amino acids can enter the tricarboxylic acid (TCA) cycle as either α-ketoglutarate or oxaloacetate, suggesting a central role of the TCA cycle in both AML and T cell ALL metabolism. Interestingly, these metabolites were not significantly increased in pre-B cell ALL, signifying the inverse it true for pre-B cells. This observation provides metabolomics evidence that is consistent with a previous study that reported downregulated expression of TCA cycle related genes in pre-B cell ALL (4). Our findings indicate that uptake and metabolism of amino acids and their derivatives is distinct for different leukemia types. Moreover, supplementation with a single metabolite can result in global changes in intracellular metabolite profiles, suggesting an influence not only as an energy substrate, but on overall metabolic pathway activity. Specifically, we conclude that the TCA cycle is more active in AML and T cell ALL and can be modulated by changing the extracellular environment, while pre-B cells are less sensitive to amino acid modulation.

(1) Meads MB, et al. Clin Cancer Res 2008;14(9):2519-2526.

(2) Ayala F, et al. Leukemia 2009;23:2233-2241.

(3) Konopleva M, et al. Drug Resist Updat 2009;12:103-113.

(4) Boag JM, et al. Leukemia 2006;20:1731-1737.

#4

Hyper activation of poly(ADP-ribose) polymerase 1 initiates large-scale metabolic changes in a cellular model of glioblastoma.

Anna M. Wilk,1 Elise Fouquerel,2 Bobbie Johnston,3 Samuel A.J. Trammell,4 Lindsay Schambeau,1 Joel F. Andrews,1 Lewis Pannell,1 Sara J. Cooper,3 Charles Brenner,4 Robert W. Sobol1. 1 _Mitchell Cancer Institute, University of South Alabama, Mobile, AL;_ 2 _Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA; _3 _HudsonAlpha Institute for Biotechnology, Huntsville, AL;_ 4 _Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA_.

PARP1 is a key enzyme of the Base Excision Repair (BER) pathway, facilitating the repair of base damage and single-strand DNA breaks. Activated PARP1 synthesizes poly (ADP-ribose) (PAR), triggering chromatin de-condensation to facilitate recruitment of BER proteins to complete repair. PARP1 activation is attenuated upon successful repair of the DNA lesion. However, unrepaired DNA breaks lead to continuous PARP1 activation and cell death. The molecular mechanism underlying PARP1 activation induced cell death was recently revealed as independent from NAD+ depletion. We have shown that PARP1 activation and PAR synthesis affect glycolysis by directly inhibiting the glycolytic enzyme, hexokinase 1 (HK1). Following on these discoveries, we decided to investigate global metabolic changes triggered by hyperactivation of PARP1. For this study, we used gas chromatography mass spectrometry (GC-MS) to quantify over 150 cellular metabolites and Multiple-Reaction Monitoring Liquid Chromatography Mass Spectrometry (MRM LC-MS) to measure NAD+ metabolites. As a model, we tested glioblastoma cells overexpressing methylpurine DNA glycosylase (MPG) to enhance the PARP1-activation response to DNA damage induced by the alkylating agent MNNG. Simultaneously, to monitor independence from the DNA damaging effect of NAD+ depletion, we utilized an inhibitor of NAD+ biosynthesis, FK866. We found that PARP1 activation leads to a strong accumulation of glucose, likely as a secondary effect of HK1 inhibition. In addition, we observed a significant change in the level of other metabolites including an increase in inosine, inosine monophosphate (IMP), cytidine and uridine levels upon PARP1 activation, suggesting an indirect effect of PARP1 activation on purine and pyrimidine metabolism. Ongoing studies will use these global approaches to unravel the complete metabolic response of cancer cells to genotoxic treatment.

#5

Using stable isotope lipidomics to identify adipocyte-induced alterations in the acute lymphoblastic leukemia lipidome.

Jonathan Tucci,1 Katy Margulis,2 Cheng-Chih Hsu,3 Wesley Dixon,2 Richard N. Zare,2 Steven D. Mittelman1. 1 _Children's Hospital of Los Angeles, Los Angeles, CA;_ 2 _Stanford University, Stanford, CA;_ 3 _National Taiwan University, Taipei, Taiwan_.

Obesity is associated with the development and progression of many cancers, including acute lymphoblastic leukemia (ALL). We have shown that ALL cells induce adipocyte lipolysis and take up adipocyte-derived free-fatty acids (FFAs). Here, we use targeted lipidomics with carbon-13 labeling to 1) determine which adipocyte-derived FFAs are taken up by ALL cells, and 2) identify the specific alterations in the ALL cell lipidomic profile caused by co-culture with adipocytes.

Mouse pre-adipocytes (3T3-L1) were differentiated into adipocytes in the presence of U13C-glucose to allow 13C incorporation into adipocyte lipids. ALL cells were then cultured alone or co-cultured over 13C-labeled or non-labeled adipocytes for 72 hours. Following co-culture, ALL cells were harvested and analyzed by nanospray desorption electrospray ionization mass spectrometry (nanoDESI-MS). Lipidomic spectra were analyzed to characterize uptake of adipocyte-derived FFAs identifying 13C-enrichment in ALL lipid moieties. The relative intensities of selected lipid peaks were compared between conditions, after normalization, to evaluate the effects of the presence of adipocytes on ALL lipidome.

Adipocytes differentiated in the presence of U13C-glucose incorporated 13C into numerous triglyceride moieties. In ALL cells co-cultured with labeled adipocytes, 13C enrichment was identified in FFA and phospholipids. Labeling of oleic acid primarily occurred in groups of two, suggesting incorporation of U13C-glucose into adipocyte FFAs through acetyl-CoA-mediated lipogenesis. Similarly, 13C enrichment of ALL cell phospholipids occurred in groups of two and three, mirroring adipocyte incorporation of U13C-glucose into phospholipids through both acetyl-CoA and glycerol. Moreover, ALL cell unsaturated FFAs had a greater 13C enrichment than saturated FFAs (Unsaturated FFAs: 14.1±5.5 vs. Saturated FFAs: 6.6±2.3, p=0.02; n=5). When co-cultured over adipocytes, ALL cells contained significantly more FFAs than ALL cells alone (Oleic Acid: 63.0±49.2 vs. 24.1±7.4, p=0.02; Stearic acid: 20.9±17.5 vs. 8.3±2.2, p=0.03; Palmitoleic Acid: 11.5±9.8 vs. 3.3±1.0, p=0.01; Palmitic Acid: 15.1±11.7 vs. 5.5±1.4, p=0.01; n=5).

Using stable-isotope lipidomics, we can effectively identify and quantify which ALL cell lipids are derived from nearby adipocytes. This methodology provides comprehensive insight into the cancer cell lipidome and can be extended to understand how genetics, the microenvironment, and treatment affect cancer lipid metabolism.

#6

Metabolomics of Gleason score in prostate tumor tissue and serum.

Kathryn Penney,1 Svitlana Tyekucheva,2 Massimo Loda2. 1 _Brigham and Women's Hospital/Harvard Medical School, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Background: Gleason score is currently the best clinical predictor of prostate cancer specific-mortality. Identifying biomarkers associated with Gleason score can improve prediction and advance the understanding of the biology of aggressive disease. Using gene expression data, we previously determined that metabolism pathways, including pyrimidine, propanoate, and beta-alanine, were differentially expressed in high and low grade tumors. The goal of this study is to identify metabolites that differ in low and high Gleason score tumors and in blood from these patients.

Methods: Metabolic profiling was performed on Gleason score 6 (n=53) and Gleason score ≥8 (n=32) radical prostatectomy tumor tissue specimens, and on serum samples from the time of diagnosis (n=30 Gleason score 6 and n=19 Gleason score ≥8) from the DF/HCC Prostate Cancer SPORE Cohort. Samples were prepared for analysis by Metabolon, Inc. with their standard solvent extraction method to recover small molecules. Metabolites were identified and quantified by gas and liquid chromatography and mass spectrometry. Missing values were calculated using k-nearest neighbor imputation, and sample values were normalized across batches. The metabolite levels were compared across Gleason score 6 and Gleason score ≥8 tumors and serum using a Wilcoxon test.

Results: In the tumor tissue, 207 metabolites were identified. Of these, 30 were present in significantly different amounts in high and low Gleason score tumors, including previously reported citrate (p=0.002) and spermine (p=0.003). In a pathway analysis, the propanoate pathway was significantly different in high and low Gleason (p=0.03), but not the pyrimidine or beta-alanine pathways (p>0.05). In serum, 15 individual metabolites had significantly different levels, including uridine (p=0.008) and N-acetylserine (p=0.0001).

Conclusions: Metabolite levels do differ in high and low Gleason score prostate tumor tissue. The novel serum results suggest that metabolites in blood may serve as biomarkers of tumor differentiation. Our ongoing research will build prediction models, and will determine if the blood metabolites can indicate the presence of high-Gleason score tumor not detected at biopsy and could serve as biomarkers for monitoring disease progression in active surveillance patients.

#7

LKB1 deficiency is associated with a unique metabolic signature in Kras mutant non-small cell lung cancer (NSCLC).

Tingyu Liu,1 Erin Sennott,1 Zhengjie Zhong,1 Mya Steadman,2 Kelly Marsh,2 Jonathan Hurov,2 Cyril Benes,1 Jeffrey A. Engelman1. 1 _Massachusetts General Hospital, Charlestown, MA;_ 2 _Agios Pharmaceuticals, Cambridge, MA_.

LKB1 deficiency is found in approximately 10% of non-small cell lung cancer harboring Kras mutation. Loss of LKB1 has been associated with increased metastatic rates and decreased survival in patients. Currently, no effective therapy has been developed against this subtype of lung cancer. LKB1 can regulate multiple cellular activities. One of the most well-studied functions is its regulation on cell metabolism through AMPK signaling pathway. How LKB1 loss globally impacts NSCLC metabolism has not been well understood yet. Using isotope-labeled substrate tracing approach, we found an increase of specially-labeled intermediates in pentose phosphate pathway (PPP) that may suggest upregulation of PPP activity. In addition, we also performed an unbiased drug screen that showed higher sensitivity to an mTOR inhibitor in Kras and LKB1 double mutant lung cancer cells relative to cancer cells under other genetic background. These data suggest that loss of LKB1 may upregulate biosynthetic reactions in Kras mutant NSCLC and render these cells sensitive to inhibitors in biosynthesis. These findings may provide new insights in developing therapeutic agents targeting KrasMT/LKB1MT NSCLC.

#8

Global and targeted metabolomic profiling of colorectal cancer progression.

Beatriz Sanchez-Espiridion,1 Lindsey White,2 Lopa Mishra,3 Gottumukkala S. Raju,3 Scott Kopetz,4 Jian Gu,1 Yuanquing Ye,1 Xifeng Wu,1 Dong Liang2. 1 _Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Pharmaceutical Sciences, Texas Southern University, Houston, TX;_ 3 _Department of Gastroenterology, Hepatology & Nutrition, The University of Texas MD Anderson Cancer Center, Houston, TX; _4 _Department of GI Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Colorectal cancer (CRC) is one of the most prevalent and deadly cancers in the world. The development of improved and robust biomarkers to enable screening, surveillance, and early detection of CRC continues to be a challenge. Patients with colorectal adenoma are at higher risk of developing colon cancer; however, noninvasive methods to identify these patients are still on demand. The aim of this study was to identify biomarkers of CRC disease progression by using metabolomic profiling of human serum samples in a multistep approach.

Methods: We performed global metabolomic profiling on 30 human serum samples from patients with colorectal adenoma, 30 CRC patients and 30 healthy controls who were matched by age, gender and ethnicity. For validation, we measured the three top differentially expressed metabolites in an additional set of 50 adenoma, 50 CRC and 50 healthy controls.

Results: Global biochemical profiles of 404 metabolites were detected, with 301 metabolites remaining after quality control procedures. In discovery phase, 50 metabolites had differential levels between colorectal adenoma, CRC and controls (P for trend <0.05), with 19 metabolites showing increased levels in CRC and adenoma in comparison to controls and 31 metabolites with decreased levels. Further exploratory analyses of these metabolites showed a key role for metabolic pathways involving urea cycle, caffeine and galactose metabolism as associated with CRC progression. The top 3 differentially expressed metabolites (Xanthine, Hypoxanthine and D-mannose) were selected for validation. Consistent with the discovery phase, CRC cases and adenoma had lower levels of Xanthine than controls (mean ± SD; 9.95 ±0.92 mg/ml vs 10.63±0.97 mg/ml; P<0.001 and 9.87±0.75 vs 10.63±0.97 mg/ml; P<0.001). The same trend was observed for Hypoxanthine (mean ± SD; 10.72 ±0.62 mg/ml vs 12.29±1.60; P<0.001 and 10.71±0.61 vs 12.29±1.60 mg/ml; P <0.001) whereas higher levels of D-mannose where observed in both CRC cases and adenoma when compared to controls (3.32±0.58 mg/ml vs 2.32 ±0.90mg/ml; P<0.001 and 3.32±0.58 mg/ml vs 2.32 ±0.90mg/ml; P<0.001). Using the median value of controls as a cut-off point, 94% of the adenoma and CRC cases showed low levels of Xanthine ( Odds Ratio (OR)=10.47, 95% confidence interval (CI) = 2.63-41.63 for adenoma; OR=38.76 and 95% CI = 6.58-228.51 for CRC ) . For Hypoxanthine, 90% of the adenoma and CRC cases showed low levels (OR =6.50 and 95% CI = 1.98--21.24 for adenoma; OR=11.19 and 95% CI = 3.28-38.21 for CRC). For D-Mannose, all adenoma cases had high levels (OR is not available due to 0 count) and 92% of CRC cases (OR = 15.99, 95% Ci=4.07-62.88, P< 0.001) had high levels of D-Mannose, compared to 50% of controls having high levels of D-Mannose.

Conclusions: Our results suggest the potential utility of the identified metabolites as new valuable biomarkers for early detection of CRC.

#9

Metabolic profiling of bladder cancer cell lines reveals molecular alterations involved in methylation and novel epigenetic phenotype.

Rashmi Krishnapuram,1 Franklin Gu,1 Salil Kumar Bhowmik,1 Suman Maity,1 Mohan Manikkam,1 Friedrich-Carl von Rundstedt,1 Vasanta Putluri,1 Yair Lotan,2 Jonathan M. Levitt,1 Seth P. Lerner,1 Cristian Coarfa,1 Arun Sreekumar,1 Nagireddy Putlurip1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX_.

Background: Bladder cancer (BCa) is primarily "carcinogen driven cancer". Epidemiological studies indicate environmental factors play a major causative role compared to genetic factors. Xenobiotic metabolism is highly perturbed and precise mechanisms involved are poorly understood during BCa progression. We identified metabolic signature that can distinguish bladder cancer from controls and reveals major alterations in phaseI/II enzymes involved in xenobiotic metabolism and suggest a key role for epigenetic modifications.

Material and Methods:

In this study, we used mass spectrometry based metabolomics profiling coupled with enrichment-based bioprocess mapping to obtain insight into biochemical alterations in bladder cancer cell lines. We further validated related gene expression using real time quantitative PCR (qPCR) and proteins using western blotting.

Results:

In this study, we used high-throughput mass spectrometry to measure over 350 compounds in seven bladder cell lines, identifying 91 metabolites which exhibited significant changes in bladder Cancer. Most importantly, methylated, hydroxylated and acetylated metabolites are altered. Interestingly, S-Adenosyl methionine (SAM) is the most prominent pathway upregulated corroborated with our previous findings obtained using patient derived metabolomic data from two independent cohorts. Second, we observe many of phaseI/phaseII metabolic enzymes including aldehyde oxidase (AOX1), cytochrome P450 1A1 (CYP1A1), CYP1B1, Glutathione S-transferase T1 (GSTT1), Glutathione S-transferase M2 (GSTM2), N-acetyl transferase I NAT1 and NAT2 are transcriptionally repressed in BCa cell line compared to benign indicating the pivotal role of methylation in gene silencing. Interestingly, we observe differential expression of polycomb group of proteins (Pcg) associated with PRC2 and PRC1 complex. Specifically, histone-lysine N-methyl transferase (EZH2) protein, which is SAM dependent histone methyl transferase and concomitant 3meHK27 trimethylated histone K27, is highly expressed in metastatic UMUC3 BCa cell line further indicating prominence of epigenetic modifications.

Conclusion:

We present an integrative pathway analysis of a metabolic gene signature which has not been previously described in the context of bladder cancer cell lines. Further mechanistic analyses reveals prominent role for methylation status and associated epigenetic modifications being played in the transcriptional repression of key xenobiotic enzymes. Collectively, our novel findings provide an opportunity for development of efficient biomarker implications and epigenetic therapy targeting BCa progression.

#10

Silibinin exhibits anti-cachectic and anti-cancerous property by modulating metabolic properties of pancreatic cancer cells.

Surendra K. Shukla,1 Aneesha Dasgupta,1 Kamiya Mehla,1 Venugopal Gunda,1 Enza Vernucci,1 Joshua Soucheck,1 Gennifer Goode,1 Ryan King,1 Anusha Mishra,1 Ibha Rai,1 Sangeetha Natrajan,1 Nina Chaika,1 Fang Yu,2 Pankaj K. Singh1. 1 _Eppley Institute for Cancer Research, UNMC, Omaha, NE;_ 2 _Department of Biostatistics, UNMC, Omaha, NE_.

Cancer cachexia is a systemic syndrome characterized by progressive weight loss of the patient due to muscle wasting and fat depletion with or without anorexia. About 80% of pancreatic ductal adenocarcinoma (PDAC) patients exhibits cachectic phenotype and it significantly contributes in mortality and morbidity of the disease. In present study we have evaluated the effect of bioactive molecule silibinin on pancreatic cancer progression and cachectic properties by utilizing in vitro as well as in vivo models of PDAC. We observed that silibinin inhibits growth and induces apoptosis in multiple pancreatic cancer cell lines in a dose-dependent manner. We also observed silibinin-mediated reduction in the expression of key glycolytic genes and inhibition of glucose uptake and lactate secretion. By performing LC-MS/MS based metabolomics, we observed that silibinin treatment leads to global metabolic alterations in pancreatic cancer cells. Pancreatic cancer cells treated with silibinin exhibited reduced expression of c-MYC level, a key metabolic regulator. Furthermore, we observed that silibinin-mediated STAT3 inhibition leads to reduced c-MYC expression. Ectopic expression of constitutively active STAT3 significantly attenuated the effect of silibinin on c-MYC expression and metabolic phenotype of pancreatic cancer cells. Silibinin treatment also inhibited tumor growth and progression of cachexia. Silibinin treatment to tumor-bearing mice also lead to increased food intake, increased grip strength and body coordination. Overall, our results demonstrate that silibinin exhibits anti-cachectic and anti-cancerous properties by inducing metabolic reprogramming in pancreatic cancer cells.

#11

Metformin exerts differential metabolic effects in ovarian cancer cell lines.

Adnan Munkarah,1 Laila Poisson,1 Seongho Kim,2 Jasdeep Chhina,1 Shailendra Giri,1 Ramandeep Rattan1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Karmanos Cancer Institute, Detroit, MI_.

Metformin is being actively repurposed for treatment of gynecologic malignancies including ovarian cancer, with various clinical trials underway. Metformin is known to alter the cancer cell metabolism, primarily by inhibiting oxidative phosphorylation and inducing glycolysis. Our aim was to investigate if metformin induces similar metabolic changes across ovarian cancer cells. Untargeted global metabolite assay, by ultra-high performance Liquid Chromatography and Gas Chromatography Mass Spectroscopy, was performed on A2780, C200, and SKOV3ip cell lines with and without metformin treatment (10mM for 48 hours). Per-metabolite comparisons were made across conditions. Interpretive analysis was performed using the KEGG molecular pathways (Kyoto Encyclopedia of Genes and Genomes) and the Ingenuity molecule library with a focus on energy pathways of glycolysis, oxidative phosphorylation and also other metabolic pathways. Additionally, glycolytic and mitochondrial respiration were measured using the Seahorse XFe Analyzer. Specific analysis of the glycolysis metabolites revealed that while glycolysis was increased by metformin in all the cells, the intracellular levels of glucose, lactose and pyruvate varied significantly across the cell lines and were differentially affected by metformin treatment. Metformin had little impact on the TCA cycle intermediates in the A2780 cells, which were significantly decreased in C200 and in contrast increased in SKOV3ip cells. Functional analysis showed the oxygen consumption rate to be significantly inhibited by metformin in all the three cell lines, while the increased fatty acid oxidation intermediates were observed across all the three cell lines albeit to a varying extent. Exploration of the global metabolite changes by metformin across the three cell lines revealed 57 common altered metabolites, of which 30 had consistent direction change, with 16 metabolite being up and 14 being downregulated by metformin treatment. Metabolite Set Enrichment analysis showed linolenic acid and methionine metabolism as most enriched in metabolites being increased by metformin, and RNA transcription and pyrimidine metabolism as most enriched in the metabolites downregulated by metformin treatment. Ingenuity analysis indicated cellular proliferation and signaling as the top common network pathway modulated by metformin. In conclusion metformin treatment had a significant and wide-spread effect on metabolism of ovarian cancer cell lines. While metformin resulted in certain consistent metabolic changes, it had cell line specific modulation on glycolysis and oxidative phosphorylation metabolites. These differential metabolic changes could indicate the degree of metformin response and suggest it to be context-dependent. Thus information about the cancer metabolism will aid in preclinical and clinical interpretation of metformin therapy in ovarian and other cancers.

#12

Combinatorial intervention with natural compounds induces key metabolic modulations for prostate cancer prevention and treatment.

Xiyuan Lu, Bo Wang, Achinto Saha, Enrique Sentandreu, Alessia Lodi, John DiGiovanni, Stefano Tiziani. _University of Texas at Austin, Austin, TX_.

Prostate cancer is one of the three most relevant cancer types in men, and it ranks second in death rate and first in newly diagnosed cancer cases according to the US cancer statistics 2015. In recent years, the impact of nutrition on cancer prevention has been increasingly recognized. Accordingly the study of natural compounds for cancer prevention and treatment has drawn attention, mainly for the low toxicity to normal tissues.

In this study, a natural compound library of 140 compounds was screened on cultured mouse prostate tumor cells from HiMyc mice (HMVP-2 cells), and ATP and reactive oxygen species (ROS) bioluminescence measurements were performed. Based on ATP and ROS results, three hits, ursolic acid (UA), curcumin (CURC) and resveratrol (RES) were selected for more in depth metabolomic and lipidomic analyses. High-resolution liquid chromatography mass spectrometry (HPLC-MS) and magnetic resonance spectroscopy (MRS) were applied for large scale untargeted metabolic and lipidomic profiling of intra and extracellular prostate cancer extracts after treatment with the three drugs and their combinations at different time points. We next performed an in vivo study on an allograft mouse model of prostate cancer by injecting HMVP-2 spheroids into FVB/N mice.

After 12 hours of treatment, 115 metabolites in KEGG database and 12 classes of lipids (664 features) were included in the study. Principal component analysis (PCA) showed that the combination of UA and CURC exerts the most profound metabolic perturbation in HMVP2 cells compared to the individual treatment or the combination of other selected hits. Further data mining showed that the CURC+UA had a synergistic effect on cell metabolism by altering metabolic pathways associated with alanine, aspartate, proline and glutamate metabolism. Moreover, key intermediates in glycerophospholipid and ceramide metabolism were highly perturbed in CURC +UA indicating a relevant response of lipid mechanism to treatment with the combination of these agents. The in vivo mouse model produces palpable tumors within 10-14 days post injection. Dietary administration of CURC+UA and UA+RES showed significant inhibition of tumor growth compared to the administration of the individual compounds, with CURC+UA yielding the most effective combination.

In summary, amongst the 140 screened compounds, CURC, UA and RES exerted the most prominent metabolic effects on prostate cancers cells, and the combined CURC+UA treatment showed a synergistic effect on cell metabolism and significantly affected key metabolic pathways active in mitochondria, most likely via lipid metabolisms.

#13

Serum caffeine metabolites and prostate cancer risk in the ATBC Study.

Shakira M. Nelson,1 Alison M. Mondul,2 Stephanie J. Weinstein,1 Demetrius Albanes1. 1 _National Cancer Institute, Rockville, MD;_ 2 _University of Michigan School of Public Health, Ann Arbor, MI_.

Background: Etiologic studies of prostate cancer have shown inconsistent associations with coffee consumption. We have previously shown increased risk with number of cups/day, with the strongest association for men consuming 4+ cups/day. The association was evident in the first 10 years of cohort follow-up, however, suggesting possible reverse causality. We conducted a serum metabolomic profiling study to biochemically re-evaluate our coffee findings and determine whether caffeine metabolites are similarly associated with prostate cancer risk.

Methods: A prospective, nested case-control analysis within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study cohort was conducted using 200 cases and 200 controls matched on age and date of baseline fasting serum collection. Sera were analyzed using ultrahigh performance liquid chromatography/mass spectroscopy (LC-MS) and gas chromatography/mass spectroscopy (GS-MS) (Metabolon, Inc.) which identified 626 known compounds, including 13 caffeine-related metabolites. Each metabolite was assessed using conditional logistic regression that estimated odds ratios (OR) and 95% confidence intervals (CI) of prostate cancer associated with a one standard deviation (1-S.D.) increment in metabolite signal strength. The analysis was also stratified by median time from baseline blood draw to prostate cancer diagnosis (<10 years vs. ≥10 years).

Results: Overall, caffeine metabolite associations with prostate cancer risk showed null results (e.g., caffeine OR 1.02, 95% CI 0.84-1.24, p=0.82; paraxanthine OR 0.99, 95% CI 0.83-1.20, p=0.96). Similar to our findings for coffee consumption, caffeine metabolites were positively associated with prostate cancer risk for men diagnosed within 10 years of blood collection. Theobromine and 1,7-dimethylurate showed the strongest signals (respectively, OR 1.69, 95% CI 1.13-2.54, p=0.01; OR 1.63, 95% CI 1.14-2.32, p=0.01). By contrast, in men diagnosed at least 10 years from baseline, caffeine metabolites were non-significantly inversely related to risk; e.g., 5-acetylamino-6-formylamino-3-methyluracil (AFMU), OR 0.77, 95% CI 0.56-1.06, p=0.10). Caffeine metabolites were positively correlated with both caffeine and coffee (median correlation coefficients, 0.78 and 0.20, respectively). Including coffee in the metabolite risk models led to little or no attenuation of the associations in either the early or later 10 year observation periods.

Conclusions: In this prospective analysis, serum caffeine metabolites were positively associated with risk of prostate cancer in men diagnosed within 10 years of blood collection. The findings provide biochemical corroboration of our previous findings for a positive coffee-prostate cancer risk association, and appear to support the possibility of reverse causality; i.e., men with subclinical tumors may increase their coffee consumption as a result of their underlying disease

#14

A switch in metabolic phenotype in vemurafenib-resistant melanoma cells to increased amino acid dependence serves as an alternate mechanism for survival.

Deeba N. Syed,1 Rahul K. Lall,1 Yong Wei Soon,1 Ahmed Aljohani,1 Changan Guo,1 Feng Liu,2 Frank L. Meyskens,2 James M. Ntambi,1 Hasan Mukhtar1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _University of California, Irvine, CA_.

Despite considerable progress in the understanding of melanoma, and an increase in overall survival, resistance ultimately develops in most patients treated with BRAF inhibitors. To study the molecular mechanism(s) involved in acquired resistance, we generated a Vemurafenib-resistant A2-1b cell line in the BRAFV600E mutated SK-Mel28 melanoma cell background. Viability assays established IC50 of parental SK-Mel28 cells as 0.2 µM, while IC50>12 µM was noted for Vemurafenib-resistant A2-1b cells. Utilizing a quantitative proteomic strategy, profiling of A2-1b versus SK-Mel28 cells resulted in positive identification of 1720 proteins (≤1% FDR). Analysis of this data set identified a subset of 13 proteins, including Enolase 2, Triose-phosphate isomerase and Glyceraldehyde-3-phosphate dehydrogenase with a role in glycolysis, gluconeogenesis and NADH repair, to be differentially regulated in resistant cells. Additional studies validated significantly decreased Enolase 1 and 2 protein expression, suppressed glucose uptake and reduced lactic acid levels in resistant A2-1b cells. The pentose phosphate pathway, in contrast to citric acid cycle intermediates, was unaffected while the latter was significantly suppressed in resistant cells. The energy status of cells was similar as evidenced by equivalent total adenylate, guanylate, NADH, NADP+ and NADPH levels. Flux experiments measuring extracelluar acidification and oxygen consumption rates demonstrated that resistant A2-1b cells are unable to efficiently utilize intrinsic or exogenous fatty acids to meet energy demands. However, A2-1b cells maintained in media deprived of arginine and lysine exhibited increased basal and maximal respiration indicative of an energy reserve that relies on amino acid metabolism. Finally, metabolomics studies showing an overall increase in the absolute concentrations of gluconeogenic and ketogenic amino acids along with significantly elevated S6Kinase expression signify a shift from glucose to amino acids as the major energy source, in resistant A2-1b cells. Our data are consistent with a model of prolonged treatment response in which de-repression of ERK-MAPK pathway activity upon emergence of resistance to Vemurafenib is associated with adaptive changes in cellular metabolism. Sensitivity to BRAF inhibitors in the context of melanoma may not be defined by a reliance on glycolysis for survival, and that switch in metabolic phenotype can serve as an alternate mechanism for cell survival.

#15

Metabolomic profiling of cell death in human lung cancer cells by a novel digitoxin analog.

Yogesh Kulkarni,1 Neelam Azad,1 Vivek Kaushik,1 Juan Sebastian Yakisich,1 Rajkumar Venkatadri,1 Clayton Wright,1 Yon Rojanasakul,2 George O'Doherty,3 Anand Krishnan V Iyer1. 1 _Hampton University, Hampton, VA;_ 2 _West Virginia University, Morgantown, WV;_ 3 _Northeastern Univrsity, Boston, MA_.

Background:

Digitoxin, a cardiac glycoside had shown considerable promise as an anti-cancer therapeutic. However, doses of digitoxin required to inhibit cancer cell proliferation lead to cardiotoxic side effects. We have previously shown that a novel analog of digitoxin, MonoD, activates apoptosis in human lung cancer cells at much lower concentrations as compared to digitoxin. We postulate that treatment with MonoD leads to distinct metabolic signatures on lung cancer cells as compared to digitoxin, which will help delineate the pathways that dictate MonoD action.

Experimental Approach:

NCI-H460 cells from identical passages were used for metabolite analysis using an optimized version of the methanol/water (80:20) protocol for metabolite extraction. The supernatant containing metabolites were suspended in 0.1% formic acid spiked with an internal standard for Q-Exactive analysis. The data was analyzed using SIEVETM 2.0 software with the component extraction algorithm that has been specifically designed for optimal data analysis in untargeted metabolomics experiments. Metabolites that were found to be significantly different between treatment groups were detected via Chemspider/HMDB database search.

Results:

We observed several metabolites along the prostaglandin synthesis pathway which were downregulated in MonoD treated lung cancer cells. Prostaglandins play a key role in the generation of the inflammatory response. Chronic inflammation has been shown to be a causative factor in a variety of cancers. Cyclooxygenase‐2 (COX-2) derived prostaglandin has been previously shown to promote tumor growth by binding its receptors and activating signaling pathways which control cell proliferation, migration, apoptosis, and angiogenesis. Using the metabolite profiling data, we posited that COX-2, a key enzyme involved in prostaglandin synthesis, in addition to NFκB, a key transcription factor of proinflammatory signaling pathway are downregulated with MonoD treatment. Immunoblotting confirmed the downregulation of COX-2 and NFκB in MonoD-treated cells.

Conclusion: Using metabolite profiling, we have elucidated an important mode of action for MonoD in lung cancer cells, and shown that MonoD exerts its anti-tumorigenic properties by downregulating key factors involved in inflammatory response.

#16

NAMPT/visfatin as a novel prognostic marker and therapeutic target in metastatic melanoma.

Valentina Audrito,1 Davide Brusa,1 Sofia La Vecchia,1 Aureliano Stingi,1 Francesca Mazzola,2 Gianna Baroni,3 Francesco Neri,1 Barbara Merelli,4 Salvatore Oliviero,1 Daniela Massi,3 Nadia Raffaelli,2 Mario Mandalà,4 Silvia Deaglio1. 1 _Human Genetics Fndn./Univ. of Turin, Turin, Italy;_ 2 _Università Politecnica delle Marche, Ancona, Italy;_ 3 _University of Florence, Florence, Italy;_ 4 _Papa Giovanni XXIII Hospital, Bergamo, Italy_.

INTRODUCTION: Treatment of BRAF(V600E) mutant metastatic melanoma (MM) with BRAF inhibitors (BRAFi) can be highly effective. However, resistance to therapy develops rapidly, with paradoxical activation of the MAP kinase signalling. For this reason novel drug combinations are being tested.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in the biosynthesis of NAD, an adenine-based dinucleotide, essential cofactor in redox reactions and a substrate of key cellular enzymes. This enzyme may also be secreted in the extracellular space (eNAMPT), where it helps establish a cyto-protective microenvironment, favouring resistance to therapy. NAMPT inhibitors are being tested as cancer therapeutics.

The aim of this work is to determine function of NAMPT in MM and in BRAFi resistant patients.

METHODS: NAMPT expression and activity in MM samples was studied using biochemical, enzymatic, immunofluorescence, immunohistochemical and ELISA assays. RNAseq and functional/metabolic analysis were also performed.

RESULTS: After establishing BRAFi-resistant melanoma cell lines, we observed increased glycolysis and mitochondrial respiration compared to sensitive cells. In line with higher metabolic needs, higher levels of NAD were measured in resistant cells, suggesting that increased NAD synthesis supports the activated metabolic phenotype.

NAMPT levels in resistant cells were markedly elevated both at the transcriptional and protein levels, confirming that this is a critical enzyme in the NAD generation. Inhibition of the BRAF/MEK axis using different agents decreased NAMPT, suggesting a direct relationship between activation of the MAPK axis and NAMPT transcription.

Data using samples from MM patients (n=80) demonstrated that i) NAMPT is up-regulated in melanoma compared to normal melanocytes and ii) NAMPT expression sharply increased upon development of BRAFi resistance (p=0.001). Moreover, eNAMPT levels were markedly elevated in MM sera compared to healthy donors or to patients with localized melanomas. Furthermore, patients with eNAMPT levels >15 ng/ml were characterized by a shorter survival. Lastly, in our cohort of patients eNAMPT decreased sharply after the initiation of therapy with BRAFi, only to increase again in patients developing resistance.

Finally, pharmacological modulation of NAMPT affects melanoma progression and responses to BRAFi. Resistant cells treated with NAMPT inhibitors, FK866 and GMX1777, reduced their proliferation rate. Moreover, NAMPT inhibition led to i) NAD and ATP levels reduction, ii) mitochondrial respiration impairment and iii) decrease of ERK1/2 and NF-kB activation.

CONCLUSIONS: Together, these findings indicate that NAMPT is overexpressed during melanoma transformation and further increased upon paradoxical activation of the MAPK axis. They also provide preliminary data to test NAMPT inhibitors in combination with BRAFi in the treatment of patients with BRAF-mutated MM.

#17

Metabolic analysis of IDH mutant gliomas in Drosophila.

Michaela Brown, Jenna Buccetti, Marla Tipping. _Providence College, Providence, RI_.

Metabolic reprogramming is a common hallmark shared by nearly all proliferating cancer cells and has emerged as an exciting new direction in cancer research. Many signaling pathways have been implicated in mechanisms leading to the shift of metabolic programs in tumors, but more recently a small number of metabolic enzymes have also been identified in this process. Genes encoding the metabolic enzymes Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) were found to be mutated in up to 70% of low-grade and medium grade gliomas, and in 15-20% of adult acute leukemia samples. These findings were the first to link the IDH gene to tumorigenesis. IDH1 and IDH2 function to irreversibly catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Although these are important metabolic enzymes, little is known about the metabolic impact harboring mutant IDH protein has on cells. Our goal is to model the IDH mutant phenotype in Drosophila glial cells for further characterization of both metabolic status, and IDH enzymatic activity. To this end, we have generated Drosophila cell lines and transformant flies that express the most commonly identified IDH mutations under control of the UAS-Gal4 system. We have also investigated the metabolic flux of Drosophila cell lines expressing IDH mutant protein, and loss of IDH function, and glycolytic enzyme expression analysis. Currently, we are investigating the metabolic flux analysis (MFA) using labeled metabolites as well as comparing the protein interactions of the wild type to the mutant IDH cells. The results of these investigations will identify potential new targets for the treatment of aggressive gliomas at the level of cellular metabolism.

#18

Galactose impacts cell growth and redox status of glioblastoma multiforme U87 cell lines.

Jan C. Lumibao,1 Vladimir Kolossov,2 Matthew T. Leslie,1 H R. Gaskins1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _Carl R. Woese Institute for Genomic Biology, Urbana, IL_.

Glioblastoma multiforme (GBM) is the most common and malignant form of brain cancer in adults, with a median survival time of 12-15 months. Characterizing the metabolic alterations of GBM is a crucial step towards identifying potential targets for treatment, with mitochondria, the electron transport chain, and associated reactive oxygen species presenting lucrative targets for therapy. However, the Warburg effect, in which cells generate ATP mainly via glycolysis despite adequate oxygen and functional mitochondria, can render cancer cells resistant to antagonists of the electron transport chain. This prompts investigations into other nodes of oncogenic metabolism such as the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase pathway which can regulate glucose metabolism. The present work defined the sensitivity of GBM U87MG, U87OE (over-expressed EGFR), and U87vIII (hyper-activated vIII mutated EGFR) cell lines to media supplemented with galactose versus conventional glucose concentrations. Cells were transfected with a mitochondrial-targeted Glrx-roGFP2 redox biosensor, a mutated GFP protein whose fluorescence reflects the local redox potential, and incubated in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with either 25 mM glucose (DMEM-Glu) or 10 mM galactose with and without 1 mM pyruvate (DMEM-Gal±Pyr). Glucose deprivation combined with galactose supplementation allows metabolic isolation of mitochondrial contributions to ATP generation because the glycolytic metabolism of galactose yields no net ATP, forcing cells to rely on increased mitochondrial respiration. Cells were exposed to differential media conditions and assessed via flow cytometry to monitor mitochondrial redox homeostasis after 24 h of media exposure. Wild type U87 cells exhibited a slight reduction upon glycolytic blockade, while mitochondria in the mutant EGFR U87OE and U87vIII cells exhibited extreme oxidation. The sulforhodamine B assay, which determines cell density based on a colorimetric measurement of cellular protein content, was performed to assess cell concentration in different media after 72 h of media exposure. Mutant EGFR U87OE and U87vIII cells experienced much greater growth inhibition and cell death compared to wild type U87MG cells. Thus, EGFR-activating mutations increase cellular dependence on glycolytic ATP generation for cell growth and redox homeostasis. Future work will build on these findings to characterize how the metabolic phenotypes and preferences of these cells are linked to their migratory behavior in an engineered 3D hydrogel environment.

#19

Neurofibromatosis type 1 (NF1) status determines sensitivity of soft tissue sarcoma and melanoma cell lines to glutaminase inhibitors.

Tahir N. Sheikh, Parag P. Patwardhan, Gary K. Schwartz. _Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY_.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic syndrome caused by a mutation in or deletion of the NF1 gene. Mutations in the NF1 gene lead to the production of a nonfunctional version of neurofibromin that cannot regulate cell growth and division. As a result, tumors such as neurofibromas can form along nerves throughout the body. The NF1 phenotype is highly penetrant and occurs in 1 in 3,000 to 4,000 people worldwide. Individuals with an altered NF1 gene are at an increased risk of developing benign and/or malignant tumors. NF1 has been shown to negatively regulate Ras activity. Ras-driven cancer cells have also been known to alter glucose and glutamine metabolism. In the present study, we evaluated the role played by NF1 status in determining the sensitivity of sarcoma and melanoma cell lines to glutaminase inhibition and its effect on Ras activity. We tested a panel of soft tissue sarcoma and melanoma cell lines that were either wild-type, null or mutant for NF1. Results from our in vitro proliferation assay showed that compared to wild-type NF1 (STS26T, LS141) cell lines, NF1 mutant (MPNST) and NF1 null (ST88 and MeWo) cell lines showed greater sensitivity to inhibition of proliferation by glutaminase inhibitors CB-839 and BPTES. Western blot analysis showed induction of apoptosis and down regulation of mTORC1 targets such as phospho-S6K and phospho-S6 by glutaminase inhibitors only in NF1 null and NF1 mutant but not wild-type NF1 cell lines. Gene silencing experiments showed that siRNA mediated knockdown of NF1 sensitizes LS141and STS26T cell lines to glutaminase inhibition. Conversely, overexpression of wild-type NF1 GRD (GAP related domain) in MeWo cell line resulted in decreased sensitivity to glutaminase inhibition when tested in a cell proliferation assay, thus, confirming the role played by NF1. Previous reports have shown that mutation or deletion of NF1 results in activation of Ras. In order to test the effect of glutaminase inhibition on Ras activity, we carried out Ras-GTP pull down assay following the treatment with glutaminase inhibitors in NF1 null and wild-type NF1 cell lines. Our results showed that glutaminase inhibition leads to down regulation of activated RAS in NF1 null but not wild-type NF1 cells. SiRNA mediated knockdown of NF1 followed by glutaminase inhibition in wild-type NF1 cell line (LS141) resulted in decreased Ras activity, further confirming our hypothesis. Results from patient derived MPNST tumor xenograft model showed a significant suppression of tumor volume when tumors were treated with glutaminase inhibitor compared to vehicle control. Taken together, our data strongly indicates that NF1 status determines the sensitivity of sarcoma and melanoma cell lines to glutaminase inhibition. Further research is warranted to explore glutaminase inhibition as potential therapy for patients with NF1 loss and/or mutation.

#20

Outlier overexpression of genes involved in fatty acid metabolism and processing define two aggressive prostate cancer phenotypes.

Sandra M. Gaston,1 Kerry Dehimer,2 James Kearns,2 Kyle-Ravi Chinsky,3 Dennis Otali,4 Denise Oelschlager,4 Soroush Rais-Bahrami,4 Jeffrey W. Nix,4 Peter Kolettis,4 George W. Adams,5 William E. Grizzle4. 1 _New England Baptist Hospital, Tufts Medical Center and Tufts Univ School of Medicine, Boston, MA;_ 2 _Tufts Medical Center, Boston, MA;_ 3 _Tufts University, Boston, MA;_ 4 _University of Alabama at Birmingham, Birmingham, AL;_ 5 _Urology Centers of Alabama, Birmingham, AL_.

Major shifts in fatty acid metabolism are part of the malignant phenotype of many types of cancer, including prostate cancer. We have used a biopsy based approach to characterize high grade prostate cancers in both African American and European American patients. This approach allows us to analyze prostate cancers from patients whose disease is too advanced for radical prostatectomy and who are thus not represented in the study specimens available from conventional biorepositories. mRNA gene expression profiling of high grade prostate cancers from biopsies revealed cancer subtypes with very high (>10 fold) levels of overexpression of fatty acid binding protein 5 (FABP5) and fatty acid synthase (FASN). Interestingly, these prostate cancer subtypes showed an either-or phenotype, with "super-overexpression" of either FABP5 or FASN but not both. These findings suggest two "outlier" prostate cancer subtypes with striking differences in the pathways that drive a shift in tumor fatty acid metabolism; one is a fatty acid synthase (FASN) dominant phenotype and the other a previously unrecognized fatty acid binding protein (FABP5) dominant phenotype. In contrast, prostate cancers with lower levels of FABP5 and/or FASN overexpression (2-4 fold) often co-overexpressed both of these genes. Immunohistochemical analyses confirmed prostate cancer FABP5 and FASN overexpression; both showed strong cytoplasmic staining. FABP5 also showed robust nuclear staining consistent with its proposed role in regulating fatty acid mediated gene expression. At both the mRNA and protein levels, the high grade prostate cancers with the highest levels of FABP5 over-expression were more common in African Americans, while the highest levels FASN overexpression were more common in prostate cancers from European Americans. These findings may provide the basis for more effective dietary interventions and targeted therapies for African American and European American patients with these two different subtypes of high grade prostate cancer.

#21

The mTORC2 target Akt is regulated in response to glutamine metabolite levels.

Estela Jacinto,1 Joseph Moloughney,1 Peter K. Kim,1 Nicole M. Vega-Cotto,1 Chang-Chih Wu,1 Thomas Lynch,1 Sisi Zhang,2 Matthew Adlam,1 Sai Guntaka,1 Po-Chien Chou,1 Joshua D. Rabinowitz,2 Guy Werlen1. 1 _Rutgers-RWJ Medical School, Piscataway, NJ;_ 2 _Princeton Univ., Princeton, NJ_.

Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and to fuel biosynthesis of macromolecules. The signals that respond to the fluctuations of these nutrients and how they control metabolic pathways remain poorly understood. mTOR, as part of mTOR complex 1 (mTORC1), responds to amino acids and plays a central role in metabolism. On the other hand, little is known on how mTORC2, consisting of the core components mTOR, rictor, SIN1 and mLST8 is regulated and its metabolic functions. The phosphorylation of the mTORC2 substrate, Akt, is enhanced in cancer cells, suggesting that mTORC2 becomes deregulated during tumorigenesis. Here we found that the activity of mTORC2 is enhanced by diminishing glutamine-derived metabolites. mTORC2 activity is required by glutamine-requiring biosynthetic pathways such as the hexosamine biosynthetic pathway (HBP). Acute nutrient withdrawal augments Akt phosphorylation but does not affect GFAT1 expression. However, extreme starvation that eventually depletes intracellular glutamine metabolites inactivates mTORC2 and downregulates GFAT1 expression. Thus, while mTORC1 senses glutamine abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and provide insights on more effective therapeutic strategies for glutamine-dependent tumors.

#22

Investigating the functional contribution of TANK binding kinase 1 to inflammation induced disease progression.

Victoria H. Burton, Rolf A. Brekken. _UT Southwestern, Dallas, TX_.

Initial stages of human pancreatic ductal adenocarcinoma (PDA) are commonly characterized by an activating mutation in K-RAS along with extensive immune cell infiltration. Direct inhibition of K-RAS through pharmacological means remains a challenge; however targeted inhibition of TANK Binding Kinase 1 (TBK1), a critical downstream effector of mutant active K-RAS is an attractive alternative. High levels of active TBK1 are associated with inflammatory disease and cancer progression. TBK1 and homolog, IKKe activate the immune response transcription factor NF-κB. In metabolically challenged mice, IKKe regulates energy balance by sustaining chronic, low-grade inflammation. We hypothesize that TBK1 signaling is also critical in metabolic regulation and is required for progression of inflammation-induced diseases such as PDA.

A kinase dead, Tbk1Δ/Δ mouse was used to determine the contribution of TBK1 to metabolic disease and PDA progression. Metabolic phenotyping experiments were performed with Tbk1Δ/Δ and Tbk1+/+ mice including a high fat diet weight study, body composition and metabolic chamber analyses. Tbk1+/+ and Tbk1Δ/Δ mice were crossed into a genetically engineered mouse model of PDA to determine the consequences of genetically removing Tbk1 on tumor development and overall survival.

Here we report that Tbk1Δ/Δ mice are significantly smaller, leaner and have less fat than Tbk1+/+ mice on high fat chow diets (HFD) for 14 weeks. Tbk1Δ/Δ mice are more active and have smaller and more abundant adipocytes relative to Tbk1+/+ mice. Additionally, Tbk1Δ/Δ mice are protected from HFD induced hypercholesterolemia and liver steatosis. White adipose tissue (WAT) from HFD fed Tbk1Δ/Δ mice exhibit an induction of brown fat gene expression suggesting that thermogenesis is a contributor to their healthier and more active phenotype. Macrophage infiltration is fairly low in WAT from Tbk1+/+ and Tbk1Δ/Δ mice on normal chow. However on HFD, WAT from Tbk1+/+ mice display a significant increase in macrophage marker expression compared to Tbk1Δ/Δ mice. These findings indicate that HFD fed Tbk1Δ/Δ mice are protected from classic phenotypes of metabolic syndrome.

In PDA, TBK1 is expressed and more active in human PDA cell lines relative to immortalized pancreatic epithelial lines and fibroblasts. Human PDA cell lines are sensitive to a small molecule inhibitor of TBK1 (compound II) in the low micromolar range. In a K-Ras driven genetic mouse model of PDA, TBK1 supports spontaneous pancreatic tumor growth as evidenced by smaller tumors in Tbk1Δ/Δ: PDA mice relative to Tbk1+/+: PDA mice. Our results suggest that TBK1 contributes to metabolic regulation and demonstrate the therapeutic potential of targeting TBK1 in pancreatic malignancies. Current work is focused on delineating the inflammatory and metabolic dysregulation in these animals and determining the precise mechanism by which TBK1 supports the progression of PDA.

#23

Caloric restriction slows tumor growth and metastases in both hormone-sensitive and hormone-resistant prostate cancers.

Robert S. Gitman,1 Meredith LaRose,1 Edouard J. Trabulsi,2 Ajay Palagani,2 Tiziana DeAngelis,1 William K. Kelly,1 Leonard G. Gomella,2 Nicole L. Simone2. 1 _Thomas Jefferson University Hospital, philadelphia, PA;_ 2 _Thomas Jefferson University Hospital, Philadelphia, PA_.

Introduction: The purpose of this study is to assess the effect of caloric restriction (CR) on prostate cancer (PCa) tumor burden, proliferation, and overall survival with and without radiation treatment (RT). Additionally, we determined if the beneficial effect of CR noted are due to a reduction in pro-inflammatory markers hypothesized to enhance PCa tumorgenesis.

Methods: To assess the effect of CR in vivo, 40 male 12 week-old NCRNu-M mice were injected with LNCaP or PC3 tumor cells. Once tumors were palpable, mice were randomly assigned to one of four treatment groups in cohorts of 10: ad libitum (AL) diet, 6.5Gy of radiation (RT), 30% reduction in caloric intake (CR), or CR+RT. Tumor growth and proliferation rate were measured 3 times per week via calipers and live bioluminescent imaging. Additionally, PCa tumor tissue were analyzed to determine if CR can exert its effect on tumor growth and metastases via IGF-1R signaling pathway.

Results: Adding CR to RT decreased tumor progression. In the PC3 murine model, when compared to AL, RT reduced tumor size by 22%, 77% with CR, and 80% with CR+RT. In the LNCaP murine model, compared with AL, CR reduced tumor size by 49% and a 55% reduction with CR+RT. Primary tumor formation began 9 weeks post tumor injection in the radiated cohort and was delayed to 15 weeks post tumor injection in the CR and CT+ RT cohorts. Additionally, time to metastasis was delayed with CR (86 days to metastasis in AL, 93 with RT, and 108 when combining CR+RT). Molecularly, CR decreased multiple members of the IGF-1R signaling pathway. The total IGF-1 and IGF-1R levels exhibited 50% reduction with CR and CR+RT but total INSR levels. CR also induced a significant decrease of pGSK3β and pAKT levels.

Conclusions: For the first time, we have shown that decreasing calories by 30% in both hormone-sensitive and hormone-refractory prostate cancer models, enhance the efficacy of radiation. Our experiments show that even CR alone and in combination with RT can improve PCa tumor proliferation rate, tumor burden, time to metastasis, and overall survival. We hypothesize that these changes are in part, due to the decrease of the IGF-1R signaling pathway. We propose that CR may be used as a novel therapeutic intervention to enhance outcomes of radiation treatment by altering the molecular profile of prostate tumors.

#24

Loss of ubiquitin-specific peptidase 18 (USP18) causes cold sensitive mice by destabilizing the critical regulator of thermogenesis: uncoupling protein-1 (UCP-1).

Xi Liu,1 Yun Lu,2 Lin Zheng,1 David J. Sekula,1 Sarah J. Freemantle,2 Ethan Dmitrovsky1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Geisel School of Medicine, Hanover, NH_.

USP18 is the major ISG15 (Interferon-Stimulated Gene 15) deconjugase that removes ISG15 from substrate proteins. We recently reported that USP18 null mice spontaneously develop leiomyosarcomas. Intriguingly, these USP18 null mice are also markedly cold sensitive as compared to their wild-type littermates. When briefly exposed to cold stimuli, USP18 null mice significantly (P < 0.05) decreased their body temperature. In contrast, wild-type mice exposed to the same experimental conditions exhibited transient changes in their body temperature. We sought to elucidate the engaged mechanism. Expression profiles of critical thermogenic regulatory proteins were examined by immunoblot analyses of brown fat tissues of USP18 null mice. Strikingly, UCP-1 expression was substantially reduced in these tissues of USP18 null versus wild-type mice. To independently confirm that reduced UCP-1 expression was caused by loss of USP18, stable knock-down of USP18 was independently achieved in a panel of murine cell lines by use of transfected small hairpin RNAs (shRNAs). The obtained results were compared to that of transfected control vectors. USP18 down-regulation by different shRNAs markedly reduced UCP-1 protein as compared to controls. As expected, engineered gain of USP18 expression in the same murine cell lines stabilized UCP-1 protein. We explored if UCP-1 destabilization was due to its complex formation with the ubiquitin-like protein ISG15. This was the expected experimental outcome since USP18 is the distinct deconjugase that removes ISG15 from complexed proteins. Immunoprecipitation assays were performed to establish this direct association. These studies revealed a complex formed between ISG15 and UCP-1 protein. Thus, engineered loss of USP18 in mice led to cold sensitive mice from repression of UCP-1. As a consequence of its complex with ISG15, the thermogenic regulator UCP-1 was destabilized. Taken together, this study extends prior work by showing a previously unrecognized link between USP18 and regulation of thermogenesis. This defines a novel role for the USP18 protease in metabolism.

#25

SCAP links glucose to lipids for tumor growth.

Chunming Cheng, Feng Geng, Jeffrey Yunhua Guo, Xiaoning Wu, Xiang Cheng, Arnab Chakravarti, Deliang Guo. _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Increased glucose consumption and elevated lipogenesis are co-occurred in malignancies. But the molecular mechanism of the link between glucose and lipid metabolism remains elusive. Our previous studies have uncovered that sterol regulatory element-binding protein (SREBP-1), an endoplasmic reticulum-bound transcription factor with a central role in lipid metabolism, is highly upregulated in glioblastoma (GBM), the most deadly brain tumor. In the current study, we found that SREBP-cleavage activating protein (SCAP), a key player regulating SREBP trafficking from ER to the Golgi and subsequent SREBP activation, links oncogenic signaling and glucose consumption to lipid metabolism. Our data showed that glucose is a critical activator for SREBP function via N-glycosylation of SCAP. N-glycosylation is a key signal to promote SCAP/SREBP complex move from ER to the Golgi and SREBP activation. Moreover, we found that EGFR/PI3K/Akt signaling activates SREBP-1 via upregulation of SCAP N-glycosylation and its protein levels through enhancing glucose uptake. Genetically silencing SCAP or mutation of the N-glycosylation sites on SCAP downregulates SREBP-1 activity and impairs GBM tumor growth. Taken together, our study revealed that glucose is a critical activator for SREBP-1 function and lipid metabolism, and SCAP integrates oncogenic signaling and fuel availability to SREBP-1-mediated lipogenesis for tumor growth. Our study also demonstrated that targeting SCAP N-glycosylation represents a promising therapeutic strategy to treat malignancies.

#26

Sensitive step in cholesterol biosynthesis reveals role for sterol metabolites in regulating growth of EGFR/KRAS-dependent tumors.

Linara Gabitova, Diana Restifo, Elizabeth Handorf, Kathy Q. Cai, Igor A. Astsaturov. _Fox Chase Cancer Center, Philadelphia, PA_.

We identified SC4MOL and NSDHL, two enzymes in the cholesterol pathway and their substrates, meiosis activating sterols (MAS), as critical regulators of receptor signaling and trafficking in normal development and in cancer. Oncogenic transformation by EGFR or RAS increases the demand for cholesterol, suggesting a possibility for metabolic interference. To test this idea in vivo, we ablated Nsdhl in adult keratinocytes expressing KRASG12D. Strikingly, Nsdhl inactivation antagonized the growth of skin tumors, while having little effect on normal skin. Loss of Nsdhl induced the expression of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, reduced the expression of low-density lipoprotein receptor (LDLR), decreased intracellular cholesterol and was dependent on the liver X receptor (LXR) α. Importantly, EGFR signaling opposed LXRα effects on cholesterol homeostasis, while an EGFR inhibitor synergized with LXRα agonists in killing cancer cells. Inhibition of SC4MOL or NSDHL, or activation of LXRα by sterol metabolites can be an effective strategy against carcinomas with activated EGFR-KRAS signaling.

#27

Understanding mechanisms of nutrient homeostasis: Amino acid availability and regulation of V-ATPase activity.

Laura A. Stransky, Michael Forgac. _Tufts University, Boston, MA_.

Cellular homeostasis requires accurate sensing of nutrient levels and subsequent regulation of biosynthetic and catabolic cellular processes. In cancer cells, metabolism is frequently disregulated to meet the increased demand for cellular building blocks to enable growth and proliferation. Thorough understanding of how homeostatic processes are regulated is necessary to determine how these processes could be manipulated to stop tumor growth. This study aims to understand amino acid homeostasis, with particular focus on the regulation of lysosomal activity, which allows protein breakdown and release of free amino acids. We have identified a novel mechanism of amino acid homeostasis whereby amino acid starvation leads to increased activity of the Vacuolar-ATPase (V-ATPase), as indicated by measurement of V-ATPase-dependent FITC-dextran fluorescence quenching in isolated lysosomes. The V-ATPase is the proton pump responsible for acidifying intracellular compartments, which is necessary for lysosomal function and protein degradation. This increased activity is due to an increase in the amount of fully assembled holoenzymes, as detected by cell fractionation and Western blotting to assess peripheral subunit association with membranes. Assembly of V-ATPase holoenzymes is a tightly controlled process. We describe the function of these changes with respect to lysosomal protein turnover and mTORC1 activation, in which the V-ATPase plays a critical role. We have also sought to characterize the signaling events that transmit the amino acid starvation signal and controls V-ATPase activity in response to changes in amino acid availability in normal and cancer cells that rely on lysosomal function. These changes do not depend on PI3K or mTORC1 activity, both of which have been linked to changes in V-ATPase activity and assembly in other response to other stimuli, such as glucose and growth factor stimulation and are highly activated across cancers. Together, these results shed light on a poorly understood aspect of cellular biology and novel mechanisms of amino acid homeostasis.

#28

Oncogenic RAS modulates glutamine's contribution to mitochondrial redox homeostasis.

Matthew T. Leslie, H. R. Gaskins. _University of Illinois, Urbana, IL_.

Enhanced glutaminolysis contributes to the metabolic reprogramming of transformed cells though it is now well established that enhanced glutamine (Gln) utilization far exceeds their anabolic needs. Oncogenic MYC and RAS contribute to a state of 'glutamine addiction', noted by increased anaplerotic flux of glutamine through the TCA cycle to support mitochondrial respiration. TCA cycle intermediates restore proliferation and prevent cell death in Gln-deprived cells and rescue the decreased proliferation and compromised viability caused by inhibition of transamination. Gln deprivation increases reactive oxygen species (ROS) in RAS and MYC transformed cells, which is abrogated by n-acetyl-cysteine. Glutamate, a product of Gln deamination by glutaminase, is a direct substrate for glutathione (GSH) synthesis in the cytosol, where higher GSH concentrations are maintained separate from mitochondria. Accordingly, we hypothesize that oncogenic RAS redirects Gln to support glutathione biosynthesis and maintain compartmentalized redox homeostasis.

A series of isogenic breast epithelial cells, which vary in RAS status as well as tumorigenicity, and well-characterized KRAS-transformed A549 lung carcinoma cells were used to study the effects of Gln deprivation. Cells were transfected with a mitochondrial-targeted Glrx-roGFP2 redox biosensor, a mutated GFP protein whose fluorescence reflects the local redox potential, and assessed via flow cytometry. TCA cycle intermediates and aminooxy acetic acid (AOA), an inhibitor of cellular transaminases that inhibits the proliferation of Gln-addicted cells, was used to define the pathways affected by RAS transformation. Monobromobimane, which binds sulfhydryls, was used to measure GSH content via flow cytometry +/- Gln supplementation.

The isogenic MCF 10Awt (nontransformed), DCIST24 Ha-RAS (transformed/not metastatic), and CA1DT24 Ha-RAS (metastatic) cell series varied in responsiveness to 24 h Gln deprivation. Resting mitochondrial redox potential in nontransformed 10A cells did not deviate after 24 h of Gln deprivation, while DCIS, CA1D, and A549 cells became oxidized. Alpha−ketoglutarate (αKG) fully abrogated mitochondrial oxidation after Gln deprivation, while aspartate and oxaloacetate did not, likely reflecting their inability to generate glutamate via the cytoplasmic transaminase GOT1. AOA reversed the αKG-mediated rescue of redox balance in Gln-deprived cells and also oxidized mitochondria in Gln-supplemented cells. Intracellular GSH was diminished (≈ 20%) upon 24 h Gln deprivation in DCIS, CA1D and A549 cells but not in parental MCF-10A breast epithelial cells.

These data demonstrate that Gln is used to maintain glutathione concentrations as well as mitochondrial redox poise in RAS-transformed cells. Reliance on Gln for redox homeostasis may yield targets to selectively predispose cancer cells to ROS as a chemotherapeutic strategy.

#29

MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation.

Jillian M. Silva,1 Marian M. Deuker,1 Bruce C. Baguley,2 Martin McMahon1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University of Auckland, Auckland, New Zealand_.

Oncogenic transformation of mutationally activated BRAF or NRAS melanocytes often requires the cooperation of genetic aberrations in the phosphoinositide 3-kinase (PI3K) pathway to promote melanomagenesis. Activating point mutations in the p110α catalytic subunit of PI3K are detected at a low frequency in both BRAF and NRAS mutant melanomas, yet the principle mechanism of this cooperation remains elusive. Thus, using BRAFV600E/PIK3CAH1047R, NRASQ61H/PIK3CAH1047R, and PIK3CAE545K mutant melanoma cells derived from metastatic melanoma patients treated with pathway-targeted inhibitors, we examined the contribution of mutational activation of PIK3CA to melanoma maintenance and signaling and the consequential response of these PIK3CA mutant cells to α-specific PI3K inhibition. Combined MEK and isoform-selective PI3K inhibition elicited more potent anti-proliferative effects and greater suppression of S-phase progression of the cell cycle compared to single-agent inhibition of either pathway. Analysis of signaling downstream of MEK or PI3K revealed that these pathways cooperated to regulate PIK3CA cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Despite the profound anti-proliferative and biochemical effects of α-specific or PI3Kβ-sparing class I PI3K inhibition in vitro, these agents elicited largely cytostatic effects on PIK3CA xenograft tumors. However, combined inhibition of PI3Kβ-sparing class I PI3K and MEK did significantly cooperate to reduce tumor growth compared to the corresponding monotherapies. Furthermore, this study provides a biochemical mechanism to explain how mTORC1/2 moderates the cooperation of MEK and PI3K signaling for the maintenance of PIK3CA mutant melanoma cell proliferation.

#30

Kras mediates amino acid metabolism during nutrient stress.

Dana Gwinn, Alejandro Sweet-Cordero. _Stanford University, Stanford, CA_.

Kras is a small GTPase that functions in the transmission of extracellular mitogenic stimuli, and is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC). Previously, Kras has been demonstrated to play a role in tumor metabolism in certain cell types and cancers, but this has yet to be analyzed in the context of NSCLC. We have analyzed how Kras changes metabolic gene expression by expressing a doxycycline-inducible hairpin against Kras in 4 NSCLC cell lines. One of the most regulated metabolism genes is asparagine synthetase (Asns), which transfers the γ-amino group of glutamine to aspartate, yielding glutamate and asparagine. We find elevated levels of Asns in Kras mutant tumors, and that expression of Asns correlates with poor patient outcome. Knockdown of Asns in the absence of exogenous asparagine in the media results in inhibition of cell growth and induction of apoptosis that is rescued by addition of asparagine. Additionally, under conditions of low glutamine levels, knockdown of Asns sensitizes cells to apoptosis, and overexpression of Asns is protective under these conditions. We also find a role for the glutaminase-activity of Asns in glutamine-mediated anapleurosis of the TCA cycle. These observations suggest Asns may be an interesting therapeutic target for NSCLC tumors with mutations in Kras. 

### Altered Glucose Metabolism in Cancer

#31

Metabolic reprogramming in ovarian cancer.

JI HEE HA, Rangasudhagar Radhakrishnan, Jeremy D. Ward, Muralidharan Jayaraman, Danny N. Dhanasekaran. _OUHSC, Oklahoma city, OK_.

Ovarian cancer is currently the most lethal gynecologic malignancy, with no new significant changes in treatment options for these patients in the last 30 years. Importantly, ovarian cancer patients have increased levels of lysophosphatidic acid (LPA), a bioactive phospholipid in ascites and serum that has been linked to driving oncogenesis and progression of ovarian cancer. Accumulating evidence has implicated Hypoxia-inducible factor-1α (HIF-1α) as a critical mediator of the glycolytic shift observed in cancer cells. However, the precise biological role of LPA in regulating HIF-1α-mediated glycolytic shift is largely unknown. Therefore, we were interested in examining the potential role of LPA in promoting metabolic reprogramming in ovarian cancer via HIF-1α signaling. In this study, we identified a novel mechanism by which LPA upregulates HIF-1α expression via Gαi2 (the gip2 oncogene) in human ovarian cancer cells. This study demonstrates that LPA induces a glycolytic shift via LPA-mediated activation of Gαi2, which causes a subsequent upregulation of HIF-1α in ovarian cancer cells. Additionally, we show that LPA-signaling via Gαi2 induces an increase in the expression of Hexokinase-2 (HK2) and Glucose transporter 1 (GLUT1), which are known targets of HIF-1α. We also demonstrate that LPA induces an increase in extracellular acidification rate (ECAR) in a dose dependent manner in both ovarian cancer cell lines and in patient-derived cells taken from the ascites fluid of ovarian cancer patients using an XFe96 analyzer. Furthermore, we found that inhibition of Rac signaling caused a reduction in LPA-induced ECAR, identifying Rac as a critical downstream component of Gαi2-medidated increase of ECAR. Moreover, using NAC, an inhibitor of redox signaling, we found that this caused a decrease in LPA-induced ECAR in SKOV3-ip cells, indicating that inhibition of redox signaling abolishes LPA-induced ECAR in ovarian cancer cells. Similarly, the use of EUK-134, a scavenger of superoxide and H2O2, also decreased LPA-induced increase in ECAR. Altogether, these results indicate that LPA regulates glycolysis through redox signaling via a Gαi2-Rac-HIF1α-dependent signaling pathway. Most importantly, the Gαi2-Rac-dependent pathway identified in this study more than likely serves as a potential driver of HIF-1α-mediated metabolic changes in ovarian cancer cells and represents a potential target for therapy in these patients.

#32

Inhibition of cancer cell growth by combined treatment with lactate dehydrogenase (LDHA) inhibitors and either phenformin or inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3).

Michael A. Lea, Yolanda Guzman, Charles desBordes. _Rutgers New Jersey Medical School, Newark, NJ_.

The tendency for enhanced glycolysis in cancer cells presents a target for chemotherapy. In previous studies we observed that proliferation of colon and bladder cancer cells can be inhibited by treatment with either phenformin or an inhibitor of PFKFB3 namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work we have examined the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15). The LDHA inhibitors that we chose to study in increasing order of IC50s were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. A synergistic anti-cancer effect of phenformin and oxamate has been reported. In the present work with colon and bladder cancer cells, additive but not synergistic growth inhibitory effects were seen with the LDHA inhibitors of which NHI-2 was effective at the lowest concentrations. Growth inhibition with PQP and PFK15 was compared in colon (Caco-2 and HT29) and bladder cancer cells (5637, HT1376, RT4, SW780, T24, TCCSUP and UM-UC-3). Apart from RT4 cells where the effects were similar, the effects were somewhat greater with PFK15 than with PQP and for both compounds the actions were seen at lower concentrations than in previous studies with 3PO. Actions on medium acidification and glucose uptake are more readily observed in the most rapidly growing cell lines. Effects of phenformin on medium pH and glucose concentration were decreased by the PFKFB3 and LDHA inhibitors that were examined. In accord with our previous studies on inhibitors of glycolysis, the increased medium acidification and glucose uptake caused by phenformin could be blocked by combined treatment with PFKFB3 or LDHA inhibitors. At the same time additive growth inhibitory effects were observed. The results supported the concept that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation while mitigating the lactic acidosis caused by phenformin as a single agent.

#33

**NSCLC cells internalize ATP** in vitro **and** in vivo **using multiple endocytotic pathways.**

Yanrong Qian, Xuan Wang, Yunsheng Li, Yanyang Cao, Xiaozhuo Chen. _Ohio University, Athens, OH_.

Upregulated glycolysis in cancer, the Warburg effect, has been studied for almost one century. However, the biological reasons for the upregulation are still debated and the effect is far from fully understood. One of the major controversies related to the effect is the role of ATP synthesis. Intratumoral ATP concentrations are found to be 10^3-10^4 times higher than those in normal tissues of the same cell origin. However, whether and how cancer cells use the abundant extracellular ATP was unknown until we recently reported that cancer cells internalize ATP by macropinocytosis (1). Extracellular ATP was found to increase intracellular ATP concentration and promote cell proliferation and drug resistance in cancer cells (1). Here we report that, using a nonhydrolyzable fluorescent ATP (NHF-ATP) as an ATP surrogate, high and low molecular weight dextrans as endocytosis tracers and fluorescence microscopy detection, cultured human NSCLC A549 and H1299 cells as well as xenografted A549 tumors were found to internalize ATP at concentrations within the reported intratumoral ATP concentrations. Endocytosis inhibitor studies show that in addition to macropinocytosis, clathrin- and caveolae-mediated endocytoses contribute to the ATP internalization, which led to a ~30% increase in intracellular ATP level after 45 min of ATP incubation. This increase cannot be accounted for by purinergic receptor signaling or increased intracellular ATP synthesis rates. Internalization of NHF-ATP and dextran exhibits time-dependent profiles parallel to the extracellular ATP-induced intracellular ATP level elevation. All these demonstrate the observed ATP increase is a result of internalization of extracellular ATP and NHF-ATP is a powerful tool for studying ATP internalization. These findings significantly deepen our understanding of the Warburg effect by showing how cancer cells in tumors take up intratumoral ATP and perform previously unrecognized biological functions. It strongly suggests ATP-sharing among cancer and stromal cells and identifies multiple novel anticancer targets (2).

1. Qian Y, Wang X, et al., Cancer Lett. 351(2014): 242-5.

2. Chen X, Qian Y, Wu S. Free. Radic. Biol. Med. 79(2015): 253-263.

#34

Monocarboxylate transporters as potential therapeutic targets in human ovarian cancer.

Amy Boyers, Ian Stratford. _University of Manchester, Manchester, United Kingdom_.

Introduction- Cancer cells have the ability to favour glycolysis for the production of lactate even in the presence of sufficient levels of oxygen. This is known as the 'warburg effect' or 'aerobic glycolysis'. As a consequence of this glycolytic switch, there is an accumulation of lactate within cells. Therefore, cells must remove this excess lactate in order to prevent cell death via intracellular acidosis. In order to do this cells use Monocarboxylate Transporters (MCTs), which are responsible for the transport of lactate in and out of cells. MCT1 and MCT4 have been found to be over expressed in many types of human cancers, including ovarian cancer.

Aims- The aim of this study was to assess how changing the expression levels of MCT1 and MCT4 effects the growth and response to therapy of four Human Ovarian cancer cell lines (Skov3, Caov3, OV90 and Ovcar3).

Methods- Cell lines were stably transfected to either over express MCT1 (Skov3, Caov3, and OV90 cells), MCT4 (OV90 cells) or to contain doxycycline inducible silencing of MCT4 (Skov3, Caov3 and Ovcar3 cells). The effect of changing the expression levels of MCT1 and MCT4 on the function of these cell lines was assessed. Assays to look at changes in cell proliferation, lactate accumulation and release, and the cytotoxicity of Paclitaxel and Carboplatin were determined in both normoxia (21% O2) and hypoxia (0.1/1% O2).

Results- Over expressing MCT1 in the three human ovarian cell lines had no effect on cell growth or lactate release in normoxia or hypoxia. Also when MCT1 was over expressed in Skov3 and Caov3 cells there were no changes in their sensitivity to Carboplatin or Paclitaxel. However, when MCT1 was over expressed in OV90 cells, they became slightly more resistant to Paclitaxel. Over expressing MCT4 in OV90 cells also had no effect on cell growth in normoxia, but significantly more lactate was released by these cells. Secondly, when these cells were grown in hypoxia they survived for a greater period of time than wild type cells. This correlated with a much higher amount of lactate being released by these cells. Finally, these cells were more resistant to Paclitaxel than both the MCT1 over expressing cell lines and their wild type counterparts.

Conclusions- Changing the expression levels of MCT1 and MCT4 in Human Ovarian cancer cells have an effect on normal cell function. This suggests that MCTs could be potential therapeutic targets in ovarian cancer.

#35

Breast cancer metabolism: Are ketone bodies energetic substrates.

Alyssa M. Dixon, Michael Weichhaus. _Chaminade University of Honolulu, Honolulu, HI_.

Cancer cell metabolism differs from non-transformed cells in that cancer cells do not employ oxidative phosphorylation for ATP-production from glucose even in the presence of adequate oxygen concentrations. This conjecture increases the cancer cell's reliance on glucose, potentially reducing the cancer cell's ability to utilize non-glucose substrates. The main alternative to glucose are high-energy breakdown products of hepatic lipid metabolism, collectively termed ketone bodies. Non-transformed cells are able to metabolize ketone bodies to form ATP, but the role of ketone bodies' metabolism in human breast cancer cells is unclear, since it relies on metabolic functions downstream of glucose metabolism. Here we aimed to determine if human breast cancer cells will utilize ketone bodies (beta-hydroxybuterate) as energy substrate in the absence of glucose.

Cell proliferation of MDA-MB-231 and MCF-7 human breast cancer cells was measured after 48 h or 72 h of treatment using an MTT-Assay. Treatment conditions were various glucose concentrations (4.5 g/l to 0 g/l) and 25 nM beta-hydroxybuterate (BHB) for all cells after overnight serum starvation.

After 48 h, cell proliferation decreased to 47% (p<0.001) in no glucose medium compared to proliferation observed for cells in 4.5 g/l glucose for MDA-MB-231 cells. Cell proliferation at glucose concentrations as low as 0.125 g/l did not change cell proliferation in these cells. After 48 h, cell proliferation of MCF-7 cells decreased to 24% (p<0.001) for cells treated with 0 g/l glucose, to 83% (p=0.0078) for cells treated with 0.125 g/l glucose and to 87% (p=0.0108) for cells treated with 0.25 g/l glucose.

After 72 h, cell proliferation decreased to 30.5% (p<0.001) after treatment with no glucose compared to proliferation observed for cells in 4.5 g/l glucose for MDA-MB-231 cells. Cell proliferation at glucose concentrations as low as 0.125 g/l did not significantly change cell proliferation in these cells. After 72 h, cell proliferation of MCF-7 cells decreased to 22% (p<0.001) for cells treated with 0 g/l glucose and to 74% (p=0.0288) for cells treated with 0.125 g/l glucose compared to proliferation observed for cells in 4.5 g/l glucose. All conditions and time points had been supplemented with 25 nM BHB.

These preliminary results indicate that human breast cancer cells are not able to use ketone bodies as energy substrates in the absence of glucose. An additional finding was also that more aggressive, triple-negative breast cancer cells are able to survive with very low glucose conditions. Pending further investigation, this may provide a rationale for dietary carbohydrate restriction as neoadjuvant therapy for breast cancer patients, but may necessitate further glucose restrictions than what can be achieved through diet alone.

#36

Light exposure at night influences host/cancer circadian regulatory dynamics, Warburg effect, and human prostate cancer progression in nude rats.

Robert T. Dauchy,1 Melissa A. Wren,1 Erin M. Dauchy,2 Steven M. Hill,1 Lin Yuan,1 Shulin Xiang,1 Yan Dong,1 Victoria P. Belancio,1 David M. Blask1. 1 _Tulane University School of Medicine, New Orleans, LA;_ 2 _Louisiana State University Health Sciences Center, New Orleans, LA_.

Current evidence indicates that rotating night shift workers have an increased risk of developing breast and prostate cancers, which have been associated with light at night (LAN)-induced circadian disruption as the principal risk factor. Previously, we demonstrated that animal room dark phase light contamination with as little as 0.20 lux (0.08 μW/cm2) suppressed the nocturnal production of the circadian oncostatic neurohormone melatonin and stimulated human breast tumor growth and metabolism. The circadian melatonin signal suppresses tumor growth and metabolism via an MT1 melatonin receptor-mediated signaling mechanism involving inhibition of aerobic glycolysis (Warburg effect) and linoleic acid (LA) uptake and conversion to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) culminating in down-regulation of the epidermal growth factor and insulin-like growth factor-1 pathways. We developed a new tissue-isolated androgen-receptor positive (AR+), castration-sensitive VCaP prostate tumor model in adult male athymic nude rats (Crl:NIH-Foxn1rnu), to test the hypothesis that nocturnal melatonin levels inhibit, while dim LAN (dLAN)-induced suppression of nocturnal melatonin production stimulates, tumor signaling, metabolic and growth activity. VCaP xenograft-bearing rats (n = 6/group) maintained on either a control light/dark cycle (LD, 12:12; 300 lux light phase intensity) or an experimental light/dark cycle (LD, 12:12dLAN (0.2 lux; dark phase intensity) for 6 weeks resulted in a 2.5-fold decrease in latency-to-onset (time of implant to first palpable mass) and 2-fold increase in tumor growth rates in experimental animals lacking a nocturnal circadian melatonin signal as compared to control animals with an intact melatonin signal. In control animals, plasma melatonin levels were high in the mid-dark phase (183.4 ± 12.8 pg/mL) and low (2.2 ± 0.4 pg/mL) in mid-light phase, while they were low throughout the 24-hr period in dLAN-exposed animals. Tumors harvested during the mid-dark phase (2400 h) revealed that cAMP levels, Warburg effect (increased glucose uptake and lactate production), LA uptake, 13-HODE production, and DNA [3H]Thymidine incorporation were all significantly elevated (P < 0.001) in dLAN as compared with the controls. Signaling pathways AKT, MEK, ERK ½, STAT3, GSK3ß, and NFκß were all phospho-activated along with increased expression of Aldo-keto reductase family 1 member C3 (AKR1C3) under dLAN conditions. AKR1C3 has been associated with intratumoral androgen synthesis and the development of castration-resistance. These findings are the first to show that the nocturnal melatonin signal inhibits, while dLAN stimulates the Warburg effect, LA metabolism and growth activity, signaling activity and AKR1C3 expression in VCaP human androgen-sensitive prostate cancer.

#37

Effect of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment.

Kishu Kitayama, Masakazu Yashiro, Tamami Morisaki, Go Masuda, Hiroakai Kasashima, Yuichiro Miki, Haruhito Kinoshita, Tastunari Fukuoka, Tsuyoshi Hasegawa, Masaichi Ohira, Kosei Hirakawa. _Osaka City University, Osaka, Japan_.

Background: Many kinds of solid tumor have heterogeneously a hypoxic environment. Cancer cells acquire characteristics that allow them to survive and proliferate in the hypoxic environment. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear. "Warburg effect" is known as anaerobic glycolysis of cancer cells, contributes tumorigenesis and tumor development. The aim of this study was to analyze the significance of glucose metabolism-related enzymes on the proliferation of gastric cancer cells in hypoxic microenvironment.

Materials and Methods: Two hypoxia-resistant cancer cell lines, OCUM-12hypo cells and OCUM-2MD3hypo cells, and their parent OCUM-12 cells and OCUM-2MD3 cells were used. OCUM-12hypo cells and OCUM-2MD3hypo cells were cloned from OCUM-12 cells and OCUM-2MD3 cells by continuous exposure to 1% oxygen, respectively. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. mRNA expression level of enolase 1 (ENO1), pyruvate kinase isozymes M2 (PKM2), and glutaminase (GLS) were evaluated by RT-PCR. Effects of inhibition of glucose metabolism-related enzymes on the proliferation and apoptosis were examined by CCK assay or FACScan analysis using siRNA (siENO1, siPKM2, and siGLS), or PKM2 inhibitor Shikonin and GLS inhibitor BPTES.

Results: Glucose metabolism-related enzymes, ENO1 and PKM2, were significantly increased in both hypoxia-resistant cancer cell lines in comparison with their parent cell lines. Expression level of ENO1, PKM2, and GLS were also significantly high in both hypoxia-resistant cancer cell lines in comparison with their parent cell lines. The proliferation of OCUM-12hypo cells and OCUM-2MD3hypo cells was significantly inhibited by the knockdown of PKM2 and GLS, but not by the knockdown of ENO1. Cell proliferation was also significantly inhibited by PKM2 inhibitor and/or GLS inhibitor. A synergistic anti-tumor effect for gastric cancer cells in hypoxia was found in combination with PKM2 inhibitor and GLS inhibitor. The combination of PKM2 inhibitor and GLS inhibitor increased apoptosis rates of hypoxic gastric cancer cells.

Conclusion: PKM2 and GLS might be important enzymes for the proliferation of gastric cancer in hypoxic microenvironment. The combined treatment with PKM2 inhibitor and GLS inhibitor produced synergistic anti-tumor effects, and might be a therapeutically promising for the treatment of gastric cancer.

#38

The clinical significance of GLUT1 expression in gastrointestinal stromal tumor.

Daisuke Kuroda, Junji Kurashige, Masaaki Iwatsuki, Tsugio Eto, Yuka Tamaoki, Mayuko Ohuchi, Kenichi Nakamura, Ryuma Tokunaga, Hironobu Shigaki, Hiromitsu Hayashi, Takatsugu Ishimoto, Yoshifumi Baba, Yasuo Sakamoto, Naoya Yoshida, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: Gastrointestinal stromal tumor (GIST) is, though it is as rare as its incidence is about 7 to 20 cases per million population per year, one of the most common non-epithelial malignant tumors of the gastrointestinal tract. Recently it was reported that Fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography-computed tomography (PET/CT) is useful in diagnosis and evaluation of malignant grade of GIST. At the same time, expression of glucose transporter 1 (GLUT1), which is a membranous protein facilitates the transport of glucose across the plasma membranes into cells, was reported to be correlated with maximum standardized uptake value (SUVmax) in PET/CT and malignant grade of various kinds of cancers. In this study, the correlation of GLUT1 expression with malignant grade in GIST was investigated.

Methods: We evaluated the protein expression of GLUT1 in immunohistochemistry in the surgically resected specimens in 67 GIST cases, and analyzed the correlation of the other clinicopathological factors.

Results: The number of cases in which the expression of GLUT1 judged to be confirmed in immunohistochemistry was 45 (73.7%). GLUT1 expression group had significantly higher preoperative SUVmax in PET/CT than GLUT1 non-expression group (p=0.03), had larger tumors in diameter (p=0.015) , and was more malignant by Fletcher classification (p<0.01). Additionally, tumor rupture rate tended to be higher in GLUT1 expression group, though there is no tendency of the correlation with tumor necrosis. Eventually, relapse free survival, as not significantly, tend to be shorter in GLUT1 expression group.

Conclusion: In our study, expression of GLUT1 is correlated with malignant grade in GIST. GLUT1 expression might be a phase of higher metabolic and malignant activity of GIST cells. GLUT1 might be a useful prognostic marker in GIST cases.

#39

UDP-N-acetylglucosamine as a regulator of cancer cell signaling and microenvironment.

Sanna Oikari,1 Satu Tiainen,2 Tiia Kettunen,2 Amro Masarwah,2 Ritva Vanninen,2 Markku Tammi,1 Päivi Auvinen2. 1 _University of Eastern Finland, Kuopio, Finland;_ 2 _Kuopio university hospital, Kuopio, Finland_.

A common feature to most cancers is their disturbed glucose metabolism: increased glucose and glutamine uptake combined with preference to utilize aerobic glycolysis, a phenomenon called Warburg effect. In addition to providing fast, yet inefficient way to produce energy, Warburg effect increases the availability of glycolysis intermediates that function as starting points of metabolic pathways facilitating the fast cell proliferation. One of the pathways involved is the hexosamine biosynthesis producing UDP-N-acetylglycosamine (UDP-GlcNAc). UDP-GlcNAc is a nucleotide sugar with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAcylation of intracellular proteins. UDP-GlcNAc is linked to cancer through its dependence on glycolysis intermediate availability and by the fact that both hyaluronan and O-GlcNAcylation are closely involved in the development and progression of various cancers. Our hypothesis is that the disturbed sugar metabolism increases the levels of UDP-GlcNAc, which implements its tumorigenic effects through hyaluronan and O-GlcNAcylation, and that these changes manifest themselves also in clinical data.

In order to test our hypothesis we collected 33 biopsy samples from breast cancer patients, and 13 healthy control samples. Clinical data and immunohistochemical samples are available from the operated patients. From these samples we have analyzed UDP-sugar content with HPLC, hyaluronan levels with ELISA-like method and by immunohistochemistry, and measured mRNA expression of key enzymes with quantitative RT-PCR.

Our results show for the first time that UDP-sugars levels are indeed elevated in breast cancer patients. The level of UDP-GlcNAc shows a 12-times increase, while other UDP-sugars were 4 to 6 times higher. To investigate the cause of differential increment of UDP-GlcNAc and other UDP-sugars, we measured the mRNA levels of genes regulating the synthesis of UDP-GlcNAc. Cancer patients had increased expression of GFAT2 while others (GFAT1, GNPDA1 and 2) remained unchanged. In accordance to our hypothesis, the increased levels of UDP-GlcNAc correlated with changes in the mRNA expression of enzymes involved in both hyaluronan synthesis and O-GlcNAcylation of proteins.

Our results show that the increased glucose uptake associated to aerobic glycolysis dramatically increases UDP-sugars in breast cancer, promoting malignant growth by increased hyaluronan synthesis and O-GlcNAc signaling.

#40

Metabolic rewiring in Rb deficient cells during cancer progression.

Susumu Kohno,1 Nobuyuki Okahashi,2 Shunsuke Kitajima,3 Sawako Suzuki,4 Tomoaki Tanaka,4 Fumio Matsuda,2 Hiroshi Shimizu,2 Chiaki Takahashi1. 1 _Kanazawa University, Kanazawa, Japan;_ 2 _Osaka University, Suita, Japan;_ 3 _Dana-farber Cancer Institute, MA;_ 4 _Chiba University, Chiba, Japan_.

Tumor suppressors control critical steps of cellular metabolism and its inactivation induces cancer specific-metabolic features including aerobic glycolysis. Glycolysis contributes to energy generation and supplying precursors of biomass, e.g., nucleotide, amino sugar, amino acid and lipids. These macromolecules were required for proliferation and cancer survival. Although aerobic glycolysis is now widely accepted as a metabolic feature of cancer, its causal relationship with cancer progression is still unclear.

In common type cancers, inactivation of RB tumor suppressor is prevalently observed during tumor progression. Previous studies demonstrated RB inactivation induces inflammation, angiogenesis, drug resistance, etc., as well as cell cycle progression. We currently focus on the impact of RB deficiency on the central carbon metabolism, e.g., glycolysis, TCA cycle and glutamine metabolism. To address this point, we established a mouse model in which RB depletion in p53-null soft tissue sarcoma cells induced undifferentiated phenotype associated with "glycolytic to glutaminolytic" switch (metabolic rewiring) most probably due to down-regulation of Pgam2 activity. Up-regulation of Pgam2 in these cells antagonized the metabolic rewiring, and suppressed spherogenic and tumorigenic activities of RB deficient cells, however did not affect cell proliferation in 2D culture conditions. Moreover, Pgam2 depletion suppressed RB-dependent myogenic differentiation. These findings suggest that RB controls tumorigenicity and differentiation by influencing central carbon metabolism via Pgam2.

#41

DCA bind to EGFR/EGFRvIII/PDK1 and affect the proliferation and growth in TMZ resistant glioblastoma model.

Kiran Kumar Velpula,1 Kamlesh Sahu,2 Jack Tuszynski,2 Maheedhara R. Guda,1 Swapna Asuthkar,1 Sarah E. Martin,1 Justin D. Lathia,3 Andrew J. Tsung1. 1 _University of Illinois College of Med. at Peoria, Peoria, IL;_ 2 _University of Alberta, Edmonton, Alberta, Canada;_ 3 _Cleveland Clinic, Cleveland, OH_.

Glioblastomas are characterized by amplification of epidermal growth factor receptor (EGFR).

Approximately half of the GBM tumors with EGFR over-expression also express a constitutively active ligand independent EGFR variant III (EGFRvIII). This phenotype represents a tumor specific target correlating with a high growth potential. Despite advances in treatment, whether surgery, chemotherapeutics or molecular targeted therapies, very few have shown promise. Temozolomide (TMZ), as standard of care alkylation therapy, demonstrates improved survival when administered with concomitant radiotherapy. Unfortunately, acquired TMZ chemo-resistance is observed in more than 90% of recurrent GBMs. To understand the mechanisms in the context of TMZ resistance, we generated an in vitro TMZ resistant model by continuous exposure of U373 cells constitutively expressing EGFRvIII (U373vIII) to 150uM TMZ for 6 months (U373vIIIR). Dicholoroacetate (DCA) is a known metabolic inhibitor of pyruvate dehydrogenase kinase 1 (PDK1). Its application to cancer has recently been revived in the realm of metabolic oncology, where reversal of the Warburg effect has resulted in decreased tumor growth. Treatment of U373, U373vIII, and U373vIIIR GBM cell lines with DCA was found to induce cytotoxicity and reduced cell survival across all cell lines. Further, micro array studies conducted on U373vIII or U373vIIIR and their subsequent treatment with DCA revealed that PDK1 is the sole target in TMZ resistant tumors expressing EGFRvIII. Additionally, we demonstrated that 1mM DCA induced mitochondrial membrane potential change as evidenced in JC-1 staining and electron microscopic studies confirming mitochondrial apoptosis. Consistent with our previous findings that EGFR interacts with PDK1, our computational analysis revealed that DCA aligns itself to the binding sites of both EGFR and EGFRvIII with strong binding affinity apart from its binding to PDK1. Clinically, expression of EGFRvIII correlated with PDK1 when compared to EGFR in GBM surgical specimens. Collectively, our studies demonstrate that principles of metabolic oncology and DCA are active in GBM treatment despite resistance providing new insight into the development of alternative treatment in TMZ failure.

#42

A new type of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and ATP-drug competition.

Xuan Wang, Yunsheng Li, Yanrong Qian, Yanyang Cao, Xiaozhuo Chen. _Ohio University, Athens, OH_.

Drug resistance is a major problem in cancer therapies and responsible for most relapse, one of the major causes of death in cancer. Several cancer drug resistance mechanisms are known, including genetic mutations in protooncogenes or tumor suppressor genes, cancer stem cells, and multi-drug resistance (MDR). Among these mechanisms, MDR is ATP-dependent. Intratumoral (extracellular) ATP levels of various cancer types are 10^3 to 10^4 times higher than those in their corresponding normal tissues but the biological significance of the high intratumoral ATP concentrations is unknown. We recently reported that extracellular ATP is internalized by cancer cells via macropinocytosis and enhances their growth and survival (1). Based on these results, we hypothesized that the internalized ATP also promotes drug resistance. Here we report that extracellular ATP, at concentrations in or below the range of reported intratumoral ATP levels, substantially increased intracellular ATP levels and promoted cancer cell drug resistance to tyrosine kinase inhibitor (TKI) sunitinib in human NSCLC A549 cells. The ATP increase and the drug resistance were mediated, at least in part, by macropinocytosis, clathrin- and caveolae-mediated endocytoses. The elevated intracellular ATP induced upregulated phosphorylation of PDGFR and proteins/enzymes in the PDGFR-mediated signaling pathways, such as Akt, mTOR, Raf and MEK, resulting in augmented cell growth and reduced apoptosis induced by sunitinib. The upregulated phosphorylation was not mediated by purinergic receptor signaling. Furthermore, in vitro, ex vivo, and in vivo, extracellular ATP partially restored phosphorylation levels of PDGRF and its downstream proteins/enzymes inhibited by sunitinib, a PDGFR inhibitor (TKI). These results strongly suggest that the extracellular ATP-increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitors for the ATP-binding site of PDGFR, enhancing the Akt-mTOR and Raf-MEK signaling pathways and promoting cancer cell survival. All these findings significantly expand our understanding of the roles of extracellular ATP in the Warburg effect (2) and drug resistance in cancer and indentify a new anti-drug resistance target.

1. Qian Y, Wang X, et al., Cancer Lett. 351(2014): 242-5.

2. Chen X, Qian Y, Wu S. Free. Radic. Biol. Med. 79(2015): 253-263.

#43

MicroRNA-130b mediates a metabolic switch to promote cutaneous squamous cell carcinoma development.

Tran N. Nguyen, Sydney Moyer, Charles H. Adelmann, Vida Chitsazzadeh, Kenneth Y. Tsai. _MD Anderson Cancer Center, Houston, TX_.

Metabolic reprogramming has been emerging as a hallmark of cancer. Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, with about 700,000 new cases annually in the U.S. We aim to investigate the role of miR-130b in reprogramming metabolism as a driver of cSCC development.

Since skin cancers have the highest mutational loads among any human cancers, we used well-controlled comparisons across tissues and across species to narrow down the number of important candidate drivers. Using the approach of matched isogenic human samples and cross-species analysis using our UV-driven hairless mouse model, we identified aberrantly expressed microRNAs (miRNAs) and their target mRNAs in order to unravel additional mechanisms behind cSCC progression. We focus on miRNAs because they regulate numerous mRNA targets and can be manipulated for cancer therapy.

We observed that miR-130b is increased in both human and mouse cSCC by 3.3-fold, and 2.6-fold, respectively. In adipocytes, miR-130b is known to reduce fat deposition and cell differentiation through targeting PPARγ. Our data and the TargetScan algorithm suggested that in keratinocytes, miR-130b suppresses PPARγ, FBP1, PGC-1α and PDK4. These genes are down-regulated in SCC tumors (compared to normal skin). Also, they are regulators of glycolysis, mitochondrial activity and lipid biosynthesis. Specifically, PPARγ promotes lipid biosynthesis; PGC-1α and PDK4 control mitochondrial activity; FBP1 suppresses glycolysis. Thus, we hypothesize that the cSCC metabolic phenotype is driven mostly by glycolysis and that miR-130b is a regulator of this metabolic switch.

We found that in cSCC cell lines, proliferation is reduced by up to 30% when miR-130b was inhibited. Also in the same setting, less glucose was consumed and less lactate was produced, suggesting that miR-130b promotes glycolysis. Realtime-PCR shows that miR-130b levels are up-regulated in SCC tumors and in SCC cell lines (compared to normal skin and primary keratinocytes, respectively). On the other hand, PPARγ and PGC-1α gene expression decreases during SCC development. To study the long-term effects of miR-130b depletion, we have also developed an inducible CRISPRi system that suppresses microRNA expression. Finally, rosiglitazone, a PPARγ agonist, suppresses the proliferation of several SCC cell lines and is under several trials for breast cancer and liposarcoma, thus suggesting that this pathway may be targetable in SCC as well. Our current studies are focused on the precise roles of PPARγ transcriptional targets, which may include FBH1, PGC-1α and PDK4 in cSCC.

#44

Hexyl-benzyl-biguanide (HBB) potently and selectively inhibits CYP3A4 epoxygenase activity and inhibits EET stabilization of mitochondrial respiration in ER+HER2- breast cancer cells, inducing glycolysis and pyruvate biosynthesis.

Zhijun Guo,1 Irina Sevrioukova,2 Eric Hanse,1 Xia Zhang,1 Ilia Denisov,3 Ting-Lan Chiu,4 Rebecca Cuellar,5 Christian Torres,4 Julia Wulfkuhle,6 Emanuel Petricoin,6 Qing Cao,1 Haitao Chu,4 Beverly Norris,4 Robert Schumacher,4 Ameeta Kelekar,1 Ian Blair,7 Jorge Capdevila,8 John Falck,9 Thomas Poulos,2 Steven Sligar,10 Gunda Georg,4 Elizabeth Amin,4 David A. Potter1. 1 _University of Minnesota Masonic Cancer Center, Minneapolis, MN;_ 2 _University of California, Irvine, Irvine, CA;_ 3 _University of Illinios, Urbana, Champaign, IL;_ 4 _University of Minnesota, Minneapolis, MN;_ 5 _University of Minnesota Masonic, Minneapolis, MN;_ 6 _George Mason University, Manassas, VA;_ 7 _University of Pennsylvania, Philadelphia, PA;_ 8 _Vanderbilt University, Nashville, TN;_ 9 _University of Texas Southwestern, Dallas, TX;_ 10 _University of Illinois, Urbana, Champaign, IL_.

Cytochrome P450 3A4 (CYP3A4) promotes ER+HER2- breast cancer cell proliferation and survival, in part, by biosynthesis of epoxyeicosatrienoic acids (EETs). EETs are known to regulate mitochondrial function in non-transformed cells, but the roles of CYP3A4 and EETs in regulation of breast cancer bioenergetics are unknown. Hexyl-benzyl-biguanide (HBB) is useful probe of CYP3A4 epoxygenase activity and selectively inhibits EET biosynthesis (IC50=9 uM vs. IC50=50 uM for CYP2C8). HBB caused depolarization of mitochondria in MCF-7 cells, while (±)-14,15-EET provided partial protection. The soluble epoxide hydrolase (sEH) inhibitor t-AUCB ameliorated inhibition of oxygen consumption rates (OCR) by HBB (20 uM), while there was no effect on extracellular acidification rate (ECAR), indicating that the primary effect of HBB is on OCR. At 30 minutes, HBB added to MCF-7 cells transiently suppressed phosphorylation of pyruvate kinase muscle isozyme 2 (PKM2) on Tyr-105, which has been reported to favor enzymatically inactive dimer over active tetramer. Suppression of phosphorylated PKM2 correlated with subsequent PKM2 tetramer formation and increase of intracellular pyruvate and extracellular lactate at 1 hour. The (±)-14,15-EET regioisomer reduced the pro-glycolytic PKM2 tetramer at 1 hour, suggesting that HBB may promote PKM2 tetramer, in part, through reduction of EET. Prolonged exposure to HBB (20 uM) in cultured cells activated phosphorylation of PKM2 on Tyr-105, but there was increased cellular necrosis correlating with reduced mitochondrial respiration and reduction of ATP stores, indicating that loss of respiration was the dominant effect. HBB inhibited the ER+HER2- MCF-7 xenograft, similar to CYP3A4 silencing. HBB promoted phosphorylation of intratumoral PKM2 on Tyr-105, consistent with long-term exposure to HBB in cultured MCF-7 cells. Notably, MCF-7 tumor response to HBB did not correlate with phosphorylation of AMPK-alpha on Thr-172, a marker of AMPK activation. Metformin (5 mM) exhibited no effect on PKM2 or its phosphorylation in cultured MCF-7 cells. Together, these results indicate that part of the inhibitory effect of HBB on ER+HER2- breast cancer is mediated through inhibition of respiration.

Significance: These results establish HBB as a useful chemical probe of respiration, with indirect effects on PKM2 regulation. HBB may also be useful as a potential therapeutic candidate for ER+HER2- breast cancer.

#45

Preventing and treating hepatic metastatic colon and pancreatic cancers by targeting cell metabolism.

AMER H. ALASADI, Jingjing Guo, Hanlin Tao, Shengkan Jin. _Rutgers, Piscataway, NJ_.

One fundamental change in cancer cells in oncogenesis is the alteration of glucose metabolism. With few exceptions, cancer cells exhibit aerobic glycolysis. A majority of pyruvate derived from glycolysis is converted to lactate instead of entering mitochondria for oxidative phosphorylation. This feature of cancer cells is also referred as "the Warburg effect". The functional significance of the inefficient metabolic mode to cancer cells is becoming clear. In essence, reduction in the flux of pyruvate into mitochondria prevents complete oxidation of glucose. This increases production of NADPH and metabolic intermediates required for biosynthesis of macromolecules essential for cell proliferation. Pyruvate flux into mitochondria can be dramatically enhanced through mitochondrial uncoupling, a process that allows protons cross the mitochondrial inner membrane without producing ATP. As a result, mitochondrial uncoupler promotes "futile" oxidation of acetyl-CoA, leading to complete glucose oxidation. Therefore, safe mitochondrial uncouplers could be potent anti-cancer agents by antagonizing the function of the Warburg effect. Here we tested this novel cancer chemotherapeutic strategy with niclosamide ethanolamine (NEN), a well-characterized mitochondrial uncoupler with an excellent safety profile, and one of its derivative (OXY-1) in culture cell models and a mouse model.

Mouse colon cancer cells (MC38) and mouse pancreatic cells (Panc02) were treated with NEN and OXY-1. At effective mitochondrial uncoupling concentrations, NEN and OXY-1 reduced cells viability, induced cell cycle arrest, and reduced clonegenicity. Moreover, NEN and OXY-1 also inhibited cell migration. These effects were associated with the activation of AMPK and reduction of NADPH/NADP ratio. We further examined the anti- cancer effect of NEN and OXY-1 on a metastatic cancer mouse model. First, we tested if the uncouplers are effective in inhibiting cancer growth. The MC38 or Panc02 cells were injected into the liver of NOD mice. Oral treatment of OXY-1 (800 ppm in food) significantly reduced tumor size and tumor incidence in mouse liver. We then tested if these drugs could inhibit tumor metastasis. MC38 or Panc02 cells were intrasplenically injected into the mice and the tumor metastasis to liver was analyzed. Oral NEN (2000 ppm in food) or OXY-1 (800 ppm in food) either totally prevented tumor metastasis to liver or drastically reduced metastatic tumor numbers and tumor volume. Taken together, our results strongly suggest that the safe and mild mitochondrial uncouplers NEN and OXY-1 could be effective in preventing and treating hepatic metastasis of colon and pancreatic cancers. Our results may provide a new and attractive way for preventing and treating these incurable tumors.

#46

The impact of monocarboxylate transporter expression on metabolic function in prostate cancer cells.

Laura Hutchinson, Amy Boyers, Amy Chadwick, Ian Stratford. _The University of Manchester, Manchester, United Kingdom_.

INTRODUCTION

The tumor microenvironment is subjected to variations in oxygen tension which results in hypoxic regions. Hypoxia in tumors is associated with radioresistance and a metastatic phenotype. Hypoxic conditions cause tumor cells to favor anaerobic metabolism, however this switch to glycolysis is also found in tumor cells under aerobic conditions, known as the 'Warburg effect'. An increased reliance on glycolysis results in large quantities of lactate, which needs to be transported out of tumor cells to maintain the intracellular pH. The monocarboxylate transporters, MCT1 and MCT4, regulate the transport of lactate in tumor cells and here we report the effect of overexpression or knockdown of these transporters on metabolism, growth and response to therapy in prostate cancer cell lines.

METHODS

Human prostate cancer cell lines were stably transfected, using lentiviral vectors, to show overexpression of MCT1 (DU145, LNCaP and PC3), overexpression of MCT4 (LNCaP), or knockdown of MCT4 (DU145 and PC3). Using these stable cell lines, the effect of overexpression or silencing of MCT1 or MCT4 was assessed in vitro. A lactate assay was used to investigate the effect on intra- and extracellular lactate under varying oxygen tensions. Effect on cell cycle progression was assessed using flow cytometry and toxicity of docetaxel was determined using MTT and SRB assays. Finally, a metabolome siRNA screen was carried out to determine if MCT4 expression was related to sensitivity to toxicity resulting from knockdown of a range of metabolic proteins.

RESULTS

Expression levels of MCT1 and MCT4 were confirmed by western blot and immunofluorescence. Silencing of MCT4 in DU145 and PC3 cell lines increased intracellular lactate under hypoxic conditions. No effect on intra- or extracellular lactate was observed in cell lines with overexpression of MCT1. Changes in expression of MCT1 or MCT4 did not appear to result in sensitization or resistance to docetaxel treatment under either normoxic or hypoxic conditions. Overexpression of MCT1 in PC3 cells caused cell cycle arrest in the G2/M phase in both normoxia and hypoxia. The metabolome screen indicated that, in wild type LNCaP cells, down-regulation of the protein product of the genes encoding 2,4-Dehydrocholesterol Reductase or Pyruvate Dehydrogenase Phosphatase Catalytic Subunit 2 was synthetically lethal. Overexpression of MCT4 resulted in protection against these lethal effects.

CONCLUSIONS

Altering the expression of MCT1 and MCT4 in human prostate cancer cells had a variety of effects on cell function. Overexpression of MCT1 caused cell cycle arrest in PC3 cells which may impact on radiosensitivity as cells are most vulnerable to radiation treatment when in the G2/M phase. The identification of two proteins that cause toxicity when silenced in LNCaP wild-type cells, but not LNCaP cells showing overexpression of MCT4, is an interesting lead for further investigation.

#48

The immunoregulatory protein B7-H3 regulates reprogramming of cancer cell glucose metabolism through reactive oxygen species mediated stabilization of HIF-1ɑ.

Sangbin Lim,1 Hao Liu,2 Luciana Barnes,1 Ming Tan1. 1 _Mitchell Cancer Institute, University of South Alabama, Mobile, AL;_ 2 _West China School of Preclinical and Forensic Medicine, Sichuan University, China_.

With the recent success of immunotherapy targeting immune checkpoints, interest in understanding the function of immune checkpoint modulators such as the B7 family proteins escalated. B7-H3 is a transmembrane glycoprotein and a member of the B7 family of immunoregulatory proteins. Extensive studies have shown that B7-H3 is important in regulating the activity of immune cells. We are among the first groups to show that in addition to immunoregulatory function, B7-H3 also has non-immunological functions, which may contribute to its role in tumor progression. Overexpression of B7-H3 is associated with tumor progression and poor patient outcome in multiple types of cancers. However, studies on how B7-H3 regulates cancer growth and metastasis are rare and the mechanisms underlying the role of B7-H3 in cancer malignancy are almost unknown. We and others have shown that B7-H3 mediates breast cancer invasion, metastasis and drug resistance and that silencing B7-H3 delays tumor growth and sensitizes cancer cells to chemotherapeutic agents in immune-independent in vitro and in vivo assays, indicating immunoregulatory B7-H3 also has immune-independent function.

In this study, we investigated the role of B7-H3 in cancer cell metabolic reprograming in vitro and in two independent xenograft mouse models. We found that B7-H3 increased glucose uptake and lactate production while it suppressed oxygen consumption in B7-H3 high-expressing cells, indicating that B7-H3 promotes Warburg effect. B7-H3 increased the protein levels of HIF-1α and its downstream targets LDHA and PDK1, which are key enzymes in glycolytic metabolism. Moreover, B7-H3 promotes HIF-1α stability via enhanced reactive oxygen species (ROS) by suppressing the activity of transcription factor Nrf2 and mitochondrial Nrf2 target genes, including antioxidant enzymes SOD1, SOD2 and PRX3. B7-H3 mediated HIF-1α stability is suppressed by ROS scavenger treatment including N-acetylcysteine and PEG-catalase. Metabolic imaging of human breast cancer xenograft in mice further demonstrated that B7-H3 enhances tumor glucose uptake and tumor growth. Together, our results show that B7-H3 regulates cancer glucose metabolism via ROS mediated-HIF1α pathway. By exploring novel functions of B7-H3, our study fills a large knowledge gap in understanding the mechanisms underlying the role of B7-H3 in tumorigenesis and cancer progression, especially in understanding the underappreciated contribution of its non-immunological functions in B7-H3 mediated cancer pathology. The knowledge gained through carrying out the project will be paradigm shifting for B7-H3 biology and will be essential for developing efficient B7-H3 targeted therapy.

#50

2-Deoxy-2-halo-D-glucose derivatives inhibition of glycolysis in pancreatic cancer.

Rafal Zielinski, Izabela Fokt, Aleksandra Rusin, Stanislaw Skora, Waldemar Priebe. _UT MD Anderson Cancer Center, Houston, TX_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most hypoxic and subsequently glycolytic tumors. Thus the inhibition of glycolysis appears to be an attractive therapeutic approach that should be explored.

Methods: A series of 2-deoxy analogs of D-glucose, namely, 2-deoxy-D-glucose (2-DG), 2-fluoro-D-glucose (2-FG), 2-chloro-D-glucose (2-CG), 2-bromo-D-glucose (2-BG), and 2,2-difluoro-D-glucose (2,2-diFG) were synthesized in our laboratory. The ability of 2-deoxy-2-halo-D-glucose derivatives to inhibit glycolysis in pancreatic cancer cell lines was tested using Seahorse 96 analyzer and XF Glycolysis Stress Test kit. The cellular ATP level was measured using CellTiter-Glo assay. Viability of cells cultured in normoxic and hypoxic conditions were assessed using MTS assay.

Results: 2,2-diFG and 2-FG were significantly more potent inhibitors than 2-DG of the Extracellular Acidification Rate (ECAR) of medium in oligomycin-stimulated PDAC cells (complete inhibition at <12.5 mM, whereas, the 100 mM is a commonly used concentration for 2-DG in Seahorse 96 experiments). Interestingly, 2-bromo (2-BG) and 2-chloro (2-CG) analogs have a limited or no effect on glycolysis at tested concentration. Consistently, both, 2,2-diFG and 2-FG, inhibited proliferation of pancreatic cancer cell lines more potently than 2-DG in either normoxic and hypoxic conditions, with the differential effects being significantly more pronounced under hypoxia.

Conclusion: Both, 2,2-diFG and 2-FG have a superior to 2-DG ability to inhibit glycolysis in vitro and should be further evaluated in vivo in the PDAC models as a potential anticancer agents.

#51

Role of MUC13 as non-hypoxic stimuli inducing HIF-1α in pancreatic cancer under normoxia.

Sonam Kumari, Sheema Khan, Subhash Chauhan, Meena Jaggi. _University of Tennessee Health Science Center, Memphis, TN_.

Objective: Pancreatic cancer is the fourth most common cause of deaths occurring due to cancer, with an overall survival rate of just 5%. MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic cancer, while an altered glucose metabolism is known to facilitate cancer cell survival and proliferation. Hypoxia-inducible factor 1 α (HIF-1 α) plays an important role in reprogramming of cancer cell metabolism by activating the transcription of genes which encode glucose transporters and enzymes involved in glycolysis. Recent reports suggest that several non-hypoxic stimuli such as lipopolysaccharides, thrombin, and angiotensin II can also increase HIF-1α under normoxia. Herein, we investigated the effects of MUC13 expression on glucose metabolism and elucidated underlying signaling mechanisms that might be involved in this process.

Methods: MUC13 null pancreatic cancer Panc-1 cells were used for the study. Glucose and Lactate assay were performed in Panc-1 cells stably expressing MUC13 (Panc-1-M13) and vector (Panc-1-V). Cell culture media after 48 hrs was collected to measure the amount of unused glucose and L-lactate concentration using glucose and Lactate assay kits. Immunoblot and semi-quantitative PCR analyses were performed to check the expression of protein and mRNA levels, respectively. Cell proliferation and colony forming assays were performed to determine the effect of MUC13 on cellular growth and cell survival.

Results: Our results demonstrate that MUC13 acts as a modulator of the glucose metabolism in pancreatic cancer cells by regulating the expression and activity of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets. We observed increased amount of L-lactate production and glucose consumption in Panc-1-M13 cells as compared to Panc-1-V cells. This facilitates metabolic alterations and help tumor cells survive and proliferate under these conditions as indicated by increased cellular growth pattern in Panc-1-M13 cells. Our results demonstrate increased expression of the downstream targets of HIF-1α, such as cell regulatory (c-Myc), and cell survival (Bcl-2) proteins, while decreased the expression of tumor suppressor/cell cycle inhibitor (p-27) proteins in Panc-1M13 cells. Additionally, an increase in the expression of a critical downstream target of HIF-1α, Glut-1 was observed in Panc-1-M13 cells.

Conclusion: Our studies indicate that MUC13 acts as a key regulator of the metabolic process and facilitates metabolic alterations in the non-hypoxic environment that help tumor cells survive and proliferate under these conditions. However, further studies are required to elucidate detailed molecular mechanisms that are involved in MUC13 mediated metabolic remodeling in pancreatic cancer cells.

#52

NF-kB promotes a Warburg metabolic profile in pediatric sarcomas.

Yuichi Ijiri, Priya Londhe, Cheryl London, Peter J. Houghton, Denis C. Guttridge. _The Ohio State University, Columbus, OH_.

In contrast to normal cells that use mitochondrial oxidative phosphorylation for ATP production in aerobic conditions, many cancer cells depend on glycolysis even in the presence of sufficient oxygen. This process is known as aerobic glycolysis or the Warburg effect. Recent reports have shown that both the classical and alternative signaling pathways of NF-κB play important roles in controlling the metabolic profile of cancer and normal cells, respectively. However, how these signaling pathways affect the metabolism of pediatric sarcomas has not yet been explored. Here, we show NF-κB activity undergoes a shift in signaling from an alternative to a classical pathway in the genesis of sarcoma subtypes, rhabdomyosarcoma and osteosarcoma. Inhibition of classical NF-κB in sarcoma cells restores alternative signaling, as well as an oxidative metabolic phenotype in vitro and in vivo. In contrast, forced expression of the alternative pathway in rhabdomyosarcoma or in osteosarcoma cells is unable to reverse NF-κB classical activity, suggesting that the activation of the classical pathway in sarcomagenesis dominates over alternative signaling. Furthermore, we observed that inhibition of NF-κB in sarcoma cells reduced the expression of the glycolytic gene, hexokinase 2 (HK2). Through chromatin immunoprecipitation (ChIP) assays, we show that HK2 is a direct NF-κB target. Moreover, deletion of HK2 by using shRNA shifts the metabolic profile in sarcoma cells away from a warburg effect. These findings demonstrate a crucial role for classical NF-κB in sarcomagenesis and suggest that NF-κB metabolically reprograms sarcoma cells by regulating HK2.

#53

Epigenetic silencing of krebs cycle metabolism in kidney cancer.

Eun-Young Kho,1 Carolina B. Livi,2 Phillip Buckhaults,3 Francesca Carobbio,4 Hye-Young Nam,4 Richard Kirkman,4 David Crossman,4 Praveen K. Vayalil,4 Shi Wei,4 Ralph J. DeBerardinis,1 Sunil Sudarshan4. 1 _U of Texas Southwestern, Dallas, TX;_ 2 _U of University of Texas Health Sciences Center at San Antonio, San Antonio, TX;_ 3 _U of of South Carolina, Columbia, SC;_ 4 _U of Alabama at Birmingham, Birmingham, AL_.

Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC) including recent large-scale transcriptomic data demonstrating loss of tricarboxylic acid (TCA) cycle enzyme expression with aggressive tumors. Using a metabolomics/transcriptomics approach, we identified loss of mRNA expression of PGC-1α and ERR-γ is associated with tumor progression and worsened outcomes in RCC patients. PGC-1α both induces and cooperates with ERR-γ to promote TCA cycle enzyme expression. Additionally, PGC-1α reconstitution in RCC cells reverses the glycolytic phenotype and suppresses invasive phenotypes. Loss of PGC-1α and ERR-γ is through DNA and histone repressive silencing of the transcription factor PRDM16. Loss of PRDM16 is among the most prominent transcriptional changes in metastatic tumor sites. PRDM16 expression in RCC cells restorers TCA cycle enzyme expression, mitigates the Warburg phenotype, and suppresses in vivo tumorigenesis. Collectively, our data uncover a transcription factor network epigenetically silenced in renal cancer resulting in suppression of mitochondrial metabolism.

#54

PI3K-directed metabolic reprogramming in thyroid cancer.

Daniela De Martino, Antonio Di Cristofano. _Albert Einstein College of Medicine, Bronx, New York, NY_.

Tumor cells reprogram their metabolic pathway to meet their energetic needs during tumor progression. Highly proliferating or transformed cells are able to switch their metabolism from oxidative phosphorylation-based energy production to aerobic glycolysis. This switch called Warburg effect is recognized as a bioenergetic signature of cancer cells.

The PI3K pathway is one of the most commonly altered signaling in human cancers and plays a primary role in the establishment of the Warburg effect by inducing the transcriptional activation of glycolytic genes, increasing the surface expression of glucose transporters, stimulating lipid synthesis.

Using a novel, clinically relevant, mouse model in which thyrocyte-specific deletion of Pten constitutively activates the PI3K signaling cascade, we have identified a previously unrecognized in vivo mechanism through which PI3K activation subverts energy metabolism in thyroid cells by repressing oxidative phosphorylation well before full neoplastic transformation takes place, leading to the coordinated downregulation of the expression of genes encoding for members of both the TCA cycle and oxidative phosphorylation pathways.

We have now extended our analysis to mice carrying the cancer-associated, constitutively active Pik3caH1047R allele, and have observed the same downregulation of TCA and oxidative phosphorylation genes in the thyroid of Pik3caH1047R heterozygous mice, suggesting that the phenotype observed in the Ptenthyr-/- mice is solely due to the activation of the PI3K pathway.

Lactate dehydrogenase is a tetrameric enzyme comprising two major subunits, A and/or B, and catalyzes the forward and backward conversion of pyruvate to lactate.

Previous data from our lab showed an increase of lactate levels in thyroids from both young, tumor-free and older Ptenthyr-/- mice, indicating an increase in glycolysis metabolic flux even before full transformation.

To test the therapeutic value of Lactate dehydrogenase inhibition both in tumor initiation and in tumor maintenance, we have undertaken a combined pharmacological and genetic approach.

Interestingly, our data suggest that pharmaceutical and genetic LDHA inhibition results in a significant reduction in the proliferation rate of different thyroid cancer cell lines.

Moreover, to test the hypothesis that the glycolytic switch is necessary for full neoplastic transformation of Pten-/- thyroids and to prove that the LDHA enzyme plays a crucial rule in this process, we have generated a new (Pten,Ldha)thyr-/- mouse model that is being currently phenotypically and molecularly characterized to assess the effect of Ldha deficiency on thyroid hyperplasia, proliferation index, lactate content, and TCA cycle/OXPHOS gene expression.

Taken together, these data pave the way to the full understanding of the metabolic landscape induced by the activation of the PI3K pathway, and to the possibility of exploring therapeutic approaches targeting metabolic deregulation in vivo.

#55

Glucose supports EGFR-mutated lung adenocarcinoma survival by sustaining EGFR stability.

Jin Kyung Rho, Yun Jung Choi, Chang-Min Choi, Jae Cheol Lee. _Asan Medical Center, Seoul, Republic of Korea_.

Tumor cells are genetically unstable, driven by genetic mutations that stimulate cancer cell proliferation and survival. These genetic mutations are generally linked to particular types of cancer. Oncogenic epidermal growth factor receptor (EGFR) is frequently mutated in non-small cell lung cancer (NSCLC), which is essential for NSCLC development and growth. However, the precise roles of EGFR in lung cancer metabolism are still unclear. Here we show that glucose (Glc) metabolism is critical for EGFR stability required for tumor growth and survival. EGFR mutation leads to a robust increase in Glc uptake, lactate production and glycolytic flux. Moreover, EGFR-mutated NSCLC cell lines are much more sensitive than EGFR WT to inhibition of Glc metabolism through EGFR degradation, suggesting that EGFR mutant NSCLCs rely more heavily than EFGR WT on aerobic glycolysis. Knockdown of EGFR in EGFR-mutated NSCLCs results in a decrease in expression level of genes that encode glycolysis enzymes, indicating that EGFR mutation leads to an increase in Glc metabolism and Glc dependence for growth and survival. Furthermore, inhibition of Glc metabolism shows a significant decrease in intracellular ATP, oxygen consumption and level of TCA cycle intermediates. Indeed, the addition of pyruvate dramatically rescues apoptotic cell death caused by EGFR degradation upon inhibition of Glc metabolism, suggesting that Glc to fuel the TCA cycle is essential for tumor survival. In addition, the inhibition of Glc metabolism overcomes T790M-mediated resistance to EGFR-TKIs. Together, our data suggest that the EGFR mutation mediates the reprogramming of Glc metabolism and this metabolic alteration represents an attractive target for new therapy of EGFR-mutated NSCLC.

#56

Suppression of 6-Phosphofructo-2-Kinase (PFKFB3) for the treatment of breast cancer.

Yoannis Imbert-Fernandez,1 Amy Clem,1 Brian Clem,1 Gilles Tapolsky,2 Sucheta Telang,1 Jason Chesney1. 1 _University of Louisville, Louisville, KY;_ 2 _Advanced Cancer Therapeutics, Louisville, KY_.

Glucose uptake by tumors is stimulated by estradiol (E2) administration in estrogen receptor positive (ER+) breast cancer patients. Notably, this E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and, therefore, downstream metabolic regulators of E2 are expected to have utility as anti-breast cancer agents. Although the stimulation of glucose metabolism by E2 has been demonstrated, relatively little is known about the precise downstream effectors required for E2 to stimulate glucose metabolism in breast cancer. We have demonstrated that the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of the glycolytic flux, is elevated in breast cancer lymph node metastases and that exposure of ER+ human MCF-7 and T-47D breast cancer cells to E2 causes a rapid increase in 14C-glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, fructose-2,6-bisphosphate (F26BP). Importantly, we also found that selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor, PFK158, markedly reduces the E2-mediated increase in F26BP, 14C-glucose uptake and glycolysis. In the current study, we sought to determine if co-administration of PFK158, with ICI 182,780 (fulvestrant), would offer a greater anti-tumor effect. In unpublished results, we demonstrated that the combination of the anti-estrogen, fulvestrant, with PFK158 results in greater regressions of MCF-7 xenografts than either drug alone in athymic BALB/c mice. In addition to regulating glucose metabolism, PFKFB3 has been found to be a regulator of the cell cycle via cyclin dependent kinases. We postulated that the combination of PFK158 with the newly FDA-approved CDK4/6 inhibitor palbociclib may result in a synergistic decrease in tumor cell growth. We found that exposure of breast cancer cells to palbociclib and PFK158 causes a synergistic increase in cell cycle arrest and apoptotic cell death. In addition, we observed a synergistic decrease in phospho-retinoblastoma protein (Rb), a key downstream target of CDK4/6. Importantly, we found that the combination of PFK158 and palbociclib did not cause an increase in cell cycle arrest or apoptosis in normal human mammary epithelial cells. Taken together, these data indicate that PFKFB3 inhibitors such as PFK158 may have clinical utility for the treatment of ER+ breast cancers when combined with anti-estrogen agents and/or CDK4/6 inhibitors.

#57

Twist1 modifies the metabolic state of breast cancer cells through transcriptional activation of a key regulator of glycolysis.

Esmeralda Casas,1 Antonio F. Santidrian,1 Mihaela Lorger,2 Ali Torkamani,1 Boris Ratnikov,3 Sheena M. Saayman,1 Julie B. Steele,1 Smith W. Jeffrey,3 Brunhilde Felding1. 1 _The Scripps Research Institute, La Jolla, CA;_ 2 _Leeds Institute of Cancer and Pathology, Leeds, United Kingdom;_ 3 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA_.

The shift from a normal to high glycolytic state (Warburg effect) is a central hallmark of almost every cancer and a strong driving force for cancer cell survival and disease progression. Importantly, studies - including our own - have shown that normalization of metabolic state in cancer cells can halt or reverse malignancy. To date, the intrinsic and extrinsic factors that drive metabolic shifts in cancer cells are not entirely clear. A better understanding of how the metabolic state of cancer cells is regulated will give us important tools to inhibit disease progression. We have established a novel model of breast cancer progression consisting of three cancer cell lines established from the primary tumor (TES1) and two sites of metastasis (TES2b and TESPE) of the same patient. We compared gene expression profiles between primary and recurrent tumor cells to identify factors that regulate disease progression and dissemination. Key differences between cells derived from primary and recurrent disease were an activation of genes involved in glycolysis and genes involved in activation and maintenance of the epithelial to mesenchymal transition (EMT) program, a powerful driver of cancer metastasis. Our analyses on a functional relationship between EMT and activation of glycolysis uncovered a direct link between the malignancy-driving EMT mechanism in breast cancer progression and metabolic reprogramming of the tumor cells. Specifically, we identified Twist1, a pro-metastatic master regulator of EMT as a direct modulator of a key glycolytic enzyme known to modulate the rate of glycolysis, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3). We found that Twist1 binds directly to the promoter region of PFKFB3 and induces expression of the enzyme. Additionally, inhibition of Twist1 expression in TES2b (TES2b-shTwist1) cells decreased glycolytic flux, while Twist1 overexpression in HMLE immortalized human breast epithelial cells (HMLE-Twist1) increased glycolytic activity and simultaneously decreased cellular respiration. Metabolomics analyses of HMLE vs. HMLE-Twist1 cells using Gas Chromatography Mass Spectroscopy (GCMS) indicated that Twist1 expression is sufficient to induce drastic changes in the overall metabolic state of the cells, including glutamine metabolism and availability of TCA cycle intermediates and amino acids. Together, our findings are significant as they underscore the importance and potency of EMT activation in cancer and uncover a novel mechanism by which EMT activation through Twist1 drives metabolic alterations that fuel cancer progression and metastasis. Results from this study will ultimately aid in the discovery and development of new paradigms to fight cancer mortality rates around the world.

#58

Metformin impacts IGF-1R/ Akt signaling and energy metabolism in lung adenocarcinoma.

Gabriela C. Lobato,1 Steve Li,2 Imad Tarhoni,1 Wen-Rong Lie,3 Jeffrey A. Borgia1. 1 _Rush University Medical Center, Chicago, IL;_ 2 _Washington University, St. Louis, MO;_ 3 _EMD Millipore, St. Charles, MO_.

Introduction: Clinical outcomes for type II diabetes patients diagnosed with NSCLC are superior for those receiving metformin relative to other diabetic medications, though the mechanism for this effect remains incompletely elucidated. We hypothesized that metformin induces changes in Akt-based signaling via AMPK that increases glycolytic flux, and adversely affects survival in cell-line models of lung adenocarcinoma. The objective for these studies is to investigate therapeutic strategies involving metformin that have the potential to synergize with strategies targeting IGF-1R/Akt signaling.

Methods: The lung adenocarcinoma cell lines, A549 and H358, were treated with 0, 1, 5, 10 mM metformin for 24, 48, and 72 hours and evaluated for changes in expression and activity of IGF-1R/Akt signaling, as well as the expression of 17 enzymes involved in glycolytic and oxidative energy metabolism, all using Luminex® immunobead assays. In parallel, MTT assays were performed for up to 5 days to document changes in cellular proliferation/ death rates. Extracellular acidification was also monitored as a measure of glycolytic flux. All findings were collated and statistically evaluated using SPSS v19 (Chicago, IL) using independent t-tests or one-way ANOVA.

Results: We observed that metformin decreased IGF-1R/Akt signaling while increasing the expression of the protein levels of the signaling intermediates. Treatment with metformin increased the expression of Akt, IRS-1, PTEN, GSK3alpha and TSC2 (p<0.05 for all). Further, metformin treatment decreased the mean ratio of phospho-/total pyruvate dehydrogenase and increased the expression of ATP Synthase (both p<0.05), and were associated (p<0.05) with a dose-dependent increase in extracellular acidification. Lower doses (0.1 mM) of metformin did not have a significant impact on cell proliferation at 5 days, but a dose-dependent decrease in cell numbers was observed, with 37% and 29% reduction in cell numbers documented (p<0.05) at 5 mM for the H358 and A549 cells, respectively.

Conclusion: This study links changes with Akt signaling with changes in glycolytic flux and oxidative metabolism. This study suggests metformin may make an attractive adjunct for advanced NSCLC patients receiving therapy that mechanistically involves targeting Akt signaling and metabolic control, such as IGF1R inhibition.

#59

Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer.

doyeon lee,1 SungSook Yu,2 Dawoon E.Jung1 Jung,1 YeoSong Lee,3 Beom Ku Choi,4 Yong Chan Lee1. 1 _Institute of Gastroenterology, seoul, Republic of Korea;_ 2 _Biomedical Science, seoul, Republic of Korea;_ 3 _Samsung Medical Research Center, seoul, Republic of Korea;_ 4 _Immune & Cell Therapy Branch, Gyeonggi-do, Republic of Korea_.

Helicobacter pylori (H.pylori) infection of the gastric mucosa is strongly associated with chronic gastritis, peptic ulcer disease and gastric cancer. Individuals infected with H.pylori possessing CagA protein produce more reactive oxygen species (ROS) and increase the risk of developing gastric cancer. Sirtuins are NAD+ dependent deacetylases that regulate cellular metabolism in response to stress. Recently, mitochondrial sirtuin, SIRT3, is known to be a tumor suppressor via its ability to suppress ROS and regulate hypoxia inducible factor 1α (HIF-1α). However, it has not been investigated whether increased ROS production by H.pylori is regulated by SIRT3 via HIF-1α regulation and is mediated through intracellular CagA. We aimed to investigate the correlation between SIRT3, ROS and HIF-1α in H.pylori infected gastric epithelial cells. In this study, we showed that SIRT3 deficient AGS cells exhibited augmented HIF-1α protein stabilization and transcriptional activity in hypoxic condition. We also found that H.pylori CagA downregulated SIRT3 activity and induced ROS production through HIF-1α stabilization, similar to hypoxic condition in gastric epithelial cells. Overexpression of SIRT3 inhibited the stabilization of HIF-1α protein and attenuated the increase in HIF-1α transcriptional activity in hypoxia. Moreover, H.pylori CagA attenuated HIF-1α stability and transcriptional activity in SIRT3 overexpressed gastric epithelial cells. Taken together, these findings provide a valuable insight into the potential role of SIRT3 in the development of gastric cancer by regulating the HIF-1α protein.

#60

PTEN expression, cholesterol metabolism, and lethal prostate cancer.

Konrad H. Stopsack,1 Travis A. Gerke,2 Lorelei A. Mucci,3 Jennifer R. Rider4. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Florida, Gainesville, FL;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 4 _Boston University, Boston, MA_.

BACKGROUND: We previously showed that mRNA expression of squalene monooxygenase (SQLE), part of the cholesterol synthesis pathway, is associated with lethal prostate cancer. In-vitro studies suggest that loss of PTEN expression and resulting PI3K pathway activation drive cholesterol ester accumulation in aggressive prostate cancers through sterol regulatory element-binding protein (SREBP) and acyl coenzyme A-cholesterol acyltransferase (ACAT1) activity. In two prospective cohorts, we studied whether lower PTEN expression is associated with cholesterol metabolism and how this relates to lethal prostate cancer.

METHODS: We analyzed men with prostate cancer from the prospective prostatectomy Health Professionals Follow-up Study and Physicians' Health Study. 105 men had lethal cancer and 284 men non-lethal disease without metastases at 8 years of follow-up. Whole-transcriptome mRNA expression profiling data was available from diagnostic prostate tumor specimens. Linear regression models were used to assess continuous and categorical associations, and Pearson correlation coefficients were calculated. Logistic regression was used to obtain odds ratios (ORs) and 95% confidence intervals (CIs) of lethal prostate cancer.

RESULTS: Both lower PTEN and higher SQLE expression were associated with higher Gleason grade (p trend < 0.001 each). Correlations of PTEN with SREBF1 (r = -0.08, p = 0.1), SREBF2 (r = -0.09, p = 0.07), and ACAT1 (r = 0.04, p = 0.4) were weak. SREBF2 was correlated with SQLE (r = 0.33, p < 0.001) but not with ACAT1 (r = -0.01, p = 0.8). Low PTEN was associated with lethal cancer (OR, 1.50 per 1 standard deviation [SD] decrease; 95% CI, 1.20 to 1.87; p = 0.001). High SQLE was associated with lethal cancer (OR 2.13 per 1 SD increase, 95% CI, 1.63 to 2.79; p < 0.001). SREBF1, SREBF2, and ACAT1 were not associated with lethal cancer (all p > 0.05). Adjusting for PTEN mildly attenuated the association of SQLE with lethal cancer (OR, 2.07; 95% CI, 1.57 to 2.73), as did additional adjustment for age, Gleason grade, and stage (SQLE: OR, 1.75; 95% CI, 1.29 to 2.40). In the lowest quartile of PTEN expression, SQLE was less strongly associated with lethal cancer (OR 1.74; 95% CI, 1.20 to 2.52) compared to the upper three quartiles (OR 2.47; 95% CI, 1.69 to 3.60; p interaction = 0.2).

CONCLUSION: Low PTEN mRNA expression and high SQLE are features of aggressive prostate cancers. However, at the time of prostatectomy, SREBF1/2 expression and resulting ACAT1 expression do not appear to be major characteristics of prostate cancers with lethal outcomes.

### Functional Genomics and Genomics of Model Systems

#61

A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.

Yuanzhong Wu, Tiebang Kang. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be generated. Recently, genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss of function screening. Here, we developed a method named Protein Stability Regulators Screening Assay (Pro-SRSA) by combining the whole genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1-DCAF8 was determined to be a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was mediated by p300/CBP and deacetylated by HDAC3, was revealed to prevent its ubiquitin-mediated degradation by proteasome. This is the first report that the acetylation as a novel posttranslational modification modulates the Cdc25A stability, and we believe that this efficient and unbiased screening method at the genome-scale will be widely used to globally identify regulators of protein stability.

#62

Functional analysis of endometrial cancer mutants of the E3 ubiquitin ligase adaptor protein, SPOP.

Fred Lozy, Mary Ellen Urick, Meghan Rudd, Deena Maurer, Daphne W. Bell. _National Human Genome Research Institute, NIH, Bethesda, MD_.

The purpose of this study is to functionally annotate endometrial cancer (EC) specific mutants of SPOP (Speckle-type POZ protein). SPOP is the substrate adaptor for the E3 ligase complex CUL3-SPOP-RBX1, which is responsible for mediating the ubiquitination, and subsequent proteasomal degradation of a number of protein substrates including the SRC3 (NCOA3/AIB1) oncoprotein. Our laboratory has previously shown that SPOP is frequently mutated in serous and clear cell ECs, two clinically aggressive subtypes of EC. Importantly, the vast majority of SPOP mutants localized within the MATH domain, which is responsible for substrate binding. We hypothesize that EC associated SPOP mutations encode loss-of-function mutants that abolish or attenuate substrate degradation.

To test this hypothesis, we initially focused our attention on five SPOP mutations we uncovered in ECs (E47K, S80R, P94A, M117I and R121Q). HEK293 cells were transiently co-transfected with a HA-tagged SRC3-expressing construct and a MYC-tagged construct expressing either vector alone, wildtype SPOP, or mutant forms of SPOP. Protein lysates from transfected cells were subjected to Western blotting to measure exogenous levels of HA-SRC3 and MYC-SPOP. Cells exogenously expressing wildtype SPOP or the E47K, P94A, M117I or R121Q mutants exhibited lower levels of HA-SRC3 than cells expressing the vector control. In contrast, cells expressing the S80R mutant had elevated levels of HA-SRC3 compared to cells expressing either wildtype SPOP or the vector control. Co-immumoprecipitation experiments are being performed to assess the substrate binding capacity of SPOP mutants.

Our findings indicate that the SPOP-S80R mutation, which is recurrent in EC, has an impaired ability to down-regulate SRC3 protein levels compared to wildtype SPOP.

Ongoing and future studies aim to elucidate the mechanism that underlies the effect of SPOP-S80R, and to functionally annotate additional SPOP mutations in EC.

#63

A transposon-based forward genetic screen for steatosis-associated liver cancer drivers.

Barbara R. Tschida,1 N. Alpay Temiz,1 Ju D. Yang,2 Vincent W. Keng,3 David A. Largaespada1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong_.

Hepatocellular carcinoma (HCC), or liver cancer, is the second leading cause of death from cancer worldwide (Ferlay et al, Lyon, France: International Agency for Research on Cancer, 2013). HCC is linked with hepatic steatosis, or fatty liver disease, the most common chronic liver disease in the US. Both hepatic steatosis and HCC are increasing in prevalence in the US. However, the molecular mechanisms that cause HCC in a steatotic liver are not entirely understood. To uncover mutations promoting cancer in fatty livers and elucidate the role of steatosis in HCC, we performed a forward genetic screen using the conditional Sleeping Beauty (SB) transposon insertional mutagenesis system in mice treated with ethanol and a choline deficient diet to induce steatosis. Mice developed hepatic steatosis and liver cancer without sex bias, modeling the pattern seen in humans in whom HCC's with steatosis as a preceding condition are represented by a nearly equal number of males and females. We found that hepatic steatosis caused a downregulation of liver Foxa1 expression and increased Foxa2 inactivating phosphorylation, thus providing a molecular explanation for the loss of gender bias in our mouse model. Transposon insertion sites were mapped from the mouse HCC tumors, and genes near or in which more insertions were observed than expected by chance were identified as common insertion sites (CIS), which we consider putative steatosis-associated liver cancer genes. Wnt/b-catenin signaling, which defines a subclass of HCC and can induce tumors in mice without sex bias, was one of the top CIS associated pathways identified. Interestingly, alpha-adrenergic and dopamine signaling were also among the top steatosis-specific CIS associated pathways, suggesting a role for the sympathetic nervous system and cAMP signaling in fatty liver-associated cancer. Further studies are underway to test the roles of the pathways and individual genes identified in this study in driving HCC. Our studies implicate Wnt/beta-catenin and sympathetic nervous system signaling in steatosis-associated HCC and suggest methods for treatment or prevention in such cases.

#64

Identification and validation of novel therapeutic targets of osteosarcoma using cutting edge technologies.

Branden Smeester, Natalie Wolf, Branden S. Moriarity. _University of Minnesota, Minneapolis, MN_.

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. The current treatment options have not changed over the last four decades and rely on tumor resection and nonspecific combination chemotherapy, resulting in a 5-year survival rate of 0-29% if clinically apparent metastases are present. Although these tumors are rare in humans, with ~900 cases annually, they are highly prevalent in canines, with over 50,000 cases annually, and have seemingly similar genetics. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS- associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR, MAPK, and ERK5 signaling pathways. We identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma. We have now begun pre-clinical testing of inhibitors of our top actionable targets to treat osteosarcoma, namely SEMA4D and CSF1R. Further, we have begun to validate top candidate metastasis genes using the CRISPR nuclease system in immortalized osteoblasts and osteosarcoma cell lines. Preliminarily we have found that loss of candidate osteosarcoma metastasis genes ANAPC1 or TOAK1 increases the migration capacity of immortalized osteoblasts, while loss of EXOC1 or GRLF increases their ability for anchorage independent growth in soft agar assay. Results of these ongoing studies will be presented.

#65

In vivo **pooled shRNA library screens identify cancer genes driving epithelial ovarian cancer.**

Michiko Kodama, Takahiro Kodama, Justin Newberg, Neal G. Copeland, Nancy A. Jenkins. _Houston Methodist Research Institute, Houston, TX_.

Background & Aims: Epithelial ovarian cancer (EOC) is the 5th leading cause of cancer-related deaths in the USA. Most cases of EOC are diagnosed at an advanced stage with peritoneal carcinomatosis (PC), which seriously affects mortality. To identify therapeutic targets for EOC, we performed in vivo loss of function screens using pooled shRNA libraries. Methods: To discover tumor suppressor genes (TSGs) involved in PC formation, the human EOC cell line, SKOV3, was transduced with a pooled shRNA library containing 42,450 shRNAs targeting 7490 druggable genes. Transduced cells that were highly evasive as measured in Boyden chamber assays were then injected intraperitonealy into female nude mice. Eighteen PC tumors that developed were then sequenced and enriched shRNAs identified. SKOV3 cells transduced with the druggable shRNA library were also injected into nude mice without further in vitro manipulation and 12 PC tumors sequenced in order to identify potential oncogenes as measured by shRNA depletion. Results: Enrichment screening of invasive cells identified 34 potential TSGs targeted by two or more shRNAs. Knockdown of the two most highly ranked genes, RAD51C and TXN, by individual shRNAs, was subsequently shown to accelerate PC formation following intraperitoneal injection of transduced SKOV3 cells. Individual knockdown of these two genes was subsequently shown to increase cell invasion in Boyden chamber assays and adhesion to the extracellular matrix in vitro. These results suggest that RAD51C and TXN are suppressors of human EOC metastasis through their negative regulation of cell adhesion and cell invasion. Finally, shRNA depletion screening identified 17 potential oncogenes. These genes included three known oncogenes, ERBB2, RAF1 and FOS, in addition to eight genes that showed a significant positive correlation between their mRNA levels and poor patient survival in human EOCs. Knockdown of the second ranked oncogene, KPNB1, significantly decreased in vitro cell proliferation in multiple human EOC cell lines, suggesting that KPNB1 may be a new potential therapeutic target of human EOC. KPNB1 knockdown in SKOV3 cells downregulated AURKA, MYBL2, CCNE1 and CDK2 mRNA levels, suggesting that KPNB1 may function by positively regulating the cell cycle. Conclusion: In vivo pooled shRNA library screens are a powerful tool for identifying novel cancer genes and new potential drug targets in human epithelial ovarian cancer.

#66

High-throughput screening using patient derived tumor cell to conduct personalized medicine.

Seung T. Kim,1 Charny Park,1 Sun Kim,1 Won Lee,2 Joon Park,1 Kyoung-Mee Kim,1 Woongyang Park,1 Jeeyun Lee,1 young park,1 Jiryeon Jang1. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Gil Medical Center, Gachon University, Incheon, Republic of Korea_.

Background:

Patient-derived cell (PDC) model is one of prospective technologies to investigate drug response for precision medicine. Previously drug sensitivity derived PDC model has been studied in colorectal cancer organoid culture or lung cancer model. They revealed that PDC model recapitulates genomic characteristic and drug high-throughput screening is amenable. We established PDC models from metastatic cancer cohort of multiple cancer types, and revealed genomic characteristics and matched drug response.

Method:

Target sequencing was performed using OncoPanel. We called SNV, and CNV from target sequencing using GATK, then clinically unassociated SNPs were eliminated. Somatic submodules were identified from integrative variant profiles using HotNet referring HPRD. Drug high-throughput screening was performed SEMCO platform. In order to identify sensitive drug and variant gene pairs, drug sensitivity was tested by ANOVA using IC50 values.

Result:

Target sequencing of exome were generated from our 165 PDC models, and drug high-throughput screening was performed from 30 PDCs and ant-cancer 15 drugs. We tried integrative analysis approaches from genomic profile including mutation and copy number alteration, and identified genomic modules related with Fanconi, Hedgehog, FGFR receptor, Cytokine-cytokine receptor pathway. Variants of known oncogenes were successfully identified in MET, BRAF, FGFR and PIK4CA. Especially novel and recurrent mutations were identified in FGFR2, and corresponding samples indicated AZD4547 sensitivity as well as known drug response case such as BRAF V600E targeting Vemurafenib.

Conclusion:

Our study explains that PDC cohort sustains cancer related pathways, and its drug response follows genomic features. It is expected that our research would contribute to facilitate precision medicine from PDC model to utilize genomics and drug response.

#67

An integrated copy number and gene expression genomics analysis and RNAi approach identifies and validates the KIFC1 kinesin as a malignant cell selective target in triple negative breast cancer.

Nirmesh S. Patel,1 Konstantinos Drosopoulos,2 Daniel Weekes,1 Elodie Noel,1 Hasan Mirza,1 Mamunur Rashid,3 Emanuele de Rinaldis,1 Fara Brasó Maristany,1 Sumi Mathew,1 Erika Francesch Domenech,1 Patrycja Gazinska,1 Farzana Noor,1 Jelmar Quist,1 Rebecca Marlow,1 Anita Grigoriadis,1 Spiros Linardopoulos,2 Andrew N. Tutt1. 1 _King's College London, London, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom;_ 3 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom_.

Triple negative breast cancers (TNBCs) lack oestrogen (ER), progesterone (PR) and human epidermal growth factor 2 (HER2) receptors, and have limited targeted treatment options. Large scale genomic and transcriptomic studies have advanced our understanding of the changes which occur in TNBCs. However, the substantial number of copy number and gene expression alterations present in TNBCs makes it difficult to identify putative drivers, biomarkers and/or therapeutic targets of the disease. To overcome this problem, we have carried out an integrative computational and RNAi based approach to identify genes required for proliferation of TNBC.

Copy number and gene expression alterations were analysed using Affymetrix Human Exon HTS1.0 and SNP6.0 data of 152 primary breast tumours enriched for a TNBC phenotype and 9 normal breast epithelium. These analyses revealed 141 candidate genes whose upregulated gene expression is copy number driven in TNBC. The functional dependence on each of these genes was subsequently examined using RNAi in an array of 17 breast cancer and non-malignant cell lines using 6-8 cell lines per gene covering the widest possible range of expression levels for that gene. We validated a malignant cell specific functional dependence on 37 of the 141 genes using this method.

STRING analysis of validated hits reveals a subset of genes involved in the process of cell division and mitosis including the previously characterised mitotic kinase TTK. Of these, we further validated KIFC1 (HSET) which is known to play a role in clustering supernumerary centrosomes, a common occurrence in breast cancer. We show that siRNA and shRNA mediated depletion of KIFC1 decreases cell viability and clonogenic ability specifically in centrosome amplified cell lines, which can be rescued upon introduction of an si/shRNA resistant KIFC1. KIFC1 depletion also produces a high level of catastrophic multipolar mitoses in centrosome amplified but not in non-amplified cell lines. Furthermore, in-vivo studies show that inducible depletion of KIFC1 suppresses tumour growth of centrosome amplified cell line xenografts.

Our work has identified and functionally validated novel drivers, and potential therapeutic targets in TNBC. The data presented here shows KIFC1, a druggable kinesin motor protein is a promising target for therapeutic intervention being expressed through gene copy gain in a significant proportion of TNBCs. We validate its role in cancer-specific amplified centrosome clustering showing KIFC1 plays an essential role in aiding the survival of breast cancer cells that have supernumerary centrosomes in both in-vitro and in-vivo contexts.

#68

Neuroblastoma drug response profiles are associated with gene expression profiles.

Jeffrey P. Bond,1 Elizabeth VanSickle,2 Thomas S. Dexhemier,3 Richard R. Neubig,3 Giselle L. Sholler2. 1 _University of Vermont, Burlington, VT;_ 2 _Helen DeVos Children's Hospital, Grand Rapids, MI;_ 3 _Michigan State University, East Lansing, MI_.

Background: High risk neuroblastoma patients have a poor response to initial chemotherapy, with 20% unable to achieve a complete response. Genomic studies have identified 6 targeted agents whose pathways have been identified in some high risk neuroblastoma tumors. The low frequency of established driver mutations, along with the variety of molecular mechanisms for cancer pathway dysregulation, suggest development of gene expression-based predictive biomarkers in addition to sequence-based biomarkers. Our platform for developing gene expression-based biomarkers includes primary neuroblastoma cell lines, high throughput quantification of cell survival, exome sequencing, and genome-wide expression profiling.

Methods: Cell survival was quantified for each of thirty primary neuroblastoma cell lines in the presence of each of six drugs (bortezomib, crizotinib, dasatinib, lapatinib, sorafenib, and vorinostat) using high throughput cell survival measurements. Titration curves were based on treatment with vehicle as well as sixteen concentrations of each drug (replicated within and between plates). Summary descriptions of the response of each cell line to each drug were obtained using the four parameter logistic model as well as the area under the curve. Exome sequences were obtained using Illumina HiSeq technology. Genome-wide expression profiles were obtained using Affymetrix GeneChip technology. Inference was based on parametric univariate and non-parametric multivariate tests.

Results: For three of the drugs (bortezomib, dasatinib, and vorinostat) the variation in response between cell lines was statistically significant (p < 0.05; F ≥3) and assay results were reproducible in the context of the differences between cell lines (for the other three drugs the estimated standard deviation of log10 EC50 ≤ 0.2). Genome-wide expression profiles were associated with variation in drug response between cell lines, for example, high PDGFRB expression indicates sensitivity to dasatinib (p < 0.001).

Conclusion: High throughput cell survival measurements provided for identification of predictive biomarkers of neuroblastoma drug sensitivity. These biomarkers are currently being studied in the PEDS-PLAN (Pediatric Precision Laboratory Advanced Neuroblastoma Therapy) clinical trial.

#69

Identification of new therapeutic targets and molecular predictors of response in oesophageal cancer.

Irene Chong,1 lauren Aronson,1 David Cunningham,2 James Campbell,1 Colm Ryan,3 Michael Davidson,2 Ian Chau,2 Alan Ashworth,4 Christopher J. Lord1. 1 _Inst. of Cancer Research, London, United Kingdom;_ 2 _The Royal Marsden Hospital, London, United Kingdom;_ 3 _University College Dublin, Dublin, Ireland;_ 4 _UCSF, San Francisco, CA_.

Purpose of the study

Oesophageal cancer is the seventh most common cause of cancer related death worldwide. Disease relapse is frequent and treatment options are limited. With the exception of HER-2 and EGFR amplification, there are currently no validated biomarkers to predict response to biological therapies in this disease. The purpose of this study was to identify new biomarker defined therapeutic approaches for patients with oesophageal cancer.

Experimental approach

We have undertaken a functional screening approach focused on the druggable kinome to generate a first map of the genetic dependencies found in oesophageal cancer. A panel of 19 oesophageal and 130 non-oesophageal cancer cell lines were screened with siRNA targeting 720 kinases to generate functional profiles. By integrating this dataset with exome sequencing and copy number data, we identified key genetic dependencies (KGDs) associated with oesophageal histology (adenocarcinoma or squamous cell carcinoma) or key driver genotypes including amplifications in c-MYC, CCND1, EGFR and ERBB2, as well as loss of functional mutations in SMAD4 and ARID1A.

Results

Interrogation of the oesophageal functional genomics dataset established oncogene addiction effects including c-MYC, which is amplified in 30% of oesophageal carcinomas. We confirmed that a novel isoform of Bruton agammaglobulinaemia tyrosine kinase gene (BTK-C) was expressed in oesophageal cancer models and that silencing of BTK resulted in selective lethality in c-MYC amplified oesophageal cell lines. To further assess the impact of BTK inhibition in oesophageal cancer, we treated a panel of oesophageal cell lines with increasing doses of ibrutinib, a small molecule tyrosine kinase inhibitor of BTK. Analysis of ibrutinib SF50 revealed that there was preferential sensitivity to ibrutinib in c-MYC amplified oesophageal cell lines. Furthermore, we observed a significant association between mutation of the chromatin remodelling factor gene SMARCA4 and dependency upon the bromodomain protein BRD4. We also identified SMAD4 kinase genetic dependencies including AKT1 and a number of its substrates (FGR, MAP3K3, CHEK1 and WEE1). We noted a densely connected group of kinases that regulate the mitotic cell cycle in the SMAD4 dependency network, and found that SMAD4 mutant cell lines exhibited an increased sensitivity to drugs, including paclitaxel, that target the mitotic checkpoint.

Conclusions

Our results support the potential of BTK small molecule inhibition as a novel therapeutic strategy in c-MYC amplified oesophageal carcinoma. Loss of function SMAD4 mutations represents a candidate biomarker of response to drugs that target the mitotic checkpoint in oesophageal cancer.

#71

Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair.

Tony Gutschner, Monika Hämmerle, Giannicola Genovese, Giulio F. Draetta, Lynda Chin. _MD Anderson Cancer Center, Houston, TX_.

The comprehensive and coordinated efforts of The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) identified the somatic genomic alterations occurring in a broad panel of human cancers. The challenge ahead is to understand the functions of these somatically altered genes.Genome editing tools like the CRISPR/Cas9 system represent a transformative technology that allows the precise engineering of cells harboring specific (point) mutations found in specific tumor subtypes, enabling the investigation of the role of a candidate cancer gene in a stringently defined genetic context. Once recruited to its DNA target sequence in cells, Cas9 induces a DNA double strand break (DSB), which is either repaired by the non-homologous end-joining pathway (NHEJ), or by homology-directed repair (HDR). The HDR pathway allows for a precise site-specific knock-in and thus functional analysis of cancer-associated sequence variants. However, HDR activity is restricted to the late S and G2 phases of the cell cycle, while the NHEJ pathway dominates DNA repair throughout the cell cycle. Thus, the error-prone NHEJ pathway out-competes the HDR pathway, which reduces the likelihood for precise insertions, deletions or base substitutions at the DSB.

Here, we devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1 resulting in a cell cycle tailored expression with low levels in G1, but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the relative rate of HDR by up to 87% at a single-copy reporter locus, and up to 42% at the endogenous MALAT1 locus compared to wild-type Cas9.

In summary, our regulatory concept takes advantage of cell-autonomous pathways to enable fluctuation of Cas9 protein levels coupled to cell cycle dynamics. This resulted in higher site-specific integration events without sophisticated manipulation of cells by small molecules or additional gene targeting reagents. Future methodological developments may enable high-resolution expression of Cas9 and alternative genome engineering proteins like Cpf1 by combining cell cycle phase-specific transcriptional and post-translational regulatory elements. This might enhance HDR efficiencies even further, and will help to make sense of cancer genomic data.

#72

Therapeutic compound discovery targeting a recurrent glioblastoma (GBM) phenotype using LINCS compounds via QUADrATiC analyses.

Shahnaz T. Al Rashid, Paul O'Reilly, Philip D. Dunne, Matthew Alderdice, Thomas J. Flannery, Kevin M. Prise, Shu-Dong Zhang, Darragh G. McArt. _Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, United Kingdom_.

Glioblastoma (GBM) is the most prevalent and aggressive of malignant primary brain tumours. Despite multi-modal therapy, including surgical resection, radiotherapy and chemotherapy, GBM continues to be uniformly lethal, with a median survival of 12-18 months from the time of diagnosis. The resistance to therapy and ultimate recurrence is thought to originate from a sub-population of cells residing in the initial tumour, identified as glioma-initiating or glioma-stem cells (GSC). These GSC have been shown to be intrinsically more invasive, with enhanced repair and drug-efflux pathways mediating resistance to chemo-radiation therapies. The purpose of this study was to use and validate a novel connectivity mapping procedure to identify candidate compounds targeting therapy-resistant and recurrent GBM. This procedure was developed by O'Reilly and colleagues ("QUADrATiC: A Scalable Approach to Repurposing FDA-approved therapeutics", under review in Bioinformatics), and in this study, was performed on publicly available NCBI GEO microarray datasets.

Based on the cancer stem cell theory whereby the resistant GSCs repopulate and are enriched in recurrent tumours, primary and recurrent GBM gene expression profiles were compared and the connections between the identified gene lists and drug responses were mapped. To validate that this connectivity mapping procedure is reliable at identifying effective treatments to target therapeutic resistance and recurrence, we tested one of the top ten compounds identified, rosiglitazone (rosi), a ligand of the peroxisome proliferator activated receptor gamma (PPARγ). The efficacy of rosi alone and in combination with the current standard of care therapies, temozolomide and radiation, at killing GSC was assessed in vitro using the clonogenic survival assay. Our data indicated that rosi significantly reduced survival of both non-stem glioma cells and GSC, alone and in combination with radiation and temozolomide. The p53 pathway was identified as a pathway by which rosi may induce permanent cell cycle arrest and cell death. Further work is ongoing to fully elucidate the downstream mechanisms of cell death. This work is proof-of-principle that the current connectivity mapping procedure can be instrumental in identifying more targeted therapeutic interventions for GBM resistance and prevent recurrence.

#73

Exploring arrayed synthetic CRISPR for functional genomic screening.

Jenille M. Tan, Scott E. Martin. _Genentech, Inc, South San Francisco, CA_.

Over the past decade, RNAi has been the workhorse for loss-of-function genomic screening. Although powerful, RNAi is hampered by a high rate of false positives driven by off-target effects. New screening technologies based on the CRISPR/Cas9 system are being rapidly adopted, and appear to be significantly less prone to off-target effects. CRISPR/Cas9 screens are conducted in pooled formats for positive and negative selection. However, this format is not amenable to many assay types (e.g., protein translocation assays). In an effort to expand and achieve a more comprehensive approach to functional genomics, we explored the use of arrayed CRISPR/Cas9 screening with synthetic CRISPR RNAs. Testing was performed by reverse transfecting synthetic CRISPR reagents into HCT 116 cells stably expressing Cas9, thereby making the workflow analogous to high-throughput siRNA screening. We first explored and optimized the system with reagents targeting genes associated with DNA replication, including geminin (GMNN). Loss of GMNN is known to result in enlarged nuclei due to aberrant DNA replication, which is easily observed and quantitated by automated microscopy. Analysis of individual cells by microscopy also enables the identification of effects that are only observed in a fraction of the population. After optimizing transfection and assay conditions, we observed CRISPR-induced phenotypes similar to siRNA knockdown. Surprisingly, these effects were observed within 72 hours and affected the vast majority of cells. These effects were even more pronounced in some clonal populations of HCT 116 Cas9. Loss of GMNN was confirmed by immunofluorescence, western blot, and enzyme mismatch cleavage assay. We then expanded this approach to other genes with similar success. By testing and validating the utility of synthetic CRISPR on cells already harboring Cas9, we show that arrayed synthetic CRISPR screening can be a valuable new tool for hit validation and screening.

#74

Targeting nuclear export for triple-negative breast cancer therapy.

Fabio Petrocca. _Boston University, Boston, MA_.

Triple-negative breast cancers (TNBC) comprise the most aggressive subtype of breast cancer. They rapidly acquire resistance to chemotherapy and are also refractory to endocrine therapy and HER2 inhibitors. In fact, no targeted therapies are currently available against these tumors. Genome sequencing studies have shown that TNBCs are exceedingly heterogeneous in genetic mutations as well as copy number variations. Yet, TNBC almost invariably harbor inactivating mutations in TP53, which seems to be a crucial event in the pathogenesis of these tumors. We recently performed a series of siRNA lethality screens in vitro (N=11 cell lines) to identify, in an unbiased way, recurrent vulnerabilities that could be harnessed as novel targets for TNBC therapy. Hits were genes whose silencing triggered massive cell death in ≥ 30% of TNBC lines tested. The screens re-discovered mitosis as a frequent Achilles' heel of these tumors (antimitotic drugs are currently a mainstay of TNBC therapy), supporting the validity of the approach. The nuclear transport gene RAN also scored among the most frequent TNBC selective dependencies. Moreover, TNBC cell lines were exquisitely sensitive to selinexor (KPT-330), a first-in-class inhibitor of exportin 1 (XPO-1) currently in phase 2 trials, both in vitro and in vivo. TNBC cells treated with selinexor died of apoptosis. In contrast, normal human primary breast epithelial progenitors (BPE) underwent cell cycle arrest (but not death) upon selinexor treatment, and were able to resume proliferation upon drug removal. The efficacy of selinexor across different TNBC models varied, suggesting that additional factors, beside the TNBC status, are at play. Preliminary data based on a genome-wide CRISPR screen for genes and networks conferring sensitivity to selinexor using a p53-mutant TNBC cell line (MDA-MB-231) implicated a number of TP53-related genes as key mediators of response. Together, these data support the notion that p53-mutant TNBC coopt nuclear export function to silence TP53-associated networks and that XPO-1 inhibitors could be a viable strategy to tackle TNBC in the clinic.

#75

CellMiner, a systems pharmacological web-application for the NCI-60 cancerous cell-lines: Updates, data integration, and translationally relevant results.

William C. Reinhold, Margot Sunshine, Sudir Varma, James Doroshow, Yves Pommier. _National Cancer Institute, Bethesda, MD_.

Understanding the influences of molecular alterations on pharmacological responses in the omic sense is at the fore of the effort to make oncology more effective and specific. At present, however, this remains a field in its infancy. The NCI-60 cancerous cell lines provide a premier set of databases for systems molecular pharmacological studies. This is due to the availability of both the robust, high-quality activity profiles generated by the Developmental Therapeutics Program (https://dtp.cancer.gov), and the panoply of molecular and phenotypic information available. The CellMiner web-application (http://discover.nci.nih.gov/cellminer) provides the user access to both of these. Activity data is available for 20,861 compounds, including 159 Food and Drug Administration (FDA)-approved, 85 clinical trial, and 443 known mechanism-of-action drugs. The molecular data includes transcript expression for 25,772 genes, genetic variants for 16,568 genes, transcript expression for 360 microRNAs, protein levels for 94 genes, DNA copy number (from aCGH) for 23,413 genes, and soon DNA methylation levels for 17,559 genes. "Cell line signatures" are provided for each of these data types, facilitating their comparison. It also provides "Pattern comparisons" for any input pattern of interest to 87,419 activity, molecular and phenotypic patterns (For CellMiner 1.7).

Here we present our CellMiner updates, examples of data integration, and translationally relevant results.

#76

KPNA7 nuclear import protein - a critical regulator of cancer cell growth.

Elisa M. Vuorinen, Nina Rajala, Hanna E. Rauhala, Anne Kallioniemi. _University of Tampere, BioMediTech, Tampere, Finland_.

Background. In eukaryotic cells, the nucleus is separated from the cytosol by the nuclear envelope, thus requiring a specific machinery for the transport of macromolecules between the two cellular compartments. The nuclear import cycle is strictly regulated and its malfunction results in incorrect localization of proteins, ultimately leading to variety of diseases including cancer.

Karyopherin alpha 7 (KPNA7) is the newest member of the karyopherin alpha family of nuclear importers. KPNA7 is mainly expressed in early embryogenesis and oocytes as well as in some cancer cells. We previously showed that silencing of KPNA7 in pancreatic cancer cell lines with high endogenous expression dramatically reduced cell proliferation and anchorage-independent growth, through a G1 cell cycle arrest and transcriptional induction of p21 expression.

Here, we further explored the functional importance of KPNA7 in a large panel of pancreatic and breast cancer cell lines and identified its cargo proteins that may be responsible for the observed phenotypes.

Methods. KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers was determined 72 to 96 h after transfection and compared to those in corresponding controls. To isolate KPNA7 cargo proteins, stable tagged KPNA7-overexpressing pancreatic cancer cell lines were established. An affinity-based protein pull-down was performed and the KPNA7-interacting partners were identified with mass spectrometry.

Results. The silencing of KPNA7 in six pancreatic and breast cell lines led to decreased proliferation in all cell lines with low to medium expression level, whereas both cancer and normal cell lines with absolutely no KPNA7 expression were not affected, thus confirming the specificity of the phenotype to KPNA7 silencing. The results were also consistent in breast cancer cell lines, implicating a role not restricted to pancreatic cancer. Interestingly, in some cell lines a distinct change from round to lobular nuclear morphology was observed, suggesting that KPNA7 may also participate in the maintenance of nuclear architecture.

The protein pull-down followed by mass spectrometry yielded multiple proteins co-purifying with KPNA7. These included several proteins involved in RNA processing and nucleopore proteins. Many of these were also involved in the regulation of cell cycle and p21 pathway, consistent with the phenotype observed in the functional studies.

Conclusions. These results implicate KPNA7 as an important regulator of cancer cell growth and suggest that it might also have other functions in the maintenance of cancer cell homeostasis. We were the first to link KPNA7 to a human malignancy and here we expand that notion beyond pancreatic cancer. We have also identified many putative KPNA7 cargos with functions matching the observed cellular phenotypes. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis.

#77

Establishment of lung cancer patient derived xenograft model from circulating tumor cells: Identification of somatic mutations associated with metastatic potentials.

Chansu Lee,1 Youngwook Kim,1 Eunkyung Bae,1 Sunghoon Cho,2 Sohyun Youn,2 Kwang-Sung Ahn,2 Bong-Seog Kim3. 1 _Samsung Biomedical Research Institute, Seoul, Republic of Korea;_ 2 _Functional Genome Institute, PDXen Biosystems Inc., Seoul, Republic of Korea;_ 3 _Veterans Health Service Medical Center, Seoul, Korea, Seoul, Republic of Korea_.

Carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, the existence and genetic alternations of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. The establishment of xenograft model using CTCs should help us to understand the biologic behaviors and genetic alternations of MICs associated the promotion of metastasis. Here, we demonstrate that xenograft model can be established by CTCs obtained from lung cancer patients and somatic mutations analyzed from xenograft model in which MICs develop metastatic tumor. After injecting mononuclear cells of lung cancer patients via tail vein, mouse was sacrificed at four weeks. After primary cultured cells were isolated from mouse lung tissues, the MICs were selected by using FASC sorting (EpCam+) and then re-injected via tail vein. And tumor formations in mouse were examined at 8 weeks. For identifying somatic mutations, target sequencing with NGS was performed in tumor DNA and cell free DNA (CF DNA) of lung cancer patients bloods. Our results indicated that nonsynonymous IDH, EGFR, KIT, ABL1, PTEN, ATM, P53, and SMAD4 were found in both tumor tissues and CF DNA of lung cancer patients' bloods. IDH and EGFR was alternative homo. Others newly found in CF DNA. For further examination of the biologic behaviors of tumor cells which harvested from xenograft model using CTCs, we cultured primary cells from lung tissues and analyzed the anti-cancer drug resistance. Our results showed that primary cultured cells (BH-007) were resistant against cisplatin and erlotinib. In assay of migration and invasion, BH-007 cells possessed high metastatic potential when compared with A549 and H23 cell lines. These data suggested that several somatic mutations affect the anti-cancer drug resistance in lung cancer patients. Our future researches will include development of patient-derived xenograft models for preclinical testing of novel therapeutics. And we develop the possible integration of CF DNA analysis and PDX model from CTCs in the design of co-clinical studies for testing in individual lung cancer patients.

#78

Systematic comparison of mRNA and phosphoproteins expression in breast cancer from cell lines to tissue.

Lijun Cheng. _Indiana University, Indianapolis, IN_.

Background:

Whether the observed variation in transcriptome can predict the variation in proteome has not been fully investigated in subtypes of breast cancer. In addition, it is highly critical to study whether trancriptome/proteome correlations can be preserved in the cell line model when it is derived from primary tumor. Here, we attempt to address the transcriptome/proteome associations in a candidate set of gene/proteins using breast cancer tumor tissues and cell lines.

Method: Reverse-phase protein arrays (RPPA) were used to measure 313 protein expressions and 46 protein phosphorylations among 421 primary breast tumors from Cancer Genome Atlas (TCGA) and 33 cell lines of breast cancer. Correlation analysis between mRNA and RPPA was conducted and compared between primary tissues and cell lines in different breast cancer subtypes. Highly concordant genes/proteins are further analyzed using pathway analysis.

Results: In the breast cancer luminal A/B subtype, high mRNA/RPPA correlations were consistently observed for ER-alpha and PR in both cell line (0.71,0.677), and primary tissue, (0.90, 0.809). In the HER2 subtype, high mRNA/RPPA correlations in EGFR and HER2 were observed in the primary tissue (0.85, 0.78), and cell (0.73, 0.65). In the basal like subtype, consistently correlated mRNA/RPPA expressions were observed in Cyclin E1, GATA3, and AR, which are (0.8, 0.83, 0.7 ) in the primary tumor tissues and (0.85, 0.59, 0.7) in the breast cancer cell lines. The overall correlation of mRNA/RPPA correlation between cell line and primary tissue is 0.71. mTOR/PI3K pathway is identificated to have a strong function for basal like subtype of breast cancer.

Conclusion: Strong mRNA/RPPA associations were observed in each breast cancer subtype, ER-alpha and PR in the luminal A/B, HER2 and EGFR in Her2+, and Cyclin E1, GATA3, and AR in the basal like. These strong mRNA/RPPA associations are highly concordant between cell lines and primary tissues. Since most of these genes are well known drug targets, the highly concordant gene/RPPA associations not only confirm the gene expression biomarkers can served as surrogates for their protein products in the drug and target selections, but also imply that breast cancer cell lines shall serve as good models for primary tumor tissues.

Keywords: Breast Cancer, Reverse-phase protein array, mRNA, Copy number variation, Cell lines

#79

Andrographolide alters genes associated with DNA repair in prostate cancer.

Ingrid Forestier-Román,1 María Sánchez-Vázquez,2 Krizia Rohena-Rivera,1 Carmen Cadilla,1 Magaly Martínez-Ferrer3. 1 _University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico;_ 2 _University of Puerto Rico Comprehensive Cancer Center, San Juan, Puerto Rico;_ 3 _University of Puerto Rico Medical Sciences Campus, School of Pharmacy, San Juan, Puerto Rico_.

Prostate cancer is the most frequently diagnosed non-cutaneous cancer and the second cause of cancer-related deaths in American men. Andrographolide, a labdane diterpenoid that is a component of the medicinal plant Andrographis paniculata, has been reported to have a wide range of biological activities including anticarcinogenic properties. In previous studies, we found that Andrographolide inhibits cell growth, tumor growth and angiogenesis in prostate cancer. Therefore, the objective of this study is to determine the mechanism of action of Andrographolide in prostate cancer. In order to determine the mechanism of action, we did gene expression profile to compare genes differentially expressed in tumors treated with Andrographolide (10 mg/kg and 25 mg/kg) and their vehicle. Tumors were developed using a xenograft model in which the prostates of SCID mice were injected with 22RV1, and mice were treated bi-weekly with Andrographolide (10 mg/kg and 25 mg/kg). Tumor tissues were collected and snap frozen. Gene expression was analyzed using the Affymetrix GeneChip® Human Gene 2.0 array. Microarray studies showed a total of 674 genes differentially expressed in tumors treated with Andrographolide (10 mg/kg) when compared to their vehicle. In addition, 218 genes were differentially expressed in tumors treated with Andrographolide (25 mg/kg) when compared to their vehicle. Genes involved in cellular processes such as cell cycle, DNA damage response, MAPK signaling, TCA cycle, glycolysis and metabolism of amino acids were differentially expressed. Ingenuity Pathway Analysis (IPA) revealed networks associated to DNA replication, recombination and repair, and post-translational modification in tumors treated with Andrographolide (10 mg/kg). Moreover, IPA revealed networks associated to cell death and survival, cellular function and maintenance, and carbohydrate metabolism in tumors treated with Andrographolide (25 mg/kg). The microarray results were confirmed by quantitative real-time PCR. Our results demonstrated that Andrographolide altered the expression of genes associated with DNA repair which could be a possible mechanism of action of its anticarcinogenic effect.

#80

Programmatic re-splicing of the hypoxia transcriptome regulates the DNA damage response.

Crispin Miller, Danish Memon, Keren Dawson, Christopher Smowton, Wei Xing, Caroline Dive. _Cancer Research UK Manchester Inst., Manchester, United Kingdom_.

Background

Hypoxia occurs within the majority of solid tumors. It has multiple impacts on tumor biology including angiogenesis, suppression of immune reactivity, enhanced receptor tyrosine kinase signaling, down regulation of DNA repair pathways, promotion of pro-survival phenotypes and increased proclivity for invasion and metastasis. Levels of hypoxia vary between and within tumors, correlate with patient outcomes, and can lead to differences in response to therapy. Despite considerable advances, our understanding of the regulatory processes that control these diverse and heterogeneous changes in cancer phenotype remain incomplete. A better understanding of how they are mediated will inform strategies for personalized medicine and promise to substantially advance our understanding of tumor heterogeneity.

Methods

Until recently, techniques for RNA-expression profiling have lacked the resolution necessary to detect genome wide changes in splicing. We exploited the increased precision offered by deep sequencing to investigate genome-wide remodelling of transcript architectures in both cell line and tumor samples. Using novel computational strategies we generated sample-specific gene models from RNA sequencing data generated over a timecourse of HCT116 colorectal carcinoma cells following a switch to 1% oxygenation and used these data to identify widespread changes in splicing. We applied similar strategies to raw RNA-sequencing data derived from TCGA.

Results

We detected the expression of thousands of novel isoforms and a systematic, pathway-dependent switch to the expression of noncoding transcripts at multiple genes, including many constituents of the Fanconi Anemia, nucleotide excision- and double strand break repair pathways. Of particular note were HDAC6 and 53BP1: critical regulators of DNA damage response. Both loci exhibited a hypoxia-dependent expression of retained-intron transcripts and a concomitant decline in protein levels, thus providing a novel and unanticipated mechanism by which hypoxia can drive genetic instability. Predictions made using our cell line model were recapitulated in a large cohort of 458 colorectal carcinomas derived from TCGA. Validation of the cell line data revealed, for the first time, a strong signature of isoform switching in which a widespread transition to noncoding expression is associated with poor patient outcome. These data were derived from independent patient biopsies sequenced by others, thus demonstrating the robustness of our findings.

Conclusions

These data therefore have clear, substantial, and immediate implications both for our understanding of the basic aetiology of solid tumors and for the development of RNA-based signatures and biomarkers in diagnostics and personalized medicine.

#81

RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines.

Hui Cheng,1 Xinping Yang,1 Anthony Saleh,1 Shaleeka Cornelius,1 Carter Van Waes,1 Zhong Chen,1 Emine Guven-Maiorov,2 Ozlem Keskin,3 Attila Gursoy,4 Ruth Nussinov2. 1 _NIH/NIDCD, Bethesda, MD;_ 2 _Cancer and Inflammation Program, Leidos Biomedical Research, Inc. Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD;_ 3 _Department of Chemical and Biological Engineering; Center for Computational Biology and Bioinformatics, Koc University, Istanbul, Turkey;_ 4 _Center for Computational Biology and Bioinformatics; Department of Computer Engineering, Koc University, Istanbul, Turkey_.

We previously observed aberrant activation of NF-kB in head and neck squamous cell carcinoma (HNSCC), however, the signaling constitutively activating NF-kB in solid cancers has not been well defined. Recently, The Cancer Genome Atlas (TCGA) project has investigated 279 HNSCC tissue specimens, and uncovered significant genomic alterations of key molecules involved in inflammation, NF-kB, and death pathways. These genetic alterations include amplification of FADD and BIRC2/3, mutations of caspase 8 and RIPKs in HPV(-), or deletion of TRAF3 in HPV(+) HNSCC tissues. Using bioinformatics analyses and protein structural and interactive tools, we identified ~60 proteins that potentially interact with these genetically altered molecules. More than 90% HNSCC tissues studied in TCGA exhibited expression or genetic alterations of these genes, of which FADD amplification and/or overexpression ranked first with 37%. We searched this group of genes among more than 20 major cancer types investigated by TCGA, among which HNSCC ranked the highest for these alterations. To further investigate and identify experimental models for functional studies of these genetic and phenotypic alterations, we have performed RNA-seq and exome DNA-seq in 15 HPV(-) and 11 HPV(+) HNSCC lines, and compared them with three normal human oral mucosa lines and 8 matched blood samples. Among the top 30 genes identified from TCGA with the alteration rate greater than 8% in cancer tissues, we found consistent expression patterns for ~57% molecules in our cell lines. To further test the function of these molecules, we established HNSCC cell lines stably transfected with a vector that contains NF-kB transcription factor response elements upstream of the α-lactamase reporter gene (Gene BLAzer). Using these NF-kB reporter cell lines, genome-wide RNAi screening assays have been performed and the regulatory and signaling molecules involved in NF-κB and death pathways in responding to TNF-α have been identified. We further validated the screening results by knockdown of 35 genes individually using two siRNAs for each genes, and found that knockdown of 16 genes significantly modulated TNF-α and Lymphotoxin β (LTβ) induced NF-κB activity and/or cell survival. We observed that TNF-α and LTβ cross-activate classical and alternative NF-κB pathways and regulate key molecules that serve differential roles in proliferation, survival and migration in HNSCC. Our data help define key molecules modulating aberrant classical and alternative NF-kB activation, as candidate molecular biomarkers for diagnosis and prognosis and targets for further preclinical and clinical investigation in HNSCC.

(Supported by NIDCD/NIH intramural projects ZIA-DC-000016, 73, 74; and NCI/NIH, under contract number HHSN261200800001E).

#82

Genomic profiling of PDX models across 14 tumor types reveals novel targeted therapeutic opportunities.

Hua Dong, Zhixiang Zhang, Baoyuan Zhang, Xuzhen Tang, Xiangnan Qiang, Yuxin Qin, Shuqun Yang, Yunbiao Yan, Norman Zhang, Qingyang Gu, Qunsheng Ji. _Oncology BU, WuXi AppTec, Shanghai, China_.

Introduction: Large efforts have been made to detect somatic alterations and corresponding transcriptome changes in cancer patients, which are expected to bring about significant improvements in precision cancer medicine. Here we report a comprehensive list of driver genes and new targets identified in a large pan-cancer cohort of 550 Patient Derived Xenograft (PDX) models across 14 tumor types.

Material and methods: For each PDX model with passages 2-4, we performed comprehensive genomic analyses, including whole exome sequencing (WES), SNP6.0 array, and RNASeq (or microarray). The genomic data were used for extrapolating genomic SNV, CNV, mRNA expression levels, and for identifying gene fusions. Genomic profiling data were generated in internal and analyzed by in-house validated pipelines.

Results: 220 PDXs were identified to have actionable driver gene mutations /and/or amplifications among 550 models. We identified novel genomic alterations in a number of the PDX models. Significant portion of the models were validated with targeted therapeutic agents (TKI, Abs etc.) and certain cytotoxic agents. All processed data could be accessed via OncoWuXi database: http://onco.wuxiapptec.com and corresponding OncoWuXi APP (App store or onco.wuxiapptec.com/oncowuxi.apk

Conclusions:

We have established a large panel of PDX models with comprehensive genomic annotation and identified novel targets, with open access database and App. These models could serve as a platform for Mouse Clinical Trial and for pre-clinical development of next generation targeted therapeutic drugs.

#83

MicroRNAs associated with the p53 tumor suppressor signaling network are differentially expressed in triple negative breast cancer cell lines.

Malcolm M. Moses, Sherette S. Godfrey, Niageria Q. Lusk, Checo J. Rorie. _North Carolina A &T State University, Greensboro, NC_.

"Triple-negative" breast cancer (TNBC) is an aggressive subtype of breast cancer that appears prevalently in African American women below the age of 35, and has no known biomarker that can be targeted for drug therapy. This aggressive breast cancer phenotype is due to its high mitotic index and high metastatic potential, and may be due to the fact that up to 80% of TNBC has a mutation in the p53 tumor suppressor gene. The p53 tumor suppressor gene signaling pathway can be mediated by microRNAs (miRNAs) which are short sequences of RNA (~22nt) that target protein coding mRNA and mark them for degradation. Characterization of the miRNA profiles in TNBC cells compared to normal breast cell lines may provide potential targets in treating TNBC. In this study, microarray analysis was performed on TNBC cell lines HCC1806, HCC70, and MB157 and the normal breast cells AG11132. The expression levels of miRNAs found to play a role in the p53 tumor suppressor gene signaling pathway were compared between the normal and TNBC cell lines. The miRNAs let-7b, let-7a, miR-29a, miR-100, let-7f, miR-34a, miR-15b, miR-21, miR-16, and miR-22 displayed the greatest differences in expression levels between the normal and TNBC cell lines, thereby implicating them as potential biomarkers for TNBC and potential drug treatment targets. These miRNAs can be studied further to determine their role in mediating the p53 tumor suppressor gene signaling pathway in these cells for their potential use in the diagnosis and treatment of TNBC.

#84

A discovery on splicing variant patterns of leucine-tRNA synthetase gene based on targeted RNA sequencing.

HyoJin Song,1 Daeyoon Kim,1 Hyejoo Park,1 Hyun Sub Cheong,2 Sunghoon Kim,3 Youngil Koh,4 Sung-Soo Yoon4. 1 _Seoul National Univ. College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Genetic Epidemiology, SNP Genetics, Inc., Seoul, Republic of Korea;_ 3 _Medical Bioconvergence Research Center, Seoul National University, Seoul, Republic of Korea;_ 4 _Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea_.

A leucine-tRNA synthetase, which belongs to the class I aminoacyl-tRNA synthetase family, is encoded by LARS gene. LARS gene functions as an intracellular leucine sensor for mTORC1, mammalian target of rapamycin C1 signaling pathway. This cytosolic gene has a catalytic role of ligating leucine amino acid to its cognate tRNA, in ATP-dependent manner. Accordingly, the protein synthesis regulation, protein translation, cell size, and autophagy are known to be reported by this gene. Therefore, a comprehensive study on LARS gene is necessary for the sake of advanced drug research and precise personalized cancer therapy, in relation to mTOR pathway, which is the root cause of cancer genesis.

We discovered the differently expressed splicing patterns in 16 transcripts in LARS gene. Alternative splicing variants of LARS gene were sequenced of 12 cancer cell lines using targeted RNA sequencing to verify their possibilities as a drug target; 12 cancer cell lines are consist of 8 hematologic cell lines and 4 solid cancer cell lines. Sequencing data of LARS gene is aligned by TopHat, and consequently transcript assembly and coverage percentage calculation are processed by Cufflinks, afterwards.

Our data, observed from the integrated analysis of these values, shows outstanding features on three transcripts: known protein coding transcript LARS-001 (ENST00000394434, known processed transcript LARS-013 (ENST00000511505), and transcript LARS-015 (ENST00000508709), which is known to retain intron sequences. First of all, the coverage percentage value of transcript LARS-001 in a multiple myeloma cell line, KMS-12-BM, was substantially different from the other cell lines. Secondly, an exceptionally high coverage percent value of the transcript LARS-013 in HL-60, an acute promyelocytic leukemia cell line, was shown. Lastly, the transcript LARS-015 showed a considerably high coverage percent value in SK-MES-1, which is a lung squamous cell carcinoma cell line.

Three distinct features of LARS transcripts, transcript LARS-001 in KMS-12-BM, transcript LARS-013 in HL-60, and transcript LARS-015 in SK-MES-1, were characterized. Interestingly, transcript LARS-001 is transcribed with no exon skipping and is only highly expressed in KMS-12-BM. On the other hand, the serial exon skipping from exon 14 to exon 26 in major transcript LARS-201 has shown commonly in 12 cell lines. This implies that LARS gene has unique patterns of alternative splicing variants, which can be consider of taking a significant role in carcinogenesis across diverse cancer types. Hence, additional analysis on exploring the biologically functional correlation between LARS gene and cancer, including cohort validation is to be demonstrated in the future research.

#85

Macrophage inflammatory protein-1β and interleukin 15 modulate gene expression patterns associated with prostate cancer progression.

Krizia Rohena-Rivera,1 Diana A. Aponte-Colón,2 María M. Sánchez-Vázquez,3 Carmen L. Cadilla,1 Magaly Martínez-Ferrer1. 1 _University of Puerto Rico Medical Sciences Campus, San Juan, PR;_ 2 _University of Puerto Rico Río Piedras Campus, San Juan, PR;_ 3 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR_.

Prostate Cancer (PCa) is the second-leading cause of cancer-related deaths in the United States. Inflammation is associated with PCa development and progression. Cytokines such as macrophage inflammatory protein-1β (MIP1β or CCL4) and Interleukin 15 (IL-15) are differentially expressed in prostate cancer patients with recurrent disease (MIP1β) or recurrence-free survival (IL-15). However, the specific pathway by which these cytokines affect PCa progression is unknown. We evaluated the changes in gene expression patterns using in vivo models of prostate cancer and microarray analysis. To generate prostate tumors in vivo we used a mouse orthotopic xenograft model. Androgen-receptor-positive (AR+) cells, 22RV1, were orthotopically injected in the anterior prostate lobes. MIP1β and IL-15 were administered bi-weekly with intraperitoneal injections during 4 weeks. Tumor tissue was collected and snap frozen for RNA extraction, and microarray analysis with the Affymetrix Human Gene 2.0 Gene Array. Differences in gene expression were analyzed with Ingenuity Pathway Analysis (IPA) software and confirmation was done via real-time PCR. Microarray analysis showed 179 genes differentially expressed between MIP1β and control. In addition, 952 genes were differentially expressed between IL-15 and control. Ingenuity Pathway Analysis revealed that MIP1β affected networks associated with cell development, proliferation and movement while IL-15 affected networks involved in lymphocyte development and movement, cell death, and the inhibition of cancer cell invasion. Knowledge of the role of MIP1β and IL-15 in the alteration of gene expression patterns is pertinent to elucidate the significance of these cytokines in prostate cancer progression.

#86

Functional effects of MutL homolog 1 promoter polymorphism in prostate cancer.

Shinichiro Fukuhara,1 Yozo Mitsui,1 Yutaka Hashimoto,1 Taku Kato,1 Ryan K. Wong,2 Varahram Shahryari,2 Soichiro Yamamura,1 Shahana Majid,1 Sharanjot Saini,1 Guoren Deng,1 Rajvir Dahiya,1 Yuichiro Tanaka1. 1 _UCSF / VA Medical Center, San Francisco, CA;_ 2 _VA Medical Center, San Francisco, CA_.

The mutL homolog (MLH1) gene protects the integrity of the genome by correcting erroneous insertions, deletions and mismatches that occur during DNA replication. Proper MLH1 expression is thus essential for the health of the cell and deficiency can result in mutation and genomic instability, which ultimately may lead to cancer. Polymorphisms in the promoter region can lead to altered gene expression and we tested the hypothesis that MLH1 promoter variants can affect levels as well as be a risk for prostatic cancer. Two polymorphic sites were analyzed, rs1800734 (A to G) and rs4647203 (G to A). A luciferase reporter construct containing the MLH1 promoter region was utilized and site-directed mutagenesis was performed to create rs1800734 and rs4647203 variants. Activity was tested using DU145, PC3 and LNCaP prostatic cells and interestingly, the rs1800734 G variant resulted in significantly decreased luciferase levels in all three cell lines compared to A (P<0.01). No difference however, was seen for the rs4647203 variant. To determine whether rs1800734 can affect clinical expression levels, benign prostatic hyperplasia specimens were evaluated for MLH1 expression by immunohistochemistry. A significantly lower H score was observed for the GG genotype compared to AA (P<0.01). To further explore the clinical relevance of rs1800734, a case-control study was performed using DNA extracted from normal healthy, BPH, and prostate cancer patients from a Japanese population by sequence-specific PCR and sequencing. Results of experiments showed a trend for the variant G allele to be associated with prostate cancer when compared to normal controls (P<0.1). However, no differences were observed between BPH and controls as well as between stages or grades of prostate cancer. The MLH1 promoter polymorphism is thus capable of altering expression and associated with prostate cancer, and these results are important in understanding its role in this disease. 

### Genomic Analysis of Cancers

#87

Clustered somatic mutations are frequent in transcription factor binding motifs in melanoma and other cutaneous malignancies.

Andrew J. Colebatch,1 Leon di Stefano,2 Stephen Q. Wong,1 Anthony T. Papenfuss,2 Grant A. McArthur1. 1 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 2 _The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia_.

We sought to explore the etiology, frequency and clinicopathologic associations of clustered noncoding hotspot mutations in cutaneous malignancies (melanoma, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC)). We did so by applying a sliding window approach which identified clusters in melanoma whole exome and genome sequencing data. These regions were then evaluated for recurrence in other skin malignancies by using whole exome data from BCC and MCC samples; motifs were extracted using a maximum expectation algorithm. We then sequenced 170 annotated melanoma clinical samples using a targeted amplicon panel which permitted analysis of clinicopathological correlations. Numerous recurrent clustered hotspot mutations were identified in melanoma whole exome and genome data sets in proximal promoters, with some gene-associated clusters present in up to 25% of cases. Mutations were clustered around canonical ETS and SP1 transcription factor binding sites and had a clear UV signature. These promoter mutations correlated with overall mutation load and NF1 mutation status, and showed significant enrichment in specific melanoma subtypes (desmoplastic and lentigo maligna melanoma), sun exposed anatomical locations, male gender, ulceration and increasing age. Identical clusters were identified in BCC and MCC samples, including several previously annotated in melanoma. In conclusion, we identify a novel class of mutation in cutaneous malignancies which rather than being defined by a particular genomic position are related to a position in a transcription factor binding motif. These mutations are also independent of cell of origin. Clinicopathologic correlation and mutation analysis support an etiologic role for chronic UV irradiation.

#88

Recurrent KRAS G12V pathogenic mutation in adenomatoid odontogenic tumors.

Carolina C. Gomes,1 Silvia F. Sousa,1 Grazielle F. Menezes,1 Thais S.F. Pereira,1 Rennan G. Moreira,1 Alessandra P. Duarte,1 Wagner H. Castro,1 Rolando A.R. Villacis,2 Silvia R. Rogatto,2 Marina G. Diniz,1 Ricardo S. Gomez1. 1 _Federal Univ. of Minas Gerais, Belo Horizonte, Brazil;_ 2 _A.C. Camargo Cancer Center, São Paulo, Brazil_.

The adenomatoid odontogenic tumor (AOT) is a benign tumor of uncertain pathogenesis. Schimmelpenning syndrome is characterized by sebaceous nevi, which occurs often on the face, associated with variable ipsilateral abnormalities of the central nervous system, including ocular and skeletal abnormalities. It results from postzygotic autosomal dominant HRAS or KRAS lethal mutations that survive by somatic mosaicism. RAS genes mutations were previously reported in lesional tissue (including nevus sebaceous) of a patient, but not in normal skin or blood leukocytes, consistent with a somatic mosaic state. Interestingly, a case of multiple AOT was reported in a Schimmelpenning syndrome patient, which prompted us to evaluate RAS genes mutations, as well as 207 cancer genes mutations in a sample of one AOT from one Schimmelpenning syndrome patient having multiple tumors (index patient). We used the Ion AmpliSeqTM Cancer Hotspot Panel to interrogate these mutations by targeted next generation sequencing. We further included 3 sporadic AOT samples to assess if they shared similar mutations with the sample of the index patient. Additionally, molecular karyotyping analysis was performed in the index patient sample, as well as in one sporadic AOT sample by using a high-density whole genome array platform (Cytoscan® HD Array). The pathogenic KRAS G12V mutation was detected in the index patient sample of AOT, and in 2 out of 3 samples evaluated. No other pathogenic mutation was detected in the AOT samples. TaqMan® Mutation Detection probes specific to the c.35G>T KRAS gene substitution and/or Sanger sequencing were used to validate the mutation in all samples and in a panel of 4 additional sporadic AOT samples. A total of 7 out of 8 AOT samples showed the KRAS pathogenic mutation. We found a few copy number variations (CNVs), most of them common variations or alterations not encompassing genes. Loss of 7p15.3 encompassing the IGF2BP3 gene was detected in the sporadic AOT sample. In conclusion, we report for the first time the recurrent activating KRAS G12V mutation in a high proportion of investigated AOTs, while no other pathogenic mutation interrogated was detected. The importance of the 7p15.3 loss in the aetiopathogenesis of this tumor type remains to be established. Despite the benign nature of AOT, our results shed some light in future molecular targeted-therapy for the lesion. Supported by: CNPq, CAPES and FAPEMIG (Brazil).

#89

Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma.

Humam Kadara,1 Murim Choi,2 Jiexin Zhang,1 Edwin Parra Cuentas,1 Jaime Rodriguez Canales,1 Stephen Gaffney,2 Zi-Ming Zhao,2 Carmen Behrens,1 Junya Fujimoto,1 Chi-Wan Chow,1 Neda Kalhor,1 Cesar Moran,1 David Rimm,2 Stephen G. Swisher,1 Don L. Gibbons,1 John V. Heymach,1 Edward Kaftan,2 Jeffrey Townsend,2 Thomas J. Lynch,2 Joseph Schlessinger,2 J. Jack Lee,1 Richard Lifton,2 Ignacio I. Wistuba,1 Roy S. Herbst2. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Yale University, New Haven, CT_.

PURPOSE: Lung adenocarcinomas (LUADs) lead to the preponderance of deaths attributable to lung cancer. We performed whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of LUADs to better understand the molecular pathogenesis of this disease and identify clinically relevant molecular markers.

METHODS: We performed WES of 108 paired surgically resected stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Additionally, ten immune related markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers Exact tests. Cox proportional hazards regression models were employed for multivariate analysis of clinical outcome.

RESULTS: LUADs in this cohort exhibited an average of 243 coding mutations per tumor. We identified 28 genes with significant enrichment for mutation. SETD2-mutant LUADS exhibited relatively poor recurrence-free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS in KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis demonstrated that PD-L1 expression was increased in smoker compared to non-smoker LUADs and, along with other immune markers, was positively correlated with somatic mutation burden. Moreover, immune marker levels including PD-L1 were elevated in TP53-mutant LUADs. In contrast, STK11 and U2AF1 mutant tumors exhibited a suppressed immune response and LUADs with PIK3CA mutations exhibited markedly decreased tumoral PD-L1 expression.

CONCLUSION: Our study highlights mutations that may impact clinical outcome and personalized strategies for immune-based therapy of early-stage LUAD patients.

#90

A cell cycle-related genomic and transcriptomic signature distinguish aneuploid and euploid acute myeloid leukemia.

Giorgia Simonetti,1 Antonella Padella,1 Marco Manfrini,1 ĺtalo Faria do Valle,2 Cristina Papayannidis,1 Carmen Baldazzi,1 Maria Chiara Fontana,1 Viviana Guadagnuolo,1 Anna Ferrari,1 Elisa Zago,3 Marianna Garonzi,4 Simona Bernardi,5 Annalisa Astolfi,6 Maria Chiara Abbenante,1 Giovanni Marconi,1 Elisa Zuffa,1 Eugenia Franchini,1 Ilaria Iacobucci,1 Michele Cavo,1 Emanuela Ottaviani,1 Nicoletta Testoni,1 Alberto Ferrarini,4 Massimo Delledonne,3 Torsten Haferlach,7 Daniel Remondini,2 Giovanni Martinelli1. 1 _Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy;_ 2 _Department of Physics and Astronomy, University of Bologna, Bologna, Italy;_ 3 _Personal Genomics, Verona, Italy;_ 4 _Department of Biotechnology, University of Verona, Verona, Italy;_ 5 _Unit of Blood Diseases and Stem Cell Transplantation, University of Brescia, Brescia, Italy;_ 6 _Centro Interdipartimentale per la Ricerca sul Cancro "G. Prodi", University of Bologna, Bologna, Italy;_ 7 _MLL Munich Leukemia Laboratory, Munich, Germany_.

Chromosome gain or loss, which is the hallmark of aneuploidy, occurs in about 10% of adult Acute Myeloid Leukemia (AML) cases (Farag et al. IJO 2002, Breems et al. JCO 2008), despite inducing a dramatic reduction of cellular fitness in non-malignant cells (Torres et al. Science 2007).

The study aimed to identify AML-specific molecular mechanisms having a causative and/or tolerogenic role towards aneuploidy.

We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 38 aneuploid (A) and 34 euploid (E) AML cases, identified according to cytogenetic analysis and SNP array (CytoScan HD, Affymetrix). Variants were called with GATK, MuTect and VarScan. We also compared the transcriptomic profile of leukemic bone marrow cells from 21 A-AML and 28 E-AML cases (HTA 2.0, Affymetrix).

A-AML showed a significantly higher mutation load compared with E-AML (median number of variants: 25 and 15, respectively, p<.001) and a specific pattern of genomic lesions. Indeed, mutations in myeloid transcription factors and chromatin modifiers preferentially occurred in E-AML (p=.04 and p<.01, respectively), while A-AML was enriched for alterations in cell cycle-related genes (p<.01), with 70% of cases carrying at least one of those mutations (vs. 35% of E-AML, p<.01). The mutated genes played different functions across cell cycle phases, including DNA replication, centrosome dynamics, chromatid cohesion, chromosome segregation and spindle-assembly checkpoint. Moreover, 29% of A-AML displayed mutations of TP53 or a TP53-related gene (DDIAS, USP10), compared with 5.9% of E-AML cases (p=.01), with enrichment for a transcriptional signature of p53 downregulation in the aneuploid cohort (p<.05). Among the differentially expressed genes, we identified a cell cycle-related signature characterized by increased CDC20 and UBE2C and reduced RAD50, ATR and CCNA1 in A-AML (p<.001), confirmed at protein level, which was able to separate A-AML and E-AML. A-AML also showed upregulation of a KRAS transcriptional signature, irrespective of KRAS mutational status (p<.05).

Our data show a link between aneuploidy and genomic instability in AML and highlight novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid phenotype. Deregulation of the cell cycle machinery and DNA damage/repair checkpoints, either through mutations, copy number and transcriptomic alterations, cooperate with leukemogenic pathways, as KRAS signaling, to develop A-AML and overcome the unfitness barrier. This evidence suggests that a number of A-AML patients may benefit from pharmacological reactivation of TP53 and inhibition of KRAS pathway.

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

#91

The mutational landscape of mucinous carcinomas of the breast.

Salvatore Piscuoglio,1 Charlotte KY Ng,1 Felipe C. Geyer,1 Carey A. Eberle,1 Elena Guerini-Rocco,1 Caterina Marchio,1 Anne Vincent-Salomon,2 Jorge S. Reis-Filho,1 Britta Weigelt1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Institut Curie, Paris, France_.

Background: Mucinous breast carcinoma (MBC) is a special histologic type of breast cancer, comprising approximately 2% of all invasive breast carcinomas. Morphologically, MBCs are characterized by clusters of tumor cells floating in large amounts of extracellular mucin. MBCs are preferentially of low histologic grade and estrogen receptor (ER)-positive and HER2-negative. MBCs have been shown to lack the hallmark 1q gains and 16q losses found in low-grade ER-positive invasive ductal carcinomas of no special type (IDC-NSTs). Here we sought to characterize the mutational landscape and copy number alterations (CNA) of MBCs by whole exome sequencing analysis.

Material and Methods: Frozen sections from 25 pure MBCs (n=13 mucinous A (paucicellular), n=12 mucinous B (hypercellular)) were subjected to microdissection to ensure a tumor cell content >85%. In addition, five cases of mixed mucinous carcinomas composed of mucinous and IDC-NST components were included in this study. In these cases, the MBC and IDC-NST components were microdissected separately. DNA samples extracted from microdissected tumors and matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000. Somatic single nucleotide variants (SNVs) were identified using MuTect and somatic insertions and deletions (indels) were identified using Strelka and Varscan2. Somatic CNAs were defined using FACETS. Mutational frequencies in MBCs were compared to those of luminal IDC-NSTs from The Cancer Genome Atlas.

Results: A median of 26 (range 5-71) non-synonymous SNVs and indels were identified per MBC. GATA3, mutated in 28% of cases, was the only significantly mutated gene as defined by MutSigCV (q<0.1). Compared to IDC-NSTs of luminal molecular subtype, MBCs were found to harbor significantly fewer mutations in TP53 (3% vs 19%, p<0.001) and PIK3CA (6% vs 41%, p<0.001). Mucinous subtypes A and B did not differ in terms of mutations and/ or CNAs, whereas MBCs of mixed phenotype harbored a greater extent of genomic instability. While MBC and IDC-NST components of mixed MBCs shared the majority of the somatic genetic alterations, indicating their clonal relatedness, mutations and CNAs restricted to either the MBC or the IDC-NST components were also identified. Furthermore, CNA analysis confirmed our previous observations that MBCs, contrary to low-grade ER-positive IDC-NSTs, lack concurrent copy number gains of 1q and 16p and losses of 16q.

Conclusions: Our data suggest that the repertoires of somatic mutations and gene CNAs of MBCs are distinct from those of luminal IDC-NSTs, and that MBCs are an entity distinct from low-grade ER-positive IDC-NSTs not only at the morphologic but also at the genetic level.

#92

Integrated analysis of copy number, sequence variant and gene expression data in kidney chromophobe cohort.

Andrea J. O'Hara, Zhiwei Che, Soheil Shams. _BioDiscovery, Hawthorne, CA_.

The Cancer Genome Atlas (TCGA) contains various types of genomic data from a wide variety of cancers, including several rare tumor types. Here we analyze copy number, sequence variant and RNA-Seq data from TCGA for kidney chromophobe (KICH). Level 1 (raw) data, rather than Level 3 (segmented) data, from TCGA was used for an integrated analysis of copy number and gene expression profiles of the two cancers. Using the copy number and SNP probes, re-processed tumor profiles were more consistent with a control set in terms of median number of copy number events, sample ploidy, and breakpoint genes than with the published level 3 TCGA data. Probe-level data were analyzed using Nexus Copy Number SNP-FASST2 algorithm (a multi-state HMM algorithm), with systematic correction applied to correct for GC biases. Additionally we performed manual baseline adjustment to correct for sample ploidy based on whole-genome B-allele frequency data for each sample. Overall, the median number of copy number events in the KICH TCGA data set was reduced from 408 (in the level 3 set) to 85. After manual inspection, more than 87% of the TCGA KICH samples available at level 3 were found to have incorrect baseline ploidy assignments. Given KICH samples are known to have low ploidy overall, this step was critical for downstream analysis. RNA-Seq results for the entire cohort were evaluated to obtain normalized relative RNA expression. Somatic sequence variation results from whole exome sequencing and relative RNA expression were integrated with the individual copy number profiles to yield integrated per-sample results across all three data modalities. Aggregate analysis indicates highly recurrent losses (50-85% of samples) on chromosomes 1, 2, 6, 10, 13, 17 and 21. Lower level recurrent losses (~15-20% of samples) were identified on chromosomes 3, 5, 8, 9, 11 and 18. Concordance analysis revealed statistically significant subsets of samples with co-occurring lower level recurrent losses. Aggregate sequence mutation analysis of chromosomes among 3, 5, 8, 9, 11 and 18 did not identify any mutational hotspots. Integration with RNA-Seq expression data from each tumor type revealed statistically significant correlations (p<0.05) with these copy number alterations, identifying potential driver genes of interest. Closer evaluation of copy number alteration and RNA-Seq expression at the individual sample level confirmed co-occurrence of significantly downregulated RNA expression within these regions of loss but no expression changes in samples without loss. This correlated with an overall difference in survival but was not statistically significant, given the overall small size of the data set. While still limiting in sample size, the TCGA KICH data series offers unique insight into a rare tumor type. Cross data integration for individual samples offers additional strength and validity to findings initially uncovered through traditional aggregate analysis.

#93

Molecular profiling of pancreatic cancer patients from a wide geographical distribution across the US.

Lola Rahib,1 Anitra Engebretson,1 Michael J. Pishvaian,2 Jonathan R. Brody,2 William A. Hoos,1 Emily E. Lyons,1 Joseph Bender,2 Emanuel F. Petricoin,2 Subha Madhavan,2 Craig Heartwell,2 Lynn M. Matrisian1. 1 _Pancreatic Cancer Action Network, Manhattan Beach, CA;_ 2 _Personalized Cancer Therapy Inc (dba Perthera), McLean, VA_.

Commercial genomic and protein-based profiling was performed on tissue from pancreatic cancer patients who enrolled in the Pancreatic Cancer Action Network's Know Your Tumor (KYT) service. The service offers molecular profiling for patients with resectable or metastatic disease, in preparation for their next treatment plan. Patients are eligible to enroll at any institution, clinic, or cancer center in the US. Biopsies, tissue retrieval and testing is coordinated by Perthera, Inc.

From 6/2014 to 11/2015 tumor biopsies, resected or archived tissue was obtained from 152 (53% male) pancreatic cancer patients and profiling including NGS/FISH (144 patients) and IHC (143 patients) was performed. The median age at biopsy or resection was 63 (25-83), 55% of the tissue came from liver, 16% from pancreas, 7% lung, 4% peritoneum, and 18% from another type of tissue. 87% of the patients were diagnosed with pancreatic ductal adenocarcinoma and the remaining with another form of confirmed or suspected pancreatic cancer.

The most identified pathogenic mutations were KRAS (85%), TP53 (72%), CDKN2A (47%), CDKN2B (24%), SMAD4 (22%), ARID1A (10%), STK11 (7%), and BRCA2 (5%). The average number of genomic alterations (likely but not confirmed to be somatic) detected was five per patient from a panel of 320 genes. Molecular modifications that are linked to a treatment option were observed in 44% of patients. Twenty four of 144 patients (17%) had mutations in at least one DNA repair gene potentially benefiting from platinum or PARP inhibitor-based therapies, including BRCA2 (5%), ATM (4%), BAP1 (4%), SMARCA4 (3%), FANC (1%), CHEK2 (1%), and PALB2 (<1%). Other actionable molecular phenotypes observed include anti-HER2 therapies targeting ERBB2 amplifications (3%) and mTOR inhibitors suitable for patients with mutations in STK11/LKB1 (7%), PTEN (3.5%), PIK3R1 (<1%), or PIK3CA (3%). Actionable alterations identified with a low frequency (<2%) include RET fusions, amplifications of FGFR, CRKL, AXL, and mutations in NTRK3, MEN1 (PNET), and BRAF. One patient harbored the BRAF V600E mutation, prompting consideration of off label melanoma-approved therapy. Overexpression of ALK by IHC was observed in one patient who chose to enroll in a ceritinib clinical trial. Six percent of patients were PDL-1 positive, and 22% were positive for PD-1 from tumor infiltrating lymphocytes, suggesting possible activity from immune checkpoint inhibitors.

To date, 7 of the 152 patients have enrolled in a clinical trial, 4 were treated with an off label treatment, 67 have yet to make their next treatment decision, and 53 are known to be deceased. KYT has successfully profiled tumors and presented targeted treatment options to patients and physicians in 33 states regardless of a patient's socioeconomic background and geographic location, moving the field a step closer towards a democratized, personalized approach to treating pancreatic cancer.

#94

Genomic landscape and clonal expansions of upper urinary tract urothelial carcinoma.

Yoichi Fujii,1 Yusuke Sato,2 Shigekatsu Maekawa,2 Hiromichi Suzuki,1 Tetsuichi Yoshizato,1 Kenichi Yoshida,1 Yuichi Shiraishi,3 Kenichi Chiba,3 Hiroko Tanaka,3 Tohru Nakagawa,2 Haruki Kume,2 Hiroaki Nishimatsu,4 Yoshikazu Hirano,4 Masashi Sanada,5 Hideki Makishima,1 Satoru Miyano,3 Yukio Homma,2 Seishi Ogawa1. 1 _Department of Pathology & Tumor Biology, Graduate School of Medicine Kyoto University, Kyoto, Japan; _2 _Department of Urology, The University of Tokyo Hospital, Tokyo, Japan;_ 3 _Laboratory of DNA information Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 4 _Department of Urology, The Fraternity Memorial Hospital, Tokyo, Japan;_ 5 _Department of Advanced Diagnosis, Clinical Research Center, Nagoya Medical Center, Nagoya, Japan_.

Backgrounds

Upper urinary tract urothelial carcinoma (UUTUC) is a relatively rare and poor prognostic cancer which accounts for 5-10% of all urothelial malignancies. Despite the histological similarity, there are epidemiological and clinical differences between UUTUC and bladder urothelial carcinoma (BUC). Compared to BUC, the molecular pathogenesis of UUTUC is poorly understood. Urothelial carcinoma is also characterized by frequent multifocal lesions, suggesting field effects from mutagenic agents in urine and/or a dissemination from the primary tumor. To reveal the origin of the multifocality as well as the molecular pathogenesis of UUTUC, we performed comprehensive molecular analysis of UUTUC with regional multiple sampling.

Materials & methods

Surgical specimens of UUTUC and matched normal samples were obtained from 57 patients with various stages who underwent nephroureterectomy. Morphologically normal urothelial epithelia were also obtained in 5 cases, all of which were confirmed to be pathologically intact by an expert pathologist. Genomic DNA was extracted from each specimen and was subjected to whole exome sequencing using Hiseq 2500 for the detection of both somatic mutations and copy number lesions.

Results

In copy number analysis, recurrent deletions in 8p, 9p and 9q were identified, where homo deletions of 9p21.3 (CDKN2A) were most frequent (40.7%) Focal amplifications in 11q13.3 (CCND1) and 12q15 (MDM2) were also found. On average, 230 somatic nonsynonymous mutations per sample were detected. The mutation signature was biased to age-related and APOBEC patterns. Mutations were most frequently observed in KMT2D (42% of cases), followed by TP53 (32%), FGFR3 (24%), KDM6A (24%), EP300 (21%) and CREBBP (13%), which were also reported to be significantly mutated in BUC. However, the frequencies of these genetic lesions were substantially different between UUTUC and BUC. For example, abnormalities in RB1, CDKN1A and PIK3CA were not detected or rare in UUTUC but have been reported to be common targets of genetic lesions in BUC. In the analysis of normal epithelia, an average of 12.7 somatic mutations per sample were found but with low variant allele frequencies between 0.05−0.1. Somatic mutations in adjacent epithelia and preoperative urine were frequently shared by primary tumor, supporting a possibility of tumor disseminations from the original sites through urinary flow. In contrast, in other cases, distant epithelia harbored driver gene mutations that were not shared by the primary tumors, suggesting the presence of a mutagenic field effect on urothelial carcinogenesis.

Conclusions

Despite the similarity in their mutation spectrum, UUTUC and BUC seemed to have distinct pathogenesis in terms of the difference in mutation frequencies. Our data suggested that both dissemination and field effect might contribute to multifocal occurrence of UUTUC. These findings will provide a new insight into the pathophysiology of UUTUC.

#95

Somatic mutations and copy number variations in cancer-associated genes in 695 ovarian cancer patients.

Elizabeth H. Stover,1 Brooke Howitt,2 Neal I. Lindeman,2 Levi A. Garraway,1 Ursula A. Matulonis1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Background: Recent genomic studies have improved molecular understanding of ovarian cancer but have had limited impact on therapeutic improvements. We sought to review somatic mutations and copy-number variations (CNVs) in cancer-associated genes in the tumors of 695 patients with ovarian, peritoneal, or fallopian tube cancer (ovarian cancer) at Dana-Farber Cancer Institute, with associated pathologic and clinical data.

Methods: Testing was performed on formalin-fixed tumor tissue without germline analysis, with IRB approval and patient consent. 310 patients underwent genotyping for 471 mutations in 41 cancer-related genes (OncoMap test). 388 patients underwent next-generation sequencing (NGS) of 300 cancer-associated genes (OncoPanel test). Most samples were primary tumors, with a small subset recurrent. The majority of tumors were adenocarcinoma/carcinoma histology.

Results: Of 310 patients tested by OncoMap, 102 had at least one variant. Among tumors with adenocarcinoma/carcinoma histology, mutations were most frequent in TP53, KRAS, PIK3CA, JAK3, MET, and CTNNB1. Gene alterations varied by subtype and supported previously reported associations including BRAF in borderline, KRAS in mucinous, PIK3CA in clear cell, and PIK3CA/CTNNB1/AKT1 in endometrioid tumors. From 388 patients tested by OncoPanel, 3903 single nucleotide variants were detected. Variants were classified by clinical pathologists according to evidence-based potential clinical relevance. 27 variants were "Tier 1" (evidence confirming clinical utility in ovarian cancer) and 127 variants were "Tier 2" (possible clinical utility in specific contexts). Tier 1 variants were most frequent in BRCA1 and BRCA2. Common Tier 2 variants included alterations in TP53, PIK3CA, KRAS, PTEN, BRCA1, BRCA2, ARID1A, and BRAF. Several alterations may be considered "actionable" in terms of eligibility for clinical trials. Finally, 227 patients tested by OncoPanel had CNV data for the panel of cancer-associated genes. CNVs were numerous with 12368 CNVs reported. CNVs were classified as 1-copy deletion (n=6033), 2-copy deletion (n=108), high amplification (n=114), and low amplification (n=6103). Among adenocarcinoma/carcinoma tumors, 2-copy deletions were frequent in RB1, PTEN, CDKN2A, and MAP2K4, and common highly amplified genes were CCNE1, KRAS, AKT2, ERBB2, and MECOM.

Conclusions: Genotyping and/or NGS of cancer-associated genes in formalin-fixed tissue from ovarian tumors is feasible and can identify potentially clinically actionable alterations. Preliminary correlations with survival and treatment response are being explored.

#96

Targeted sequencing from endoscopic ultrasound-guided fine needle aspirates of pancreatic ductal adenocarcinoma.

Joo Kyung Park,1 Kwang Hyuk Lee,1 Jong Kyun Lee,1 Kyu Taek Lee,1 Woong-Yang Park,2 Dae-Soon Son3. 1 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 2 _Samsung Genome Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 3 _Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology, Samsung Electronics, Seoul, Republic of Korea_.

Next-generation sequencing (NGS) that enables the analyses of massively parallel sequences of DNA can advance the understanding of the underlying molecular pathophysiologies of cancer. Such recent genomic analyses have revealed a complex mutational landscape in surgical specimens of PDAC. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is used in the diagnosis of pancreatic cancer. Several genomic analyses using EUS-FNA in PDAC have been undertaken in a small number of patients. This study evaluated the feasibility of targeted NGS of specimens obtained using EUS-FNA to clarify genetic markers associated with the prognosis of pancreatic cancer. One hundred sixty six patients who underwent EUS-FNA with pathologically confirmed PDAC were included. Genomic DNA was extracted and quality control metrics of DNA analytes were measured. The specimens that passed a quality control test (N=101, 61%) underwent NGS using a customized cancer panel (CancerSCAN) enriched in the exons of 83 genes. Clinical prognostic factors associated with survival in PDAC were age, performance status, CA 19-9 level, curative resection, chemotherapy, tumor mass size, tumor location, stage and metastasis site in univariate analysis (P=0.020, <0.001, 0.015, 0.022, <0.001, <0.001, 0.031, 0.024 and 0.028, respectively). Multivariate Cox proportional-hazards analysis revealed chemotherapy (P < 0.001, hazard ratio (HR) = 5.7, 95% CI, 2.8 to 11.3) and tumor mass size (P=0.001, HR=0.4, 95% CI, 0.2 to 0.7) as independent prognostic factors. Each patient had different numbers of genetic alterations ranging from 0 to 17, and 99% of the patients had at least a single genetic alteration. KRAS mutation was found in 86 patients (86%); KRAS variant allele frequency ranged from 2 to 79%. The frequencies of major gene mutations responsible for PDACs were KRAS 86%, CDKN2A 22%, TP53 72%, SMAD4 25% and BRCA 14%. Genetic alterations associated with survival were ARID1A, AURKB1, CDK4, ERBB4, HNF1A, JAK3, MPL, PIK3R, PTCH1, STK11 and TOP1 in univariate analysis. In multivariate analysis, CDK4, HNF1A, MPL, PIK3R, PTCH1, STK11 and TOP1 mutations were independently associated with overall survival (P=0.011, P=0.001, P=0.001, P=0.005, P=0.041, P=0.050 and P=0.003, respectively). KRAS gene (variant allele frequency 50%) and the EphB4 mutation were significantly associated with PDAC metastasis. ATRX and TERT genes were significantly associated with the pattern of metastasis in PDACs. ABL1 and BRAF mutations were significantly associated with stable disease as well as partial response to chemotherapy (P =0.047 and P =0.015, respectively). Targeted sequencing using EUS-FNA specimens in PDAC is feasible and shows similar genomic profiles in surgical specimens. Novel genes associated with survival, metastasis and chemotherapy response in PDAC were identified.

#97

A novel PTPRK-FILIP1 fusion gene in invasive mucinous adenocarcinoma of lung without known driver mutations.

Sung-Hye Hong,1 Geon Kook Lee,1 Sang Hoon Song,2 Seung Youn Lee,2 Jin Young Kim,1 Ji-Youn Han1. 1 _National Cancer Center, Gyeonggi-do, Republic of Korea;_ 2 _TheragenEtex Bio Institute Inc., Suwon, Republic of Korea_.

Invasive mucinous adenocarcinoma (IMA) accounts for 2 to 10% of all adenocarcinoma cases of the lung and has a poor prognosis. The KRAS mutation is the most commonly detected genetic alteration in IMAs. Recent progress has permitted the identification of novel fusions such as NRG1. In an effort to further discovery of new driver oncogenes, we have carried out whole-transcriptome sequencing of IMA. To exclude IMA cases with known driver oncogenes, we performed targeted sequencing using the Ion AmpliSeq Cancer Hotspot Panel. We also tested other currently known fusion genes in lung cancer including CD74-NRG1, TPM3-ROS1, SDC4-ROS1, SLC34A2-ROS1, CD74-ROS1, EZR-ROS1, LRIG-ROS1, KIF5B-RET, CCDC6-RET1, EZR-ERBB4, TRIM24-BRAF, KIAA1468-RET, EML4-ALK, and KIF5B-ALK using RT-PCR. Finally we found 10 IMA cases without known driver oncogenes and performed transcriptome paired-end sequencing (the Illumina HiSeq platform). We utilized and compared 3 bioinformatic pipelines, FusionMap, deFuse, and ChimeraScan to identify fusion events. Of 10 IMA cases tested, we found 2 novel fusions, PTPRK-FILIP1 and CEP85L-RNGTT, from a never-smoking female patient. They were shared across all three fusion-finder and never reported in TCGA fusion gene data portal. Only PTPRK-FILIP1 was showed an in-frame fusion between exon 2 of PTPRK and 5' UTR of FILIP1. It was confirmed by RT-PCR analysis and Sanger sequencing. This precise fusion was not present in the matched normal tissue. The reading frame is preserved that both the start codon of PTPRK and FILIP1. However, PTPRK mRNA expression is increased until exon 2 because of the fusion includes only two exons of PTPRK. Otherwise, expression analysis showed significant increase of FILIP1 mRNA expression in the affected tumor pair sample. The protein product from FILIP1 is closely associated with the protein filamin A, which is associated with tumor proliferation, cell migration, and metastasis. Many studies have reported that filamin A as a poor prognostic factor. Considering its implication in tumor proliferation, cell migration, and metastasis, PTPRK-FILIP1 could be a novel target for IMA. This is the first report of PTPRK-FILIP1 fusion gene in IMA without known driver mutations. Further functional validation of FILIP1 fusion is planned.

#98

The somatic mutational landscape of the mitochondrial genome in prostate cancer: evaluation of clinical impact.

Julia F. Hopkins,1 Veronica Y. Sabelnykova,1 John Watson,1 Lawrence E. Heisler,1 Junyan Zhang,2 Michael Fraser,2 Theodorus van der Kwast,3 Robert G. Bristow,2 Paul C. Boutros1. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;_ 3 _Department of Pathology and Laboratory Medicine, Toronto General Hospital/University Health Network, Toronto, Ontario, Canada_.

Prostate cancer remains the most prevalent and second most lethal non-skin cancer in men. Whole genome studies have provided important insights into specific driver genes, however most of these studies have not assessed one key portion of the genome: the mitochondrial genome. To gain a complete understanding of the most commonly-diagnosed sub-groups of prostate cancer: low- and intermediate-risk localized disease, we surveyed the mitochondrial genomes from next-generation sequencing (NGS) data of over 300 tumour samples from prostate cancer patients. These samples were mainly from prostate cancer patients with clinical Gleason Scores of 3+3, 3+4 and 4+3. All had at least 5 years of follow-up data (median > 8 years), allowing identification of clinical associations with identified somatic mutations via Cox Proportional Hazards modeling and machine-learning. Recurrent somatic mutations in mtDNA were identified, and these were associated with clinical outcomes. One third of patients were found to have a somatic mtDNA mutation. These mutations appear to be associated with age of patient. The mtDNA region with the majority of mutations was the regulatory D-loop region, although certain proteins had high numbers of mutations. Those somatic mutations occurring within the coding regions in general were nonsynonymous. Specific identified candidate somatic mutations were validated via Sanger sequencing. Clinical associations between somatic were also integrated with existing copy-number alteration (CNA) biomarkers using machine learning methods to evaluate performance. mtDNA mutations were also compared to identified aberrations (CNA, PGA, SNVs) within the nuclear genome to determine correlations between the two genomes, in addition to other somatic mutations or altered-expression in nuclear-encoded mitochondrial proteins. Taken together, these data demonstrate a key role for mitochondrial mutations in driving prostate cancer.

#99

Ovarian cancer specific therapeutic vulnerability.

Kosuke Yoshihara, Ryo Tamura, Kazuaki Suda, Tatsuya Ishiguro, Takayuki Enomoto. _Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan_.

High-grade serous ovarian cancer comprises approximately 40% of epithelial ovarian cancer cases and is the most aggressive histologic type. Because this type of cancer usually presents as advanced stage disease at the time of diagnosis, the clinical outcome in patients with advanced stage ovarian cancer remains poor.

A recent study has identified that homozygous deleted essential-redundant genes generate therapeutic vulnerabilities in glioblastoma multiforme. On the other hand, a pan-cancer analysis of somatic copy number alterations demonstrates that ovarian cancer has high frequent somatic copy number alterations compared to other tumor types including glioblastoma. Therefore, we hypothesized that ovarian cancer has also therapeutic vulnerabilities leading to development of new therapeutic strategies. To discover ovarian cancer specific vulnerability, we used DNA copy number and mRNA expression data submitted to the Cancer Genome Atlas. First, we performed GISTIC 2.0 to detect copy number alterations and found 4107 genes with homozygous deletion in at least 1% of 562 ovarian cancer samples. Of them, 3060 genes had at least one paralogue gene defined by Ensembl. We focused on metabolism pathway to extract 182 metabolism-related genes. By comparing gene expression between homozygous deletion samples and others using unpaired t-test, we identified 73 genes that were significantly down-regulated in homozygous deletion samples (p < 0.05). Next, we used copy number data submitted to Cancer Cell Line Encyclopedia to discover 41 candidate genes that were homozygously deleted in at least one ovarian cancer cell line. These findings suggest that cancer specific vulnerability exists not only glioblastoma multiforme but also other tumor types including ovaryan cancer and that further functional validation of 41 candidate genes might lead to development of new therapeutic strategies in ovarian cancer in the near future.

#100

The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers.

Gabriel S. Macedo, Kathleen A. Burke, Salvatore Piscuoglio, Charlotte K. Y. Ng, Felipe C. Geyer, Luciano G. Martelotto, Anastasios D. Papanastatiou, Maria R. De Filippo, Anne M. Schultheis, Edi Brogi, Mark E. Robson, Hannah Y. Wen, Britta Weigelt, Jorge S. Reis-Filho. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: BRCA1 or BRCA2 germline mutations account for a substantial proportion of hereditary breast cancers. The majority of BRCA1-associated breast cancers are of triple-negative phenotype (estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-negative) and harbor TP53 somatic mutations. BRCA2-associated cancers are less homogeneous and often ER-positive. Systematic analyses of the profiles of somatic genetic alterations in BRCA1- and BRCA2-breast cancers have yet to be reported. Here we sought to determine the repertoire of somatic mutations and copy number alterations (CNAs) in breast cancers occurring in patients with BRCA1 or BRCA2 germline mutations. .

Methods: Eleven BRCA1- and five BRCA2-associated breast cancers were microdissected. DNA was extracted from microdissected frozen tumor-normal pairs and subjected to targeted capture massively parallel sequencing using the MSK-IMPACT platform, which includes all exons and selected introns and regulatory regions of 410 key cancer genes. Somatic single nucleotide variants, small insertions and deletions, CNAs and the cancer cell fraction (CCF) of each alteration were defined using state-of-the-art bioinformatics algorithms.

Results: 82% (9/11) and 18% (2/11) of BRCA1-breast cancers were triple-negative and ER-positive/HER2-negative, respectively. All BRCA2-cancers were ER-positive, of which one was HER2-positive. Sequencing analysis revealed a median of four (2-11) and two (0-6) non-synonymous somatic mutations in BRCA1- and BRCA2-breast cancers, respectively. Within BRCA1-breast cancers all but one (10/11, 91%) harbored TP53 clonal somatic mutations, and clonal somatic loss of the BRCA1 wild-type allele was found in 8 of these cases. The BRCA1-breast cancer lacking these alterations was ER-positive and the only case harboring a PIK3CA mutation. Additional clonal mutations identified in BRCA1-breast cancers included somatic mutations affecting EGFR and RB1. Subclonal mutations in known cancer genes (e.g. GATA3 and PTEN) were also identified, suggesting intra-tumor genetic heterogeneity. All BRCA2-breast cancers analyzed displayed clonal loss of the wild-type of BRCA2, but no gene was found to be recurrently mutated.

Conclusions: BRCA1- and BRCA2-breast cancers are both characterized by clonal inactivation of the BRCA1 and BRCA2 wild-type alleles, respectively, which likely constitute early genetic events. Within BRCA1-breast cancers, TP53 mutations are highly recurrent and clonal, and may precede somatic loss of the BRCA1 wild-type allele, as the latter was subclonal in one case harboring a clonal TP53 somatic mutation.

#101

Identification of pancreatobilliary cancer specific fusion genes in patient tissues.

Kahee Kim,1 Dawoon E. Jung,2 Mi-kyoung Seo,3 Sang Woo Kim,3 Si Young Song4. 1 _Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Republic of Korea;_ 2 _Institute of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Yonsei Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 4 _Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea_.

Gene fusion occurs when a part of one gene fuses with or attaches to a part of another gene by genome rearrangement and the result in gene fusion may possess oncogenic properties; fused gene may be translated into a unique protein that may promotes cancer properties. Pancreatic cancer and bile duct cancer are one of the most lethal cancer types with low 5-year survival rates, but lack of proper diagnostic or prognostic markers. Our aim was to investigate pancreatobilliary- specific fusion genes found in patient's specimens. We used TRIzol to extract total RNA from seven pancreatic cancer tissues and normal tissues from the same patients, and five bile duct cancer tissues and normal tissues from the same patients. Total RNA was assessed for RNA sequencing and the result data was analyzed using ChimeraScan to detect gene fusion. To identify the cancer somatic fusion gene, the fusion occurred in normal tissues and previously reported fusion genes found in normal tissues were excluded and the fusion occurred in coding region was included. As a result, we found 87 pancreatic cancer tissue specific- and 52 bile duct cancer tissue specific- fusion genes. We then selected fusion genes that either one of the gene has been reported to express in cancer tissues. We confirmed the expression of selected fusion genes by RT-PCR in cancer and normal tissues as well as in cancer cell lines. As a result we observed novel pancreatobilliary cancer specific fusion genes. These findings indicate the presence of novel fusion genes as well as its possible application for the early diagnosis or prognosis of pancreatic and bile duct cancer.

#102

Pan-cancer analysis of sex differences in somatic mutation profiles.

Constance H. Li, Syed Haider, Paul C. Boutros. _Ontario Institute for Cancer Research, Toronto, Ontario, Canada_.

Sex influences the clinical and pathological characteristics of a number of cancers including those of the lung, colon and kidney. Men and women differ in incidence rate, response to treatment and survival - even after controlling for confounding factors such as smoking status, age and weight. Some gene-specific sex differences have been identified in these cancers, but there has not been a rigorous, genome-wide analysis of the mutational differences in tumors between men and women. We fill this gap, providing a comprehensive assessment of sex-associated differences in cancer genomics.

We leverage resources from the International Cancer Genome Consortium-The Cancer Genome Atlas pan-cancer project, which includes genomic, transcriptomic and epigenomic profiles of 25,000 tumours and whole genome sequences of 5,000 tumours. Using this data, we analyzed mutational differences between the sexes in 15 tumor types.

We use both univariate (t-test, proportions test) and multivariate (multinomial logistic regression) statistical techniques, along with bioinformatics and pathway analyses. We found distinct sex-associated genomic differences in the copy number aberration (CNA) profiles of six tumor types and in pan-cancer analysis. We observe higher genome instability in male-derived tumors and sex-biased mutations in specific genes and across genomic intervals. Using multivariate modeling to control for confounding variables reinforces the significance of sex in these genomic regions and also reveals additional sex-biased CNAs. Ongoing work with mRNA abundance data suggests an association between sex-differences in CNAs and mRNA abundance both in cis and in trans. Further investigation into affected genes and pathways may reveal fundamental differences in how tumors develop in men and women and provide explanations for reported clinical and pathological sex differences.

#103

Gene expression based subtypes of colorectal cancer.

Kaja G. Berg, Ina A. Eilertsen, Sharmini Alagaratnam, Stine A. Danielsen, Arild Nesbakken, Anita Sveen, Ragnhild A. Lothe. _Institute for Cancer Research, Oslo, Norway_.

Colorectal cancer is a heterogeneous disease and improved molecular stratification of patients is warranted for precise treatment. Several gene expression classification systems are reported in the literature, albeit many have shown low reproducibility in external validation. A robust random forest classification system based on gene expression profiles was recently proposed, suggesting the existence of four biologically different subtypes with associations to prognosis (CMS1, CMS2, CMS3 and CMS4) (Guinney,J., et al., Nat Med, 2015). By applying the CMS classifier to high-quality gene expression profiles from a consecutive series of >400 stage I-IV primary colorectal cancers we are able to reproduce results from Guinney et al in an independent cohort. Analyses of mutational hotspots in BRAF / KRAS and the complete coding sequence of TP53 confirms enrichment of BRAF mutations in CMS1 (p<0.001), TP53 mutations in CMS2 and KRAS mutations in CMS4 (p<0.001). We further validate high levels of somatic copy number aberrations (SCNAs) in CMS2 and CMS4 compared to CMS1 and CMS3 (pair-wise comparisons, fraction of genome affected by SCNAs: CMS1 vs CMS2: p<0.001; CMS1 vs CMS4: p<0.01; CMS2 vs CMS3: p<0.001; CMS3 vs CMS4: p<0.01). Additionally we explore within-subtype variations with respect to SCNAs in the CMS2 and CMS4 subtypes. Altogether our results validate the CMS system as a robust classification system for colorectal cancer.

#104

Investigation of genetic architecture of multiple myeloma by next generation sequencing.

Kerry Deutsch,1 Fang Yin Lo,1 Claire Olson,1 Sharon Austin,1 Kellie Howard,1 Amanda Leonti,1 Lindsey Maassel,1 Christopher Subia,1 Tuuli Saloranta,1 Nicole Christopherson,1 Kathryn Shiji,1 Shradha Patil,1 Stuart Schwartz,2 Peter Papenhausen,2 Steven Anderson,3 Anup Madan1. 1 _Covance, Seattle, WA;_ 2 _Laboratory Corporation of America® Holdings, Research Triangle Park, NC;_ 3 _Covance, Durham, NC_.

Myeloma is a genetically heterogeneous disease and is sub-classified based on the presence of structural variants and genetic mutations. Structural variants/copy number changes are historically identified by traditional methods such as karyotyping and fluorescence in situ hybridization (FISH). Although microarray based genome wide analyses greatly improve the resolution of structural variation, they may be limited by probe density. Consequently, identification of structural variation may be insensitive to specific disrupted gene(s), neglecting the sequence complexity that might underlie these rearrangements. Determination of the specific breakpoints of structural variants at the nucleotide level is required for a better understanding of the genetic causes and to enhance the development of therapeutics for patients. The emergence of Next-Generation Sequencing (NGS) technology has led to the identification of structural variants in the genome at a higher resolution relative to currently used cytogenetic methods. We analyzed DNA extracted from a set of patients with multiple myeloma, who had Affymetrix SNP array (~2.7 million probes) data, by whole exome sequencing (WES) at 100X coverage on the Illumina HiSeq platform to identify the full spectrum of associated genomic aberrations. Sequence data was mapped to the hg19 reference sequence and analyzed by various in-house developed and open source data analytic tools. Additionally, a custom sequence analysis pipeline was written to interrogate chromosomal deletions and translocations in these samples. Our analysis showed that ~43% (6/14) of patients have deletions in chr17p and/or chr13q. We further confirmed structural variants using the Integrative Genomics Viewer (IGV). These data indicate the efficacy of WES for the precise determination of translocation and inversion breakpoints. In addition, we were able to identify single nucleotide variants (SNVs) and insertions/deletions (indels) in these samples. We then used the Ingenuity Variant Analysis (IGV) program to identify clinically actionable variants. These datasets are being further analyzed by various pathway analysis tools to define possible pathogenic mechanisms in multiple myeloma.

#105

Bioinformatic analysis of RNA-Seq data to search for novel diagnostic/prognostic biomarkers of pancreatic ductal adenocarcinoma.

Omar J. Sosa, Vinícius F. Paixão, João C. Setubal, Eduardo M. Reis. _University of Sao Paulo, Sao Paulo, Brazil_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly human malignancies. The only curative treatment available is the surgical removal of the tumor in early stages of the disease. Current methods for early detection and treatment are poor, justifying more studies in this field. We aim to generate a high-resolution catalog of the PDAC transcriptome, to reveal transcriptional alterations associated with the malignant phenotype of PDAC. To that end 28 patient matched samples of PDAC and nontumor adjacent pancreatic tissue were processed for the production of rRNA subtracted cDNAs libraries and subsequently sequenced in the Illumina HiSeq platform producing 17 million 100 nt paired-end reads per sample on average. We are implementing an informatics pipeline to detect and evaluate the differential expression in PDAC of well-annotated genes, including long noncoding RNAs (lncRNAs). In addition, we will search for novel lncRNAs and alternative splicing isoforms of protein-coding genes expressed in pancreatic tissues. Following the initial quality control (FastQC) and trimming of low quality regions (Trimmomatic), reads from each sample were aligned to the human genome (TopHat2) and a count table with the number of reads per gene for each sample was created (HTSeq). For evidence of changes across experimental conditions we use DESeq2, identifying 310 genes up-regulated and 354 down-regulated (p-adjusted<0.001 and lfc<|3.3|), including 156 lncRNAs. The differentially expressed genes detected are involved in pathways related to cancer, regulation of cell cycle process, membrane and secreted proteins. De novo transcript assembly (Trinity) revealed a number of yet unannotated transcripts and isoforms expressed in pancreatic tissues. Altogether, the transcript catalog generated herein provides a valuable resource for the identification of novel putative candidate biomarkers to improve the early detection or evaluation of therapeutic response in PDAC.

Work supported by FAPESP, CNPq and CAPES.

#106

Transcriptionnal profiling of upper-tumor urothelial carcinomas.

Gabriel Malouf,1 Roger Mouawad,2 Xiaoping Su,3 Hui Yao,3 Eva Comperat,1 Morgan Roupret,1 Jean-Philippe Spano,1 David Khayat1. 1 _Salpetrier Hospital, IUC du cancer, Pierre et Marie Curie University, Paris, France;_ 2 _Salpetrier Hospital, Lab of Fondation Avec, IUC du cancer, Pierre et Marie Curie University, Paris, France;_ 3 _M.D Anderson Cancer Center, Houston, TX_.

Background: Upper-tumor urothelial carcinomas (UTUC) represent 5% among all urothelial tumors. The most frequent histological variant is transitional cell carcinomas (TCC). UTUC might arise within renal pelvis or ureters. Several studies suggested aggressive behavior of tumors arising within the ureter as compared to those which develop within renal pelvis. However, whether there is a biological basis of this difference remains unknown.

Methods: We performed RNAsequencing on fourteen UTUC including 6 cases arising in the ureter and 8 in renal pelvis. Library prep followed by next-generation sequencing were done using Illumina Hiseq platform. Unsupervised clustering using differentially expressed genes was used to identify cancer subtypes. Identified subtypes were correlated with clinico-pathological features. Pathway analysis was performed using Gene Set Enrichment Analysis (GSEA).

Results: Unsupervised clustering of gene expression revealed two clusters. Surprisingly, all tumors (n=5/5) encompassing C1 were located in the ureters as compared to only one (n=1/9) in C2 cluster (p=0.003). No difference was observed between C1 and C2 clusters according to age, gender, grade, muscle-invasion status and stage. Using stringer cut-off (fold change ≥ or ≤-2 and FDR<0.05), 34 genes were found to be differentially expressed between the 2 clusters. GSEA revealed negative enrichment of several pathways related to B and T-cell activation in C1 relative to C2 cluster. This was the case for NAIVE_TCELL_VS_DC_UP (p=0.01; FDR=0.16) and NAIVE_BCELL_VS_NEUTROPHIL_UP (p=0.002; FDR=0.12). Among the most overexpressed genes in C1 cluster, we identified the serine/threonine-protein kinase gene SGK2 (Fold change=17; FDR=0.004) as a putative therapeutic target. Of note, AIM2 which is part of the inflammasome with a key role in tumorigenic reversion was one of the most down-regulated genes (fold change= -24; FDR=0.02) in C1 cluster.

Conclusion: Our report suggests that transcriptional profiling of UTUC arising in the ureters and renal pelvis might be distinct. The observed difference might be related to immune response. Validation of these data in a larger independent cohort with analysis of the immune infiltrate is required.

#107

Unsupervised analysis of cancer-cell intrinsic transcriptional traits defines a new classification system for colorectal cancer with improved predictive and prognostic value.

Andrea Bertotti,1 Claudio Isella,1 Sara E. Bellomo,1 Francesco Brundu,2 Francesco Galimi,1 Elisa Ficarra,2 Livio Trusolino,1 Enzo Medico1. 1 _University of Torino, Candiolo, Italy;_ 2 _Politecnico di Torino, Torino, Italy_.

Recent work built on published transcriptional subtypes of colorectal cancer (CRC) has shown that tumor stromal content heavily impacts CRC classification, with clinical and biological implications. Lineage-dependent transcriptional components of stromal origin could therefore dominate over more subtle, yet relevant, gene expression traits inherent to cancer cells. Based on the notion that in patient-derived xenografts (PDXs) stromal cells of the human tumor are substituted by their murine counterparts, we assessed cancer-cell intrinsic transcriptional features by human-specific expression profiling on 515 PDXs from 244 CRC patients. We identified five CRC intrinsic subtypes (CRIS) endowed with molecular, functional and phenotypic peculiarities, many of which were not previously evidenced: CRIS-A was a MIN-like, KRAS-mutated, and secretory subtype with sustained glycolytic metabolism and inflammatory traits; CRIS-B grouped a subset of poorly differentiated tumors with active TGFβ signaling and EMT features; CRIS-C clustered KRAS wild-type CIN tumors exhibiting elevated EGFR pathway activity and MYC copy number gains; CRIS-D tumors were characterized by a typical stem phenotype with high WNT signaling, associated with strong enrichment in IGF2 amplification/overexpression and FGFR autocrine stimulation; CRIS-E again displayed WNT-related features, but associated with a Paneth-like phenotype and a TP53-mutated genotype. CRIS subtypes successfully categorized independent sets of primary and metastatic CRC tumors, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances. Integration of stroma-centered transcriptional profiles with cancer-cell intrinsic classification by CRIS further enhanced prognostic significance.

#108

Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas.

Hio Chung Kang,1 Hong Kwan Kim,2 Sharon Lee,3 Pedro Mendez,1 James Kim,3 Gavitt Woodard,1 Kuang-Yu Jen,4 Li Tai Fang,1 Kirk Jones,4 David Jablons,1 Il Jin Kim1. 1 _Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA;_ 2 _Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 3 _CureSeq Inc, Brisbane, CA;_ 4 _Department of Pathology, University of California San Francisco, San Francisco, CA_.

Malignant pleural mesothelioma (MPM) is a rare malignancy with a highly unfavorable prognosis. A strong link has been established between increased risk for MPM and exposure to asbestos or erionite. As asbestos had widely been used in different industries, the incidence of MPM in the United States is expected to steadily rise and peak with about 70,000 new MPM cases over the next 20 years. Median survival has ranged from 10-17 months. But the underlying genetic mechanism is not fully understood. Moreover, genetic alterations and causes for multiple primary cancer development including MPM are unknown.

We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. The development of multiple primary malignancies including a rare MPM led us to search for underlying genetic alterations. Interestingly, most mutations identified in the MPM patient were highly enriched for the mutant allele, suggesting a homozygous alteration or deletion of wild-type allele when minimal contamination of normal pleura in the MPM is taken into consideration. We found that most variants identified showed a high frequency of mutant alleles, except variants on chromosomes 7, 16 and 20. Analysis of tumor allelic ratio to normal using all variants in exome sequencing revealed that this mesothelioma showed genome-wide allelic loss or loss of heterozygosity (LOH), which appears to be distinct from other cancers that have many genetic alterations with focal allelic loss. To the best of our knowledge, this type of extensive genome-wide allelic loss has not been described in MPM.

Whole exome sequencing analysis revealed the MPM had R282W, a key TP53 mutation, and genome-wide allelic loss or loss of heterozygosity, a distinct genomic alteration not previously described in MPM. We identified frequent inactivating SETDB1 mutations in this patient and in 77 additional MPM patients (mutation frequency: 9%, 7/78) by targeted deep sequencing.

In summary, we identified genome-wide allelic loss in a patient who had MPM and two additional primary cancers, results which suggest that careful analysis in exome sequencing is needed to detect genome-wide deletion in MPM samples with or without multiple primary cancers. The high frequency of mutations in SETDB1 that we found suggests that this and other histone-related genes are important in MPM.

#109

Multiple different mutations of same cancer genes in the same tumors from lung adenocarcinoma patients.

Yan Xing,1 Jianjun Zhang,2 Jun Li,2 Jianhua Zhang,2 Alexandria T. Phan,1 Jenny C. Chang,1 Andrew Futreal2. 1 _Houston Methodist Hospital, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Previous studies have demonstrated multiple different mutations of the same cancer genes in different subclones (multiple-hit) of the same tumors suggesting cancer gene convergence. By analyzing publically available large dataset, we sought to investigate whether multiple-hit phenomenon reflects a propensity for specific routes of tumorigenesis in certain patients with lung adenocarcinomas.

Methods: Whole exome sequencing data from the Cancer Genome Atlas (TCGA) lung adenocarcinoma study was queried and nonsilent somatic mutations (stop-gain, stop-loss, frameshift, splicing site and nonsynonymous) were analyzed. Bootstrapping was performed to investigate whether multiple-hit (≥ 2 mutations of the same gene in the same tumor) phenomenon is enriched in cancer genes (from Cancer Gene Census (CGC), Catalogue Of Somatic Mutations In Cancer (COSMIC) database excluding "Translocation only" genes) compared to all other genes (background). Using stage-stratified Cox proportional hazards regression, we examined the association between multiple-hit mutation status in well-known cancer genes and overall survival (OS) in patients who had complete staging and follow up information in the most recent TCGA survival dataset (updated on 4/10/2015). Subclonal analysis is in process to assess the clonal relationship among different mutations of the same genes in the same tumors.

Results: Somatic mutation data from 538 lung adenocarcinomas was analyzed. In total, 17,059 genes showed at least one nonsilent mutation in at least one tumor and 9,151 genes (54%) demonstrated multiple different nonsilent mutations in at least one tumor (multiple-hit). Of the 419 CGC cancer genes, 323 (77%) demonstrated multiple-hit phenomenon (p < 0.001). Totally 142,253 nonsilent mutations were identified in any genes, 27,616 (19%) of which were multi-hit mutations (background). Compared to this, 2,414 of 9,297 (26%) of nonsilent mutations identified in the 419 CGC cancer genes were multiple-hit mutations, which is significantly higher than background (26% vs 19%, p<0.001). Bootstrap resampling was performed with 10,000 samples confirmed above finding (p<0.01). Multivariate survival analyses suggested that multiple-hit mutation status was associated with worse OS for EGFR (HR 3.27, p=0.03, in patients with stage III/IV, median OS was 0.52 vs. 3.5 years) after adjusted by stage.

Conclusions: Cancer gene convergence in lung adenocarcinomas suggests certain genes and/or pathways may be critical for carcinogenesis in certain patients. Multiple different mutations of certain cancer genes may have impact on clinical outcomes. Further studies are warranted to further investigate the clinical implications of multi-hit phenomenon and their potential role in identifying novel cancer genes.

#110

Molecular profiling of Tudor domain-containing proteins identifies PHF20L1 as a candidate oncogene in breast cancer.

Yuanyuan Jiang,1 Lanxin Liu,1 Wenqi Shan,1 Zeng-Quan Yang2. 1 _Wayne State University, Detroit, MI;_ 2 _Wayne State University, Barbara Ann Karmanos Cancer Institute, Detroit, MI_.

Tudor domain-containing proteins (TDRDs), which recognize and bind to methyl-lysine/arginine residues on histones and non-histone proteins, play critical roles in regulating chromatin architecture, transcription, genomic stability, and RNA metabolism. Dysregulation of several TDRDs have been observed in various types of cancer. However, neither the genomic landscape nor clinical significance of TDRDs in breast cancer has been explored comprehensively. Here, we performed an integrated genomic and transcriptomic analysis of 41 TDRD genes in breast cancer (TCGA and METABRIC datasets) and identified associations among recurrent copy number alterations, gene expressions, clinicopathological features, and survival of patients. Among seven TDRDs (PHF20L1, ARID4B, SETDB1, LBR, TDRKH, TDRD10, and TDRD5) that had the highest frequency (>10%) of gene amplification, the plant homeodomain finger protein 20-like 1 (PHF20L1) was the most commonly amplified (17.62%) TDRD gene in TCGA breast cancers. Different subtypes of breast cancer had different patterns of copy number and expression for each TDRD. Notably, amplification and overexpression of PHF20L1 were more prevalent in aggressive basal-like and Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. Furthermore, knockdown of PHF20L1 inhibited cell proliferation in PHF20L1-amplified breast cancer cell lines. PHF20L1 protein contains N-terminal Tudor and C-terminal plant homeodomain domains. Detailed characterization of PHF20L1 in breast cancer revealed that the Tudor domain likely plays a critical role in promoting cancer. Mechanistically, PHF20L1 might participate in regulating DNA methylation by stabilizing DNA methyltransferase 1 (DNMT1) protein in breast cancer. Thus, our results demonstrated the oncogenic potential of PHF20L1 and its association with poor prognostic parameters in breast cancer. Furthermore, our findings provide strong foundations for further research and therapeutic targeting of PHF20L1 or other TDRDs in breast cancer.

#111

Recurrent mutations of CDKN2A genes characterize worse prognosis of HPV positive and negative oropharyngeal squamous cell carcinoma.

Jongkeun Lee, Dongwan Hong, Eun Kyung Lee, Yuh-Seog Jung, Weon Seo Park. _National Cancer Center, Goyang-si, Republic of Korea_.

Background: Oropharyngeal squamous cell carcinoma (OSCC) exhibits unique clinical and molecular characteristics, depending on its association with high-risk human papillomavirus (HPV) or other carcinogen like smoking1. Considering significant subset of patients with OSCC still exhibits poor response to contemporary treatments2, which frequently cause significant toxicity, a more detailed understanding of these poor responders, especially adopting current precision genomic technology, could provide a clue for developing better treatment.

Methods: In this study, we gathered the somatic point mutations and mRNA expression data of 520 OSCC patients from The Cancer Genome Atlas (TCGA) and then investigated recurrent somatic point mutations. To genes having the recurrent and same type point mutations, in addition, we check out the mRNA expression and analyzed a survival analysis.

Results: We found 7 somatic point mutations of CDKN2A gene, which having recurrent patients and only nonsense SNVs. Out of these mutation, 6 recurrent point mutations of CDKN2A gene have highly expressed on PIK3CA, FGFR2 and FGFR3 genes, as well as showed a significant deterioration on survival outcome.

Conclusion: Recurrent nonsense mutations of CDKN2A mostly (6/7, 85.7%) derived an abnormal regulation of other genes, such as PI3KA, FGFR2, and FGFR3. Moreover, this pattern of mutations significantly characterizes ominous prognosis in HPV positive and negative OSCC. Our result might provide better insights for characterizing the subgroup of OSCC and eventual development of precision strategy for this disease subset.

References

1. Licitra, L. et al. High-risk human papillomavirus affects prognosis in patients with surgically treated oropharyngeal squamous cell carcinoma. J Clin Oncol 24, 5630-6 (2006).

2. Ang, K.K. et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 363, 24-35 (2010).

#112

Genetic alterations in molecular tumor subgroups of malignant pleural mesothelioma.

Lisa Quetel, Robin Tranchant, Clément Meiller, Sandrine Imbeaud, Annie Renier, Françoise Le Pimpec-Barthes, Jessica Zucman-Rossi, Marie-Claude Jaurand, Didier Jean. _INSERM U.1162, Paris, France_.

Background: Malignant pleural mesothelioma (MPM) is a very aggressive neoplasm linked to asbestos exposure. In spite of research efforts to develop more effective therapeutic approaches, its prognosis remains poor, because of the absence of curative treatment. The identification of molecular profiles by omic analyses is a powerful approach to better define MPM subclasses and new targeted therapies. A recent transcriptomic analysis allowed to define a robust MPM classification and to identify two subgroups, C1 and C2, linked to histology and survival. The main finding was to separate epitheloid MPM, the most frequent histologic subtype, within these two subgroups. Epitheloid MPM with a worse survival prognosis were classified in the C2 subgroup. To better define the heterogeneity of MPM and establish a more precise classification, we determined genetic alterations in C1 and C2 MPM.

Material and Methods: Using a gene candidate approach, we performed Next-Generation Sequencing (Miseq, Illumina) in 84 MPM including 49 MPM primary cultures and 35 frozen tumors to identify genetic alterations in specific genes with a 200X depth. We selected 22 genes including key altered genes in mesothelial carcinogenesis (NF2, CDKN2A, CDKN2B BAP1, TP53 and LATS2) previously sequenced by Sanger method, genes mutated at low frequency in MPM (KRAS, HRAS, EGFR⋯) and genes recently suggested as altered in MPM (CUL1, KMT2D, SETD2⋯).

Results: Previous mutations identified by Sanger sequencing in our collection have been confirmed. The results showed an enrichment in C>T transitions, which was already observed in an exome sequencing study in MPM. We identified 93 variants inducing protein structure modification. Among them, 70% were deleterious to the function of the protein (deletion/insertion, splice-site mutation, substitutions nonsense and damaging missense predicted by SIFT or Polyphen). For most of the genes, the mutation frequency was consistent with literature data. The highest alteration frequency was found in CDKN2A, CDKN2B, NF2 and BAP1 genes (over 25%), lower frequencies were found in TP53, LATS2, KMT2D and SETD2 genes (5% to 20%). The alteration frequency of four genes was different between C1 and C2 tumor subgroups. BAP1 mutations were more frequent in C1 MPM, as we showed previously. We also identified new relevant mutations notably in the methyltransferases SETD2 and KMT2D that are also associated to C1 MPM. Mutations in TP53 were significantly associated with C2 MPM.

Conclusion: We defined the genetic alterations signatures of C1 and C2 tumor subgroups. Mutations in enzymes involved in chromatin organization seem to be characteristic of the C1 subgroup. Interestingly, TP53 mutations, which are known to provide tumor aggressiveness, are found in the C2 subgroup gathering patients with the worse prognosis. Ongoing analysis of 83 additional MPM would allow to confirm statistically all the associations we observed.

#113

Novel fusion transcripts identified by RNAseq cooperate with somatic mutations in the pathogenesis of acute myeloid leukemia.

Antonella Padella,1 Giorgia Simonetti,1 Anna Ferrari,1 Giulia Paciello,2 Elisa Zago,3 Carmen Baldazzi,1 Viviana Guadagnuolo,1 Cristina Papayannidis,1 Valentina Robustelli,1 Enrica Imbrogno,1 Nicoletta Testoni,1 Massimo Delledonne,3 Ilaria Iacobucci,1 Tiziana Clelia Storlazzi,4 Elisa Ficarra,2 Pier-Luigi Lollini,1 Giovanni Martinelli1. 1 _Univeristy of Bologna, Bologna, Italy;_ 2 _Politecnico di Torino, Dept. of Control and Computer Engineering, Torino, Italy;_ 3 _Personal Genomics, Verona, Italy;_ 4 _Univeristy of Bari, Bari, Italy_.

Acute Myeloid Leukemia (AML) is a highly heterogeneous disease and somatic mutations and fusion genes have a crucial diagnostic, prognostic and therapeutic role in AML. However, the leukemogenic potential of the fusions, their cooperation with somatic mutations and their prognostic role are still unknown.

The aim of the study was to identify novel rare gene fusions having a causative role in leukemogenesis and to identify cooperative somatic mutations as potential targets for personalized therapies in AML cases with rare and poorly described chromosomal translocations.

We performed RNAseq on bone marrow samples from 5 AML patients (#59810, #20, #84, #21 and #32). The presence of gene fusions was assessed with deFuse and Chimerascan. Putative fusion genes were prioritized using Pegasus and Oncofuse to select biologically relevant fusions. WES was performed on samples #59810, #20 and #21 and variants were detected using the software Mutect and GATK.

The CBFβ-MYH11 chimera was identified in sample #84, with inv(16), thus confirming the reliability of our analysis.

Sample #59810 carried the fusion transcript ZEB2-BCL11B (Driver Score, DS=0.7), an in-frame fusion and a rare event in AML associated with t(2;14). The fusion transcript showed 3 splicing isoforms and ZEB2 and BCL11B transcripts were upregulated in patient's blasts, compared with 53 AML samples with no chromosomal aberrations in the 14q32 region. The WT1-CNOT2 chimera was also detected, which is a novel out-of-frame fusion (DS= 0.008) related to t(11;12) translocation. WES analysis showed SNVs in TET2 and BCOR genes.

In sample #20 we identified two different fusion transcripts: CPD-PXT1 (DS= 0.07), which was the reciprocal fusion product of t(6;17) translocation, and SAV1-GYPB, which was cryptic at cytogenetic analysis (DS= 0.8). Interestingly, the latter fusion involved a tumour suppressor gene. Moreover, mutations in BCOR and NRAS were detected by WES.

RNAseq on sample #21 identified a novel fusion event between chromosomes 19 and 7, involving the genes OAZ and MAFK (DS= 0.9). The 3' partner gene is a transcription factor, a functional class recurrently altered in AML. This patient showed SNVs in CBL and SMC1A genes.

Finally, no chimeras were confirmed in sample #32 having a t(12;18) translocation.

Our data suggest that fusion events are frequent in AML and a number of them cannot be detected by current cytogenetic analyses. Gene fusions cooperate with somatic mutations in the genomic landscape driving AML pathogenesis and its heterogeneity. Moreover, the results indicate that different approaches, including WES, RNAseq bioinformatic and statistic tools need to be integrated in order to identify potential targets for personalized therapies.

Acknowledgements: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project.

#114

Oncogenomic analysis of Merkel cell carcinoma.

Tara Gelb, Kenneth Daily, Amy Coxon, Isaac Brownell. _Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD_.

We used an integrative oncogenomic approach to investigate the molecular etiology of Merkel Cell Carcinoma (MCC) in an effort to identify novel therapeutic targets and biomarkers for this highly aggressive neuroendocrine skin tumor. Because MCC is rare and poorly understood, there are currently no effective treatment options for patients with advanced disease. Approximately 80% of MCC tumors have Merkel Cell polyomavirus (MCV) DNA integrated into the host genome, and viral oncogenes are thought to drive carcinogenesis. Moreover it has been reported that MCV-negative MCC samples have an increased rate of mutations relative to MCV-positive tumors. Taken together, this suggests that MCV-positive and negative MCCs are driven by different mechanisms. We used whole exome sequencing and bioinformatic analyses to identify somatically dysregulated genes and pathways in MCC tumor samples. Additionally, we used a comparative genomic hybridization array to identify genomic amplifications and deletions. Whole transcriptome profiling was achieved using microarrays and RNA-sequencing. As the same tumor samples were used in each of these platforms, we were able to systematically correlate sequence variants with copy number changes and gene expression to identify high priority targets for further study. Interestingly, despite differences in driver mutations, we found relatively few differences in the global gene expression of MCV-positive and MCV-negative MCC tumors. Overall, our integrative oncogenomic approach has identified novel therapeutic targets for the treatment of MCC that are currently undergoing functional validation and preclinical testing.

#115

In silico **evaluation of DNA-damage-inducible transcript 4 gene (DDIT4) in the outcome of several malignant tumors.**

Joseph A. Pinto,1 Christian Rolfo,2 Leny Bravo,3 Williams Fajardo,3 Zaida D. Morante,1 Silvia Neciosup,1 Luis Mas,1 Alfredo Aguilar,1 Carlos Castañeda,1 Luis Pinillos,1 Carlos Vallejos,1 Henry Gomez1. 1 _Oncosalud, Lima, Peru;_ 2 _Phase I – Early Clinical Trials Unit, Antwerp University Hospital & Center for Oncological Research (CORE), Antwerp, Belgium; _3 _Universidad Privada San Juan Bautista, Lima, Peru_.

Objectives: Although DDIT4 gene encodes a protein whose main action is to inhibit mTOR. There are not previous reports relating this gene with the clinical outcome in cancer patients. Our aim was to explore the influence of this gene in the clinical characteristics and the outcome of malignant tumors.

Methods: We evaluated in an in silico study of publicly available datasets, influence of DDIT4 expression in the clinical outcome in terms of either disease-free survival (DFS), progression free survival (PFS) or overall survival (OS), according to the availability of this variable in the dataset. KM Plotter (http://kmplot.com/) and SurvExpress (http://bioinformatica.mty.itesm.mx/) were used to evaluate the influence of DDIT4 gene in the outcome (either DFS, PFS or OS). cbioportal.org was used to explore structural gene changes. The median of expression discriminate two groups at different risk. Twelve types of cancer were evaluated including. Acute Myeloid Leukemia, Brain Cancer, Breast Cancer, Bone Cancer, Cervical Cancer, Head and Neck Cancer, Hematological Cancer, Liver Cancer, Lung Cancer, Pancreatic Cancer, Ovarian Cancer and Prostate Cancer

Results: DDIT4 had low structural alterations rates (0-5.1%); however, in breast tumors xenografts, gene amplification occurs in 17.4%, indicating a key role in tumor progression. High levels of DDIT4 was significantly associated with a worse outcome in breast cancer (n=4142), P=3x10-14 (HR=1.64, CI95%: 1.44-1.87). In this malignancy, high DDIT4 expression was associated with the triple negative phenotype and higher pathological complete response rates. In addition High DDIT4 level was associated to a worse outcome in acute myeloid leukemia (n=168), P=3.47x10-5 (HR=2.31, CI95%:1.55-3.43), glioblastoma multiforme (n=538), P=0.005809 (HR=1.31, CI95%:1.08-1.59); ovarian cancer (n=1648), P=0.0096 (HR=1.2, IC95%:1.04- 1.37); head and neck squamous cell carcinoma (n=283), P=0.03347 (HR=1.49, IC95%:1.03-2.15) and lung adenocarcinoma (n=866), P=0.0038 (HR=1.4, CI95%: 1.02-1.91). In contrast, a high level of DDIT4 was associated with a better prognosis in gastric cancer (n=641), P=1.7x10-6 (HR=0.62, CI95%:0.5-0.75) and lung squamous cell carcinoma (n=675), P=0.0015 (HR=0.42, IC95%:0.24-0.73).

Conclusion: Although DDIT4 structural alterations are infrequent, its dysregulation impact in the clinical outcome in several types of cancer. DDIT4 is an attractive target to improve the therapeutic strategies, mainly those related with mTOR inhibition.

#116

Study of TERT promoter and FGFR3 mutations in upper-tumor urothelial carcinomas.

Roger Mouawad,1 Souheyla Bensalma,2 Xiaoping Su,3 Frederick Allanic,2 Eva Comperat,4 Morgan Rouprêt,4 JeanPhillipe Spano,4 Gabriel Malouf,4 David Khayat4. 1 _AP-HP, Salpetriere Hospital, Lab of Avec Foundation,IUC, Pierre et Marie Curie University, Paris, France;_ 2 _Lab of Avec Foundation, IUC, Pierre et Marie Curie University, Paris, France;_ 3 _M.D. Anderson Cancer Center, Houston, TX;_ 4 _AP-HP, Salpêtrier Hospital, IUC, Pierre et Marie Curie University, Paris, France_.

Background: Mutations in the promoter of the telomerase reverse transcriptase (TERT) and fibroblast growth factor receptor 3 (FGFR3) genes represent the most recurrent somatic alterations in urothelial carcinomas of the bladder. However, data regarding frequency and prognostic values of these alterations in upper-tumor urothelial carcinoma (UTUC) remain limited.

Methods: DNA was extracted from 31 UTUC including 12 cases arising in the ureter and 19 in renal pelvis. Somatic mutations of TERT promoter and FGFR3 gene were assessed by Sanger sequencing. The generated sequences were analyzed by seqscape and mutational status of each of those genes were correlated with clinico-pathological tumor features. Disease-free survival and overall survival were estimated using the Kaplan-Meier method.

Results: Median patient's age was 72 years (range: 41-83) with a male predominance (90.3%). Mutation status of TERT and FGFR3 were obtained in all tumors. Out of 31 UTUC, 81% were classified as high-grade including 56% which were muscle-invasive (≥pT2). TERT and FGFR3 mutations were detected in 19 (61%) and 14 (46%) tumors, respectively. Of note, 8 (26%) out of them harbored both mutations. In the TERT-mutated subgroup, we observed higher incidence of men as compared to TERT non-mutated subgroup (p=0.04); in addition, we did not identify any other significant differences regarding other clinico-pathological features. This was also the case for FGFR3 mutations, although we observed a higher frequency in tumors arising in the ureter (66.6%) as compared to those located in renal pelvis (28.5%) (p=0.11). Neither mutations of FGFR3 nor TERT were associated with progression-free survival and patient's overall survival.

Conclusion: Our study identified a high rate of somatic mutations in FGFR3 and TERT which were similar between muscle-invasive and non-muscle invasive tumors as well as between tumors arising in the ureter or renal pelvis. As targeted inhibitors are being investigated in this setting, these results might have implications for patient's management.

### Genomic Profiling of Cancers

#117

Genetic landscapes of relapsed and refractory diffuse large B cell lymphomas.

Nathalie Johnson,1 Sarit Assouline,1 Miguel Alcaide,2 Arezoo Mohajeri,2 Celia Greenwood,1 Yasser Riazalhosseini,1 Koren Mann,1 Ryan Morin2. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Simon Fraser University, Burnaby, British Columbia, Canada_.

Purpose: Relapsed or refractory diffuse large B cell lymphoma (rrDLBCL) is fatal in 90% of patients and yet little is known about its biology.

Experimental Design: Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions.

Results: Based on the a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3, CCND3, NFKBIZ and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ. We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B cell-type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes.

Conclusions: This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL.

#118

A comprehensive profile of the genomic architecture of curable prostate cancer.

Michael E. Fraser,1 Theodorus van der Kwast,1 John McPherson,2 Colin C. Collins,1 Yves Fradet,3 Bernard Tetu,3 Alain Bergeron,3 Rob G. Bristow,1 Paul C. Boutros2. 1 _Univ. of Toronto Ontario Cancer Inst., Toronto, Ontario, Canada;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _Universite Laval, Quebec City, Quebec, Canada_.

There is an urgent need to develop novel biomarkers of treatment response for precision medicine in prostate cancer, particularly in the setting of localized, non-indolent disease, which represents the vast majority of cases at initial clinical presentation. The Canadian Prostate Cancer Genome Network (CPC-GENE) - a member of the International Cancer Genome Consortium - aims to identify multi-modal prognostic and predictive signatures of therapeutic outcome based on intrinsic tumour genomics, transcriptomics, and epigenomics, which are being incorporated into novel clinical trials of treatment escalation/de-escalation for image-guided radiotherapy and/or radical prostatectomy. Herein we report the results of the largest prostate cancer whole-genome sequencing study to date, consisting of 194 patients and whole-exome sequencing of 479 patients with localized, potentially curable disease; a sample size that allows for saturating discovery of genes mutated at ≥1% frequency. We show that tumours treated by curative local therapy have a paucity of clinically-actionable mutations relative to high-risk localized or metastatic disease, but instead harbor a significant number of recurrent non-coding aberrations and genomic rearrangements, including a novel inversion - and an associated reduction in gene expression - of the PTEN tumour suppressor gene on chromosome 10. The median tumour contains three distinct driver mutations, with >86% of tumours possessing at least one known driver. Importantly, multiple driver aberrations are associated with patient outcome, including copy number aberrations and methylation events, but notably excluding well-described recurrent events such as TMPRSS2:ERG fusions and SPOP point mutations. Localized hypermutation events (e.g. kataegis) were detected in >20% of tumours, and were strongly enriched for aggressive disease. Taken together, these data provide a comprehensive analysis of the genomic landscape of localized, non-indolent prostate cancer. Moreover, our results strongly suggest that specific molecular aberrations may serve as useful biomarkers for improved pre-treatment stratification of patients with localized, low/intermediate risk disease into escalation/de-escalation protocols, and support the development of clinical trials to assess this hypothesis, based on patient-specific molecular profiles.

#119

Genomic analysis of remnant gastric adenocarcinoma.

Edaise M. da Silva, Helen H. Won, Dianne Torrence, Daniel Coit, Michael F. Berger, Laura Tang. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Introduction: Prior to pharmaceutical treatment of gastroduodenal ulcer disease, partial gastrectomy was performed for the management of ulcer disease complicated by bleeding (PUD). The proposed mechanism for the development of gastric cancer in this setting is the chronic irritation of the mucosa by duodenal reflux into the gastric remnant. The aim of this study is to characterize the genetic profile of remnant gastric carcinoma (RGC).

Material and Methods: Thirty cases of RGC developed in the setting of prior partial gastrectomy for PUD and without neoadjuvant chemotherapy were retrospectively collected from the institution database. Histopathological evaluation was performed to ensure >80% tumor cellularity with matching normal gastric mucosa prior to DNA extraction. Ultra-deep targeted sequencing of the thirty matched pairs was performed using the MSK-IMPACT assay (Integrated Mutation Profiling of Actionable Cancer Targets) to identify mutations and copy number alterations in a common set of 410 cancer-associated genes. MSK-IMPACT data from previously sequenced 45 sporadic gastric carcinomas (GC) was used as comparison.

Results: The cohort consisted of 22 (73%) males and 8 (27%) females with the mean age of 73 years. The median interval between PUD and RGC resections was 34.8 years (7-60 years). By Lauren's classification, 60% of the tumors were intestinal, 23% diffuse and 17% mixed. Most of the tumors (47%) were poorly differentiated and stage II (40%). MSK-IMPACT sequencing revealed 299 total somatic alterations occurring in 148 of 410 targeted genes with a median number of non-synonymous somatic mutations and copy number events of 3 and 1 per patient, respectively. Of the 207 total mutations, 27.5% were hotspot COSMIC and 23.2% were loss-of-function mutations. Recurrent genomic events included mutually exclusive mutations in TP53 (33%) and amplifications in MDM2 (26%), alterations in the ERBB family genes (37%), ARID1A (16%), KMT2D (13%), CDH1 (13%), and CDK4 (13%). In addition, actionable alterations in PIK3CA (10%), FGFR1 (4%), PTEN (4%), and concurrent hotspot KRAS mutations and inactivation of p15/p16 (10%) were observed. Compared to the sporadic GC cases, RGC cases presented differences in the landscape and recurrence of alterations.

Conclusion: The pathogenesis of RGC is likely associated with the prolonged exposure of the gastric mucosa to gastroduodenal contents. The mean age of patients may also exert effects on the development of the RGC. Based on our sequencing results, RGC may present a genetic profile distinct from that of sporadic gastric tumors. Assessment of these alterations identified 30% of patients with potential therapeutic targets for the treatment of this disease and deserves further investigation.

#120

Estrogen and progesterone receptor expression in different molecular uterine leiomyoma subclasses.

Netta Mäkinen,1 Annukka Pasanen,2 Hanna-Riikka Heinonen,1 Kati Kämpjärvi,1 Simona Bramante,1 Miika Mehine,1 Jyrki Jalkanen,3 Oskari Heikinheimo,4 Jari Sjöberg,4 Ralf Bützow,2 Lauri Aaltonen1. 1 _Department of Medical and Clinical Genetics, Medicum and Genome-Scale Biology Research Program, Research Programs Unit, University of Helsinki, Helsinki, Finland;_ 2 _Department of Pathology, The Laboratory of Helsinki University Central Hospital (HUSLAB), Helsinki University Central Hospital and Medicum, University of Helsinki, Helsinki, Finland;_ 3 _Department of Obstetrics and Gynecology, Central Finland Central Hospital, Jyväskylä, Finland;_ 4 _Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland_.

Uterine leiomyomas (ULs) are benign, estrogen- and progesterone-dependent smooth muscle tumors of the uterus. They are among the most common human neoplasms with an estimated prevalence of 20-40% in women of reproductive age, but also percentages as high as 77% have been reported. Although benign, ULs form a major burden to women's health. Approximately 25% of women with ULs have clinically relevant lesions, which cause morbidity and thus require treatment. As a consequence, ULs are the leading cause of hysterectomy worldwide. Despite the high prevalence and socio-economic impact of ULs, the molecular mechanisms underlying the growth and development of these lesions remain largely unknown. Familial, epidemiological, and cytogenetic studies indicate that genetic factors play a central role in the development of these lesions. Our recent findings, derived from the use of high-throughput technologies, have pointed to at least three distinct molecular UL subclasses, each candidate subclass displaying a characteristic genetic driver aberration as well as global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, and FH (fumarate hydratase)-deficient ULs. Although the majority of ULs show estrogen and progesterone receptor (ER and PR) positivity on protein level, a subgroup of these tumors are ER- or PR-negative with an estimated frequency of up to 10%. The aim of this study is to assess the potential correlation between steroid receptor expression and different molecular UL subclasses. The study material consists of 1100 ULs collected from hysterectomy patients during surgery in Helsinki University Central Hospital and Central Finland Central Hospital, Finland. All ULs are systematically screened for MED12 mutations by Sanger sequencing. Because overexpression of HMGA2 typically arises through a chromosomal translocation, HMGA2-overexpressing ULs as well as FH-deficient tumors are tentatively identified using a high-density customizable Infinium® HumanCore-24+ BeadChip with 4000 additional custom markers that cover known genes, candidate regions, and single-nucleotide variations related to UL genesis. Also ULs without any known genetic driver aberrations are included in the study. ER and PR protein expression are analyzed by immunohistochemistry. Currently, molecular classification of the tumors plays no role in choosing treatment, but ULs are treated as a single entity. Different molecular subclasses may, however, use different molecular pathways, have different clinical outcome, or response to treatment. Identification of ER- and PR-negative ULs and their potential association with the genetic driver aberrations may provide important new information on the molecular mechanisms underlying UL genesis. Increased knowledge is also essential for developing new targeted treatments and improving management of the condition.

#121

Analysis and comparison of somatic copy number alterations in gastric cardia adenocarcinoma and gastric non-cardia adenocarcinoma.

Manjiao Liu,1 Zhen Liu,1 Jian Bai,1 Hong Cai,2 Yang Ke,2 Changqing Zeng1. 1 _Beijing Institute of Genomics, Beijing, China;_ 2 _MOE Key Laboratory of Carcinogenesis and Translational Research, Laboratory of Genetics, Peking University, Beijing, China_.

Gastric adenocarcinoma is one of the most common cancer type worldwide. Anatomically it may raise from the cardia or non-cardia part of the stomach and previous studies have shown differential susceptibilities loci and prognosis between gastric cardia adenocarcinoma (GCA) and gastric non-cardia adenocarcinoma (GNCA). To better understand the genomic alterations that drive gastric adenocarcinoma and to investigate the differences of genetic aberration between GCA and GNCA, we analyzed somatic copy-number aberrations in 205 gastric adenocarcinomas resected samples, including 114 GCAs and 91 GNCAs using Illumina Human OmniZhongHua BeadChips. We identified 49 significantly recurrent focal alterations in gastric adenocarcinoma. In addition to those previously reported in stomach cancer, several novel focal changes were also observed, including gain of 11p11.2, 6q23.3, 12p11.1, 5p15.33 and loss of 8p11.22, 9p22.2, 18q22.1, 15q21.2, 17q23.3. A few known or potential oncogenes such as TERT, MYB, and IRS2, were included in the focal amplified regions that were not reported in gastric cancer before. Also, a few potential tumor suppressors were seen in these focal loss regions, including DOK6, BRIP1 and NDST4. Especially, HDC, a gene involved in gastric acid secretory, was harbored in the newly identified focal loss region, 15q21.2. Three focal changes, loss of 20p12.1 (containing MACROD2), 6q26 (containing PARK2), and gain of 11p11.2 (containing CRY2), were identified to correlate to the post-surgery overall survival, which may deserve to be further investigated as the potential prognosis markers. Comparison of somatic copy number alternation patterns revealed that in chromosome arm level, 16p and 5q had distinct copy number status between GCA and GNCA samples. The frequencies of three novel focal changes were significantly different between GCA and GNCA. Furthermore, KRAS amplification, a well characterized gastric adenocarcinoma prognosis marker was associated with worse clinical outcome in GNCA, but not in GCA.

Taken together, these results provided novel candidate oncogenes or tumor suppressors for gastric adenocarcinoma and different features in structural alterations between GCA and GNCA, which potentially inform novel opportunities for targeted therapeutic interventions.

#122

Comparative analysis of small cell lung cancer and other pulmonary neuroendocrine tumors.

Julie George,1 Lynnette Fernandez-Cuesta,1 Vonn Walter,2 Neil Hayes,2 Roman Thomas1. 1 _Department of Translational Genomics, University of Cologne, Cologne, Germany;_ 2 _UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC_.

Small cell lung cancer (SCLC) is accounting for 15% of all lung cancer cases and belongs to the family of pulmonary neuroendocrine tumors. This tumor entity additionally includes large-cell neuroendocrine carcinomas (LCNEC) and pulmonary carcinoids (PCA), which occur in 3% and 2% of all lung cancer patients, respectively. Whereas lung carcinoids are clinically benign, SCLC and LCNEC are characterized as high-grade malignant tumors, which are associated with heavy smoking and a poor 5-year survival rate of less than 5%.

We aimed to characterize the genomic alterations in neuroendocrine lung tumors and performed whole genome, whole exome and transcriptome sequencing on up to 110 SCLC cases, over 40 pulmonary carcinoids and over 50 LCNEC tumors.

SCLC and LCNEC tumors reveal high mutation rates with an average of 9.5 and 8.6 mutations per megabase, respectively. Our sequencing studies have shown that SCLC tumors are characterized by bi-allelic inactivation of TP53 and RB1 in almost 100% of the cases. Recurrent significant genomic alterations affected among others TP73 (13%) and NOTCH family genes (25%). Further studies in a pre-clinical mouse model confirmed NOTCH as tumor suppressor and as a regulator of neuroendocrine differentiation in SCLC (George et al., 2015).

LCNEC tumors showed TP53 and RB1 alterations in up to 20% of the cases. Additionally, LCNECs were found with STK11 and KEAP1 mutations, which occurred mutually exclusive to RB1 alterations (P<0.05).

In light of these distinct mutational characteristics, we analyzed the transcriptome sequencing data of neuroendocrine lung tumors in comparison to lung adenocarcinomas (LUAD) and lung squamous cell carcinomas (LUSQ). An unsupervised clustering approach led to the identification of five expression clusters: LUAD, LUSQ and PCA formed distinct transcriptional classes; the majority of SCLC and LCNEC tumors clustered into two subgroups. While a few LCNEC tumors clustered with LUAD, LUSQ and PCA, RB1-mutated LCNEC tumors predominantly clustered with SCLC samples (P <0.05). KEAP1 and STK11 mutated LCNECs did not segregate with a particular histological tumor type.

Neuroendocrine lung tumors confirmed high expression levels of neuroendocrine markers. A detailed comparison of LCNEC and SCLC tumors revealed that the majority of the SCLC and a few LCNEC tumors were found in a transcriptional group that showed high expression of the neuroendocrine lineage transcription factor ASCL1, gastrin releasing peptide (GRP) and DLL3, which was less pronounced in transcriptional subsets formed by the majority of LCNEC tumors. Patient tumors that clustered to the transcriptional class marked by higher ASCL1 expression were identified to have a worse overall survival (P<0.05).

In summary, these findings point to transcriptional differences in SCLC and LCNEC tumors and emphasize the precise analysis of these histological tumor types with respect to their molecular biology and therapeutic treatment option.

#123

Multiplatform molecular profiling of invasive lobular breast cancer.

Raquel A. Nunes,1 David Arguello,2 Zoran Gatalica,2 Sandeep Reddy,2 Sandra M. Swain1. 1 _Washington Hospital Ctr., Washington, DC;_ 2 _Caris Life Sciences, Phoenix, AZ_.

Introduction: Invasive lobular breast cancer (ILC) is the second most common subtype of invasive breast cancer accounting for 10% of breast cancer diagnosis. ILC has particular histological and clinical characteristics and a distinct response to therapy. Characterizing the molecular alterations in ILC may lead to an improved understanding of its biology and provide new therapeutic options. The purpose of this study is to describe the molecular profile of ILC and compare it to the one of invasive ductal cancer (IDC).

Methods: Three-hundred and thirty nine pure ILC specimens profiled from January 2012 - November 2015 were evaluated (Caris Life Sciences, Phoenix, AZ). Multiplatform profiling consisted of gene sequencing (next generation sequencing [NGS]), gene amplification (CISH or FISH), and protein expression (immunohistochemistry [IHC]). Molecular characteristics of estrogen receptor (ER) positive and human epidermal growth receptor factor 2 (HER2) negative pure ILC (n= 236) and IDC (n=286) were compared.

Results: By IHC, ER expression was present in 87.7% (277/316), progesterone receptor in 59.6% (198/313), HER2 in 3.5% (11/313), androgen receptor in 87% (262/301), PD-L1 in 8.1% (12/148) and PTEN in 63.3% (198/313). Amplifications were detected in MYC (7.7%, 2/26), EGFR (8.3%, 2/24), ERBB2 (4.5%, 13/290) and TOP2A (1.3%, 3/236). Mutations were detected in AKT1 (4.7%, 9/191), ATM (3.7%, 7/190), BRCA1 (4.2%, 4/96), BRCA2 (9.5%, 9/95), ERBB2 (7.5%, 14/186), PIK3CA (54.5%, 103/189), PTEN (7.9%, 15/189), and TP53 (13.4%, 25/186). A comparison of ER-positive/HER2-negative invasive lobular and ductal carcinomas revealed significant differences in AR expression (89.7% vs 79.6%, p = 0.0022), and mutations in CDH1 (10.1% vs 0.0%, p = 0.0001), ERBB2 (8.2% vs 2.1%, p = 0.0079), and TP53 (10.3% vs 31.8%, 0.0001).

Conclusions: Multiplatform testing of this large series of ILC reveals recurrent alterations and a distinct molecular profile when compared to IDC. These support the definition of ILC as biologically distinct entity. High AR expression and high rates of dysregulation along the PIK3CA/AKT/mTOR pathway are consistent with recent reports in the literature.

#124

Molecular pathogenesis of an advanced cutaneous T-cell lymphoma.

Linghua Wang,1 Xiao Ni,2 Kyle R. Covington,1 Liu Xi,1 David A. Wheeler,1 Madeleine Duvic2. 1 _Baylor College of Medicne, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of cutaneous T-cell lymphoma (CTCL). MF is primarily a benign disease often confined to skin. MF, in about 10% of cases, can progress to SS or transform to large-cell histology. SS, the leukemic variant of CTCL, is defined as erythroderma on ≥ 80% of the skin plus ≥1000/μL circulating malignant T-cells. SS is very aggressive with a median overall survival of only 2-5 years. So far, effective treatment options for CTCLs are still very limited and the underlying genetic basis remains incompletely characterized. Here, we performed integrated genomic analyses of 105 patients with de novo or transformed SS, using whole-exome, targeted exon, transcriptome sequencing and SNP array.

Frequent somatic alterations were identified in TP53, CARD11, CCR4, PLCG1, TNFRSF1B, CDKN2A, ARID1A, RPS6KA1 and ZEB1. Activating CCR4 and CARD11 mutations were detected in nearly one third of patients. CCR4 is important for T-cell migration into the skin. All CCR4 mutations were either nonsense or frameshift and located at the C-terminus. CARD11 is required for T cell receptor (TCR)-mediated activation of NF-κB signaling. About half of the CARD11 mutations were located in the domain previously reported in diffuse large B cell lymphoma (DLBCL). The remaining mutations were located at a highly conserved new hotspot. As was previously suggested in DLBCL, therapies targeting CARD11 signaling could benefit SS patients. PLCG1 is a key modulator of TCR signaling. In addition to the recently reported S345F hotspot mutation, we identified a new hotspot located in the C-terminal of the protein. The mutation frequency of the new hotspot was significantly higher in patients with prior history of MF, suggesting the possibility that PLCG1 may play a role in transformation of MF to SS. Focal deletion of ZEB1 was observed in over half of the patients. ZEB1 is suggested to be critical for early T-cell development. ZEB1 downregulation contributes to resistance to TGF-β1-mediated growth suppression in adult T-cell leukemia/lymphoma derived cell lines. Loss of ZEB1 may play a similar role in SS pathogenesis. IL32 and IL2RG were strongly upregulated in nearly all patients. IL32 protein expression is correlated with the mRNA expression and the density of Sézary cells in patients' blood. IL32 accelerates the proliferation of CTCL cell lines through mitogen-activated protein kinase and NF-κB-mediated signaling. IL32 could become a target that is specific to SS patients. Surprisingly, the UVB mutation signature was detected in most of the cases, indicating that the malignant clonal population arose from a cell that spent significant time in the superficial layers of the skin and challenging the view that SS originated from the circulating memory T-cell rather than those resident in the skin.

Our results showed profound disruption of key T-cell signaling in SS patients and suggested potential targets for novel therapies.

#125

Whole-exome sequencing identifies NF-kappaB pathway regulators frequently mutated in nasopharyngeal carcinoma.

Hong Zheng, Wei Dai, Arthur KL Cheung, Josephine MY Ko, Rebecca Kan, Bonnie WY Wong, Merrin ML Leong, Maria L. Lung. _The University of Hong Kong, Hong Kong, Hong Kong_.

Introduction

Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy with a high prevalence in Southeast Asia. To date, the genomic abnormalities leading to the pathogenesis of NPC remain unclear. Thus, we sought to characterize the mutational landscape in NPC tumors using next-generation sequencing approaches and to identify significantly mutated genes and pathways.

Methods

124 NPC primary tumors were examined to define the mutational landscape with whole-exome sequencing (WES) and targeted re-sequencing. Mean target coverage of tumor and blood samples was 70X and 49X in WES, and 190X and 68X in targeted resequencing, respectively. Somatic SNPs and INDELs were called with MuTect and VarScan2, respectively. MutSigCV was applied to identify potential driver events in tumorigenesis. Verification rate for somatic mutations was 95%. The functional consequences of mutations in candidate genes were evaluated by the luciferase promoter, cell proliferation, and colony formation assays.

Results

The mutation rate of NPC is relatively low, with a median of 0.9 somatic mutations per megabase. Mutational signature analysis revealed two signatures in NPC, the ubiquitous signature in cancer characterized by C>T transitions predominantly occurring at NpCpG trinucleotides and the APOBEC-related signature characterized by C>G and C>T mutations at TpCpN trinucleotides, which is related to the innate immune APOBEC family of cytidine deaminases.

MutSigCV analysis identified significantly mutated genes, NFKBIA, TP53, CYLD, KMT2D, DMXL1, KMT2C, GPR144, RYR2, BOD1L1, AKAP9, and CEP192, with q values less than 0.1. Pathway and gene ontology analysis identified several pathways/terms with enriched somatic mutations including cell cycle phase transition, chromatin modification, cell death, immune response, p53 pathway, viral carcinogenesis, and the canonical NF-κB signaling pathways. TP53 is the most frequently mutated gene (7.3%, 9/124). Almost all somatic mutations fall into the DNA binding domain of TP53, including well-known hotspot and gain-of-function mutations.

Multiple loss-of-function (LOF) mutations were detected in NF-kB negative regulators, including NFKBIA (encodes IκBα protein), CYLD, and TNFAIP3. Mutations in NFKBIA were shown to alter the tumor suppressive function of IκBα.

Conclusions

In this study we detected an APOBEC-related signature in NPC. Several NF-kB negative regulators, including NFKBIA and CYLD, were mutated in a subset of NPC primary tumors, which may contribute to pathogenesis of NPC through NF-kB signaling pathway. These data provide an enhanced road map for understanding the molecular basis underlying NPC and also provide insight for exploring new therapies.

Acknowledgement

This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China Grant number AoE/M006/08 to MLL.

#126

The transcriptome landscape of high-risk neuroblastoma.

Jun S. Wei,1 Shile Zhang,1 Young K. Song,1 Shahab Asgharzadeh,2 Sivasish Sindiri,1 Xinyu Wen,1 Rajesh Patidar,1 Jaime M. Guidry Auvil,1 Daniela S. Gerhard,1 Robert Seeger,2 John M. Maris,3 Javed Khan1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _The Children's Hospital Los Angeles, Los Angeles, CA;_ 3 _The Children's Hospital of Philadelphia, Philadelphia, PA_.

Despite the success of multimodal therapies, the mortality and morbidity remains substantial for patients with high-risk neuroblastoma (NBL). Sequencing of paired tumor/normal DNA of NBL has revealed a low somatic mutation burden and few recurrent somatically-mutated genes. Here we hypothesize that the integrated analysis of DNA sequencing with whole transcriptome sequencing (WTS) in patients with high-risk NBL tumor will yield valuable insights into the biology of this disease. We performed WTS of 139 NBLs (118 high-risk stage 4 and 21 stage 4S tumors) which had whole genome sequencing or whole exome sequencing of case-matched tumor/normal pairs through the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. We identified expressed mutations, fusion genes, and correlations between gene expression and clinical parameters of patients such as survival to provide understandings of high-risk NBL biology.

Out of 1500 protein-coding changing somatic nucleotide variants detected by DNA sequencing, 614 variants (41%) were also detected in the transcriptome. Twenty-four genes known to be recurrently mutated in NBL showed the exact mutations in their transcriptome as seen in the DNA, including ALK (9.4%), ATRX (2.2%) and MYCN (1.4%). Fusion gene analysis identified in-frame fusions involving ALK (n=2) and FOXR1 genes (n=4). All tumors positive for ALK- and FOXR1-fusions expressed transcripts containing ALK or FOXR1 sequences at much higher levels (>10 folds) than those without fusion in these respective genes. Consensus clustering using tumor gene expression profiles revealed 4 subgroups with distinct survival probability. Among them, several molecular signatures including MYC activation and tumor microenvironment were observed. Intriguingly, 58% tumors without MYCN-amplification showed a MYC activation signature significantly associated with worse overall survival (p=0.0017). Further examination of these tumors with the MYC activation signature revealed different somatic alterations including MYCN P44L mutations, high expression of other MYC family members (MYC and MYCL), mutations in the RAS pathway, and FOXR1 fusions. Interestingly, a gene expression signature representing tumor-associated macrophages (TAM) and regulatory T-cells significantly correlated with a worse outcome in NBLs with normal MYCN copy number, similar to that seen in tumors with the MYC activation signature. In contrast, NBL patients with tumors showing a signature of cytotoxic T-cells and B-cells have better outcomes. Furthermore, tumors of the latter subgroup express significantly more somatic SNVs comparing to the other two subgroups of worse outcome with MYC activation or TAM signatures, suggesting that expressed neo-antigens may elevate cytotoxic T-cell response in these tumors. Our study suggests that patients with high-risk neuroblastoma may benefit from immune-based therapies including check point inhibitors in the future trials.

#127

The genomic landscapes of inflammatory bowel disease-associated colorectal cancers.

Ana I. Robles,1 Giovanni Traverso,2 Ming Zhang,3 Nicholas Roberts,3 Mohammed A. Khan,1 Christine Joseph,3 Gregory Lauwers,4 Florin Selaru,5 Maria Popoli,3 Xiquan Ke,6 Ralph H. Hruban,7 Stephen J. Meltzer,6 Kenneth W. Kinzler,3 Bert Vogelstein,3 Curtis C. Harris,1 Nickolas Papadopoulos3. 1 _Laboratory of Human Carcinogenesis, NCI-CCR, National Institutes of Health, Bethesda, MD;_ 2 _Division of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 3 _Ludwig Center, Johns Hopkins University School of Medicine, Baltimore, MD;_ 4 _Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 5 _Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD;_ 6 _Division of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, MD;_ 7 _Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD_.

Long duration inflammatory bowel disease (IBD) increases the risk for colorectal cancer (CRC). Mutation analysis of limited numbers of genes has suggested that CRCs arising in an IBD background differ from those that do not. We sought to characterize the genetic landscape of these tumors through whole-exome sequencing.

Thirty-one IBD patients with CRC were identified, including 15 with ulcerative colitis, 14 with Crohn's disease and 2 with indeterminate colitis. Whole-exome sequencing was performed on micro-dissected tumor and matched non-neoplastic formalin-fixed paraffin-embedded (FFPE) tissues. Somatic alterations were identified by comparing matched specimens. Mutation prevalence in sporadic CRC was obtained from previously published exome-sequencing studies.

An average of 11.6 Gb were sequenced per sample. The depth of quality coverage of the targeted region was 29 to 100-fold in the tumors; and 17- to 123-fold in non-tumor samples. Two specimens harbored somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1 and MSH6 and exhibited hypermutable phenotype. The remaining cases had a median mutation rate of 1.33 mutations/Mb and an average of 71 alterations per sample. The pattern of base changes in non-hypermutated tumors showed a preponderance of C to T transitions (62%), a majority of which (48% of all mutations) occurred at 5′-CpG-3′ sites. An excess of A to C transversions at AA dinucleotides, particularly in a context of AAG trinucleotides, was also noted. This pattern has been previously observed in esophageal and gastric cancers, both of which are also tied to chronic inflammation. There were no apparent differences in the spectrum of substitutions between tumors arising in UC and CD patients. We identified 28 genes recurrently mutated in three or more non-hypermutated samples. TP53 was the most commonly mutated gene, with incidence prevalence similar to sporadic CRC (63% of cases), but with different mutation spectrum. APC and KRAS were mutated at significantly lower rates than in sporadic CRCs (13% and 20% of cases, respectively). Genes mutated more commonly or uniquely in IBD-CRC, included SOX9 and EP300, encoding proteins in the WNT pathway, NRG1, encoding an ERBB ligand, and IL16, encoding a cytokine. Three cases featured hotspot PIK3CA mutations. Likely oncogenic driver gene mutations in BRAF, CTNNB1, and CREBBP were found in one case each. Mutated genes were enriched for gene ontologies associated with cell communication, cell-cell signaling, and cell adhesion. Our study also revealed recurrent mutations in components of the Rho/Rac GTPase network, suggesting a role for non-canonical WNT signaling in IBD-CRC.

CRCs arising in IBD patients demonstrate a unique molecular profile, which provides clues to their etiology. Our study also sets the stage for improved early detection of IBD-associated CRCs based on their unique genetic compositions as well as for the tailoring of therapies in this patient population.

#128

Comprehensive molecular characterization of 412 muscle-invasive urothelial bladder carcinomas: final analysis of The Cancer Genome Atlas (TCGA) project.

John N. Weinstein,1 Seth P. Lerner,2 David J. Kwiatkowski,3 Gad Getz,4 Jaegil Kim,5 Hikmat A. Al-ahmadie,6 Andrew D. Cherniack,5 Guangwu Guo,5 Rehan Akbani,1 Katherine A. Hoadley,7 William Y. Kim,7 Gordon Robertson,8 Andrew J. Mungall,9 Toshinori Hinoue,10 Peter W. Laird,10 Jonathan E. Rosenberg,6 Joaquim Bellmunt,5 Dean F. Bajorin,6 Margaret B. Morgan,1 Chad J. Creighton,2 Dmitry Gordenin,11 Joshua M. Stuart,12 Xiaoping Su,1 Michael C. Ryan,13 Jeffrey S. Damrauer,14 Wei Zhang,1 Yuexin Liu,1 Yiling Lu,1 Nikolaus Schultz,6 Raju Kucherlapati,15 Gordon B. Mills,1 Donna E. Hansel,16 Brian D. Robinson,17 Bodgen A. Czerniak,1 Victor E. Reuter6. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Broad Institute of Massachusetts Institute of Technology and Harvard University Cambridge, Boston, MA;_ 5 _Broad Institute of Massachusetts Institute of Technology and Harvard University Cambridge, Cambridge, MA;_ 6 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 7 _UNC Lineberger Comprehensive Cancer Center; University of North Carolina Chapel Hill, Chapel Hill, NC;_ 8 _British Columbia Cancer Agency, Vancouver, BC;_ 9 _British Columbia Cancer Agency, Vancouver, British Columbia, Canada;_ 10 _University of Southern California Epigenome Center, Los Angeles, CA;_ 11 _Mechanisms of Genome Dynamics Group, Research Trianble Park, NC;_ 12 _University of California at Santa Cruz, Santa Cruz, CA;_ 13 _In Silico Solutions,, Fairfax, VA;_ 14 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 15 _Harvard Medical School, Boston, MA;_ 16 _University of California at San Diego, San Diego, CA;_ 17 _Weill Medical College of Cornell University, New York, NY_.

Introduction: In 2014, TCGA's Bladder Cancer Working Group presented a preliminary integrated molecular analysis of 131 muscle-invasive urothelial carcinomas (Nature 507:315, 2014). We now report on the entire cohort of 412 fresh-frozen, chemotherapy-naïve tumors. Included in the analysis were paired blood and/or tumor-adjacent tissue samples. This is the largest sequencing project on bladder cancer to date. After strict clinical and pathologic quality control, tumors were analyzed for DNA copy number variants, somatic mutations, DNA methylation, mRNA, microRNA and (phospho-) protein expression, transcript splicing, gene fusions, viral integration, APOBEC mutagenesis, pathway perturbation, clinical correlates, and histopathology.

Results: There was a high overall somatic mutation rate (8.0/Mb), with a median of 245 and mean of 348 coding-region mutations per sample. That is the third highest mutation rate among the cancer types profiled by TCGA (after cutaneous melanoma and non-small cell lung cancers). We identified 54 genes as significantly mutated, compared with 32 in the original report on 131 tumors. TP53 mutations were the most common (49%), and also quite common were mutations in a number of chromatin-modifying genes, including MLL2 (29%), KDM6A (26%), ARID1A (25%), MLL3 (19%), EP300 (15%), CREBBP (12%), and MLL (11%). Other cancer-related genes showing frequent mutations included PIK3CA (22%), RB1 (17%), FGFR3 (14%), STAG2 (14%), ATM (14%), ELF3 (12%), FAT1 (12%), SPTAN1 (12%), ERBB2 (12%), ERBB3 (11%), ASXL2 (10%), ERCC2 (9%), CDKN1A (9%), TSC1 (8%), CDKN2A (7%), RHOB (6%), NFE2L2 (6%), PARD3 (6%), FAM47C (5%), RBM10 (5%),HRAS (5%), KRAS (4%), and PTEN (3%). High mutation burden was associated with improved outcome (p=0.0004). APOBEC mutagenesis explained 70% of the mutation burden and was associated with survival. Gene silencing by promoter hypermethylation was identified in 167 genes with at least 5% frequency in the cohort. The previously identified four mRNA expression subtypes were again found in the complete set of 412 tumors, and the proportions of samples in each subtype were similar to the previous proportions. Reverse-phase proteomic array analysis of 344 of the samples revealed clusters associated with diagnostic subtype, pathological stage, and grade but not with smoking history or non-muscle invasive status.

Conclusions: This integrated molecular analysis of 412 TCGA tumor samples largely validates and considerably extends observations from the initial cohort of 131 patients. The larger cohort significantly increased our power to detect lower-frequency aberrations that were not identified in the original cohort. The results provide a robust basis for further functional studies of bladder cancer biology and also provide additional incisive information for the identification of molecular targets for therapy.

#129

Integrated genomic analysis of survival outliers in glioblastoma.

Sen Peng,1 Harshil Dhurv,1 Brock Armstrong,1 Jeffrey Kiefer,2 Bodour Salhia,2 Julianna Ross,1 Christophe Legendre,2 Selene Virk,3 Andrew E. Sloan,3 Quinn T. Ostrom,3 Jill Barnholtz-Sloan,3 Nhan L. Tran,1 Michael E. Berens1. 1 _Cancer and Cell Biology Division, The Translational Genomics Research Institute (TGen), Phoenix, AZ;_ 2 _Integrated Cancer Genomics Division, The Translational Genomics Research Institute (TGen), Phoenix, AZ;_ 3 _Case Western University, Cleveland, OH_.

Despite the general poor prognosis for patients with GBM, a proportion survives well beyond the median survival of 12-14 months following diagnosis. To elucidate molecular features associated with disproportionately protracted survival, we conducted deep genomic comparative analysis of a cohort of patients receiving standard therapy (surgery plus concurrent radiation and temozolomide) wherein "GBM outliers" were identified: patients who responded (long-term survivor, LTS) versus those who failed rapidly (short-term survivor, STS). The datasets enabled interrogation for signatures indicative of tumor vulnerability. Whole genomic, transcriptomic and epigenetic analyses of 18 patients, including 8 LTS with an average 30 months overall survival (OS) and 10 STS with an average of 7 months OS were performed to capture single nucleotide variants (SNVs), indels, translocations, intra-chromosomal rearrangements, copy number variants, along with DNA methylation and mRNA expression. LTS and STS cases showed equal proportion of 7p11.2 (EGFR) amplification and 9p21.3 (CDKN2A) deletion. However, LTS GBM showed frequent chromosomal gains in 4q12 (PDGFRA and KIT) and 12q14.1 (CDK4) and deletion in 19q13.33 (BAX, BCAT2 and CD33), whereas, STS GBM showed frequent deletion in 9p11.2 (FOXD4L2 and AQP7P3) and 22q11.21 (HIC2). In addition, LTS GBM showed a 2-fold increased copy number alteration (specifically deletion) as compared to STS GBM. By gene expression analysis, supervised clustering using the CIN70 signature (prognostic) showed an increased expression in STS GBM. Overall, whole genome methylation analyses showed that STS GBM tumors harbor more hypomethylation in probes situated in -200 bp of transcription start site (TSS) and both exon 1 and 5'UTR region. A methylation signature consist of 89 probes distantly separate LTS from STS GBM tumors. We posit that genomic instability (broadly inclusive) is associated with vulnerability of GBM to standard therapy; conversely, genomic instability coupled with genetic and epigenetic signatures may identify patients where up-front entry into alternative, targeted regimens would be a preferred, more-efficacious management.

Supported by a grant from the Ben & Catherine Ivy Foundation.

#130

International Cancer Genome Consortium (ICGC).

Jennifer L. Jennings, Thomas J. Hudson. _Ontario Inst. for Cancer Research, Toronto, Ontario, Canada_.

The International Cancer Genome Consortium (ICGC) was established to bring together researchers from around the globe to comprehensively analyze the genomic, transcriptomic, and epigenomic changes in 50 different tumour types or subtypes that are of clinical and societal importance across the globe (International network of cancer genome projects. Nature 464, 993-998 (15 April 2010)). As of November 2015, the ICGC has received commitments from researchers and funding organizations in Asia, Australia, Europe, North America and South America for 89 project teams in 17 jurisdictions to study more than 25,000 tumour genomes. Processed data is available via the Data Coordination Centre (http://dcc.icgc.org) based at the Ontario Institute for Cancer Research and is updated semi-annually. The November 2015 release (Version 20) includes datasets from 66 ICGC projects. In total, ICGC data release 20 comprises data from 14,767 cancer genomes. The Pan-Cancer Analysis of Whole Genomes (PCAWG) project of the ICGC and The Cancer Genome Atlas (TCGA) is coordinating analysis of more than 2,800 cancer genomes, with the extensive use of cloud computing. Because of the very large size of the pan-cancer dataset, with 5,000 whole genome sequences, PCAWG is using a distributed compute cloud environment (generated by computing centres in the USA, Europe and Asia) that meets the project's technical requirements and the bioethical framework of ICGC and its member projects. Each genome is being characterized through a suite of standardized algorithms, including alignment to the reference genome, uniform quality assessment, and the calling of multiple classes of somatic mutations. Scientists participating in the research projects of PCAWG are addressing a series of fundamental questions about cancer biology and evolution based on these data. The first phase of ICGC, which is slated for completion in 2018, has focused on developing extensive catalogs of tumour genomic information. The proposed second phase, ICGCmed, will link genomics to clinical information and health, including lifestyle, patient history, response to therapies, and underlying causes of disease, for a broad spectrum of cancers, including preneoplastic lesions, early cancers and metastases. The goal will be to accelerate the movement of genomic information into the clinic to guide prevention, early detection, diagnosis, and prognosis, and provide the information needed to match a patient's disease to the most effective combinations of therapy. The ICGC develops policies and quality control criteria to help harmonize the work of member projects located in different jurisdictions. Data produced by ICGC projects are made rapidly and freely available to qualified researchers around the world via the data cloud and through the ICGC Data Coordination Center at (http://dcc.icgc.org). More information can be found on www.icgc.org.

#131

Allelic imbalance analysis of uterine carcinosarcoma: An inquiry into the dual nature of the neoplasm.

Aditya S. Deshpande,1 Zachary Weber,2 Raed Sulaiman,3 Natasha Flier,3 Cheryl Ageton,3 Mary Fagerness,3 Joseph Sulaiman,3 Gareth E. Davies,2 David Starks,3 Luis Rojas-Espaillat,3 Paul A. Scheet,1 Erik Ehli2. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Avera Istitute for Human Genetics, Sioux Falls, SD;_ 3 _Avera Cancer Institute, Sioux Falls, SD_.

Uterine carcinosarcoma (UCS), also known as Malignant Mixed Mullerian Tumor, is a neoplasm of the female genital tract that shows histological features of both carcinoma and sarcoma. Some studies have indicated that UCS arises from sarcomatous differentiation of high-grade carcinoma while others have confirmed a bi-clonal nature of the tumor. Given these differences, further investigation in the origin of this tumor with newer approaches and technologies is warranted. In this study we determined the allelic imbalance (AI) profiles of the two components of UCS and compared the AI events that were shared by, or differed between, them. Samples were obtained from 10 patients diagnosed with UCS and were preserved in formalin fixed paraffin embedded (FFPE) blocks. The two components (carcinoma & sarcoma) were identified and micro-dissected, and whole-genome DNA micro-arrays were applied to the isolated DNA. We then applied a sensitive computational method that combines haplotype information with SNP array data to identify somatic segmental copy number variations and copy-neutral loss of heterozygosity (cnLOH). After we obtained the AI events we filtered small events (<2 MB) to rule out germline duplications. We then obtained events that are common to both components by using a criterion of 80% reciprocal overlap. All such events were classified as a gain, loss or cnLOH. We found that, on an average, 80% of the events found in the carcinoma component are common with the sarcoma component. Conversely, only 64% of events seen in the sarcoma component are found in the carcinoma. This may be in part due to the fact that the sarcoma component usually showed greater number of events and most of these events were larger than the corresponding carcinoma component. Also the sarcoma component showed an increased degree of allelic imbalance than the counterpart. We then looked into particular regions of known tumor suppressor and oncogenes and identified loss in the TP53 gene region (60% of tumors), gains in PIK3CA and PTEN gene regions (40% of tumors), among other events. Our analysis showed that the sarcomatous component shared major portions of the AI profile with the carcinomatous component, but also showed additional events and a greater degree of AI. One explanation for this observation can be that the sarcomatous component retains the AI profile of the carcinomatous component and also gathers additional imbalance. This suggests directionality to the development of UCS: that it arises by sarcomatous differentiation of already existing carcinoma of the uterus. We are pursuing additional assessments of point mutations and expression profiles to complement our existing analyses.

#132

Comprehensive genomic analysis of human papillomavirus-associated oral cancers.

David E. Symer, Keiko Akagi, Kevin R. Coombes, Weihong Xiao, Robert K. L. Pickard, Amit Agrawal, Maura L. Gillison. _Ohio State Univ. Comp. Cancer Center, Columbus, OH_.

Human papillomavirus-associated oral cancers comprise a distinct disease that is increasing in frequency in Western countries. Recent genomic landscape studies of oral cancers and of HPV-associated cervical cancers identified the most commonly mutated genes in each. Here we report a comprehensive analysis of 83 HPV-positive oropharyngeal cancers studied by whole genome sequencing (WGS), and an additional 46 tumors evaluated by the Cancer Genome Atlas project (TCGA) by exome sequencing. We confirmed recurrent mutations in previously reported, frequently mutated candidate driver genes including PIK3CA, as well as several novel gene targets, each occurring recurrently at frequencies of more than approximately 3%. Our WGS data demonstrated recurrent focal genomic instability associated with HPV insertional mutagenesis. We observed dramatic changes in genomic copy number variants (CNVs) at chromosome 3q as well as numerous other chromosomal regions that are recurrently gained or lost in HPV-positive tumors. RNA-Seq data from this collection of oral cancers defined differentially expressed transcript levels and other molecular alterations associated with single nucleotide variants (SNVs), CNVs and other genomic structural variants in the tumors. Together, our results reveal a unique pattern of somatic variants that distinguishes HPV+ oral cancers both from HPV- oral cancers as well as other HPV+ tumors arising in distinct tissues including the uterine cervix. These data shed new light on the molecular pathogenesis of oral cancers, and provide new opportunities for development of diagnostic and therapeutic strategies for affected patients.

#133

Integrated molecular characterization of uterine carcinosarcoma in The Cancer Genome Atlas (TCGA) project.

Rehan Akbani,1 The Cancer Genome Atlas Research Network, Douglas A. Levine2. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

We used array- and sequencing-based technologies to perform an integrated genomic, epigenomic, transcriptomic, and proteomic characterization of the rare tumor type uterine carcinosarcoma (UCS) within the context of The Cancer Genome Atlas (TCGA) project using 57 cases that had stringent histopathologic review. Cohort samples had extensive copy number alterations and highly recurrent somatic mutations. Nearly all (91%) cases had TP53 mutations, similar to ovarian and serous uterine carcinomas, and frequent mutations were also found in PTEN, PIK3CA, PPP2R1A, FBXW7, and KRAS, similar to endometrioid and serous uterine carcinomas. Transcriptome sequencing identified a strong EMT gene signature in a subset of 17 (30%) cases, and cases with EMT signatures had decreased expression of mir-200 family members that was attributable to epigenetic alterations at miRNA promoters. The range of EMT signatures scores in UCS was the largest among all the TCGA tumor types studied. UCS shared proteomic features with both gynecologic carcinomas and non-gynecologic mesenchymal-like tumors. Our results indicate that UCS tumors share many features with serous-like endometrial carcinomas, including frequent TP53 mutations and extensive somatic copy number alterations, though with greater EMT features. Multiple somatic mutations and copy number alterations in genes that are therapeutic targets were identified. There was a high degree of mutational clonality, consistent with tumors being derived from a single cell of origin. Taken together, these data suggest that while some UCS tumors develop from an endometrioid lineage, the majority likely de-differentiate from a serous precursor, potentially accounting for their clinical aggressiveness and poor response to treatment.

#134

Mutational landscape of breast cancers from PALB2 germline mutation carriers.

Salvatore Piscuoglio,1 Charlotte KY Ng,1 Y Hannah Wen,1 Arto Mannermaa,2 Paolo Peterlongo,3 Carlo Tondini,4 Marketa Janatova,5 Teo Soo Hwang,6 Pei-Sze Ng,6 Lai-Meng Looi,7 William Foulkes,8 Georgia Chenevix-Trench,9 Britta Weigelt,1 Melissa C. Southey,10 Marc Tischkowitz,11 Jorge S. Reis-Filho,1 PALB2 Interest Group. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _University of Eastern Finland, Kuopio, Finland;_ 3 _Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy;_ 4 _Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy;_ 5 _Charles University in Prague, Prague, Czech Republic;_ 6 _Cancer Research Malaysia, Subang Jaya, Malaysia;_ 7 _University of Malaya, Kuala Lumpur, Malaysia;_ 8 _McGill University, Montreal, Quebec, Canada;_ 9 _QIMR Berghofer Medical Research Institute, Brisbane, Australia;_ 10 _University of Melbourne, Melbourne, Australia;_ 11 _University of Cambridge, Cambridge, United Kingdom_.

Introduction: The PALB2 gene encodes the partner and localizer of BRCA2 protein, which interacts with BRCA1/2 and is involved in homologous recombination DNA repair. Germline mutations in PALB2 are associated with an increased risk of breast cancer, with a cumulative risk of 35% by age 70 in female PALB2 mutation carriers. Whether the PALB2 wild-type allele is lost in the development of PALB2 breast cancers has yet to be defined. Further, the repertoire of somatic genetic alterations in these tumors is currently unknown. In this study we sought to characterize the genomic landscape of PALB2 breast cancers and to define the differences in the repertoire of somatic genetic alterations and mutational signatures between PALB2 and BRCA1 and BRCA2 breast cancers.

Material and Methods: Representative samples from nine breast cancers from patients with known PALB2 germline mutations were microdissected. DNA samples from microdissected tumors and matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000. Somatic mutations were defined using MuTect and insertions and deletions using Strelka and Varscan2. Driver mutations were defined by state-of-the-art bioinformatics methods. Mutational signatures were defined using non-negative matrix factorization. Copy number alterations (CNAs) and regions with loss of heterozygosity were determined using FACETS. The mutational frequency of breast cancers from PALB2 germline mutation carriers was compared to that of breast cancers from BRCA1 (n=11) and BRCA2 (n=10) germline mutation carriers from The Cancer Genome Atlas study.

Results: Three patients harbored germline frame-shift PALB2 mutations (2 S1169fs, 1 T841fs), five displayed truncating mutations (3 W1038* and 2 Q775*) and 1 harbored a missense mutation (W1140G, of uncertain significance). Somatic loss of the PALB2 wild-type allele was found in 3 cases, in 2 of which the loss was caused by CNAs and in 1 case it was caused by a somatic PALB2 Q479* mutation. A median of 65 somatic mutations (range 45-223) and a median of 1 driver mutation (range 0-3) were identified per tumor. Cancer genes mutated in PALB2 breast cancers included TP53 (n=2), PIK3CA (n=2), NF1 (n=1) and NCOR1 (n=1). Six cases displayed mutational signatures consistent with the aging process; the BRCA signature was not found in any of the cases analyzed. Breast cancers from PALB2 mutation carriers had fewer somatic TP53 mutations than BRCA1 breast cancers (2/9, 22% vs 9/11, 82%, p=0.02). No difference in the repertoire of somatic mutations between PALB2 and BRCA2 breast cancers was observed.

Conclusion: Unlike breast cancers from BRCA1 and BRCA2 mutation carriers, the majority of breast cancers from PALB2 mutation carriers lacked somatic loss of the wild-type allele and none displayed a BRCA mutational signature. No highly recurrently mutated gene was identified, but pathogenic mutations in driver genes (TP53, PIK3CA, NF1 and NCOR1) were found.

#135

Mutational landscape of uterine and ovarian carcinosarcomas reveals new recurrently-mutated histone core genes as drivers of epithelial-mesenchymal transition.

Gulden Menderes, Siming Zhao, Carlton L. Schwab, Salvatore Lopez, Jonathan D. Black, Emiliano Cocco, Stefania Bellone, Dan-Arin Silasi, Elena Ratner, Masoud Azodi, Babak Litkouhi, Peter E. Schwartz, Joseph Schlossinger, Richard Lifton, Alessandro Santin. _Yale University School of Medicine, New Haven, CT_.

Objective:

Carcinosarcomas (CS) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. While an increasing body of evidence supports the origin of both CS elements from a common epithelial cell that undergoes sarcomatous dedifferentiation, limited information is currently available about the role of somatic mutations (SNV) and copy number variations (CNV) in these rare tumors. Accordingly, the objective of our study was to evaluate the genetic landscape of uterine and ovarian CSs by whole-exome-sequencing (WES).

Methods:

We analyzed the mutational landscape of 63 fresh frozen CS biopsies and 5 primary CS cell lines by WES, most of which were matched to normal DNA from the same patients. We also performed multi region WES in separate carcinoma and sarcoma areas to resolve the genetic architecture and evolutionary histories of CS. Finally, in transfection experiments we validated somatic histone 2A and 2B mutations as potential drivers of epithelial-mesenchymal transition (EMT).

Results:

Our results demonstrated the presence of two major genetic types of uterine CSs [i.e., uterine serous carcinoma-like (USC) profile and endometrioid carcinoma-like profile (EAC)] and provided evidence that both subtypes, as well as ovarian CS, arise from sarcomatous transformation of carcinomas. In addition to recurrent mutations in cancer genes previously identified in USC and EAC such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4/Mi2b and BCOR, we found an excess of mutations in histones H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone cluster containing these genes. We also found frequent deletions comprising TP53 and MBD3, (a member with CHD4/Mi2b of the NuRD-chromatin-modification-complex), and frequent amplification of chromosome segments containing PIK3CA, CCNE1, TERT and c-MYC. Amplifications of the histone cluster and TERT were significantly more frequent in CS than their epithelial counterparts. Stable transgenic expression H2A and H2B in USC cell lines demonstrated that mutant, but not wild type H2A and H2B increased expression of markers of epithelial-mesenchymal transition (EMT), suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages resulting in these two components.

Conclusions:

Our results provide new insight into the origins and development of uterine and ovarian CS and defined the genetic landscape of this type of malignant tumor. These findings also suggest new therapeutic targets for these highly aggressive neoplasms.

#136

Refining the molecular profile of colorectal tumors to expand prevention and treatment opportunities.

Catherine S. Grasso,1 Eve Shinbrot,2 Ming Yu,3 Max Liesersen,4 Mark Chaisson,5 Andrew Chan,6 Charles Connolly,1 James Dai,7 Margaret Du,1 Charles Fuchs,6 Levi Garraway,6 Marios Giannakis,6 Tabitha Harrison,1 Li Hsu,7 Jeroen Huyghe,1 Jasmine Mu,8 Shuji Ogino,6 Colin Pritchard,9 Stephen Salipante,9 Wei Sun,7 Syed H. Zaidi,10 Ni Zhao,7 William Grady,3 Ben Raphael,4 Thomas Hudson,10 David Wheeler,11 Ulrike Peters1. 1 _Dept. of Cancer Prevention, Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Baylor College of Medicine — Human Genome Sequencing Center, Houston, TX;_ 3 _Dept. of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _Dept. of Computer Science, Brown University, Providence, RI;_ 5 _Dept. of Genome Sciences, University of Washington, Seattle, WA;_ 6 _Dana-Farber Cancer Institute, Boston, MA;_ 7 _Dept. of Biostatistics, Fred Hutchinson Cancer Research Center, Seattle, WA;_ 8 _Cancer Program, The Broad Institute, Boston, MA;_ 9 _Dept. of Laboratory Medicine, The University of Washington, Seattle, WA;_ 10 _Ontario Institute of Cancer Research, Toronto, Ontario, Canada;_ 11 _Dept. of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX_.

The completion of The Cancer Genome Atlas (TCGA) project for colorectal cancer (CRC) is ushering in a new phase of identifying treatment strategies tailored to the molecular profile of each person's tumor. Precision medicine approaches to cancer treatment rely on the identification of molecular profiles that can be used to identify effective therapies and can be used in a targeted sequencing setting to make treatment decisions.

The initial TCGA colorectal effort included 276 samples and focused on integrating data from exome sequencing with genome-wide DNA copy number alterations (CNAs), DNA methylation, and mRNA and microRNA expression. Since then a total of 626 samples have been completed with the potential to refine CRC subtypes, identify novel mutated pathways, and further functional understanding. Such a large data set presents opportunities to identify new recurrent drug targets and to stratify patients into groups that are predictive of treatment response. However, large data sets also present substantial challenges, since hand-curation becomes intractable, while computational tools can be overwhelmed by hypermutation and copy number changes.

Here we present a comprehensive molecular analysis of all 626 TCGA colorectal cancer samples, including exome sequencing, CNAs, DNA methylation, and mRNA expression. For each data type, we identified recurrently altered genes. Using MutSigCV on 525 samples yielded 27 and 87 significantly mutated genes in non-hypermutated and hypermutated samples, respectively, a substantial increase over the 15 and 17 somatically recurrently mutated genes identified using MutSig in non-hypermutated and hypermutated samples, respectively, in the previously published TCGA colorectal study. For example, PTEN, a known tumor suppressor, was not reported as significantly recurrently mutated in the initial TCGA non-hypermutated set; however, it was in the larger non-hypermutated set, demonstrating the power of a larger data set for assessing the significance and relative frequency of mutations in the context of known subtypes.

In addition, we integrated the somatic mutation data, copy number data, LOH data, and hyper-methylation data to identify genes, like MLH1, that are recurrently disrupted by different mechanisms. We also considered somatic mutations that are likely gain-of-function mutations based on nonrandom clustering; and we used recurrent indels to identify loss-of-function drivers in samples positive for microsatellite instability (MSI). We further classified each sample using the previously identified subtypes -- BRAF+, KRAS+, APC+, CTNNB1+ (beta-catenin+), TGFBR2/SMAD4+, PTEN+ and PIK3CA+, and R-spondin fusion positive, as well as CpG Island Methylator Phenotype (CIMP) and MSI - in order to refine the relevant molecular signatures driving CRC etiology and thereby prevention and treatment paradigms.

#137

Development and application of a 62-gene panel for assessment of somatic sequence and structural variants in tumor DNA derived from non-Hodgkin lymphoma patients treated in a phase 1 clinical trial with the EZH2 inhibitor tazemetostat.

Scott R. Daigle,1 Samuel Angiuoli,2 Sian Jones,2 Scott Ribich,1 Heike Keilhack,1 Mark Sausen,2 Peter T. Ho,1 Stephen J. Blakemore1. 1 _Epizyme, Inc., Cambridge, MA;_ 2 _Personal Genome Diagnostics, Baltimore, MD_.

Tazemetostat is a small molecule inhibitor of the histone methyltransferase EZH2 and is currently in phase 2 clinical trials in relapsed refractory Non-Hodgkin's Lymphomas (RR-NHL) including diffuse large B cell and follicular lymphoma. We report the development and application of a NHL targeted sequencing panel designed to identify molecular variants, including specific somatic sequence mutations (single base and insertion/deletion), amplifications and translocations in both tumor and cell free circulating tumor DNA (ctDNA). Analysis of somatic alterations in both tissue and ctDNA collected pre-dose enables determination of molecular variants that may correspond with clinical activity, while longitudinal analysis of ctDNA sampled on therapy could help monitor patients for minimal residual disease and emergence of acquired resistance. In collaboration with Personal Genome Diagnostics (PGDx) a panel of 62 NHL specific genes was designed to selectively analyze regions of the genome previously identified as somatically altered in NHL. DNA derived from matched tumor tissue and plasma were screened utilizing this panel using the Illumina HiSeq 2500 platform with 100 bp paired-end reads. Average target coverage for the tissue panel was 1,250-fold while coverage for the ctDNA was approximately 20,000-fold and 3,700-fold for sequence mutations and structural alterations, respectively. Data were analyzed using PGDx's validated cancer genome analysis algorithms that allow for reliable identification of mutations with high sensitivity and specificity. Validation of both the tumor and ctDNA panels was performed using tumor and plasma specimens previously characterized for sequence mutations, amplifications, translocations, and microsatellite instability. For archive tumor, analyses of cell line specimens with an experimental tumor purity of 20-100% using 50-100ng of DNA yielded sensitivity and specificity of 100% for detection of 358 previously characterized sequence mutations and structural variants. Similar ctDNA analyses using fragmented cell line and plasma derived DNA with an experimental tumor purity of 0.10%-25.0% using 9-167ng of DNA yielded a sensitivity of 100% for detection of over 100 genetic variants. Following successful validation of the panel we proceeded to sequence tumor tissue from 11 patients and ctDNA from 16 NHL patients enrolled in the tazemetostat phase 1 clinical trial. Tumor tissue from these patient samples had been previously sequenced using a smaller 39 gene panel. We observed high concordance with 100% of variants detected within the shared gene set of 33 genes between the historic data and our new 62 gene panel. We will report the landscape and concordance of genetic alterations identified through next-generation sequencing analyses of tumor and cell-free DNA.

#138

Solid papillary carcinoma with reverse polarization are driven by IDH2 and PI3K pathway mutations.

Sarah Chiang,1 Britta Weigelt,1 Huei-Chi Wen,1 Salvatore Piscuoglio,1 Luciano G. Martelotto,1 Charlotte K Y Ng,1 Jörg Balss,2 Gabrielle Baker,3 Kimberly S. Cole,4 Andreas von Deimling,5 Julie M. Batten,6 Jonathan D. Marotti,7 Hwei-Choo Soh,8 Benjamin L. McCalip,9 Raymond S. Lim,1 Kalliopi P. Siziopikou,10 Randi Burke,11 Song Lu,12 Xiaolong Liu,13 Tarek Hammour,14 Edi Brogi,1 A. John Iafrate,6 Jorge S. Reis-Filho,1 Stuart J. Schnitt15. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _German Consortium of Translational Cancer Research (DKTK), Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _Department of Pathology, University of Chicago, Chicago, IL;_ 4 _Memorial Sloan Kettering Cancer CenterDepartment of Pathology, Montefiore Medical Center and Albert Einstein College of Medicine, New York, NY;_ 5 _Department of Neuropathology, Institute of Pathology, INF 224, Ruprecht-Karls-University Heidelberg, Heidelberg, Germany;_ 6 _Department of Pathology, Massachusetts General Hospital, Boston, MA;_ 7 _Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH;_ 8 _Pathology North, North Shore Private Hospital, Sydney, Australia;_ 9 _Miami Valley Hospital, Dayton, OH;_ 10 _Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL;_ 11 _Kaiser Permanente, Baldwin Park, CA;_ 12 _Department of Pathology, Mon General Hospital, Morgantown, WV;_ 13 _ABQ Health Partners, Albuquerque, NM;_ 14 _Maine Medical Center, Portland, ME;_ 15 _Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA_.

Background: Solid papillary carcinoma with reverse polarization (SPCRP) is a rare breast cancer subtype, whose most striking morphologic feature is the presence of back-to-back columnar epithelial cells with nuclei situated in an apical rather than in the normal basal location. We sought to characterize the morphologic and genetic landscape of SPCRP and determine whether it represents a distinct subtype of breast cancer underpinned by highly recurrent disease-specific genetic alterations.

Material and Methods: Archival sections of 13 SPCRPs were subjected to microdissection. DNA samples extracted from microdissected tumor and matched normal samples were subjected to whole exome sequencing (n=2) on an Illumina HiSeq2000, and DNA samples from tumors were further subjected to targeted massively parallel sequencing (n=1), SNaPshot profiling (n=7) and/or Sanger sequencing. State-of-the-art bioinformatics methods were used to define somatic mutations. Non-malignant breast epithelial MCF10A cells with and without somatic PIK3CA H1047R knock-in were used to assess the functional impact of the mutations identified by sequencing of SPCRPs in two- and three-dimensional cell culture systems.

Results: Ten of 13 (77%) SPCRPs were found to harbor IDH2 R172 hotspot mutations, and eight of these cases had concurrent pathogenic mutations affecting PIK3CA or PIK3R1, in particular PIK3CA H1047R hotspot mutations. Functional monolayer and three-dimensional cell culture studies demonstrated that IDH2 R172 and PIK3CA mutations constitute likely drivers of this tumor and contribute to its unusual nuclear polarization phenotype.

Conclusions: Our results suggest that SPCRP is a distinct subtype of breast cancer underpinned by IDH2 hotspot mutations in conjunction with mutations affecting canonical genes of the PI3K pathway. Given that IDH2 hotspot mutations have not been described in breast cancer to date, these may be used as a diagnostic ancillary marker for SPCRPs. Furthermore, IDH2 mutations may serve as a potential therapeutic target in SPCRP patients with disseminated disease.

#139

Novel gastric cancer cell lines established from diffuse type gastric cancer patients for potential subgroup-specific therapy.

Tae Soo Kim,1 Kyu Hyun Park,1 Woo Sun Kwon,1 Won Suk Lee,1 In Hye Jeong,1 Sun Kyoung Kang,1 Hyun Myong Kim,1 Sun Young Rha,2 Hyun Cheol Chung2. 1 _Song-Dang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Song-Dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

In vitro culture system is a long-standing useful tool in the study of cell biology for developing new therapeutic modalities and discovering new anti-cancer drugs. Therefore establishment and characterization of cell lines are very important in vitro study. However, establishment of GC patients derived cell lines are very challenging, especially with diffuse type which has different biology and worse prognosis from intestinal type.

Yonsei Cancer Center (YCC) has been consistently worked to build human cancer cell lines, and resulting in over 30 novel cancer cell lines from metastatic gastric cancer patients. Most of YCC GC cell lines were established by primary culture of peritoneal fluid from metastatic gastric cancer patients. Interestingly, YCC-16 cells were isolated from the peripheral blood of gastric cancer patients. 20 cells were established from diffuse type GC, and 21 cells were from signet ring cell type.

In this study, we evaluated 29 YCC GC cell lines and 23 GC cell lines from other sources (ATCC, KCLB, and JCRB). For molecular characterization of 52 GC cell lines, we analyzed copy number variant (CNV), single number variant (SNV) and gene expression using whole-exome sequencing (WES) and RNA sequencing. The 7 cell lines (YCC-17, 18, 19, 20, 29, 34, 36) were compared with their germline variants of paired PBMC. Also, these cell lines were investigated expression of proteins related to cancer progression and the sensitivity of chemotherapies and diverse molecular targeted drugs/antibodies. We evaluated the tumorigenesis with soft-agar assay and invasiveness by transwell assay.

In addition to previous novel EBV infected cell line (YCCEL1/YCC-10, J Gen Virol. 2013), we observed amplification and overexpression of receptor tyrosine kinase (RTK) including HER2 (YCC-19, 32, 33, 38, 42), EGFR (YCC-11, 21), Met (YCC-31, 42), and FGFR2 (YCC-28, 30); confirming that protein was overexpression by Western blot in 20/52 (38.5%). Interestingly, amplification of RTKs was detected mutually exclusive pattern with other RTK amplification. Furthermore, RTK amplified cell lines were shown to be sensitive to target specific small molecules such as BGJ398. Also, we identified cell line specific genetic variations including ARID1A which might be the potential new targets in diffuse type GC cell lines. Then we could subgroup the cell lines based on 4 subtypes of TCGA.

In conclusion, our GC cell line bank with genomic and biological characteristics based on the clinical information, would be useful in subgroup specific target selection, drug screening and mechanism evaluation for improving GC treatment.

#140

LSAMP gene deletion is associated with rapid disease progression in prostate cancer of African American men.

Albert Dobi,1 Gyorgy Petrovics,1 Hua Li,1 Shyh-Han Tan,1 Tanja Stümpel,2 Denise Young,1 Shilpa Katta,1 Qiyuan Li,3 Kai Ying,1 Bernward Klocke,2 Lakshmi Ravindranath,1 Indu Kohaar,1 Yongmei Chen,1 Dezső Ribli,4 Korbinian Grote,2 Hau Zou,5 Joseph Cheng,5 Clifton L. Dalgard,6 Shimin Zhang,7 István Csabai,4 Jacob Kagan,8 David Takeda,9 Massimo Loda,9 Sudhir Srivastava,8 Matthias Scherf,2 Martin Seifert,2 Timo Gaiser,10 David G. McLeod,11 Zoltan Szallasi,9 Reinhard Ebner,5 Thomas Werner,2 Isabell A. Sesterhenn,7 Matthew Freedman,9 Shiv Srivastava1. 1 _Center for Prostate Disease Research, Uniformed Services University of the Health Sciences and Walter Reed National Military Medical Center, Rockville, MD;_ 2 _Genomatix, Munich, Germany;_ 3 _Medical College, Xiamen University, Xiamen, China;_ 4 _Eötvös Loránd University, Budapest, Hungary;_ 5 _CytoTest, Rockville, MD;_ 6 _Uniformed Services University, Bethesda, MD;_ 7 _Joint Pathology Center, Silver Spring, MD;_ 8 _NCI, Bethesda, MD;_ 9 _Dana–Farber Cancer Institute, Boston, MA;_ 10 _Universitätsmedizin Mannheim, Mannheim, Germany;_ 11 _Center for Prostate Disease Research, Uniformed Services University of the Health Sciences and Walter Reed National Military Medical Center, Bethesda, MD_.

INTRODUCTION: Disproportionately higher rates of prostate cancer (CaP) incidence and mortality have been reported among African American (AA) men. Although oncogenic TMPRSS2-ERG gene fusion and deletion of the PTEN tumor suppressor gene are established cancer driver gene alterations in CaP, they are known to be more prevalent among men of European ancestry. By utilizing carefully annotated specimens, this study focused on the discovery of recurrent genomic alterations in CaP of AA men in comparison to Caucasian Americans (CA).

METHODS: Genomic DNA from clinically localized primary prostate tumors (Gleason 6 or 7 with primary pattern 3) and matched peripheral blood lymphocytes of seven AA and seven CA patients, were analyzed by paired-end sequencing on Illumina Genome Analyzer IIx to a depth of 30x. Following alignment to reference genome, somatic alterations on tumor DNA that include single nucleotide variants (SNVs), insertion and deletions (Indels), structural variations, copy number variations, and inter- and intra-chromosomal translocations of tumor DNA sequence were identified. To confirm prevalent genomic deletions we performed FISH analysis on a tissue microarray constructed from 42 AA and 59 CA tumor and normal samples of an independent cohort. Frequently deleted loci were further validated by analysis of TCGA CaP SNP array data from 41 AA and 279 CA prostate tumors.

RESULTS: A comparative evaluation of whole genome sequences of AA and CA CaP revealed a prevalent deletion of the LSAMP locus of chromosome 3q13.31 in AA CaP. These observations were confirmed by SNP array and FISH assays in independent cohorts of specimens. AA CaP patients with LSAMP deletion showed rapid disease progression. In contrast to higher frequency of LSAMP deletion, significantly lower frequencies of PTEN and ERG alterations were noted in CaP of AA men. Furthermore, CaP genomes of AA men displayed a higher rate of inter-chromosomal rearrangements than those from CA men.

CONCLUSIONS: We highlight distinct features of AA and CA CaP genomes including common CaP driver genes (TMPRSS2- ERG, PTEN) and define a novel recurrent deletion of the LSAMP locus. This study underscores the need for careful evaluations of cancer genomes in underrepresented populations in the global context with implications for precision medicine strategies.

#141

Gene expression profile differences between early- and late-onset colorectal adenocarcinoma.

Valentine N. Nfonsam, Jana Jandova. _University of Arizona, Tucson, AZ_.

Introduction: Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer related deaths in the US. The overall incidence of CRC in the US has decreased over the past three decades, yet recent literature indicates increase in incidence among individuals younger than 50. These early-onset CRC tumors (EO) tend to be more aggressive and advanced at initial diagnosis. As the etiology of EO CRC is not understood yet, the aim of this study was to elucidate gene expression profiling in EO CRC and show its molecular uniqueness compared to late-onset (LO) CRC.

Methods: Two cohorts of patients with sporadic EO CRC (age under 50) and LO CRC (age over 65) tumors were identified. Tumors and their matching non-involved tissue samples with equal representation of colon and rectal neoplasms from twelve EO patients and twelve LO patients were obtained. Patients with Lynch syndrome, familial adenomatous polyposis and inflammatory bowel disease were excluded. De-paraffinized tissues were macro-dissected from FFPE sections, RNA isolated and used for nanoString nCounter PanCancer Pathways Panel gene expression analysis to quantify transcript levels of 770 genes representing 13 canonical cancer pathways. Statistical analysis was performed using the Gene Expression R-script module within the nCounter software v2.6. A gene was considered to be above background if the average count for the target gene was greater than the average counts for the eight negative control genes and if the P value of the t-test was less than 0.05.

Results: We analyzed a total of twenty four tumor samples, six EO and six LO colon tumors and six EO and six LO rectal tumors. Their expression profiles were then compared to their matching non-involved tissues in order to identify genes that are unique to colon and rectal neoplasm, respectively. Out of 770 PanCancer Panel pathway genes assayed, 98 genes were uniquely expressed in EO colon tumors with 73 genes being up- and 25 down-regulated with a fold change higher than two. 77 genes were uniquely expressed in EO rectal tumors, with 57 of them up-regulated and 20 down-regulated. Further statistical analysis revealed that, from 265 genes differentially expressed specifically in EO colon tumors, changes in expression of 147 genes were statistically significant (p<0.05). Similarly, from 275 genes differentially expressed in EO rectal tumors, 82 showed statistically significant alterations in expression.

Conclusions: Results of this study suggest EO CRC as a distinct molecular subtype which is characterized by unique molecular events compared to LO disease. Further studies using larger cohorts of patients are needed to validate these findings. Such studies might offer the possibilities of coming up with novel molecular markers to enhance newer, faster and noninvasive detection modalities for young patients with CRC tumors.

#142

Mutation and immune profiles in early-stage lung squamous cell carcinoma.

Murim Choi,1 Humam Kadara,2 Jiexin Zhang,2 Edwin Parra Cuentas,2 Jaime Rodriguez Canales,2 Stephen G. Gaffney,1 Zi-Ming Zhao,1 Carmen Behrens,2 Junya Fujimoto,2 Chi-Wan Chow,2 Neda Kalhor,2 Cesar Moran,2 David Rimm,1 Stephen Swisher,2 Don L. Gibbons,2 John V. Heymach,2 Edward Kaftan,1 Jeffrey Townsend,1 Thomas J. Lynch,1 Joseph Schlessinger,1 J. Jack Lee,2 Richard Lifton,1 Roy S. Herbst,1 Ignacio I. Wistuba2. 1 _Yale University, New Haven, CT;_ 2 _UT MD Anderson Cancer Center, Houston, TX_.

PURPOSE: Lung squamous cell carcinoma (LUSC) accounts for 20-30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We sought to perform whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of early-stage LUSCs to increase our understanding of the pathobiology of this malignancy.

METHODS: Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n=108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers was done on a subset of LUSCs (n=91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using ANOVA and Fisher tests. Cox proportional hazards regression models were used for statistical analysis of clinical outcome.

RESULTS: This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response.

CONCLUSION: Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

#143

Mutational landscape of colorectal adenomas reveals potential signatures for progression.

Shu-Hong Lin,1 Gottumukkala S. Raju,2 Chad Huff,1 Jian Gu,1 Jiun-Shen Chen,1 Scott Kopetz,3 David G. Menter,3 Ernest Hawk,1 Lopa Mishra,4 Yuanquing Ye,1 Xifeng Wu1. 1 _Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Gastroenterology, Hepat, & Nutr, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX; _3 _Department of Gastrointestinal (GI) Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Department of Gastroenterology, Hepatology & Nutrition, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction: Colorectal cancer (CRC) continues to rank as one of the most common cancers in the U.S. and around the world. Colorectal adenoma is the well-established precursor of CRC, yet only a very small proportion of adenomas progress to CRC. Therefore, identification of biomarker associated with adenoma progression is vital for risk stratification and development of intervention strategies. We hypothesize that genomic analysis of adenoma will lead to better understanding of tumorigenesis and identification of novel biomarkers.

Methods: We carried out whole exome sequencing (WES) on tissues and paired blood in 14 sessile serrated adenoma (SSA) and 36 tubulovillous adenoma (TVA) patients. Frequently mutated genes from TVA along with significantly mutated genes of TCGA colorectal adenocarcinoma were further investigated in an additional 100 pairs of TVA by targeted sequencing.

Results: 1) WES revealed similar somatic mutation frequency but distinctive mutated genes in TVA and SSA. 2) All adenomas included in WES were non-hypermutators with mutation frequency ranged from 0.55 to 3.71 (average: 1.49) non-silent somatic mutations per million base, which is significantly lower than that of TCGA non-hypermutators that ranged from 0.48 to 10.11 (average 4.47, p < 2.2x10^-16). 3) MutSigCV v1.4 discovered shared and unique significantly-mutated genes (SMG) in TVA and SSA reflecting difference in biology. 4) Targeted sequencing confirmed the findings in WES with a better estimate of population frequency for genes of interest. 5) Unsupervised clustering achieved high discrimination between adenoma and adenocarcinoma. 6) A subset of mutations that best discriminate adenoma and adenocarcinoma were identified by random Forest.

Conclusions: We discovered a set of genes with distinctive mutation pattern between adenoma and adenocarcinoma. We are conducting functional analysis to explore the potential of these genes as progression driver and risk predictors. We predict that the findings of this project will help lay the foundation to inform development of surveillance program and preventive intervention in CRC.

#144

Gene expression analysis of HPV negative and HPV positive penile cancer.

María Sánchez-Vázquez,1 Krizia Rohena-Rivera,2 Carlos Pérez-Ruiz,2 Antonio Puras-Báez,2 Carmen Cadilla,2 Magaly Martínez-Ferrer2. 1 _University of Puerto Rico Comprehensive Cancer Center, San Juan, PR;_ 2 _University of Puerto Rico Medical Sciences Campus, San Juan, PR_.

Penile cancer is a disease with a high morbidity and mortality among developing countries. Although penile cancer is a relatively uncommon cancer in developed countries, its incidence is nearly four times higher in Puerto Rico when compared with other racial and ethnic groups in the United States. Approximately 40% to 45% of penile cancers are HPV-related. The etiology of penile cancers is not completely understood and additional research is necessary to fully delineate the sequence of molecular events involved in both HPV mediated and non-HPV mediated pathways leading to penile cancer. The objective of this study is to determine the genome-wide expression profiles in HPV mediated and non-HPV mediated penile cancer. Penile cancer fresh tissue was obtained from surgically intervened cases. The global gene expression profile was identified and analyzed using the Affymetrix GeneChip® Human Gene 2.0 array. Microarray analysis showed a total of 158 genes differentially expressed between HPV mediated and non-HPV mediated penile cancer. Ingenuity Pathway Analysis (IPA) revealed alterations in the celular movement, cancer, lipid metabolism, carbohydrate metabolism, interleukin, TGF-B, integrin, PI3-kinase and MMP signaling pathways. The microarray results were confirmed by quantitative real-time PCR for some representative genes. Functional and pathway annotation using bioinformatics tools helped to identify specific pathways. Knowledge of the biological pathways and networks of the genes that are significantly expressed or altered are pertinent to understanding the molecular basis of the penile cancer.

#145

Comprehensive sequencing analyses of uterine and ovarian carcinosarcoma.

Seiichi Mori, Osamu Gotoh, Yuko Sugiyama, Kazuyoshi Kato, Yutaka Takazawa, Tetsuo Noda. _Japanese Foundation of Cancer Research, Tokyo, Japan_.

Carcinosarcoma (CS) of the uterus or the ovary, known as malignant mixed Muellerian tumor, is a biphasic neoplasm composed of malignant epithelial (carcinoma) and mesenchymal (sarcoma) elements. CS is a rare disease (two per 100,000 women) and exhibits poor prognosis (5-year survival rates: uterus and ovary; 31 and 7.5 %, respectively). Treatment regimen for CS follows to that for endometrioid adenocarcinoma (EC) since CS has been considered as an aggressive form of high-grade EC. However, CS has a significantly worse prognosis than EC. Identification of a molecular target and/or a molecular mechanism in tumorigenesis from a large cohort study may improve unfavorable outcomes. Toward this aim, we perform targeted resequencing analysis with a panel of 596 genes, which were previously implicated as critical not only for uterine, ovarian or sarcomatus oncogenesis but also for molecular targets in therapeutics. Through analyses of 84 cases of uterine and ovarian CS, consensus clustering with deregulated pattern of CS genome identified four genomic subtypes that correlate with genetic or epigenetic alterations, histopathology and patient outcomes. Moreover, potentially actionable alterations were found in a substantial number of genes (including PIK3CA, MTOR, KRAS and CCNE1). This study would provide deep insights into the development of novel diagnostic and personalized treatment for CS patients.

#146

Genomic analysis of endometrial stromal sarcoma of uterus.

Youn Jin Choi, Soo Young Hur, Jong Sup Park, Keun Ho Lee, Yoon Kyung Lee. _The Catholic University of Korea, Seoul, Republic of Korea_.

Although recurrent gene fusions such as JAZF1-JJAZ1 are considered driver events for endometrial stromal sarcoma (ESS) development, other genomic alterations remain largely unknown. In this study, we performed whole-exome sequencing, transcriptome sequencing and copy number profiling for five ESSs (three low-grade ESS (LG-ESS) and two undifferentiated uterine sarcomas (UUSs)). All three LG-ESSs exhibited either one of JAZF1-SUZ12, JAZF1-PHF1 and MEAF6-PHF1 fusions, whereas the two UUSs did not. All ESSs except one LG-ESS exhibited copy number alterations (CNAs), many of which encompassed cancer-related genes. In UUSs, five CNAs encompassing cancer-related genes (EZR, CDH1, RB1, TP53 and PRKAR1A) accompanied their expressional changes, suggesting that they might stimulate UUS development. We found 81 non-silent mutations (35 from LG-ESSs and 46 from UUSs) that included 15 putative cancer genes catalogued in cancer-related databases, including PPARG and IRF4 mutations. However, they were non-recurrent and did not include any well-known mutations, indicating that point mutations may not be a major driver for ESS development. Our data show that gene fusions and CNAs are the principal drivers for LG-ESS and USS, respectively, but both may require additional genomic alterations including point mutations. These differences may explain the different biologic behaviors between LG-ESS and UUS. Our findings suggest that ESS development requires point mutations and CNAs as well as the gene fusions.

Clinical and histologic characteristics of five endometrial stromal sarcomas

---

|

Age | Histopathology grade | Diagnosis | Specimen status | TNM

Case 1 | 32 | Low-grade | LG-ESS | Primary | T1bN0M0

Case 2 | 34 | Low-grade | LG-ESS | Primary | T1bN1M0

Case 3 | 57 | Low-grade | LG-ESS | Primary | TxNxM1

Case 4 | 65 | High-grade | UUS | Metastatic | T2aN0M0

Case 5 | 57 | High-grade | UUS | Metastatic | TxNxM1

### Intratumor Heterogeneity and Resistance

#147

Exome sequencing identifies frequent histone methyltransferase mutations in metastatic SCC.

Amanda Ewart Toland,1 Dawn Allain,1 Jessica Gillespie,1 Priyaharsini Nagarajan,2 Sara Peters,1 Hatice Gulcin Ozer,1 Thomas Olencki,1 Ayse Selen Yilmaz,1 Theodoros Teknos1. 1 _Ohio State University, Columbus, OH;_ 2 _University of Texas, MD Anderson, Houston, TX_.

Although only a small proportion of cutaneous squamous cell carinomas (cSCC) metastasize, approximately 3900-8800 individuals in the United States die of metastatic cSCC each year. Little is known about the molecular events that lead approximately 2-6% of cSCCs to metastasize. Treatment efforts are hampered by a lack of ability to predict which lesions are more likely to recur or metastasize as these known correlates do not have high specificity for prediction. Furthermore, there is no FDA approved therapy that is specifically indicated for metastatic cSCC. Deep-sequencing efforts have identified targetable mutations and pathways for several cancers. Exome and targeted sequencing of primary cSCCs led to the discovery that inactivating mutations in NOTCH pathway genes occur in over 75% of tumors; however NOTCH mutations are not indicative of metastasis. Published studies identified frequent mutations in KMT2C and KMT2D in cSCCs with aggressive features. To identify mutations that are unique to or enriched in metastatic samples which may help to identify novel therapeutics for treating aggressive cSCCs, we performed exome sequencing of 37 samples consisting of matched normal DNAs, primary tumors and metastatic cSCCs. Many of the mutations found in the metastatic samples were also observed in the matched primary tumors. We found an enrichment of mutations in chromatin remodeling genes. Epigenetic regulatory genes mutated in the metastatic samples included KMT2D (64%), KMT2C (44%), SETD2 (45%), KAT6A (36%), EP300 (18%), and KDM6A/UTX (9%). We performed additional targeted deep-sequencing to evaluate whether candidate genes were frequently mutated in non-aggressive cSCCs. Initial analyses of targeted-sequencing of 10

non-aggressive primary cSCCs and 7 additional metastatic cSCCs as well as data from published exome studies on non-aggressive cSCCs suggest that chromatin remodeling genes are mutated at a much higher rate in metastatic cSCCs. These data provide potential pathways for therapeutic intervention for this deadly cancer.

#149

Integrated analysis of intratumor heterogeneity with genetic and epigenetic aberrations during colorectal cancer evolution.

Shinya Kidogami,1 Atsushi Niida,2 Ryutaro Uchi,1 Yusuke Takahashi,3 Masaki Mori,3 Satoru Miyano,2 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu, Japan;_ 2 _Human Genome Center Institute of Medical Science University of Tokyo, Tokyo, Japan;_ 3 _Graduate School of Medicine, Osaka University, Suita, Japan_.

[background] Intratumor heterogeneity (ITH) is presumably generated by branching clonal evolution of cancer cells. Recently, a multiregional sequencing approach, which sequences DNA sampled from geographically separated regions of a single tumor, has revealed branched evolution and ITH. In this study, we present genetic and epigenetic analysis of ITH in a series of nine colorectal cancers

[material and method] We conducted analysis of samples from geographically separated regions from nine colorectal tumors. From each of the nine tumor, we obtained 5-21 multiregional samples, which were 75 samples in total, together with 9 paired normal mucosa samples. For two cases, samples from liver metastases were obtained. We performed exome sequencing and copy number (CN), methylation, and mRNA expression array profiling.

[Result] Each of the multiregional mutation profiles harbored founder and progressor mutations; founder mutations are shared by all regions while progressor mutations are not. We found that mutations in well-known driver genes such as APC, KRAS, and FBWX7 were acquired as founder mutations, while mutations in PIK3CA recurrently occurred as progressor mutations, suggesting that PIK3CA mutations are a late event in the evolution of colorectal cancer. We counted each category of mutation and then identified a correlation between the number of founder mutations and the age of the patients. Our analysis showed that C > T transitions at CpG sites are more prominent in founder mutations than in progressor mutations. Then, the multiregional CN profiles showed that amplifications of 7p, 13q, 10q, 20p, and 20q frequently occurred across all samples in multiple tumors, namely as founder CN alterations. Finally, our data showed that hypermethylations in CpG islands were more prominent in founder methylations than in progressor methylations, suggesting that CpG island hypermethylations mainly occur in the early phase of colorectal cancer evolution.

[Conclusion] In this study, our integrated analysis revealed not only extensive ITH, but also the evolutionary histories of the nine tumors. In particular, by focusing on founder and progressor mutations, we identified clues for decoding the life history of the tumors.

#150

Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system.

Grace X.y. Zheng, Tarjei Mikkelsen, Jessica Terry, Phillip Belgrader, Paul Ryvkin, Ryan Wilson, Tobias D. Wheeler, Zachary Bent, Geoff McDermott, Solongo Ziraldo, Alexander Wong, Michael Schnall-Levin, Ben Hindson. _10X Genomics, Inc., Pleasanton, CA_.

Advances in single cell RNA quantification techniques have enabled comprehensive study of subpopulations of cells within a heterogeneous population. The application of single cell quantification techniques to oncology is helping to elucidate the complex variability in genetic and epigenetic interactions that occur within tumor cells and their microenvironment. However, current single cell RNA-sequencing methods are limited by their reliance on costly infrastructure and laborious experimental protocols. We developed the GemCode Platform, which combines microfluidics with molecular barcoding and custom bioinformatics software to enable 3' mRNA counting from thousands of single cells. Here we utilized the GemCode Platform to profile primary cells from healthy donors and cancer patients.

Cell lines and cancer samples were obtained from commercial sources. Single cells, reagents and a single gel bead containing barcoded oligonucleotides were encapsulated into picoliter-sized droplets using the 10X Genomics GemCode Platform. The platform achieved extremely high cell loading efficiency (> 50%), enabling the creation of libraries from precious samples. Lysis and barcoded reverse transcription of RNAs from single cells were performed inside each droplet. High quality next generation sequencing libraries were finished in a single bulk reaction. The GemCode software suite was utilized for processing, interactive analysis and visualization of single cell gene expression data.

We demonstrated single cell behavior through mouse- and human cell mixing experiments with a low doublet rate of <1%, making the platform suitable for profiling of rare cancer cell populations. We profiled >40,000 peripheral blood mononuclear cells from healthy donors and detected all major subpopulations (i.e., B cells, CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, monocytes) in similar proportions to those previously reported in the literature. Notably, the high-throughput nature of the platform enabled resolution of finer sub-structures such as CD4+ effector memory cells and CD4+ central memory cells. Experiments comparing cells isolated from patients with hematologic malignancies (such as CLL, AML and CML) with whole bone marrow from healthy donors further demonstrate the power of single cell profiling for characterizing disease-associated changes in complex tissues.

We demonstrate the ability to perform high-throughput gene expression profiling of mRNAs in single cells. The high-throughput platform enables detection of rare cells in a heterogeneous tumor population. Moreover, efficient cell loading enables analysis of clinically relevant sample types with limited cell input. An integrated single cell mRNA analysis will lead to novel insights into the molecular characteristics of individual cancer cells and provide targets for therapeutic intervention.

#151

Modeling the rise of intratumoral heterogeneity in growing, static, and regressing human colorectal polyps.

Chelsie K. Sievers,1 Luli Zou,1 Perry J. Pickhardt,1 Kristina A. Matkowskyj,1 Dawn Albrecht,1 David H. Kim,1 Fouad J. Moawad,2 Brooks D. Cash,2 Mark Reichelderfer,1 Michael A. Newton,1 Richard B. Halberg1. 1 _University of Wisconsin, Madison, WI;_ 2 _Walter Reed National Military Medical Center, Bethesda, MD_.

Benign adenomatous colon polyps are thought to be transformed into cancers through the stepwise accumulation of mutations. However, not all polyps progress. A significant number remain static in size, regress, or resolve completely. The mechanisms underlying these differential fates are unknown, and currently there are no biological characteristics that can reliably predict which polyps will grow or progress into invasive cancer. To determine the mutational landscape, targeted next generation sequencing was performed on a unique collection of small (6-9mm) colorectal polyps with known growth rates based on interval imaging with CT colonography. To determine spatial location of identified mutations within a polyp, micro-dissection was performed followed by quantitative PCR to validate low frequency mutations. The mutational landscape of small polyps is varied both within and among individual polyps. Polyps carried 0-3 pathogenic mutations with the most frequent being in APC (67%, 32/48), KRAS/NRAS (17%, 8/48), BRAF (17%, 8/48), FBXW7 (10%, 5/48), and TP53 (8%, 4/48). Additionally, 13% (6/48) contained driver mutations at varied mutant allele frequencies, indicating the presence of subclonal populations. In silico modeling of tumor growth was used to determine the likely size at which additional driver mutations arose in order to observe those varied frequencies. This model of colon tumor growth was adapted so that mutations occur with a given probability of 10-5, which may change the fitness positively or negatively, and the lineage from these mutant subpopulations was tracked during tumor growth. In silico polyps were sectioned and mutant allele frequency was recorded and compared to the frequencies observed from the targeted sequencing of human polyps. Contrary to the slow step-wise accumulation of mutations theory, these data indicate small colonic polyps can have multiple pathogenic mutations in crucial driver genes that arise early in a tumor's existence. Understanding the molecular pathway of tumorigenesis and clonal evolution in polyps that are at risk for progressing to invasive cancers will allow us to begin to better predict which polyps may be more likely to progress into adenocarcinomas and which patients are predisposed to developing advanced disease.

#153

Patterns of genetic progression in high grade prostatic intraepithelial neoplasia.

Seung-Hyun Jung, Sun Shin, Sug Hyung Lee, Yeun-Jun Chung. _Catholic University of KOREA, Seoul, Republic of Korea_.

High grade prostatic intraepithelial neoplasia (HGPIN) is considered a neoplastic lesion that precedes prostate cancer (PCA). Genetically, HGPIN is known to have some driver mutations such as TMPRSS2-ERG fusion and 8p loss. However, we still do not know how the whole picture of somatic mutations in HGPIN is and what are added during the progression to PCA. To identify the genomic differences between HGPIN and PCA, we analyzed 20 regions of paired HGPIN and PCA from 6 patients by whole-exome sequencing and array-comparative genomic hybridization. As expected, the number of total mutations and CNAs of HGPINs were significantly fewer than those of PCAs. Mutations in FOXA1 and CNAs (1q and 8q gains) were detected in both HGPIN and PCA (common), suggesting their roles in early PCA development. Mutations in SPOP, KDM6A, and KMT2D were PCA-specific, suggesting their roles in HGPIN progression to PCA. The 8p loss was either common or PCA-specific. HRAS and FOXA1 mutations were PIN-specific. Our data show that PCAs are direct descendants of HGPINs in most cases that require more genomic alterations to progress to PCA. The nature of heterogeneous HGPIN population that might attenuate genomic signals should further be studied. Our results provide a clue to explain the long latency from HGPIN to PCA and provide useful information for clinical decision making in the genetic diagnosis of HGPIN and PCA.

#154

Defective calcium signaling pathway highlights the mutational landscapes of liver metastasis from colorectal and breast cancer.

Fangfang Song,1 Xiangchun Li,2 Yuexin Liu,3 Hong Zheng,1 Haixin Li,1 Jun Wang,2 Wei Zhang,3 Kexin Chen1. 1 _Tianjin Medical University Cancer Institute and Hospital, Tianjin, China;_ 2 _BGI-shenzhen, China;_ 3 _The University of Texas MD Anderson Cancer Center, TX_.

Background

Imaging-based early detections of colorectal cancer (CRC) and breast cancer (BC) have significantly improved clinical outcome of these two common cancers. However, when metastasis to distant organs (e.g. liver) occurs, few therapeutic options are currently available. Recent advances in the next-generation sequencing technologies have revolutionized our possibilities to precisely profile all the genetic changes involved in the evolutionary process of individual cancers. To gain insight into genetic and molecular underpinning of metastasis, we performed whole exome sequencing of paired primary and metastases samples from seven CRC/BC patients. Candidate gene mutations were orthogonally validated by targeted sequencing on 28 paired samples including an additional 21 matched primary and metastatic tumors.

Results

We found that the mutational spectra of metastases were highly heterogeneous. A pairwise comparison between primary and metastatic samples indicated many point mutations were exclusively enriched either in primary tumor or metastases or were present in a sharing pattern between them, showing a lesion-specific genetic difference. Besides the known and novel mutations in APC, TP53, KRAS genes, we discovered 64 significantly mutated genes that were associated with liver metastases from CRC or BC, 9 of which were metastasis specific. Notably, genetic aberrations in calcium signaling pathway genes (GPR98, SYT6, TACR3, CYSLTR1, RYR3, and CACNA1A) were somatically mutated in liver metastases in 29% of CRC and BC cases, indicating that calcium-dependent signaling plays a key role in distant metastasis.

Conclusions

These results begin to define the genetic basis underlying liver metastasis from CRC/BC and identify potential therapeutic targets.

#155

The analysis of the genomic evolution of squamous cell carcinoma of the lung.

Arthur Krause,1 Thomas Lorber,1 Valeria Perrina,1 Tanja Dietsche,1 Michael Barrett,2 Christian Ruiz,1 Lukas Bubendorf1. 1 _Institute for Pathology, University Hospital of Basel, University of Basel, Basel, Switzerland;_ 2 _Mayo Clinic in Arizona, Scottsdale, AZ_.

Background:

Genomic tumor heterogeneity is one reason for recurrence and resistance to cancer therapy. It is still not known what leads to the relapse or the emergence of metastasis in individual patients. To contribute to answer this question, we investigated the evolutionary patterns in squamous cell carcinoma of the lung (SCC) in 10 patients with multiple matched tumors (primary vs recurrence/metastasis).

Methods:

To identify the genomic profile, frozen tissues from SCC patients were subjected to multiparameter ploidy profiling (MPP). Tumor populations were sorted based on ploidy (DNA-content using DAPI) and p40 was used to control for diploid tumor populations. Subsequent genomic profiling by array comparative genomic hybridization (aCGH) and targeted massive parallel sequencing (Ion Torrent Comprehensive Cancer Panel with an all-exon coverage of 409 cancer genes) was applied.

Results:

The usage of MPP enabled the determination of the clonal composition of the SCC and define genomic characteristics. Due to flow sorting the tumor populations were effectively isolated and existence of distinct clonal populations was indicated. Aneuploid and diploid tumors were successfully revealed using the additional marker p40. Therefore, the absence of non-malignant cells increased the precision in the genomic analyses

Conclusion:

The study uncovered the composition of human SCC in the sense of different clonal populations, which harbor private genomic aberrations and mutations confirmed by aCGH and NGS. Even if our data is still preliminary we provide evidence that applying MPP increases the precision of aCGH and sequencing analysis. Genomic analyses of sorted pure tumor population allows to reconstruct the clonal evolution and facilitates the understanding of tumor heterogeneity and their evolutionary pattern, which could have potentially an impact on therapeutic response or metastasis development.

#156

Integrated exome and transcriptome sequencing of primary lung cancers and paired distant metastases.

Jianjun Zhang, Chia-Chin Wu, Jianhua Zhang, Junya Fujimoto, Xingzhi Song, Xizeng Mao, Huadong Sun, Sahil Seth, Rebecca Thornton, Marcus Coyle, Latasha Little, Curtis Gumbs, Carmen Behrens, Chi-Wan Chow, Erik Sulman, Ganesh Rao, Stephen Swisher, Ignacio Wistuba, John Heymach, Andrew Futreal, Daniel Gomez. _The UT MD Anderson Cancer Center, Houston, TX_.

Background: The precise molecular mechanisms underlying metastasis of nonsmall cell lung cancers (NSCLC) are largely unknown. Two recent studies comparing genomic landscapes of primary NSCLC tumors and paired brain metastases suggested branched evolution, where all metastatic and primary tumors shared a common ancestor yet continued to evolve independently. The integrated genomic and transcriptomic profiles of primary NSCLC and metastases have not been studied in any details.

Methods: We performed whole exome sequencing (WES) and RNA sequencing (RNA-seq) of surgically resected primary tumors and paired distant metastases from 7 patients with NSCLC.

Results: Totally, 6,945 somatic mutations, including 1,702 non-silent (stop-gain, stop-loss, frameshift, splicing site and nonsynonymous) mutations were identified by WES. Metastases trended to have larger mutation burdens compared to paired primary tumors, although the difference was not statistically different (average 595 mutations per tumor in primary tumors versus 852 mutations per tumor in metastases, respectively, p = 0.54). On average, 51% of all mutations (24% to 93%) were shared between primary tumors and metastases. We identified 14 canonical cancer gene mutations in this cohort of patients, defined as mutations that lead to amino acid changes identical to those found previously in cancer genes or disrupting mutations in tumor suppressor genes, all of which were shared between primary tumors and paired distant metastases. In addition, metastases resembled paired primary NSCLC tumors closely in regard to somatic copy number aberration profiles and mutation signatures. Pathway analysis from RNA-seq data demonstrated that 25 of the 35 signal transduction pathways that were significantly down regulated in metastases relative to primary NSCLC tumors were related to immune activation. Validation study with a larger patient cohort is in progress.

Conclusions: Although branched evolution is a common phenomenon during metastasis of NSCLC, majority of canonical cancer gene mutations are probably early molecular events likely acquired before metastasis initiates. Mutation mechanism may be determined early during carcinogenesis and preserved during cancer evolution even at the metastatic sites. Immune suppression may be one characteristic feature of cancer cells of metastatic capacity.

#157

Single-cell DNA sequencing identifies a late dissemination model in metastatic colorectal cancer.

Marco L. Leung, Anna Casasent, Yong Wang, Emi Sei, Nicholas Navin. _University of Texas MD Anderson Cancer Ceneter, Houston, TX_.

Metastatic colorectal cancer (mCRC) is a devastating disease with only 11% 5-year survival rate for stage IV patients. The genomic basis of metastasis has been difficult to study, in part due to the extensive intratumor heterogeneity at both the primary and metastatic tumor sites. Previous studies have applied bulk next-generation sequencing (NGS) methods, which have limited ability to resolve intratumor heterogeneity. To address this problem, we developed a highly-multiplexed single cell DNA sequencing method that combines flow-sorting of single nuclei, multiple-displacement-amplification, low-input library preparation, library barcoding, targeted capture and NGS to generate high-coverage data from single tumor cells. To increase throughput, we performed targeted single-cell DNA sequencing using a panel of 201-cancer genes to analyze single cells from primary tumors and liver metastases from two mCRC patients. In each patient, we sequenced 90 single tumor cells from the primary and metastatic tumor, to delineate the clonal architecture of the tumor and reconstruct their phylogenetic lineages. In addition, we sequenced populations of tumor cells at high coverage depth (200X).

Our data identified a large number of nonsynonymous mutations that evolved in the root nodes during the earliest stages of primary tumor evolution and were maintained in all single cells during the clonal expansion of the tumor mass. We also identified a small number of mutations that were specific to the liver metastases, which are likely to play an important role in metastatic dissemination. Using the single cell data, we constructed phylogenetic trees, which revealed branched evolution at both organ sites. Collectively, our data suggest that both mCRC patients are consistent with a late-dissemination model, in which the primary tumors evolved for a long period of time prior to the dissemination of clones to distant organ sites. This model has important clinical implications, by suggesting that surgical intervention or therapeutic treatment of the primary CRC tumor can prevent metastasis, since single tumor cells do not disseminate at the earliest stages of tumor growth

#158

Clonal evolution and genomic tumor heterogeneity in non-small cell lung cancer deciphered by multiparameter ploidy profiling.

Thomas Lorber,1 Tanja Dietsche,1 Valeria Perrina,1 Darius Juskevicius,1 Arthur Krause,1 David Mueller,1 Michael Barrett,2 Christian Ruiz,1 Lukas Bubendorf1. 1 _University Hospital Basel, Basel, Switzerland;_ 2 _Mayo Clinic, Scottsdale, AZ_.

BACKGROUND

Genomic intra- and intertumor heterogeneity is one main reason for relapse and resistance to therapy. There is a shortage of studies characterizing the level of genomic intertumoral heterogeneity of known cancer genes in multiple longitudinal biopsies of individual patients. Additionally, a workflow to deconvulate the intermixture of tumor and stromal components is missing. To overcome these limitations and to decipher the genomic heterogeneity and clonal evolution, we developed a method and applied it on matched (primary-recurrence/metastasis) non-squamous, non-small cell lung cancers (NSCLC).

METHODS

Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin and paraffin embedded (FFPE) tissues, followed by multiparamter flow sorting. Sorted populations were subjected to genomic profiling by high resolution array comparative genomic hybridization (aCGH) and massively parallel sequencing (MPS). DAPI allowed to seperate populations by ploidy and anti-TTF1 antibodies was used to control for tumor nuclei. Array-CGH, combined with ploidy, was used to retrieve genome wide copy numbers. The Comprehensive Cancer Panel that covers all-exons of 409 cancer genes was applied on all sorted tumor and stromal populations to detect somatic mutations and their variant allelic frequency (VAF).

RESULTS

MPP was successfully applied to 44 frozen or FFPE tissue specimens from 19 patients. Array-CGH and MPS of TTF1-negative, normal cells were concordant to germline controls. Sequencing revealed that 50% of mutations are shared between primary tumors and metastases. Except for one patient, mutations with VAF>0.3 are shared between primary and metastasis. The variant allelic frequency was significantly higher in shared mutations than mutations that were private to one tumor. Besides common activating mutations in EGFR and KRAS we found biallelic inactivation in tumor suppressor genes like KEAP1, NF1, STK11 and TP53. Two clonal evolutionary patterns were found: 1) early and 2) late divergence. Matched tumors without any shared mutations were classified as unrelated primary tumors.

CONCLUSION

The power of MPP is to increase the precision of downstream analysis, due to the sorting of pure populations of tumor cells. It permitted to infer the clonal evolution of tumor populations with unprecedented confidence. The low level of genomic heterogeneity of mutations with VAF>0.3 in this study is in line with recent data from Zhang et al., who showed that the poor precision of low depth sequencing (<277x) contributes to an overestimation of genomic heterogeneity (Zhang et al., Science 2014). Integrational analysis of ploidy, chromosomal aberrations and mutations in 409 cancer genes allowed to draw the evolutionary picture of each patient's disease. MPP appears as a promising tool for identification of genomic vulnerabilities that could be exploited for tailored treatment.

#159

Analysis of resistance to the combination of a PI3K- and Parp-inhibitor using a genomic sequencing approach.

Sheida Nabavi,1 Ashish Juvekar,2 Nicholas Wang,3 Lewis C. Cantley,4 Gerburg M. Wulf2. 1 _University of Connecticut, Storrs, CT;_ 2 _Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA;_ 3 _Oregon Health and Science University, Portland, OR;_ 4 _Weill Cornell Cancer Center, New York, NY_.

The objective of this study is to investigate the genomic and transcriptonal changes that are associated with resistance to treatment with the combination of a PI3K-inhibitor (NVP-BKM120) and a PARP-inhibitor (Olaparib). Previous studies have shown that the combination of a PI3K-inhibitor and a PARP-inhibitor synergistically reduced the growth of BRCA-related xenograft tumors derived from patients with TNBC. However, in clinical practice, primary and secondary resistance to this combination is observed. We hypothesize that acquired resistance is the result of a genomic evolution resulting from the selection pressure exerted by the drug treatment.

We used a genetically engineered mouse model, K14-Cre BRCA1f/fp53f/f, where primary tumors had been propagated in Cre-negative littermates and treated to point of resistance with NVP-BKM120 or NVP-BKM120 plus Olaparib. For each of the three tumors, we analyzed exome sequencing and RNA-seq using Illumina technology at the stage of sensitivity (C), resistance to PI3K-inhibition (B) and resistance to the combination (BO); i.e. for each individual tumor a parental clone and its drug-resistant subclones, obtained in vivo, were analyzed. We used the Mutect software tool to call somatic mutations, VarScan2 to call somatic copy number variations, Cuffdiff for differential expression analysis, and TophatFusion. Data were integrated to identify genes for functional analyses using MetaCore and MSigDB software tools.

We found that tumors resistant to the combination drug treatment in general had fewer gross genomic alterations (CNVs) than parental tumors, indicative of evolution of a less variable subclone. However, these tumors also displayed a higher number of non-synonymous mutations than their parental clone. Pathway analysis showed that somatically mutated genes in PI3K-inhibitor resistant tumors were highly enriched for antigen processing and presentation, while those of tumors resistant to the combination treatment were enriched for histone modification pathways. Notably, the tumors resistant to PI3K-inhibitor alone were enriched for Insulin-processing pathways. Genomic losses in PI3K-inhibitor treated tumors enriched for the estrogen receptor pathway (ESR) in breast cancer and DNA damage pathways.

In conclusion, our experimental design of analyzing isogenic tumors resistant to PI3K- or combined PI3K- and Parp-inhibitors enabled us to identify genomic alterations and pathways that explain the evolution to drug resistance.

#160

Identification of the carcinoma and immune cells in the breast cancer by single-cell RNA sequencing.

Woosung Chung,1 Hye Hyeon Eum,1 Kyu-Tae Kim,1 Kyung-Min Lee,2 Arum Jo,1 WonShik Han,2 Hae-Ock Lee,1 Woong-Yang Park1. 1 _Samsung Genome Institute, Seoul, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea_.

Summary: Breast cancer is a heterogeneous type of cancer accompanying extensive immune cell infiltration. Although its mortality rate has been decreasing with early diagnosis and introduction of molecular targeted therapies, it is still one of the world leading cause of cancer death in women. In breast cancer, molecular subtyping is used for the patient stratification and treatment decisions, emphasizing the importance of inter-patient tumoral heterogeneity in the tumor cell behavior. Tumor cells within a patient may also manifest intra-tumoral heterogeneity, which may cause treatment resistance. Single cell gene expression profiling would reveal both inter- and intra-tumoral heterogeneity in breast cancer encompassing tumor cells and associated stromal and immune cells.

Experimental Process: From 4 different subtype primary breast cancer and 2 lymph node metastases, single cells were captured and amplified through C1™Single-cell Auto Prep System (Fluidigm) with the SMARTer Ultra Low RNA Kit. Sequencing libraries were constructed with the Nextera XT DNA sample Prep Kit and sequenced through a Hiseq2500 (Illumina) as 100-bp paired end mode of the TruSeq Rapid PE Cluster kit and Truseq Rapid SBS kit.

Computational Process: RNA sequencing reads were aligned to the modified human genome reference using 2-pass mode of STAR_2.4.0b, and transcript per million (TPM) values were obtained as the relative gene expression levels of single cells through RSEM v1.2.17.

Results: As most breast cancers originate from the mammary epithelium, we distinguished carcinoma cells from associated non-tumor cells by epithelial cell adhesion molecule (EPCAM) signature genes. We validated the distinction with chromosomal gene expression patterns and gene set enrichment analyses using tumor, stromal, or immune genesets. With or without removal of non-tumor cells, TNBC tumor cells showed the highest gene expression heterogeneity, recapitulating the known inter-patient heterogeneity. TNBC tumor cells from a single patient were categorized into 6 different TNBC-subtypes, further demonstrating intra-tumoral heterogeneity. In addition, immune cells identified in the TNBC tumor were naïve B cells and regulatory T cells, suggesting non-activated status of the immune system.

Conclusions: The high resolution gene expression profiling revealed features of breast cancer transcriptome which may provide clues for molecular directed therapies targeting the tumor or the immune compartment.

#161

Single-cell RNA sequencing presents explicit expression signatures in primary breast cancer.

Hye Hyeon Eum,1 Hae-Ock Lee,1 Woosung Chung,1 Kyu-Tae Kim,1 Kyung-Min Lee,2 Arum Jo,1 WonShik Han,2 Woong-Yang Park1. 1 _Samsung Genome Institute, Seoul, Republic of Korea;_ 2 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea_.

Intratumoral heterogeneity has emerged as a problem to be resolved for precise diagnosis and effective treatment of cancer. Breast cancer is one of the most heterogeneous cancer in its molecular subtypes and its intratumoral genomic diversity has been demonstrated as well. Single-cell sequencing approach would reveal heterogeneity in gene expression signatures and the common gene expression characteristics across tumor cells within a tumor. In this study, we isolated live individual cells and amplified cDNAs using C1™Single-cell Auto Prep System (Fluidigm) then performed RNA sequencing using HiSeq2500 System (Illumina). Single-cell transcriptomes were acquired from four primary tumor (ER+/HER2-, ER-/HER2+, ER+/HER2+ and triple negative breast cancer) and two matched lymph node metastases (ER+/HER2+ and triple negative breast cancer). Because the tumor tissue isolates may include stromal and immune cells in the tumor microenvironment, we defined 'epithelial cell adhesion molecule signature' to filter out non-tumor populations (Poster# presented by Woosung Chung et. al) After the removal of non-tumor cells, collected tumor single-cell transcriptomes largely recapitulated bulk tumor-expression profiles. The ER+/HER2- tumor cells showed highly activated estrogen receptor-associated pathways. Although both ER-/HER2+ and ER+/HER2+ tumors had the HER2 gene amplification and overexpression, HER2/HER3 pathway is activated only in ER-/HER2+ tumor cells. The association of gene expression for cancer stem cells and chemoresistance, such as IL8 and CXCL1, with HER2/HER3 signaling pathway activation was prominent. By contrast, the ER+/HER2+ tumor cells had highly activated estrogen receptor signaling, presenting them as the luminal type. The triple negative breast cancer cells highly expressed adhesion molecules and proteases suggesting their invasive nature, yet demonstrated extensive gene expression heterogeneity. Collectively these data revealed the power of single cell gene expression profiling in the precise characterization of tumor cell properties. The explicit gene expression signatures for patient specific tumors may be defined as subtype-specific signatures and helpful to unmask targets for molecular directed therapies.

#162

Genomic analysis of uterine aspirates improves diagnosis and captures the intratumor heterogeneity in endometrial cancer.

Alba Mota,1 Eva Colas,2 Pablo Garcia-Sanz,1 Irene Campoy,2 Alejandro Rojo-Sebastian,3 Luis Chiva,3 Sonsoles Alonso,3 Sonia Gatius,4 Antonio Gil-Moreno,2 Xavier González-Tallada,4 Berta Díaz-Feijoo,2 Patrycja Ziober Malinwska,5 Marcin Bobiński,5 Jaume Reventos,2 Xavier Matias-Guiu,4 Gema Moreno-Bueno6. 1 _Universidad Autónoma de Madrid, Madrid, Spain;_ 2 _Vall d'Hebron Research Institute, Barcelona, Spain;_ 3 _MD Anderson Cancer Center Madrid, Madrid, Spain;_ 4 _Hospital Universitari Arnau de Vilanova, Lleida, Spain;_ 5 _Medical University of Lublin, Lublin, Poland;_ 6 _Universidad Autónoma de Madrid/Fundacion MD Anderson International, Madrid, Spain_.

Background: Endometrial cancer (EC) is the most common cancer in female genital tract in developed countries. Although the majority of ECs are diagnosed in early stages, with a 5-year overall survival of around 80%, an early detection of these tumors is crucial to increase survival of patients given that advanced tumors are associated with poorer outcome. Furthermore, the correct assessment of pre-clinical diagnosis is also decisive to guide the surgical management and treatment of the patient.

Results: Genetic analysis of uterine aspirates identifies the mutational profile of endometrial cancers and represents the intra-tumor heterogeneity found in the analysis of multiple tumor regions from hysterectomy.

Conclusions: The current study reveals the potential of the genetic analysis of uterine aspirates as a pre-operative tool to diagnosis, even in biopsies with insufficient material, with a sensitivity of 93.7% and a specificity of 91.7%. Additionally, genetic analysis of uterine aspirates allows to capture the intra-tumor heterogeneity identified in ECs.

#163

Genetic difference between multicentric carcinogenesis and intrahepatic metastasis of hepatocellular carcinoma.

Yutaka Midorikawa,1 Shogo Yamamoto,2 Kenji Tatsuno,2 Hiroki Ueda,2 Shingo Tsuji,2 Genta Nagae,2 Tadatoshi Takayama,1 Hiroyuki Aburatani2. 1 _Nihon Univ., Tokyo, Japan;_ 2 _Univ. of Tokyo, Tokyo, Japan_.

Multiple hepatocellular carcinoma (HCC) is categorized into 2 types; multicentric hepatocarcinogenesis (MC) and intrahepatic metastasis (IM). Although discrimination of the types of multiple HCC is of clinical importance, it is quite difficult to determine it correctly even after histological diagnosis. In order to classify multiple HCC into MC and IM, we compared pairs of multiple HCC samples from 12 patients who were clinically diagnosed MC, 6 patients with IM with regard to molecular aberrations, and 2 patients with pairs of primary HCC and extrahepatic metastasis such as adrenal grand and lung metastasis. Exome sequence showed that 125 mutations (90.6%) per patient with IM were common, which were consistent with the result of a pair of primary HCC and extrahepatic metastasis. On the other hand, 138 somatic mutations per tumor, and no common somatic mutations were identified in the 7 patients with MC. Especially only a few passenger mutations were different between IM specimens suggesting that IM pairs developed from the common ancestor. In contrast, 5 patients who clinically diagnosed MC had common mutations (41.7%), which indicates that pairs of HCC of these patients were genetically IM. Notably, mutation signatures obtained from exome sequence data of 2 nodules from the same patients with IM were similar, while those from patients with MC were different by profiling analysis. On the other hand, methylation profile shows that 2 HCC samples from the same patients with MC as well as IM were classified into the same cluster; the methylation status from the different ancestor is similar, and not altered through carcinogenesis. Taken together, comparison of mutations in a pair of HCCs makes it possible to classify multiple HCCs into MC and IM, which is difficult to be correctly determined in clinical practice, and is available to make treatment plans for multiple HCC patients. Furthermore, methylation status of 2 nodules of MC from same patient were similar, while those of mutation signatures were different, which suggests that the epigenomic changes are the earlier event prior to the genomic alterations.

#164

Clonal evolution in noncancerous esophageal mucosa in normal and cancer-bearing individuals.

Akira Yokoyama,1 Hiromichi Suzuki,1 Tetsuichi Yoshizato,1 Kosuke Aoki,1 Yusuke Shiozawa,1 Youichi Fujii,1 Yusuke Sato,1 Nobuyuki Kakiuchi,1 Sugi Kin,1 Keisuke Kataoka,1 Kenichi Yoshida,1 Hideki Makishima,1 Yusuke Amanuma,2 Shinya Oohashi,2 Yuichi Shiraishi,3 Kenichi Chiba,3 Hiroko Tanaka,3 Brown J.B.,4 Masashi Sanada,5 Shigeru Tsunoda,6 Sachiko Minamiguchi,7 Yoshiharu Sakai,6 Hironori Haga,7 Tsutome Chiba,8 Satoru Miyano,3 Manabu Muto,2 Seishi Ogawa1. 1 _Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan;_ 2 _Department of Clinical Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan;_ 3 _Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan;_ 4 _Center for Medical Education, Graduate School of Medicine, Kyoto University, Kyoto, Japan;_ 5 _Department of Advanced Diagnosis, Nagoya Medical Center, Nagoya, Japan;_ 6 _Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan;_ 7 _Department of Diagnostic Pathology, Graduate School of Medicine, Kyoto University, Kyoto, Japan;_ 8 _Graduate School of Medicine, Kyoto University, Kyoto, Japan_.

Background

Esophageal squamous cell carcinoma (ESCC) represents the most common form of esophageal cancer worldwide, especially in East Asia, where alcohol drinking and smoking have been implicated in the field carcinogenesis of ESCC. However, the oncogenic process therein has been poorly understood in terms of gene mutations.

Patients & Methods

A total of 100 samples, including cancer, dysplastic, and non-dysplastic esophageal tissues, were obtained from 24 individual with (N=14) or without (N=10) ESCC (a median of 2.5 samples per case: 1−29) either by endoscopy or surgery and were subjected to whole exome sequencing (WES). An additional paired cancer/non-caner samples from 32 patients was analyzed by targeted sequencing (TS). All samples were analyzed for copy number alterations (CNAs) using SNP array- and/or digital sequencing-based karyotyping.

Results

In WES, clonal evolution in esophageal epithelia, as determined by the presence of somatic mutations, was detected in 21 of 21 cancer, 12 of 12 dysplastic, and 63 of 67 non-dysplastic samples, where the mean number of mutation per sample showed a significant trend to increase in cancer (65) and dysplastic samples (50) compared to non-dysplastic samples (13) (P=2.1×10-11). CNAs, especially those involving CDKN2A, CCND1, YAP1, and EGFR, were frequently affected in cancer samples, but rarely so in non-dysplastic samples. Non-dysplastic samples tended to have smaller allelic burden and therefore, clone size, compared to dysplastic and cancer samples (P=2.2×10-16). Mutations had a predominant age-related signature in non-dysplastic samples but increasing APOBEC3A/3B patterns was observed in cancer and dysplastic samples. Shared mutations were found only within cancer tissues but never among dysplastic or non-dysplastic samples, suggesting the latter lesions are clonally independent from each other. In accordance with previous reports, TP53 mutations were found in 21/21 cancer samples and also found in dysplastic (11/12) and non-dysplastic samples at a lower frequency (26/67). Strikingly, non-dysplastic samples harbored a very high frequency of NOTCH1 mutations (51/67), which were also found in cancer (3/21) and non-dysplastic (8/12) samples but at much lower frequencies (P=6.6×10-7). TS of validation samples confirmed the trend of higher NOTCH1 (84% vs. 25%) and lower TP53 mutation rates (38% vs. 100%) in non-dysplastic samples compared to cancer samples. The number of mutations in non-dysplastic samples was higher in drinkers than non-drinkers. Multiple NOTCH1 mutations were more common in cancer patients and drinkers than non-drinkers.

Conclusion

Clonal proliferation in non-cancer esophageal epithelia is common even in non-ESCC cases and extensive in ESCC cases. NOTCH1 and TP53 mutations play major roles in clonal evolution in common but may have differential impacts on esophageal carcinogenesis, which is likely to be shaped by APOBEC-induced mutations and CNAs.

#165

Single-cell transcriptome analysis guides tailored combinatorial therapeutics in refractory kidney cancer.

Kyu-Tae Kim,1 Hye Won Lee,2 Hae-Ock Lee,1 Hye Jin Song,3 Da Eun Jeong,4 Sang Shin,4 Hyunho Kim,5 Yoojin Shin,5 Do-Hyun Nam,6 Byong Chang Jeong,7 David G. Kirsch,8 Kyeung Min Joo,9 Woong-Yang Park1. 1 _Samsung Genome Institution, Seoul, Republic of Korea;_ 2 _Institute for Future Medicine, Samsung Medical Center, Seoul, Republic of Korea;_ 3 _Departments of Anatomy and Cell Biology, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 4 _Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences & Technology, Seoul, Republic of Korea; _5 _School of Mechanical Engineering, Korea University, Seoul, Republic of Korea;_ 6 _Departments of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 7 _Departments of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 8 _Departments of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC;_ 9 _Departments of Anatomy and Cell Biology, Sungkyun, Seoul, Republic of Korea_.

Metastatic renal cell carcinoma (mRCC) evolves from primary RCC (pRCC) and harbors multiple subpopulations with distinct molecular and phenotypic features. Such underlying intratumoral heterogeneity (ITH) imposes difficulties in designing marker-based clinical trials because targeted mono-therapy eliminates a specific subpopulation of tumor cells while leaving others unharmed. Accordingly, a rational combination strategy that minimizes the survival of the drug-resistant subpopulation in a given heterogeneous tumor is essential for long-term therapeutic efficacy. Here, we examined the ITH of a paired mRCC and pRCC using single-cell mRNA sequencing (scRNA-seq) to identify specific tumor cell populations with drug target pathway activation. From the single cell expression profiles, we found the highly activated status of the EGFR and Src signaling pathways in the mRCC compared to the pRCC, with supporting in vitro high-throughput drug screening results. Distinct features of intratumoral expression variability across mRCC single cells that were masked in the bulk measurement prompted us to test the co-targeting strategy for the EGFR and Src pathways with increased likelihood for complete response. This combinatorial strategy showed significantly better treatment effects on mRCC-derived xenograft platforms in vitro and in vivo than monotherapies. Taken together, our findings show clinical implications of scRNA-seq in designing effective treatment regimens for overcoming treatment failure to conventional monotherapies, and also provide novel insights to the unmet clinical needs in effective personalized treatments.

#166

Optimizing the detection of subclonal somatic variants with VarDict.

Zhongwu Lai,1 Brad Chapman,2 Miika Ahdesmäki,3 Oliver Hofmann,4 Justin Johnson,1 Jonathan Dry1. 1 _AstraZeneca, Waltham, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _AstraZeneca, Cambridge, United Kingdom;_ 4 _University of Glasgow, Glasgow, United Kingdom_.

Detection of somatic variants in tumor tissues is far from a solved problem. Heterogenous tumor materials complicate variant assessment in low frequency subclonal populations, which can play an important role in the development of drug resistance. Loss of heterozygosity, copy number variation and other ploidy changes can further change the expected distribution of variant metrics.

We demonstrate improvements in the specificity and sensitivity of variant calling algorithms by optimising linear combinations of widely used filtering criteria such as allelic frequency, depth and p-value distributions. We specifically improve SNP and indels from the freely available VarDict caller (https://github.com/AstraZeneca-NGS/VarDictJava) and show that these optimisations can be generalised across a wide range of allelic frequencies. We validate our findings against synthetic and in vitro standards such as the ICGC-TCGA DREAM challenge truth set. Our proposed filtering strategy is widely applicable to other variant callers, and the updated filters for VarDict dramatically improve sensitivity and precision on low frequency variants which are crucial for our ability to reconstruct likely tumor populations from short read sequencing data and to study tumor evolution.

#167

Inferring subclonal relationships between multiple metastases from rapid autopsy of a single melanoma patient.

Soroush Samadian, Prashant Bavi, Dianne Chadwick, Arnavaz Danesh, Mark Dowar, Tiantian Li, Madura Siva, Michael Roehl, Anthony m. Joshua, Trevor J. Pugh. _Princess Margaret Cancer Center, Toronto, Ontario, Canada_.

Background: Despite recent advances in personalized cancer therapeutics, little impact has been made on curing advanced melanomas over the last several decades. Patients with metastatic melanoma generally have a poor prognosis; survival is limited and typically the 5-year survival rate is less than 15% in patients with metastatic disease. Dysregulation of BRAF signaling has been shown to be one of these key drivers of the pathogenesis of disease in a large subpopulation (~40%) of patients. Although targeted therapy with BRAF and MEK inhibitors is the mainstay of therapy for recurrent/metastatic melanomas, this treatment modality provides a temporary respite. Often resistant tumor clones(carrying mutations in oncogenes) emerge, followed by a rapid progression, causing the patients to succumb to the disease. As such, in this study, we aim to understand inter- and intra- tumor heterogeneity that leads to resistance to therapy.

Methods: We conducted deep exome sequence analysis (250X coverage) of a collection of 8 metastatic melanoma tumor samples collected by rapid autopsy from a patient who became resistant to Vemurafinib despite harboring sensitizing BRAF p. V600E mutations. We used whole exome sequence analysis to detect somatic mutations and copy number variants in every metastatic tumor. To infer subclonal populations present within each metastasis, we applied a combination of clonal inference tools to determine subclonal structures based on somatic point mutations (PyClone) and copy number alterations (TITAN). Subsequently, we applied clustering algorithms to the inferred populations to construct inter-tumor phylogenies.

Results: We found that 89% of somatic point mutations were shared across all metastatic sites and were primarily clonal. Conversely, the cellular prevalence inferred from copy number alterations showed a greater degree of subclonal evolution. We found a clear relationship between the physical locations of tumors and shared subclonal patterns, whereby metastases from lung, brain and omentum metastases clustered distinctly from those in rectum, small intestine, peritoneum, diaphragm and mesentery. To look for shared patterns of subclonal variation underlying metastatic disease and resistance to therapy, we are currently extending our analysis to an additional six patients, each with tissues from 7-9 metastatic sites collected by rapid autopsy.

Conclusion: Our analysis of multiple metastatic sites from a single patient by deep exome sequence analysis found most somatic mutations were shared across tumor sites and subclonal evolution appears to be driven primarily by acquisition of copy number alterations. Tumors in near proximity shared greater genomic similarity compare to distant sites. Ongoing analysis of additional patients is required to uncover recurrent mechanisms of resistance to targeted therapies in this disease.

#168

Sample handling impacts genomic heterogeneity assessments and transcriptomic analyses in blood samples.

Emily A. Stevens,1 Lan W. Beppu,2 Zaneta J. Holman,2 Jordan L. Smith,2 Brent Wood,3 Jerald P. Radich,2 Janis L. Abkowitz,3 H. Joachim Deeg,1 Amy L. Paguirigan2. 1 _Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of Washington, Seattle, WA_.

We aimed to assess the degree to which common hematopoietic sample handling and nucleic acid processing can modify the results of transcriptomic and genetic heterogeneity studies, and ultimately obscure the underlying biology.

Given the numerous cell types in the hematopoietic system and their variable longevity, it is critical for accurate results to ensure similar cell populations are compared between patients, and that the presumed disease lineage is included in the sample. Through a series of paired samples, we have begun to investigate the potential impact that sample processing, storage, and nucleic acid extraction may have on data analysis, particularly in myeloid disease studies. Peripheral blood and bone marrow samples from normal donors, patients with idiopathic cytopenia of undetermined significance, myelodysplastic syndrome, and acute myeloid leukemia were included in this study. Conditions tested included fresh vs residual clinical specimens, whole blood vs density gradient separation, cryogenic storage, flow cytometric sorting, and column- or Trizol- based nucleic acid extraction methods. RNA sequencing and targeted DNA sequencing were performed on the resulting extracted nucleic acids and we characterized the results from the various populations and processing types.

Current bioinformatic approaches to describe clonality in whole blood/marrow samples require a number of assumptions that may not be true across all hematopoietic disease. Our results suggest that using density gradient based methods or flow cytometry sorting leads to significant differences in the apparent clonal composition, including number of predicted clones (i.e. clusters of variants at similar allele frequency) and the specific clonal identity. Our data suggest experimentally validated methods are needed to deconvolute the clonal heterogeneity in whole blood and marrow samples.

An example of the impact of sample type/processing on gene expression is the result comparing two patient cohorts via two blood collection types from each person at the same timepoint. Using a false-discovery rate cutoff of 1%, we identified 543 genes differentially regulated between the two groups using mononuclear cell fractions and 528 for PAXgene tubes. However, only 59 genes were common in both sets, highlighting the impact sample type has on observed expression differences.

Clearly, if we are to improve our ability to identify biologically relevant genes that are differentially expressed or concurrently mutated in clones in heterogeneous hematological malignancies, additional understanding of the underlying population compositions will be critical. Identifying effects of sample processing, storage, and nucleic acid extraction on results of genetic and genomic studies will also improve reproducibility. We will use the results here to ensure optimal patient sample processing for our upcoming translational projects.

#169

Drug resistance gene expression pathways in primary refractory breast cancer.

Charlene Kan, Shikha Parsai, Stefanie Avril, Janice Lyons, Hannah Gilmore, Junran Zhang, Lyndsay Harris. _University Hospitals Seidman Cancer Center, Cleveland, OH_.

Purpose/Objective:

Breast cancer is a collection of diseases with distinct molecular subtypes with differing prognoses. Clinically, there is a group of therapy resistant breast cancers characterized by resistance to conventional chemo- and radiotherapy with short overall survival and poor prognosis, which we termed primary refractory breast cancer (PRBC). We hypothesized that PRBC is characterized by specific genomic changes that may contribute to therapy resistance. The aim of this study was to identify changes in gene expression profiles of PRBC compared to triple negative (TNBC) and ER+/-PR positive (HR) breast cancer cohorts.

Methods:

PRBC was defined as patients with an initial diagnosis of Stage I-III breast cancer who either I) developed metastatic disease within 6 months of completing treatment or II) died of breast cancer within two years of initial diagnosis. Formalin-fixed paraffin-embedded (FFPE) tissue was obtained under an IRB approved protocol 01-13-43c. RNA was extracted from macro-dissected FFPE tumor tissue. Gene expression profiling was performed with Affymetrix Human Transcriptome Array 2.0 and gene signatures were analyzed using a single sample gene set enrichment methodology. Gene signatures of PRBC patients were compared to signatures of two TNBC and HR cohorts with gene expression data generated by identical methodology.

Results:

A total of nineteen samples from eight patients had sufficient tumor material to obtain RNA. The median age at diagnosis was 44.3 years, 62.5% were triple negative. At initial diagnosis the stage ranged from IA to IIIC. Sixty-three percent received neoadjuvant chemotherapy. The median time to relapse was 8.4 months and median time to death 1.7 years. Genomic analysis identified highly significant (p<10-6) increased expression of ABC transporters, hypoxia, interferon, DNA repair and AKT signatures compared to control cohorts.

Conclusions:

PRBC is characterized by significant upregulation of multiple gene expression signatures important in drug resistance including ABC transporters, hypoxia, AKT and DNA repair pathways. Confirmation of these results would allow the development of alternative treatment strategies for PRBC to help improve the devastatingly poor prognosis of this subgroup of patients.

#170

Dissecting components of intercellular communication in a heterogeneous tumor.

Balaji Santhanam, Yee Him Cheung, Vartika Agrawal, Konstantin Volyanskyy, Nevenka Dimitrova. _Philips Research North America, Cambridge, MA_.

Background: Recent advancements in single-cell sequencing technologies have enabled the study of complex biological systems such as tumors. They provide quantification of the extent and nature of the underlying heterogeneity and its impact on the disparate levels of response to therapy. The roles of receptor-ligand signaling in intercellular communication during embryogenesis and, in multicellular development and function is fairly well understood. Intercellular communication within the heterogeneous tumor mass, may proceed through the coordinated action of ligands and their cognate receptors. However, this phenomenon has not been explored in detail, especially in the context of tumor heterogeneity. We propose that a systems-level understanding of ligand- and receptor-states would shed novel insight in to dysregulated pathways in tumorigenesis and perhaps treatment.

Data: By performing single-cell RNA sequencing (RNA-seq) on freshly resected and dissociated primary glioblastomas (GBMs), Bradley Bernstein and colleagues (Patel AP et al, Science 2014) provided novel insights into the underlying heterogeneity at the molecular level. We used the data obtained from 430 cells analyzed by single-cell RNA-seq from five patient samples. The data were mean-centered, log-transformed and included about 6000 genes.

Methods: For our analysis we relied on a comprehensive list of ligand-receptor pairs from Ramilowski JA et al (Nature Communications 2015) and used 137 pairs for which data was available. Based on levels of their expression, we quantized ligand-receptor states and categorized each pair into one of four states. We adopted an unsupervised approach using hierarchical clustering to identify tightly grouped clusters. In order to test for the robustness of the clustering, we used multiscale bootstrapping and identified groups with ITGB1, LRP1, CD44, EGFR as high confidence clusters. We performed these analyses both globally and on individual samples to account for patient genotype differences.

Results: We observed that most of the tightly grouped clusters were ligand-receptor pairs that shared the same receptor. Global clustering revealed enrichment of receptors (ITGB1, ITGA5, EGFR and FGFR1) in pathways relating to signal transduction of L1 and L1CAM interactions. LRP1 and ITGB1 clusters were found in all five samples and pathways relating to hemostasis and extracellular matrix organization, respectively, were enriched.

Discussion: Bernstein et al observed that several receptors and ligands associated with GBM showed mosaic expression even among cells within a given patient sample. We broadened the scope and specifically posed the hypothesis that networks of ligands and receptors are involved in intercellular communication critical to tumor progression and pathogenesis. These molecules may also serve as good therapy targets and more detailed analyses is required to explore this possibility.

#171

Comparative genomic analyses of synchronous colorectal cancers by exome sequencing.

Ulrika A. Hänninen,1 Tomas Tanskanen,1 Riku Katainen,1 Jan Böhm,2 Minna Taipale,3 Jussi Taipale,3 Jukka-Pekka Mecklin,4 Netta Mäkinen,1 Lauri A. Aaltonen1. 1 _Genome-Scale Biology Research Program and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland;_ 2 _Department of Pathology, Jyväskylä Central Hospital, University of Eastern Finland, Jyväskylä, Finland;_ 3 _Genome-Scale Biology Research Program, University of Helsinki, Helsinki, Finland and Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden;_ 4 _Department of Surgery, Jyväskylä Central Hospital, University of Eastern Finland, Jyväskylä, Finland_.

Colorectal cancer (CRC) is the third most common cancer type and the second leading cause for cancer deaths in the Western world. Around 3.5% of CRC patients have more than one primary colorectal carcinoma at the time of diagnosis. These so-called synchronous colorectal cancers (SCRC) are more common in men and have been associated with predisposing conditions, such as hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatosis polyposis (FAP) and inflammatory bowel disease. Such conditions account for slightly more than 10% of SCRC cases. The pathological and clinical features of the tumors have been widely studied. The underlying molecular mechanisms in SCRC have not been well described, though. The research has mainly focused on single factors such as microsatellite instability (MSI) status or APC-, KRAS-, and p53-mutations.

By exome sequencing a tumor pair and corresponding normal sample from 23 patients, we aim to study the extent of genetic overlap between the synchronous tumors to assess if they derive from a common progenitor. The tumor data will be filtered against the normal tissue data to verify the somatic origin of the observed mutations. The comparison of the mutation profiles in each tumor pair will take into account the number of shared variants that occur in the exact same positions in the coding region. If the tumors share a high number of changes in exactly the same base it would suggest they have a common origin. Different mutation patterns, with possibly a few coincidental shared mutations, may support a stochastic process in the genesis of the two tumors. We will also identify commonly mutated genes - those that are mutated in both tumors of a synchronous pair or among synchronous pairs of different patients.

The patient material derives mainly from a consecutive, population-based colorectal cancer collection "Suolisyöpä Keski-Suomi 2000-2010" from Jyväskylä, Finland. In addition three SCRC-cases were included from our in-house collections. Knowledge on the molecular basis of SCRC, which haven't been studied to this extent before, provides insight on how these tumors arise and allows for development of more personalized treatments.

#172

Managing metastatic breast cancer via serial monitoring with circulating cell-free tumor DNA next generation sequencing testing.

Laura Austin,1 Rebecca Nagy,2 Oliver Zill,2 Richard B. Lanman,2 AmirAli Talasaz,2 Massimo Cristofanilli3. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Guardant Health, Redwood City, CA;_ 3 _Northwestern University, Chicago, IL_.

Background: Metastatic breast cancer (MBC) is an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Patients with treatment-refractory MBC may benefit from tumor genomic evaluation using next generation sequencing (NGS). Furthermore, analysis of circulating tumor DNA (ctDNA) in patients with advanced disease offers the possibility of non-invasive molecular monitoring.

Methods: A patient with MBC was tested at each progression with a ctDNA NGS panel (Guardant360™) that includes all NCCN-recommended somatic genomic variants for solid tumors and sequences complete exons of >50 genes to report single nucleotide variants (SNVs), fusions, amplifications, and indels with high sensitivity and ultra-high specificity (>99.9999%). The patient was diagnosed with invasive breast cancer at age 44 and treated with surgery and hormonal therapy. At age 61, she had axillary adenopathy and liver metastases. Treatment details are in Table 1.

Results: ctDNA analysis was performed at the time of metastatic diagnosis and at 5 additional time points over the course of treatment. All samples revealed an ERBB2 exon 19 indel (p.Leu755_Glu757delinsSer), and multiple SNVs and gene amplifications. ERBB2 amplification was seen in 4 of 6 samples. Mutant allele fractions (Table 1) correlated with clinical response to treatment and progression.

Conclusions: Analysis of ctDNA in this patient identified an ERBB2 exon 19 indel, which are present in 2-4% of non-small cell lung cancers but 1-2% in breast cancer. Treatment with anti-HER2 monoclonal antibody or dual anti-EGFR/ERBB2 tyrosine kinase inhibitor therapies may show clinical benefit. ctDNA analysis can detect emergence of actionable resistance mutations with the advantage of serial evaluation, allowing capture of inter- and intra-tumor heterogeneity and illustration of molecular progression and response.

Table 1.

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Blood Draw | Disease status at time of blood draw | Mutant allele fraction of ERBB2 indel (* = ERBB2 amplification) | Treatment

1 | Initial diagnosis | 9% | trastuzumab, pertuzumab and docetaxel (TPD), cycle 1

2 | Stable disease | 1%* | TPD cycle 2

3 | Progressing | 10%* | TPD cycle 3

4 | Progressing | 62%* | trastuzumab, emtansine (TDM1) 3 cycles

5 | Progressing | 70%* | vinorelbine, trastuzumab, everolimus

6 | Partial response | 1% | flourouracil, epirubicin, cyclophoasphamide

#173

Whole exome analysis of HER-2 positive human breast cancers: Molecular mechanisms underlying response to neoadjuvant therapy with trastuzumab.

Marco La Ferla,1 Paolo Aretini,1 Cristian Scatena,2 Michele Menicagli,1 Francesca Lessi,1 Sara Franceschi,1 Luca Cantini,3 Generoso Bevilacqua,4 Antonio Giuseppe Naccarato,4 Andrea Fontana,3 Chiara Maria Mazzanti1. 1 _Fondazione Pisana per la Scienza - ONLUS, Pisa, Italy;_ 2 _Division of Pathology - Dept of Laboratory Medicine University Hospital of Pisa, Pisa, Italy;_ 3 _University Medical Oncology Unit II, Azienda Ospedaliero-Universitaria Pisana (AOUP), Pisa, Italy;_ 4 _Division of Pathology, University Hospital of Pisa, Pisa, Italy_.

Background: HER2 receptor is overexpressed in 20-30 % of BC and it has been chosen to be an effective therapeutic target. Trastuzumab (Herceptin), a humanized monoclonal antibody, has been approved by FDA to be anti-HER2 therapy, but the mechanism by which Trastuzumab exerts its antitumor activity is not fully understood. Despite that almost 50% of HER2 Positive BC patients do not respond to Trastuzumab based therapy or become resistant during the therapy. An understanding of Trastuzumab resistance mechanisms would be a helpful tool in the development of rational drug combinations to circumvent resistance and allow better selection of patients likely to respond. The aim of this study is the correlation of the mutational profile of HER-2 positive patients with complete response and partial response to the neoadjuvant therapy with Trastuzumab and evaluation of possible molecular factors predictive of response.

Matherials and Methods: We perfomed, so far, whole-exome DNA sequencing (Ion Proton system) to analyze primary FFPE biopsy and primary surgical removed tumors of HER2 positive BC patients specifically selected for different response to the neoadjuvant therapy with trastuzumab and all ER and PR negative: 3 Full Responders (FR) with a complete disappearance of the tumor mass after the therapy and 2 Partial Responders (PR) with a partial shrinkage of the tumor mass, which is then removed by surgery. More patient samples are in the process to be analyzed by whole exome analysis for a total of 20 patients (10 each group).

Results: We identified gene mutations in SYNE2, ANKRD44, CEP350 and FAM175a genes present in the biopsy of PR patients and their presence rise in the post therapy samples. Interestingly these mutations rise in the post therapy samples while are nearly absent in the FR patients. On the other side we found some variants variants in MACC1, MAPK1, MAPK1, BEST3, COL21a1, LARP1B and MYOM1 genes present in the FR samples and absent in the PR samples. Thanks to literature analysis we found that some of these variants are already associated with Trastuzumab resistance and sensitivity.

Conclusions: We found that some genes have a reduction/disappearance or an increase of the mutant allele after treatment. Our findings indicate a possible involvement of a combination of gene mutations associated with resistance to the Trastuzumab therapy for the PR patients. The final gold is the creation of a gene panel implicated in resistance and/or in the complete response to Trastuzumab. Moreover other genes which resulted differentially expressed between PR and FR HER2+ patients are in the process of being investigated. However functional studies, as well as association studies in a larger patient dataset are needed to explore our hypothesis.

#174

Isolation of primary human tumor cells significantly reduces bias in molecular analysis and improves culture of target cells.

Lena Willnow,1 Stefan Tomiuk,1 Jutta Kollet,1 Stefan Wild,1 Silvia Rüberg,1 Claudius Fridrich,2 Peter Mallmann,2 Frauke Alves,3 Philipp Ströbel,3 Dominik Eckardt,1 Andreas Bosio,1 Olaf Hardt1. 1 _MILTENYI BIOTEC GMBH, BERGISCH GLADBACH, Germany;_ 2 _University Hospital Cologne, Cologne, Germany;_ 3 _University Medical Center Göttingen, Göttingen, Germany_.

Solid tumors are infiltrated by cells of non-tumor origin, including heterogeneous lymphocyte subpopulations, fibroblasts, and endothelial cells. The amount and composition of infiltrating cells is highly variable and patient dependent, which makes analyses of primary tumor samples difficult. The contaminating cells lead to hybridization of non-tumor cell derived mRNA molecules to probes on microarrays and a significant reduction of sensitivity caused by measurement of irrelevant signals during next-generation sequencing or proteome analysis can be expected. In addition, the culture of human tumor cells is frequently hampered by fibroblasts overgrowing the target cells, which bias assays such as drug sensitivity tests. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from primary tissue. This procedure is based on the comprehensive depletion of cells of non-tumor origin by combining automated tissue dissociation and magnetic cell sorting (MACS). A negative selection strategy enables the isolation of the tumor cell population without knowledge of surface protein expression on these cells. Even tissues that initially contain low numbers of tumor cells (< 20%) reach purities of higher than 95% in less than 20 minutes. Here, we have applied this method to isolate human tumor cells from primary and metastatic ovarian carcinoma and thymoma specimens. The purified human ovarian carcinoma tumor cell fraction was further used for the isolation of CD133+ cancer stem cells. Cultivation of human tumor cells was more consistent with prior removal of contaminating stromal cells than without. Whole genome expression profiling of bulk human tumor tissue and matched purified tumor cells showed a significant reduction in stroma specific gene expression signatures while tumor specific signatures were enriched. Moreover, the comparison of expression profiles of isolated cancer stem cells to their parent population of purified tumor cells indicate that different tumor cell subpopulations could be characterized more precisely than if the unpurified fraction was used as reference. This suggests a reduced risk of misinterpretation when only purified tumor cells are evaluated or used as a control for smaller subpopulations. Whole exome sequencing of the same samples further verified the benefit of tumor cell purification from stromal contamination. The sequence read heterogeneity was dramatically reduced in the purified

samples. The reduction of non-tumor cell derived DNA, therefore, led to the detection of a higher number of tumor specific SNPs and INDELS, and consequently in a higher confidence in the results, in particular for mutations only present in a subset of tumor cells. Taken together, removal of non-tumor cells strongly improves their subsequent culture and molecular analysis of primary human tumor tissue.

#175

Transcriptomic and epigenetic dynamics of breast cancer progression in the MMTV-PyMT mouse model.

Ying Cai, Ruben Nogales-Cadenas, Quanwei Zhang, Jhih-rong Lin, Wen Zhang, Zhengdong Zhang. _AECOM, bronx, NY_.

Using RNA-sequencing and ERRBS (enhanced reduced representation bisulphite sequencing), we identified genes involved in breast cancer malignant progression.

Human breast cancers have complex molecular mechanisms of progression and metastasis. To improve diagnosis and drug development, it is critical to identify the genes and molecular pathways involved in tumor progression and malignant transition. Using the polyoma middle T antigen (PyMT) mouse model, we profiled gene expression from four different stages of tumor progression, including hyperplasia, adenoma/MIN (mammary intraepithelial neoplasia), early and late carcinoma. We identified a large number of differentially expressed genes in tumors compared with normal mammary gland, enriched in E2F targets and G2M checkpoint genes. Using the temporal information, we found Myc, Sox4 and Casp9 were associated with tumor progression. Using co-expression network analysis, we identified signature modules of panels of genes, revealing novel insights into molecular mechanisms of breast tumorigenesis. For example, Mcm6 and Ncapd2, hub genes in a DNA replication and cell cycle module, predict metastasis in human samples. We also found the Hells, Hmgb2 and Cit genes to be increased in expression in both mouse and human TCGA (The Cancer Genome Atlas) datasets.

We complemented these studies by profiling DNA methylation using ERRBS in the same samples. We identified a large number of differentially methylated cytosines (DMCs), enriched in cis-regulatory elements as well as near genes in cancer-related pathways. A small subset of the DMCs was well conserved between the progressive stages of tumorigenesis, with the greatest shift occurring with the transition to malignancy. We also observed a global hypomethylation and a shift towards a subset of loci with hypermethylation with tumor progression. Expression of some tricarboxylic acid (TCA) cycle genes was negatively correlated with DNA methylation, which may reflect altered metabolic regulation in carcinoma. We then constructed a co-methylation network and discovered a module with decreased DNA methylation in tumor that was enriched with genes from WNT and MAPK signaling pathways. In addition, we compared our results to the human TCGA breast carcinoma dataset and found the epigenetic modules to be overall distinctive between the two species.

Altogether, our gene expression, DNA methylation and network analyses with the PyMT mouse model shed new light on transcriptional and epigenetic dynamics during breast cancer malignant progression and reveal gene modules that may regulate this biological process.

### Kinases and Phosphatases

#176

Development of 1- and 3-phosphohistidine monoclonal antibodies utilized as novel tools to study histidine phosphorylation.

Wayne A. Speckmann, Mark Santos, Reshmie Shea, Sheri Geiman, Arazo Saadat, James Hoberg, Wei Zheng. _EMD Millipore, Temecula, CA_.

Phosphorylation of proteins is one of the most common forms of post-translational modification (PTMs) and is a mechanism for regulating enzyme activity or protein function. There are several amino acids that can be phosphorylated but despite its importance in cancer and tumor metastasis histidine phosphorylation (pHis) has been highly overlooked. One of the key reasons is the inherent challenge associated with studying pHis legitimately, due largely in part to the lack in availability of specific antibodies. Unlike serine, threonine, and tyrosine which all form acid-stable phosphoester (P-O) bonds upon phosphorylation, histidine forms heat and acid-labile phosphoramidate (P-N) bonds. Because of the instability of the P-N bond, it is nearly impossible to assess the prevalence of pHis accurately.

Here, we outline the development of selective, sequence independent anti-1-pHis and anti-3-pHis monoclonal antibodies which have addressed this current need for specific antibodies to detect the phosphorylation of histidine. The antibodies developed specifically recognize 1-pHis and 3-pHis and are isomer specific. In addition, they do not cross-react with phospho-tyrosine (pTyr), whereas, the first and second generation "pan-pHis" polyclonal antibodies against 3-pHis have been reported to do so. Moreover, it has been suggested that pHis could be as abundant as pTyr, comprising of approximately 1% of all known phosphorylation in cells, making specific monoclonal antibodies for detection highly desirable.

Validation data of the four monoclonal phospho-Histidine antibodies developed (clones SC50-3 and SC1-1 for assessment of 1-pHis activities; clones SC56-2 and SC39-6 for assessment of 3-pHis activities) demonstrate the specificity and broad utility of these monoclonal antibodies when identifying pHis substrates, making them useful tools in a variety of immunological and biological assays.

#177

Src family kinase Fyn promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer.

Karen Cavassani, Murali Gururajan, Margarit Sievert, Peng Duan, Michael Freeman, Gina Chia-Yi Chu, Neil Bhowmick, Leland Chung, Edward Posadas. _Cedars Sinai Medical Center, Los Angeles, CA_.

FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PCa cells lines treated with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients.

#178

PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer.

Fara Braso-Maristany,1 Simone Filosto,1 Steven Catchpole,1 Rebecca Marlow,1 Jelmar Quist,1 Erika Francesch Domenech,1 Gianmaria Liccardi,2 Leila Zakka,1 Violeta Serra,3 Albert Gris,3 Maggie Cheang,2 Nirmesh Patel,1 Anna Perdrix Rosell,1 Patrycja Gazinska,1 Elodie Noel,1 Jonathan Watkins,1 Pierfrancesco Marra,1 Anita Grigoriadis,1 Andrew Tutt1. 1 _King's College London, London, United Kingdom;_ 2 _The Institute of Cancer Research, London, United Kingdom;_ 3 _Vall d'Hebron Institute of Oncology, Barcelona, Spain_.

Triple-Negative Breast Cancers (TNBCs) are aggressive, associated with poor prognosis and lack targeted therapies, mainly due to the absence of suitable molecular targets. The genomic region 6p21-p25 is recurrently amplified in TNBCs and encompasses the PIM1 oncogene.

This study interrogated genomic breast cancer datasets and identified gene copy number-driven increase of PIM1 expression in TNBC. To understand the role of PIM1 in malignant phenotypes of TNBCs, functional studies were carried out in breast cancer and non-malignant mammary epithelial cell line models. RNA interference and rescue-of-function experiments revealed addiction to PIM1 kinase for cell population growth and apoptosis protection in TNBC cells, absent in non-malignant cells. In cells sensitive to PIM1 knockdown, we observed a subsequent reduction of the expression of the anti-apoptotic proteins BCL2 and MCL1. Moreover, exogenous expression of BCL2 prevented apoptosis caused by PIM1 knockdown. BH3-profiling analysis further confirmed that PIM1 blocks apoptosis elicited through the mitochondrial pathway in TNBC cell lines. The activity of PIM1 in other cancers has been closely linked to the regulation of c-MYC, an "un-targetable" oncogene that drives malignancy and that is frequently amplified and highly expressed in primary TNBCs and post-chemotherapy residual disease. Importantly, we found that PIM1 expression associates with several MYC-transcriptional signatures in TNBCs and mediates phosphorylation of c-MYC Ser62 and Histone-H3 Ser10, key targets for MYC-driven transcription. Exogenous expression of MYC rescued the phenotypes caused by PIM1 knockdown, providing mechanistic insights for the observed addiction. Finally, the therapeutic potential of targeting PIM1 was assessed using AZD1208, a small molecule inhibitor of the PIM kinase family. AZD1208 inhibited growth and sensitized TNBC derived cell lines, cell line xenografts and PDXs to chemotherapy by increasing apoptosis.

We therefore identify PIM1 as a driver of malignancy in TNBC, illustrating relationships with MYC-activation and regulation of anti-apoptotic BCL2 and MCL1 proteins. PIM1 inhibitors could provide a potential therapeutic to abrogate chemotherapy resistance in TNBCs.

#179

Atypical protein kinase C inhibition in prostate cancer cells: a study of ICA-1 and ACPD.

Andre H. Apostolatos. _University of South Florida, Tampa, FL_.

Aggressive and metastatic prostate cancers are highly lethal tumors and are one of the leading causes of death among men. Certain prostate cancers are highly lethal tumors due to the emergence of therapy-resistant cancer cells. There is a gap in knowledge relating to which intracellular chemicals confer the emergence of therapy-resistant prostate cancer cells, which intracellular chemicals are responsible for prostate cancer survival and which chemicals regulate intracellular signaling pathways in prostate cancer. To address this critical problem, we investigated two drugs that act as inhibitors of atypical Protein Kinase C Iota. The objective of this study was to test the effectiveness of ACPD as an atypical PKC inhibitor and ICA-1 as an inhibitor of PKC iota. [The Inter. J. Biochem. & Cell Biol. 43:784-794(2011)] DU-145 and PC-3 prostate carcinoma cells and non-malignant prostate RWPE-1 cells were each cultivated in separate flasks. They were treated with drugs of interest for three consecutive days. The cells were counted before and after the treatments and a statistical analysis was performed. The cells were lysed and levels of PKC iota and PKC zeta were measured by Western blotting and immunoprecipitation. Our preliminary results show that treatment of DU-145 and PC-3 showed a statistically significant decrease in cells that was proportional to the concentration in both drugs. Treatment of the RWPE-1 cells showed no statistically significant change in population. The Western blotting showed both drugs decreased PKC iota. In conclusion ACPD and ICA1 are effective inhibitors of PKC iota and PKC zeta and consequently significantly reduced the neogenesis of the prostate DU-145 and PC-3 carcinoma cells while having negligible effect on the non-malignant prostate RWPE-1 cells.

#180

Identification and characterization of ATM substrates in mitosis.

Rebecca J. Boohaker, Qinghua Zeng, Bo Xu. _Southern Research, Birmingham, AL_.

The Ataxia-Telangiectasia Mutated (ATM) Kinase, mutation of which is responsible for the autosomal recessive disorder Ataxia-Telangiectasia, is an essential element for cells to deal with DNA double-strand breaks. In the absence of DNA damage, it has also been recognized that ATM has a role in optimizing the spindle assembly checkpoint during mitosis. In the process, ATM is activated at a lower level (compared to the DNA damage response) which requires phosphorylation by the Aurora-B kinase. Known ATM targets in mitosis include kinetochore proteins Bub1 and Mad1. However, it has become clear that ATM-mediated phosphorylation in mitosis is distinct from that in the DNA damage response. Therefore, we performed an unbiased assay to identify ATM substrates in mitosis in order to study the functional significance of mitotic ATM activation. To achieve our goal, we utilized a stable isotope labeling by amino acid (SILAC) /Peptide IP with phosphor-specific SQ/TQ antibodies and mass spectrometry (MS). About 18 proteins were identified as substrates of mitotically activated ATM in the assay. We then conducted bioinformatic analyses for the identified proteins using PANTHER and Ingenuity programs. With many proteins having their known functions in mitosis, we found a much smaller number, but highly interconnected, pool of proteins in the ATM substrate list as compared to that of the DNA damage response. Among them, we validated several phosphorylation events using an in vitro kinase assay. Furthermore we present data to demonstrate the functional significance of ATM-mediated phosphorylation. Taken together, our data provide a comprehensive picture of how mitotically activated ATM signals to downstream targets to fine-tune the mitotic checkpoint.

#181

Constitutive IRAK1/4 kinase activation contributes to NF-kB activity and chemoresistance in pancreatic cancer.

Daoxiang Zhang, Lin Li, Hongmei Jiang, Jinsheng Yu, Brett Knolhoff, Richard Head, Albert C. Lockhart, David G. DeNardo, Andrea Wang-Gillam, Marianna B. Ruzinova, Kian-Huat Lim. _Washington Unversity School of Medicine, St Louis, MO_.

Constitutive activation of the NF-κb transcription factor is a major molecular mechanism that contributes to the aggressive behavior and treatment resistance of pancreatic cancer. Understanding the molecular mechanisms that activate NF-κB will provide novel therapeutic opportunities to improve the dismal outcome of pancreatic cancer patients. In the present study, we showed that the Interleukin-1 Receptor-Associated Kinases 1 and 4 (IRAK1 and IRAK4) are constitutively activated in a majority of pancreatic cancer cell lines and patients samples, and are major drivers of NF-κB activity. Notably, we found that constitutive phosphorylation of IRAK4 is associated with poor patient prognosis. Suppression of IRAK1 and IRAK4 with small molecule inhibitor or RNA-interference in pancreatic cancer cells significantly reduces NF-κB activity, three-dimensional growth, invasiveness, production of inflammatory, and augment their sensitivity to chemotherapeutic agents in vitro. Notably, we showed that the NF-κB activity of pancreatic cancer cell with IRAK4 ablated using CRISPR technology can be restored with wild-type, but not kinase-dead IRAK4 mutant, supporting development of IRAK4 inhibitor as a novel therapeutic agent in pancreatic cancer. Silencing of IRAK1 or IRAK4, and more potently both, significantly abrogated the tumorigenic potential of human and murine pancreatic cancer cells as xenograft in mice. Lastly, we showed that IRAK1/4 inhibitor augments the therapeutic effect of gemcitabine in tumor-bearing mice by suppressing proliferation and increasing apoptosis of neoplastic cells, and reducing stromal fibrosis. Together, our data established IRAK4 kinase inhibitors as a promising novel class of targeted agent in pancreatic cancer.

#182

Dual inhibition of the MEK5 and PI3K pathways most effectively reduces proliferation and migration in triple negative breast cancer cells.

Thomas Wright, Christopher Raybuck, Katheryn Wendekier, Jane E. Cavanaugh. _Duquesne University School of Pharmacy, Pittsburgh, PA_.

Aberrations in the MAPK/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide-3-kinase (PI3K) pathways have been linked to increased proliferation and survival in triple negative breast cancer (TNBC) cells. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. Promising combinations of MEK and PI3K inhibition have been evaluated in phase I clinical trials for various cancer types. However, these clinical trials have had limited efficacy and have yet to encompass the MEK5/ERK5 pathway, which has been shown to promote cell survival. The goal of this study was to examine the crosstalk between the MEK1/2, MEK5, and PI3K pathways and determine the most promising combination of the MEK1/2, ERK5, and PI3K inhibitors, U0126, XMD8-92, and LY294002, respectively, in a diverse panel of triple negative breast cancer cell lines: BT549, MDA-MB-231, and MDA-MB-468. Our results indicate that dual inhibition of the MEK5 and PI3K pathways significantly reduced both proliferation (45.53%) and migration (39.37%) in MDA-MB-231 TNBC cells. In contrast, inhibition of ERK1/2 alone or in combination with PI3K or ERK5 inhibition yielded mixed responses. Interestingly, dual inhibition of the MEK5 and PI3K pathways increased the activity of the MEK1/2 pathway. These data suggest that crosstalk between these kinases occurs and dual inhibition of PI3K and ERK5 may be a novel therapeutic approach for treating TNBC.

#183

Inhibition of BRAF kinase alone in BRAF V600E-mutated undifferentiated thyroid carcinoma results in no growth arrest.

Md. Atiqur Rahman, Ali Salajegheh, Haleh Vosgha, Hamidreza Maroof, Robert A. Smith, Alfred KY Lam. _Griffith University, Gold Coast, QLD, Australia_.

Background: Undifferentiated thyroid carcinoma is the most aggressive type of thyroid carcinoma. It is refractory to most of the conventional therapies and its median overall survival is less than six months. Mutation in BRAF gene was found around 25% of undifferentiated thyroid carcinoma. Mutated BRAF particularly the V600E mutated BRAF auto-activates the BRAF-MEK proliferation-survival cell signalling pathway. The objectives of our current study were to inhibit the BRAF kinase and investigate its effect on undifferentiated thyroid carcinoma proliferation.

Materials and methods: 8050C, a homozygous BRAF V600E mutate undifferentiated thyroid carcinoma cell line, was used to develop anti-BRAF shRNA stable cell line. Quantitative real time polymerase chain reaction (qPCR) and western blot were performed for ratifying the BRAF inhibition efficiency of stable cell line. Proliferation and colony formation assay were done to see the effect of BRAF inhibition on undifferentiated thyroid carcinoma proliferation. In addition, impact of BRAF inhibition on proliferation cell signalling pathways were checked through western blot analysis.

Results: Efficiency of BRAF knockdown was around 87% which resulted in approximately 83% inhibition of BRAF kinase (p < 0.05). About 54% proliferation inhibition (p < 0.05) was observed in the BRAF inhibited group compare to control group in the first week. But the proliferation rate in the BRAF inhibited group increased over the subsequent weeks and we did not observed any sign of cell growth arrest. In addition, colony formation assay revealed approximately 13% reduction of colony formation ability (p < 0.05) in BRAF inhibited group compare to control group. Western blot analysis of signalling kinases discovered reduction of around 72% MEK kinase activity after BRAF kinase inhibition. As we observed increasing trend of proliferation after an initial sharp drop in cell proliferation after BRAF kinase inhibition, we hypothesized and analysed the alternative PI3K-AKT proliferation pathway. Surprisingly we found about 63% increase of AKT kinase activity in the BRAF inhibited group compare to control group (p < 0.05).

Conclusions: Inhibition of BRAF kinase reduced cell proliferation initially, but this proliferation inhibition did not lead to cell growth arrest at the end. Increased AKT kinase activity counteracted the effect of BRAF kinase inhibition as AKT kinase has the ability to contribute in overall cell proliferation. Therefore, both kinases need to be inhibited simultaneously for cell growth arrest in undifferentiated thyroid carcinoma.

#184

The interaction of TLK1 and NEK1 in Prostate cancer as key regulators of the DDR and in mitosis.

Zachary M. Connelly, Arrigo DeBenedetti. _LSUHSC Shreveport, Shreveport, LA_.

Background: The Tousled Like Kinases (TLKs) are involved in DNA repair, the DDR, and also in mitotic segregation. However, only a few substrates of TLK1 have been identified thus far, such that the mechanism of action of these kinases remains largely unknown. We have recently identified the proteome target of TLK1 that consists of 165 proteins identified with high confidence. Among these, stands out prominently NEK1, and member of the NIMA family of protein kinases that is specifically involved in the DDR and in mitotic segregation, making NEK1 and ideal target to explain several of the functions of TLK1. Specifically, NEK1 is involved in the early response of the DDR such that it regulates the activity of the effector kinases Chk1 and Chk2, while specific inhibitors of TLK1 lead to prolonged S phase arrest and Chk1 activation after treatment of cells with doxorubicin or hydroxyurea (HU). We have now begun to investigate the relationship between TLK1 and NEK1.

Results: The strong interaction between TLK1 and NEK1 was established by reciprocal coIP. We also found that the interaction is cell cycle regulated in DU145 cells, with strongest interaction in S and G2/M phases after synchronization of cells with HU. The expression of NEK1 in the most common human PCa cell lines was also established, and the protein was found to be variably expressed and phosphorylated. The activity of NEK1 was also found to be cell cycle regulated using immunoprecipitated NEK1 and casein as the substrate. Again maximal activity was found in S and phases of the cell cycle. The activity of NEK1 and TLK1 is regulated by the DDR, and NEK1 activity was increase after treatment of cells with doxorubicin. However, an inhibitor of TLK1 suppressed the increased activation, reinforcing a strong relation between TLK1 and NEK1 activity. Recombinant TLK1B stimulates the activity of recombinant NEK1 using casein as a substrate. Further suggesting that TLK1B is a regulator of NEK1 activity in cells. LNCaP cells when treated for 4 hours in charcoal stripped medium, have much higher NEK1 activity.

Conclusions: We have identified, first through a proteomic screen, a strong interaction between TLK1 and NEK1. The activity of the two proteins throughout the cell cycle and in response to DNA damage is exactly coregulated, and we propose that TLK1 is a direct regulator of NEK1 activity. TLK1 and NEK1 are highly expressed in PCa samples compared to normal prostate cells (Atlas of human cancers), and we propose a novel axis that is upregulated in PCa that leads to resistance to DNA damaging agents/treatments (including androgen deprivation) and to the induction of mitotic aberrations that are a common occurrence in cancer cells. Since TLK1B expression is induced by androgen deprivation (ADT), we suggest that NEK1 activity is also increased following ADT via the PI3K/AKT/ mTOR/eIF4E pathway.

#185

Role of phosphorylation in Lmtk3 activation and its contribution in breast cancer progression.

Giulia Lucchiari,1 Hua Zhang,1 Joao Nunes,1 Yichen Xu,1 Arnhild Grothey,2 Justin Stebbing,1 Georgios Giamas2. 1 _Imperial College London, London, United Kingdom;_ 2 _University of Sussex, Brighton, United Kingdom_.

LMTK3 is an oncogenic receptor tyrosine kinase implicated in various types of cancer including breast, lung, gastric and colorectal. We have already demonstrated the contribution of LMTK3 in invasion, migration and transcriptional regulation as well as its involvement in endocrine resistance in breast cancer. Despite the significant progress in understanding the role of LMTK3 in human tumourogenesis, the signalling pathways implicated in LMTK3 regulation still remain to be elucidated.

Protein phosphorylation play an essential role in regulating intracellular signal transduction pathways involving almost every aspect of cell activity. In silico phosphoproteomic analysis predicted several potential LMTK3 phosphorylation sites targeted by different kinases including Cyclin-dependent kinase 5 (CDK5), a cytoplasmic proline-directed serine/threonine kinase that is commonly overexpressed in many solid tumours. Interestingly, recent work performed by two different groups showed that CDK5 is a regulator of LMTK1 and LMTK2 in neuronal cells, resulting in axonal outgrowth and potentially influencing a number of neurophysiological processes.

By performing a variety of molecular/cellular and biochemical experiments we confirmed the ability of CDK5 to phosphorylate LMTK3 in vitro and identified its exact phosphorylation sites. Moreover, we investigated the consequences of CDK5 phosphorylation on various LMTK3 processes, amongst which its stability, sub-cellular localization, its ability to interact with chromatin and others. In addition, the clinical correlation of CDK5 and LMTK3 expression in cell lines and patients' samples were assessed. In aggregate, we describe a new cellular pathway encompassing CDK5 and LMTK3 that results in breast cancer tumour progression.

Our data will be presented.

#186

Functional characterization of ERK3 mutants existing in human cancers.

Hadel Alsaran, Lobna Elkhadragy, Weiwen Long. _Wright State University, Dayton, OH_.

Extracellular signal-Regulated Kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer, and plays an important role in promoting cancer migration and invasion. While the link between ERK3 and cancers has been recognized, little is known about ERK3 mutations in cancer progression. In this study, we have investigated ERK3 mutations on R64 and L290 that have been found in cancers of lung, large intestine and skin (COSMIC database). Importantly, mutations of R64C, R64H, L290P and L290V all are located in the kinase domain of ERK3. In order to characterize these mutations, we generated ERK3 mutants with each of these point mutations, and then overexpressed them in HeLa cells with or without stable knockdown of endogenous ERK3. Notably, we found that in comparison with wild type ERK3, all of these cancer-related mutations lead to reduction of phosphorylation at S189 within the activation loop (an indicator of ERK3 kinase activity). While activation loop phosphorylation is well known to result in activation of the classical MAPKs, its effects on the activity and functions of ERK3 remain elusive. To study the functional impact of these ERK3 mutations on cancer cell invasiveness, we performed trans-well migration assays using HeLa cells overexpressing each of these mutants or the wild type ERK3. In line with previous finding, overexpression of ERK3 increased cell migration. Interestingly, both L290P and L290V mutations significantly increased ERK3's ability in promoting cell migration, whereas R64C and R64H mutations resulted in a decrease in cell migration. These results suggest that S189 phosphorylation within the activation loop does not correlate with ERK3's function in promoting cancer cell motility. To conclude, we report for the first time the functional characterization of cancer-related ERK3 mutations in modulating cancer cell migratory properties.

#187

ASN0007, a potent ERK1/2 inhibitor with strong antitumor activity in multiple RAS mutant models.

Sanjeeva P. Reddy,1 Dhanalakshmi Sivanandhan,2 Raghava Reddy Kethiri,2 Sreekala Nair,2 Purushottam Dewang,2 Mohd Zainuddin,2 Krishnakumar Vaithilingam,2 Roger A. Smith,1 Sandeep Gupta,1 Scott K. Thompson1. 1 _Asana BioSciences, Bridgewater, NJ;_ 2 _Jubilant Biosys Ltd, Bangalore, India_.

The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), play a critical role in the RAS/RAF/MEK signaling pathway which is quite frequently activated by mutations in upstream targets such as BRAF, RAS and receptor tyrosine kinases. Most of the resistance mechanisms to BRAF and MEK inhibitors ultimately lead to increase in phosphorylation of ERK1/2 suggesting the importance of this node in the RAS/RAF/MEK pathway even in the resistance setting. Therefore, inhibition of ERK1/2 offers a promising strategy to address both innate and acquired resistance to BRAF and MEK inhibitors in various solid tumors. In order to inhibit this critical node of the RAS/RAF/MEK pathway, we discovered and characterized ASN007, a novel compound that shows potent inhibition of ERK1/2 kinases in a biochemical assay with low single-digit IC50 values. In mechanistic cell-based assays, ASN007 inhibited the phosphorylation of ERK1/2 substrates such as pRSK1, FRA1 and Elk1 in various cell lines. As expected, the compound did not have any effect on the phosphorylation of ERK1/2. ASN007 showed strong anti-proliferative activity in both BRAF and RAS mutant cell lines. Importantly, when compared to other ERK1/2 inhibitors such as BVD-523 and GDC-0994, ASN007 showed potent antiproliferative activity in a much broader panel of KRAS, NRAS and HRAS mutant cell lines representing various histological tumor types. The compound also caused cell cycle arrest at the G0-G1 phase and induced apoptosis in both BRAF and RAS mutant cell lines. In a daily oral dosing regimen, ASN007 demonstrated strong inhibition of tumor growth in multiple BRAF and KRAS mutant xenograft models in mice and was well tolerated at efficacious doses. ASN007 has a good pharmacokinetic profile in pre-clinical species and does not exhibit significant inhibition of CYP enzymes or hERG channel. Based on its profile in preclinical studies, ASN007 is expected to show strong efficacy in BRAF and RAS mutant cancers. ASN007 is currently in preclinical development.

#188

Isoform-specific AKT functions in thyroid carcinoma development.

Michela Ranieri, Antonio Di Cristofano. _Albert Einstein College of Medicine, Bronx, NY_.

Mammalian cells contain three genes that encode three AKT isoforms (AKT1-3). The three genes have >85% sequence identity, and possess conserved threonine and serine residues that are critical for full activation. All AKT isoforms seem to have identical or similar substrate specificity, and it is not clear whether they possess different functional specificities in vivo.

Using conditional, organ-specific isoform deletion in a model of PI3K-dependent neoplastic transformation and progression, we are dissecting the molecular and biological roles of AKT1 and AKT2.

Thyroid-specific deletion of Pten (Ptenthy-/- mice) results in thyroid hyperplasia at birth which then slowly progresses to solid nodular lesions and finally to invasive carcinomas.

Simultaneous thyroid-specific deletion of Akt1 and/or Akt2 demonstrates that both AKT1 and AKT2 are necessary to confer proliferative ability to Pten-/- thyrocytes in an additive manner. However, with age, (Pten,Akt1)thy-/- mice have a significantly higher incidence of thyroid lesions compared to (Pten,Akt2)thy-/- mice. In addition, deletion of Akt2 is more effective than that of Akt1 to extend survival and reduce lesion aggressiveness in Ptenthy-/-/KrasG12D mice.

Transcriptomic and proteomic analyses have revealed that AKT2 loss rescues most of the expression and activation changes induced, in vivo, by Pten deletion, while AKT1 loss has more restricted effects.

Futhermore we have identified and are characterizing subsets of genes that are differentially regulated by AKT isoforms.

These data point to the existence of dramatically different molecular pathways associated with each AKT isoform in the thyroid, and represent a strong rationale for the development of less toxic, more effective treatments based on isoform-specific inhibitors.

#189

Protein kinase C alpha suppresses AKT activation in endometrial cancer.

Alice H. Hsu,1 Kathryn J. Curry,2 Kang-Sup Shim,3 Peter Frederick,2 Carl D. Morrison,2 Baojiang Chen,1 Subodh M. Lele,1 Takiko Daikoku,4 Sudhansu K. Dey,5 Gustavo Leone,6 Adrian R. Black,1 Jennifer D. Black1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _Ohio State University, Ohio, OH;_ 4 _Kanazawa University, Kanazawa, Japan;_ 5 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH;_ 6 _Ohio State University, Columbus, OH_.

Endometrial cancer (EC) is the most common gynecological malignancy and the fourth leading cancer in women in the United States. Hyperactivation of the PI3K/AKT pathway is observed in ~90% of EC cases and this pathway is the most prominent driver of endometrial carcinogenesis. Mutations in multiple PI3K/AKT pathway components (including PTEN, PIK3CA, PIK3R1, AKT) often coexist in EC, indicating that EC development requires full, unopposed activation of PI3K/AKT signaling. In this study, we identified PKCα as a negative regulator of AKT activation that suppresses endometrial carcinogenesis. Our analysis revealed that PKCα protein and mRNA are reduced or lost in 50% of EC cell lines and ~60% of human ECs. PKCα deficiency was also observed in hyperplastic endometrial lesions in murine models with allelic knock-in of mutant Pten (ptenΔ4-5/+, ptenC124R/+ and ptenC129E/+) or endometrial-specific Pten deletion (ptenpr-/-, ptenltf-/-), indicating that loss of PKCα is an early event in endometrial carcinogenesis.

Mechanistic analysis has further revealed that loss of PKCα is independent of changes in PI3K/AKT signaling; however, the enzyme acts as a potent negative regulator of the PI3K/AKT pathway in EC cells. PKC agonists reduced AKT phosphorylation/activity in EC cells that retain PKCα, via a PP2A-dependent mechanism. Conversely, PKCα knockdown led to increased AKT phosphorylation. Notably, while PKC agonists did not affect AKT in EC cells with loss of PKCα, exogenous expression of the enzyme in these cells restored the inhibitory effect of the agonists on PI3K/AKT signaling. Collectively these data indicate that PKCα has a restraining effect on PI3K/AKT signaling in EC cells, providing a basis for its loss during endometrial tumor progression.

Physiological relevance of these findings was established using cell lines, patient samples and mouse models. PKCα restoration in human EC cells decreased expression of oncogenes such as cyclin D1 and Id1 and abolished their anchorage-independent growth, supporting a tumor suppressive role for the isozyme in this tissue. Analysis of PKCα expression in patient specimens determined that loss of PKCα expression correlates with high grade disease. Finally, in mice heterozygous for mutant Pten, PKCα knockout led to a 3-fold increase in endometrial tumor burden at three months, confirming a role of PKCα as a tumor suppressor in the endometrium.

Taken together, our results provide evidence that PKCα 1) negatively regulates AKT signaling and the expression of important oncogenes in EC cells; and 2) functions as tumor suppressor in the endometrium whose loss is associated with disease progression. Thus, PKCα signaling represents a promising biomarker for risk stratification in early stage disease and may provide insight into therapeutic strategies for late stage disease.

Supported by NIH grants CA036727, CA016056, and DK60632.

#190

**Mechanism of** CDKN3 **overexpression in cancer.**

Chao Fan,1 Lu Chen,1 Qingling Huang,1 Tao Shen,2 Eric A. Welsh,1 Jamie K. Teer,1 W Douglas Cress,1 Jie Wu2. 1 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL;_ 2 _Peggy and Charles Stephenson Cancer Center, Oklahoma City, OK_.

The cyclin-dependent kinase inhibitor 3 (CDKN3) is a dual specificity protein tyrosine phosphatase (PTP) known to dephosphorylate CDK1/2 on the activating Thr-161/Thr-160. Paradoxically, CDKN3 mRNA is often overexpressed in human cancer. It was reported that CDKN3 overexpression may be associated with splicing variants or mutations that produce dominant-negative CDKN3. We found that CDKN3 is overexpressed in non-small cell lung cancer (NSCLC) and that CDKN3 overexpression is prognostic of poor overall survival in three cohorts of lung adenocarcinoma. We next analyzed CDKN3 transcripts in a panel of 24 cell lines and lung adenocarcinoma tissues and also examined CDKN3 mutations in the Cancer Genome Atlas (TCGA) and the Moffitt Cancer Center's Total Cancer Care® (TCC) projects. Two CDKN3 transcripts were detected in all samples. These CDKN3 transcripts represent the full length CDKN3 mRNA and a normal transcript lacking exon 2, which encodes an out of frame 23-amino acid peptide with little homology to CDKN3. CDKN3 mutations were very rare and no disruptive mutation was found. Significantly, in synchronized cell cultures, CDKN3 mRNA and protein were elevated during the mitosis phase of cell cycle. Furthermore, CDKN3 expression had the highest correlation with mitotic-associated genes in lung adenocarcinoma tissues. Knocking down of CDKN3 prolonged the mitotic phase of cell cycle. Thus, CDKN3 overexpression in lung adenocarcinoma is not attributed to alternative splicing or mutation but is due to increased mitotic activity. Our data also suggest that CDKN3 is important for mitotic exit.

#191

LMTK3 is a novel regulator of oncogenic KIT in KIT-mutant cancers.

Lillian R. Klug, Jeffery W. Tyner, Michael C. Heinrich. _Oregon Health and Science University, Portland, OR_.

Multiple cancers, such as gastrointestinal stromal tumors (GIST) and melanoma, have been shown to be caused by somatic activating mutations in the receptor tyrosine kinase KIT. The major cause of death in patients with advanced KIT-mutant cancers is due to KIT tyrosine kinase inhibitor-resistant metastatic disease. Resistance develops almost exclusively because of secondary mutations within KIT, highlighting the importance of KIT in the proliferation and survival of these tumors. Therefore, strategies that affect KIT protein expression and maturation may be therapeutic. In order to identify regulators of KIT that may represent novel targets, we performed an siRNA screen in multiple KIT-mutant cancer cell lines. From these screens we identified lemur tyrosine kinase 3 (LMTK3) as a novel KIT regulator and potential target in KIT-mutant cancers. LMTK3 has been implicated as an important protein in multiple cancers, including leukemia, breast, and colon cancer. However, the oncogenic function of LMTK3 is not entirely clear. Despite its name, lemur tyrosine kinase 3 is, in fact, a serine/ threonine kinase. Beyond its enzymatic kinase function, LMTK3 has also been shown to have important scaffolding functions. LMTK3 has not been previously known to interact with KIT, but does regulate the stability of other receptors. We have found that LMTK3 silencing reduced the viability of all KIT-mutant GIST and melanoma cells tested to date, including those with ATP binding pocket and activation loop resistance mutations. Importantly, LMTK3 silencing decreased the viability of KIT-mutant cells specifically, as silencing of LMTK3 in cells lacking KIT mutations did not affect their viability. Further, we found the decrease in cell viability was due to induction of apoptosis, as seen by cleavage of PARP within 96 hours of LMTK3 silencing. Because these cells depend so heavily on KIT and loss of KIT signaling results in cell death, we hypothesized that LMTK3 silencing may be affecting this pathway. Indeed, with LMTK3 silencing there was a significant decrease in phosphorylated KIT (Y719), which was accompanied by decreased phospho-AKT (S473). We also observed a robust decrease in total KIT protein levels. However, there was no decrease in KIT transcript levels, suggesting that LMTK3 plays a role in regulating KIT at the protein level. While other KIT protein regulators, such as HSP90, have been suggested as therapeutic targets for GIST, the ubiquitous functions of these targets makes these treatments sub-optimal, often causing toxicity. Silencing of LMTK3, on the other hand, specifically kills KIT-mutant cancer cells. Furthermore, as a kinase, inhibition of LMTK3 by a small molecule may be possible, and we are in the process of identifying LMTK3 inhibitors. LMTK3 is a positive regulator of oncogenic KIT in GIST and melanoma and represents a novel, tractable target in KIT-mutant cancers.

#192

Targeting the protein kinase Hunk in HER2+ resistant breast cancer.

Joelle Zambrano,1 Kendall Phelps-Polirer,2 Melissa Abt,1 Elizabeth Yeh1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Clemson University, Clemson, SC_.

Despite the initial success of common HER2 inhibitors used in the clinic, including trastuzumab and lapatinib, HER2 positive (HER2+) breast cancer cells often acquire resistance through mechanisms that continue to remain poorly understood. A number of signaling molecules, which act downstream of HER2, including PI3K, Akt, JNK, and Hormonally-Upregulated Neu-associated Kinase (Hunk), are thought to contribute to resistance. Our recent studies indicate that targeting Hunk via shRNA in HER2+breast cancer cells that are sensitive to HER2 inhibitors (BT474) and cells that are resistant (JIMT) leads to increased caspase-3 activity in vitro and impaired tumor growth in vivo. In addition, we find that targeting Hunk not only sensitizes HER2+ cells to apoptosis, but also impairs autophagy, as indicated by decreased LC3BII expression, the lipidated form of LC3B which signifies active autophagy. Because elevated Hunk protein expression correlates with poorer prognosis, and since Hunk knockdown impairs autophagy, it is possible that Hunk acts to upregulate autophagy thereby contributing to the development of resistance. We have also found that the kinase JNK plays a role in resistance in HER2+ breast cancer cells, and demonstrate that JNK inhibition via a pan-JNK inhibitor impairs tumor growth of JIMT cells. We further show that co-targeting of JNK and HUNK results in greater cell death and significantly reduced tumor growth than either individual treatment in the JIMT-1, HER2+ resistant cell line, as well as a HER2+ line that was engineered to be lapatinib resistant, BT474-LR. Overall, our results indicate that targeting Hunk in HER2+ breast cancer not only alters autophagy and impairs tumor growth, but that co-targeting Hunk and JNK shows greater therapeutic effects on reducing tumor growth and inducing cell death.

#193

Mechanism of the AMPK-dependent activation of eukaryotic elongation factor 2 kinase (eEF-2K).

David H. Giles, Chris M. Crittenden, Jennifer S. Brodbelt, Kevin N. Dalby. _The University of Texas at Austin, Austin, TX_.

Eukaryotic elongation factor 2 kinase (eEF-2K) negatively regulates the rate of protein translation by phosphorylating and inactivating elongation factor 2 (eEF-2). Activation of eEF-2K by AMP-activated protein kinase (AMPK) contributes to the adaptive response of cancer cells to nutrient deprivation, promoting cancer cell survival and tumor progression. However, the mechanism by which AMPK activates eEF-2K is poorly described. Using LC-MS/MS, we confirmed previous findings that AMPK phosphorylates eEF-2K on three sites in vitro: Ser78, Ser366, and Ser398. Previous reports indicate that only Ser398 is phosphorylated by AMPK in cells, suggesting phosphorylation of Ser78 and Ser366 (which are known to inhibit eEF-2K via other kinases) are in vitro artifacts. To test the notion that phosphorylation of Ser398 activates eEF-2K, we purified recombinant eEF-2K S78/S366A, which cannot be phosphorylated at Ser78 or Ser366. Incubation of eEF-2K S78/S366A with AMPK led to significant phosphorylation of eEF-2K after four hours but did not alter eEF-2K activity against a peptide substrate. We then purified a form of eEF-2K S78/S366A in which Ser398 is ~90% phosphorylated (eEF-2K SSA-pS398), as shown by LC-MS/MS and Western blot. The activity of eEF-2K SSA-pS398 against a peptide substrate was unchanged compared to a non-phosphorylated control, as was its activity against full-length eEF-2. Similarly, the EC50 and affinity of eEF-2K S78/S366A for Ca2+/calmodulin (CaM), which eEF-2K requires for activity, were unchanged by Ser398 phosphorylation. Phosphorylation of Ser398 did not alter the rate of autophosphorylation of eEF-2K S78/S366A at Thr348 or Ser500, which modulate its activity and dependence on Ca2+/CaM, respectively. Overall, our in vitro data suggests that AMPK may not directly regulate eEF-2K through phosphorylation of Ser398. In cells, mTOR activates several kinases, including p70S6K, that phosphorylate and inactivate eEF-2K. AMPK inhibits mTOR through phosphorylation of TSC2 and Raptor, suggesting AMPK could relieve eEF-2K from mTOR-mediated inhibition by suppressing mTOR activity. In line with this notion, we found that treatment of MDA-MB-231 breast cancer cells with Torin1 (a selective inhibitor of mTOR) or AICAR (an AMP-mimetic that activates AMPK) led to similar levels of eEF-2K activation, and activation of AMPK with AICAR reduced levels of mTOR activity. Combined application of Torin1 and AICAR resulted in levels of eEF-2K activity similar to those obtained with either compound separately. Taken together, our data suggests that the AMPK-dependent activation of eEF-2K likely involves the ability of AMPK to inhibit mTOR and relieve eEF-2K from mTOR-mediated inhibition, and questions the role of Ser398 phosphorylation in activating eEF-2K directly.

#194

Increased PKR expression inhibits differentiation of hematopoietic stem cells.

Richard L. Bennett,1 Xiaodong Cheng,1 Michael T. Byrne,2 Amalin Asous,1 Mohini Patel,1 Zeel Patel,1 W. Stratford May1. 1 _University of Florida, Gainesville, FL;_ 2 _Vanderbuilt University Medical Center, Nashville, TN_.

Acute myelogenous leukemia (AML) is characterized by a differentiation block at an early stage of hematopoiesis that results in the accumulation of immature myeloid cells and an impaired production of blood cells. This parallels findings in solid tumors that demonstrate differentiation stage is often associated with tumor behavior and generally an immature tumor is more aggressive than the more differentiated counterpart. Thus, new

research designed to more fully understand the mechanisms of cellular differentiation may improve our understanding of neoplastic transformation and identify new therapeutic targets. Recently our laboratory has determined that increased expression and activation of the interferon-inducible, dsRNA-activated kinase (PKR) is associated with shorter remission duration and worse survival for patients with AML. PKR is a key mediator of the cellular response to inflammatory stress that has been found to be upregulated by chronic inflammation and aging. Since we have previously reported that mice expressing a PKR transgene (TgPKR) in hematopoietic cells have a reduced number of bone marrow (BM) Lin-, Sca1+, cKit+ hematopoietic stem/progenitor cells (HSPCs) and develop a dysplastic BM with an increased frequency of BM blasts, we investigated whether PKR may affect HSPC differentiation. Results indicate that BM of TgPKR mice contains a significantly reduced number of multipotent progenitor cells (MPPs) that are more frequently arrested in G0/G1 than wild type (WT) mice. In contrast, BM of PKR knockout mice (PKRKO) have an increased frequency of proliferating MPPs (S+G2/M) but a decreased frequency of both Long-term (LT) and Short-term (ST) HSCs compared to either WT or TgPKR mice. Although LT-HSCs of TgPKR mice are more frequently quiescent in vivo they remain functional in vitro as demonstrated by nearly double the colony forming capacity compared to BM of PKRKO mice after >4 weeks in long term culture initiating cell (LTC-IC) assays. In addition, when differentiation of the myeloid cell line K562 is induced by PMA treatment, cells with reduced PKR expression by siRNA knockdown display an increased rate and extent of megakaryocyte differentiation compared to control cells. These findings suggest that increased PKR expression has a previously unrecognized function to inhibit hematopoietic cell differentiation. This may account for why TgPKR mice accumulate quiescent LT-HSCs and develop BM dysplasia with age. Furthermore, these results suggest that increased PKR expression/activation as observed in high-risk MDS, AML and other hematologic malignancies may contribute to leukemogenesis, at least in part, by blocking hematopoietic cell differentiation. In addition, these new findings may be applicable to solid tumors since increased PKR signaling has also been reported to be associated with a more rapid and aggressive course of tumor progression in breast cancer, melanoma and colon cancer.

#195

Defining the role of PAK7 variants in melanoma.

Kyle M. LaPak, Michael A. Gross, Denny C. Vroom, Greg B. Lesinski, William E. Carson. _The Ohio State University, Columbus, OH_.

Melanoma mortality is directly linked to its profoundly metastatic nature and inherent resistance to conventional chemotherapy. For this reason, increased understanding of the molecular mechanisms driving melanoma progression is of utmost importance. The p21-activated serine/threonine kinases (PAKs) are frequently mutated or overexpressed in cancer and have recently been implicated in tumor metastasis, proliferation, and apoptotic resistance. This observation has inspired drug discovery efforts to limit PAK activity; yet, mechanistic understanding of how PAKs (especially Type II PAKs) contribute to tumor initiation and progression is still evolving. PAK7, a type II PAK, is mutated in 16-18% of all melanomas. Melanoma-associated PAK7 mutations occur throughout the gene and have unknown biochemical and physiological consequences. The objective of this study was to determine how tumor-associated PAK7 variants mechanistically contribute to melanoma initiation and progression. First, we characterized PAK7 protein expression in a panel of 39 melanoma cell lines and primary melanocyte cultures. PAK7 levels were similar in both tumorigenic and non-tumorigenic cells and failed to correlate with the status of common melanoma driver mutations (e.g. NRASQ61, BRAFV600). In an array of 101 human tumor biopsies, PAK7 expression did not significantly change with advancing melanoma stage; however, PAK7 levels were higher in regional lymph node biopsies when compared to primary skin lesions. Since mutation is the predominant mechanism affecting PAK7 in melanoma, we investigated the biochemical and physiological consequences of common PAK7 variants. 11 distinct PAK7 mutations were stably expressed at low levels in VMM39 (melanoma) and NCI-H441 (lung) cells. Using these clones, changes in the phosphorylation of downstream PAK7 targets associated with cellular migration (p120), proliferation (CRAF), and apoptotic resistance (BAD, MDM2) were examined. In addition, we defined phenotypic alterations in cellular morphology, proliferation, migration, and apoptotic resistance associated with the expression of each mutant. These data allowed us to classify PAK7 variants of unknown significance into groups associated with specific cellular outcomes. To our knowledge, this is the first study to functionally characterize a wide variety of melanoma-associated PAK7 mutations and represents a critical first step in understanding how this kinase contributes to tumor initiation and progression.

#196

The oncogenic role of PRL-3 in multiple myeloma through regulation of Src kinase family members.

Pegah Abdollahi,1 Esten Nymoen Vandsemb,1 Magnus Aassved Hjort,2 Kristine Misund,1 Toril Holien,1 Torstein Baade Rø,2 Tobias Schmidt Slørdahl,3 Magne Børset4. 1 _K. G. Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway;_ 2 _K. G. Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Department of Pediatrics, St Olavs University Hospital, Trondheim, Norway;_ 3 _K. G. Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Clinic of Medicine, St Olavs University Hospital, Trondheim, Norway;_ 4 _K. G. Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Department of Immunology and Transfusion Medicine, St Olavs University Hospital, Trondheim, Norway_.

Introduction: Phosphatase of regenerating liver 3 (PRL-3) is a dual specificity phosphatase and its up-regulation in cancer cells is related to poor prognosis. We have previously described PRL-3 as a downstream target of IL-6 in multiple myeloma (MM) by demonstrating that it was upregulated in response to this cytokine. The Src kinase family (SFK) is composed of 8 members and regulates proto-oncogenic cellular pathways. As it has been reported that the SFK members Lyn, Fyn and Hck are important in signal transduction of IL-6 signals in MM cells and that Src is reported to be the major kinase regulated by PRL-3 in colon cancer, we evaluated effects of PRL-3 on activation of Fyn, Lyn, Hck and Src in MM.

Methods: By retroviral transduction in the IL-6-dependent MM cell line INA-6,we generated functional PRL-3(INA-PRL-3) overexpressing cells, and control cells expressing catalytically inactive mutant PRL-3 (C104S) or empty vector (Mock). We measured global tyrosine (Y) phosphorylation (P) by immunoblotting using (P-Y1000) antibody. We measured the expression of 4 SFK members by qRT PCR and immunoblotting. Activity of SFK members was evaluated using MILLIPLEX MAP8-plex Human SFK kit. We confirmed MILLIPLEX result for Src by immunoblotting with antibody against P-Y416 Src (activating phosphorylation site). We also measured Src activity after inhibiting PRL-3 by PRL-3 inhibitor I or shRNA. Finally, we showed the influence of PRL-3 on cell growth and cell sensitivity to two Src inhibitors (PP2 and Su6656) by using CellTiter-Glo Luminescent Cell Viability Assay.

Results: INA-PRL-3 had more global P-Y than C104S and Mock cells in the absence of IL-6 and showed a pattern of P-Y more similar to that of the parental INA-6 cells grown in the presence of IL-6. C104S and INA-PRL-3 cells showed lowest and highest activity, respectively, of Lyn and Src. Stimulation of cells with IL-6 increased active Lyn and Src but still C104S had lowest activity. We did not observe significant difference in activity of Fyn and Hck. Measuring total amount of 4 SFK members showed up-regulation of total Hck and Fyn in C104S cells in mRNA and protein level. This indicated that inactive PRL-3 in C104S cells leads to less SFK activity compared to both INA-PRL-3 and Mock cells. Increased total amount of Hck and Fyn in C104S could be the result of diminished negative feedback regulation of expression of SFK members by their active forms. INA-PRL-3 showed more P-Y416 Src also by immunoblotting and its inhibition by PRL-3 inhibitor or shRNA decreased Src activity. INA-PRL-3 had higher growth rate than both C104S and Mock cells. This difference was more prominent in the absence of IL-6 and high SFK activity in INA-PRL-3 could be one of the possible reasons for this. INA-PRL-3 also showed high sensitivity to both PP2 and Su6656.

Conclusion: Inhibition of PRL-3 and SFK members could be considered as a possible treatment for MM.

#197

Phospholipids regulate the localization and oncogenic potential of protein tyrosine kinase 6 in prostate cancer.

Darren J. Wozniak, Ben Hitchinson, Wenjun Bie, Vadim Gaponenko, Angela L. Tyner. _University of Illinois at Chicago, Chicago, IL_.

Background: Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular SRC-related tyrosine kinase that does not possess canonical localization signals. Lack of targeting yields flexibility in its intracellular localization and access to substrates. PTK6 is observed in the nuclei of prostate epithelial cells where it promotes differentiation and inhibits growth. PTK6 is activated at the plasma membrane in PTEN-deficient mouse prostate tumors where the concentration of PIP3 is high due to PTEN loss of function. Membrane-associated PTK6 induces cell transformation, the epithelial-mesenchymal transition, and tumor cell metastasis. In human prostate cancer, active PTK6 associates with the plasma membrane and its increased expression correlates with metastasis and poor survival. Our studies aimed to characterize how the mislocalization and activation of PTK6 in prostate cancer is regulated.

Results: Based on our observation that loss of PTEN induces accumulation of PTK6 at the membrane, we hypothesized that PTK6 binds to the plasma membrane secondary messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Amino acid sequence alignment predicts H52, Y53, and K54 of PTK6 as a potential PIP3 binding site, which resembles the lipid-binding HYK domain of tyrosine kinases BTK and Abl. Using NMR saturation transfer difference (STD) experiment, we observed that the SH2 domain of PTK6 contains a binding site for inositol trisphosphate (IP3) a soluble PIP3 mimic. As predicted, statistical analysis identified Y53 of the PTK6 HYK motif within the SH2 domain as a residue with substantial chemical shift perturbations (CSPs) induced by independent titrations with PIP3 analogs IP3, IP4, and 1,2-dihexanoyl PIP3 in 15N HSQC, as compared to controls. Using autophosphorylated and unphosphorylated PTK6, NMR STD experiments show that only active (pY342) PTK6 binds to IP3. Treatment of prostate cancer cells with the PI3K inhibitor wortmannin reduces active PTK6 at the plasma membrane.

Conclusions: Our data demonstrate that active PTK6 is recruited to the plasma membrane through direct interactions with PIP3. Membrane association places PTK6 in close proximity to its oncogenic substrates AKT, FAK and BCAR1, thereby promoting tumor progression. These findings may facilitate identification of novel PTK6 inhibitors that block its association with the membrane, while not affecting its tumor suppressive role in the nucleus.

This work is funded by NIH grant 1R01CA188427 to ALT and VG.

#198

The phosphatase PRL-3 is expressed in primary B-ALL cells and is important for adhesion and migration.

Magnus Aassved A. Hjort, Pegah Abdollahi, Esten Vandsemb, Tobias Schmidt Slørdahl, Bendik Lund, Magne Børset, Torstein Baade Rø. _NTNU, Trondheim, Norway_.

Introduction: Phosphatase of regenerating liver -3 (PRL-3) is a dual specificity phosphatase, and is associated with aggressiveness and metastatic disease in a number of solid tumors. Less is known about its role in hematological cancer, but we have previously shown that PRL-3 is expressed in primary multiple myeloma (MM) cells and MM cell lines. More recently PRL-3 is shown to be expressed in acute myeloid leukemia (AML), and associated with poor prognosis. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite progress in treatment and survival, treatment related toxicity is a major concern. Therefore, it is a need for new clinical markers and therapy targets to optimize the treatment. We hypothesized that PRL-3 is expressed, and have oncogenic functions in B-ALL, and can be a possible clinical marker and a novel drug target.

Methods: Primary ALL cells from peripheral blood were obtained from The Regional Biobank of Central Norway, and PRL-3 mRNA expression was measured with qRT-PCR. The PreB-ALL cell lines Reh and MHH-CALL-4 were used for further experiments. IL-7 (100 ng/mL) were used for stimulation. ShRNA against PRL-3 was used to knock down PRL-3 in Reh (shPRL-3) and for empty vector control (shMOCK) by use of lentiviral transduction. To study adhesion we used fibronectin (20 µg/mL) and stimulated with IL-7, IL-8 and IGF-1 (all 100 ng/mL) for 45 min in 37°C. Migration was studied with transwell permeable plates (pore size 3 µm) using SDF1α (75 ng/mL) as a chemoattractant. Further we examined the effect of the small molecular inhibitor PRL-3 Inhibitor I on ALL cell lines. Apoptosis and cell viability was determined by Annexin-FITC/-PI with flow cytometry, and by CellTiter-Glo® Luminescent Cell Viability assay kit, after 24-72 hours incubation with PRL-3 Inhibitor I.

Results: 89% (16 of 18) of primary B-ALL samples expressed PRL-3, and 33 % (6 of 18) at high levels. Stimulation with IL-7 induced expression of PRL-3 in a dose-dependent manner in Reh and MHH-CALL-4. The knock down of PRL-3 by shRNA was 80 % efficient. Reh shMOCK cells were significantly more adherent to fibronectin after stimulation with IL-7, IL-8 and IGF-1 compared to Reh shPRL3. The cells' ability to migrate towards a SDF1α gradient were significantly reduced in Reh shPRL-3 cells compared with Reh shMOCK. PRL-3 inhibitor I also reduced the viability and induced apoptosis in Reh cell line in a dose-dependent manner, but not in MHH-CALL-4 cell line, at a concentration of 40-80 µM.

Conclusion: PRL-3 was expressed in 89 % of primary B-ALL samples. Knock down of PRL-3 clearly reduced cell adhesion and migration. PRL-3 inhibitor I induced apoptosis and reduced cell viability in Reh, but not in MHH-CALL-4. This study indicates that PRL-3 could be important in the pathogenesis of B-ALL, and might be a suitable target for treatment in the future.

#199

Evaluation of multiple EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants using in vitro modeling.

Hanako Hasegawa. _Keio University, Shinjuku-ku, Japan_.

Purpose:

We are able to use multiple EGFR-TKIs to treat patients with lung cancer harboring EGFR mutations. However, there is no clear guideline regarding which EGFR-TKIs should be used for which mutations. The purpose of this study is to establish an in vitro model to determine the "therapeutic window" of EGFR-TKIs against various types of EGFR mutations, including several EGFR exon 20 insertion mutations.

Experimental design:

We evaluated drug sensitivity and downstream signaling of human lung cancer cell lines harboring EGFR mutations (PC9, H3255, PC9-ER, BID007 and H1975) and Ba/F3 cells exogenously expressing mutated or wild type EGFR for the 1st (erlotinib), 2nd (afatinib), and 3rd (osimertinib and rociletinib) generation EGFR-TKIs using MTS assay, apoptosis assay and western blotting, simultaneously.

An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFRs.

Results:

The model of mutation specificity identified wide therapeutic windows of afatinib for exon 19 deletions and L858R and those of osimertinib and rociletinib for T790M positive mutations in human cell lines and Ba/F3 cells. In human cell lines and Ba/F3 cells harboring exon 19 deletions or L858R, afatinib showed the lowest IC50 and most potently inhibited the phosphorylation of EGFR, AKT, and ERK. Afatinib and 3rd generation EGFR-TKIs (osimertinib and rociletinib), effectively inhibited the cell growth and the phosphorylation of downstream signaling of EGFR in T790M positive cells. For EGFR exon 20 insertion mutations, osimertinib was potent and presented the most wide therapeutic window.

Conclusion:

This model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations including EGFR exon 20 insertions.

#200

PDLIM2 phosphorylation in determining breast cancer phenotype.

Orla T. Cox, Janina Berghoff, Deirdre A. Buckley, Rosemary O'Connor. _University College Cork, Cork, Ireland_.

Several genes that are involved in cancer progression are induced by Insulin-like Growth Factor-1 (IGF-1). One of them encodes the PDZ-LIM protein PDLIM2, which is localized at the cytoskeleton and nucleus in epithelial and hematopoietic cells. PDLIM2 is repressed in certain cancers including breast cancers, but it is highly expressed in invasive cancer cells in triple negative breast cancer (TNBC), where it has also been associated with poor survival. PDLIM2 regulates the stability and activity of transcription factors including beta catenin, STATs, NFκB and IRFs and has been associated with ubiquitination and destabilization of proteins such as NF-κB, E-cadherin, and p27KIP1. However, PDLIM2 itself is also regulated by posttranslational modifications, but how this controls PDLIM2 activity is still largely unclear.

Therefore, this study aimed to investigate PDLIM2 phosphorylation and its effect on PDLIM2 sub-cellular location and function in transcription factor regulation. The PDLIM2 protein sequence includes multiple potential phosphorylation sites and it migrates as several distinct phosphatase-sensitive species in SDS PAGE and 2D electrophoresis, indicating phosphorylation at several residues. Using cellular fractionation and immunofluorescence we show that PDLIM2 accumulates in the nucleus in response to cell stimulation with IGF-1 or TGFβ. Moreover, we show that inhibitors of Rho Kinases suppress PDLIM2 phosphorylation in response to these stimuli. In addition, we demonstrate that mutation of the predicted Rho Kinase phosphorylation sites in PDLIM2 reduce protein stability, an effect that can be reversed by application of proteasomal inhibitor.

Overall, our results indicate that phosphorylation by Rho kinases plays an important role in the subcellular location and regulation of PDLIM2 function, which opens a new avenue in targeting PDLIM2 activity in invasive cancer cells.

#201

Tribble 3 as integrator of metabolic stresses during lung cancer progression.

Abeer Almiman, Jamila Adom, Yande Guo, Jiuhui Wang, Daotai Nie. _Southern Illinois University School of Medicine, Springfield, IL_.

Introduction: Tribbles pseudokinase 3 (TRIB3) has been identified as a stress related gene. Recent studies reported the increased expression of TRIB3 in cancers and its correlation with the poor prognosis in cancer patients. However, other studies suggest TRIB3 as a tumor suppressor due to its inhibitory activities toward PI3K/Akt and Mek-Erk signaling. In the present study, we aim to investigate the expression of TRIB3 in lung cancer cells and whether it plays a role in lung cancer progression and responses toward chemotherapy.

Methods: TRIB3 expression was determined by RT-PCR and Western blot. A lentiviral vector was used to overexpress TRIB3 in lung cancer cells. Small hairpin RNA constructs targeting TRIB3 were used to deplete TRIB3 expression. A CRISPR-CAS9 construct with guiding sequence matching to TRIB3 gene was used to knock out its expression. Cell proliferation, survival, and migration were determined using standard techniques.

Results: Through in silico analyses, we found that high level of TRIB3 RNA was associated with poor survival of lung cancer patients. In lung cancer cells (NCI-H358), TRIB3 expression was markedly stimulated during metabolic stresses or ER stresses. Overexpression of TRIB3 in cells led to decreased cell proliferation and increased arrest at G0/G1 phase, even when the cells were not under overt metabolic stresses. RT-qPCR analyses of cell cycle proteins revealed an upregulation of p27, a CDK inhibitor, in cells overexpressing TRIB3. Overexpression of TRIB3 reduced Akt phosphorylation at threonine 308. Depletion or knockout of TRIB3 led to increased cell growth but cells became more sensitive to stressors, leading to increased apoptotic cell death.

Conclusion: Our study reveals complex roles of TRIB3 in lung cancer progression. Induced by metabolic stresses, TRIB3 can block cell cycle progression through p27 in adaptive responses. Further studies are needed to determine whether and how TRIB3 functions as a metabolic gatekeeper during lung cancer progression.

#202

Depletion of PTP4A3 phosphatase disrupts colorectal cancer cell adhesion and extracellular matrix interactions.

Kelley E. McQueeney,1 Paula Pekic,1 Joseph M. Salamoun,2 Jennifer Ahn,1 Elizabeth R. Sharlow,1 Peter Wipf,2 John S. Lazo1. 1 _University of Virginia, Charlottesville, VA;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States and the world and the 5-year survival rate in the United States of ~60% highlights the need for better treatment options. The central challenge in the design and development of drugs to treat CRC has been the validation of novel drug targets and the generation of new therapies aimed at those targets. The goal of this project is to better understand the role of one such potential CRC molecular target. Protein Tyrosine Phosphatase 4A3 (PTP4A3) is currently among the most consistently up-regulated proteins seen in colorectal tumors but it suffers from an incomplete understanding of its substrate(s) and of its participation in tumor formation and progression. To clarify how PTP4A3 expression impacts cell behavior, we isolated primary colon tumor epithelial cells from Ptp4a3fl/fl mice. PTP4A3 was deleted in vitro by adenoviral infection of Ptp4a3fl/fl cells with Cre recombinase accompanied by a green fluorescent protein (GFP) marker. As a control, the Ptp4a3fl/fl cells were infected with GFP alone. Virally infected cells were selected by fluorescence activated cell sorting. In the sorted cells, the expression of Cre resulted in a complete lack of PTP4A3 protein by western blotting and undetectable Ptp4a3 mRNA as determine by quantitative RT-PCR. Analysis of these genetically similar cells revealed that Ptp4a3-/- tumor cells had significantly reduced colony formation and migration as compared to Ptp4a3fl/fl cells. Notably, the growth rate of Ptp4a3fl/fl and Ptp4a3-/- cells was similar when cultured on plastic in a 2D monolayer. In contrast, Ptp4a3fl/fl spheroids grew significantly faster than the Ptp4a3-/- cells when cultured in Matrigel. Moreover, while the Ptp4a3fl/fl cells were able to form cohesive spheroids when cultured in cell repellent plates, there was a striking inability of the Ptp4a3-/- cells to form these 3D structures. In addition, treatment of the Ptp4a3fl/fl cells with a potent and selective PTP4A3 phosphatase inhibitor, thienopyridone, phenocopied the appearance of the Ptp4a3-/- cells grown in cell repellent plates. These results suggest PTP4A3 expression-dependent changes in tumor cell behavior may be rooted in a disruption in the adhesion and extracellular matrix-signaling axis. This hypothesis was supported by significant changes in the gene expression profile of adhesion and extracellular matrix proteins in the Ptp4a3-/- CRC cells. The isogenic Ptp4a3fl/fl and Ptp4a3-/- CRC cells provide an invaluable tool to elucidate further the function of PTP4A3 and its link to adhesion and the extracellular matrix interactions.

#203

Single cell imaging of kinase inhibitor-induced effects in breast cancer cell lines.

Caitlin Mills,1 David Andrews,2 Peter Sorger1. 1 _Harvard Medical School, Boston, MA;_ 2 _Sunnybrook Research Institute, Toronto, Ontario, Canada_.

There is need for improved targeted therapies for the treatment of breast cancer. Kinase inhibitors are candidates to be used in this context. With the goal of uncovering specific vulnerabilities in differing cell lines, we treated five breast cancer cell lines representing triple negative (BT20, MDAMB231, and Hs578T), hormone receptor positive (MCF7), and Her2 amplified (SKBR3) disease and the non-malignant MCF10A line with a panel of 105 kinase inhibitors covering a broad range of targets. Cells were treated with doses between 0.1 and 10 µM for 24 hours, at which time they were stained with DRAQ5 (DNA) and TMRE (mitochondrial membrane potential). Live-cell images were acquired using a high throughput, confocal Opera microscope. Cell segmentation, based on the DRAQ5 staining, and feature extraction (intensity, morphology, and texture) were performed with Acapella software. Over 300 features were extracted for ~1.5 million cells. Analytical methods have been applied to identify those treatments that induced significant changes to the cells. Over half of the kinase inhibitors queried had a significant effect within 24 hours in all cell lines at 10 µM. Cell cycle inhibitors had the most common effects across cell lines. Cell line specific effects were also uncovered, for example, MDAMB231 cells were the most sensitive to MAPK inhibitors. These, early time point, results have been compared with the effects of the same inhibitors on cell growth to better understand the mechanisms leading to cell line and pathway specific effects.

#204

Disruption of the RACK1/PP2A complex results in a decrease in the adhesion, proliferation, migration and invasion capabilities of breast cancer cells.

Maeve Kiely,1 Rosemary O'Connor,2 David Adams,3 George S. Baillie,4 Patrick A. Kiely1. 1 _University of Limerick, Limerick, Ireland;_ 2 _University College Cork, Cork, Ireland;_ 3 _Heriot-Watt University, Edinburgh, EH14 4AS, United Kingdom;_ 4 _University of Glasgow, Glasgow, United Kingdom_.

Conflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. PP2A has a well-established role as a tumour suppressor within signalling pathways but is also known to play a pro-carcinogenic role in certain circumstances [1]. RACK1 has been identified as a direct binding partner of PP2A to regulate cell migration and stabilize PP2A activity [2]. Our objective was to further characterise the interaction between PP2A and RACK1 in breast cancer cells. The PP2A holoenzyme is assembled in MCF-7 cells and we found that both the C subunit and A subunit of PP2A are assembled on the RACK1 scaffold. We used immobilized peptide arrays representing the entire PP2A-Catalytic protein to identify amino acids on the C subunit of PP2A that are required for the binding of RACK1. Once identified, these sites were mutated and expressed in the context of the full length PP2A C subunit protein and stable cell lines were generated. When the RACK1/PP2A interaction was disrupted, cells exhibited reduced PP2A phosphatase activity, confirming the role for RACK1 in stabilizing PP2A activity. We used the stable cell lines to determine that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of this breast cancer cell model. The work has significantly advanced our understanding of the RACK1/PP2A complex and indicates a pro-carcinogenic role for the RACK1/PP2A complex. This work has highlighted a novel mechanism to target PP2A activity and may provide a potential therapeutic target in the treatment of breast cancer.

1. Kiely, M. and P.A. Kiely, PP2A: The Wolf in Sheep's Clothing? Cancers, 2015. 7(2): p. 648-669.

2. Kiely, P.A., et al., Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and β1 integrin and for tumor cell proliferation and migration. Journal of Biological Chemistry, 2008. 283(34): p. 22952-22961.

#205

Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay.

Lu Chen, Anthony J. Saporita, Wen-Rong Lie, Reeti Maheshwari, Melissa Schluter, Jehangir Mistry, Joseph Hwang. _MilliporeSigma, St. Charles, MO_.

A novel multiplex immunoassay was developed for examining acquired resistance to Receptor Tyrosine Kinase (RTK) inhibitors. RTKs are transmembrane proteins which act as receptors for growth factors, neurotrophic factors and other extracellular signaling molecules. These cell-surface kinases are activated by extracellular ligands leading to receptor dimerization and tyrosine phosphorylation at specific residues in the cytoplasmic tails to initiate RTK-mediated signal transduction. Of the >500 known protein kinases in the human genome there are approximately 60 RTKs. They are central components of cell signaling networks and play crucial roles in normal physiological processes and disease processes ranging from diabetes to cancer. Many RTKs, such as EGFR, HER2, c-Kit, PDGFR and VEGFR, have been used as targets for drug development and RTK-targeted therapies have illustrated the utility of these treatments for selected cancers. However, in many cases, compensatory RTK signaling enables cancer cells to acquire resistance to RTK inhibitors that selectively target a single RTK. For example, some EGFR and HER2 inhibitors have led to resistance and are associated with increased expression of IGF1R. In order to understand the mechanisms of resistance to RTK inhibitors, we have developed a bead-based multiplex immunoassay capable of simultaneously detecting phosphorylation of 18 different RTKs. First we demonstrate detection of RTK phosphorylation in response to growth factor stimulation using various cell lines. Next we examined the specificity of two inhibitors targeting EGFR and HER2. These inhibitors specifically reduced growth factor-stimulated phosphorylation of EGFR and HER2, without inhibiting the activation of other RTKs. Although we did not observe compensatory activation of other RTKs in response to the two EGFR and HER2 inhibitors we tested, this study demonstrates the feasibility of using this novel 18-plex RTK panel for examining the mechanism of resistance to single RTK inhibitors. In summary, the RTK multiplex panel allowed for simultaneous detection of multiple tyrosine phosphorylated RTKs in a specific, sensitive, and reproducible manner.

### Mitochondria, Autophagy, and Metabolic Vulnerabilities

#206

Cytochrome c oxidase subunit 5a (COX5A) is identified as a potential therapeutic target for lung cancer with high therapeutic index through a pooled shRNA screen.

Toshio Kato,1 Mitsuo Sato,1 Masashi Kondo,1 Tomohiko Kakumu,1 Naoyuki Yogo,1 Tetsunari Hase,1 Masahiro Morise,1 Yoshionori Hasegawa,1 John D. Minnna,2 Luc Girard2. 1 _Nagoya University, Nagoya, Japan;_ 2 _Hamon Center for Therapeutic Oncology Research and the Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, TX_.

[Background]Recent advances in high throughput analysis of genetic and epigenetic alterations revealed numerous changes in lung cancer cells. It is vital to sort out genes that substantially contribute to oncogenic properties of cancer cells from these numerous altered genes by performing a functional screening because such genes could serve as valuable therapeutic targets. To this end, we performed a screening with a pooled shRNA library for genes critical for survival and/or proliferation in lung cancer cells. [Methods and Results]A pooled shRNA library comprising 5,000 genes, with 3 to 7 constructs per gene, were transduced in NCI-H460 lung cancer cell line. Subsequently, genomic DNA was extracted from the transduced cells and numbers of shRNA for each gene were quantified by next generation sequencing. Through a stringent filtering process we narrowed down genes to a list of 24 candidates as potentially target genes. Next, we integrated data of genome-wild gene expression and copy number analyses of a panel of lung cancer cell lines to narrow down to genes more relevant to lung cancer. Through these processes, we listed up several candidate genes. Among them, we focused on Cytochrome c oxidase subunit 5a (COX5A), one of the nuclear encoded subunits of cytochrome c oxidase, the terminal protein of the mitochondrial electron transport chain. Importantly, COX5A knockdown didn't suppress growth of HBEC3, a normal control cell line, suggesting that therapeutics of targeting COX5A may have high therapeutic index.

[Conclusions]Our results suggest that inhibiting COX5A may be an attractive strategy for the treatment of NSCLC with high therapeutic index.

#207

Regulation of mitochondrial biogenesis in primary human clear cell renal cell carcinoma cells.

Helén Å. Nilsson, Martin E. Johansson. _Lund University, Malmö, Sweden_.

Clear cell renal cell carcinoma, ccRCC, is the most prevalent type of kidney cancer. In over 90% of ccRCCs the tumor suppressor protein von Hippel Lindau is inactivated, leading to a pseudohypoxic phenotype with vast effects on angiogenesis as well as energy metabolism. In accordance with the Warburg effect, aerobic glycolysis increases, while mitochondrial oxidative phosphorylation is decreased.

We have previously published data showing that also the number and activity of mitochondria is strikingly reduced in primary human ccRCC cells compared to normal kidney epithelial cells. This was not seen in any of the established RCC cell lines included in the study. We are now continuing these studies by investigating the regulation of mitochondrial biogenesis in primary human ccRCC cells, and how the number and activity of mitochondria influences the tumorigenic properties of these cells.

RNA has been isolated from ccRCC tissue areas identified as mitochondria high or low. The expression profile in these samples will be analyzed by RNA sequencing.

Human primary ccRCC cells obtained from patient nephrectomies are kept in culture and the regulation of mitochondrial biogenesis in these cells is investigated. How the mitochondrial content affects tumorigenic properties such as proliferation, migration and resistance to apoptosis is also investigated.

Preliminary data indicate that primary ccRCC cells have an absent or severly limited capability to increase the number of mitochondria on metabolic demand, and that the regulation of mitogenesis is defective.

These results point towards a malfunctioning energy sensing and mitochondrial biogenesis in ccRCC cells that might be part of the explanation to the very low mitochondrial content in these cells.

#208

BPM 31510-induced alteration in Complex II activity is functionally linked to cell death activation pathway in a preclinical model of triple-negative breast cancer.

Tulin Dadali,1 Anne R. Diers,1 Arleide Lee,1 Ezer Benaim,1 Joaquin J. Jimenez,2 Stephane Gesta,1 Vivek K. Vishnudas,1 Rangaprasad Sarangarajan,1 Niven R. Narain1. 1 _Berg, LLC, Framingham, MA;_ 2 _University of Miami Miller School of Medicine, Miami, FL_.

Although only 15-20% of total breast cancer diagnoses are of the triple-negative breast cancer (TNBC) subtype, they account for a significant portion of the mortality rate due to their more aggressive phenotype and a high risk of reoccurrence. Metabolic rewiring supports breast cancer progression and metastasis, particularly in ER-negative and triple-negative (TNBC) breast tumors. Thus, we examined the effects of BPM 31510, a metabolic-modulating agent in clinical trials for solid tumors, in in vitro and in vivo ER-negative and TNBC models. BPM 31510 EC50/EC>90 values were determined for a panel of the breast cancer cell lines and compared to non-tumorigenic MCF12A cells in vitro, and the MDA-MB231 and SkBr-3, TNBC and ER-negative models respectively, were found to be the most sensitive to BPM 31510. Treatment with BPM 31510 (EC50 and EC>90 doses) resulted in a time- and dose-dependent decrease the viable cell population (PI- and Annexin V-negative) and a concomitant increase in cells in early and late apoptosis (PI-negative and PI-positive Annexin V-positive cells, respectively), suggesting that BPM 31510 activates regulated cell death pathways. Consistent with the in vitro data, MDA-MB231 tumor-bearing mice had smaller tumors after 30 days of treatment with BPM 31510 and increased cleaved caspase 3 staining in resected tumors. In vitro, BPM 31510-dependent breast cancer cell death was preceded by mitochondrial membrane potential depolarization (TMRE flow cytometry) and alterations in mitochondrial respiration characterized by a consistent, dose-dependent decrease in succinate (Complex II)-fueled respiration with more varied responses to BPM 31510 in cells provided the Complex I substrates (pyruvate or palmitoyl carnitine). To investigate the role of Complex II in BPM 31510-mediated cell death, pharmacological inhibitors of the dicarboxylate site (malonate) and Qp site (atpenin A5) of Complex II were used in combination with BPM 31510 to assess the resultant effects on cell death in MDA-MB231 cells. Co-treatment with malonate significantly attenuated BPM 31510-mediated cell death while atpenin A5 did not affect BPM 31510-induced cell death, indicating succinate oxidation at the dicarboxylate site of Complex II is required, in part, for induction of cell death by BPM 31510. Together, these data demonstrate BPM 31510 has a potent anti-cancer activity in preclinical breast cancer models and define a functional link between Complex II activity and the mechanism of action for BPM 31510.

#209

The effect of heme influx on initiation and tumorigenesis of NSCLC.

Sagar Sohoni, Md Maksudul Alam, Chantal Vidal, Jagmohan Hooda, Li Zhang. _University of Texas at Dallas, Richardson, TX_.

Heme is a central molecule for mitochondrial function and for all processes involved in oxygen utilization. Heme serves as a prosthetic group or as a cofactor for a number of oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Heme also directly regulates the synthesis, translocation and assembly of these enzyme complexes. Most, if not all, human cells can synthesize and uptake heme from the circulation. A number of epidemiological studies have shown that high heme intake is associated with increased risk of cancer, including lung cancer. Recent studies carried out in our lab showed intensified mitochondrial respiration and increased levels of heme and hemoproteins in non-small-cell lung cancer (NSCLC) cells. Together, these experimental and epidemiological studies strongly suggest that heme is an "oncometabolite." To assess the status of heme metabolism in cancer cells, we performed a series of experiments in NSCLC cells and compared the results with an immortalized normal lung cell line, HBEC30KT. We used Zinc protoporphyrin, an analogue of heme to measure the level of heme uptake. We found that heme uptake as well as heme synthesis are significantly elevated in all NSCLC cells compared to HBEC. The rate of heme degradation was also measured in these cell lines. Previously, our lab demonstrated that lowering intracellular heme levels selectively decreases oxygen consumption in NSCLC cells and inhibits cell migration and colony formation. Experiments are underway to test ways to alter intracellular heme availability and characterize their effects on NSCLC tumor growth and metastasis.

#210

Targeting the mitochondrial DNA polymerase gamma with 2'3'-dideoxycytidine as a novel therapeutic strategy for acute myeloid leukemia.

Sanduni Liyanage, Rose Hurren, Xiaoming Wang, Neil Maclean, Rebecca Laposa, Aaron Schimmer. _University of Toronto, Toronto, Ontario, Canada_.

A subset of AML cells display unique mitochondrial characteristics such as increased mitochondrial DNA (mtDNA), mt mass, and sensitivity to inhibition of mtDNA transcription and protein translation. Given these unique mitochondrial characteristics, we evaluated the effect of inhibiting mtDNA replication by targeting the mtDNA polymerase gamma (POLG). POLG is a nuclear-encoded gene that replicates and repairs mtDNA. POLG mRNA is increased in leukemia cell lines and a subset of AML patients.

We evaluated the preclinical efficacy of targeting POLG using the nucleoside analog 2',3'-dideoxycytidine (ddC), an FDA-approved anti-retroviral drug that cross-reacts with POLG.

Treatment with ddC at 500nM depleted mtDNA by >90%, decreased mtDNA-encoded COXI and COXII proteins and basal oxygen consumption, and induced apoptosis in AML cell lines (OCI-AML2, TEX, K562). ddC further decreased cell viability in a subset of primary AML cells, while normal peripheral blood stem cells (PBSC's) were resistant to ddC in vitro.

Next, we assessed the preclinical efficacy and toxicity of ddC in an OCI-AML2 xenograft model of human AML. Treatment with 35mg/kg of i.p. ddC induced tumor regression and decreased tumor mass by >75% compared to vehicle. No toxicity was observed including changes in liver enzymes or organ histology. We also observed reductions in COXI and COXII protein in tumors but not liver excised from treated mice.

We next investigated the mechanism for preferential sensitivity to ddC in AML cells. A two-fold greater decrease in mtDNA content was observed in AML cells treated with ddC compared to PBSC's, in part explaining the heightened sensitivity. However, the increased sensitivity to ddC in AML was not due to increased mtDNA turnover as mtDNA depletion after ethidium bromide treatment was comparable between AML and PBSC's.

Therefore, we investigated the role of ddC uptake and metabolism. ddC is transported by nucleoside transporters (ENT's, CNT's) and activated by cytoplasmic nucleoside kinases to ddC-triphosphate (ddC-TP). Analysis of Haferlach et. al. (2010) dataset demonstrated that a subset of AML cells have higher mRNA expression of hENT2 and hCNT1, and the nucleoside diphosphate kinases NME1,2,3,5 compared to normal hematopoietic cells. Using mass spectrometry, we observed higher levels of ddC and ddCTP in sensitive AML cells compared to resistant cells. Lastly, knockdown of the deoxycytidine kinase rescued ddC cytotoxicity in TEX cells.

In summary, targeting POLG with ddC displays anti-leukemic activity in both in vitro and in vivo models of AML. The preferential toxicity of ddC is due, in part, to increased rates of uptake and activation in AML along as well as their increased sensitivity to inhibition of oxidative phosphorylation. Thus, POLG inhibitors such as ddC may have clinical utility in a subset of AML.

#211

The mitochondrial protein MTP18 enhances chemosensitivity by promoting mitochondrial fission.

Lynn Htet Htet Aung, Peifeng Li. _University of Illinois at Chicago, Chicago, IL_.

Background: Chemotherapy plays a major role in cancer therapy. Yet, one of its greatest obstacles is the development of chemotherapy resistance. Hence, understanding the molecular basis of chemoresistance is crucial to improve its therapeutic outcome. A close relationship between mitochondrial dynamic and cellular apoptosis has recently been identified. Mitochondrial fission is necessary for initiation of cellular apoptosis, whereas mitochondrial fusion is able to inhibit apoptosis. However, the association of mitochondrial fission and chemo-sensitivity has not been widely explored. Recently, mitochondrial protein MTP18 has been shown to promote mitochondrial fragmentation in various cancer cell lines. However, its role in apoptosis and chemotherapeutic resistance remains elusive. Here, we aimed to detect 1) whether MTP18 is able to regulate mitochondrial fission and apoptosis in gastric cancer cells, and 2) by which mechanism it regulates these events.

Methods and Results: Doxorubicin is one of the most widely used chemotherapeutic agents in variety of cancers. Mitochondrial staining with MitoTracker Red CMXRos revealed doxorubicin was able to induce mitochondrial fission both in vitro and in vivo gastric cancer models. We observed doxorubicin led to a decreasing level of MTP18 expression in gastric cancer cell lines, and this effect was concentration- and time-dependent. To understand the role of MTP18 in doxorubicin-induced mitochondrial fission and apoptosis, endogenous MTP18 expression was knockdown using MTP18-shRNA. Strikingly, doxorubicin could not induce mitochondrial fission in gastric cancer cells transfected with MTP18-shRNA. On the other hand, upon enforced expression of MTP18, the same concentration of doxorubicin sensitized a significantly higher number of gastric cancer cells to undergo mitochondrial fission and apoptosis. These findings suggest that MTP18 is required for doxorubicin-induced mitochondrial fission and apoptosis. In exploring the molecular mechanism of MTP18, we found that MTP18 enriched dynamin-related protein Drp1 accumulation in mitochondria and mediated the signal of doxorubicin to induce mitochondrial fission. Drp1 accumulation in mitochondria caused the release of cytochrome-c to cytosol and subsequently promoted apoptosis.

Conclusion: Taken together, our findings suggest that MTP18 induces mitochondrial fission and enhances apoptosis in gastric cancer cells, by promoting Drp1 accumulation in mitochondria. Thus, it is compelling us to conclude that MTP18 enhances doxorubicin sensitivity of gastric cancer cells by targeting mitochondrial fission machinery.

#212

BPM 31510 synergizes gemcitabine efficacy in pancreatic adenocarcinoma via mechanism independent of its anti-Warburg influence on metabolism.

Tulin Dadali,1 Anne R. Diers,1 Rakib Ouro-Djobo,1 Justin Bourdelais,1 Ezer Benaim,1 Bianca Jambhekar,1 Tony E. Walshe,1 Vivek K. Vishnudas,1 Joaquin J. Jimenez,2 Rangaprasad Sarangarajan,1 Niven R. Narain1. 1 _Berg, LLC, Framingham, MA;_ 2 _University of Miami Miller School of Medicine, Miami, FL_.

Pancreatic adenocarcinoma (PanCa) is associated with poor prognosis and overall survival. Current first-line therapeutics are cytotoxic agents targeting DNA based mechanistic end-points for efficacy, their use limited by dose associated toxicity. There is a critical need for therapeutics with novel mechanisms amenable to combination with current standard-of-care chemotherapy to improve outcomes. BPM 31510 is a drug that targets cellular metabolism networks, effectuating an anti-Warburg effect in cancer. The documented high metabolic phenotype observed in PanCa provided rationale for investigation of BPM 31510 alone and in combination with gemcitabine in in vitro and in vivo PanCa models. Based on BPM 31510 EC50/EC>90 values for MIA-PaCa-2 and Panc-1 PanCa cell lines in vitro, the PanCa cells were significantly more sensitive to BPM 31510 compared to fibroblasts. BPM 31510 treatment in a time- and dose-dependent manner decreased the PI- and Annexin V-negative (viable) population and a concomitant increase in the percentage of Annexin V-positive cells, with PI indicative of early and late apoptosis. The anti-cancer activity of BPM 31510 was assessed in vivo using MIA-PaCa-2 tumor-bearing immune-compromised mice. Treatment with increasing doses of BPM 31510 (0.5-50 mg/kg IP, 3X/week) significantly improved survival outcomes, with the highest dose extending median survival by more than 36 days compared to saline control. Moreover, combined treatment with BPM 31510 and gemcitabine (150 mg/kg IV, 1X/week, given on cycles, 3 weeks on 1 week) resulted in further extension of median survival over either treatment alone. The mechanistic underpinnings for the enhanced efficacy of combination treatment were explored in vitro. In MIA-PaCa-2 cells, co-treatment with BPM 31510 and gemcitabine increased indices of regulated cell death higher than observed for either treatment alone. In contrast, although treatment with either BPM 31510 or gemcitabine alone increased caspase-3 activity, co-treatment did not enhance caspase-3 activation, suggesting that BPM 31510 augments gemcitabine cytotoxicity through independent mechanisms. In fact, BPM31510, and not gemcitabine, increased the mitochondrial uncoupling efficient ratio and Stateapparent in MIA-PaCa-2 cells. Nonetheless, co-treatment with BPM 31510 and gemcitabine synergistically decreased the mitochondrial membrane potential (ΔΨm) in cells prior to cell death. Taken together, these data indicate that BPM 31510-driven bioenergetic alterations are separate from the effects of gemcitabine; however, their effects converge at the mitochondrion to dissipate ΔΨm and activate regulated cell death. The data suggests that combination of BPM 31510 with gemcitabine in pancreatic cancer will effectuate an efficacy response via independent mechanisms with improvement in therapeutic outcome.

#213

Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells.

Wojciech Senkowski, Malin Jarvius, Kim Kultima, Jenny Rubin, Mats Gustafsson, Peter Nygren, Rolf Larsson, Mårten Fryknäs. _Uppsala University, Uppsala, Sweden_.

Inhibition of mitochondrial oxidative phosphorylation (OXPHOS) has recently emerged as a promising strategy for treatment of therapy-resistant cancer cells. These cells often reside within hypoxic tumor regions, where nutrient concentrations are low. Recently, OXPHOS inhibitors have been demonstrated to be toxic to quiescent, nutrient-deprived cells in multicellular tumor spheroids. Such spheroids, formed without medium exchange over the culture period, can serve as an appropriate model to mimic quiescent in vivo tumor regions. These spheroids exhibit low cell proliferation and comprise necrotic cores, contrary to commonly used spheroids cultured with frequent medium change.

We here aimed to characterize how quiescent cells respond to OXPHOS inhibition and thereby identify processes that could be co-targeted for enhanced toxicity. We treated HCT116 colon cancer cell line, grown as monolayer cultures and spheroids, with a range of OXPHOS inhibitors (n=10, including FDA-approved drugs, e.g. nitazoxanide) and other compounds (n=14) at escalating doses and in 4 biological replicates. Then, we obtained global gene expression profiles (n=1149, including 144 vehicle controls) of all treatment conditions using L1000 Gene Expression Profiling method.

We found that upon exposure to OXPHOS inhibitors cells grown as nutrient-deprived spheroids significantly and in dose-dependent manner upregulate expression of genes involved in biosynthesis of cholesterol. This response was not observed for spheroids cultured with medium change or monolayer cell cultures. Thus, we were interested if simultaneous exposure to OXPHOS inhibitors and statins, inhibitors of mevalonate (cholesterol precursor) synthesis, would result in enhanced cytotoxic effects in quiescent, metabolically stressed cells.

We here demonstrate that combination of OXPHOS inhibitors and statins results in pronounced synergistic cytotoxicity in metabolically stressed spheroids. This effect was observed for various classes of OXPHOS inhibitors and different types of statins, indicating that the observed synergy was not a result of off-target effects. This notion was further strengthened by the finding that mevalonate largely abrogated the synergistic effects.

In conclusion, we here report that statins enhance the toxic effects of OXPHOS inhibitors in quiescent, metabolically stressed cells. Our results can serve as a foundation for further studies on targeting therapy-resistant and nutrient-deprived cancer cells by inhibition of OXPHOS. We also demonstrate, for the first time, that the L1000 Gene Expression Profiling can be used to study 3D cell cultures. Importantly, our findings underscore the importance of using a relevant cellular model for target discovery endeavors.

#214

Intracellular ATP depletion in cancer cells by a synthetic ATP-binding protein.

Eddie Khav, Bernardo Chavira, Matt Hamada, Nagaraj Vinay Janthakahalli, Shaleen Korch. _Midwestern University, Glendale, AZ_.

Introduction: Altered cellular energy metabolism is a crucial characteristic of most cancers regardless of tissue or cellular origin. In contrast to normal cells, which generate most of their adenosine triphosphate (ATP) through oxidative phosphorylation, cancer cells are dependent on aerobic glycolysis to meet energy demands (Warburg effect). However, recent studies demonstrate an upregulation in mitochondrial activity (O2 consumption and ATP production) when cancer cells are subjected to stress, such as radiation/chemotherapy treatment. To understand the significance of ATP produced by oxidative phosphorylation in cancer cells, a synthetic ATP-binding protein, DX, was targeted to the mitochondria in HeLa cells to reduce bioavailable ATP. Hypothesis: Reduced available ATP, due to DX in the mitochondria of HeLa cells, will negatively affect cellular metabolism, viability and chemoresistance. Methods: For controlled expression, DX was cloned into the pcDNA™5/TO expression vector under the control of a tetracycline (Tet)-regulated promoter. The DX construct was designed to translocate into the matrix of the mitochondria using the mitochondria targeting sequence and the 3' UTR of a nuclear SOD2 gene. Cultured T-REx™ HeLa cells (which stably express the Tet-repressor protein) were transfected with pcDNA™5/TO::DX-MITO. As a control, T-REx™ HeLa cells were transfected with the parent plasmid, pcDNA™5/TO, and cultured under identical conditions. RT-PCR was performed to confirm DX expression, while western blot analysis was performed to confirm DX localization to the mitochondria. The impact of DX on cell viability and morphology was assessed using a tetrazolium-based colorimetric assay, caspase 3/7 assay, and by light/fluorescent microscopy. To correlate phenotypic change with DX activity, the level of bioavailable ADP /ATP was measured at specific time points following DX induction. Results: RT-PCR and western blot analysis confirmed DX expression and translation in HeLa cells, respectively, following Tet induction. Cells expressing DX experienced a significant reduction in viability and alteration in morphology compared to control HeLa cells. Contemporaneous with DX expression, bioavailable ATP decreased and the levels of caspases 3/7 increased suggesting that a majority of cells undergo apoptosis in response to energy crisis. Conclusion: Reduced intracellular ATP levels, mediated by a synthetic ATP-binding protein, negatively affects the viability of cervical cancer cells. The consequence of this on biochemical pathways, mitochondrial function and cell viability requires further investigation. We suggest DX, could be used to control and regulate energy metabolism and represents a new approach for cancer chemotherapy.

#215

Metabolic and mitochondrial consequences of p53 upregulation and their relation to cell fate outcomes.

Yau Christina, Katya Frazier, Daniel Rothschild, Shona Mookerjee, Martin Brand, Gary Scott, Christopher C. Benz. _Buck Institute for Research on Aging, Novato, CA_.

Background: p53 regulates a plethora of biological processes including cellular metabolism and mitochondrial function. Using the small molecule MDM2 agonist MI-63, we previously demonstrated differential p53 upregulation and induction of global metabolomics profiles in malignant MCF-7 and non-malignant MCF-10A mammary epithelial cells undergoing different cell fates (apoptosis vs. cytostasis). In particular, p53 upregulation suppressed glycolytic metabolite pools in MCF-10A, while increasing their levels in MCF-7 cells. We postulated that this opposing metabolic response may be linked to their different cell fate outcomes. In this study, we evaluated the association between p53 induced effects on live cell metabolic flux and cell fate using different p53wt cell lines subjected to comparable p53 upregulation.

Methods: We employed four p53wt cell lines: MCF-7, ZR-75-1, SJSA-1 and MCF-10A. Cell fate outcomes upon p53 upregulation (24hr, 10μM MI63) were assessed by Western blotting for cytostasis (p21) and apoptosis markers (cleaved PARP, PUMA). Expression changes in enzymes involved in proline metabolism were similarly measured. Real-time measurements of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) under control and MI-63 treatments were assessed by the Seahorse Extracellular Flux Analyzer, and specific acidification contributions from respiration and glycolysis to total ECAR were computed.

Results: p53 upregulation induced apoptosis in the malignant MCF-7, ZR-75-1 and SJSA-1 cells, but not in non-malignant MCF-10A cells. MI-63 treatment decreased basal OCR in MCF-7 cells (t-test p =0.046), but not the other cell lines. No significant differences in maximum OCR were observed between control and MI-63 treatments in any of the cell lines tested. As well, p53 upregulation did not produce any significant change in total ECAR. However, in MCF-7 cells, glycolytic ECAR was significantly increased (MI-63/Control: 4.36, Tukey test p = 0.001), consistent with our earlier metabolomics conclusions. In contrast, no significant changes in glycolytic ECAR were produced in MCF-10A, ZR-75-1 or SJSA-1 cells (MI-63/Control: 1.27, 1.32 and 1.33 respectively). Interestingly, p53 upregulation induced expression of proline dehydrogenase (PRODH) in all three malignant cell lines, but not in MCF-10A cells; and coordinated changes in other proline catabolizing enzymes (P5C dehydrogenase, P5C reductase) were seen in MCF-7 cells.

Conclusion: These findings indicate that p53 induced effects on oxygen consumption and glycolysis are cell context dependent but are not likely responsible for the different cell fate outcomes observed following p53 upregulation. Rather, our evidence suggests a potential mechanistic link between p53 induced effects on proline metabolism and cell fate outcomes, indicating that this stress-activated mitochondrial pathway deserves further study.

#216

The effect of UCP2 upregulation on cellular redox status and mitochondrial respiration.

Annapoorna Sreedhar, Yunfeng Zhao. _Louisiana State University Health Sciences Center, Shreveport, LA_.

Uncoupling protein 2 (UCP2) is a mitochondrial protein present in the inner mitochondrial membrane. They belong to the family of anion mitochondrial carriers. UCP2 are thought to have natural antioxidant effect by attenuating the generation of superoxides.

Since cancer cells exhibit elevated oxidative stress, they can effectively adapt to this situation by upregulating UCP2. As a matter of fact, UCP2 is overexpressed in many forms of cancer including prostate, breast, lung and head & neck. Not surprising, our early studies demonstrate that knockout of UCP2 suppresses skin tumor formation in a multistage skin carcinogenesis model.

The purpose of this study was to detect the impact of UCP2's upregulation on mitochondrial redox status and mitochondrial respiration, using the well characterized tumor promotion model, JB6p+ cells. Our results have demonstrated that UCP2 overexpression enhanced protein expression and activity levels of manganese superoxide dismutase (MnSOD), a mitochondrial-resident enzyme that governs the egression of superoxides. To assess the impact of MnSOD upregulation on mitochondrial redox status, levels of superoxides and hydrogen peroxides were detected. Our results indicated that MnSOD upregulation maintained steady flow of hydrogen peroxides originating from mitochondria. We further examined the effect of UCP2 upregulation-induced MnSOD expression on mitochondrial respiration and glycolytic rate. In agreement with our data, cells overexpressing UCP2 had significantly higher oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). In conclusion, our data suggests that UCP2 promotes carcinogenesis via decreasing superoxide generation whereas promoting hydrogen peroxide production via elevating MnSOD. Collectively, our results support the hypothesis that enhanced UCP2 expression sustains metabolic switch in cancer cells.

#217

Mitochondrial reprogramming regulated cancer pathway in triple negative breast cancer.

Jun H. Park,1 Sajna Vithayathil,1 Danli Wu,1 Vasanta Putluri,1 Pi-Lin Sung,1 Efrosini Tsouko,2 Vadiraja B. Bhat,3 Cristian Coarfa,1 Daniel E. Frigo,2 Michael T. Lewis,1 Arun Sreekumar,1 Patricia Yotnda,1 Chad J. Creighton,1 Nagireddy Putluri,1 Lee-Jun C. Wong,1 Benny A. Kaipparettu1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Houston, Houston, TX;_ 3 _Agilent Technologies, Wilmington, DE_.

Compared to other subtypes of tumors, triple negative breast cancers (TN BCa) currently suffer from limited knowledge on its etiology and treatment options. Transmitochondrial cybrids (cybrid) and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in TN BCa. Analysis of cybrids and established BCa cell lines showed that metastatic TN BCa maintain high levels of ATP through fatty acid β-oxidation and activate Src oncoprotein by its autophosphorylation. Inhibition and induction of β-oxidation including the shRNA mediated knockdown strategies, and analysis of patient derived xenograft (PDX) models confirmed the role of mitochondrial β-oxidation in Src activation and metastasis. Analysis of BCa clinical data further reaffirmed the role of mitochondrial β-oxidation in Src regulation and their significance in BCa metastasis. This study is innovative in showing the mitochondrial reprogramming mediated regulation of a major cancer pathway by its post-translation modification.

#218

Ewing sarcoma gene, EWS, role in mitochondrial homeostasis.

Jun Hong Park. _Institute for Basic Science, Ulsan, Republic of Korea_.

Peroxisome proliferator-activated receptor γ coactivator (PGC-1α) is a central regulator of mitochondria biogenesis, function and cellular energy metabolism. PGC-1α protein is intrinsically unstable but the mechanism by which PGC-1α stability is regulated is not well understood. Here, we report that a multifunctional protein EWS (Ewing sarcoma) is critical for PGC-1α protein stability and mitochondrial homeostasis. EWS inactivation results in a rapid degradation of PGC-1α via ubiquitin-dependent proteasome pathway and reduces mitochondrial abundance. Re-introduction of EWS in Ews-deficient cells restores PGC-1α expression and mitochondrial anomalies. In the absence of Ews, expression of E3 ubiquitin ligase, FBXW7 (F-box/WD40 domain protein 7), is increased and depletion of Fbxw7 in Ews-null cells restores PGC-1α expression and mitochondrial density. Consistent with these findings, mitochondrial abundance and activity are significantly reduced in brown fat and skeletal muscles of Ews-deficient mice and expression of slow myosin heavy chain is significantly decreased in skeletal muscles of Ews-/- mice compared to controls. Furthermore, Ews-null mice display reduced expression of mitochondrial biogenesis, respiration and fatty acid β-oxidation genes in the liver and abnormally high serum lactate levels. These results demonstrate a novel role of EWS in mitochondrial and cellular energy homeostasis by controlling PGC-1α protein stability.

#219

PHF20 induced necrotic like cell death mediated by ROS via enhanced mitochondrial biogenesis.

Jisoo Park,1 Yuwen Li,2 Gyeyeong Kong,1 Kisun Mun,1 Hyunji Lee,1 Dohoon Kim,1 Quangdon Tran,1 Ming Wang,1 Bingjie Peng,1 Youngeun Hong,1 Gang Min Hur,1 Jongsun Park1. 1 _Chungnam National Univ. College of Med., Daejeon, Republic of Korea;_ 2 _Fourth Military Medical University, Shaanxi, China_.

The importance of PHD finger protein 20 (PHF20) in the cellular process is recently emerging as a regulator for the p53 and NF-kB signaling as well as the reprograming process of induced pluripotent stem cell. In this study, we have provided the clear evidence that PHF20 exhibit the novel function in tumorigenesis by regulating the mitochondria biogenesis. Overexpression of PHF20 increases mitochondria biogenesis via up-regulation of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α) in HCT116 cells. PHF20 overexpression enhances mitochondrial respiratory capacity and oxygen consumption, leading to the accumulation of reactive oxygen species (ROS). Accumulated ROS by PHF20 causes the necrotic like cell death in HCT116 cells. The measurement of mitochondrial ROS in the cells expressing PHF20 revealed that excessive production of mitochondrial ROS facilitates the formation of mitochondrial permeability transition pore, resulting in the loss of mitochondrial membrane potential. PHF20-mediated ROS accumulation damages DNA and leads to over-activation of PARP but not caspase. Antioxidants and PARP-1 inhibitor are able to rescue PHF20 induced cell death, whereas z-VAD, pan-caspase inhibitor, had no effects. Immunohistochemical analysis of various human cancer revealed that PHF20 expression is highly elevated in most of tumor compared to normal tissue. Taken together, PHF20 appears to be tumor-specific antigen and PHF20-mediated PGC1-α upregulation is responsible for the elevation of intracellular ROS generation, resulting the necrotic like cell death. Thus, this study provided the new concept for treating glioma patients and novel pharmacological target in various cancers including glioblastoma and hepatocellular carcinoma

#220

SR4 uncouples mitochondrial oxidative-phosphorylation, modulates AMPK-mTOR signaling, and inhibits proliferation of HepG2 hepatocarcinoma cells.

Sharad S. Singhal, James Figarola, Jyotsana Singhal, Joshua Tompkins, David Horne, Sanjay Awasthi, Arthur Riggs. _City of Hope, Duarte, CA_.

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration with the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as promising targets for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential (MMP) in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol, but not cyclosporin A, and were non-existent in mitochondria-DNA depleted HepG2 (po) cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase (ANT) and uncoupling proteins, decreases MMP, and promotes swelling of valinomycin-treated mitochondria in potassium acetate media. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin (mTOR) signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down regulation of cell cycle, mitochondrial and oxidative-phosphorylation pathway transcripts at 24 h post treatment. Collectively, our studies demonstrate that SR4's previously reported indirect activation of AMPK and in-vitro anticancer properties, as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity (Supported by NIH grant CA 77495 and Beckman Research Institute of the City of Hope).

#221

Mitochondrial genome and the risk of lung cancer: The Multiethnic Cohort.

Li Tao,1 Yuqing Li,1 Sung-Shim Lani Park,2 Kenneth B. Beckman,3 Christian Caberto,4 Annette Lum-Jones,4 Loic Le Marchand,4 Daniel O. Stram,2 Richa Saxena,5 Iona Cheng1. 1 _Cancer Prevention Institute of California, Fremont, CA;_ 2 _Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA;_ 3 _University of Minnesota Genomics Center, Minneapolis, MN;_ 4 _Epidemiology Program, University of Hawaii Cancer Center, Honolulu, HI;_ 5 _Department of Genetics and Medicine, Harvard Medical School, Boston, MA_.

Background: The human mitochondrial genome (mtDNA) encodes for 13 essential polypeptides of the mitochondrial respiratory complex. Maternally-inherited variation in mtDNA may influence cancer development by altering mitochondrial proteins and complexes involved in the oxidative phosphorylation (OXPHOS) process. In lung cancer, Complex I appears frequently altered, which has been linked to increased cellular proliferation and invasion, and overproduction of reactive oxygen species. Few studies have investigated the associations between mtDNA variants and lung cancer risk.

Methods: Within the 16 kb mtDNA, we tested 186 mitochondrial single nucleotide polymorphisms (mtSNPs) located in 13 genes that comprise the 4 complexes of the OXPHOS pathway and mtSNPs for rRNA and tRNA in 773 lung cancer cases and 10,491 controls from the Multiethnic Cohort. Logistic regression was conducted to examine the odds ratio (OR) and corresponding 95% confidence interval (CI) for mtSNPs and haplogroups associated with lung cancer risk. Associations were adjusted for age, sex, maternal self-reported race/ethnicity, principal components of global ancestry, smoking status, and the number of cigarettes smoked per day. The sequence kernel association test was conducted for pathway analysis, adjusting for the same covariates. Stratified analysis was conducted by self-reported maternal race/ethnicity—African American (AA), Japanese American (JA), Latino (LA), European American (EA), and Native Hawaiian (NH).

Results: Overall, the most significant lung cancer association was seen with mt15629 (OR: 0.36, 95% CI: 0.22-0.60; p=8x10-5 that reached a Bonferroni significance threshold of p=2.7x10-4). This association was largely driven by AA (OR: 0.41, 95% CI: 0.24-0.71; p=1.6x10-5; MAF=0.05) and LA (OR: 0.14, 95% CI: 0.03-0.54; p=4.8x10-5; MAF=0.007) data with no evidence of heterogeneity in effects by race/ethnicity. Mt15629 was monomorphic in JA, EA, and NH populations. Similar patterns of associations were seen across histological cell types (small cell carcinoma, squamous cell carcinoma, and adenocarcinoma). Furthermore, mt15629 showed no evidence of heterogeneity in effects by smoking status and was not associated with smoking status or cigarette pack years. Haplogroup L1 was associated with lung cancer risk among LAs (OR: 0.14, 95% CI: 0.04-0.48; p=0.002) for which mt15629 is one of the defining haplogroup mtSNPs. Pathway analysis showed an association between the cumulative effect of 161 polymorphic mtSNPs among LAs (p=0.002); also the OXPHOS pathway (p=0.002), 4 mitochondrial complexes (p=0.001-0.049) and 10 of 15 mitochondrial genes (p=0.001-0.049) were associated with lung cancer risk among LAs.

Conclusion: Our findings demonstrate an association between specific variants in the mitochondrial genome and lung cancer risk, particularly among Latinos. The mechanism underlying this association appears to be independent of smoking status.

#222

Inhibitors of mitochondrial transcription (IMT) specifically affect cancer cell proliferation.

Tim Bergbrede,1 Axel Choidas,1 Raffaela Di Lucrezia,1 Sascha Menninger,1 Anke Unger,1 Koch Uwe,1 Peter Nussbaumer,1 Nina Bonekamp,2 Emily Hoberg,3 Victor Posse,3 Andrea Felser,4 Maria Falkenberg,3 Nils Göran Larsson,2 Claes M. Gustafsson,3 Bert M. Klebl1. 1 _Lead Discovery Center GmbH, Dortmund, Germany;_ 2 _Max Planck Institute for Biology of Ageing, Cologne, Germany;_ 3 _University of Gothenburg, Gothenburg, Sweden;_ 4 _Karolinska Institutet, Stockholm, Sweden_.

Mitochondria contain a unique DNA genome (mtDNA) that encodes a number of key components of the respiratory chain. Levels of mtDNA gene expression correlate with the metabolic requirements of mammalian cells. Recently, the role of mitochondrial function for the development and progression of cancer has been highlighted. Oncogenes have been identified to redirect mitochondrial functions to increase cancer cell survival. Increased levels of oxidative phosphorylation have also been implicated in cancer cell resistance towards drugs. In agreement with this notion, compounds that inhibit respiration may specifically target such resistant cells. Finally, it was demonstrated that inhibition of mitochondrial gene expression leads to apoptosis in rapidly dividing cells. Based on these observations, we present a completely novel principle different from known electron transfer chain blockers to treat cancer by inhibiting mtDNA transcription. To this end a study to identify and characterize inhibitors of mitochondrial transcription (IMTs) with lead-like properties was initiated. A high throughput biochemical screening campaign was performed identifying small molecular weight inhibitors of PolRMT, the mitochondrial RNA polymerase, which controls mtDNA transcription. Upon hit optimization, potent compounds with biochemical and cellular IC50s in the low nanomolar range have been identified. IMTs displayed good physicochemical and pharmacological properties. They have been shown to be inactive on other RNA polymerases proving their excellent specificity. Testing IMTs in assays for cancer cell proliferation revealed potent inhibition of sensitive cell lines (double digit nanomolar range). Interestingly, none of the tested primary non-cancer cell lines was affected by the IMTs, indicating a good therapeutic window. Testing selected IMTs in pharmacokinetic studies in vivo revealed high exposures and no detectable adverse effects (single and repeated dosing). Medicinal chemistry optimization will help to identify candidate compounds for efficacy testing in xenograft models.

#223

Comparison of human-specific versus cross-reactive Complex I inhibitor for in vivo tumor efficacy.

Sven Christian, Carolyn Algire, Wolfgang Schwede, Jeffrey S. Mowat, Alexander Ehrmann, Stephan Menz, Marcus Bauser, Andrea Haegebarth. _Bayer Pharma AG, Berlin, Germany_.

Mitochondria are both key regulators of energy supply and apoptotic cell death. The mitochondrial electron transport chain (ETC) consists of four enzyme complexes that transfer electrons from NADH to oxygen. During electron transfer, the ETC (Complex I to IV) pumps protons into the inter-membrane space, generating a gradient across the inner mitochondrial membrane that is used by Complex V to drive ATP synthesis. Recent publications have shown that tumor cells harboring specific mutations (LKB1, mIDH and others) are more sensitive to Complex I inhibition, compared to cells that do not have these mutations. We have identified an optimized human/mouse cross-reactive Complex I inhibitor that allows profiling of Complex I inhibitors in pharmacological models. We have pursued different approaches based on the literature, an unbiased screen and in-house results generated with the human-specific Complex I inhibitor BAY 872243 to identify sensitive in vivo tumor models. However, using the cross-reactive Complex I inhibitor we were unable to identify sensitive models apart from weakly sensitive LKB1-deficient tumors (A549, G361) when animals were treated at maximum tolerated dose (MTD). In addition, all approaches for combination therapy failed to improve efficacy in vivo. Direct comparison of human-specific Complex I inhibitor BAY 87-2243 and cross-reactive inhibitor BAY179 in a sensitive LKB1-deficient melanoma model, G361, demonstrated that inhibition of Complex I specifically in the tumor is a valid approach as it results in tumor growth inhibition of ~50%. However, cross-reactive compounds do not reach exposures at MTD to generate comparable effects.

#224

The role of the microphthalmia associated transcription factor in autophagy regulation in melanoma.

Katrin Moller,1 Sara Sigurbjornsdottir,1 Remina Dilixiati,1 Solveig H. Brynjolfsdottir,1 Lionel Larue,2 Vesteinn Thorsson,3 Eirikur Steingrimsson,1 Margret H. Ogmundsdottir1. 1 _University of Iceland, Reykjavik, Iceland;_ 2 _Institute Curie, Paris, France;_ 3 _Institute for Systems Biology, Reykjavik, Iceland_.

Autophagy is considered a therapeutic target in various cancer types, including melanoma. The transcription factors TFEB and TFE3 have been shown to play a key role in autophagy regulation in various cell types by affecting gene expression required for this process. A close relative to these factors is the transcription factor MITF; often termed the master regulator of melanocyte development and function. MITF activity has also been shown to be important for various stages of melanoma formation and progression.

Analysis of The Cancer Genome Atlas expression data from melanoma tumors reveals that MITF expression correlates with lysosomal and autophagy gene expression in metastatic tumors. Furthermore, analysis of expression data from 23 melanoma cell lines shows that MITF expression correlates with lysosomal and autophagy gene expression, and the analysis of available MITF ChIP sequencing data shows that MITF binds the promoters of lysosomal and autophagy genes. This binding was experimentally verified by ChIP experiments in 501Mel melanoma cells. MITF over-expression and knock down experiments show effects of MITF on lysosomal and autophagy gene expression, as well as an effect on the autophagy activity.

Autophagy is high in melanoma and has been linked to tolerance against cancer treatment. We have found that MITF regulates autophagy genes and activity in melanoma cell lines. In light of both MITF and autophagy being considered a therapeutic target in melanoma, this relationship needs to be taken into consideration.

#225

**Allelic variations in** MnSOD **and** GPx-1 **affect metabolism, mitochondrial membrane potential and expression of signaling proteins.**

Dede N. Ekoue, Soumen Bera, Emmanuel Ansong, Peter Hart, Virgilia Macias, Andre Kajdacsy-Balla, Marcelo Bonini, Alan M. Diamond. _University of Illinois at Chicago, Chicago, IL_.

MnSOD detoxifies superoxide and impacts tumor biology by generating H2O2 which can diffuse through the mitochondrial membrane and affect metabolism and apoptosis. The selenium-dependent enzyme GPx-1 localizes to both the cytoplasm and the mitochondria and reduces H2O2 to water and may therefore modulate the impact of MnSOD on carcinogenesis. A val to ala polymorphism in codon 16 of the MnSOD gene has been shown to be associated with increased prostate cancer risk in men with the lowest level of dietary antioxidant intake and individuals who consumed less dietary antioxidants, including selenium, had the greatest risk of prostate cancer (Li et al, 2005). A polymorphism in the GPx-1 gene resulting in a leucine (L) instead of a proline (P) at position 198 has frequently been reported to be associated with elevated cancer risk. To examine the molecular mechanism behind the epidemiological observation that genotypes in GPx-1 gene modify elevated risk of cancer associated with MnSOD genotypes, MCF-7 human breast cancer cells null for GPx-1 and having negligible levels of endogenous MnSOD were transfected with GPx-1 and MnSOD allele-specific expression constructs to determine outcomes related to cellular signaling and metabolism. MnSOD and GPx-1 alleles differentially interacted to modulate the expression of the anti-oxidant stress response regulator Nrf2, the cell adhesion protein E-Cadherin, the cell signaling protein pAkt, the anti-apoptotic protein Bcl-2 and the mitochondria located deacetylase Sirt3. Also, co-expression of the GPx-1A7L and either MnSODval or MnSODala increased oxidative phosphorylation as measured by O2 uptake using the Seahorse XF24 XF analyzer, which simultaneously determines relative mitochondrial respiration and glycolysis. In order to assess whether GPx-1 and MnSOD allelic variants modulate mitochondrial membrane potential, CMX-ROS fluorescence was measured. Independent and irrespective of which allele was evaluated, MnSOD and GPx-1 individually decreased mitochondrial potential as compared to that seen using MCF-7 control cells. In addition, co-expression of GPx-1A7L with either MnSODval or MnSODala further decreased membrane potential above that observed for either MnSOD and GPx-1 alone. In order to determine if MnSOD and GPx-1 levels are associated with a higher risk for biochemical recurrence of prostate cancer after radical prostatectomy, immunohistochemistry of human prostate tissue cores will be performed. Preliminary results indicate that a high MnSOD/ GPx-1 ratio was observed in prostate cancer tissue compared to adjacent normal tissue.

#226

PI3K pathway inhibition induces a different metabolic response in NSCLC cells harboring WT and G12C mutant KRAS.

Elisa Caiola, Laura Brunelli, Mirko Marabese, Roberta Pastorelli, Monica Lupi, Massimo Broggini. _IRCCS- Istituto Di Ricerche Farmacologiche 'Mario Negri', Milan, Italy_.

Lung cancer is the most common cancer worldwide and, because of its high fatality, it also represents the most common cause of cancer related deaths. Non-small-cell lung cancers (NSCLCs) constitute about 85% of lung cancers and the 5-year survival rate is currently below 20%. Only a limited number of NSCLC patients, carrying alterations in specific genes, can benefit from small molecules therapies, highlighting the need for new treatment strategies to improve the outcome of the remaining patients.

KRAS is among the most frequently mutated oncogene in NSCLC and 90% of the mutations affect codon 12, with the G12C substitution being the most frequent. KRAS mutations in NSCLC are supposed to be associated to a poor prognosis and poor response to chemotherapy. Despite KRAS being one of the earliest known oncogenic drivers in NSCLC, it is considered practically undruggable. Mutations in KRAS gene lead to the activation of PI3K/akt/mTOR pathway, whose effective inhibition remains a challenging clinical target.

Since PI3K/akt/mTOR pathway and KRAS oncogene mutations have both a role in cancer cell metabolism, we investigated whether the activity of PI3K/akt/mTOR inhibitors (BEZ235 and BKM120) in cells harboring different KRAS status is related to their effect at metabolic level.A targeted metabolomics approach was applied to a robust and well characterized isogenic system to profile the metabolic adaptations occurring in KRAS-G12C and KRAS-wild type (WT) cells in response to treatment with BEZ235 and BKM120. KRAS-G12C and KRAS-WT harboring cells displayed similar sensitivities to BEZ235 and BKM120 treatments. Metabolomics analysis indicated that the different KRAS isoforms induced a distinct metabolic response to PI3K inhibitor treatments. In particular, impairment of glutamine and serine metabolism, in KRAS-G12C and KRAS-WT respectively, was observed after pharmacological blockade of the PI3K signaling. In addition, PI3K inhibitors caused autophagy in KRAS-WT isoform, but not in KRAS-G12C, where a striking decreased in ammonia production was found as probable consequence of glutamine metabolism impairment. At doses relevant for autophagy, differences in apoptosis and cell cycle phase distribution were not detected in KRAS-WT and KRAS-G12C clones.

These preliminary results would set the basis for more effective therapeutic combinations possibly exploiting the different metabolic response to PI3K/akt/mTOR inhibitors in NSCLC cells harboring a different KRAS status.

#227

Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma.

Viola Lanier. _Morehouse School of Medicine, Acworth, GA_.

Background: The tissue microenvironment plays a major role in tumor development and recurrence. It is known that leptin secreted by cancer and adipose cells can modify the tumor stroma, which is composed of immune, fibroblast, and endothelial cells (EC). Leptin affects both normal and cancer associated fibroblasts. We have previously shown that leptin can modify EC directly via an increase of angiogenic features; which was linked to VEGFR signaling and Notch expression independent of VEGF. It is hypothesized that leptin induces a communication between fibroblast and endothelial cells via induction of Notch signaling, which may be related to exosome production.

Methods: Non-expressing Ob-R fibroblasts, and fibroblasts transfected with Ob-R plasmid, were used to investigate leptin's actions on Notch activation. Fibroblasts were challenged with leptin (0.6, 1.2, 6.2 nM) and an inhibitor of Notch signaling activation (DAPT). Fibroblast culture supernatants were added to and human umbilical vein endothelial cells (HUVEC) cultures. Notch proteins and RNA expression and molecular markers of activated fibroblasts and derived exosomes were determined via Western blot, RT-PCR, and multiplex analyses, respectively. EC angiogenic features were also determined.

Results: Our findings suggest that leptin induces fibroblast activation, which triggers the expression of Notch and the development of angiogenic features by EC.

Conclusions: A novel mechanism is proposed by which leptin promotes tumor stroma development via Notch activation and a crosstalk between fibroblasts and EC. Cancer therapies targeting leptin signaling could be a new strategy for interfering with cellular processes that promotes changes of tumor stroma towards a more aggressive tumor environment.

#228

Tumor suppressor functions of BNIP3 and mitophagy in pancreatic ductal adenocarcinoma.

Aparajita H. Chourasia, Maya Z. Springer, Kay F. Macleod. _University of Chicago Ben May Department for Cancer Research, Chicago, IL_.

The role of mitochondrial dysfunction in cancer is an emerging research field fuelled by the growing realization that mitochondrial dynamics are subject to oncogenic control, with mitochondrial biogenesis and mitophagy in particular modulated directly or indirectly by key oncogenes (c-Myc, B-Raf, K-ras) and tumor suppressors (p53, pRB). Work from our lab has focused on understanding how defects in mitophagy influence tumorigenesis with a particular focus on how BNIP3 loss during tumor progression influences disease outcome. BNIP3 functions as a dimer at the outer mitochondrial membrane to target mitochondria for degradation at the autophagolysosome through its interaction with processed LC3 at nascent phagophores. BNIP3 is a transcriptional target of HIF-1, E2F and FoxO3A transcription factors and is induced by activated Ras and suppressed by the RB and p53 tumor suppressors. Various studies have shown BNIP3 expression to be induced at early stages of human cancers and then lost as cancers progress to malignancy. In particular, epigenetic silencing of BNIP3 in human pancreas cancer has been correlated with reduced median survival. However, the mechanisms by which loss of BNIP3 may be contributing to cancer progression are not well delineated and a better understanding of how BNIP3-dependent mitophagy modulates pancreatic cancer in particular could provide new insight to the development of this deadly disease. We set out to examine how loss of BNIP3 affects the progression of pancreatic ductal adenocarcinoma using human cell lines and genetically engineered mouse models. We show that loss of BNip3 markedly accelerates tumor initiation and progression to invasive PDAC in the Pdx1-Cre;LSL-KRasG12D mouse model with metastases to the liver and lungs evident in BNip3 null mice but not in age-matched wild-type mice. Supporting a role for BNIP3 in suppressing pancreatic tumorigenesis, we also showed that re-expression of exogenous BNIP3 in Miapaca-2 cells (in which endogenous BNIP3 is silenced) resulted in reduced growth while knockdown of BNIP3 in CFPAC1 cells (that express BNIP3) promotes cell growth. Work described here examines the underlying mechanisms explaining these tumor suppressor functions of BNIP3 in pancreatic ductal adenocarcinoma.

#229

A metabolic switch controls cell fate decision-making in intestinal differentiation downstream of the tumor suppressor adenomatous polyposis coli (apc).

Imelda T. Sandoval,1 Richard Glenn C. Delacruz,1 Braden N. Miller,1 Christeena Satterfield,1 Kristophor Olson,2 Shauna Hill,3 Holly Van Remmen,3 Jared Rutter,2 David A. Jones1. 1 _Functional and Chemical Genomics, Oklahoma Medical Research Foundation, Oklahoma City, OK;_ 2 _Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT;_ 3 _Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK_.

Mutation in the tumor suppressor Adenomatous Polyposis Coli (APC) is the primary initiating mutation in a majority of colon tumors. Recent studies have shown that APC positively correlates with Mitochondrial Pyruvate Carrier 1 (MPC1), a crucial player in pyruvate metabolism and has been reported to repress the Warburg effect and growth in colon cancer cells. Utilizing the zebrafish to examine the relationship between apc and mpc1, we found that mpc1 expression is reduced in embryos harboring a genetic mutation (apcmcr) or diminished expression (apc mo) of apc. Antisense morpholino knockdown of mpc1 in wild type embryos (mpc1 mo) resulted in an array of developmental defects that recapitulated phenotypes of impaired apc function including failed intestinal differentiation. This was accompanied by mitochondrial dysfunction in mpc1 mo as evidenced by a decrease in metabolic respiration and triglyceride levels. We also observed altered mitochondrial function and dysregulation of enzymes involved in pyruvate metabolism in apcmcr and apc mo. Moreover, hMPC1 RNA rescued intestinal differentiation in both zebrafish models of apc deficiency. Meta-analyses using TCGA human tumor samples corroborated our findings in the zebrafish. Our data demonstrate a novel role for apc in pyruvate metabolism through regulation of mpc1 that drives normal intestinal differentiation and support the broader idea that metabolic changes can dictate cell fate programs.

#230

The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation.

Feven Tameire, Maria A. Monroy, Constantinos Koumenis. _University of Pennsylvania, Philadelphia, PA_.

The ability of cancer cells to adapt to non-cell autonomous (extrinsic) and cell autonomous (intrinsic) stresses is critical for maintaining cell viability and growth. The Integrated Stress Response (ISR) pathway is essential in cancer cell adaptation to extrinsic stresses such as hypoxia and nutrient deprivation both in vitro and in vivo. The ISR kinases, PERK and GCN2, phosphorylate eIF2α, transiently inhibiting protein translation during times of stress. Phosphorylation of eIF2α promotes the preferential translation of the transcription factor ATF4, which activates a gene expression program that enhances uptake and synthesis of amino acids, antioxidant defense and chaperone expression to promote recovery from nutrient deprivation and ER stress. Here, we demonstrate the importance of ISR signaling in MYC oncogene induced intrinsic stress both in vitro and in vivo.

We previously reported that activation of PERK, during c-Myc induction is vital for activating cytoprotective autophagy and suppressing c-Myc induced apoptosis. Here, we demonstrate GCN2 is also robustly activated during c-Myc induction. GCN2 activation after c-Myc induction regulates ATF4 transcription and translation. ATF4 was necessary for survival and suppression of oxidative stress during c-Myc activation. We have identified ATF4 targets that regulate amino acid uptake and involved in amelioration of oxidative stress. Furthermore, siRNA mediated knockdown or genetic knockout of GCN2 was synthetic lethal with c-Myc activation. Interestingly, PERK is still able to phosphorylate eIF2α in the absence of GCN2. However, eIF2α phosphorylation was completely inhibited in the absence of both kinases suggesting that these kinases compensate for each other and both are required for optimal phosphorylation of eIF2α after c-Myc activation. Consistent with our in vitro data, we observe higher levels of p-eIF2α and ATF4 in lymphoma cells compared to WT B cells in vivo in a mouse model of Myc driven lymphoma. These findings highlight a novel mechanism of stress response pathway activation to support c-Myc driven tumorigenesis involving an amino acid sensor kinase GCN2.

#231

Tumor microenvironment regulate cancer cells metabolomics.

Hongyun Zhao,1 Lifeng Yang,1 Joelle Baddour,1 Abhinav Achreja,1 Vincent Bernard,1 Tyler Moss,2 Juan C. Marini,3 Thavisha Tudawe,1 Anthony S. Lucas,4 Hector Alvarez,4 Sonal Gupta,4 Sourindra N. Maiti,5 Laurence Cooper,5 Donna Peehl,6 Prahlad T. Ram,2 Anirban Maitra,4 Deepak Nagrath1. 1 _Rice University, Houston, TX;_ 2 _Department of Systems Biology, University of Texas, MD Anderson, Houston, TX;_ 3 _Baylor College of Medicine, Houston, TX;_ 4 _Departments of Pathology and Translational Molecular Pathology, Ahmad Center for Pancreatic Cancer Research, Houston, TX;_ 5 _Department of Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _Stanford University, Stanford, CA_.

Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment (TME) in cancers development. Altered cellular metabolism is a hallmark of cancer. Until now, much of the published literature has focused on neoplastic cell-autonomous processes for these metabolic adaptations and less is known for the causes due to the interactions between cancer and its microenvironment. Here, we demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. Through 13C-labeled experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells and help them proliferate well. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA cycle intermediates, and these molecules are avidly utilized by cancer cells for central carbon metabolism. In addition to the direct provision of exogenous metabolites as cargo, we also provide evidence that metabolites derived from exosomes can promote tumor growth under nutrient deprivation conditions. Taken together, our work reveals a novel role for the TME in regulating the metabolic adaptation in cancer cells and uncovers the underlying mechanism of this regulation.

#232

Pharmacological and genetic dissection of one carbon pathway provides novel insights into generation, transformation and utilization of one carbon units.

Vipin Suri,1 Nello Mainolfi,1 Adam Friedman,1 Mikel P. Moyer,1 Greg Ducker,2 Joshua D. Rabinowitz,2 Mark Manfredi1. 1 _Raze Therapeutics, Cambridge, MA;_ 2 _Princeton University, Princeton, MA_.

Tumorigenesis, tumor progression and metastasis require metabolic rewiring to support the need for rapid biomass accumulation, reduction of oxidative stress and epigenetic remodeling. One carbon (1C) metabolism provides essential substrates for such metabolic rewiring and enzymes of 1C pathway are frequently overexpressed in tumors consistent with the increased demands on these biochemical pathways. While 1C pathway modulators such as antifolates are efficacious, the clinical utility of antifolates is limited by their inability to distinguish between the metabolic requirements of proliferating cells vs transformed cancer cells. Precise targeting of 1C metabolism in transformed cells can potentially lead to highly effective and safe anticancer therapies.

The enzymes of 1C metabolism are organized in a spatially distinct set of parallel reactions in the cytosol and the mitochondrion. The core 1C machinery extracts 1C units from donors such as serine onto a folate backbone. The oxidation state of the folate associated 1C unit can then be manipulated according to the needs of the 1C utilization reactions. The quantitative contribution of cytosolic vs mitochondrial pathway, regulation of 1C metabolism and biochemical basis of partitioning of 1C units between different downstream reactions is not fully understood. To understand the role of the mitochondrial and cytosolic pathways in the generation and transformation of 1C units, we generated potent and selective inhibitors of MethyleneTetraHydroFolate Dehydrogenase Type 2 (MTHFD2) and dual inhibitors of Serine HydroxyMethyl Transferase (SHMT) 1 and 2. We also generated knockout cells lacking various enzymes of mitochondrial and cytosolic 1C pathway. The combined pharmacological-genetic approach allowed quantitative dissection of the contribution of cytosolic and mitochondrial pathways. Interestingly, genetic disruption of the mitochondrial pathway dramatically increased sensitivity of cells to inhibition of SHMT1 while deletion of SHMT1 increased the sensitivity to MTHFD2 inhibitors suggesting interdependence and flexibility between the mitochondrial and cytosolic 1C pathway arms. The effects of 1C inhibitors on cell viability could be rescued by other 1C donors as well as 1C pathway products, further informing on the critical products of 1C metabolism and potential escape mechanisms.

Our observations show that cancer cells can utilize multiple routes to generate 1C units and identify the contribution of specific enzymes. Pharmacological inhibition of some of these enzymes strongly affects cell viability and combination of inhibitors of cytosolic and mitochondrial 1C enzymes in the same or different molecules can be particularly effective. In addition, genetic lesions in the mitochondrial or cytosolic 1C arms can also sensitize tumors to inhibitors of specific 1C pathway enzymes.

#233

Identification of cancer vulnerabilities to metabolic perturbation using genome wide CRISPR screens.

Megha Chandrashekhar,1 Michael Arreger,2 Ashwin Seetharaman,2 Traver Hart,2 Jason Moffat1. 1 _Donnelly Center for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada;_ 2 _Donnelly Center for Cellular and Biomolecular Research, Toronto, Ontario, Canada_.

The CRISPR-Cas9 system provides an effective way to introduce targeted loss-of-function mutations in mammalian cells. The advance that the CRISPR-Cas9 technology brings to human genetics sets the stage for identifying cellular fitness genes that operate either globally (core fitness genes) or specifically within a particular genetic background or environmental context (context-specific fitness genes). In tumors, this is the foundation for the concept of synthetic lethality as genes required in tumor cells but not in adjacent normal tissues should make ideal therapeutic targets with high effectiveness and minimal side effects. Towards this goal, we have developed a second-generation CRISPR gRNA library of 176,500 guides targeting 17,661 human protein-coding genes. We used the library to screen five human cell lines, representing a cross-section of wild type and cancer tissues, to identify genes whose knockouts induce significant fitness defects. Our screens accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and identify novel context-specific vulnerabilities. Interestingly, we identified a specific dependency on mitochondrial activity and we validated this using various complex I inhibitors. This strongly supports the idea that oxidative phosphorylation (OXPHOS) dependency - a clear exception to the Warburg effect - is a targetable weakness of some tumors. In order to further understand this metabolic vulnerability, we performed a synthetic lethal screen to identify sensitizers of OXPHOS inhibition. Our screen revealed that inhibition of mitochondrial OXPHOS sensitizes the cells to loss of other metabolic pathways such as glycolysis, pentose phosphate pathway and lipid biosynthesis. Furthermore, loss of the cytosolic aspartate aminotransferase GOT1 was found to be synthetic lethal with perturbation of OXPHOS. This is consistent with the essential role of the electron transport chain in cell proliferation, which is to enable aspartate synthesis. Additionally, we also find novel genes, whose loss of function might alleviate the affect of OXPHOS perturbation. Our findings demonstrate that CRISPR-Cas9 screens enable a high-resolution view of the genetic vulnerabilities of a cell that may represent therapeutic opportunities in cancer. Further, synthetic lethal chemical-genetic screens can reveal novel functional drug combinations, which will enhance the efficacy of targeted therapies.

#234

Mitochondrial morphology as a biomarker of cancer phenotype and drug response.

Randy J. Giedt,1 Paolo Fumene Fergulio,1 Divya Pathania,1 Katherine S. Yang,1 Aoife Kilcoyne,1 Claudio Vinegoni,1 Tim J. Mitchison,2 Ralph Weissleder1. 1 _Massachusetts General Hospital/ Harvard Medical School, Boston, MA;_ 2 _Harvard Medical School, Boston, MA_.

Mitochondria, critical organelles in quiescent and dividing cells, have been shown to vary in number, mass, and shape. While previous work has focused on understanding molecular mechanisms underlying changes in normal and immortalized cells, less is known about longer-term mitochondrial behavior such as morphological patterns in individual cells and overall populations, the heterogeneity and functional consequences of these patterns, and ultimately how potential mitochondrial heterogeneity can be exploited in the clinic. Indeed, while previous studies have been conducted illustrating mitochondrial dysfunction and morphological alterations in certain tumor types, more comprehensive analyses across tumor and cell types, as well as from cells derived from individual patients, have yet to be conducted. A primary impediment to this work is the technical challenge of consistently assaying mitochondrial morphology in diverse cell types where fixation procedures, collection techniques, and differing optical qualities between samples make potential analyses a challenging application.

We have developed an image based analytical technique to phenotype mitochondrial morphology in different cancers, including in vitro cancer cell lines and patient derived cancer cells. Our methodology is based on a combination of novel, clinically relevant collection and fixation techniques, and advanced image processing and computer learning analysis methodologies. Our results demonstrate that i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; ii) a given phenotype is characteristic of a cell population and fairly constant over time; iii) mitochondrial patterns correlate with cell metabolomics measurements and iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells.

#234A

Aberrant sodium current contributes to constitutive mTOR activity in malignant melanoma cells.

Benjamin Gallant,1 Nicole Lizza,1 Ryan Garrity,1 Audrey Madigan,1 Zachary Jost,1 Alfredo Gonzalez,1 Jeanine Justiniano,1 An Xie,2 Yinsheng Wan1. 1 _Providence College, Providence, RI;_ 2 _Brown University, Providence, RI_.

Transformation from disciplined melanocytes to untamed melanoma cells remains an enigma. Our previous studies have demonstrated that melanoma cells are more resistant to oxidative stress and melanoma cells exhibit constitutive mTOR activity. Surprisingly, further studies have failed to present the expression and activity of EGFR using conventional anti-EGFR antibodies. We hypothesized that constitutive mTOR activity in melanoma cells may be due to mutated EGFR variants and membrane channel activities. Using patch clamping technique, we have shown that melanoma cells (WM 266-4) but not human skin melanocytes exhibit Na+ current which is blocked by TTX. Interestingly, mTOR inhibitor, rapamycin, blocks Na+ current. Western blot and confocal microscopy data further revealed that Na+/Ca2+ exchanger blocker KB-R7943 (KBR), and L-type Ca2+ channel blocker Nifedipine (NIF) inhibits mTOR activity in melanoma cells in a dose dependent manner. To further characterize Na+ channels, we used commercially available channel antibodies. Western blot analysis data showed that melanoma cells but not human melanocytes express Na+ v1.5 and Na+ v1.6 and NCX3. Functional studies also indicated that KBR and NIF inhibit melanoma cell proliferation and migration. Taken all together, our data suggest that aberrant Na+ current contributes to constitutive mTOR activity in melanoma cells and channel blockers may be potential for the treatment of melanoma.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Cellular Processes and Responses to Therapy

#235

AXL inhibition leads to a reversal of a mesenchymal phenotype sensitizing cancer cells to targeted agents and immuno-oncology therapies.

Katherine K. Soh, Wontak Kim, Ye Sol Lee, Peter Peterson, Adam Siddiqui-Jain, Steven L. Warner, David J. Bearss, Clifford J. Whatcott. _Tolero Pharmaceuticals, Inc., Lehi, UT_.

Mesenchymal properties and the epithelial-to-mesenchymal transition (EMT) contribute to the initiation and progression of many tumor types and ultimately can lead to drug resistance and highly aggressive disease. It is becoming increasingly clear that the more mesenchymal characteristics cancer cells acquire the more resistant they become to standard chemotherapy, targeted agents, and even immune checkpoint inhibitors. We have been exploring the role of the receptor tyrosine kinase, AXL, and its related TAM family members, in promoting the mesenchymal phenotype in cancer cells and how these effects promote drug resistance and escape from immune surveillance. TP-0903, a potent AXL inhibitor, leads to a reversal of the mesenchymal phenotype in multiple cancer models. Following TP-0903 treatment, we observed changes in mRNA expression using RT-qPCR and protein expression using standard immunoblotting that are consistent with a reversal of the mesenchymal phenotype. Upon treatment with TP-0903 cancer cells possessed lower motility and a decrease in anchorage-independent growth, both hallmarks of a mesenchymal cell. In vivo models of erlotinib-resistant non-small cell lung cancer (NSCLC) were utilized to demonstrate TP-0903 single agent activity in highly mesenchymal models; however, more importantly, treatment with TP-0903 was able to sensitize this highly refractory model to erlotinib. AXL function and tumor mesenchymal characteristics also provide mechanisms for the cancer cells to evade immune surveillance. This is achieved by the role that AXL plays in detecting neighboring apoptotic cells resulting in the engulfment of dead cells (efferocytosis) and the associated debris in order to prevent the immune system's exposure to auto-antigens under normal physiological conditions or exposure to cancer-associated neo-antigens in a tumor. Inhibition of AXL by TP-0903 can potentially inhibit tumor-associated efferocytosis leading to a stronger immunogenic response to the tumor. Indeed, results demonstrated synergy when TP-0903 was combined with an anti-PD-L1 agent in a syngeneic triple negative breast cancer mouse model. Interestingly, during the evaluation of TP-0903 in models of EMT, we detected dramatic change in the expression of the retinoic acid (RA) metabolizing protein CYP26A1, suggesting that AXL inhibition leads to changes in RA metabolism. Our data suggest that AXL induces a transition to a mesenchymal phenotype in cancer cells through the suppression of RA signaling and that TP-0903 can rapidly reverse this phenotype by signaling through RA causing the cell to revert to a more differentiated state. Due to its ability to reverse the aggressive mesenchymal phenotype of cancer cells, TP-0903 is a promising agent with the potential to have single agent activity and combined synergy with targeted anti-cancer agents and immunotherapies.

#236

Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors alone or together with AR antagonists in advanced prostate cancer.

Leandro S. D'Abronzo,1 Michael Crapuchettes,2 Ryan E. Beggs,2 Swagata Bose,1 Salma Siddiqui,3 Yu Wang,1 Blythe Durbin-Johnson,1 Chong-Xian Pan,1 Paramita Ghosh1. 1 _UC Davis, davis, CA;_ 2 _Department of Veteran's Affairs, Mather, CA;_ 3 _Department of Veteran's Affairs, davis, CA_.

The standard of care for patients with recurrent prostate cancer (PCa) is the use of androgen receptor (AR) antagonists, but the treatment ultimately fails, resulting in the development of castration resistant PCa (CRPC). Patients with CRPC are frequently continue to express an active AR, despite castration resistance, and AR inhibitors remain effective in these patients for several months. We previously showed that upregulation of mammalian target of rapamycin (mTOR) activity upon use of AR antagonists contributed to acquired resistance to this therapy, and that a combination of an mTOR inhibitor and an AR antagonist overcame resistance to AR antagonists alone (Wang et al, Oncogene, 2008;27(56):7106-17). Based on our data, a Phase II clinical trial was conducted to determine the efficacy of the combination of the mTOR inhibitor RAD001 and the AR antagonist bicalutamide in bicalutamide-naïve CRPC patients (ClinicalTrials.gov: NCT00814788). This study, which was recently concluded, showed a response rate of 75% with this combination with the historical control of 25%. The overall goal of this project was to define pathways that results in resistance to combinations of mTOR and AR inhibitors in patients with CRPC.

Comparison of various mTOR inhibitors: the mTORC1 inhibitor RAD001, a mTORC1/C2 dual inhibitor INK128 and a mTORC1/C2/PI3K triple inhibitor BEZ-235 either alone or in combination with AR antagonists bicalutamide and enzalutamide in various prostate derived cell lines including C4-2, PC-346C, 22Rv1 and CWR-R1, identified cells that were resistant (CWR-R1, PC-346C) vs those that were sensitive (22Rv1, C4-2) to these inhibitors. Investigation of the base-line molecular profile of these cells demonstrated that those that expressed high levels of phosphorylated form of eIF4E (S209) were resistant to mTOR inhibitors. Downregulation of eIF4E phosphorylation by siRNA resulted in sensitivity of CRPC cells to the combination of the mTOR inhibitors with AR antagonists. Investigation of the mechanism by which eIF4E phosphorylation levels increased in certain CRPC cells but not in others revealed that expression and transcriptional activity of the AR negatively correlated with the levels of eIF4E phosphorylation. In cells with high basal levels of phospho-eIF4E, bicalutamide further increased eIF4E phosphorylation, whereas those with low eIF4E levels were not further affected. The ability of AR inhibition to suppress eIF4E phosphorylation was mediated by MAP kinase interacting kinase (Mnk), and the ability of some cells to phosphorylate eIF4E, but not others, correlated with the levels of Mnk phosphorylation. Based on these studies, we predict that patients with high basal PSA who express low levels of Mnk phosphorylation are the ones who are likely to respond to the combination of an mTOR inhibitor and an AR antagonist.

#237

**Enrichment of AXL-high/ MITF-low melanoma cells in the presence of MAPK inhibitors** in vitro **.**

Ken Dutton-Regester, Levi Garraway. _Dana Farber Cancer Institute, Boston, MA_.

BRAF and MEK inhibitors have become a standard of care for patients with metastatic BRAF mutant (V600) melanoma. Despite their success, 10- 20% of patients exhibit 'intrinsic' resistance and fail to respond to treatment. Recently, distinct transcriptional profiles have been associated with sensitivity to these drugs with intrinsically resistant melanomas exhibiting an AXL-high/ MITF-low phenotype (1). Using melanoma cell lines from the Cancer Cell Line Encyclopedia with matched gene expression data, we confirmed that AXL-high/ MITF-low cell lines had increased resistance to both BRAF (Dabrafenib) and MEK (Trametinib) inhibitors, either singly or in combination. Using flow cytometry with an AXL antibody, we observed that AXL-high/ MITF-low resistant cell lines frequently exhibited a high percentage of AXL+ cells (≥90%) while AXL-low/ MITF-high sensitive cell lines showed the opposite (≤5%). We hypothesized that the small percentage of AXL+ cells in the sensitive cell lines may be responsible for mechanisms of adaptive or acquired resistance (a feature that is frequently observed with the use of BRAF and MEK inhibitors in vitro and in the clinic). To explore this, we cultured sensitive cell lines in vitro for 5 days in the presence of combined Dabrafenib and Trametinib (DT) and performed flow cytometry to determine if there was a change in the percentage of AXL+ cells. All sensitive cell lines exhibited an enrichment of AXL+ cells with increasing concentrations of DT (maximum increase compared to DMSO control across individual cell lines ranged from 10% to 85%). It is unknown whether this phenomenon could be explained by a clonal selection or 'plastic' epigenetic reprogramming process and as such, we are currently performing experiments to determine this. Looking to the future, identifying potential dependencies of AXL-low/ MITF-high melanomas will deepen our understanding of the biology supporting this resistant state and provide a platform for the design of future clinical intervention strategies in this subset of patients.

1) Konieczkowski et al. 2014. Cancer Discovery. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. 4(7):816-27.

#238

Androgen insensitivity syndrome germline loss-of-function mutations in the androgen receptor that acquire somatic gain-of-function in prostate cancer.

Philip A. Watson,1 Minna D. Balbas,1 Zeda Zhang,1 Taslima F. Ishmael,1 Kayla E. Lawrence,1 John Wongvipat,1 Sophie D. Sawyers,1 Yi-Mi Wu,2 Dan Robinson,2 Yang Shen,3 Arul M. Chinnaiyan,2 Charles L. Sawyers1. 1 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 2 _University of Michigan School of Medicine, Ann Arbor, MI;_ 3 _Texas A &M University, College Station, TX_.

Genomic alterations in the androgen receptor (AR) commonly occur in patients with advanced prostate cancer resistant to androgen deprivation therapies. While still retaining androgen sensitivity, recurring AR mutations become drivers of antiandrogen resistance by causing alterations in the ligand binding pocket that enables one specific antiandrogen to bind that particular mutant receptor in an agonist conformation, thereby aberrantly activating AR transcriptional activity. We chronically treated antiandrogen-sensitive preclinical prostate cancer models (LNCaP-AR and CWR22Pc) with antiandrogens followed by high-throughput sequencing of the AR exons 2-8 to search for novel mutations that may be associated with antiandrogen resistance. The mutation A597T in the DNA binding domain was found in four LNCaP-AR xenograft tumors from mice receiving enzalutamide or ARN-509, while the ligand binding domain mutation P893S was uncovered in CWR22Pc cells treated in vitro with combined androgen depletion and bicalutamide. Recent whole exome sequencing studies of clinical prostate cancer identified single patients with each of these mutations at high allele frequencies and without concurrent AR amplification (www.cBioPortal.org). A597T occurred in a Gleason 6 primary tumor, whereas P893S was found in a patient with metastatic castration resistant prostate cancer who had received bicalutamide during the course of treatment. Remarkably, both A597T and P893S were earlier reported as germline loss-of-function mutations in patients with androgen insensitivity syndrome. Using an AR reporter assay in AR-null cells coupled with overexpression of these AR mutants, we confirmed that P893S is not activated by the androgen dihydrotestosterone (DHT), but nevertheless is potently stimulated by bicalutamide. In silico modeling showed that in contrast to the wild-type AR, cofactor-recruiting helix 12 of the ligand binding domain of P893S substantially drifted away from an agonist conformation when bound to DHT but was able to adopt the active conformation when simulated with bicalutamide. Importantly, overexpression of AR-P893S in CWR22Pc cells conferred bicalutamide resistance in vivo. These findings highlight an unexpected contextual element to the function of AR mutants, and suggest that rare or private AR mutations may nonetheless act as drivers of clinical progression.

#239

Alteration of drug sensitivity according to the induction and reversion of epithelial-to-mesenchymal transition (EMT) in human lung adenocarcinoma cell lines harboring an EGFR mutation.

Ryota Kurimoto,1 Shunichiro Iwasawa,1 Takahiro Ebata,1 Tsukasa Ishiwata,1 Ikuo Sekine,2 Yuji Tada,1 Koichiro Tatsumi,1 Shuhei Koide,1 Atsushi Iwama,1 Yuichi Takiguchi1. 1 _Chiba University, Chiba, Japan;_ 2 _Tsukuba University, Ibaraki, Japan_.

Introduction: EMT phenotype is known to relate to drug resistance and immune tolerance of cancer. However, mechanism underling these phenomena, including the mechanism of the induction and reversion of EMT, are not fully understood.

Methods: EGFR mutated human lung adenocarcinoma cell lines, HCC-827 and PC-9, were treated with TGF-ß and/or FGF-2 to induce EMT. EMT was reversed by treating the induced EMT cells with either of PP242 (mTOR inhibitor), metformin and DMSO. The phenotypic alterations according to the induction and reversion of EMT were accessed by a wound healing assay for mobility, flow cytometry for cell cycle, MTT and apoptosis assays for drug sensitivity to cisplatin and gefitinib, and RT-PCR and fluorescence IHC for PD-L1 expression.

Results: The treatment with combination of TGF-ß/FGF-2 showed an efficient increase in expressions of mesenchymal markers and slug in both cell lines, and a decrease in E-cadherin expression in HCC827. In immunoblotting analyses, the Smad3 pathway in PC-9, and the Smad3, MEK/Erk and mTOR pathways in HCC-827 were involved in the induction of the EMT. The induced EMT was accompanied by enhanced cell motility and accumulation in the G0/G1 phases in both cell lines. The induced EMT also made the cells less sensitive to gefitinib in both cell lines, and to cisplatin in HCC-827. Increased PD-L1 expression was observed in both cell lines. Treatment of the induced EMT cells with PP242, metformin and DMSO reverted the altered expressions of epithelial and mesenchymal markers, cell motility and cell cycle. These agents reverted the EMT to different extents and through different pathways, depending on the cell lines. Reversion of the EMT using each of the 3 agents partly restored drug sensitivity, and suppressed PD-L1 expression.

Conclusion: Inducing EMT by treatment with TGF-ß/FGF-2 is an effective way to acquire drug resistance and up-regulation of PD-L1. Reversion of the EMT, therefore, has a potential to overcome drug resistance and immune tolerance of cancer.

#240

Functional significance of co-occurring mutations in PIK3CA and MAP3K1 in breast cancer.

Alvaro Avivar-Valderas,1 Robert McEwen,2 Amir Taheri-Ghahfarokhi,3 Ambar Ahmed,4 Daniel Stetson,4 Brian Dougherty,4 Marcello Maresca,3 Kevin Hudson,1 Francisco Cruzalegui1. 1 _AstraZeneca, Macclesfield, United Kingdom;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _AstraZeneca, Mölndal, Sweden;_ 4 _AstraZeneca, Waltham, MA_.

PI3Kα signalling pathway is frequently hyper-activated in breast cancer (BrCa) with mutations of PIK3CA (~45%) frequently found in luminal A BrCa samples. Genomic studies have also uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (~8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples and co-occurring mutation of PIK3CA and MAP3K1 are found in ~11% of PIK3CA mutants (TCGA). How these two alterations cooperate to promote tumorigenesis and impact the sensitivity to PI3Kα(AZD8835) and AKT (AZD5363) inhibitors is currently unknown. Using siRNA targeting and precise gene editing we have knocked down MAP3K1 in PIK3CA mutant cell lines to create in vitro models reflecting the mutational status of PIK3CA and MAP3K1 in BrCa patients. MAP3K1-deficient cells exhibited increased proliferation rate and decreased sensitivity to AZD8835 compared with parental control cells. In addition, mechanistic analysis revealed that siRNA mediated depletion of MAP3K1 enhances AKT phosphorylation and downstream signalling (i.e. pPRAS40, pFoxO1/FoxO3a) and provides resistance to AZD8835-mediated pathway inhibition. Using in vitro 3D-breast cancer organoid models we have found that MAP3K1 depletion increases overall acinar volume and counteracts AZD8835-mediated reduction of acinar growth. MAP3K1 deficient acini exhibited high pAKT levels in the presence of AZD8835 compared with control acini validating observations in 2D. Remarkably, whereas in control acini pAKT+ cells were restricted to the basal compartment, MAP3K1 deficient acini also exhibited pAKT+ cells in the luminal compartment suggesting anoikis resistance which is a hallmark in early stages of tumorigenesis. Elucidating the mechanistic basis for these clinically co-occurring mutations will provide insights into the mechanisms contributing to the oncogenic role of the PI3K pathway and future patient selection strategies.

#241

Up-regulation of PD-L1 in melanoma determines resistance to BRAF and MEK inhibitors, induces a more aggressive phenotype and is partially mediated through post-transcriptional mechanisms involving miR-17-5p.

Davide Brusa,1 Aureliano Stingi,1 Valentina Audrito,2 Francesca Orso,3 Sara Serra,2 Roberta Buonincontri,2 Francesco Neri,1 Gianna Baroni,4 Barbara Merelli,5 Romina Nassini,4 Daniela Massi,4 Salvatore Oliviero,6 Daniela Taverna,3 Mario Mandalà,5 Silvia Deaglio2. 1 _Hugef-Torino, Torino, Italy;_ 2 _Hugef and University of Turin, Torino, Italy;_ 3 _University of Turin, Torino, Italy;_ 4 _University of Florence, Florence, Italy;_ 5 _Papa Giovanni XXIII Hospital, Bergamo, Italy;_ 6 _Hugef-Torino and University of Turin, Torino, Italy_.

Introduction: The therapy of metastatic melanoma (MM) was radically changed by the introduction of BRAF inhibitors (BRAFi). Even if highly effective in the short term, patients invariably develop resistance in the long term. For this reason other inhibitors as well as alternative or complementary therapeutic strategies are being tested in these patients. Among the immune checkpoint targets of clinical importance is PD-1, which is expressed by T cells and which binds to the PD-L1 ligand, which may be expressed by melanoma cells. We and others showed that PD-L1 is an independent negative prognostic marker for patients with MM.

Methods: BRAFi-/MEKi-resistant melanoma cell lines were generated by treating cells with increasing concentrations of BRAFi or MEKi. Resistance, viability and aggressiveness were analyzed by MTT, migration and wound healing assays. Results were confirmed using xenograft models. Resistant cell lines were compared using RNAseq to identify enriched genetic pathways involved in the resistance. Luciferase reporter assay analysis was used to study the direct interactions between the PD-L1 and miR-17-5p.

Results: By comparing responses to BRAFi in PD-L1+ and PD-L1- variants of the A375 cell line, we found that PD-L1 expression conferred resistance to BRAFi or MEKi. Conversely, silencing of the molecule restored sensitivity to these drugs. Resistant melanoma cell lines acquired PD-L1 expression and were characterized by a specific genetic profile, with the modulation of genes controlling cell movement and immune responses. Consistently, these cells showed a more aggressive behavior both in vitro and in xenograft models. PD-L1 silencing in resistant cells decreased invasive properties and restored expression of HLA-II molecules.

PD-L1 up-regulation was only partly dependent on the paradoxical activation of the MAPK pathway, which characterized resistant cells. In addition, we found that resistance to BRAFi and MEKi down-modulated miR-17-5p, which showed an inverse correlation with PD-L1. Transfection of miR-17-5p into BRAFi-resistant cell lines induced the down-modulation of PD-L1, reduced the aggressive behavior of the cells and partially restored sensitivity to BRAFi. Finally, in the plasma of patients with MM, miR-17-5p was inversely correlated with expression of PD-L1 in the tumor tissue.

Conclusions: These data demonstrate a direct link between expression of PD-L1 and resistance to BRAFi, as well as to a more aggressive behavior of melanoma cells. Furthermore, we define a novel post-transcriptional circuit responsible for PD-L1 up-regulation, based on a direct interaction between miR-17-5p and PD-L1 mRNA. Lastly, miR-17-5p plasmatic levels show an inverse correlation with PD-L1 expression by tumor cells, suggesting that they may be useful in monitoring disease outcome and drug sensitivity.

#242

Deciphering the role of PTEN as a predictive biomarker to next generation isoform-selective PI3K inhibitors in PIK3CA mutant breast cancer cells.

Heather M. Moore, Heidi M. Savage, Mark R. Lackner, Timothy R. Wilson. _Genentech, South San Francisco, CA_.

The phosphoinositide-3 kinase (PI3K) pathway is one of the most frequently activated signaling pathways in human cancer and represents a promising therapeutic target. Taselisib (GDC-0032), a potent and selective inhibitor of the PI3K alpha, delta and gamma isoforms, but less potent in the beta isoform, displays increased sensitivity in models that are driven by mutant PIK3CA (Ndubaku CO et al, J Med Chem, 2013). Selective pressure caused by, small molecules such as PI3K inhibitors will ultimately lead to clinical resistance and disease progression. Recently, a published mechanism of acquired resistance to the PI3K inhibitor alpelisib (BYL-719, a PI3K alpha-selective inhibitor) reported that genomic loss of the tumor suppressor PTEN led to clinical progression in a metastatic breast cancer patient harboring an activating PIK3CA mutation (Juric D et al, Nature, 2014). As taselisib and alpelisib are next generation PI3K inhibitors, we sought to determine if there are any differences to drug sensitivity in PIK3CA mutant cells that have concomitant loss of PTEN. To investigate this hypothesis, we first measured viability in breast cancer cells, each with differing genetic backgrounds treated with two PI3K inhibitors, taselisib and alpelisib. We found that PIK3CA mutant and PIK3CA mutant/PTEN null cells were sensitive to treatment when compared to wild-type and PTEN null cells. However, PIK3CA mutant/PTEN null cells displayed higher IC50 values for both inhibitors when compared to PIK3CA mutant cells. To control for differences in genetic backgrounds, we next tested whether knockdown (KD) of PTEN expression in PIK3CA mutant breast cancer cells would promote resistance and reactivate the PI3K signaling cascade even in the presence of a PI3K inhibitor. Transient, siRNA-targeted KD of PTEN in the PIK3CA mutant breast cancer cell lines EFM19 and T47D conferred resistance to alpelisib after 6 days of drug treatment, an observation that was less pronounced in taselisib treated cells. Additionally, PTEN KD in DMSO-treated cells led to an increase in cell proliferation and enhanced downstream PI3K signaling as measured by phosphorylated AKT and S6 proteins when compared to non-targeting siRNA controls. The reactivated, pro-survival signaling with PTEN KD was maintained upon treatment with both inhibitors when compared to control cells, an observation that was more pronounced in alpelisib treated cells. These data suggest that PIK3CB can compensate for PIK3CA inhibition and that PTEN loss could lead to clinical resistance to alpelisib, but may not constitute a resistance mechanism to taselisib. As both molecules have entered phase 3 studies in estrogen receptor positive breast cancer, understanding how biomarker-defined genotypes respond to next generation PI3K inhibitors is critical for identifying patients most likely to gain therapeutic benefit.

#243

Investigating microtubule growth dynamics in patient-derived metastatic prostate cancer cells.

Alexandre Matov,1 Johan de Rooij,2 Jay Gatlin,3 Julia Rohrberg,1 Nikta Ahmad,1 Rahul Aggarwal,1 Jeff Simko,1 Andrei Goga,1 Charles Ryan,1 Torsten Wittmann1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _University Medical Center Utrecht, Utrect, Netherlands;_ 3 _University of Wyoming Laramie, Laramie, WY_.

Advanced prostate cancer (PC) is treated primarily by means of chemotherapy with one of the clinically approved microtubule (MT)-targeting agents (MTAs): paclitaxel, docetaxel and cabazitaxel. The mechanism of action of the MTAs on the cellular level is to bind to MTs and inhibit MT-dependent intracellular pathways, ultimately leading to cell death. In PC, MTAs are the only chemotherapy class shown to improve survival, however, there is high variability of response to MTA chemotherapy among patients as well as emergence of resistance to drug action which hampers the clinical efficacy. Clinicians need additional information to match individual patient tumors to the most effective drugs or refrain from treating patients that will not respond. However, the precise mechanism of action or resistance development to MTAs is not understood, which represents a critical gap in our knowledge to select the best treatment. Our hypothesis is that there are inherent differences in tumor MT dynamics among individual patients which dictates their response to treatment with specific MTAs. To this end, we envision that a systematic characterization of PC MT dynamics and their response to MTAs will allow chemotherapy customization and prolong survival. Specifically, our approach consists of the analysis of metastatic castrate resistant PC (mCRPC) organoids (Gao D. et al, 2014) derived from bone (acetabulum, vertebrae), soft tissue (salvage prostatectomy, lymph nodes), pleural effusion and circulating tumor cells biopsy samples obtained from metastatic patients who have received hormonal therapy and the MTA docetaxel or hormonal therapy alone or no treatment. We apply a quantitative single-cell image analysis algorithm (ClusterTrack) to measure a panel of twelve distinct MT dynamics parameters (a MT dynamics signature). Preliminary analysis suggests differences between different mCRPC organoids. MT tracking results indicate a correlation of decreased MT growth rate with increased Op18/stathmin expression. In this context, we hypothesize that differential expression of MT regulating genes affects specific parameters of MT dynamics, thus determining the sensitivity to MTA treatment.

#244

Paclitaxel increases the assembly and function of the tumor microenvironment of metastasis in breast cancer.

George S. Karagiannis, Allison H. Harney, Yarong Wang, Jessica Pastoriza, Jeanine Pignatelli, Jesus Anampa, Joseph A. Sparano, Joan G. Jones, David Entenberg, John S. Condeelis, Maja H. Oktay. _Albert Einstein College of Medicine, Bronx, NY_.

Chemotherapy induces influx of bone marrow-derived progenitors such as mesenchymal stem cells, endothelial progenitors and proangiogenic monocytes into the primary tumor to promote angiogenesis. Thus it is feared that chemotherapy may potentiate tumor cell invasion and metastasis. Here, we show that paclitaxel delays tumor growth in several mammary carcinoma mouse and human breast cancer models, yet it significantly increases the density of microanatomical sites called "tumor microenvironment of metastasis" (TMEM) that are responsible for tumor cell intravasation and dissemination of breast cancer. The TMEM site consists of a Mena-overexpressing cancer cell in direct contact with a Tie2hi/VEGFhi macrophage and an underlying endothelial cell. Mice treated with paclitaxel have significantly more circulating tumor cells (CTCs) and metastatic foci when compared to vehicle-treated animals indicating that the chemotherapy-induced TMEM are active in assisting tumor cell intravasation. Moreover, syngeneic transplantation of Dendra2+/PyMT tumors into FVB recipients showed significantly higher incidence of Dendra2+ cells in the lung, following paclitaxel administration. In parallel experiments, paclitaxel induced the influx of macrophages and intravasation of cancer cells as observed using intravital imaging of MMTV-PyMT-Dendra2/Cfms-CFP mice, in which blood vessels were visualized with Quantum dots. Furthermore, paclitaxel treatment in experimental mice caused a significant increase in the expression of Mena at the gene and protein levels. PCR assays for total Mena (PanMena) or specific Mena isoforms (MenaINV, Mena11a) revealed that this increase was particularly attributed to the invasive Mena isoforms [i.e. MenaINV and MenaCalc (Menacalc = PanMena - Mena11a)]. These pre-clinical data are supported by the findings from a cohort of 10 breast cancer patients who received neoadjuvant dose-dense paclitaxel followed by doxorubicin/ cyclophosphamide. Of these tumors, 7/10 patients had more than 2-fold increase in TMEM density following neoadjuvant chemotherapy regimen. Moreover, chemotherapy produced an acute increase of up to 150-fold in MenaINV expression in 3/7 and up to 5.5-fold in MenaCalc in 3/4 patients who underwent serial fine needle aspiration (FNA) biopsy before and after 1-2 doses of either neoadjuvant paclitaxel or doxorubicin-cyclophosphamide. This is provocative because an increase in either MenaCalc score or TMEM density are independently associated with increased risk of distant recurrence in breast cancer patients. In conclusion, our data indicate that paclitaxel treatment induces intravasation-mediated dissemination of breast cancer cells in rodents and in certain clinical scenarios in humans by promoting increases in MenaCalc expression and TMEM intravasation sites.

#245

The master neural transcription factor BRN2 is an androgen suppressed driver of neuroendocrine differentiation in prostate cancer.

Jennifer Leah Bishop,1 Daksh Thaper,1 Sepideh Vahid,1 Randy Jama,1 Kirsi Ketola,1 Soojin Kim,1 Alastair Davies,1 Arkhjamil Angeles,1 Barinder Sangha,1 Hidetoshi Kuruma,1 Alexander W. Wyatt,1 Martin E. Gleave,1 Dong Lin,1 Colin C. Collins,1 Yuzhuo Wang,1 Himisha Beltran,2 Amina Zoubeidi1. 1 _Vancouver Prostate Ctr., Vancouver, British Columbia, Canada;_ 2 _Weill Cornell College of Medicine, New York, NY_.

Neuroendocrine prostate cancer (NEPC) is an incurable, rapidly progressing and lethal disease. Treatment for NEPC is extremely limited; indeed, it is increasingly recognized as a highly therapy resistant tumor variant that evolves from castration-resistant prostate cancer (CRPC) in a subset of patients treated with potent androgen receptor (AR) pathway inhibitors like enzalutamide (ENZ). Importantly, mechanisms by which the AR directly controls the emergence of NEPC from CRPC under the selective pressure of ENZ remain elusive. As the AR is the cornerstone therapeutic target in men with CRPC, understanding its contribution to the development of NEPC is critical to better implement current standard-of-care therapies such as ENZ, and to identify novel therapeutic targets for this incurable disease.

Hallmarks of NEPC are resistance to ENZ and the loss or reduced activity of the AR, suggesting the AR controls the emergence of NEPC from CRPC. Thus, we hypothesized that a consequence of ENZ treatment and resistance in CRPC is relief of AR-mediated suppression of factors that drive NEPC. To investigate this, we developed a model of prostate cancer that mimics clinical progression to CRPC and ENZ resistance (ENZR CPRC). Mirroring what is observed in some patients who progress on ENZ, 25% of our ENZR CRPC tumors and derived cell lines showed strong reduction in classic activity of the AR and a NEPC phenotype. By interrogating NEPC-like ENZR CRPC and ENZ-treated CRPC tumors and cell lines with RNA-Seq and mechanistic in vitro and in vivo studies, as well as human tumors with RNA-Seq and IHC, we identified the master neural transcription factor BRN2 as a central and clinically relevant driver of NEPC differentiation in CRPC that is controlled by the AR.

Specifically, we found AR binding in the BRN2 enhancer directly represses BRN2 transcription, which is sufficient and required for NEPC marker expression and aggressive growth of ENZR CRPC or ENZ-treated CRPC. Our data also reveal a striking overlap of AR and SOX binding motifs in the enhancer region of BRN2 that allow the AR to competitively inhibit BRN2 from interacting with and regulating SOX2, another cell-fate determining transcription factor associated with NEPC, and that BRN2-SOX2 interaction further drives NEPC differentiation. Importantly, we found BRN2 is highly expressed in human NEPC and metastatic CRPC, especially in patients with low AR activity, highlighting its clinical relevance to disease that is difficult to treat with mainstay therapies.

These data underscore the consequences of potent AR inhibition in CRPC, revealing a novel mechanism of AR-dependent control of NEPC via direct suppression of BRN2 and its regulation of, and interaction with SOX2. Meeting a critical unmet medical need for patients, this work uncovers BRN2 as a clinically relevant potential therapeutic target to prevent emergence of NEPC from ENZR CRPC.

#246

Role for ATP7A copper transporter in cisplatin resistance, tumorigenesis and metastasis.

Vinit Shanbhag, Nikita Gudekar. _University of Missouri, Columbia, MO_.

Copper is a trace metal with a ready capacity to gain or donate electrons. This property is harnessed by numerous enzymes to perform vital functions in the body. In humans, copper is required for various biochemical processes including cellular respiration, connective tissue development, iron transport, and pigmentation. The same redox property which makes copper useful can also have deleterious effects if the proper balance is not maintained. Hence a delicate balance of copper should be maintained in the body. Cellular copper homeostasis is maintained by several different proteins. Ctr1 (copper transporter 1) has high affinity for copper and aids in copper uptake into the cell. ATP7A and ATP7B are copper exporting ATPases, which share 60% identity, are functionally homologous. Under low intracellular copper concentrations, ATP7A is localized to the TGN (trans-Golgi network), where it transports copper to newly synthesized cuproenzymes. Under elevated copper concentrations, ATP7A traffics to post TGN vesicles and to the plasma membrane, subsequently releasing the copper load by fusion with the plasma membrane.

There are emerging studies implicating a role of ATP7A in resistance against the platinum-based chemotherapy agent, cisplatin, which is widely used to treat cancer. Previous studies have correlated ATP7A expression to the sensitivity of cultured cells to cisplatin. Here, we report that deletion of the Atp7a gene in isogenic RAS-transformed mouse embryonic fibroblast (MEF) cells not only confers sensitivity to cisplatin, but markedly prevents tumor formation in the absence of chemotherapy agent. Deletion of Atp7a in the breast cancer cell line, 4T1, was also found to suppress tumorigenesis and markedly reduce metastasis to the lungs. The ATP7A knockout 4T1 cells showed a significant decrease in Lysyl Oxidase (LOX) activity as compared to the 4T1WT cells. Given the numerous studies showing that LOX family involved in breast tumor metastasis, ATP7A might play a role in tumor progression by virtue of its requirement for metallating LOX. The ATP7A knockout 4T1 cells exhibited reduced survival as compared to the 4T1WT cells in the presence of physiological concentrations of hydrogen peroxide or hypoxia, suggesting that hyper-accumulation of copper sensitizes these cells to ROS production. Taken together, these findings identify the ATP7A copper transporter at the nexus of platinum-drug resistance, tumorigenesis and metastatic pathways, underscoring its potential as a therapeutic drug target at multiple stages of carcinogenesis.

#247

Understanding the role of tyrosine 1101 in the nuclear translocation of the epidermal growth factor receptor.

Bailey G. Flanigan, Mari Iida, Toni M. Brand, Deric L. Wheeler. _University of Wisconsin-Madison, Madison, WI_.

Triple-negative breast cancer (TNBC) is a subclass of breast cancers that are negative for the estrogen receptor, the progesterone receptor, and HER2. Currently, TNBCs have poor prognosis and very few proven molecular targets. One promising target in TNBC has been the epidermal growth factor receptor (EGFR), as it is overexpressed in as many as 80% of TNBCs. Strikingly, however, antibody-based therapy directed against the EGFR (cetuximab) in TNBCs has had modest clinical impact. Over the last decade, advances in EGFR biology have established that the EGFR functions in two distinct signaling pathways: classical membrane-bound signaling and the nuclear EGFR signaling pathway. We previously demonstrated that EGFR nuclear translocation is dependent on Src Family Kinases (SFKs) and phosphorylation of tyrosine 1101 (Y1101) on the cytoplasmic tail of the EGFR. In these studies, translocation of the EGFR to the nucleus led to activation of the nuclear EGFR signaling pathway, which resulted in cetuximab resistance. Resistance could be reversed by simultaneously targeting both the nuclear EGFR signaling pathway and the classical EGFR signaling pathway through the inhibition of its nuclear transport (blockade of SFKs) and with cetuximab, respectively. Despite the identification of nuclear EGFR as a functional molecular target in TNBC, the role of Y1101 in EGFR translocation has yet to be identified.

In this study, we seek to better understand the role of Y1101 in the nuclear translocation of EGFR in TNBC. Using a battery of TNBC cell lines, we have created isogenic pairs that overexpress either wild type EGFR (EGFR-WT) or EGFR with a mutant Y1101 (EGFR-Y110F). Using electron and super-resolution confocal microscopy as well as cellular fractionation, we have shown that EGFR-Y1101 undergoes normal endocytosis upon EGF stimulation in multiple cell lines, but unlike EGFR-WT, EGFR-Y1101F deposits around the perinuclear region. These data suggest that EGFR-Y1101F is able to undergo normal translocation to the perinuclear space, but that amino acid Y1101 is necessary for direct nuclear entry, potentially due to its interactions with nucleoporins. Current work is focused on the identification of nucleoporins with which EGFR-Y1101 may interact. In addition, continued work focuses on allelic mutation using CrisprCas9. Ultimately, the long-term goal of this project is to understand how Y1101 mediates EGFR nuclear translocation. With this information, we will then aim to develop allosteric inhibitors of Y1101 that may abrogate EGFR nuclear translocation and lead to a novel therapeutic which targets the nuclear EGFR signaling pathway in TNBC.

#248

Metformin sensitizes endometrial cancer cells to chemotherapy by inhibiting IDH1-TET1 pathway.

Mingzhu Bai,1 Zhenbo Zhang,1 Yinhua Yu,2 Youji Feng1. 1 _Shanghai General Hospital, Shanghai, China;_ 2 _Ob/Gyn Hospital of Fudan University, Shanghai, China_.

Chemotherapy-resistance is the major obstacle for endometrial carcinoma patient administration. Accumulated evidences indicate that metformin sensitizes cancers from several different sites to chemotherapy drugs and reverses progestin resistance in endometrial cancer. Our group has demonstrated that metformin reverses progestin resistance and enhances the rate of cell-killing induced by chemotherapeutic agents in endometrial cancer cells by downregulating GloI expression. Recent study has revealed that aberrant expression of IDH1 contribute to chemotherapy resistance. However, the role of IDH1 in metformin induced chemotherapy sensitivity is not well characterized. As a critical enzyme of the tricarboxylic acid (TCA) cycle, IDH1 catalyzes isocitrate to produce α-ketoglutarate (α-KG), a substrate of TET1, which control TET1-mediated epigenetic modulation of the target genes, including the chemotherapy-resistant genes. In the present study, we have found that IDH1 is overexpressed in endometrial cancers compared with benign endometrial lesions and normal endometria by immunohistochemical analysis of specimens (200 cases of endometrial cancers, 10 cases of benign endometrial lesions and 10 cases of normal endometria). Tissue microarray immunohistochemical detection showed the expression of IDH1, TET1 and 5hmc was positively correlated in different pathological types of endometrial tissue. Interestingly, we found that metformin reduces expression of IDH1 at protein levels with a time- and dose-manners in endometrial cancer cells. Furthermore, overexpression of IDH1 increased TET1 at protein level and blocked metformin-sensitized endometrial cancer to chemotherapy drugs, whereas decrease IDH1 expression lessened TET1 expression and paralleled with the attenuated chemotherapy-resistance. Mechanistic investigations revealed that TET1 involved in metformin-enhanced chemotherapy sensitivity via IDH1 by western blotting and DNA dot blotting assays, this may attribute to that reduced production of α-KG resulted from metformin-suppressing IDH1 is not enough to start TET1-mediated epigenetic modulation of chemotherapy resistant genes. Overall, our findings demonstrate that IDH1 regulates α-KG level and TET1 expression, which in turn affecting metformin mediated endometrial cancer chemotherapeutic sensitivity. Our study suggests IDH1 is a potential target to overcome cisplatin and paclitaxel resistance in endometrial cancer.

#249

Breast cancer cell vesiculation is driven by calpain: implications in cancer therapy.

Jack Taylor, Ritu Jaiswal, Mary Bebawy. _The University of Technology Sydney, Sydney, Australia_.

Microparticles (MPs) are small membrane vesicles (0.1 to 1 µm in diameter) released from the plasma membranes of most cell types, (malignant and non-malignant) upon cellular activation or during apoptosis. We discovered that MPs provide a "non-genetic" mechanism for the acquisition of multidrug resistance and increased metastatic capacity in tumour cell populations, whereby they serve as vectors in the intercellular transfer of functional cancer proteins and nucleic acids. By this mechanism, MPs effectively confer the transfer, dissemination and dominance of within cancer cell populations within a matter of hours.

The aim of this study was to define the pathways governing membrane vesicle biogenesis in malignant and non-malignant cells with the aim of identifying cancer specific mediators, which could serve as novel therapeutic targets in preventing microvesicle induced deleterious traits in cancer.

We preformed a comparative analysis using the non-malignant human brain endothelial cell line (HBEC-D3) cells and drug sensitive and resistant human breast adenocarcinoma cells (MCF-7) and (MCF-7/DX) respectively. Cell surface topography and vesiculation pits were visualized at rest, and in the +/- calcium ionophore, A23187 and the Calpain inhibitor ALLM using contact mode Atomic Force Microscopy (Nanowizard, JPK Instruments, Germany) on fixed cells. We observed that at rest, cells from both malignant cell lines were high vesiculators relative to the HBEC-D3 cells. In the presence of ALLM we observed a direct inhibition of vesiculation in the malignant cells whilst vesiculation was enhanced in the non-malignant cells. Upon the release of calcium from intracellular stores using A23817, we observe an increase in vesiculation across all cell types, however to a greater extent in the HBEC-D3 cells. Interestingly, only upon the release of intracellular calcium in HBEC-D3 cells do we see an inhibition of vesiculation by ALLM. These results clearly demonstrate an alternative pathway governing vesiculation at rest for the non-malignant HBEC-D3 cells. Whereas contrary to this, vesiculation at rest in malignant cells appears to be calpain dependent. Future work aims to validate these findings and examine the presence of distinct pathways of vesiculation in malignant cells relative to non-malignant cells. These results have important implications in defining novel strategies to selectively targeting malignant cells and for the circumvention of deleterious traits acquired through intercellular exchange of extracellular vesicles.

#250

Interleukin-6 as potential predictive marker for therapeutic effect of Gefitinib in patients with advanced non-small-cell lung cancer harboring EGFR mutations.

Tomoki Tamura, Katsuyuki Hotta, Yuka Kato, Takehiro Tanaka, Kouichi Ichimura, Kadoaki Oohashi, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Mitsune Tanimoto, Katsuyuki Kiura. _Okayama University, Okayama-shi, Kita-ku,Okayama, Japan_.

Background: Although epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitors (TKIs) are the key drug in patients with EGFR-mutant (mt) Non-small-cell Lung Cancer (NSCLC), some of them can not respond well to its therapy. An overexpression of Interleukin (IL)-6 in tumor cells is postulated as a potential mechanism for such resistance or low sensitivity to EGFR-TKI in the preclinical models (PNAS 2010). Here, we evaluated clinically if tumor IL-6 level can be predictive for the effect of EGFR-TKI therapy, and evaluated the expression of IL-6 in EGFR-mt NSCLC cell-lines, and the effect of EGFR-TKI.

Methods: A total of 52 pts with advanced EGFR-mt NSCLC who had received gefitinib (G) were retrospectively assessed. The protein expression of IL-6 in the tumor cells was immunostained. Each specimen was assessed independently by 2 physicians (TT and YK) and 2 pathologists (KI and TT), and judged as positive if ≥ 50% of 100 tumor cells were stained positively (BJC 1999). Serum IL-6 level was measured by CLEIA in 11 (21%) of 52 pts.

Next we checked 13 EGFR-mt NSCLC cell lines. Enzyme linked immunosorbent assay (ELISA) and real time PCR (RT-PCR) of cell culture supernate were peformed.

Results: Patients demographics were as follows: 24 men; median age, 66 yrs; PS 0-1, 48; stage IV, 22; Ad, 49; exon19, 29. Of these, 24 (46%) and 28 (54%) were defined as IL-6-postitive (group P) and IL-6-negative (group N), respectively. Group P had worse PFS (75% v 92% at 6m; p < 0.05), which was retained in the multivariate analysis (HR: 2.38; 95%CI: 1.00-5.68; p=0.05). In contrast, PFS in the platinum-based chemotherapy did not differ in groups P and N (p=0.47). The serum IL-6 level ranged from 0.75 to 23.80 pg/ml (median: 2.90 pg/ml), which correlated neither to that in the tumor cells (regression coefficient: 1.69, p = 0.29) nor PFS in gefitinib therapy (p = 0.44).

The IL-6 level in cell culture supernatant was ranged from 1.46 to 1219.75 pg/ml (median: 85.84 pg/ml). 2 of 13 cell lines showed especially high IL-6 level; IL-6 level of 11-18 was 1090.91 pg/ml and H1650 was 1219.75 pg/ml. RT-PCR also showed the high expression level of IL-6 mRNA in 11-18 and H1650 compared with other cell lines. 11-18 and H1650 showed low sensitivity to G therapy.

Conclusions: Pts in group P benefited less from G therapy.

In vitro experiment suggested that secretion of IL-6 may confer the less sensitivity to EGFR-TKIs. These data suggest that the expression of IL-6 lead to early acquisition of resistance to EGFR-TKIs in EGFR-mt tumors.

#251

Stem cell altruism may serve as a novel drug resistance mechanism in oral cancer.

Bidisha Pal,1 Reza Bayat-Mokhtari,1 Hong Li,1 Rashmi Bhuyan,1 Joyeeta Talukdar,2 Sora Sandhya,2 Anupam Sarma,3 Wael Tasabehji,1 Seema Bhuyan,2 Sukanya Gayan,1 Amal Ch Kataki,3 Debabrata Baishya,4 Herman Yeger,5 Dean W. Felsher,6 Bikul Das6. 1 _Forsyth Institute, Cambridge, MA;_ 2 _KaviKrishna Laboratory, Guwahati Biotech Park, Guwahati, India;_ 3 _B Borooah Cancer Institute, Guwahati, India;_ 4 _Gauhati University, Guwahati, India;_ 5 _Hospital for Sick Children, Toronto, Ontario, Canada;_ 6 _Stanford University School of Medicine, Stanford, CA_.

Background: The mechanism of oral squamous cell carcinoma resistance to platinum-based chemotherapy is not clearly known. Previous studies indicated that glutathione (GSH), a cellular antioxidant may detoxify cisplatinum (CDDP), a commonly used chemotherapy agent in oral cancer. Our previous research in human embryonic stem cells (hESCs) indicated the altruistic behavior of ABCG2+ hESCs that secrete high level of GSH to protect other hESCs exposed to oxidative stress (Das B et al. Stem Cells, 2012). Here we investigated if CDDP exposure lead to altruistic stem cell reprogramming of ABCG2+ oral squamous cell carcinoma cells and subsequent GSH-mediated resistance against CDDP. Methods: Two oral squamous cell cancer cell lines SCC-25 and SCC-15 were treated with 3-10 uM CDDP for three-days, and then subjected to flow cytometry and immunomagnetic sorting based evaluation of ABCG2+ cells. The conditioned media (CM) obtained from ABCG2+ cells were examined for GSH content. The CM treated cancer cell lines were examined for resistance against CDDP toxicity. Next, the post-CDDP treated ABCG2+ cells were examined for enhanced stemness phenotype that corresponds to altruistic stem cell phenotype (Das B et al, Stem Cells 2012). Results: We found that CDDP treatment increases the ABCG2+ self-renewal capacity of SCC-25 and SCC-15 cells as measured by serial transplantation assay. The CM of the post-CDDP treated cells exhibited high level of GSH. When the SCC-15 and SCC-25 cells were treated with CM plus CDDP, the cancer cells exhibited 10-15-fold increase in resistance against CDDP toxicity. Next, the post-CDDP treated SCC-25 and SCC-15 cells exhibited enhanced stemness reprogramming phenotype characterized by very high HIF-2alpha, Sox-2 and Nanog transcriptional activity. Furthemore, we found increased expression of EMT (epithelial mesenchymal transition) marker expression including Snail, Twist and vimentin as evaluated by flow cytometry. siRNA HIF-2alpha treatment led to marked loss in the in vivo self-renewal capacity of ABCG2+ SCC-25 and SCC-15 cells. We then noted that post-CDDP ABCG2+ cells exhibited reversible state, as after two weeks of culture, most of the cells either differentiated or underwent apoptosis. Conclusions: These results indicate that oral cancer cells exhibit altruistic defense mechanism to resist the toxicity of CDDP. The altruistic defense mechanism involved high secretion of GSH. Thus, we suggest that similar to bacterial altruism as a mechanism of drug resistance, stem cell altruism may also serve as a novel mechanism of drug resistance in cancer.

#252

CXCL7-CXCR2 axis as a novel prognostic factor in myeloid cell associated glioblastoma.

Bhagelu R. Achyut, Adarsh Shankar, Meenu Jain, Kartik Angara, ASM Iskander, Roxan Ara, Ali S. Arbab. _Georgia Regents University, Augusta, GA_.

Glioblastoma (GBM), a grade IV glioma, is considered highly vascular and invasive subtype. GBM is most lethal during first year after initial diagnosis despite surgical resection, radiotherapy and/or chemotherapy. Antiangiogenic treatments (AATs) against VEGF-VEGFR pathways resulted into increased GBM tumor growth with marked hypoxia and increased recruitment of bone marrow derived cells (BMDCs) to the tumor microenvironment (TME), without any survival benefits.

Preclinical studies have found that AAT failures were associated with increased bone marrow-derived tumor-associated myeloid cells (TAMCs) in GBM. Depletion of CSF1R+ myeloid cells using GW2580 decreased recruitment of TAMCs in the tumor, and reduced GBM growth. Interestingly, AAT increased the expression of CXCL7 chemokine, and CSF1R blockade decreased CXCL7 in GBM microenvironment. In addition, CXCL7 expression was correlated with number of tumor infiltrating bone marrow derived cells, phospho-ERK MAPK expression, and proliferation of bone marrow cells in GBM.

Here, we looked at the prognostic significance of CXCL7 and its receptor CXCR2 in human GBM. We performed systematic human data analysis (www.oncomine.com), which revealed that CXCL7 is highly expressed (43 to 150 fold) in normal BM compared to other normal organs. This suggests that CXCL7 is required for BM cell survival and migration both in normal and tumor conditions. In GBM datasets, CXCL7 mRNA expression in tumor was increased (> 4.5 fold) in GBM patients (n=27) compared to normal brain (n=4). CXCL7 mRNA expression was seen more in stroma (n=4) than tumor cells (n=5). Overall survival status analysis showed that patients with GBM who died early (n=28) had significantly higher CXCL7 mRNA expression compared to living patients (n=10). In addition, increased CXCL7 mRNA expression was associated with the GBM tumor recurrence (n=8) compared to primary occurrence (n=8). Interestingly, CXCL7 mRNA expression was correlated with the CXCR2 mRNA expression (CXCL7 receptor) both in normal (0.7, n=71) and brain tumor tissues (0.6, n=68). Similarly, CXCR2 mRNA expression was increased in GBM (n=78) compared to other brain tumors. CXCR2 mRNA expression was seen more in stroma (n=4) than tumor cells (n=5). Patients with GBM, who died early (n=19) had significantly higher CXCR2 mRNA expression compared to living patients (n=10). In addition, increased CXCR2 mRNA expression was associated with the GBM tumor recurrence (n=8) compared to primary occurrence (n=8). However, data regarding the CXCL7 and CXCR2 expression in response to AATs is lacking in human cancer datasets.

In summary, we identified CXCL7-CXCR2 axis as a novel prognostic factor in GBM. However, studies are required to investigate CXCL7-CXCR2 regulation in myeloid cell survival/ polarization in TME.

#253

Tetrandrine promotes prostate cancer cell apoptosis in part by up-regulation of death receptors.

Gauri Shishodia, Sweaty Koul, Qin Dong, Sergey Slepenkov, Hari K. Koul. _Louisiana State University Health Sciences Center/FWCC, Shreveport, LA_.

Introduction: Tumor necrosis factor-related apoptosis-inducing-ligand (TRAIL) is a member of the tumor necrosis factor superfamily. TRAIL, a valuable agent for cancer therapy, induces apoptosis in the majority of cancer cells, but development of resistance limits its clinical use in many malignancies including prostate cancer. We have demonstrated that Tetrandrine derivatives (TET), promote apoptosis in PCa cells in vitro and in vivo. In the present study, we investigated the ability of TET to sensitize tumor cells to TRAIL. We also investigated the role of two-death receptors- DR4 and DR5 in overcoming TRAIL resistance in prostate cancer cells.

Methods: Prostate cancer cell lines LNCaP and LNCaP derived C4-2b were used for this study. Cell lines were treated with TET and TRAIL alone or in various combinations of these two for different time points. mRNA levels were quantitated via Real Time-PCR and changes in protein expression were analyzed by Western Blot. Cytotoxicity was evaluated using the Crystal violet and MTT survival assay. Cells were transfected with Lentiviral-shRNA to generate stable DR4 and DR5 single and double knockdown cells. Apoptosis assay was done using BD Pharmingen FITC Annexin-V Apoptosis Detection kit I.

Results: LNCaP and C4-2b cells did not show a significant response following exposure to TRAIL. Exposure of LNCaP and C4-2b cells to TET resulted in increased mRNA and protein levels of death receptors DR4, DR5. Pretreatment of both cell lines with TET for 12h, followed by TRAIL treatment for another 24h increased the apoptosis inducing potential of TRAIL in these cells. Exposure of TET treated cells in vitro to soluble h-TRAIL in combination with TET resulted in synergistic apoptosis response. shRNA targeting DR4 and DR5 significantly reduced the levels of DR4 and DR5 protein. There was a reduced expression of DR4 and DR5 in the knock down cells in response to TET in time-dependent and concentration-dependent manner. In comparison to scrambled cells, DR4, DR5 single knockdown and DR4/DR5 double-knock down cells showed reduced sensitivity to TRAIL and TET. These cells were less apoptotic as compared to scrambled LNCaP cells as revealed by our apoptosis assay data. Taken together these data suggest that TET induced sensitization of PCa cells to TRAIL is mediated in part by DR4/DR5.

Conclusion: Our results suggest that TET induces apoptosis in part by inducing the expression of DR4 and DR5, and provide the proof-of-principle for a novel therapeutic strategy to sensitize cancer cells to apoptosis.

Acknowledgement: Financial support from following Sources is gratefully acknowledged: (VA Merit Award 1BX001258; NCI RO1-CA161880); Carroll W. Feist Endowed Chair Funds, FWCC support and Chair commitment LSUHSC-Shreveport).

#254

Lung cancer cells growing under prolonged period of serum starvation display increased stemness.

Rajkumar Venkatadri, Juan Sebastian Yakisich, Neelam Azad, Anand Krishnan V. Iyer. _Hampton University, Hampton, VA_.

Purpose: Resistance to anticancer drugs is a common cause of failure in chemotherapy. We recently found that adherent cells growing under prolonged periods (7-12 days) of serum starvation become highly resistant to conventional anticancer drugs such as Paclitaxel, Hydroxyurea and Colchicine but retain sensitivity to therapeutic levels of the cardiac glycoside Digitoxin.

Objectives: The main goal of this study is to characterize the biological properties of cells growing under prolonged periods of serum starvation (PPSS), and to elucidate the underlying mechanism of chemoresistance to Paclitaxel and sensitivity to Digitoxin with particular emphasis on Sox2 and the PI3K/AKT signaling pathway.

Experimental setup: Distribution of cell cycle was characterized by flow cytometry. Cell viability was assessed using the MTT assay. Western blotting was employed to evaluate the expression of several key protein markers of cancer stemness (Sox2, Wnt), multidrug resistance (MDR1, ABCG2), epithelial mesenchymal transition (Vimentin), and cell survival (Bcl-2, XIAP, PI3K/AKT).

Results: Cells grown under PPSS showed a higher proportion of cells in G1/S and G2 compared to cells growing under routine culture conditions (RCCs). Western blot analysis showed that these cells expressed higher levels of Sox2, MDR1, ABCG2, Bcl-2 compared to cells growing under RCCs. Pretreatment of cells for 3 h with Verapamil or Sorafenib (MDR1 and ABCG2 inhibitors, respectively) followed by co-incubation with Paclitaxel for 72 h did not overcome Paclitaxel resistance. Obatoclax (a Bcl-2 inhibitor), Wortmannin or LY294002 (PI3K inhbitors) also failed to overcome Paclitaxel resistance. It was interesting to observe that cells growing under PPSS were highly resistant to Obatoclax, Wortmannin and LY294002 compared to cells growing under RCCs. However, these cells were highly sensitive to treatment with Digitoxin.

Conclusions: It is likely that multiple mechanisms (increased stemness, overexpression of multidrug resistance protein and prosurvival proteins) contributes to the resistance of cells under PPSS to Paclitaxel, Obatoclax and PI3K inhibitors. Preliminary results indicates that Digitoxin exerts its antiproliferative effect in part by downregulating Sox2. However, the lack of effect of Wortmannin and LY294002 on Paclitaxel resistance suggests that this effect is not via the PI3K/AKT pathway. Adherent cells growing under PPSS provide a simple yet powerful system to screen for drugs targeting cancer stemness.

#255

Docetaxel inhibits testosterone mediated activation of the AR pathway by SLCO1B3.

Ellen S. de Morree,1 Ashraf Aghai,1 Alice A. Gibson,2 Ron H.J. Mathijssen,1 Eric A.C. Wiemer,1 Alex Sparreboom,3 Wytske M. van Weerden,1 Ronald de Wit1. 1 _Erasmus University, Rotterdam, Netherlands;_ 2 _Ohio State University, Rotterdam, OH;_ 3 _Ohio State University, Ohio, OH_.

Background

Docetaxel is the first line of chemotherapy in metastatic castration resistant prostate cancer (mCRPC) patients. More than 50% of prostate cancer tumors express the solute carrier organic anion transporter family member 1B3 (SLCO1B3/OATP1B3), an uptake transporter that actively transports docetaxel into the cell. Interestingly, SLCO1B3 is also a transporter of endogenous substrates, such as testosterone. As the androgen receptor (AR) pathway is still active in mCRPC patients, we hypothesize that docetaxel can interact with the uptake of testosterone through SLCO1B3, which would result in decreased AR-pathway activation and subsequent tumor growth inhibition.

Materials and Methods

SLCO1B3 overexpressing cells (CHO-1B3) were used for uptake experiments with radioactively labeled hormones, such as estradiol-β-glucuronide (E2G). We stably transfected prostate cancers cells (PC346C-DCC and PC346C-DCC-G) with SLCO1B3 or with a green fluorescent protein (GFP) tag only. An androgen responsive element (ARE)-luciferase assay was created as a sensitive measure of AR pathway activation. ARE-activity and prostate specific antigen (PSA) production were analyzed with an ELISA assay after exposure to 1nM testosterone for 30 minutes with or without preincubation with docetaxel 10 μM in SLCO1B3 overexpressing and GFP- tagged control cells.

Results

SLCO1B3 overexpressing cells showed testosterone-mediated activation of the AR pathway: ARE-activity increased 6.4-fold and PSA production 7-fold in SLCO1B3-overexpressing cells as compared to control cells. Pre-incubation of CHO-1B3 cells with docetaxel resulted in reduced uptake of E2G to the level observed in control cells. Similarly, after exposure to testosterone and docetaxel ARE-activity was significantly decreased in SLCO1B3-overexpressing cells compared to that of controls.

Conclusion: Docetaxel inhibits the activation of the AR pathway via SLCO1B3. This is a novel anti-androgenic effect exerted by docetaxel in addition to inhibition of the AR translocation that we demonstrated previously. Experiments are ongoing to investigate if testosterone uptake via SLCO1B3 is inhibited by docetaxel.

#256

Analysis of combined drug effects on hENT1 and hENT4 in pancreatic cancer cells.

Stancy J. Joseph, Beverly Word, Beverly Lyn-Cook. _NCTR/FDA, Jefferson, AR_.

Pancreatic cancer is the 4th leading cause of cancer death and diagnosis usually occurs at late stages due to the lack of symptoms and early detection, making surgical intervention almost unfeasible due to low survival rates. Pancreatic cancer patients have one of the worst prognoses among all cancer types with a 5 year survival rate of less than 5%. Despite significant improvement in understanding molecular and epigenetic changes of this disease, the prognosis and management remained unchanged. Gemcitabine, a deoxycytidine nucleoside analog, is the golden standard of advanced pancreatic cancer treatment for patients with locally advanced or metastatic cancer of the pancreas. Patients treated with gemcitabine can, however, eventually develop resistance to this drug. Previously published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C) though up-regulation of the human equilibrative nucleoside transporter 1 (hENT1). hENT1 (SLC29A1) is the major drug transporter for gemcitabine. One of the drugs currently being investigated for treatment of pancreatic cancer is metformin. Metformin is most commonly used for the treatment of type 2 diabetes mellitus and has exhibited both chemopreventive and chemotherapeutic activities in preclinical human pancreatic cancer cells and animal models. Metformin has been found to be transported by another member of the equilibrative nucleoside transporter (ENT) family named hENT4 (SLC29A4). The current study examined the combined drug effects of gemcitabine, metformin and I3C on hENT1 and hENT4 in pancreatic cancer cells. Several pancreatic cell lines were examined for cell viability, drug synergy and modulation of hENT1 and hENT4 expression when treated with either gemcitabine, metformin, I3C or in combination for 24h and 72h. The results varied for each of the cell lines. After 24h and 72h treatment there was a significant decrease in cell viability when pancreatic cancer cells were treated with metformin or gemcitabine or 500µM I3C alone or in combination. The decrease in cell viability for these treatment conditions was also time dependent, where pancreatic cancer cells treated for 72h exhibited lower cell viability compared to 24h treatment. Pancreatic cancer cells treated with 250µM I3C alone or in combination did not decrease cell viability after 24h; however after 72h the cell viability varies based on the cell line. In most cases the drug activity exhibited antagonistic effect in combination therapy treatment. hENT1 and hENT4 expression levels varied between cell lines, where gemcitabine resistant cell lines exhibited lower hENT levels. Our initial findings showed that gemcitabine may also modulate hENT4 expression in gemcitabine sensitive cell lines and I3C modulations hENT4 expression levels.

#257

Evaluation of organic cation transporter 1 (OCT1, SLC22A1) as transporter for sorafenib.

Claudia Neul,1 Sharyn D. Baker,2 Alex Sparreboom,2 Elke Schaeffeler,1 Stefan Laufer,3 Matthias Schwab,4 Anne T. Nies1. 1 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, and University of Tübingen, Stuttgart, Germany;_ 2 _Division of Pharmaceutics & Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH; _3 _Dept. of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Eberhard Karls University Tübingen, Tübingen, Germany;_ 4 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart and Dept. of Clinical Pharmacology, Institute of Experimental and Clinical Pharmacology and Toxicology, University Hospital Tübingen, Stuttgart, Germany_.

The multikinase inhibitor sorafenib is approved for the treatment of advanced clear-cell renal and hepatocellular carcinomas, yet, patients' survival is prolonged for only several months (Wilhelm et al., Mol Cancer Ther 2008). The understanding of this poor efficacy remains incomplete. One reason for treatment failure may be the high interindividual variability in pharmacokinetics of the orally-given sorafenib. Membrane transporters may contribute to pharmacokinetics of sorafenib and its metabolites as recently reported for the hepatic solute carrier (SLC) organic anion transporter family member 1B1 (OATP1B1) and OATP1B3 (Zimmerman et al, Clin Cancer Res 2013). In contrast, the role of organic cation transporter 1 (OCT1), which is also a major hepatic uptake transporter (Nies et al, Handb Exp Pharmacol 2011), for cellular uptake of sorafenib is controversial (Hu et al, Clin Cancer Res 2009; Swift et al, Drug Metab Dispos 2013; Herraez et al, Hepatology 2013). We therefore sought to comprehensively elucidate the impact of OCT1 on cellular sorafenib uptake by using a combination of different in vitro cell culture models and in vivo approaches involving experiments with Oct1 knockout (-/-) mice. Firstly, we analyzed cellular accumulation of radiolabeled sorafenib by the mammalian cell line HEK293 stably expressing OCT1. Despite overexpression of a functional OCT1 transporter, cellular uptake of neither sorafenib nor its active metabolite sorafenib N-oxide was increased. Next, we studied sorafenib and sorafenib N-oxide pharmacokinetics in mice with a homozygous genetic deletion of the Oct1 transporter. The deficiency of Oct1 had no influence on plasma and hepatic sorafenib and sorafenib N-oxide concentrations. Finally, we investigated sorafenib transport into the human hepatocellular carcinoma cell lines HepG2 and HuH7. Although neither OCT1 mRNA nor protein was detected, a considerable cellular accumulation of sorafenib was observed in both cell lines. Expression profiling of a panel of 55 relevant SLC drug uptake transporters identified several potential novel candidates mediating sorafenib accumulation.

We conclude from these findings that cellular uptake of sorafenib and sorafenib N-oxide is independent of OCT1 and that other, as yet unidentified, transport mechanisms are responsible for cellular sorafenib uptake.

Supported by the Robert Bosch Foundation, Stuttgart, Germany and the ICEPHA Grant Tübingen-Stuttgart, Germany.

#258

Is P53 the up-and-coming predictive biomarker for volasertib treatment in NSCLC.

Jolien Van den Bossche,1 Filip Lardon,1 Christophe Deben,1 Ines De Pauw,1 Vanessa Deschoolmeester,1 Evelien Smits,1 Pol Specenier,2 Patrick Pauwels,2 Marc Peeters,2 An Wouters1. 1 _Antwerp University, Wilrijk, Belgium;_ 2 _Antwerp University Hospital, Edegem, Belgium_.

Introduction: Polo-like kinase (Plk1), a key regulator of cell division, is considered as an attractive target for mitotic intervention. The observed Plk1 overexpression in several tumor types, including non-small cell lung cancer (NSCLC), has led to the development of small molecule inhibitors. Moreover, previous studies suggested an interplay between Plk1 and the tumor suppressor P53. This led to the hypothesis that the P53 status might be predictive for the response to Plk1 inhibition. Therefore, we investigated the cytotoxic effect of volasertib in NSCLC cell lines with a different P53 background, under normoxia and hypoxia.

Material and methods: A549 (TP53 wild type) was transduced with a P53 shRNA lentiviral vector to obtain the P53 deficient sub-cell line A549-920 or with an empty vector as control (A549-NTC). In addition, NCI-H1975 cells were used as a TP53 mutant (R273H) cell line. Cells were treated with 0-85nM volasertib (24-72h). Cell survival was assessed using the sulphorhodamine B assay and IC50-values were calculated using WinNonlin software. The effect of Plk1 inhibition (0-20nM) on cell cycle distribution (24h) and apoptosis induction (48h) was determined flow cytometrically. In addition, we investigated the potential of volasertib to inhibit cell migration using a Transwell system. All experiments were performed under both normoxia and hypoxia (<0.1% O2).

Results: Plk1 inhibition established a dose-dependent growth inhibition under both normoxia and hypoxia. Interestingly, a reduced sensitivity to volasertib treatment (24h) was observed in P53 deficient cells (A549-920, IC50: 27.59±5.77 nM) compared to P53 wild type cells (A549-NTC, IC50: 17.87±0.40 nM) (p<0.001). Except for the NCI-H1975 cells, a decreased effect of volasertib was observed under hypoxia (p<0.001). After treatment, a G2/M phase block was induced in all cell lines (p<0.004), albeit more pronounced in A549-920 cells compared to A549-NTC cells (p=0.06). Under hypoxia, this mitotic arrest was only detected using high volasertib concentrations (12.5–20nM) (p<0.04). Furthermore, a sub-G1 peak could be observed after treatment with high volasertib concentrations, suggesting apoptosis induction. Indeed, an increase in PI-stained cells was detected 48h after Plk1 inhibition in all cell lines (p<0.042). Remarkably, induction of cell death was higher in P53 wild type cells compared to P53 deficient cells (p<0.02). Transwell experiments, investigating the potential of volasertib to inhibit cell migration, are currently being analyzed.

Conclusion: Our results show that there is a difference in response to Plk1 inhibition in cancer cells with and without functional P53. While P53 deficient/mutant cells were mostly arrested in the G2/M phase, cell death was induced in P53 wild type cells. These data lead to the hypothesis that P53 might be a predictive marker for response to Plk1 inhibition.

#259

Modulation of doxorubicin actions on HCC cells by insulin-like growth factor-I.

Brian I. Carr,1 Maria Grazia Refolo,2 Rosalba D'Alessandro,2 Nicola Carella,2 Caterina Messa,2 Aldo Cavallini2. 1 _iBG Biomedicine and Genome Institute, Izmir, Turkey;_ 2 _IRCCS De Bellis Medical Center, Castellana Grotte, Italy_.

Tumor microenvironment is increasingly understood to influence tumor biology in many tumor types, including HCC. We recently showed that platelets could modulate Doxorubicin (Dox)-mediated HCC growth inhibition (Refolo MG et al. Modulation of doxorubicin mediated growth inhibition of hepatocellular carcinoma cells by platelet lysates. Anticancer Agents Med Chem. 2014;14:1154-60). Platelets contain cytokines and growth factors, including IGF-1, PDGF, FGF, EGF and serotonin. We found that Insulin-like growth factor-1 (IGF-1) shifted the Dox growth-inhibitory dose-response in the range of 0.1-1.0 µM on Hep3B, HepG2, PLC/PRF/5 human HCC lines, all of which became less sensitive to both Dox-mediated inhibition of growth and apoptosis. Similar antagonism by IGF-1 was found for Dox-mediated inhibition of cell migration (scratch assay) and invasion (Boyden chamber). This antagonism of Dox effects on cell growth, migration and apoptosis by IGF1, was blocked by GSK1838705A, an IGF-1R inhibitor. Furthermore, when platelet extracts were used to antagonize Dox inhibition of growth and migration, the platelet effects were also blocked by addition of GSK1838705A to the cultures, showing that the platelet effects were largely mediated by IGF-1. Dox caused a decrease in the levels of anti-apoptosis markers Bcl-2, Bcl-xL and survivin as seen on WB, whereas levels of all three anti-apoptosis markers were increased by IGF-1 action. These actions of IGF-1 were also blocked by GSK 1838705A. Furthermore, Dox caused a decrease in the levels of P-ERK, P-p38 and P-STAT3 (ser727). These signaling molecule decreases were also reversed by the presence of IGF-1.

Conclusions. Dox actions on HCC cells can be antagonized by platelets and specifically by platelet-associated IGF-1. IGF-1 antagonists appear to be attractive enhancers of sensitivity to Dox actions.

### Combination Chemotherapy

#260

Quercetin protects from doxorubicin induced vascular toxicity but impairs its cytotoxic profile in breast cancer cells.

Hanan A. Assiri,1 Fahad A. Al-Abbasi,1 Hany M. El-Bassossy,1 Mohamed A. El-Moselhy,2 Ahmed M. Al-Abd1. 1 _King Abdulaziz Univ., Jeddah, Saudi Arabia;_ 2 _Ibn Sina National College for medical Studies, Jeddah, Saudi Arabia_.

The use of anti-cancer adjuvant therapy is rationalized by potentiating the efficacy and/or protecting from major side effects of chemotherapeutics. Doxorubicin is an effective anthracycline anticancer agent for breast cancer despite its dose-limiting cardiovascular toxicity. Quercetin is a flavonoid compound with potent antioxidant and cardiovascular protective activity. The purpose of this study is to investigate the influence of quercetin on the cytotoxic profile of doxorubicin in breast cancer cell lines on the top of ameliorating doxorubicin induced acute vascular toxicity. Incubation of isolated aortic rings with doxorubicin (10 uM) significantly increased contractile response of aorta to phenylephrine and decreased its relaxation response to acetylcholine. Quercetin significantly protected from doxorubicin induced vascular toxicity showing reduced contractile and increased relaxation responses of aorta to doxorubicin. On the other hand, quercetin combination significantly resulted in increasing the IC50 of doxorubicin from 0.4±0.03 to 0.8±0.06 µM in MCF-7 cells with combination index indicative of strong antagonism (CI-value = 3.2). The IC50 of doxorubicin did not change after combination with quercetin in MDA-MB-231 cells (70±10 and 76.3±4 nM, respectively), however the CI-value was 1.98 which is indicative of antagonism as well. Only in T47D cells, quercetin combination with doxorubicin was additive (CI-value = 1.17) and the IC50 of doxorubicin was significantly dropped from 0.74±0.05 to 0.36±0.003 µM. Quercetin did not affect total ROS level induced by doxorubicin, however, it significantly impaired the immediate phase of intracellular ROS generation by doxorubicin within MCF-7 cells from 125.2±3.6% to 102.5±3.9% of control cells. Yet, quercetin synergized doxorubicin cytotoxicity against MCF7 cells under hypoxic condition and significantly decreased its IC50 from 8.9±0.7 to 0.9±0.02 µM (CI-value = 0.2). Further investigation showed that the combination of doxorubicin with quercetin significantly increased the concentration of pro-caspase 3 compared to doxorubicin alone in T47D and MDA-MB-231 cell lines. Cell death mechanism was further investigated using annexin-V/PI differential staining. Quercetin combination did not significantly increase the percent of apoptotic or necrotic cells compared to treatment with doxorubicin alone in all cell lines under investigation. In addition, quercetin induced significant accumulation of cells at S-phase as well as in G2/M-phase in both MCF-7 and MDA-MB-231 cell lines. Quercetin did not affect cell cycle distribution in T47D cells. In conclusion quercetin despite its potent vascular protective activity against doxorubicin, it shows antagonistic to boarder line-additive interactions with doxorubicin in different breast cancer cell lines.

#261

Quinacrine in endometrial cancer: repurposing an old antimalarial drug.

Eleftheria Kalogera, Debarshi Roy, Ashwani Khurana, Susmita Mondal, Xiaoping He, Sean C. Dowdy, Viji Shridhar. _Mayo Clinic, Rochester, MN_.

Although the majority of patients with endometrial cancer (EC) are diagnosed early when disease is confined in the uterus and prognosis is excellent, there is a subset of patients with advanced, metastatic, recurrent or persistent after surgery disease with dismal prognosis. Chemotherapy has a critical role for these patients. Carboplatin (carbo) and paclitaxel (taxol) is the standard first-line chemotherapy in EC with response rates as low as 50% and even lower for recurrent disease. Furthermore, many patients will eventually relapse with chemo-resistant disease and very poor prognosis. As the chemotherapeutic options for these patients are limited and of questionable efficacy, it is of paramount importance that novel agents are identified to increase or restore chemo-sensitization to platinum-based chemotherapy.

Quinacrine (QC) is an inexpensive antimalarial drug with a predictable safety profile which recently surfaced as a promising anticancer agent thought to be associated with decreased risk of developing chemo-resistance through targeting multiple pathways simultaneously. While prior investigators have provided evidence that QC exerts strong anticancer activity against a wide range of solid tumors, there are no studies focused on QC in EC.

In our study, QC exhibited strong synergism in vitro when combined with cisplatin, carbo or taxol with the highest level of synergism being observed in the most chemo-resistant EC cell line. Neither QC monotherapy (QC every other day for 3 weeks) nor standard chemotherapy (carbo+taxol for 3 cycles, on days 3, 7, 11) significantly delayed tumor growth in the mouse xenografts. Only combination (standard chemo + QC every other day during carbo+taxol treatment) and maintenance (combination regimen followed by QC every other day until end of study) significantly augmented carbo+taxol antiproliferative effect as evidenced by the significant decrease in tumor burden and delayed tumor growth in xenografts. Combination treatment was associated with a 14-day prolongation of median survival compared to carbo+taxol (68 vs. 54 days). Maintenance therapy with QC was proven superior to carbo+taxol as it resulted in long-term stabilization of disease and further prolongation of overall survival; in fact, median survival in the maintenance group was not reached as the mice were surviving at the end of study. QC alone, in combination with carbo+taxol or as maintenance was well-tolerated with no issues of weight loss compared to control mice. A yellow skin discoloration was noted during QC treatment which was entirely reversible within few days upon QC discontinuation.

In conclusion, QC exhibited significant antitumor activity against EC cell lines in vitro and was successful in stabilizing disease and prolonging survival as maintenance therapy in chemo-resistant EC mouse xenografts. This data suggests that QC may be an important adjunct to standard chemotherapy for patients with recurrent EC.

#262

Combination therapy of folate-targeted chemotherapeutics with anti-PD-1 antibody against folate receptor-positive tumors in immunocompetent murine models.

Yingjuan June Lu, Leroy W. Wheeler, Vicky Cross, Elaine Westrick, Alex Lloyd, Christopher P. Leamon. _Endocyte, Inc., West Lafayette, IN_.

The PD-1/PD-L1 (programmed cell death protein 1; programmed death-ligand 1) pathway plays a key role in immunological tolerance and autoimmunity. PD-1 is primarily expressed on activated T and B cells while its receptor, PD-L1, is expressed in a variety of cell types including resting lymphocytes, DCs, and macrophages. Recently, multiple human solid tumor types (melanoma, NSCLC, RCC, ovarian, and colorectal cancer) have been found to overexpress PD-L1 within its tumor microenvironment. More importantly, PD-1/PD-L1 blocking agents (pembrolizumab, nivolumab, lambrolizumab, MPDL3280A) have been found active as a single agent or in combination with chemotherapy and some have produced durable clinical responses. On the other hand, human epithelial tumors are also known to overexpress the high-affinity folate receptor (FR)-alpha that is capable of bringing folate-linked drugs into the cell cytosol via endocytosis. Using flow cytometry, we assessed PD-L1 expression in various FR-positive syngeneic mouse tumor models (M109, Renca, L1210A, 4T1-Cl2, ID8-Cl15). PD-L1 expression was found to vary significantly among different tumor types and could be found not only on CD45- malignant cells but also on tumor stromal cells such as F4/80+CD11b+ tumor-associated macrophages. Using a rat anti-mouse PD-1 antibody (clone RMP1-14), we studied in-vivo efficacy of a folate-targeted chemotherapy in combination with PD-1/PD-L1 blockage in PD-L1-expressing tumor models. In syngeneic models with high PD-L1 expression and tumor-infiltrating lymphocytes, a synergistic and curable anti-tumor effect was observed and the animals developed tumor-protective immunity against rechallenge. Thus, our preliminary results suggest that folate-targeted chemotherapeutics may also be enhanced by PD-1/PD-L1 blockage by means of overcoming tumor-mediated immune suppression

#263

Epicatechin protects from doxorubicin induced cardiotoxicity without affecting its cytotoxic profile in breast cancer cells.

Ohoud Y. Alshehri,1 Fahad A. Al-Abbasi,1 Hany M. El-Bassossy,2 Hossam M. Abdallah,3 Ahmed M. Al-Abd4. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Zagazig Univ., Zagazig, Egypt;_ 3 _Cairo University, Cairo, Egypt;_ 4 _National Research Center, Giza, Egypt_.

Doxorubicin is an effective anthracycline anticancer agent for breast cancer despite its dose-limiting cardiovascular toxicity. Epicatechin is a flavonoid compound with potent antioxidant activity.. The purpose of this study is to investigate the influence of epicatechin on the cytotoxic profile of doxorubicin in breast cancer cell lines on the top of ameliorating doxorubicin induced cardiotoxicity. Rats received doxorubicin (12 mg/kg, single iv injection) with or without epicatechin (12 mg/kg, daily, oral) for 6 days then cardiac electrocardiographic was recorded. Doxorubicin administration induced cardiac ischemia which was reflected by increased QT and QTc intervals (1.76 and 1.8 folds, respectively); and increased heart rate. Epicatechin co-administration completely blocked doxorubicin induced ECG abnormalities. Epicatechin combination significantly decreased the IC50 of doxorubicin from 0.55±0.03 to 0.24±0.01; from 0.7±0.036 to 0.56±0.017, and from 0.07± 0.017 to 0.04±0.014 µM in MCF-7, T47D and MDA-MB-231 breast cancer cell lines, respectively. In addition, epicatechin significantly increased doxorubicin cytotoxicity against MCF-7 cells under hypoxic condition and decreased its IC50 from 8.9±1.2 to 6.0±1.2 µM. In MDA-MB-231, epicatechin significantly blocked p-gp efflux activity and enhanced intracellular accumulation of p-gp probe (rhodamine) by 3-4 folds at concentration range (3-100 µM). Further investigation showed that the combination of doxorubicin with epicatechin significantly increased the concentration of pro-caspase 3 compared to doxorubicin alone in T47D and MDA-MB231 cell lines. Cell death mechanism was further investigated using annexin-V/PI differential staining. Epicatechin combination did not significantly increase the percent of apoptotic or necrotic cells compared to treatment with doxorubicin alone in MCF-7 and MDA-MB-231 cells. In T47D, epicatechin combination with doxorubicin significantly increased cell apoptosis and necrosis compared to doxorubicin treatment alone from 1.5±0.5 to 4.6±0.7% and 8.0±1.7 to 12.8± 7.9%, respectively. In addition, epicatechin combination with doxorubicin decreased pro-caspase-3 concentration from 16.7±1.1 fg/cell to 9.2±2.6 fg/cell. Finally, epicatechin did not affect cell cycle distribution induced by doxorubicin. In conclusion, epicatechin significantly protected from doxorubicin induced cardiotoxicity and marginally improved its cytotoxicity against breast cancer cells.

#264

The cell cycle checkpoint kinase 1/2 inhibitor, LY2606368 with PARP inhibition results in synergistic cytotoxicity in high-grade serous ovarian cancer (HGSOC) at lower than physiologically administered concentrations.

Yeong-ran Ahn, Takuhei Yokoyama, Minshu Yu, Nicolas Gordon, Elise C. Kohn, Jung-min Lee. _NIH, Bethesda, MD_.

Background: Chk1/2 are major cell cycle regulators in p53-deficient tumors, such as HGSOC. Chk1 plays a critical role in DNA repair, facilitating the BRCA2-RAD51 interaction by phosphorylating the BRCA2 C-terminal domain and Thr309-RAD51. Clinical activity with the Chk1/2 inhibitor, LY2606368 mesylate monohydrate (LY), has been observed in solid tumors. We hypothesize that Chk1 inhibition would sensitize HGSOC to PARP inhibition (PARPi) by preventing nuclear RAD51 foci formation, thus impairing DNA repair. We investigated potential synergy of the combination of LY and the PARPi, olaparib (O), in HGSOC cell lines, testing lower concentrations than clinically attained.

Materials and Methods: We examined cytotoxicity, DNA damage, and nuclear RAD51 foci formation by LY and/or O using XTT assay, comet assay and immunofluorescence (IF), in 3 HGSOC cell lines: two BRCA1/2 wild type (CAOV3, OV90), and one with BRCA2 mutation (PEO1). We examined a dose range based on clinically achievable concentrations of LY (0.53μM–1.34μM) and O (7.8μM–11.0μM) and calculated IC50 concentrations for cytotoxicity. The combination index (CI) was calculated to evaluate synergism. DNA damage and nuclear RAD51 foci formation were examined. DMSO was used as vehicle control. All experiments were performed in at least three replicates in all cell lines. Results are presented as mean ± SEM.

Results: LY alone yielded cytotoxicity in CAOV3, OV90, and PEO1 with IC50 6.34nM, 35.2nM, 12.6nM, respectively. O 5μM monotherapy resulted in 47%, 13%, and 69% cytotoxicity in CAOV3, OV90, and PEO1. LY/O combination showed 51%, 58% and 82% cell injury in CAOV3, OV90 and PEO1. CI values indicated cytotoxicity synergism with the combination. LY/O (5nM/5μM) treatment showed greater DNA damage than O alone in OV90 and PEO1 (p<0.05), and a similar trend was observed in CAOV3 (20.2% ±3.10 vs. 6.71% ±1.08, p=0.05). The mean percentage of cells with ≥ 5 nuclear RAD51 foci decreased with LY/O (5nM/5μM) combination therapy compared to O alone in CAOV3 (9.03% ±1.85 vs. 36.3% ±7.45, p<0.01) and OV90 (3.66% ±1.84 vs. 22.9% ±3.08, p<0.01), but not in PEO1 (1.26% ± 1.26 vs. 3.73% ±3.73). Ongoing work includes cell cycle analysis and immunoblotting to examine biochemical changes in DNA repair and cell cycle checkpoint pathways.

Conclusions: Our preliminary results suggest synergistic activity of the combination of LY and PARPi in HGSOC cell lines using concentrations that are lower than clinically attainable in patients.

#265

Cotargeting EGFR, STAT3 and Src-Notch pathways: a promising approach to improve the efficacy of EGFR-TKIs in the treatment of NSCLC patients.

Niki Karachaliou,1 Imane Chaib,2 Sara Pilotto,3 Jordi Codony,4 Xueting Cai,5 Xuefei Li,6 Ana Drozdowskyj,7 Carles Codony,4 Andrés Felipe Cardona,8 Guillermo López-Vivanco,9 Alain Vergnenègre,10 José Miguel Sánchez,11 Mariano Provencio,12 Filippo de Marinis,13 Enric Carcereny,2 Noemí Reguart,14 Rosario García-Campelo,15 Silvia Marin,2 Cristina Teixido,4 Isabella Sperduti,16 Sonia Rodríguez,4 Roger Estrada,17 Raimon Puig de la Bellacasa,17 José Luis Ramírez,18 Miguel Angel Molina-Vila,4 Caicun Zhou,6 Peng Cao,5 Patrick Ma,19 Trever Bivona,20 Rafael Rosell18. 1 _Instituto Oncológico Dr Rosell (IOR), Quirón-Dexeus University Institute, Barcelona, Spain;_ 2 _Institut Catala d'Oncologia. Hospital Germans Trias i Pujol, Badalona, Spain;_ 3 _Azienda Ospedaliera Universitaria Integrata , University of Verona, Verona, Italy;_ 4 _Pangaea Biotech, Quirón-Dexeus University Institute, Barcelona, Spain;_ 5 _Hospital of integrated traditional Chinese and Western Medicine, Nanjing, China;_ 6 _Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Shangai, China;_ 7 _Pivotal, Madrid, Spain;_ 8 _Clinical and Translational Oncology Group-FIMAC, Bogotá, Colombia;_ 9 _Hospital de Cruces de Barakaldo, Vizcaya, Spain;_ 10 _Service de Pathologie Respiratoire et d'Allergologie, CHU, Limoges, France;_ 11 _Hospital La Princesa, Madrid, Spain;_ 12 _Hospital Puerta de Hierro, Madrid, Spain;_ 13 _Istituto Europeo di Oncologia (IEO), Milano, Italy;_ 14 _Hospital Clínic, Barcelona, Spain;_ 15 _Hospital A Coruña, A Coruña, Spain;_ 16 _Regina Elena National Cancer Institute, Rome, Italy;_ 17 _Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain;_ 18 _Institut Catala d'Oncologia, Univ. Hospital Germans Trias i Pujol, Badalona, Spain;_ 19 _West Virginia University, Lung Cancer Research, VA;_ 20 _UCSF Helen Diller Familiy Comprehensive Cancer Center, San Francisco, CA_.

Intrinsic or acquired resistance limits the clinical effectiveness of EGFR tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer (NSCLC) patients (p) with EGFR mutations. One of the signaling mediators downstream of activated EGFR is signal transducer and activator of transcription 3 (STAT3). Not only does gefitinib not inhibit STAT3, but it also augments STAT3 tyrosine phosphorylation. EGFR blockade enriches lung cancer stem cells (CSCs) through NOTCH3-dependent signaling. A co-receptor of IL-6 (gp130) associates with Src and triggers activation of YAP and NOTCH. Our study is designed with three parallel objectives: firstly, to demonstrate that single EGFR TKI treatment cannot abrogate STAT3 and Src in EGFR mutant NSCLC cell lines; secondly, to examine whether the combination of gefitinib with compounds that target STAT3, (TPCA-1) and Src (saracatinib), suppresses the mechanisms of resistance; thirdly, to identify biomarkers in clinical tumor samples that may help us predict the outcome of EGFR TKIs and design effective combination therapies. Cell viability assay (MTT), western blotting, quantitative-real time PCR (qRT-PCR) and aldefluor assay-flow cytometry were used. We found that gefitinib increases pSTAT3 Y705 in PC-9 cells (that harbor the exon 19 deletion) in a time- and dose-dependent manner. Nine days after gefitinib treatment STAT3 mRNA level was significantly elevated. PC-9 cells showed dramatic increase in the fraction of ALDH+ cells upon treatment with gefitinib. TPCA-1 increased sensitivity to gefitinib in the PC-9 cells. Combination of gefitinib with TPCA-1 abrogated pSTAT3 Y705 but neither inhibited pPaxillin Y118 (Src induced) and pYAP S127 nor prevented the increment in the ALDH+ CSCs subpopulation. The triple combination of gefitinib, TPCA-1 and saracatinib was highly synergistic and abrogated pSTAT3 Y705, pPaxillin Y118 and pYAP S127. We performed qRT-PCR at baseline tumor samples of 64 EGFR mutant NSCLC p treated with first line EGFR TKIs and found that high expression of STAT3 and YAP were significantly correlated with shorter median progression-free survival (mPFS). mPFS was 9.6 months (m) (95% CI, 5.9 to 14.1) for p with low STAT3 and 18.4m (95% CI, 8.8 to 30.2) for p with high STAT3 mRNA expression (P<0.001). mPFS was 9.6 months (95% CI, 7.7 to 15.2) for p with low YAP and 23.4 months (95% CI, 13.0 to 28.1) for p with high YAP mRNA expression (P=0.005). A combined STAT3 and YAP risk group model was constructed since the mRNA expression of the 2 transcripts was weakly correlated (r=.0.15; P=0.305). mPFS was 25.7 months for p with low STAT3 and YAP mRNA (95% CI, 8.5 to 60.9), 9.4 months for p with high STAT3 and YAP mRNA (95% CI, 2.8 to 15.2), and 14.1 months for others (95% CI, 8.2 to 23.4) (P=0.004). Single EGFR TKI treatment can no longer be considered adequate for p with EGFR mutant lung cancer and a clinical trial co-targeting STAT3 and Src is warranted.

#266

The G-quadruplex ligand EMICORON potentiates the antitumor efficacy of chemotherapy on colon cancer experimental models.

Manuela Porru,1 Simona Artuso,1 Luca Pompili,2 Carla Caruso,3 Armandodoriano Bianco,4 Marcella Mottolese,1 Carla A. Amoreo,1 Annamaria Biroccio,1 Carlo Leonetti1. 1 _Regina Elena National Cancer Institute, Rome, Italy;_ 2 _Regina Elena National Cancer Institute and University La Tuscia, Rome and Viterbo, Italy;_ 3 _University La Tuscia, Viterbo, Italy;_ 4 _University "Sapienza", Rome, Italy_.

Background. G-quadruplex (G4) structures, present at the telomeric ends of chromosomes and in the promoters of a wide range of genes important in cell signaling, gained interest as therapeutic targets and several small molecules able to bind and stabilize G4 structures have been developed. However, due to the poor drug-like properties and/or selectivity profile none of the G4 ligands has progressed through the drug discovery pipeline. Our group recently showed that the novel G4 ligand EMICORON exhibited a favourable pharmacological profile, was well tolerated in mice and elicited antitumor efficacy against advanced experimental models of colon cancer. Based on these results, our aim was to study the ability of EMICORON to increase the efficacy of the standard chemotherapy for human colon cancer.

Matherials and Methods. We assessed the in vitro cytotoxic activity of EMICORON in combination with SN-38, 5-Fluorouracil and Oxaliplatin by clonogenic and 3D tumor-spheroid formation assay on HT29 colon cancer cells. The combination index (CI) values indicating synergism (CI 0.9 1.2) was evaluated. In vivo experiments were performed in mice bearing HT29 tumors treated with Irinotecan followed by EMICORON in one or two cycles of administration. Moreover, efficacy of the standard FOLFIRI (5-Fluorouracil, Leucovorin and Irinotecan) or FOLFOX (5-Fluorouracil, Leucovorin and Oxaliplatin) was evaluated in two different colon cancer patient-derived xenografts (PDXs).

Results and conclusions. The exposure of HT29 cells to the combination of EMICORON with chemotherapeutics resulted strongly synergistic when the drugs were administered following the sequence SN-38→EMICORON, EMICORON→5-Fluoruracil or EMICORON→Oxaliplatin, while the opposite sequences were additive or slightly antagonistic. The high activity of SN-38 followed by EMICORON was confirmed in the HT29-formed spheroids. When mice bearing HT29 xenografts were treated with EMICORON or Irinotecan, a tumor weight inhibition (TWI) of 35 or 65 % was observed respectively, while the treatment with Irinotecan followed by EMICORON resulted more effective with a TWI of 80%. This marked antitumor effect of the combination produced an increase in overall survival of mice of 85%. Interestingly, the administration of a second cycle of treatment produced an impressive increase of survival of mice to 114%. Then, we tested the efficacy of FOLFOX or FOLFIRI therapeutic regimens on two colon cancer PDXs and as expected these treatments were very effective in reducing the tumor mass and delaying the tumor regrowth, but the relapse of the disease was observed in all the mice. In conclusion, our results demonstrating that EMICORON is able to increase the potency of any single drug, provide a compelling argument to suggest that the integration of EMICORON in standard chemotherapeutic regimens could be a highly valuable strategy.

#267

Preclinical investigation of the HSP90 inhibitor, ganetespib, in combination with FDA-approved cytotoxic agents for their potential anti-tumor effects on human neuroendocrine tumors.

Chung Wong,1 Evan Vosburgh,1 Arnold Levine,2 David Proia,3 Eugenia Xu1. 1 _Raymond & Beverly Sackler Foundation, New Brunswick, NJ; _2 _Institute for Advanced Study, Princeton, NJ;_ 3 _Synta Pharmaceuticals Corporation, Lexington, MA_.

The gastroenteropancreatic neuroendocrine tumor (GEP-NET) system is comprised of a heterogeneous group of tumors with increasing incidence. Current standard of care cytotoxic agents have limited efficacy, which necessitates the need for innovative therapeutic approaches. Heat shock protein 90 (HSP90) is overexpressed in a wide range of tumor types including human pancreatic neuroendocrine tumors (PanNETs). Ganetespib is a second-generation HSP90 inhibitor that is well tolerated in cancer patients (dosed into >1500 patients) and is currently being evaluated in several investigator sponsored clinical trials including acute myeloid leukemia (AML), ovarian cancer, breast cancer, and other tumor types.

Here, we show that ganetespib inhibits the proliferation of NET cells and induces apoptosis in vitro with potency comparable to another second-generation HSP90 inhibitor (NVP-AUY922), but with potency two- to seven-fold more than first generation inhibitors (17-AAG, IPI-504) in BON-1, CM and H727 cell lines, and thirty-fold more in QGP-1 cell line. In mice bearing PanNET tumor xenografts, single agent ganetespib reduced the growth of tumors without signs of toxicity. Tumors from ganetespib treated animals demonstrated reduced phospho-AKT and phospho-ERK expression, and elevated HSP70 expression, supportive of exposures necessary for functional activity. In an effort to identify clinically meaningful agents that could further potentiate the antitumor activity of ganetespib, we performed in vitro combination screens evaluating proliferation in four NET cell lines with ganetespib and forty FDA approved anticancer agents. Ganetespib showed synergistic effects when combined with inhibitors of mTOR, topoisomerase I or II, or DNA synthesis. Results from our ongoing in vivo combination studies with ganetespib and these three classes of anticancer agents will be presented.

#268

PARP3 inhibitors in cancer therapy.

Bahram Sharif-Askari, David Davidson, Lilian Amrein, Lawrence Panasci. _Faculty of Medicine, Department of Oncology, McGill University, Segal Cancer Center, Lady Davis Institute, Montréal, Quebec, Canada_.

Poly-ADP-ribose-polymerase (PARP1) is an important regulator of DNA damage response (DDR). After induction of certain types of DNA damage, including nicks and double strand breaks (DSB)s, PARP1 is rapidly recruited to altered DNA sites were its catalytic activity produces protein-conjugated long-branched-poly-ADP-ribose (PAR) chains. These protein modifications result in the recruitment and activation of multiple proteins involved in DNA repair. Although there is considerable research describing the characteristics and activity of PARP1/2, there are 15 other PARP family members that are less well characterized. Of these PARP3 has been shown to have a distinct role in DNA repair. This protein has been shown to have a critical role in DSB resolution and to interact with partner proteins know to function in classical nonhomologous end joining (NHEJ) such as DNA-PKcs, Ku70, and Ku80.

In order to examine the role of PARP3 vis-à-vis chemotherapy, we utilized the specific PARP3 inhibitor, ME-0328 in combination with activated cyclophosphamide (4HC) in the chronic lymphocytic leukaemia (CLL) cell lines, MEC1 and MEC2.

Results from MTT cytotoxicity assays showed that the 50% inhibitory concentration (IC50) (50% of control) of 4HC in MEC-1 cells was 12.9μM. When used in combination with a nontoxic concentration (2μM) of the specific PARP3 inhibitor ME-0328 the IC50 of 4HC was reduced to 3.3μM. In MEC-2 cells the IC50 of 4HC was 10.8μM and was reduced to 2.1μM in the presence of ME-0328 at 2μM. ABT-888, a specific PARP1/2 inhibitor in clinical trials had a minimal effect on 4HC cytotoxicity in these cell lines. 4HC results in interstrand crosslinks (ICLs) which are believed to be repaired by homologous recombination. Intriguingly, PARP3 negatively regulates class switch recombination via activation-induced cytidine deaminase in B-lymphocytes. This may be involved in ME-0328 sensitization of 4HC in B-lymphocytic malignancies.

Given these preliminary results we hypothesize that the PARP3 inhibitor, ME-0328 will sensitize B-lymphocytic cancers to chemotherapies. To explore this hypothesis this project will pursue the following objectives:

1. Characterize the selective sensitization of ME-0328 with 4HC and/or bendamustine (an ICL-inducing drug utilized in the treatment of B-lymphocyte malignancies) in B-lymphoma and CLL cell lines plus CLL clinical samples.

2. Determine the mechanisms by which ME-0328 sensitizes CLL cells to 4HC and/or bendamustine (i.e. reduction of PARP1 or 3 activity and/or altered activity of known DNA repair proteins (Rad51, DNA-PK, H2AX) and/or altered activation-induced cytidine deaminase).

3. Determine the in-vivo activity of 4HC and/or bendamustine with or without ME-0328 in a Rag2 /-γcc-/- xenograft model using the MEC1-CLL cell line.

The inhibition of PARP3 may increase the sensitivity of tumor cells to DNA damaging chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use.

#269

The role of BRCA1 and AEG1 mRNA expression in advanced non-small-cell lung cancer (NSCLC) patients (p) with EGFR activating and pretreatment T790M mutations receiving the combination of erlotinib plus bevacizumab (E+B) in the BELIEF trial.

Rafael Rosell,1 Niki Karachaliou,2 Ana Giménez-Capitán,3 Carles Codony-Servat,3 Oliver Gautschi,4 Enriqueta Felip,5 Alessandra Curioni-Fontecedro,6 Solange Peters,7 Santiago Ponce-Aix,8 Martin Früh,9 Miklos Pless,10 Sanjay Popat,11 Sinead Cuffe,12 Paolo Bidoli,13 Adolfo Favaretto,14 Roswitha Kammler,15 Urania Dafni,16 Zoi Tsourti,16 Miguel Angel Molina-Vila,3 Rolf A. Stahel17. 1 _Institut Catala d'Oncologia, Univ. Hospital Germans Trias i Pujol, Badalona, Spain;_ 2 _Instituto Oncológico Dr Rosell (IOR), Quirón-Dexeus University Institute, Barcelona, Spain;_ 3 _Pangaea Biotech, Quirón-Dexeus University Institute, Barcelona, Spain;_ 4 _Cantonal Hospital Lucerne, Oncology Department, Lucerne, Switzerland;_ 5 _Vall d'Hebron University Hospital, Barcelona, Spain;_ 6 _Division of Internal Medicine and Clinic of Oncology, University Hospital, Zurich, Zurich, Switzerland;_ 7 _Centre Hospitalier Universitaire Vaudois (CHUV), Département d'Oncologie, Lausanne, Switzerland;_ 8 _Hospital Universitario 12 de Octubre, Lung Cancer Unit, Madrid, Spain;_ 9 _Cantonal Hospital St. Gallen, Oncology and Hematology, St Gallen, Switzerland;_ 10 _Cantonal Hospital Winterthur, Medical Oncology, Winterthur, Switzerland;_ 11 _Royal Marsden Hospital, Medical Oncology Unit, London, United Kingdom;_ 12 _St James's Hospital and ICORG (All Ireland Cooperative Oncology Research Group), Dublin, Ireland;_ 13 _Ospedale San Gerardo, Monza, Italy;_ 14 _Istituto Oncologico Veneto, IRCSS, Padova, Italy;_ 15 _ETOP Coordinating Office, Berne, Switzerland;_ 16 _Frontier Science Foundation – Hellas, Athens, Greece;_ 17 _University Hospital Zurich, Clinic of Oncology, Zurich, Switzerland_.

The BELIEF trial (NCT01562028) examined the efficacy of E+B for the 1st line treatment of European p with advanced NSCLC harboring exon 19 deletion or exon 21 L858R epidermal growth factor receptor (EGFR) mutations with or without pretreatment T790M. Median progression-free survival (mPFS) was 13.8 months (m) (95% CI 10.3-16.2) for the 109 p enrolled in the study. mPFS was 16m (95% CI 13.1-not estimable [NE]) and 10.4m (95% CI 9.2-15.6) for the 37 T790M(+) p and 72 T790M(-) p, respectively (P=0.089). We have previously shown that low BRCA1 levels neutralize the negative effect of pretreatment T790M and are associated with longer PFS to erlotinib. Low astrocyte elevated gene 1 (AEG1) levels are associated with longer PFS to erlotinib. A secondary objective of the BELIEF trial was the assessment of the prognostic impact of BRCA1 and AEG1 mRNA expression in the baseline tumor tissue.

We assessed the baseline mRNA expression levels of BRCA1 and AEG1 and correlated them with PFS and response in the 109 EGFR-mutant NSCLC p of the BELIEF trial. BRCA1 and AEG1 mRNA levels were estimable by quantitative-real time PCR in 46 and 61 p respectively. Expression levels were divided into two groups according to their median.

No statistically significant associations were found among the expression of the two biomarkers and gender, smoking status, histology, PS or type of EGFR mutation. The association of BRCA1 and AEG1 mRNA with PFS did not differ according to T790M status (interaction P=0.81 and 0.42, respectively). Among the T790M (+) p, those with low BRCA1 mRNA had a longer mPFS of 24.6m (95% CI: 4.9-NE) compared to 15.4 m (95% CI: 2.7-NE) for p with high BRCA1 mRNA; P=0.63. For all and T790M (-) p, BRCA1 levels did not differentiate mPFS to E+B (low vs high BRCA1 mRNA, 11.0 m [95% CI: 6.0-NE] vs 9.5 m [95% CI: 7.2-NE]; P=0.99 and 6.9 m [95% CI: 2.1-NE] vs 9.4 m [95% CI: 4.1-NE]; P=0.71, respectively). Similarly, among the T790M (+) p, those with low AEG1 expression had a longer mPFS of 24.6m (95% CI: 4.9-NE) compared to 15.4 m (95% CI: 5.2-NE) for those with high AEG1 mRNA; P=0.93. For all and T790M(-) p, AEG1 levels did not differentiate mPFS to E+B (low vs high AEG1 mRNA, 10.3 m [95% CI: 8.4-24.6] vs 15.4 m [95% CI: 6.0-33.9]; P=0.90 and 10.3m [95% CI: 8.4-NE] vs 13.3m [95% CI: 4.7-33.9] P=0.67). No statistically or clinically significant associations were found among BRCA1, AEG1 mRNA levels and response to E+B.

The BELIEF trial reconfirms our previous findings that pretreatment EGFR T790M is present in 34% of the patients. E+B is associated with a prolonged mPFS of 24.6m for EGFR-mutant p with both pretreatment EGFR T790M and low BRCA1 mRNA expression, as observed in our previous study with erlotinib alone. Similar results were observed for AEG1 levels though the interaction was not statistically significant for either biomarker.

#270

Synergistic effect with APR-246 and standard chemotherapy in small cell lung cancer cells carrying smoking-associated TP53 mutations.

Nina Mohell, Åsa Fransson, Jessica Alfredsson, Mikael von Euler, Ulf Björklund, Lars Abrahmsen. _Aprea AB, Solna, Sweden_.

Background: Although smoking increases risk for many tumor types, no malignancy is more closely linked to tobacco exposure than small cell lung cancer (SCLC); 90% of SCLC patients are smokers. Carcinogenic compounds in cigarettes increase cancer susceptibility due to formation of DNA adducts, leading to oncogenic mutations. Etoposide in combination with cisplatin or carboplatin is the standard chemotherapy for SCLC. However, most SCLC patients eventually die of chemotherapy-refractory disease. Mutation in the tumor suppressor gene TP53 is one of the main causes for resistance, and occurs in more than 70% of SCLC patients (http://p53.free.fr). APR-246 (PRIMA-1MET) is the first clinical-stage small molecule that reactivates mutant p53 by inducing its wild-type conformation triggering apoptosis. It has been tested in a First in Human clinical trial in hematological malignancies with encouraging results (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study in combination with platinum-based therapy in recurrent ovarian cancer is ongoing (AACR abstract #CT204, 2015). We have previously observed strong synergy with APR-246 and platinum compounds in TP53-mutant drug-resistant ovarian cancer cells (Mohell et al. Cell Death and Disease 6, 2015). The aim of the present study was to investigate whether APR-246 synergizes with standard chemotherapy in SCLC cells carrying smoking-associated and/or lung cancer-specific TP53 mutations. Methods: Cell viability was tested using CellTiter-Glo assay, TP53 status with Sanger sequencing and single strand conformation analysis, and p53 protein level with Western blotting. Combination Index (CI) was calculated according to Additive model. Results: We observed synergistic (CI<0.8) or strong synergist effect (CI<0.5) with APR-246 and cisplatin in SCLC cell lines carrying smoking-associated (often G>T transversion) and/or lung cancer specific homozygous TP53 mutations, including NCI-H2195 (V157F, 4.3% of all SCLC tumors), NCI-H1048 (R273C, 1.78%) and NCI-H889 (C242S, 1.07%). Synergy was also observed in H196 cells with hotspot R175H mutation (2.14%), while additive effect (CI=0.8-1.2) was found in NCI-H1882 (R273L, 1.78%) and NCI-H187 (S241C, 0.36%) cells. Synergy was also observed with etoposide in NCI-H2195 cells. Moreover, APR-246 sensitized the NCI-H2195 cells to cisplatin; the IC50 value decreased about 2-fold at clinically relevant concentration of APR-246. All tested SCLC cell lines had medium or high level of p53. Further studies with other conventional drugs are ongoing. Conclusions: Treatment with APR-246 in combination with cisplatin or etoposide resulted in synergy or strong synergy in SCLC cells carrying lung cancer-specific and/or smoking-related TP53 mutations. Our results suggest that combination treatment with APR-246 and standard chemotherapy can provide significantly improved treatment of TP53-mutant SCLC.

#271

Influence of autophagy modulation on synergistic interactions of lapatinib and mTOR targeted agents in HER2-amplified lapatinib resistant breast cancer models in vitro and in vivo.

Wieslawa H. Dragowska,1 Sherry A. Weppler,1 William Wei Chu,1 Norman S. Chow,1 Jenna S. Rawji,1 Ashleen S. Prasad,1 Karen A. Gelmon,2 Sharon M. Gorski,3 Marcel B. Bally4. 1 _Experimental Therapeutics Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 2 _Medical Oncology, BC Cancer Agency; Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada;_ 3 _Michael Smith Genome Sciences Centre, BC Cancer Agency; Department of Molecular Biology and Biochemistry, Simon Fraser University, Vancouver and Burnaby, British Columbia, Canada;_ 4 _Experimental Therapeutics Laboratory, BC Cancer Agency, Faculty of Pharmaceutical Sciences, Department of Pathology and Laboratory Medicine, University of British Columbia; Centre for Drug Research and Development, Vancouver, British Columbia, Canada_.

Background: Resistance against HER2 targeted agents ultimately limits the therapeutic success in patients with HER2-positive breast cancer. It was shown that PIK3CA mutations contribute to lapatinib resistance and achieving control of a downstream PI3K/mTOR signaling is necessary for optimal effectiveness of HER2 blockade. We and others have shown that catalytic mTORC1/2 inhibitors reverse lapatinib resistance and inhibit growth of HER2-overexpressing breast cancer models in vitro and in vivo. However, activity of these targeted agents is hindered by the compensatory and adaptive mechanisms that arise to assure cell survival. One of the pro-survival responses is cytoprotective autophagy induced by lapatinib and mTORC1/2 inhibitors. Thus, we examined whether impairing autophagy could augment activity of lapatinib/mTORC1/2 inhibitors combinations in lapatinib-resistant breast cancer models. Methods: The combination of lapatinib with catalytic mTORC1/2 inhibitors KU-0063794 (KU) or AZD2014 (AZD) was evaluated in vitro and in vivo in lapatinib resistant PIK3CA mutated HER2-overexpressing/amplified MDA-MB-361, JIMT-1 and MDA-MB-453 breast cancer models in the presence of siRNA-based (Atg7, Beclin-1) and pharmacological (hydroxychloroquine (HCQ)) inhibitors of autophagy. Results: In vitro lapatinib/mTORC1/2 combinations elevated autophagy to a greater extent that either compound alone. Genetic or pharmacological inhibition of treatment-induced autophagy further decreased cell viability, suggesting that autophagy was playing a cytoprotective role in this context. In vivo, lapatinib and AZD combinations achieved effective tumor growth inhibition of 98%, 111% and 152% in MDA-MB-361, JIMT-1 and MDA-MB-453 models respectively, however addition of HCQ did not significantly enhance this therapeutic response (p>0.05). Conclusion: Negligible effects of HCQ in vivo in tumors treated with lapatinib/AZD combinations may be attributed to ineffective inhibition of autophagy-mediated survival signals that, if significantly blocked, could increase efficacy of the treatment. Utilizing carrier nanotechnology to optimize delivery of HCQ to the tumor site and molecular analysis of HCQ-engendered off target effects on survival and proliferation pathways in tumor tissue are being pursued.

#272

Metformin enhances the anti-prostate cancer activity of abiraterone and enzalutamide.

Yi Xie, Linbo Wang, Arif Hussain. _University of Maryland, Baltimore, MD_.

The standard treatment for advanced castration sensitive prostate cancer (CSPC) is androgen deprivation therapy (ADT), generally in the form of suppression of gonadally produced androgens via medical or surgical castration. The selective inhibitor of androgen biosynthesis Abiraterone (Abi), and the androgen receptor (AR) antagonist Enzalutamide (MDV3100), are second line hormonal therapies used to treat PC patients failing ADT. However, these drugs provide treatment response for a limited duration of time due to the development of resistance to them. Included among the mechanisms leading to resistance to ABI or MDV is the expression of the AR variant AR-V7. Metformin, a widely used oral anti-diabetic drug, has been reported to reduce prostate cancer mortality in PC patients with type II diabetes. In this study, we explored the effects of ABI, MDV and metformin in a pre-clinical model of PC. Both Abi and MDV inhibited cell proliferation and the expression of prostate specific antigen (PSA) in androgen-sensitive LNCaP cells. However, neither induced cell death, and in fact MDV appeared to actually inhibit apoptosis in LNCaP cells. Both agents increased AR and AR-V7 expression, which in part may account for development of resistance to ABI or MDV. In contrast, metformin inhibited AR, AR-V7 and PSA expression in a dose-dependent manner, and induced apoptotic cell death in PC cells. Metformin, in combination with ABI or MDV, further enhanced apoptosis and inhibited expression of PSA than it did as single agent. Interestingly, at near physiological concentrations metformin induced apoptosis in a caspase-independent manner rather than by a caspase-dependent apoptotic program that is reported to occur with high doses of metformin. In conclusion, Abi or MDV inhibits AR signaling and PC cell proliferation, but also induces AR and AR-V7 expression. Metformin decreases AR and AR-V7 expression and induces apoptotic cell death. The combination of metformin with androgen biosynthesis/AR inhibitors results in caspase-independent apoptotic cell death in addition to inhibition of both cell proliferation and AR signaling. Further studies will be needed to evaluate the clinical efficacy of these combination treatments in patients with PC.

#273

Inhibition of EGFR/STAT3/CEBPD axis reverses cisplatin cross-resistance with paclitaxel in urothelial carcinoma of urinary bladder.

Ju-Ming Wang. _Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University (NCKU), Tainan, Taiwan_.

Cisplatin (CDDP) is frequently used in combination chemotherapy with paclitaxel (PTX) in the treatment of urothelial carcinomas of the urinary bladder (UCUB). The development of CDDP cross-resistance with PTX has been suggested and remains a challenge that hinders successful UCUB treatment. Consequently, elucidating the mechanisms underlying CDDP-induced anticancer drug resistance is imperative, and this knowledge will aid in the development of novel strategies. CEBPD expression was maintained in post-operative chemotherapy patients, and this expression was responsive to CDDP even in CDDP-resistant UCUB cells. Upon CDDP treatment, CEBPD activated ABCB1 and ABCC2. The EGFR/STAT3 pathway contributed to CDDP-induced CEBPD expression in UCUB cells. Treatment with gefitinib and S3I-201 significantly decreased CEBPD expression and enhanced sensitivity of CDDP-resistant NTUB1/P cells to CDDP and PTX. Our results demonstrate the risk of CEBPD activation in CDDP-resistant UCUB cells and suggest a potential therapeutic strategy for treating patients with UCUB and UCUB with CDDP-induced PTX resistance using CDDP in combination with either gefitinib or S3I-201.

#274

Assessing combinations of chemotherapy in alveolar soft part sarcoma cell lines.

Justine Clark, Elliot Kahen, Diana X. Yu, Christopher L. Cubitt, Daniel M. Sullivan, Damon R. Reed. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

Background: Alveolar Soft Part Sarcoma (ASPS) is a rare soft tissue sarcoma subtype occurring mainly in children and young adults. Malignant tumors develop in the connective tissues, typically in the lower extremities of the legs and thighs, but can also be found in the head and neck regions. ASPS is slow growing, highly angiogenic, and may have unique metabolic properties. ASPS is characterized by an unbalanced translocation between the ASPSCR1 gene (whose product tethers GLUT4 transporters) and the TFE3 gene (encoding a transcription factor). The resulting ASPSCR1-TFE3 fusion gene is responsible for an aberrant transcription factor that is thought to be involved in the pathogenesis of the disease. Methods to assess a number of different agents in combination are needed for practical use in clinical trials.

Methods: Here, we seek to examine the therapeutic activity of single agent and combinations of epigenetic, anti-angiogenic and a diverse mix of other mechanisms of action agents using high-throughput screening platforms and well-established cell line models for ASPS (ASPS-1 and ASPS-KY). We designed stringent screening conditions that assess the compounds at clinically-relevant concentrations and time of exposures that mimic the in vivo pharmacokinetics in an effort to maximize the translational potential of these results to the clinical setting. 54 agents were screened across the 2 cell lines with attention to existing clinical data and angiogenesis inhibitors. The cell titer glo assay was used to determine cell viability at three clinically achievable concentrations for all agents. The most promising 8 agents were then studied in all two-drug combinations to evaluate for synergy.

Results: The results of our screening showed that several agents significantly reduced cell viability at clinically relevant concentrations and exposure times with belinostat (an HDAC inhibitor) and dinaciclib (a CDK 1, 2, 5 and 9 inhibitor) in particular having good activity. Several combinations with doxorubicin showed good efficacy and synergy. Belinostat and doxorubicin affected both ASPS-KY and ASPS-1 cell lines similarly, with an average fraction affected (FA) of 0.87. The combination index (CI) indicated strong synergy in the ASPS-KY (CI = 0.09) and some synergy in ASPS-1 (CI = 0.74). Similarly, dinaciclib and doxorubicin produced a FA of 0.85 with good synergy (CI of 0.51-0.66). In addition, the combination of belinostat and tivantinib showed additive effects in both cell lines (CI of 0.88-1.2) with a FA of ~0.8. These findings suggest potentially promising opportunities for developing combinational approach to treating childhood soft tissue sarcomas.

#275

Combining low dose microtubule targeting agents with belinostat potentiates cytotoxic response in HDACi resistant diffuse large b-cell lymphoma.

Aaron P. Havas, Anvi Bhakta, Kameron Rodrigues, Catharine L. Smith. _University of Arizona, Tucson, AZ_.

Patients diagnosed with Diffuse Large B-cell lymphoma have an overall 60% five-year survival rate. New therapeutic approaches are needed to effectively treat aggressive forms of DLBCL that are refractory to the standard treatment or that relapse within two years of treatment. Histone deacetylase inhibitors (HDACi) are novel therapeutics that are well tolerated in humans and are being extensively evaluated in combination with other therapeutics against hematologic malignancies. Rational selection of companion therapeutics has been difficult due to the cell type-specific mechanisms of HDACi action. In order to address this, we have developed a pre-clinical model system of sensitivity and resistance to HDACi-induced cytotoxicity in DLBCL cell lines that share characteristics with aggressive DLBCL tumors. We previously reported that HDACi resistance is associated with reversible arrest in G1 that involves sustained up-regulation of cyclin-dependent kinase inhibitors. In the current study we demonstrate that HDACi-sensitive cell lines undergo mitotic arrest prior to anaphase in response to treatment with the approved HDACi, belinostat, consistent with activation of the spindle assembly checkpoint (SAC). In contrast, HDACi-resistant cell lines are capable of completing mitosis in the presence of belinostat. To force SAC activation in HDACi resistant cell lines, we used low dose microtubule targeting agents (MTA) vincristine and paclitaxel to induce maximal mitotic arrest with minimal cytotoxicity. The combination of these low dose MTAs and belinostat efficiently caused SAC failure, mitotic slippage, and apoptosis in a synergistic manner. A key mechanism associated with resistance to MTAs is their ability to induce aneuploid cell populations that survive and undergo endoreduplication. The addition of belinostat eliminated the accumulation of a vincristine-induced aneuploid population. Pan-caspase inhibition in conjunction with belinostat and vincristine co-treatment resulted in a sustained survival of the aneuploid population. Thus belinostat triggers apoptosis in the aneuploid cells to enhance the cytotoxic effects of vincristine. Our study identifies the use of low dose MTA/HDACi combination as a potential therapeutic strategy for treatment of relapsed or refractory DLBCL.

#276

Enhancing standard chemotherapy response by targeted inhibition of MAPK signaling pathways in experimental pancreatic cancer.

Niranjan Awasthi, Sheena Monahan, Margaret A. Schwarz, Roderich E. Schwarz. _Indiana University School of Medicine, South Bend, IN_.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all solid tumors, with an overall survival rate of less than 6%. Gemcitabine (Gem) remained the standard of care agent over the past 16 years despite limited clinical benefits. Nab-paclitaxel (NPT), a water-soluble albumin-bound formulation of paclitaxel, has recently shown greater efficacy in advanced PDAC. Nab-paclitaxel combination with gemcitabine is now standard of care for advanced PDAC. More than 90% of PDAC tumors harbor an activating mutation in the KRAS oncogene. Since to date no therapeutics have effectively targeted this oncogene product, alternative strategies focus on inhibition of downstream effectors of KRAS signaling pathways. Trametinib (Tra) is a small molecule inhibitor of the RAF-MEK-ERK (MAPK) signaling pathway that is a well-described mediator of KRAS induced transformation and tumorigenesis. In the present preclinical study, we evaluated combination treatment benefits of the standard chemotherapeutics with trametinib to define a novel therapeutic strategy for PDAC.

Median animal survival over controls (20 days) in human intraperitoneal PDAC xenografts was significantly improved by NPT (33 days, a 65% increase, p=0.0004), Gem (26 days, a 30% increase, p=0.002), NPT+Gem (39 days, a 95% increase, p=0.0001) and Tra (31 days, a 55% increase, p=0.0004) therapy. Survival was further increased by addition of trametinib to chemotherapy: NPT+Tra (median: 37 days, a 85% increase, p=0.0001), Gem+Tra (34 days, a 70% increase, p=0.0001) and NPT+Gem+Tra (49 days, a 145% increase, p<0.0001). Treatment of subcutaneous tumor-bearing mice with chemotherapy and trametinib resulted in significant tumor growth inhibition. Net tumor growth in different therapy groups over two weeks varied between controls (432.6 mm³), NPT (105.3 mm³), Tra (184 mm³), NPT+Tra (81 mm³), NPT+Gem (37.3 mm³), and NPT+Gem+Tra (-8.1 mm³). Mean tumor weight (in g) in different therapy groups was as follows: controls 0.38±0.06, NPT 0.23±0.06, Tra 0.31±0.03, NPT+Tra 0.23±0.06, NPT+Gem 0.15±0.05 and NPT+Gem+Tra 0.11±0.05. In vitro studies demonstrated inhibition in PDAC cell (AsPC-1, Panc-1, Mia PaCa-2, CFPAC) proliferation by treatment with NPT+Gem, trametinib, and combination. Immunoblot analysis revealed that trametinib effects were accompanied by decrease in phospho-ERK expression and increase in the expression of apoptosis-related cleaved caspase-3 protein. These findings suggest that the effects of the standard chemotherapeutic regimens can be enhanced through specific inhibition of downstream components of the MAPK signaling pathway, which clinically could lead to improved PDAC therapy effects.

#277

Broad-spectrum anticancer activities of the biguanides metformin and buformin in combination with 2-deoxy-glucose or WZB-117 in human lung cancer cells.

Juan Sebastian Yakisich, Neelam Azad, Anand V. Iyer. _Hampton University, Hampton, VA_.

Purpose: The biguanides Metformin (MET) and to a lesser extent Buformin (BUF) have recently been shown to exert anticancer effects. In particular MET targets cancer stem cells (CSCs) in a variety of cancer types. These compounds have not been extensively tested for combination therapy.

Objectives: In this study we investigated in vitro the anticancer activity of MET and BUF alone or in combination with 2-deoxy-D-glucose (2-DG), or WZB-117 (WZB), which are a glycolysis and a GLUT-1 inhibitor, respectively, in H460 human lung cancer cells growing under three different culture conditions with varying degrees of stemness: 1) Routine Culture Conditions (RCCs), 2) Floating Lung Tumorspheres (LTSs) that are enriched for stem-like cancer cells and 3) Adherent cells under prolonged periods (8-12 days) of serum starvation (PPSS): These cells are highly resistant to conventional anticancer drugs such as Paclitaxel, Hydroxyurea and Colchicine and display increased level of stemnes markers.

Experimental setup: For cells growing under RCCs and under PPSS, cell viability was measured by the MTT assay. Viability of LTS was evaluated by the CCK assay. Clonogenicity was evaluated by the colony formation assays.

Results: As single agents MET, BUF, 2-DG and WZB-117 potently inhibited the viability of cells growing under RCCs. These results were confirmed by the colony forming assay. Both MET and BUF showed a strong synergistic effect when used in combination with 2-DG. A weak potentiation was observed when used with WZB-117. Under RCCs H460 cells were more sensitive to MET and BUF and WZB-117 compared to non-tumorigenic Beas-2B cells. While LTSs were less sensitive to each single drug, both MET or BUF in combination with 2-DG showed a strong synergistic effect and reduced cell viability to similar levels compared to the parental H460 cells. Adherent cells growing under PPSS were also less sensitive to each single drug and MET and BUF showed a strong synergistic effect on cell viability in combination with 2-DG.

We are currently evaluating the generation of reactive oxygen species (ROS) and the signaling pathways involved in the antiproliferative effects of these agents as single drugs as well as in combination.

Conclusions: Overall our data demonstrates that combination of BGs with either 2-DG or WZB-117 have a "Broad - spectrum" anticancer activities targeting cells growing under a variety of cell culture conditions with varying degrees of stemness. These properties may be useful to overcome the chemoresistance due to intratumoral heterogeneity found in lung cancer.

#278

No cell left behind: Residual ovarian spheroids drive recurrence and are sensitive to the pro-oxidant elesclomol.

Ian S. Goldlust,1 Kelli Wilson,1 Ludmila Szabova,2 Xiaohu Zhang,1 Lesley Mathews-Griner,3 Maria Vias,4 Anna Piskorz,4 Rory Stark,4 Lee Mendil,4 Monica Kasbekar,1 John Braisted,1 Rajarshi Guha,1 Crystal McKnight,1 Paul Shinn,1 Donna Michelle-Smith,4 Zoe Weaver Ohler,2 Mindy Davis,1 Udo Rudloff,2 Sam Michael,1 Madhu Lal-Nag,1 Scott Martin,5 Christina Annunziata,2 Marc Ferrer,1 James D. Brenton,4 Craig Thomas1. 1 _NIH/NCATS, Bethesda, MD;_ 2 _NCI/CCR, Bethesda, MD;_ 3 _Novartis, Rockville, MD;_ 4 _University of Cambridge/Cambridge Institute, Cambridge, United Kingdom;_ 5 _Genentech, San Francisco, CA_.

Sphere forming cells persist in the ascitic fluid of patients with high-grade serous ovarian cancer after first-line therapy and likely contribute to relapse and metastasis. These residual tumor spheres, which are enriched for cancer cells by up to 95% based on detectable TP53 mutations, are slow growing, resistant to platinum-based chemotherapy, and remain a large obstacle towards durable remission. Screening established, rapidly dividing monolayer cell lines in proliferation assays has failed to produce chemotherapeutics capable of eradicating this slow growing population. To identify a consolidation therapy that targets these residual tumor cells we screened nearly 2000 mechanistically annotated, approved and investigational drugs in three cell lines (PEO1, PANC1, A375M) cultured in spheroid and conventional monolayer. To elucidate targetable genes and pathways responsible for spheroid maintenance we performed a whole-genome RNAi screen against PEO1 in both culture conditions. Our pharmacological and genetic profiling provided mechanistic insight into the baseline changes that occur when cells are grown in three dimensional, anchorage independent conditions and identified susceptibilities specific to spheroid populations. Consistent with residual disease, cultured spheres were resistant to many common chemotherapeutics, most notably proteasome inhibitors, but were highly sensitive to the pro-oxidant elesclomol. We verified elesclomol's activity in ex vivo cancer spheroids extracted from the ascitic fluid of patients with advanced high-grade serous ovarian cancer using a simple, culture-free technique. Expression profiling of cultured and ex vivo spheres revealed broad downregulation of genes involved in cell cycle signaling (AURKA, AURKB, PLK1, CDK1, CCNA2, CCNB1, CCNB2, GMNN, CHEK1). Treatment with elesclomol induced expression of chaperone proteins (HSP6, HSP7, HSPA1A, HSPA1B, DNAJA4, DNAJB1), metallothionein proteins (MT1F, MT1M, MT1P2, MT1X, MT2A), and genes involved with the oxidative stress response (HMOX1, ABCB1, SLC7A11) consistent with increased reactive oxygen species caused by high intracellular Cu2+. To pursue this drug as a consolidation therapy in vivo, we evaluated elesclomol's toxicity, pharmacokinetic properties, and efficacy in a murine model for recurrent high-grade serous ovarian cancer. By targeting residual tumor cells with elesclomol after successful treatment with platinum-based therapeutics we hope to prevent recurrence.

#279

Combinatorial strategies for glioblastoma using brain tumor-initiating cells: targeting the JAK/STAT and EGFR pathways.

Hema A. Luchman,1 Katharine V. Jensen,1 Ahmed Aman,2 Samuel Weiss1. 1 _Univ. of Calgary, Calgary, Alberta, Canada;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada_.

Glioblastoma multiforme (GBM), characterized by an aggressive clinical course, therapeutic resistance, and striking molecular heterogeneity, remains incurable. Recent evidence, from our group and others, indicates that the JAK2/STAT3 pathway is an important mediator of tumor cell survival, growth, and invasion in GBM. We investigated the efficacy of a novel JAK2 inhibitor, pacritinib, in brain tumor initiating cell (BTIC) lines to evaluate its potential use in the treatment of GBM patients. In a Phase III study of patients with myelofibrosis, pacritinib demonstrated manageable toxicity and clinically and statistically improved patient spleen volume and reported outcomes. Treatment with pacritinib resulted in on-target JAK2/STAT3 inhibition at 1-2μM and dramatically reduced BTIC proliferation, regardless of endogenous MGMT promoter methylation or EGFR, PTEN, and TP53 mutational status. Pacritinib in combination with temozolomide, the current standard of care agent for GBM, prolonged survival over either drug alone in orthotopically xenografted NOD-SCID mice. We tested the hypothesis that combinatorial targeting of the JAK2/STAT3 pathway and other oncogenic drivers would be effective in GBM. A large number of GBMs have EGFR alterations and, despite poor clinical translation to date, EGFR inhibition remains of therapeutic relevance. Interestingly, EGFR inhibition leads to activation of survival-signalling pathways such as STAT3, diminishing the effectiveness of EGFR inhibition. We find that concurrent inhibition of JAK2/STAT3 and EGFR signalling may be an effective, clinically relevant therapeutic strategy for GBM. We examined the in vitro actions of pacritinib on BTICs in combination with clinically relevant EGFR inhibitors (erlotinib, afatinib, lapatinib and AZD9291). Combinatorial treatment with pacritinib and EGFR inhibitors showed striking responses, with lowered IC50s and BTIC viability. The combinatorial actions of EGFR and STAT3 inhibition were particularly effective in BTIC lines with EGFR activating vIII and missense point mutations. On-target activity was demonstrated with reduced phospho-EGFR, phospho-STAT3 and effectors of both pathways. In vivo pharmacokinetic and pharmacodynamic studies demonstrated that pacritinib and the EGFR inhibitors afatinib and AZD9291 penetrate the brain and have on-target activity. Ongoing in vivo studies using orthotopic xenograft BTIC models will determine whether combinatorial inhibition of these two pathways will provide survival benefit. Further studies are aimed at investigating whether combinatorial inhibition of other pro-oncogenic pathways, using the BTIC model both in vitro and in vivo, may be effective strategies in GBM.

#280

**Synergistic** in vitro **activity of MOR209/ES414 in combination with enzalutamide.**

Toddy Sewell,1 Michelle Blake,1 Jane A. Gross,1 Jan Endell,2 Johannes Weirather,2 John W. Blankenship1. 1 _Emergent BioSolutions Inc., Seattle, WA;_ 2 _MorphoSys, Planegg, Germany_.

Metastatic castration-resistant prostate cancer (mCRPC) presents a daunting therapeutic challenge for patients who eventually progress after androgen ablation therapy. Although newer AR antagonists show a survival benefit, these patients ultimately relapse. Thus, it is critical to identify new therapies that will work in combination with AR antagonists or in AR-antagonist resistant settings. MOR209/ES414 is a bispecific ADAPTIR™ (modular protein technology) molecule currently under investigation in a phase 1 clinical trial in mCRPC, including patients that have progressed on enzalutamide. In pre-clinical studies, MOR209/ES414 has previously been shown to redirect T cell cytotoxicity against cells expressing Prostate Specific Membrane Antigen (PSMA). Here, we aimed to determine whether enzalutamide affects PSMA expression on CRPC cells in vitro, and could alter the effectiveness of MOR209/ES414 at inducing T-cell mediated tumor cell death.

To address these questions, we utilized a CRPC cell line, 22Rv1, which has low expression of PSMA and is resistant to enzalutamide. Treatment of 22Rv1 cells with 10 µM enzalutamide for 3 weeks resulted in increased PSMA cell surface expression as measured by flow cytometry. 22Rv1 cells exposed to enzalutamide were also more sensitive to MOR209/ES414-mediated T-cell lysis, with a 25% increase in cytotoxicity over a 4 hour chromium release assay compared to 22Rv1 cells that were not exposed to enzalutamide. A concomitant decrease in EC50 for MOR209/ES414-mediated lysis from 0.8 pM to 0.5 pM was also observed in 22Rv1 cells exposed to enzalutamide.

MOR209/ES414 and enzalutamide were next tested in combination using a PSMA-positive, enzalutamide-sensitive cell line, LNCaP cultured with primary T cells over several days. Suboptimal doses of MOR209/ES414 and enzalutamide were added to the cultures, and surviving LnCaP cells were quantitated 4 days later. Enzalutamide did not interfere with the ability of MOR209/ES414 to target T cells to LNCaP cells, and Combination Index analysis showed the activity of MOR209/ES414 and enzalutamide to be synergistic in vitro. These studies provide a rationale for further examination of combining MOR209/ES414 with enzalutamide, even in enzalutamide-resistant settings.

#281

Bendamustine has the biochemical properties of an alkylating agent that synergizes with nucleoside analogues in chronic lymphocytic leukemia.

Sara EF Kost,1 Eric DJ Bouchard,2 Élise LaBossière,1 Xibiao Ye,3 Michelle L. Queau,1 William S. Liang,2 Versha Banerji,2 Spencer B. Gibson,2 Sachin Katyal,2 James B. Johnston2. 1 _CancerCare Manitoba, Winnipeg, Manitoba, Canada;_ 2 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 3 _Centre for Healthcare Innovation, Winnipeg, Manitoba, Canada_.

Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and may function as an alkylating agent (eg, chlorambucil, CLB) while sharing structural similarities to a nucleoside analogue (eg, fludarabine, FLU). Enhanced cell death has been observed with the combination of BEN and FLU in primary CLL cells in vitro, but both BEN and FLU are marrow-toxic, potentially limiting the clinical value of this combination. While the nucleoside analogue pentostatin (PEN) is active in CLL and less marrow-toxic than FLU, it is unknown whether it produces synergy when combined with BEN. PEN acts by inhibiting adenosine deaminase, resulting in the accumulation of deoxyadenosine (dADO), which is toxic to CLL cells. In the present study, we evaluated the activity of BEN against CLL cells in vitro, as compared to CLB, FLU or dADO/PEN, alone and in combination. Cell death was assessed using annexin V/7-ADD staining and the MTT assay. We observed that BEN, CLB, FLU, or dADO/PEN induced apoptosis, the extent of which was time- and concentration-dependent. In general, cross-resistance was observed to all agents, with previously treated patients or those with a del 17p being most resistant. Enhanced cell killing was seen when combining BEN or CLB with FLU or dADO/PEN, with the extent of synergy being similar for FLU or dADO/PEN. To correlate mechanism of cell death as a result of treatment with these agents, we analyzed the expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). An increase in DR5 (but not DR4) surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed following treatment indicating that all agents induced death through the death receptor and mitochondrial apoptotic pathways. However, using γH2AX and the alkaline comet assay, FLU produced a greater number of DNA double-strand breaks (DSB) than BEN or CLB. Thus, all these agents induce apoptosis through the mitochondrial and death receptor pathways with cross-resistance being observed in most cases. BEN is more similar to CLB in producing fewer DNA DSBs than FLU and demonstrating synergy when combined with FLU or dADO/PEN. The latter observation suggests that combining BEN with PEN might be useful clinically.

#282

Pan-RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4 and 6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

Shih-Hsun Chen, Youyan Zhang, Robert D. Van Horn, Tinggui Yin, Lysiane Huber, Teresa F. Burke, Xueqian Gong, Wenjuan Wu, Shripad Bhagwat, Sean Buchanan, Richard P. Beckmann, Ramon V. Tiu, Sheng-Bin Peng. _Eli Lilly and Company, Indianapolis, IN_.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, including melanoma, lung, colorectal, and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor currently in phase I clinical trial, has been shown to inhibit cell proliferation of RAS- or BRAF-mutant tumor cells in vitro and xenograft tumor growth in vivo. An unbiased screen for compounds that synergize with LY3009120 in RAS/BRAF mutant cancers identified inhibitors of CDK4 among the top hits. In this study, we found that combined inhibition of RAF and CDK4 and 6 by LY3009120 and abemaciclib cooperatively reduced viability of tumor cells with KRAS, NRAS or BRAF mutation in vitro. In animal models, the LY3009120 and abemaciclib combination exhibited synergistic regression of tumor growth in multiple xenograft models with KRAS, NRAS, or BRAF mutation. Molecular mechanistic analysis revealed that pan-RAF inhibitor treatment suppressed the cyclin D1 upregulation which was mediated through CDK4 and CDK6 inhibition by abemaciclib, and the combination treatment cooperatively demonstrated more complete inhibition of RB phosphorylation. These results were further verified by CDK4 and CDK6 siRNA knockdown and another CDK4 and CDK6 selective inhibitor palbociclib. Importantly, the more complete phospho-RB inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combinational treatment led to more significant cell cycle G0/G1 arrest and apoptosis of tumor cells. These preclinical findings suggest that the combinational inhibition of RAF and CDK4 and CDK6 signaling by LY3009120 and abemaciclib is synergistic and should be further studied compared to single agents in the treatment of cancer in patients with KRAS, NRAS or BRAF mutations.

#283

PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances pemetrexed antitumor efficacy in a human nonsquamous NSCLC xenograft model.

Renee Clift, Jessica A. Cowell, Susan J. Zimmerman, Mathieu Marella, Ping Jiang, Arnold B. Gelb, Michael J. LaBarre, Daniel C. Maneval, Curtis B. Thompson, Xiaoming Li. _Halozyme Theraputics, San Diego, CA_.

Introduction: Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that has been shown to accumulate at high levels in several cancers, including non-small cell lung cancer (NSCLC). The accumulation of HA in NSCLC is associated with a more aggressive phenotype and shortened overall survival. In a prevalence analysis using tissue microarrays of archival human specimens (N=194) stained by affinity histochemistry, we demonstrated that 55% of adenocarcinomas, 74% of squamous cell carcinomas and 83% of large cell carcinomas accumulated HA. The engineered enzyme PEGylated recombinant human hyaluronidase PH20 (PEGPH20) removes HA from the tumor microenvironment. PEGPH20-based therapies are being evaluated in clinical studies of several solid tumors, including NSCLC (NCT02346370, NCT02563548). Herein, we evaluated the hypothesis that PEGPH20 combination therapy could enhance the efficacy of pemetrexed (PEM), a folate antimetabolite approved by FDA as a first-line treatment in combination with cisplatin, against locally advanced and metastatic NSCLC in patients with non-squamous histology.

Methods: We evaluated PEGPH20 plus PEM in a NSCLC xenograft model. First, the human NSCLC adenocarinoma cell line A549 was transduced with hyaluronan synthase-3 (HAS3) to generate the A549/HAS3 cell line. Next, A549/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors size reached ~250 mm3, mice were staged into six groups: (1) vehicle; (2) PEGPH20, (3) PEM daily, (4) PEM weekly, (5) PEGPH20+PEM daily or (6) PEGPH20+PEM weekly. Vascular volume, hypoxia and tumor growth inhibition (TGI) were assessed using conventional methods. Affinity histochemical staining for HA was performed on formalin-fixed paraffin-embedded tissue sections after harvest.

Results: A549/HAS3 tumors grew faster than parental A549 tumors in vivo. PEGPH20 monotherapy significantly reduced tumoral HA, increased vascular volume and reduced hypoxia in tumors. Further, TGI with PEGPH20+PEM, weekly or daily, was 29.7 or 31.1 %, respectively, whereas in either PEGPH20 alone or PEM alone, both were <9%. Staining of A549/HAS3 tumors showed increased accumulation of HA as compared with the parental A549 xenografts.

Conclusion: These data suggest that addition of PEGPH20 to pemetrexed increases the efficacy of PEM and support further investigation of PEGPH20 plus PEM treatment in NSCLC tumors of non-squamous histology that accumulate HA.

#284

IT-139 holds potential for combination therapy.

Tara Lee Costich,1 Jyothi Sethuraman,1 Rick Crouse,2 Suzanne Bakewell1. 1 _Intezyne Technologies, Tampa, FL;_ 2 _Yale University, New Haven, CT_.

IT-139, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is a ruthenium based small molecule that has successfully completed a phase I clinical trial in 41 patients with mild to moderate systemic toxicities. There were no dose limiting hematologic toxicities observed. IT-139 holds potential for continued clinical development because of its aqueous solubility, long half-life and manageable toxicity. Stable disease was observed in 11 of the 41 patients of the Phase I clinical trial, but we believe IT-139's future clinical development will be in combination therapy. In vitro studies revealed potential for additive and synergistic combinations. Few in vivo combination studies were completed previously due to challenges associated with administration. Here we show that by overcoming the administration challenges in our animal models we were able to identify a dependable maximum tolerated dose (MTD), optimize dosing regimens for combination xenograft efficacy studies and extend the length of treatment in efficacy studies. In an HCT116 subcutaneous model, increased efficacy was demonstrated when IT-139 was dosed in combination with oxaliplatin over oxaliplatin alone. In an A549 lung carcinoma subcutaneous model, increased efficacy was observed when IT-139 was dosed in combination with cisplatin over cisplatin alone. Increased toxicity from the combined treatment was observed after the initial treatment and the chemotherapeutic dose was reduced for the duration of the study. However, clinical pharmacokinetic results confirmed IT-139's long plasma half-life and metabolic studies have confirmed its high affinity for plasma proteins. Based on these data we staggered the dosing of IT-139 by 24 hrs allowing us to dose both IT-139 and a chemotherapeutic agent at their MTDs. This dosing regimen saw an increase in efficacy in an HT-29 colorectal xenograft model treated with IT-139 and the anti-metabolite 5-FU over treatment with 5-FU alone. We also observed improved efficacy with IT-139 dosed weekly instead of the q4d schedule, suggesting that the pharmacokinetics of IT-139 allows less frequent dosing without compromising efficacy. The toxicology report from IT-139 dosed at 50 mg/kg shows microscopic alterations in the liver and kidney. Minimal to moderate hepatocellular alterations were noted in periportal regions at 48 hrs. Renal tubular necrosis was noted at 24, 48 and 72 hrs, but tubular regeneration was noted at 48 and 72 hrs time points. In the combination therapy (IT-139 at 50 mg/kg plus oxaliplatin) mice there were no results in the liver and kidneys to suggest exacerbation of the noted alterations. There were no observations of either liver or kidney toxicity in the Phase I human study. These findings further attest to the potential of IT-139 in combination therapy, but suggest further investigation to identify the optimal dosing regimen. Current mechanism of action studies will collaborate and support our in vivo studies for the future success of IT-139's return to the clinic.

#285

Disulfiram, a dual MGMT and aldehyde dehydrogenase inhibitor, sensitizes ER-positive breast cancer cells to temozolomide and cyclophosphamide.

George C. Bobustuc,1 Deborah Donohoe,1 Alisher Holmuhamedov,1 Srivenugopal Kalkunte,2 Santhi D. Konduri1. 1 _Aurora Health Care, Milwaukee, WI;_ 2 _Texas Tech University Health Sciences Center, Amarillo, TX_.

Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including breast cancer. MGMT is a DNA repair protein leading to chemo resistance. MGMT expression directly correlates with ER expression and tamoxifen resistance. Aldehyde Dehydrogenase (ALDH) activity, as a progenitor/stem cell marker, while it has been reported to inversely correlate with MGMT expression in other cancers, has also been linked to chemotherapy and radiation resistance.

Methods: We have tested the effect of Antabuse (disulfiram, DSF), as a dual MGMT and ALDH inhibitor, at nontoxic doses, in combination with Temozolomide (TMZ) or/and Cyclophosphamide (CP) on ER+ breast cancer cells.

Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases ER+ breast cancer cell growth (MCF7, T47D and ZR75) in a dose-dependent manner. DSF further sensitizes breast cancer cells to TMZ or/and CP and significantly inhibits breast cancer growth without causing unwanted side effects on the normal breast epithelial cells. Dose effect and isobologram studies confirm synergistic activity of DSF + CP and moderate synergism for DSF + TMZ. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP), significantly inhibits expression of MGMT, aldehyde dehydrogenase, ERα, E2F and BIRC5 gene (survivin) - all involved in breast cancer tumorigenesis. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) caused significant apoptosis in breast cancer cells. In a dose dependent manner, DSF inhibited colony formation, effect which was further enhanced by addition of TMZ/CP (DSF ± TMZ/CP). Similarly, DSF alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) decreased the metastatic potential of breast cancer cells.

Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and ALDH significantly enhances the antitumor effect of alkylators as CP and TMZ in ER+ breast cancer

#286

Preclinical study: HDAC inhibition combined with gemcitabine for the treatment of leiomyosarcoma.

Gonzalo Lopez,1 Edwin Choy,2 Shreyaskumar Patel,3 Hemant Bid,1 Danielle Braggio,1 Dina Lev,4 Raphael Pollock1. 1 _The Ohio State University, Columbus, OH;_ 2 _Dana-Farber/Harvard Cancer Center, Boston, MA;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Sheba Medical Center, Tel Aviv, Israel_.

Objective

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastasic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of pan-HDACi alone and in combination with gemcitabine in LMS.

Methods

Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included a pan-HDAC inhibitor (HDACi) and nucleoside analog, gemcitabine (Gem). MTS and clonogenic assays were used to evaluate the effect of HDACi on LMS cell growth. Cell cycle FACS and annexin V PI/FACS analysis were used to determine the effects of HDACi on cell cycle and apoptosis, respectively. Compusyn software was used to determine in vitro synergy studies for the combination of HDACi + Gem. A LMS xenograft model in SCID mice was used to test the impact of HDACi alone, Gem alone and the combination of HDACi + Gemcitabine.

Results

Pan-HDAC inhibition abrogated LMS cell growth and clonogenic potential. HDACi induced G2/M cell cycle growth arrest and enhanced apoptosis in LMS cell lines. The combination of HDACi + Gem exhibited a synergistic effect in LMS cells in vitro. Similarly, HDACi combined with Gem resulted in superior anti-LMS effects in vivo.

Conclusion

LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of pan-HDAC inhibition combined with gemcitabine for the treatment of LMS.

#287

CPX-351 cytotoxicity against fresh AML blasts is increased for FLT3-ITD+ cells and correlates with drug uptake and clinical outcomes.

Max Gordon,1 Paul Tardi,2 Lawrence D. Mayer,2 Jeffrey Tyner1. 1 _Oregon Health and Science University, Portland, OR;_ 2 _Celator Pharmaceuticals, Vancouver, British Columbia, Canada_.

Background: CPX-351, a synergistic ratio of cytarabine (Cyt) and daunorubicin (Daun) co-encapsulated in a nano-scale carrier, has demonstrated improved complete remission and overall survival rates compared to conventional Cyt + anthracycline treatments in two randomized clinical trials and is currently being evaluated in a Phase 3 trial vs 7+3 Cyt:Daun. To determine whether these effects are attributable to liposome-mediated altered drug PK or specific AML blast genotypes/phenotypes, we investigated the ex vivo cytotoxic potency of CPX-351 against fresh AML blast samples. Cytotoxicity results were correlated with patient characteristics as well as CPX-351 cellular uptake and molecular phenotype status including FLT3-ITD, NPM1 and CEBPα. Methods: Peripheral blood was obtained at diagnosis from patients with AML. White blood cells were isolated on Ficoll gradients. Cells were incubated over graded concentrations of CPX-351 for 72 hr at which time cell viability was assessed by MTS assay. Cell viability values were normalized to patient-matched untreated cells. Patient-specific IC50 values were calculated and correlated with clinically-relevant disease features. Uptake of intact CPX-351 liposomes by AML cells was determined by monitoring cells for daunorubicin fluorescence by flow cytometry. Results: CPX-351 cytotoxicity was evaluated in 53 AML patient specimens. CPX-351 IC50 values ranged from 0.03:0.006 uM to 10:2 uM (Cyt:Daun), and all values were significantly lower than the 72 hour plasma drug concentration of 60:12 uM Cyt:Daun observed in patients receiving CPX-351. Sensitivity to CPX-351 was similar regardless of cytogentic risk, NPM1 and CEBPα status as well as whether the patients went on to achieve a CR or PD upon receiving standard 7+3 Cyt:Daun after blast samples were taken. Surprisingly, AML blasts exhibiting the FLT3-ITD phenotype exhibited some of the lowest IC50 values and as a group were 5-fold more sensitive to CPX-351 than those with wild type FLT3. Flow cytometric analysis revealed a correlation between uptake of CPX-351 into AML blasts and its cytotoxic potency. Taken together, the data are consistent with clinical observations where CPX-351 retains significant anti-leukemic activity in AML patients exhibiting high-risk characteristics that are typically associated with poor outcomes when treated with conventional regimens. Conclusions: The profile of ex vivo AML blast sensitivity to CPX-351 mirrors the efficacy profile observed clinically and may provide a means to identify specific AML patient genotypes/phenotypes that could benefit most from CPX-351 treatment. The increased sensitivity of FLT3-ITD+ blasts to CPX-351 is an example of how such analyses may identify additional AML patient populations warranting further clinical investigation.

#288

Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel.

Courtney L. Jarvis, Thomas Nelius, Dalia Martinez-Marin, Srirupa Cheerla, Stéphanie Filleur. _Texas Tech University Health Sciences Center, Lubbock, TX_.

Despite recently approved novel agents, taxane-based chemotherapy remains the major therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC). Still mCRPC continues to be incurable. Furthermore, patients often experience severe side effects, prompting a re-evaluation of conventional regimen. Past studies have demonstrated promise for Low-Dose Metronomic (LDM) chemotherapy; schedule defined as the frequent administration of low doses of chemotherapeutic drugs with no prolonged drug-free breaks. Yet relative activities of LDM taxanes and combinations with known anti-neoplasic agents have to be investigated. Using CRPC cell lines (PC3, Du145 and the LNCaP-derivative CL1), we have shown that cabazitaxel-treated cells have a significantly lower EC50 compared to docetaxel, with Du145 cells presenting the greatest differences. In cell cycle analysis, both low-dose taxanes increased the sub-G1 cells population. However, the sub-G1 increase was significantly greater in cabazitaxel- than in docetaxel-treated Du145 cells, but not in PC3 and CL1. Accordingly, plasma membrane Annexin V elevation occurred in Du145 cells at lower doses of cabazitaxel than docetaxel validating a higher efficacy due to increased apoptosis. As other possible cell death mechanisms, autophagy and necrosis were investigated. Beclin1 expression levels remain unchanged in all three cell lines. In contrast, necrosis was stimulated in all the taxanes-treated cell lines in a dose dependent manner. In vivo, LDM cabazitaxel was significantly more efficient in delaying tumor growth than docetaxel. This effect was markedly increased when cabazitaxel was combined with the angio-inhibitor and anti-tumor Pigment Epithelium-Derived Factor (PEDF). Other results showed that PEDF and LDM chemotherapy combination induces more phagocytosis of CRPC cells when compared to single treatments. In conclusion, our data demonstrate a higher efficacy of cabazitaxel on CRPC both in vitro and in vivo, and suggest that LDM taxane chemotherapy/PEDF combination could be used as a novel therapeutic strategy for CRPC.

#289

Melphalan and XPO1 inhibition are synergistic in pre-clinical models of multiple myeloma.

Joel G. Turner, Jana L. Dawson, Daniel M. Sullivan. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

Introduction:

Multiple myeloma (MM) accounts for about 10% of all hematologic malignancies, with estimated numbers of new cases and deaths for 2015 in the US at 26,850 and 11,240, respectively. Significant increases in response/survival have been seen over the past several years; however, MM remains incurable and patients ultimately die from progressive disease refractory to anti-myeloma therapy. There is clearly a need for additional effective therapies.

Materials and Methods:

Human MM parental and melphalan (MEL) resistant cell lines were treated with XPO1 inhibitors (XPO1i) KPT330 or KPT8602 +/- MEL and assayed for apoptosis and viability by flow cytometry. Treated cells were assayed for DNA damage by comet assay and phospho-H2AX protein expression. XPO1/MEL treated cells were assayed for p53, NFkB, IKKα, FANCF, and FANCL by Western blot. Cells from patients with newly diagnosed or relapsed/refractory MM treated with XPO1i/MEL were assayed for apoptosis. NOD/SCID-gamma mice with MM tumors were treated with XPO1i/MEL and assayed for tumor growth, survival, and toxicity.

Results:

MM cell viability was decreased synergistically and apoptosis increased by XPO1I/MEL treatment in all MM cell lines tested. XPO1/MEL drug combination significantly increased DNA damage when compared to either MEL or XPO1 alone, as shown by comet assay and increased phospho-H2AX expression. Western blot showed that XPO1i treatment of MM cells increased p53 and decreased NFkB, IKKα, FANCF, and FANCL. MEL-resistant MM cell lines were found to be sensitized by XPO1i to MEL as shown by apoptosis assay (20-fold). CD138+/light chain+ MM cells from newly diagnosed and relapsed/refractory MM patients were also sensitized (5-10 fold) by XPO1i to MEL (apoptosis assay). NOD/SCID-gamma mice challenged with MM tumors demonstrated a strong synergistic antitumor effect in XPO1i/MEL-treated mice, with increased survival and no significant toxicity.

Conclusions:

XPO1i's improved the response of human MM cell lines and patient MM cells to MEL in vitro and ex vivo. It is possible that this synergy may be due to increased nuclear p53 in combination with decreased NFkB and IKKα and decreased DNA repair proteins FANCL and FANCF reversing MEL resistance by the Fanconi Anemia/BRCA pathway. These preliminary data suggest that XPO1i's augment MEL-induced DNA damage and may also block the repair of the DNA damage—both of which could result in synergistic cell kill. Combination therapies using XPO1i, especially the clinical compounds KPT330 (selinexor) and KPT8602 +/- MEL, may significantly improve the treatment outcomes of MM. 

### Mechanisms of Drug Resistance 1

#290

NF1 loss modulates cellular fitness in KIT-mutant gastrointestinal stromal tumor (GIST).

Adrian Marino-Enriquez, Bin Li, Nacef Bahri, Alexandra Lauria, Yuexiang Wang, Jonathan A. Fletcher. _Brigham and Women's Hospital, Harvard Medical School, Boston, MA_.

Patients with inoperable GIST obtain substantial clinical benefit from imatinib and other KIT/PDGFRA inhibitors. However, secondary resistance develops in 90% of patients within 18-24 months. Reactivation of oncogenic signaling pathways at time of progression is most often due to secondary mutation in KIT or PDGFRA, but downstream RAS or BRAF mutations are occasionally alternative mechanisms of KIT/PDGFRA-inhibitor resistance.

We identified deleterious NF1 mutations in 3 KIT-mutant GIST samples from patients progressing on imatinib. In genomic scale loss-of-function shRNA genomic screens, designed to identify genes involved in imatinib resistance, a shRNA targeting NF1 was the most enriched shRNA in imatinib-treated GIST cells out of 54,000 shRNAs (NF1 gene score 29/11,238). To evaluate the contribution of NF1 loss to KIT-mutant GIST resistance, NF1-deficient GIST models were generated from 2 patient-derived, KIT-mutant GIST cell lines, GIST882 and GIST-T1, by sequential cotransfection with Cas9 and 2 independent NF1-targeting CRISPR-sgRNAs.

Complete loss of NF1 protein was observed after 5-10 passages due to on-target heterogeneous genomic deletion at the NF1 locus. Orthogonal validations were obtained using stable shRNA-mediated NF1 knockdown with similar results in the same GIST cell lines. NF1-deleted cells retained expression of mutant KIT but were resistant to imatinib (IC50 196 and 500nM, significantly increased from baseline 34 and 110nM, for GIST-T1 and GIST882, respectively). Notably, after interruption of imatinib treatment, NF1-deficient GIST cells showed reduced proliferation (69% and 77% growth reduction at day 7 off imatinib, for GIST-T1 and GIST882, t test p<0.001) despite continuous expression of activated KIT and downstream signaling effectors.

In conclusion, acquired NF1 loss induces TKI resistance in KIT-mutant GIST cells but results in reduced cellular fitness in the absence of KIT-inhibitor treatment. These findings suggest that NF1-deficient GIST subclones that expand on imatinib treatment can be destabilized by intermittently discontinuing KIT-inhibitor therapy.

#291

Single cell analyses reveal resistance heterogeneity in EGFR mutant NSCLC.

Matt J. Niederst,1 Huidan Zhang,2 Haichuan Hu,1 Naiwen Cui,2 Yamei Cai,2 Hillary Mulvey,1 Zofia Piotrowska,1 Lecia Sequist,1 David A. Weitz,2 Jeffrey A. Engelman1. 1 _MGH Cancer Center/Harvard Medical School, Charlestown, MA;_ 2 _Harvard School of Engineering and Applied Sciences, Cambridge, MA_.

In EGFR mutant lung adenocarcinomas, the activating mutation to EGFR is the founder alteration, present in every cancer cell. Most patients with this genetic alteration respond to EGFR tyrosine kinase inhibitors, although the average duration of the response is only one year before the cancer develops acquired resistance. Efforts to identify the mechanisms underlying the resistance in patients have led to the identification of a resistance mechanism in the majority of cases. However, preliminary data is emerging indicating that multiple resistance mechanisms can be present within an individual patient's disease, and thus, classifying resistant disease with a single mechanism is an oversimplification. Here, utilizing single cell approaches on patient samples, we demonstrate heterogeneity within individual biopsies with respect to resistance mechanisms, epigenetic state, and sensitivity to TKI. Additionally, we observe that the fraction of T790M-positive cells is predictive of response to 3rd generation EGFR inhibitor treatment, suggesting that the degree of heterogeneity has important clinical implications. As therapies designed to target singular resistance mechanisms become more widely implemented, assessing heterogeneity will become increasingly critical in order to choose the most effective therapy for the patient.

#292

Integrated analysis of ibrutinib resistance in chronic lymphocytic leukemia.

Inhye E. Ahn,1 Adam Albitar,2 Chingiz Underbayev,1 Sarah Herman,1 Xin Tian,1 Susan Soto,1 Maryalice Stetler-Stevenson,1 Irina Maric,1 Mohammed Z. Farooqui,1 Maher Albitar,2 Adrian Wiestner1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _NeoGenomics Laboratories, Irvine, CA_.

BACKGROUND: Ibrutinib covalently binds to Bruton's tyrosine kinase, inhibits B-cell receptor signaling, and is active in chronic lymphocytic leukemia (CLL). Progressive disease (PD) on ibrutinib has been reported due to histologic transformation or mutations of BTK or PLCG2. Here we report integrated analysis of the clinical and molecular characteristics of CLL pts who progressed on ibrutinib (PD group).

METHODS: Under a phase II investigator-initiated trial (ClinicalTrials.gov, NCT01500733) 84 CLL pts with TP53 aberration (deletion 17p or TP53 mutation) or age ≥65 were treated with ibrutinib 420mg daily. Samples from the PD group were tested for mutations of BTK and PLCG2 by a high-sensitivity assay using branched and locked nucleic acids. In pts with mutations at PD, stored samples from earlier time-points were also sequenced.

RESULTS: 13 (15.5%) of 84 pts progressed at a median follow up of 24 months. 3 of 4 early PDs (≤12 months) were due to histologic transformation, while 8 of 9 late PDs (median 34.9 months) were due to CLL. PFS was inferior in subgroups with TP53 aberration, unmutated IGHV, advanced Rai stage, high β-2 microglobulin and relapsed/refractory disease (log-rank p<0.05 for all tests). 8 pts with progressive CLL were subsequently treated with small molecules targeting PI3K or Bcl-2, and 6 were alive to date (longest follow-up 15 months). Two types of non-synonymous mutations at BTK exon 15 (C481S, C481R) and five types of non-synonymous mutations at PLCG2 exon 19, 20 and 24 (R665W, P664S, P664L, S707Y, L845F) were identified in 8 out of 9 pts having progressive CLL. No mutation was found in pts with transformation and in one pt with progressive CLL. Concomitant BTK and PLCG2 mutations were found in 5 out of 8 pts (62.5%). Mutations pre-dating clinical PD were identified in stored samples from 6 pts as early as 13 months before progression (range 1.8-13.0). The median time to the first detected mutation was 23.1 months (range 5.4-34.7). Mutational complexity increased over time as reflected by increasing types of mutations (n=3) and allele frequencies (n=3) at later time-points. Both PD with progressive CLL and non-PD groups showed equivalent depth of best response in peripheral blood (PB) and bone marrow during treatment (p>0.05). At PD, tumor burden increased by 2 to 32-fold from nadir based on PB flow cytometry.

CONCLUSION: Most pts with progressive CLL on ibrutinib carry BTK and/or PLCG2 mutations. Concurrent mutations of BTK and PLCG2 are common at progression, and either or both of these mutations can be acquired many months before clinical progression. In cases with detectable mutations but without clinical progression, pts can benefit from prolonged treatment with ibrutinib until clinical progression occurs. Upon clinical progression with CLL, pts can still show responses to alternative targeted agents.

#293

EGFR inhibition generates drug-tolerant persister cells by blocking AKT activity and thus inactivating Ets-1 function.

Janyaporn Phuchareon, David W. Eisele, Frank McCormick, Osamu Tetsu. _University of California, San Francisco, CA_.

In non-small-cell lung cancer (NSCLC)—the leading cause of cancer death worldwide—about 10-20% harbor mutations in epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK). Although treatment with EGFR tyrosine kinase inhibitors (TKIs) had shown promise, drug resistance has been the most important determinant limiting its success. Recent studies have identified the mechanisms of drug resistance to TKIs, but the origin of acquired resistant cells remains to be elucidated. We consider that they must arise from a small subset of surviving populations after EGFR inhibition. Thus, we have studied the mechanism by which the subset remains viable after EGFR inhibition, despite cell death in the vast majority. Our study demonstrates that EGFR inhibition in lung cancer cells generates a drug-tolerant subpopulation by blocking AKT activity and thus inactivating Ets-1 function. The remaining cells enter a dormant, non-dividing state because of the inhibited transactivation of Ets-1 target genes cyclins D1, D3, and E2. Moreover, Ets-1 inactivation inhibits transcription of dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated, which combines with c-Src to renew activation of the Ras/MAPK pathway, causing increased cell survival by accelerating Bim protein turnover. Conversely, inhibition of elevated ERK1/2 by the addition of a MEK inhibitor enhances programmed cell death through rewiring apoptotic signaling. These observations may explain why a small subset of quiescent cells can tolerate TKIs, and might suggest that combined treatment of TKI and MEK inhibitor could overcome drug resistance in the population.

#294

The role of the histone deacetylase HDAC 8 in mediating BRAF/MEK inhibitor metastatic dissemination and resistance.

Michael Emmons, Rashed Rab, Ritin Sharma, John Koomen, Keiran Smalley. _H. Lee Moffitt Cancer Ctr. & Res. Inst., Tampa, FL_.

BRAF/MEK inhibitor resistance frequently results in increased phosphorylation of EphA2 at S897, which increases the invasive potential of melanoma cells in a ligand-independent manner. Through use of an unbiased phosphoproteomic screen, we identified HDAC phosphorylation as also being increased following the acquisition of BRAF/MEK inhibitor resistance. Treatment with the pan-HDAC inhibitor LBH 589 inhibited EphA2 phosphorylation at S897 and reduced tumor invasion, indicating a role for HDACs in the regulation of the resistance-associated metastatic phenotype. A comparison of HDAC protein levels in matched BRAF inhibitor sensitive/resistant cell lines showed that HDAC6, HDAC8, and HDAC11 protein levels were more highly expressed in BRAF resistant cell lines with HDAC8 being upregulated in 5 of 6 cell line pairs. shRNA knockdown of HDAC8 in the BRAF/MEK inhibitor resistant cell line WM164RR resulted in a decreased in pEphA2 as well as a decrease in pAKT at S473, which is known to directly bind to and phosphorylate EphA2 at S897. Reduced expression of HDAC8 in WM164RR cells resulted in a decrease in the invasive potential of the resistant phenotype as well as a resensitization of these cells to the BRAF inhibitor vemurafenib. HDAC8 overexpression in drug naïve cells led to increased melanoma cell invasion in a 3D spheroid and a matrigel invasion model. Overexpression of HDAC8 was also found to decrease the level of apoptosis following BRAF inhibitor treatment and was associated with increased number of clones in a colony formation assay. Conversely, knocking down HDAC8 with shRNA led to an increase in vemurafenib-mediated apoptosis. Mechanistically, it was found that HDAC8 overexpression increased the speed of adaptive MAPK signaling following BRAF inhibition and that this in turn limited the expression of pro-apoptotic BIM. Overall, these results indicate a dual role for HDAC8 in mediating BRAF/MEK inhibitor resistance and metastatic dissemination. Increased levels of HDAC8 caused an increase in invasion as well as a decrease in vemurafenib-induced apoptosis in BRAF inhibitor sensitive cell lines. Our studies suggest that selective HDAC8 inhibition may be one strategy to limit adaptation to both BRAF and BRAF/MEK inhibitor therapy.

#295

The cytosine deaminase APOBEC3B affects responses to therapy in estrogen receptor positive breast cancer cells.

Kelly S. LaPara,1 Emily Law,2 Reuben Harris,3 Douglas Yee4. 1 _Masonic Cancer Center, University of Minnesota, Minneapolis, MN;_ 2 _Biochemistry, Molecular Biology and Biophysics Department, Masonic Cancer Center, Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN;_ 3 _Biochemistry, Molecular Biology and Biophysics Department, Masonic Cancer Center, Howard Hughes Medical Institute, University of Minneosta, Minneapolis, MN;_ 4 _Masonic Cancer Center, University of Minnesota, Minneapolis, MN_.

Endocrine therapy, chemotherapy, or both represent the mainstay of therapy for estrogen receptor positive (ER+) breast cancer. However, acquired (secondary) drug resistance is a major clinical problem. We identified APOBEC3B (A3B), a DNA cytosine deaminase, as a source of DNA mutagenesis driving tumor evolution and contributing to poor clinical outcomes in several cancers by catalyzing genetic changes required for drug resistance and metastasis. A3B accounts for up to half of the mutational load in breast carcinomas expressing this enzyme. High levels of A3B correlate with poor clinical outcomes in tamoxifen treated patients. To evaluate the role for A3B in resistance to therapy, we used the ER+ MCF-7L breast cancer cell line. These cells have modestly elevated levels of endogenous A3B and we suppressed A3B levels by shRNA and enhanced levels by transduction. In vitro, alterations in A3B levels do not affect monolayer growth or cell doubling times. However, in vitro response to tamoxifen was prolonged in MCF-7L cells with suppressed A3B. In a xenograft model, MCF-7 cells with suppressed A3B remained responsive to tamoxifen for a longer period of time compared to wild type cells. Cells with overexpression of A3B had a very short period of tamoxifen suppression compared to wild-type cells. These data suggest high A3B levels are associated with a shorter period of response to tamoxifen. To evaluate the role for A3B in chemotherapy resistance, we treated cells with 5-fluorouracil. In contrast to tamoxifen therapy, cells with elevated A3B levels remained responsive to fluorouracil for longer periods of time compared to wild-type cells. Since A3B converts cytosine to uracil (C-to-U), we speculate that A3B enzymatic activity might enhance sensitivity to a fluoropyrimidine. Thus, the ability of A3B to affect therapeutic resistance in breast cancer may be dependent on the type and mechanism of drug treatment.

#296

Targeting the melanosome: overcoming MAPK-inhibitor resistance in melanoma.

April A.N. Rose,1 Matthew G. Annis,1 Dennie T. Frederick,2 Zhifeng Dong,1 Lawrence Kwong,3 Tibor Keller,4 Thomas Hawthorne,4 Ian R. Watson,1 Keith T. Flaherty,2 Peter M. Siegel1. 1 _Goodman Cancer Research Centre, Montreal, Quebec, Canada;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Celldex Therapeutics, Needham, MA_.

Background: The duration of survival benefit from MAPKi in B-raf mutant melanoma (MEL) is limited by the near universal development of resistance (RES). Recently, MiTF, a transcription factor and master regulator of melanocyte differentiation - was identified as a hub of MAPKi RES. Given its dual role as both a tumor promoter and inducer of MEL senescence, MiTF is a problematic therapeutic target. Pigmentation is a MiTF-dependent feature of MELs correlates with poor survival. We have interrogated the MAPKi/MiTF-dependent regulation of a panel of MITF-regulated melanosomal differentiation antigens (MDAs) for their potential as therapeutic targets to overcome MAPKi RES.

Methods: The TCGA MEL gene expression data set was interrogated. B-raf mutant MEL cells were exposed to dabrafenib (D), or trametinib (T). qRT-PCR, Immunoblot or FACS were used to assess MiTF and MDA expression. Serial biopsy tissue and serum samples from MEL patients taken before treatment, during treatment, and at progression with MAPKi therapy were obtained, and analyzed by IHC and qRT-PCR, RNAseq, and ELISA. Transient siRNA-mediated knockdown was used to assess the requirement for MiTF, in MAPKi-mediated induction of MDAs, including GPNMB. To assess the interaction between MAPKi and CDX011 (C), a GPNMB- targeted antibody drug conjugate, mice bearing A375 and WM2664 xenografts were treated with T or DT + C in combination.

Results: MiTF is strongly correlated with MDAs, including GPNMB in gene-expression datasets derived from human MEL tumors. A MiTF driven MDA-gene-signature correlates with poor survival among 291 melanoma patients. MAPKi treatment increased MiTF and MDAs in MEL cells, and induced pigmentation in vivo. This phenotype was reversible as MEL acquired RES to MAPKi. Knockdown of MiTF leads to abrogation of MAPKi-mediated GPNMB induction. GPNMB promotes MEL invasion in vitro. MiTF and MDA were elevated in tumors and serum from patients receiving MAPKi treatment and returned to baseline in tumors that acquired RES. The addition of C to either T or DT led to, decreased pigmentation, prolonged tumor regression and inhibited the development RES to T or DT treatment in vivo. Tumors that acquired RES to DT displayed increased c-Met expression and MAPK activation; tumors treated with DT+C failed to develop this phenotype.

Conclusions: MAPKi induce MDA in a MiTF-dependent manner in MEL. This process leads to melanocytic differentiation and increased pigmentation, which is subsequently reversed as a tumor acquires RES to MAPKi. This demonstrates the plasticity of MEL in response to MAPKi. The pigmented phenotype is less proliferative but more invasive which is in part mediated by GPNMB. This transient phenotype may allow tumors to acquire characteristics (ie. c-Met upregulation and MAPK re-activation) which contribute to MAPKi RES. Targeting a pro-invasive MDA, GPNMB with CDX-011 subverts this plasticity and impairs the development of MAPKi RES.

#297

Epigenetic activation of IGF1R signaling promotes resistance to momelotinib and MEK inhibition in KRAS-driven lung cancer.

Shunsuke Kitajima, Hajime Asahina, Jillian D. Cavanaugh, Ting Chen, Ashley A. Merlino, Thai C. Tran, Kwonk-Kin Wong, David A. Barbie. _Dana-Farber Cancer Institute, Boston, MA_.

KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), yet remains resistant to targeted therapy. Previously, we reported that RAL-TBK1 activation downstream of KRAS promotes an autocrine cytokine circuit that fuels tumorigenesis. Interruption of this signaling by treatment with momelotinib, an inhibitor of TBK1/IKKε and JAK kinases, together with MEK inhibition, was effective in the aggressive Kras-p53 (KP) mouse lung cancer model. These findings have prompted a human clinical trial of momelotinib and trametinib in advanced refractory KRAS mutant NSCLC. Because this combination leads to acquired resistance in mice, we sought to understand additional pathways that could compensate for TBK1/JAK and MEK inhibition. Receptor tyrosine kinase (RTK) profiling of A549 cells treated with momelotinib revealed delayed activation of IGF1 receptor (IGF1R) signaling. Indeed, IGF1 supplementation partially rescued momelotinib-induced growth arrest of A549 cells, while combination of momelotinib with the dual IGF1R/insulin receptor (IR) inhibitor linsitinib cooperated to impair cell viability, which was more pronounced in KRAS/STK11 (encoding LKB1) mutant cell lines compared with LKB1 wild type cell lines. Triple momelotinib/trametinib/linsitinib therapy also improved tumor shrinkage in Kras-Lkb1 (KL) mice compared with KP mice. However, chronic treatment with this triple combination required pulse therapy due to toxicity, and was inferior to momelotinib/trametinib dosed daily, which itself promoted deep responses in KL mice lasting 6 weeks.

In parallel, we analyzed mediators of acquired resistance to chronic momelotinib and MEK inhibitor exposure in A549 cells. Interestingly, IGF1 expression was dramatically upregulated in resistant cells, in contrast to IGF2, and enhanced autocrine activation of IGF1R signaling. Importantly, both IGF1 upregulation and inhibitor resistance were completely reversible after drug withdrawal for several passages, yet reappeared quickly upon drug readdition, suggesting epigenetic reprogramming. Consistent with this hypothesis, whereas global H3K27 histone acetylation was decreased in resistant cells, this epigenetic mark was strongly enriched at the IGF1 promoter and linked tightly with both IGF1 levels and drug resistance. Treatment of resistant A549 cells with the BRD4 inhibitor JQ1 completely suppressed IGF1 expression and downstream IGF1R activation, and impaired cell viability. Combination JQ1 treatment also prevented momelotinib/MEK inhibitor induction of IGF1 in A549 cells, synergized to reduce outgrowth of resistant cells, and was well tolerated as daily triple combination therapy in mice. Together, these findings suggest the potential of BET inhibition to synergize with and prevent the development of acquired resistance to momelotinib and MEK inhibitor treatment in KRAS mutant NSCLC.

#298

Amplification of mutant ERBB2 drives resistance to the irreversible kinase inhibitor neratinib in ERBB2-mutated breast cancer patients.

F Javier Carmona,1 David Hyman,1 Gary Ulaner,1 Joe Erinjeri,1 Nancy Bouvier,1 Helen Won,1 Richard Cutler,2 Al Alani,2 Michael Berger,1 José Baselga,1 Maurizio Scaltriti1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Puma Biotechnology, CA_.

Overexpression of human epidermal growth factor receptor 2 (ERBB2), often accompanied by ERBB2 gene amplification, is present in ~25% of early-stage breast cancers. In a relatively small subset of breast cancer patients (4,000 cases each year in the US) ERBB2 is alternatively activated by gain-of-function mutations without gene amplification and receptor overexpression. These somatic mutations frequently occur in the kinase domain of ERBB2 and induce receptor phosphorylation, PI3K/AKT pathway hyperactivation, and malignant transformation. Breast cancer patients bearing ERBB2-mutated tumors are candidates for treatment with the irreversible kinase inhibitor neratinib. Although this therapeutic strategy is showing remarkable activity in a significant proportion of patients, emergence of acquired resistance inevitably occurs. The aim of this study is to identify the possible mechanisms of acquired resistance to neratinib therapy in ERBB2-mutated breast cancers. As part of the ongoing phase II SUMMIT basket study of neratinib in ERBB2-mutant cancers (NCT01953926), we have identified a common genomic alteration in tumors from three patients that progressed following initial benefit from neratinib treatment. Targeted exome sequencing of biopsies collected at time of disease progression revealed increased copy number of the ERBB2-mutant allele. In one case, the resistant lesions were confirmed to be avid for zirconium-89-trastuzumab and the patient responded to subsequent antibody-based anti-ERBB2 therapy. In an attempt to confirm that increased expression of the mutant allele was sufficient to limit neratinib sensitivity, we measured the antitumor activity of this agent in MCF10A cells stably expressing several activating mutations of ERBB2 compared to cells expressing equal levels of the wild type receptor. We found that higher concentrations of neratinib were needed to inhibit the proliferation of ERBB2-mutant cells compared to wild-type. Further, enhanced receptor activity in the ERBB2-mutant cells correlated with increased formation of ERBB2/ERBB3 dimers, activation of the PI3K/AKT pathway and in vivo tumorigenic potential. Combined ERBB2 and ERBB3 inhibition efficiently inhibited phosphorylation of ERBB2/ERBB3 and cell proliferation. We plan to overexpress the ERBB2-mutant allele in cell lines/organoids obtained from ERBB2-mutant patient-derived xenografts, phenocopying our results from the clinical specimens. We hypothesize that this manipulation will be sufficient to induce increase ERBB2/ERBB3 dimerization and resistance to neratinib. In summary, we believe that amplification of mutated ERBB2ERBB2 promotes increased ERBB2/ERBB3 dimerization, ERBB3 activation, and subsequent downstream signaling activation. Dual ERBB2/ERBB3 blockade may be a potential strategy to delay or prevent resistance to neratinib in ERBB2-mutant breast tumors.

#299

Patient-derived tumor microenvironment models uncover non-autonomous TKI resistance mechanisms in NSCLC.

Haichuan Hu, Matthew Niederst, Jeffrey Engelman. _MGH Cancer Center, Charlestown, MA_.

Background: Tyrosine kinase inhibitors (TKI) have yielded great responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations, however these and other targeted therapies are limited by intrinsic and acquired drug resistance. Previous studies looking into relapsed samples largely expanded our understanding of tumor autonomous resistance mechanisms. In this study, we aimed to decipher the non-autonomous mechanisms in vitro by recapitulating the in vivo tumor microenvironment from patient-derived models.

Method: Cancer-associated fibroblast cells are isolated directly from EGFR mutant and ALK translocated NSCLC biopsies. Over 20 patient-derived fibroblast (PDF) models have been established for studying the tumor microenvironment's effect on TKI response.

Result: We found that many, but not all, PDF cells exert significant resistance to TKIs. Furthermore, there is considerable variability in the degree and secreted factor / mechanism by which they are protecting the tumor cells. These variations are further correlated with patient in vivo response.

Conclusion: Together, our results indicate that PDFs are clinically relevant models for deciphering non-autonomous resistance mechanisms, that they are heterogeneous in protecting cancer cells from TKI treatment, and that the resistance mediated by PDFs can be overcome by specific therapeutic combinations.

#300

Inhibition of oncogenic MAPK signaling in melanoma triggers SOX2-dependent adaptive drug resistance.

Stefanie Flueckiger,1 Andrea Aloia,1 Lukas Frischknecht,1 Amanda Tuozzo,1 Davide Croci,1 Olga Shakhova,2 Christian Britschgi,2 Reinhard Dummer,2 Mitchell Paul Levesque,2 Wilhelm Krek1. 1 _ETH Zurich, Zurich, Switzerland;_ 2 _University Hospital of Zurich, Zurich, Switzerland_.

Treatment of metastatic melanoma with BRAFV600E-specific small molecule inhibitors (BRAFi) provides dramatic clinical relief response but fails to suppress long-term tumor growth in the long-term. Extensive studies have uncovered identified a number of resistance-causing genetic alterations that , which are acquired upon continued drug exposure. However, little is known about the changes in gene expression programs changes during the critical time before drug resistance is manifested on at the genomic level. These changes may lead to adaptive drug resistance and hence, represent vital mechanisms by which tumor cells survive the acute drug insult prior to acquisition of resistance-causing mutations. Using a combination of RNAseq and molecular analyses, we find that acute inhibition of oncogenic MAPK signaling by BRAFi or MEKi in established and primary patient-derived melanoma cell cultures, leads to a rapid induction of the stem cell maintenance and pluripotency factor SOX2. Immunohistochemical analysis of Sox2 expression in melanoma sections derived from BrafV600E/Pten-/--mice treated for 36 hours with BRAFi/MEKi confirmed the emergence of a Sox2-positive melanoma subpopulation in vivo. SOX2 Ectopic ectopic expression of SOX2 protected melanoma cells from the anti-proliferative effects of BRAFi indicative ofindicating a role for SOX2 in mediating drug resistance. Furthermore, siRNA-mediated depletion or overexpression experiments of SOX2 unveiled revealed that it is necessary and sufficient for the expansion of a drug-tolerant SOX2+CD24+ cell population. This subpopulation is characterized by increased migratory and invasive behavior as well as an increased proliferative potential in the presence of BRAFi when compared to SOX2+CD24- cells. These results imply that BRAFi treatment induces a SOX2-dependent cellular program with features of adaptive drug resistance. Suppression of drug-dependent induction of SOX2 or its downstream effector pathways could potentially extend the clinical efficacy of BRAFi.

#301

Identification of NRAS isoform 2 overexpression as a novel mechanism facilitating BRAF inhibitor resistance in malignant melanoma (MM).

Megan Duggan,1 Andrew Stiff,1 Gonzalo Olaverria Salavaggione,1 Maryam Bainazar,1 Nicholas Latchana,1 Joseph Markowitz,2 Albert de la Chapelle,1 Ann-Kathrin Eisfeld,1 William Carson1. 1 _The Ohio State University Medical Center, Columbus, OH;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Activating mutations in BRAF are found in 50% of melanomas and are associated with poor outcomes due to constitutive activation of the mitogen activated protein kinase (MAPK) pathway. Treatment with BRAF inhibitors (BRAFi), such as vemurafenib, is effective but resistance can develop. A major mechanism of resistance involves feedback loop-mediated increased expression of the NRAS gene. Recently, we discovered the NRAS transcript is differentially spliced to give 5 novel isoforms (isos), which differ in their expression, cellular localization and downstream effects. We hypothesized that differential expression of one or more of the isos may contribute to the development of BRAFi resistance. We assessed iso-specific NRAS expression in 14 MM cell lines and melanocytes, and noted a curious expression profile of iso2: while undetectable in melanocytes and BRAFwt MM cells, its abundance was increased in BRAFmut cells, and it was the only NRAS iso highly overexpressed in 3 BRAFi resistant cell lines (p<0.01). Iso2 is characterized by an additional exon (exon 3b) compared to canonical NRAS iso1. To explore the possible role of NRAS iso2 in MM, BRAFmut A375 cells were engineered to overexpress NRAS isos 1, 2 and empty vector (EV). Iso2 overexpression led to significantly increased MM cell proliferation in vitro, and surprisingly also increased proliferation during treatment with BRAFi (p<0.001). In a xenograft model, athymic nude mice with A375 tumors stably overexpressing iso1, iso2 or EV were treated with oral BRAFi (50 mg/kg) or PBS for 21 days. Iso 2 overexpressing tumors did not respond to in vivo treatment with BRAFi and grew faster than their iso 1 overexpressing and EV counterparts, as quantified by IVIS imaging, which resulted in a significantly shortened survival of both BRAFi and PBS treated iso2 mice. In contrast, overexpression of canonical iso1 did not affect BRAFi sensitivity. Strikingly, shRNA-mediated knockdown of iso2 in BRAFi resistant cells resulted in decreased survival compared to iso1 or scramble knockdowns. Downstream signaling analysis utilizing immunoblots and confocal microscopy indicated a decrease in MAPK signaling and an increase in PI3K signaling in iso2 overexpressing cells compared to iso1 overexpressing cells. Protein complex immunoprecipitation of iso2 in COS-7 cells validated a binding affinity of NRAS iso2 to both PI3K and BRAF/RAF1. Since MAPK signaling is strongly reduced in iso2 expressing cells, this may suggest an allosteric inhibition mechanism, which prevents binding and activation of BRAF by canonical NRAS iso1.

This study indicates for the first time that NRAS iso2 may play a role in BRAFi resistance by facilitating alternative survival signaling through the PI3K pathway in the presence of MAPK pathway inhibition. Targeting NRAS iso2 may be a beneficial treatment strategy in the prevention and management of BRAFi resistance in melanoma.

#302

ECM/Integrin signaling promotes resistance to the combination of HER2 and PI3K inhibitors in HER2+, PIK3CA-mutant breast cancer.

Ariella B. Hanker, Monica Valeria Estrada, Junfei Zhao, Feixiong Cheng, Preston D. Moore, Darren Tyson, Violeta Sanchez, Brent N. Rexer, Melinda Sanders, Zhongming Zhao, Thomas P. Stricker, Carlos L. Arteaga. _Vanderbilt University Medical Center, Nashville, TN_.

HER2 amplification and activating mutations in PIK3CA, the gene encoding the p110α subunit of PI3K, often co-occur in breast cancer. We generated a transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. In these mice, PIK3CAH1047R accelerates HER2-mediated tumor formation and promotes resistance to HER2 inhibitors (Hanker et al. PNAS 2013). HER2+/PIK3CA tumor growth was inhibited by treatment with the HER2 antibodies trastuzumab and pertuzumab in combination with the pan-PI3K inhibitor BKM120 (TPB). We sought to discover mechanisms of acquired resistance to the triple therapy by long-term treatment of established HER2+/PIK3CA tumors. Tumor transplants derived from a transgenic HER2+/PIK3CA tumor were initially growth inhibited by TPB. After several weeks, a subset of transplants (3/11) resumed growth in the presence of continuous TPB therapy. TPB-resistant tumors were cross-resistant to the combination of T + P + BYL719 (a p110α-specific inhibitor).

Whole exome sequencing did not identify acquired somatic alterations in TPB-resistant tumors, including in HER2. However, RNA-seq revealed significant transcriptional upregulation of extracellular matrix (ECM) genes and genes involved in cell adhesion, including collagens, tenascins, and thrombospondins. Likewise, trichrome staining revealed a significant increase in collagen fibers and IHC analysis confirmed increased Tenascin expression in the TPB-resistant tumor stroma. In addition, western blot analysis revealed increased expression of an activated form of integrin β1, a substrate for ECM ligands such as collagen, as well as P-SrcY416 (activated by integrins/focal adhesion). We also found that transcription of many of these genes is induced by short-term TPB treatment in human breast cancer cell lines by qRT-PCR.

Interestingly, primary tumor cells derived from TPB-resistant tumors no longer displayed resistance when grown in vitro. These cells regained TPB resistance when re-introduced into mice. Plating primary tumor cells on growth factor-reduced Matrigel or on Collagen I-coated plates restored resistance, suggesting that the ECM directly promotes TPB resistance. We are currently investigating whether inhibition of Integrin/Src signaling reverses TPB resistance. We are also exploring whether components of the ECM are altered in residual disease specimens from HER2+ breast cancer patients treated with neoadjuvant anti-HER2 therapies. Our data suggest that upregulation of ECM/integrin/Src signaling contributes to resistance to combinations of HER2 and PI3K inhibitors, and strongly support the growing body of literature indicating that components of the tumor microenvironment promote resistance to targeted therapies.

#303

Compensatory up-regulation of NEK9 contributes to resistance to PLK1 inhibition in triple-negative breast cancer cells.

Nelson K. Y. Wong, Devon Mitchell, Mohamed K. Khan. _BC Cancer Research Centre, Vancouver, British Columbia, Canada_.

Patients with triple-negative breast cancers (TNBCs) have very poor prognosis in terms of relapse and overall survival. There is currently no targeted therapy for this heterogeneous group of patients and it presents a pressing unmet medical need. Recent data suggest that Polo-like kinase 1 (PLK1) is a plausible target for treating TNBCs, and many small-molecule inhibitors are currently under development for targeting this kinase. Since clinical data have shown that single-agent treatment often leads to resistance and relapse of the cancer, it is essential to identify potential resistance mechanisms of TNBCs to PLK1 inhibition such that combination therapy can be rationally designed to increase treatment efficacy.

We have selected ten individual cell clones from a TNBC cell line, MDA-MB-231, through long-term incubation of the cells with increasing concentrations of a PLK1 inhibitor BI 2536. We hypothesized that increased expression and/or activities of other kinases can compensate for the PLK1 inhibition. To identify these kinases, we screened the expression levels of >500 human kinases in the resistant clones in comparison to the parental cells, using the Nanostring platform, and generated a list of candidate kinases. Among the candidates, NEK9, a downstream effector of PLK1, was confirmed to be over-expressed in all resistant cell clones through Western blotting. MDA-MB-231 parental cells were then infected with a lentiviral over-expression vector that contains NEK9 and GFP. When a mixed population of NEK9-over-expressing cells (GFP+) and parental cells (GFP-) were incubated in the presence of BI 2536, increased proportion of GFP+ cells and increased mean GFP fluorescence intensity was observed over time through flow cytometry. Increase of NEK9 expression was also confirmed with Western blotting in the mixed cell culture experiment, indicating the selection of the NEK9-over-expressing cells in the presence of BI 2536.

In conclusion, we have constructed a list of over-expressed kinase candidates that may contribute to the resistance to PLK1 inhibition. We further identify that over-expression of NEK9 contributes to the resistance phenotype. Our data prompt the need to further study the biology around NEK9 and suggest the possibility of combination therapy through inhibition of PLK1 and NEK9 in treating TNBCs.

#304

HER2 basal tumors have frequent mutation of HER3 and are more resistant to HER2-targeted therapy.

Vinay Varadan,1 Salendra Singh,1 Hannah Gilmore,1 Shikha Parsai,1 George Somlo,2 Maysa Abu-Khalaf,3 William Sikov,4 Lyndsay N. Harris1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _City of Hope, Duarte, CA;_ 3 _Yale University, New Haven, CT;_ 4 _Brown University, Providence, RI_.

Background: HER2 tumors are heterogeneous at both the clinical and molecular levels (Harris et al 2007, Varadan, AACR 2015). We and others have identified a distinct molecular subtype termed 'HER2 basal' that demonstrates lower response to preoperative trastuzumab and is characterized by overexpression of the 'basal-like' signature genes (Harris, CCR 2007; Carey, JCO 2015). We sought to better understand the molecular underpinnings of the HER2 basal phenotype by performing whole exome sequencing (WES) in a cohort of HER2 tumors treated with trastuzumab-containing preoperative therapy.

Methods: A multicenter trial (BrUOG 211B) was conducted to determine predictors of pathologic complete response (pCR) to trastuzumab (T)-containing preoperative therapy. Sixty HER2+ Stage II-III patients were enrolled and assigned to either a single dose of T or nab-paclitaxel (N) followed by T+N+carboplatin (TNC). Fresh tumor core biopsies were taken at baseline and 2 weeks after the initial dose of T or N. PAM50 subtyping was performed using gene expression data from patient tumor biopsies and tumors were classified into HER2-Enriched, HER2-Luminal and HER2-Basal subtypes based on ER/PR IHC values and relative expression of the proliferation-associated genes within the PAM50 gene list. WES was performed on a total of 86 samples (49 baseline and 37 from the post brief-exposure timepoint), with each sample sequenced at an average depth of 90X. Somatic mutations (single nucleotide variants and short indels) were assessed using Genome Analysis Toolkit (GATK) UnifiedGenotyper followed by a robust pipeline comparing multiple databases to eliminate germline variants. Copy-number estimation was performed on the WES data using a novel algorithm, 'ENVE' (Varadan, Genome Med, 2015) that robustly identifies somatic copy-number alterations.

Results: WES profiles of HER2 basal tumors (n=6) revealed lower average copy number for HER2 (2.5 vs 5.8) and were less likely to have high-level amplification (>4 copies per cell) of other amplicons (e.g. 11q13, 20q13) with the exception of 8q24. The so-called 'Myc' amplicon was co-amplified in all subtypes relatively equally (3/6 in HER2-B, 11/19 in HER2-E, 10/23 in HER2-L). HER2-B tumors were also characterized by higher frequency of mutations in Rb (2/6 versus 1/42; P = 0.0376) and HER3 (4/6 vs 2/42; P= 0.0011) compared with other subtypes and frequent alteration of p53 (4/6 versus 17/42; P = 0.22). pCR to preop TNC was HER2-B 3/8, HER2-E 10/20, HER2-L 6/21, not significant in this small cohort. The presence of HER3 mutations suggests a potential mechanism for the previously reported poor clinical response to preoperative anti-HER2 therapy in HER2 basal tumors.

Conclusions: The HER2 basal subtype is more likely to exhibit mutations in HER3 and is less likely to respond to trastuzumab-based therapy. Larger cohorts are required to confirm these findings and trials of other HER2 targeted agents in this subtype should be considered.

#305

SRC mediates intrinsic resistance to BRAF(V600E) inhibition in colon cancer.

Jean-Philippe F. Coppé,1 Changjun Wang,1 Diede Brunen,2 Anirudh Prahallad,2 Ana Ruiz-Saenz,1 Danislav Spassov,1 Bo Pan,1 Aleksandra Olow,1 Denise Wolf,1 Christina Yau,1 Christian Posch,1 Evelyn Lee,1 Miki Mori,1 Katherina Chua,1 Julia Malato,1 Donghui Hwang,1 Paul Phojanakong,1 Zhongzhong Chen,3 Byron Hann,1 Lamorna Brown-Swigart,1 Mark Moasser,1 René Bernards,2 Laura van 't Veer1. 1 _University of California at San Francisco, San Francisco, CA;_ 2 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 3 _The State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China_.

BACKGROUND. Colon cancer patients harboring a BRAF(V600E) oncogenic lesion have poor prognosis, and show very limited response to small-molecule inhibitor PLX4032 (Vemurafenib).

METHODS. To investigate mechanisms of intrinsic resistance causing the limited therapeutic effect of PLX4032 in BRAF(V600E) colorectal cancer, we performed a high throughput kinase activity mapping (HT-KAM) screen to search for kinases whose activity is induced upon BRAF(V600E) inhibition.

RESULTS. We found that proto-oncogenic SRC family kinases are activated upon PLX4032 treatment, and act in parallel to (and independently of) EGFR and HER3 pathways. The mechanism of reversible and intrinsic resistance to BRAF(V600E) inhibition was confirmed via exogenous genetic alteration of SRC kinases and regulators of SRC kinases functions. SRC feedback activation was conserved in BRAF(V600E) thyroid cancer cells, and could be efficiently targeted in non-mutated RAS, BRAF(V600E)-only cancer cells. SRC kinases-targeting therapies Dasatinib or Saracatinib combined with Vemurafenib, and with or without Gefinitib or Lapatinib, efficiently restored therapeutic response in a large series of BRAF(V600E) colorectal and thyroid cancer cells, including ones otherwise resistant to dual PLX4032+Gefinitib or PLX4032+Lapatinib therapies, in cell survival and colony formation assays. Both Dasatinib or Saracatinib treatments showed strong synergy with BRAF(V600E) inhibition, and with or without Gefinitib, in an in vivo pre-clinical tumor progression model.

CONCLUSION. Our kinase-network exploration strategy HT-KAM defined a pivotal role for SRC kinases in regulating intrinsic resistance to Vemurafenib therapy. Given the highly aggressive and chemotherapy-resistant nature of BRAF-mutated colorectal cancers, the new dual and triple kinase-targeting combination therapies uncovered in this study, will deserve further clinical evaluation.

#306

Analysis of differentially expressed exosomal genes from ibrutinib resistant diffuse large B cell lymphoma.

Neeraj Jain, Lalit Sehgal, Tamer Khashab, Felipe Samaniego. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of the patients develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. Selective pressure provided by chemotherapy causes aberrant gene expression and selection of pre-existing population of chemo-resistant cells. These chemo-resistant cells can also transmit chemo-resistance to chemo-sensitive cells through exosomal transfer of molecules (miRNA, mRNA and proteins). In this study, we have analysed differentially expressed mRNA and miRNA from syngeneic ibrutinib (an irreversible BTK inhibitor, FDA approved) resistant DLBCL cells and from their isolated exosomes. Five DLBCL-GCB and five ABC subtype were analysed for their sensitivity with ibrutinib. We found three ABC subtype and one GCB subtype (OCL-LY1) were sensitive to ibrutinib. We have generated syngeneic ibrutinib drug resistant model from sensitive cells (at least 10 fold increase in IC50 value) by continuous culturing them in the presence of ibrutinib with gradual increase in drug concentration in each passage. Ibrutinib sensitive DLBCL cells when cultured with the syngeneic resistant cells in transwell apparatus, acquired resistance to ibrutinib. We also analysed the sensitivity of ibrutinib resistant cells with other conventional drugs for non-Hodgkin lymphomas. We further tested the expression of drug resistant genes in syngeneic resistant cells by real time PCR analysis. Later, we isolated the total RNA and miRNA species from DLBCL ibrutinib sensitive, their syngeneic resistant cells and from respective exosomes and analysed for differential expression by micro array analyses. Thus in conclusion, our study demonstrates that chemo-resistance can be transferred from resistant cells to sensitive cells via exosomal horizontal transmission of miRNA and mRNA in diffuse large B cell lymphoma cells. Moreover, our study identified potential targets that can be used to overcome chemo-resistance in diffuse large B cell lymphoma.

#307

GPR120/FFAR4 activation by fatty acid 16:4(n-3) plays a key role in resistance to chemotherapy.

Julia Houthuijzen,1 Ilse Oosterom,2 Brian Hudson,3 Akira Hirasawa,4 Laura Daenen,2 Chelsea McLean,1 Steffen Hansen,5 Marijn van Jaarsveld,1 Daniel Peeper,1 sahar Jafari Sadatmand,1 Jeanine Roodhart,2 Chris van de Lest,6 Trond Ulven,5 Kenji Ishihara,7 Graeme Milligan,3 Emile Voest1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _University Medical Center Utrecht, Utrecht, Netherlands;_ 3 _University of Glasgow, Glasgow, United Kingdom;_ 4 _Kyoto University, Kyoto, Japan;_ 5 _University of Southern Denmark, Odense, Denmark;_ 6 _University Utrecht, Utrecht, Netherlands;_ 7 _National Research Institute of Fisheries Science, Kanazawaku, Japan_.

Although chemotherapy is designed to eradicate tumor cells it also has a significant impact on normal tissues. These host-responses can have large effects on the efficacy of treatment and overall survival. Fatty acids are increasingly recognized to play important signaling roles. 12-S-HHT and 16:4(n-3) are two platinum-induced fatty acids (PIFAs) that induce systemic resistance to a broad range of DNA-damaging chemotherapeutics. PIFAs exert their chemoprotective effect on tumor cells via an indirect mechanism involving splenic F4/80+/CD11blow macrophages. Here we identified GPR120 on mouse and human splenic macrophages as the relevant receptor for 16:4(n-3). GPR120 (FFAR4) is a free fatty acid receptor that binds medium- to long-chain fatty acids and omega-3 fatty acids and is involved in anti-inflammatory response and metabolic control. Although both GPR40 (FFAR1) and GPR120 can be activated by 16:4(n-3) in vitro, only inhibition or genetic loss of GPR120 was able to block 16:4(n-3)-mediated chemoresistance in vivo. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in splenic macrophages resulting in the production and secretion of resistance-inducing lysophosphatidylcholine 24:1 (LPC(24:1)). Taken together, we identified a novel function for GPR120. Activation by 16:4(n-3) leads to enhanced cPLA2 activity and production of LPC(24:1) resulting in chemotherapy resistance.

#308

Interrogating Goldilocks: Searching for mediators of oncogene overdose in engineered BRAFV600E inhibitor resistant melanoma cells.

Daphne R. Pringle, Martin McMahon. _Huntsman Cancer Institute/University of Utah, Salt Lake City, UT_.

The phenomenon of oncogene overdose has been exhibited in patient-derived melanoma xenografts, and clinical trials are underway to determine if intermittent BRAFV600E inhibitor (BRAFi) treatment may take advantage of this phenomenon to benefit melanoma patient outcomes. While oncogene overdose has been described in many settings, molecules that mediate this phenomenon of are poorly characterized. This abstract describes the generation and initial testing of a melanoma cell system to identify the mediators downstream of BRAFV600E hyperactivity that mediate oncogene overdose.

BRAFi resistant cell lines were generated from YUMM1.1 (BRAFV600E, PTENNull, INK4A-ARFNull) mouse melanoma cells. To generate BRAFi resistant cell lines, retroviruses harboring known human BRAFi resistance alleles were used to infect YUMM1.1 cells. Infected cells were selected directly in the appropriate antibiotic and 1 μM LGX818, a BRAFV600E specific inhibitor currently in clinical trials. Resulting drug resistant populations were single cell cloned and expression of transduced proteins verified. To probe the ability of resistance alleles to lead to oncogene overdose and growth arrest, colony forming assays, cell counting assays, cell cycle analyses, BrdU incorporation assays and immunoblots of relevant cellular pathways were performed.

Results indicate that expression of alleles that confer resistance to LGX818, as well as to inhibition of other nodes in the MAPK pathway, does not lead to cross-resistance to inhibitors of other pathways such as the PI3K pathway. Cells expressing some resistance alleles (e.g. BRAFV600E), but not others (e.g. NRASQ61K), demonstrate oncogene overdose when LGX818 is withdrawn. Data from cell cycle analyses suggest that, unlike the G1 cell cycle arrest seen in parental cells exposed to BRAFi, cells exhibiting oncogene overdose show no aberrations in their cell cycle state, despite reduced DNA synthesis as measured by BrdU incorporation, suggesting a distinct mechanism of arrest. Immunoblot analyses of critical signaling pathways show that mTORC signaling to the protein synthesis machinery is activated upon oncogene overdose, suggesting a potential role for this pathway, and its amino acid sensing function, in oncogene overdose induced arrest. The role of protein synthesis is under further investigation.

These data suggest that not all mechanisms of BRAFi resistance are equivalent in their ability to elicit oncogene overdose. It has become clear that oncogene overdose induced growth arrest is inherently more complicated than G1 cell cycle arrest seen in drug naïve melanoma cells or in primary human or mouse cells. Further elucidation of mediators of the 'Goldilocks effect' of BRAFV600E signaling in melanoma and in other cellular contexts has the potential to shed light on alternative mechanisms to induce melanoma cell growth arrest, even after the development of resistance.

#309

Mechanisms of resistance to trastuzumab emtansine in gastric cancer.

Juliette Sauveur,1 Abdelkamel Chettab,1 Eva matera,1 Aurore Cleret,1 Ariel Savina,2 Charles Dumontet1. 1 _Cancer Research Center of Lyon, Lyon, France;_ 2 _Institut Roche, Paris, France_.

Background: Overexpression of Human Epidermal growth factor Receptor 2 (HER2) in cancer was associated with poor outcome and high chances of recurrence before the development of anti-HER2 agents. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate targeting HER2 based on its antibody component trastuzumab. After internalization of T-DM1/HER2, Lys-MCC-DM1, a tubulin binding agent is released in the cytoplasm. In spite of therapeutic advances, acquired resistance to treatment remains a major obstacle. A better understanding of the mechanisms of resistance to T-DM1 is necessary to improve treatment regimens for HER2-overexpressing cancer patients. We have developed and characterized resistance models to T-DM1 in order to describe underlying resistance mechanisms and develop alternative strategies.

Methods: To develop in vitro T-DM1 resistance models, HER2-overexpressing esophageal adenocarcinoma cell line OE-19 was exposed to increasing doses of T-DM1 several months in the absence or presence of cyclosporin A (CsA, an inhibitor of MDR1 transporter). Resistance models were called OE-19 TR (exposed to T-DM1) and OE-19 TCR (exposed to T-DM1 and CsA). To characterize the resistance models, sensitivity to T-DM1 was assessed by MTT assay, Annexin V-PI staining and xCELLigence. Cell cycle distribution was assessed by propidium iodide using flow cytometry. Cross-resistance was studied by MTT assay. Expression of HER2, ABC transporters and tubulin was studied by rt-qPCR, flow cytometry and/or Western Blot. An expression profile of sensitive and resistant cells to T-DM1 was performed using a pan-genomic Illumina cDNA array.

Results: After prolonged exposure of OE-19 cells to T-DM1, IC50 value is 18-fold higher in TR cells and 21-fold higher in TCR cells and both models become less sensitive to T-DM1-induced apoptosis. T-DM1 induced G2/M arrest in OE-19 sensitive cells but not in resistant cells. We found that resistant cells become less sensitive to trastuzumab but remain sensitive to other HER2-targeted therapies such as lapatinib, as well as to a variety of chemotherapy agents. Among the resistant cells, a subpopulation with increased expression MDR1 was identified in OE-19 TR and TCR. Concerning the expression of the main targets of T-DM1 (HER2 and tubulin); we found that HER2 expression and total α and β tubulin expression and content remained unchanged. However the isoforms β2 and β3 were overexpressed in both resistant models. A transcriptomic approach of T-DM1 resistant cells showed modification in adhesion junctions and focal adhesions.

Conclusion: Prolonged exposure of the OE-19 cell line to T-DM1 resulted in resistance to this immunoconjugate. We are working on describing the resistance mechanisms to T-DM1, which are important to understand cancer progression in patients receiving this therapy. In the long term, we hope this study will allow proposing agents that benefit T-DM1-refractory cancer patients.

#310

Mechanism of sorafenib resistance in acute myeloid leukemia.

Oscar Lindblad,1 Eugenia Cordero,1 Alexandre Puissant,2 Lucy Macaulay,1 Nuzhat N. Kabir,3 Jianmin Sun,1 Karin Haraldsson,1 Åke Borg,1 Fredrik Levander,1 Kimberly Stegmaier,2 Kristian Pietras,1 Lars Rönnstrand,1 Julhash U. Kazi1. 1 _Lund University, Lund, Sweden;_ 2 _Harvard Medical School, Boston, MA;_ 3 _KN Biomedical Research Institute, Barisal, Bangladesh_.

Activating mutations in FLT3 occur in up to 35% of patients with AML and correlate with poor prognosis. Therapy directed against FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. Dual PI3K/mTOR inhibitors have displayed promising results in the treatment of solid tumors, and of hematological cancers. In this report we describe that a dual PI3K/mTOR inhibitor is effective against sorafenib-responsive, and -resistant, AML cell lines both in vitro and in vivo. We generated two cell lines by sustained treatment with sorafenib. Parental cell lines carry the FLT3-ITD mutation and are highly responsive to FLT3 inhibitors, while sorafenib-resistant cell lines display resistance to multiple FLT3 inhibitors. Next generation sequencing did not show any significant difference in the mutational burden in between responsive and resistant cell lines. While next generation sequencing identified FLT3-D835Y with an allele-depth of 67:37 in a resistant cell line, Sanger sequencing and protein mass-spectroscopy did not identify any acquired mutations in the kinase domain of FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, while it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer dependent on FLT3. Gene expression analysis of sorafenib-sensitive and -resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway that correlated with the resistant phenotype, which was further supported by phospho-specific-antibody array analysis. The selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation of resistant cell lines as well as induces apoptosis. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway results in FLT3-inhibitors-resistance and a dual specific PI3K/mTOR inhibitor is an effective treatment in both tyrosine kinase inhibitor sensitive and resistant AML.

#311

**Novel role for MIG-6 in mediating TKI resistance in** ALK **-rearranged lung cancer.**

Huan Qiao,1 Yingjun Yan,1 Matthew A. Smith,2 Eric B. Haura,2 Marc Ladanyi,3 Christine M. Lovly1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Moffitt Cancer Center, Tampa, FL;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

INTRODUCTION: Patients with ALK-rearranged non-small cell lung cancer (NSCLC) derive significant anti-tumor responses when treated with ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, the first-in-class ALK TKI. However despite the high response rates to these agents, acquired resistance to ALK TKI therapy remains a significant barrier to overcome in order to maximize therapeutic responses in patients with ALK+ lung cancer. In crizotinib-resistant tumors, EGFR activation has been demonstrated to mediate acquired resistance in several independent studies. The tumor suppressor gene product MIG-6 (encoded by ERFFI1) acts as a natural inhibitor of signaling through EGFR (ErbB1) and other ErbB family members. However, a role for MIG-6 in ALK+ lung cancer has not yet been elucidated. Here, we investigated the regulation of human MIG-6 in ALK+ lung cancer cells.

DESIGN: MIG-6 expression, protein interaction and phosphorylation status were evaluated in ALK+ lung cancer cell lines and human tumor samples. The impact of MIG-6 loss and gain of function on ErbB receptor activity and on cell proliferation/survival were determined in several models of TKI-sensitive and TKI-resistant ALK+ lung cancer.

RESULTS: MIG-6 protein level is regulated by EML4-ALK fusion protein at both transcriptional and post-translational levels. Genetic or pharmacologic knock-down of ALK significantly reduced MIG-6 protein levels in ALK+/TKI sensitive cells. We established a novel interaction between MIG-6 and ALK fusion proteins (both EML4-ALK and other fusion partners), and this association was dependent on ALK kinase activity. MIG-6 can also be tyrosine-phosphorylated by EML4-ALK and be threonine-phosphorylated through EML4-ALK-dependent MAPK activation. The reduced MIG-6 protein level was accompanied by increased total and phosphorylated ErbB family members (EGFR, HER2, and HER3) in ALK+/TKI sensitive lung cancer cells. Consistent with this observation, MIG-6 protein level is also attenuated in ALK+/TKI resistant cells, following the decreased EML4-ALK activity, while EGFR signaling activity is remarkably up-regulated in these resistant cells. Crizotinib-resistant cells were re-sensitized upon exposure to EGFR/HER2 inhibitors in combination with crizotinib. In addition, MIG-6 reconstitution impeded the development of early adaptive resistance in crizotinib-sensitive cells and was also able to inhibit the proliferation of crizotinib-resistant cells.

CONCLUSION: Our study presents an in-depth mechanistic understanding of how ErbB family members, such as EGFR, are up-regulated at the time of crizotinib resistance and provides insights into the early escape mechanisms tumor may have to evade ALK TKI therapy.

#312

Evidence of EZH2 dependent and independent mechanisms of tazemetostat treatment emergent resistance in models of diffuse large B cell lymphoma.

Carly T. Campbell,1 Jeff S. Jasper,2 Scott R. Daigle,1 Scott A. Ribich,1 Heike Keilhack,1 Jesse S. Smith,1 Peter T. Ho,1 Stephen J. Blakemore1. 1 _Epizyme, Cambridge, MA;_ 2 _Q2 Solutions, Morrisville, NC_.

Tazemetostat is a small molecule inhibitor of the histone methyltransferase EZH2 and is currently in phase 2 clinical trials including relapsed refractory Non-Hodgkin Lymphomas (RR-NHL) Diffuse Large B Cell Lymphoma and Follicular Lymphoma. In the phase 1 clinical trial RR-NHL patients demonstrated positive clinical activity with a favorable safety profile. Acquired mutations in the D1 domain (I109K, Y111D, Y111L) and the SET domain (Y661D) of EZH2 have recently been reported as a mechanism of resistance to non-tazemetostat small molecule EZH2 inhibition. Given the clinical activity observed in the phase 1 tazemetostat clinical trial and these reports of pre-clinical EZH2 inhibitor induced resistance we embarked on investigations of the potential of tazemetostat to induce resistance in NHL cell lines. An EZH2 Y641F mutant DLBCL cell line WSU-DLCL2 was exposed to 1 µM tazemetostat, a dose which is 100 fold greater than the naïve line's 11 day growth IC50. After 8 weeks, growth of the tazemetostat treated cells matched that of the control cells. Subsequent increases in dose of tazemetostat up to 10 µM did not yield any changes in growth rate of the treated cells. EZH2 wild-type PMBCL cell line U2940 was exposed to a step wise increase in tazemetostat concentration for 7 weeks and finally maintained at the naïve cell line 11 day proliferation IC50 of 10 µM, with minimal effects on cell growth. Tazemetostat resistant cell lines were screened for acquired EZH2 mutations using a full coding next generation sequencing assay, with a mean depth of 17,126 across all positions. Sequencing results showed the resistant U2940 had gained mutations in EZH2 similar to those previously identified, a heterozygous Y661N mutation and a low frequency mutation of Y111H, consistent with a subclonal mutation. These acquired mutations have been reported to interfere with the binding of EZH2 inhibitors, which supports the minimal reduction of H3K27me3 as measured by ELISA in the resistant U2940 after treatment with tazemetostat. In contrast, after induction of tazemetostat resistance WSU-DLCL2 retained equipotent sensitivity to reduction of H3K27me3 by ELISA. Correspondingly, sequencing of EZH2 in the resistant WSU-DLCL2 line did not identify any additional mutations. These findings suggest continued target engagement with tazemetostat in the resistant WSU-DLCL2, and that a novel bypass mechanism may be engaged to confer resistance. In an attempt to identify the mechanism of resistance in the WSU-DLCL2 line, whole exome sequencing and RNA sequencing has been performed. Detailed mutational, gene expression and pathway analysis will be performed on these data to investigate mechanisms of treatment emergent resistance to tazemetostat. Understanding these mechanisms may guide hypotheses for rational combinations and provide direction for future preclinical and potentially clinical studies.

#313

Type II interleukin 1 receptor-modulated SETBP-1/SET/PP2A/p-ERK signaling reduces the therapeutic activity of regorafenib against human colorectal cancer cells.

Ai-Chung Mar,1 Chun-Ho Chu,2 Shung-Haur Yang,3 Jeng-Kai Jiang,3 Chung-Wai Shiau,4 Te-Chang Lee1. 1 _Institute of Biomedical Sciences, Taipei, Taiwan;_ 2 _Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan;_ 3 _Division of Colon and Rectal Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan;_ 4 _Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan_.

Regorafenib, a newly approved multi-kinase inhibitor by US FDA, has recently demonstrated overall survival benefits in late-stage colorectal cancer (CRC) patients. We have preliminarily observed that the expression type II interleukin 1 receptor (IL1R2), an IL-1 decoy receptor, was closely associated with regorafenib resistance in human CRC cells. We also found that IL1R2 expression was associated with poor prognosis and 5-year survival of CRC patients. In this study, we conducted experiments to elucidate the mechanisms underlying which IL1R2 is involved in regorafenib resistance. We first demonstrated that silencing of IL1R2 in HT29 cells overcame its resistance to regorafenib, whereas ectopic expression of IL1R2 in HCT116 cells reduced its sensitivity to regorafenib in either in vitro or in vivo assay systems. In addition, we established regorafenib-resistant DLD-1 cells (DLD-1-R) by long-term culturing DLD-1 cells in the presence of regorafenib, and we observed that protein expression of IL1R2 was increased in DLD-1-R cells compared to the parental DLD-1 cells. Furthermore, our results showed that IL1R2 expression was positively associated with p-ERK levels, which are crucial for survival of cancer cells. Pretreatment of HT29, IL1R2-overexpressing HCT116, and DLD-1-R cells with MEK/ERK inhibitor U0126 significantly reversed their regorafenib resistance in in vitro and in vivo systems. Mechanistically, we revealed that increased p-ERK levels in IL1R2-overexpressing cells were due to the decrease of PP2A activity instead of its expression levels. Since PP2A is a phosphatase of ERK, we further demonstrated that IL1R2 mediated through transcriptionally enhancing the expression of SETBP-1. The formation of SETBP-1/SET/PP2A complex decreased the activity of PP2A. Taken together, our present study identified IL1R2 was a potential prognostic and staging biomarker of CRC patients. Significantly, we also demonstrated a new molecular mechanism involved in regorafenib resistance and the combination of regorafenib and MEK/ERK inhibitor is a rationale regime to overcome regorafenib resistance in CRC patients.

#314

Cell cycle phase-specific drug resistance as an escape mechanism of melanoma cells.

Kimberley A. Beaumont,1 David S. Hill,1 Sheena M. Daignault,2 Danae M. Sharp,1 Brian Gabrielli,2 Wolfgang Weninger,1 Nikolas K. Haass2. 1 _The Centenary Institute, Newtown, Australia;_ 2 _The University of Queensland Diamantina Institute, Woolloongabba, Australia_.

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic areas within tumors contain clusters of cancer cells that arrest in the G1 cell cycle phase. It is conceivable that cancer cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. Here, we have used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of G1-arrest on clinically used melanoma drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1-arrest, are resistant to induced cell death by the proteasome inhibitor bortezomib and the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to MAPK pathway inhibition. Of clinical relevance, pre-treatment of melanoma cells with a MAPK pathway inhibitor resulted in resistance to temozolomide or bortezomib, while in contrast pre-treatment with temozolomide did not result in resistance to the MAPK pathway inhibitor. In summary, we have established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies.

#315

Potential role of androgen receptor in aromatase inhibitor resistance.

Caleb Hunt, Sarah E. Lock, Cynthia E. Shannon, Nichole M. Varela-Gonzalez, Randolph K. Larsen, Amanda J. Schech. _St. Mary's College of MD, St. Mary's City, MD_.

Clinical assessment of breast cancer tumors has shown that androgen receptor (AR) is expressed in more than 75% of cases, independent of molecular subtype. Specifically, clinical studies have shown that estrogen receptor (ER) and AR discordance correlates with endocrine resistance. Preclinical studies show that downregulation of ER results in a lack of estrogen mediated growth which is concurrent with upregulation of AR and its mediated gene targets. In addition, forced expression of AR was shown to induce resistance to tamoxifen, even when estrogen receptor was still expressed. Furthermore, ER positive breast cancer cells made resistant to tamoxifen express higher levels of AR and show sensitivity to abiraterone acetate. Together, these results implicate AR in endocrine resistance and suggest AR as a potential therapeutic target in a subset of these endocrine resistant cancers. The aim of this study was to better understand the role of AR in aromatase inhibitor (AI) resistance through the use of cell lines made resistant to AI through xenograft mouse modeling. In this study, MCF7 cells, MCF7 cells transfected with the human aromatase gene (MCF7Ca), and their aromatase inhibitor (AI) resistant counterparts, were analyzed for changes in AR and ER expression by qPCR and Western blot. Sensitivity of these cells to AR targeting agents was assessed by MTT assay. MCF7Ca cells displayed decreased expression of AR and increased expression of ER when compared to MCF7 cells. AI-resistant cell lines displayed modulation of ER and AR expression when compared to their AI-sensitive counterparts. Upregulation of AR was accompanied with increased AR transcriptional activity, as demonstrated by increased expression of PSA. Treatment of AI-resistant cells with AR targeting agents displayed increased sensitivity when compared to AI-sensitive cells. Together, these results suggest that upregulation of AR may contribute to AI-resistance, and that targeting AR in AI-resistant breast cancer may have therapeutic relevance.

#316

FTY720 enhances the efficacy of paclitaxel and of carboplatin in drug-resistant ovarian cancer cells.

Kelly M. Kreitzburg,1 Ronald D. Alvarez,1 Karina J. Yoon,1 Charles N. Landen2. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _University of Virginia, Charlottesville, VA_.

Objectives: Our long-term goal is to develop effective therapies for ovarian cancer by targeting molecules that contribute to drug resistance in this tumor type. Preliminary RNA-seq data were generated using a patient-derived xenograft model. These data identified the sphingosine-1-phosphate (S1P) signaling pathway as one of the pathways most affected by carboplatin and paclitaxel, the current standards of care for ovarian cancer. We then examined whether a synthetic analog of sphingosine, FTY720, enhanced the cytotoxicity of platinum and taxane resistant ovarian cancer cell lines.

Methods: To assess the efficacy of FTY720 + carboplatin and of FTY720 + paclitaxel, we exposed three pairs of parental and drug resistant human ovarian cancer cell lines to each combination. The cell lines used were: SKOV3-ip1 and SKOV3-TR (taxane resistant), A2780 and A2780-CP20 (platinum resistant), and HeyA8 and HeyA8-MDR (taxane-platinum resistant). We used alamarBlue cell proliferation assays to determine the efficacy of FTY720 as a single agent or in combination with paclitaxel or carboplatin, and immunoblots to assess the expression of proteins involved in the S1P pathway.

Results: FTY720 as a single agent decreased cell viability in a dose-dependent manner in all three pairs of cell lines, with IC50 values ranging from 5 to 8 μM. When cells were exposed to the IC50 of FTY720 + a range of concentrations of carboplatin or paclitaxel, FTY720 had little effect in any of the three parental cell lines and in SKOV3-TR cells. Interestingly, however, FTY720 decreased the IC50 of carboplatin 15- to 16-fold and the IC50 of paclitaxel 20- to 90- fold in A2780-CP20 and HeyA8-MDR cells, respectively. We also observed that FTY720 altered the expression of sphingosine kinase2, S1P lyase, and acid ceramidase, each of which contributes to the ceramide-sphingosine-S1P rheostat that tightly regulates expression of S1P pathway proteins.

Conclusions: Our data show that FTY720 sensitized drug resistant ovarian cancer cell lines A2780-CP20 and HeyA8-MDR to carboplatin and to paclitaxel 15- to 90-fold. The data suggest that the S1P pathway may contribute to carboplatin and paclitaxel resistance in this cell type. We propose that proteins of the S1P pathway merit further study as effective molecular targets in drug resistant ovarian cancer cells.

#317

Microtubule polymerizing effects of ellagic acid reduce efficacy of taxane chemotherapy.

Jillian N. Eskra, Alaina Dodge, Maarten C. Bosland. _University of Illinois at Chicago, Chicago, IL_.

Ellagic acid (EA) is a polyphenolic compound found in berries, pomegranates and other foods, which has cancer preventive and therapeutic properties. EA inhibits proliferation, induces apoptosis, blocks cell cycle progression and inhibits ABC drug transporters. Based on these anti-cancer activities of EA, we hypothesized that EA could enhance the efficacy of taxane chemotherapy on prostate cancer cells and inhibit drug resistance. We used castration-resistant 22Rv1, VCaP, and C4-2 prostate cancer cells and androgen-independent PC-3 cells to study effects of EA on efficacy of the taxane drug cabazitaxel (CBZ). Cells were treated with EA (0.1 µM - 30 µM) and after 72 hr proliferation was measured using the SRB assay. EA inhibited proliferation at levels greater than 10 µM. At 30 µM the greatest inhibition was observed in the most proliferative cell lines (22Rv1: 66.7%, PC-3: 82.7%), with a more modest effect on the slower growing cells (VCaP: 25.2%, C4-2: 31.7%). We next tested if growth inhibitory concentrations of EA could enhance the microtubule stabilizing effects of CBZ. In a cell-free assay, CBZ (1 nM - 1 µM) dose dependently increased tubulin polymerization, and EA (10 µM) increased polymerization by 61.0%. However, we observed an antagonistic response with the combination of CBZ and EA, reducing polymerized tubulin by 13.9% compared to CBZ alone. To confirm these results in cell culture, we treated 22Rv1 cells with EA (10 µM) and CBZ (10 nM) and determined levels of polymerized tubulin by western blotting. We observed a 1.6 fold increase in polymerized tubulin by EA, a 5.5 fold increase by CBZ, which was reduced to a 4.6 fold increase by combination of CBZ with EA. Contrary to our hypothesis, these results suggest that EA may interfere with taxanes, leading to a reduction in drug effectiveness. Additionally, the ability of EA alone to induce polymerization suggests the possibility that the growth inhibitory activity of EA is a consequence of its effects on microtubule dynamics, potentially through an interaction with taxane binding site of tubulin. This is consistent with our observation that EA has the greatest growth inhibitory effect on very proliferative cell lines, since microtubule targeting agents are most effective at killing rapidly dividing cells. To our knowledge, this is the first evidence of microtubule targeting as an anticancer mechanism of EA. We also studied the effects of EA on drug efflux to determine if EA could inhibit development of taxane drug resistance. We treated C4-2 cells with EA (0.01 µM - 30 µM) and measured calcein retention as marker of the activity of the P-glycoprotein (P-gp) pump. There was a dose dependent increase in calcein retention by EA, with a 1.9 fold increase at the highest concentration (30 µM), suggesting an inhibition of P-gp pump activity with increasing amounts of EA. Thus, EA may increase intracellular taxane levels, but at the same time reduce their microtubule-polymerization effects.

#318

Trigonella foenum (fenugreek) inhibited cancer stem cell properties and increased sensitivity to Doxorubicin resistant ZR-75-1 human breast cancer cell line by targeting mutant p53 and Notch-4.

Ahmed S. Sultan,1 Amira S. Fayala,2 Marwa Elkamel,2 Reem Bakir,2 Fatma Foad2. 1 _LCCC-GUMC, Washington, DC;_ 2 _Alexandria University, Alexandria, Egypt_.

Despite advances in detection and therapies, breast cancer is the most common malignant disease in women worldwide. Doxorubicin is one of the effective agent for breast cancer treatment, but the resistance to it is representing a major obstacle for breast cancer treatment.

Herein, we established a breast cancer cell line that showed a significant resistance to doxorubicin at clinically relevant concentrations. The epithelial breast cancer cell line ZR-75-1 was sequentially treated with doxorubicin for several treatment cycles. In consequence, we obtained chemoresistant cells displaying a mesenchymal-like phenotype, ZR-75-1-DR. The ZR-75-1-DR cells showed an increase in breast cancer stem cells (BCSC) activity, mutant p53 but not wild type, NOTCH-4 and microRNAs, small noncoding RNAs, expression compared to parental cells, suggesting their expression may contribute to the evolvement of BCSCs. Understanding the early molecular mechanisms of cross talk among mutant p53, Notch-4 and microRNAs in resistant to doxorubicin is crucial to provide better therapeutic strategies for breast cancer progression.

Recently, we showed that Trigonella foenum (Fenugreek) extract (FCE), a traditional herbal plant with anti-tumor activity, induced apoptosis in HepG2 cells mediated by up-regulation of wild P53. To investigate the possible effect of FCE in the expression levels of mutant p53, Notch-4 and miRNAs in ZR-75-1-DR and parental cell lines, cells were pre-treated with or without FCE and/or doxorubicin for different time intervals. miRNAs were quantified using qRT-PCR and p53, Notch-4 expression and BCSC phenotype were evaluated by western blot analysis and immunohistochemistry.

Our results revealed that FCE treatment showed a significant decrease in cell viability, clonogenicity and invasion capacity with a decrease in expression of mutant p53 and Notch-4 in ZR-75-1-DR compared to untreated cells. Furthermore, the inhibition of mutant p53 and Notch-4 expression was associated with a significant decrease in miR-21 and miR-142 with an increase in expression of let-7a and miR-34a. Furthermore, ZR-75-1-DR pre-treated with FCE showed an increase in chemosensitivity to doxorubicin at low doses with reduction in expression of stemness factor, ALDH1, compared to the parental and doxorubicin treated cells. In vivo, knockdown both mutant p53 and NOTCH-4 showed a significant regression in tumor size of ZR-75-1-DR xenografts compared to those of parental cells and control lentivirus after combined treatment with FCE and doxorubicin, but not with doxorubicin alone.

All in all, our data introduced FCE as a promising non-toxic herbal, which has fourteen bioactive compounds and therapeutic potential to reduced cancer stem cell properties and increased sensitivity to doxorubicin treatment, by directly targeting mutant p53, NOTCH4 and microRNAs.

#319

Role of GPR110 on tumorigenesis and metastasis in HER2+ breast cancer in the context of anti-HER2 drug resistance.

Debashish Sahay,1 Raksha Bhat,1 Motthafar Rimawi,2 C. Kent Osborne,2 Rachel Schiff,2 Meghana V. Trivedi1. 1 _Department of Pharmacy Practice and Translational Research, University of Houston College of Pharmacy, Houston, TX;_ 2 _Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX_.

Drug resistance is a major limitation of treatment regimens targeting the human epidermal growth factor receptor-2 (HER2) in breast cancers that overexpress this protein. Therefore, identification of novel drug targets to overcome anti-HER2 drug resistance is an unmet clinical need. Our previous studies have suggested that GPR110 may be a potential target for anti-HER2 drug resistance and tumorigenicity. Furthermore, GPR110 is an orphan receptor in the adhesion G-protein coupled receptor family, members of which have shown a role in cancer metastasis. In this study, our objectives were (1) to assess the gene expression of GPR110 using the Taqman RT-PCR assay in anti-HER2 drug resistant and in cancer stem-like cell population of various HER2+ breast cancer cell line models and (2) to investigate the effects of GPR110 gene knockdown on tumorigenesis and metastasis using various cell-based assays in HER2+ breast cancer cell lines and their anti-HER2-resistant derivatives. Cells transfected with non-targeting and 3 independent GPR110-targeting siRNAs were used to assess anchorage-independent cell growth, mammosphere formation, and cell invasion and migration. We found that GPR110 gene expression was significantly higher (>/= 2-fold) in lapatinib-resistant (LR), trastuzumab-resistant (TR), and lapatinib + trastuzumab-resistant (LTR) derivatives of BT474, SKBR3 and UACC812 cell lines compared to the parental cells. GPR110 gene expression was also significantly higher (>/= 1.9-fold) in ALDH+ versus ALDH- cell populations of BT474, SKBR3, MDA-MB-361 and HCC1569 cell lines. In addition, GPR110 gene knockdown led to a 55-85% (p<0.05) decrease in anchorage-independent cell growth in BT474 and SKBR3 LTR derivatives but not in parental cells. A significant reduction in mammosphere count upon GPR110 knockdown was also seen in LTR derivatives (50-70%, p<0.05) and not in parental BT474 cells. Interestingly, GPR110 knockdown in SKBR3 parental cells resulted in a significant reduction in cell invasion (40-50%, p<0.05) and migration (40-50%, p<0.05) suggesting a potential role of GPR110 in tumor cell dissemination. Overall, these results demonstrate that GPR110 is overexpressed in multiple anti-HER resistant derivatives as well as breast cancer stem-like cell population of various HER2+ breast cancer cell line models. Our studies also suggest a role of GPR110 in tumorigenesis specifically in the context of anti-HER2 drug resistance and in metastatic processes. Future in vivo studies will assess GPR110 as a novel drug target to overcome anti-HER2 drug resistance and combat metastasis in HER2+ breast cancer. 

### Novel Antitumor Agents

#320

Inhibition of adenosine A2A receptor (A2AR) by CPI-444 enhances CD8+ T cell killing of a HER-2/neu expressing murine tumor.

Blake A. Scott,1 Todd Armstrong,2 Elizabeth M. Jaffee2. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Johns Hopkins University School of Medicine Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD_.

Immunotherapies such as checkpoint inhibitors that block immune suppressive mechanisms are transforming the way some cancers are treated. The identification of additional mechanisms that suppress immune cell function is stimulating the search for other immune modulating agents that are additive or synergistic with current immune checkpoint inhibitors or are capable of overcoming mechanisms of resistance and tolerance. Extracellular adenosine and its signaling through A2AR increases the activity of regulatory T cells (Tregs), and attenuates tumor-specific CD4+/CD8+ T cells. In this study we evaluated the role of a small-molecule inhibitor of A2AR in enhancing antitumor immunity against murine Her-2/neu (neu)-expressing breast cancer. We examined whether an A2AR inhibitor would enhance the activity of cancer specific CD8+ T cells when given with a T cell inducing vaccine. To test this, neu-expressing mammary tumor bearing HER-2/neu transgenic (Neu-N) mice were treated with low dose cyclophosphamide (Cy) to deplete Tregs followed one day later with a granulocyte-macrophage colony-stimulating factor (GM-CSF) and neu-expressing whole cell vaccine (GVAX). Mice were treated with the A2AR inhibitor, CPI-444, by oral gavage daily for twelve days. Mice also received adoptively transferred high-avidity naïve neu-specific CD8+ T cells intravenously one day after vaccination. Mice were monitored for tumor progression, and tumor-infiltrating lymphocytes (TIL) were harvested from additional mice at multiple time points and analyzed for T cell activation by flow cytometry. Inflammatory changes in the tumor microenvironment were assessed by immunohistochemistry. We found that T cells adoptively transferred to mice together with Cy, a neu-targeted vaccine and CPI-444 are more effective at reducing established tumors and delaying tumor progression when compared with mice treated with Cy, vaccine, adoptively transferred T cells and vehicle control. Of particular note, mice receiving the same treatment but with CPI-444 in lieu of vehicle had a significant mean difference in tumor area of 6.446mm2 (P<0.05) when compared to the vaccine and vehicle control cohort. When compared to the tumor alone cohort, mice receiving the full therapeutic regimen had a significant mean difference in tumor area of 13.59mm2 (P<0.001). Mechanistic studies are underway to assess the activity of the adoptively transferred T cells in the TIL. These studies may provide the rationale to test an A2AR inhibitor in combination with adoptively transferred T cells and/or vaccines in patients with cancer.

#321

S49076, a novel kinase inhibitor of MET, AXL and FGFR, demonstrates marked in vitro and in vivo efficacy in hepatocellular carcinoma.

Annemilai Tijeras-Raballand,1 Adrien Pasteur-Rousseau,2 Matthieu Martinet,1 Philippe Bonnin,2 Marc Pocard,3 Sébastien Banquet,4 Valérie Cattan,4 Eric Raymond,5 Armand de Gramont6. 1 _AAREC filia Research, Boulogne-Billancourt, France;_ 2 _Inserm U965, Clinical Physiology-Functinnal Investigations, Lariboisière University Hospital, Paris, France;_ 3 _Inserm U965, Digestive and Cancer Surgery Department, Lariboisière University Hospital, Paris, France;_ 4 _Innovation Therapeutic Pole, Institut de Recherches Internationales Servier, Suresnes, France;_ 5 _Hopitaux Universitaires Paris-Nord Val de Seine, HUPNVS, Paris, France;_ 6 _AAREC Filia Research and Drugs Evaluation Laboratory, Department of Oncology, CHUV, Lausanne, Switzerland_.

Introduction: Hepatocellular carcinoma (HCC) is the second cause of cancer-related deaths worldwide. While sorafenib may improve overall survival in advanced HCC, new therapies are needed. Both preclinical and clinical data support a role of MET and AXL receptor tyrosine kinases in HCC progression. We thus evaluated S49076, a novel MET/AXL/FGFR inhibitor in HCC cell lines in vitro and in a transgenic mouse model of HCC.

Materials and Methods: MET and AXL expressions were assessed in a panel of HCC cell lines by western blot. Downstream signaling pathways were investigated in the presence of HGF, GAS6 and S49076. Cell invasion was evaluated on matrigel assay. Transgenic mice developing stage-defined HCC were treated with S49076 (50mg/kg). Tumor response was evaluated measuring liver volume by echo-doppler and the number of tumor nodules at intermediary (12weeks) and final sacrifice (16 weeks).

Results: Both MET and AXL are expressed in the majority of HCC lines screened. Notably, a high level of AXL was observed in the SK-HEP1 cells which were therefore selected for further investigation. MET and AXL pathways were strongly stimulated by their respective ligands, HGF and GAS6. Combination of HGF and GAS6 led to marked activation of ERK. S49076 inhibited MET and AXL pathway activation and HGF- and GAS6- induced invasion at nanomolar concentrations. In vivo anti-tumor efficacy of S49076 was demonstrated in a transgenic MET-expressing mouse model of HCC.

Conclusion: S49076 displayed strong MET and AXL pathway inhibition and anti-invasive properties in SK-HEP1 HCC cells. Moreover, S49076 demonstrated antitumor activity in a mouse model of HCC. Together, these results would support development of S49076 as an innovative treatment in HCC patients.

#322

Selective inhibition of MYC expression by a small molecule G-quadruplex stabilizer.

Elena C. Leon,1 John K. Simmons,1 David Calabrese,2 Wendy DuBois,1 Kenneth Felsenstein,1 Lindsey B. Saunders,2 Shuling Zhang,1 Aleksandra Michalowski,1 Frank Gonzalez,1 Beverly A. Mock,1 John S. Schneekloth2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Cancer Institute, Frederick, MD_.

MYC overexpression or amplification is commonly seen in a wide variety of tumor types, including multiple myeloma. Although MYC appears to be an important driver of the oncogenic phenotype, the helix-loop-helix topology, short half-life, and rapid replenishment of its encoded protein make it a particularly challenging drug target. However, the promoter region of the MYC gene contains a G-quadruplex (G4) DNA secondary structure that can be stabilized to block transcription. Recently, we reported the identification of a compound from a small molecule microarray screen that selectively binds the MYC G4, compared with other oncogenes that also contain G4s, and inhibits MYC mRNA and protein expression, leading to cell cycle arrest in human multiple myeloma cell lines (ACS Chem Biol. 2015). Here we describe the in vitro and in vivo evaluation of a more potent analog that retains the selectivity of the original compound for the MYC G4 sequence. After observing strong growth inhibitory activity of this new compound in a diverse panel of human cancer cell lines, we evaluated its pharmacokinetic and toxicity profiles in mice. Preliminary pharmacokinetic studies showed promising serum bioavailability and exposure properties when administered either intravenously or intraperitoneally. The compound was well tolerated in a month long, dose-escalation toxicity study in mice receiving daily administration of the compound. No adverse effects were observed during the study. In an assessment of short term in vivo activity, MYC expression was inhibited in multiple myeloma xenografts. More long-term studies to evaluate the anti-tumor activity of the compound are currently in progress. Thus, our data provide evidence that small molecule stabilization of the MYC G4 can drive transcriptional silencing of oncogenic MYC expression both in vitro and in vivo.

#323

SKI-178, a single agent co-targeting sphingosine kinase 1 and microtubule dynamics, as a therapeutic strategy for treatment of acute myeloid leukemia.

Taryn E. Dick,1 Jeremy A. Hengst,1 Laura M. Geffert,1 Robert F. Paulson,2 Hong-Gang Wang,1 David F. Claxton,1 Mark Kester,3 Thomas P. Loughran,3 Jong K. Yun1. 1 _Penn State University College of Medicine, Hershey, PA;_ 2 _Penn State University, University Park, State College, PA;_ 3 _University of Virginia, Charlottesville, VA_.

Acute myeloid leukemia (AML) is a heterogeneous and rapidly progressing blood cell cancer caused by numerous cytogenetic alterations. Although significant improvement in treatment of AML has been made, the unfortunate reality is that currently available treatments are largely ineffective for most AML patients. Thus, there is a critical need for new therapeutic targets and agents for the treatment of AML.

The sphingolipid metabolic pathway is an untapped source of new therapeutic targets for the treatment of AML. Sphingosine Kinase 1 (SphK1) plays a central role in the sphingolipid metabolic pathway as the key enzyme regulating the intracellular equilibrium between pro-apoptotic Ceramide (Cer) and pro-mitogenic/pro-survival Sphingosine-1-phosphate (S1P), a.k.a. the "Sphingolipid Rheostat". As SphK1 activity increases in the cell, pro-mitogenic/pro-survival S1P signaling predominates and AML cells become dependent upon S1P signaling (non-oncogene addiction), while depletion of Cer levels makes them more resistant to chemotherapies.

We previously developed a novel SphK1-selective inhibitor (SKI-178) that is potently cytotoxic to multiple AML cell lines including multi-drug resistant lines. Our recent, thorough examination of the apoptotic mechanism-of-action of SKI-178, including direct target engagement assays employing the Cellular Thermal Shift Assay (CETSA) revealed that SKI-178 also acts as a colchicine binding site directed microtubule disrupting agent (MDA). Numerous studies have demonstrated that agents that promote Cer accumulation, including SphK inhibitors, synergistically induce apoptosis, in combination with MDAs by the simultaneous activation of pro-apoptotic Bcl-2 family proteins and inhibition of anti-apoptotic Bcl-2 family proteins, respectively. SKI-178 uniquely accomplishes these two separate cellular effects as a single agent.

We examined the therapeutic efficacy of SKI-178 in three mouse models of AML. Using a retro-viral transduction model of the MLL/AF9 t(9;11)(p22;q23) translocation, we have shown that SphK1 is necessary for the development of MLL/AF9-driven AML and that SKI-178 effectively induces complete remission of AML in this model system. Separately, in a human AML cell line (MOLM-13; MLL/AF9+, FLT3-ITD) xenograft model, SKI-178 significantly extended survival relative to vehicle controls. Lastly, employing a Patient Derived Xenograft model of a human primary AML sample (FLT3-ITD, NPM1+), SKI-178 also significantly extended survival relative to vehicle treated cohorts. In all 3 models, SKI-178 was well tolerated and did not affect normal hematopoiesis. Together, these data demonstrate the therapeutic efficacy of a strategy co-targeting SphK1 inhibition and microtubule dynamics and suggest that SKI-178 should be further developed as a novel therapeutic agent for AML.

#324

Targeting RNA-polymerase I using CX-5461 as a mechanism for treating chemotherapy resistant epithelial ovarian cancer.

Robert Cornelison,1 Zachary C. Dobbin,2 Ashwini A. Katre,2 Dae Hoon Jeong,2 Yulia Petrova,1 Danielle C. Llaneza,1 Adam D. Steg,2 Yinfeng Zhang,2 David A. Schneider,2 Charles N. Landen1. 1 _University of Virginia School of Medicine, Charlottesville, VA;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

BACKGROUND: One of the first identified hallmarks of neoplastic transformation was an increased rate of ribosome biogenesis, and increased activity of RNA polymerase I (RNA pol I) is observed in most human cancers. Ribosome biogenesis is an energy intensive and tightly regulated process, and recent studies have shown that CX-5461, a potent and specific RNA pol I inhibitor, has selective toxicity to cancer cells. Here we demonstrate that CX-5461 is an effective anti-proliferative agent in epithelial ovarian carcinoma cells with an increased efficacy in chemoresistant ovarian cancer cells compared to their sensitive parental lines.

METHODS AND RESULTS: Ovarian cancer cells were demonstrated to be highly sensitive to RNA pol I inhibition by MTT assay, with an IC50 range from 25nM to 2uM in 13 different ovarian cancer cell lines, and an IC50 of 3uM in the nontransformed immortalized normal surface epithelial line HIO-180. Notably, chemoresistant cell populations were generally more sensitive to CX-5461, compared to matched chemosensitive lines. IC50 in HeyA8MDR was 80nM compared to 2000nM in parental HeyA8, and was 100nM in SKOV3TRip2 compared to 400nM in SKOV3ip1. CX-5461 induces G2/M arrest (averaging 52.7% in G2 compared to 32.5% in controls), primarily through mitotic catastrophe, inducing an accumulation of multinucleated senescent cells. DNA damage pathways are activated, operating through ATM/ATR, as demonstrated by rescue with caffeine and ATR specific inhibitors. In an effort to identify why chemoresistant cells might be more susceptible, ribosomal synthesis processes were examined. Chemoresistant cells were found to have 2-4-fold increased Pol I occupancy on rDNA by ChIP, and increased rRNA synthesis by isotopic incorporation, while ribosome abundance and overall translation efficiency, measured by sucrose gradient fractionation, were unchanged. Treatment of five separate patient-derived xenograft models (PDX) with CX-5461 showed a complete response in one model, partial 55% reduction in tumor size in a second, stable disease in a 3rd, and progression in two models.

CONCLUSIONS: CX-5461 shows high activity in ovarian cancer cell lines and patient-derived xenograft models, with a preferential effect on chemoresistant cells. Enhanced sensitivity to the compound is due at least in part to cancer cells' increased demand for rRNA synthesis. RNA pol I inhibitors represent an exciting new treatment option that may have preferential effect on the chemoresistant population.

#325

Evaluating the consequences of MCT1 inhibition in Burkitt lymphoma following treatment with AZD3965.

Richard A. Noble,1 Natalie Bell,1 Helen Blair,1 Huw D. Thomas,1 Simon Bomken,1 Susan E. Critchlow,2 Stephen R. Wedge1. 1 _Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom;_ 2 _Oncology Innovative Medicines Unit, AstraZeneca, Cambridge, United Kingdom_.

Many tumors display an altered metabolic phenotype with an increased reliance on glycolysis resulting in a greater production of the waste product, lactate [1]. Use of glycolysis, as opposed to oxidative phosphorylation, represents a less efficient means of ATP generation but provides a selective advantage to cancer cells in that it rapidly supplies intermediates to support anabolic pathways necessary for increased cellular proliferation. Lactate efflux, is facilitated by monocarboxylate transporters 1-4 and is essential to prevent feedback inhibition of major energy generating pathways. A sub-group of cancers express only MCT1 and are therefore exclusively reliant on this transporter to export lactate. Elevated MCT1 expression has previously been identified in Burkitt lymphoma (BL), a highly aggressive form of non-Hodgkin's lymphoma characterised by MYC translocations [2].

We have studied the effects of a potent, selective and orally available inhibitor of MCT1, AZD3965, in models of BL that predominantly express MCT1. MCT1 inhibition was associated with intracellular lactate accumulation in vitro and a significant growth inhibitory effect (AZD3965 72h GI50 <10 nM). MCT1 inhibition was also associated with a number of changes in intracellular metabolites, including increases in TCA cycle and early glycolytic intermediates, indicating potential feedback mechanisms following lactate accumulation.

A measurable increase in lactate accumulation was also detectable in vivo following acute treatment with AZD3965 (100 mg/kg p.o.) using NOD/LtSz-scid IL-2Rγ -/- (NSG) mice bearing subcutaneous CA46 human xenografts. The efficacy of AZD3965 was subsequently investigated in vivo using a more clinically representative form of this BL xenograft model. Here, CA46 cells were engineered to express firefly luciferase (SLIEW lentivector) and then inoculated intravenously into NSG mice. Tumor engraftment was confirmed 6 days after inoculation by the detection of luciferase-based bioluminescence using the IVIS® Spectrum. Oral treatment with either AZD3965 (100mg/kg) or vehicle (both administered twice-daily Monday to Friday and once-daily at weekends) was then initiated for a period of 24 days. Bioluminescence was measured 3 days after the start of treatment and at weekly intervals to determine total tumor burden. Tumor growth relative to control xenografts was inhibited significantly by AZD3965 treatment (>99% P<0.005 Student's two-tailed t-test). In support of this efficacy, post-mortem macroscopic measurements revealed greatly reduced tumor cell engraftment of bone marrow and spleen in MCT1 inhibitor treated animals.

AZD3965 has potent growth inhibitory activity in both in vitro and in vivo models of Burkitt lymphoma that are reliant upon MCT1 for lactate transport.

[1] Science (1956) 123, 309-314

[2] Cancer Res (2014) 74, 908-920

#326

Discovery and in vitro and in vivo characterization of a novel, small-molecule Ras inhibitor class.

Adam B. Keeton,1 Bing Zhu,1 Kevin J. Lee,1 Joshua C. Canzoneri,2 Sara C. Sigler,1 Ashley S. Lindsey,1 Veronica Ramirez-Alcantara,1 Luciana Barnes,1 Tyler E. Mattox,1 Kate McConnell,1 Kristy L. Berry,1 Jacob Valiyaveettil,1 Xi Chen,1 Michael R. Boyd,2 Gary A. Piazza1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _ADT Pharmaceuticals, Inc., Orange Beach, AL_.

Introduction: Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Through iterative screening and chemical optimization using a phenotypic assay designed to select for Ras inhibitors, we identified a novel series of compounds that potently and selectively inhibit the growth of tumor cells harboring activated Ras relative to cells lacking activated Ras. Several compounds in the series have favorable drug-like properties with strong antitumor activity in a mouse K-Ras mutant tumor model.

Methods: Viable cell number was measured using a luminescent indicator of ATP. Disruption of Ras-Raf binding was determined by pre-incubating GST-Raf beads with cell lysates in the presence of test compounds for 30 min. Ras activation was measured by Ras pull-down and western blotting using an anti-Ras antibody. Cell cycle arrest was measured by DNA content. Antitumor activity was determined in a subcutaneous mouse tumor model involving K-Ras mutant HCT116 colon tumors. Mice were treated with Ras inhibitors administered ip for 14 days bid at a dose of 2.5 or 5 mg/kg.

Results: Low nanomolar concentrations of DC070-547 inhibited the growth of multiple tumor cell lines harboring activated K-Ras, N-Ras or H-Ras with a selectivity index greater than 100-fold over cells lacking activated Ras. Ras selectivity was confirmed by transfecting human HT29 colon and H322 lung tumor cells that lack activated Ras with mutant H-Ras, thus inducing sensitivity to DC070-547. The compound also inhibited Ras-Raf binding as evident by Ras-pull down assays of lysates from tumor cells treated with DC070-0547 at concentrations that inhibit tumor cell growth. DC070-547 caused G2 phase cell cycle arrest and induced apoptosis selectively in tumor cells containing activated Ras. Cultured epithelial cells derived from normal colon, mammary, and lung tissues were essentially refractory to treatment.The Ras inhibitors were evaluated for antitumor activity in a subcutaneous mouse model involving K-Ras mutant human HCT116 colon tumors. Treatments were well tolerated and completely suppressed tumor growth with the effect being sustained for at least six weeks after treatment was discontinued. Complete tumor regression was also apparent in some of the treated mice.

Conclusion: While Ras is widely considered to be non-druggable, we have identified a novel series of compounds that potently and selectivity inhibit the growth of tumor cells harboring activated Ras. With promising drug-like properties, these compounds represent a first in class series of Ras inhibitors from which several prospective drug development candidates have been identified.. These results support further preclinical development and future Phase I/II clinical evaluation in patients with Ras-driven cancers.

#327

Anti-tumor effect and destabilization of R175H-mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy.

Liz J. Hernandez Borrero, Shengliang Zhang, David Dicker, Wafik El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

Tumor suppressor p53 is a master regulator of genotoxic and cellular stress signals, controlling cell fate by transcriptionally activating genes involved in DNA repair, cell cycle arrest, and apoptosis. p53 is mutated in over half of human cancers and this is associated with tumor development and chemotherapy resistance. TP53 gene mutations can result in the abolishment of p53 contact with DNA and disruption of p53 structural conformation. These mutations not only prevent p53 to exert its normal tumor suppressive functions but can result in gain-of-function activity, acquiring oncogenic characteristics. Therefore, altering the stability of mutant p53 protein is an attractive therapeutic strategy in cancer cells. We investigated small molecules that modulate mutant p53 stability and restore the p53-signaling pathway. We identified a small molecule, CB002, as a candidate for restoration of the p53 pathway in mutant p53-bearing cancer cells. Three colorectal cancer cell lines: SW480, DLD-1, HCT116-R175H and the RXF393 renal cancer cell line, were treated with different concentrations of CB002 at various time points. Cell lines exposed to CB002 showed an increase in apoptotic and cell death markers, such as NOXA/DR5 induction, cleaved caspases and PARP, as early as 16 hrs. CB002 decreased mutant p53 stability in structural conformation-mutant bearing cells (HCT116 R175H and RXF393). Our data suggests that CB002 stimulates an increase of p53 target genes and promotes expression of pro-apoptotic proteins. R175H p53 mutant protein expression was largely rescued by the co-treatment with MG132, a proteasomal inhibitor, implicating a role for the ubiquitin proteasome system. Furthermore, we observed significant autophagy induction upon CB002 treatment, as indicated by LC3 conversion and p62 levels. Autophagy inhibition decreased levels of cell death markers and increased the stability of the R175H-mutant p53, suggesting that autophagy might be required for CB002-induced cell death and mutant p53 turnover. Therefore, we hypothesize that CB002 is capable of degrading mutant p53 and restoring the p53 pathway through p53 family members in colorectal cancer cells. Currently we are investigating p53 degradation mechanism by CB002, the role of p53 family members in the restoration of the pathway and autophagy on mutant p53 stability. Hence, our results provide an insight on effective p53 pathway activation through the use of small molecules.

#328

Antitumor activity of the oncolytic peptide LTX-315 in syngeneic tumor models.

Ketil André Camilio,1 Baldur Sveinbjørnsson,2 Sylvie Maubant,3 Guillaume Serin,3 Jean-François Mirjolet,3 Francis Bichat,3 Øystein Rekdal2. 1 _Institute of Medical Biology, Tromso, Norway;_ 2 _Lytix Biopharma AS, Oslo, Norway;_ 3 _Oncodesign S.A., Dijon Cedex, France_.

Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in a number of species as a defense mechanism for eukaryotic cells against pathogens and are an integral part of the innate immune system. CAPs have a broad spectrum of activities including antimicrobial and anticancer while being less cytotoxic toward non-malignant cells. The potential therapeutic application against cancer has spawned an interest in developing oncolytic agents that display a new mode of action to overcome the potential drug resistance associated with other current therapeutics. The anticancer effects of CAPs are still under investigation, but several peptides have already exhibited a promising potential with cytotoxic activities against a broad spectrum of tumor cells. Oncolytic peptides exert their activity through either a membranolytic mode of action or an interaction with intracellular targets, or a combination of both.

LTX-315 (K-K-W-W-K-K-W-Dip-K-NH2), a novel oncolytic peptide developed by Lytix Biopharma AS has the potential to adopt an amphipathic helical coil structure. In vitro studies have demonstrated that it was highly effective against both drug-resistant and drug-sensitive cancer cells from several organ origins, with lower toxicity toward normal cells. LTX-315 was designed for intratumoral treatment of transdermal lesions. Previously, LTX-315 has been shown to induce complete regression of B16 melanomas and long lasting antitumor immune responses. Histological analyses of treated tumors revealed extensive hemorrhagic necrosis and infiltration of CD3+ T cells. Moreover, mRNA levels of inflammatory cytokines such as IL1β, IL6 and IL18 were found to be increased in the tumor tissue after LTX-315 treatment. The treatment did also prevent lung metastasis in mice re-challenged with B16F1 cells intravenously. Due to its oncolytic mode of action, LTX-315 induces immunogenic cell death through the release of danger-associated molecular pattern molecules and tumor antigens.

Recently, we have demonstrated that when subcutaneously established EMT-6 tumors (inoculated into both flanks of the animal) were treated intratumorally with LTX-315, an antitumor response was observed with a T/C ratio of 17% 19 days post start of treatment. Furthermore, an abscopal effect of LTX-315 on the untreated tumor was also reported but only when it was combined with anti-PD-L1 antibody. At the end of study, 50% of mice that had received the combination therapy were still alive vs 30% and 40% in the groups treated with LTX-315 or anti-PD-L1 antibody alone, respectively. In conclusion, LTX-315 seems to be an ideal combinations partner for immune checkpoint inhibitors.

#329

Reprogramming glucose metabolism and energy production with a small molecule HJC0152 suppresses breast cancer development and progression to metastasis.

Hao Zou,1 Na Ye,2 Hui Pang,1 Dan Zhang,1 Ruping Yan,1 Haijun Chen,2 Guoshuai Cai,1 Lili Wang,1 Zhengduo Yang,1 Haiying Chen,2 Grace Xu,1 Yingchao Zhang,1 Ritu Arora,3 Ming Tan,3 Yongchang Wei,1 Jia Zhou,2 Qiang Shen1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of Texas Medical Branch, Galveston, TX;_ 3 _University of South Alabama Mitchell Cancer Institute, Mobile, AL_.

Currently there are no targeted therapeutic strategies for estrogen receptor (ER)-negative breast cancer (ENBC), which constitutes 30-40% of breast cancer cases and is prone to metastasize and recur. Distant metastasis accounts for 90% of cancer-associated deaths. The majority of deaths from breast cancer are caused by distant metastasis developed in lung, liver, bone, or brain. However, to date it remains a challenge and unmet need to treat existing metastasis and block new metastasis in cancer patients. Dysregulated glucose and energy metabolism is critically involved in the development and progression of various cancers via promoting aberrant cell growth, malignant transformation and metastasis, but the potential role of glucose/energy metabolism in ENBC progression and metastasis has scarcely been explored heretofore, thus representing a key knowledge gap and a potential avenue for anticancer targeting. A number of anticancer metabolic and biogenetic therapies have been developed, yet none of them has progressed to clinical use, due to their limited potency, specificity or drug properties such as toxicity and poor bioavailability. We recently identified a novel small molecule HJC0152 that significantly suppresses ENBC xenograft tumor growth and blocks ER-negative mammary tumor development in mouse models. HJC0152 treatment for 24-72 hours differentially modulates protein expression of HK1, PFK-L, PFKFB2, ENO2, PDH, PDK1, PGAM1 and ALDOA in a time-dependent manner. HJC0152 also regulates the transcription of genes involved in glucose and mitochondrial energy metabolism, including the subunits of mitochondrial respiratory chain complexes. Functional assessments of mitochondrial complexes demonstrate that HJC0152 significantly inhibits Complexes IV but increases Complex V (ATP synthase) function, while Complexes I and II function is minimally affected. Migration and invasion of MDA-MB-231 cells are significantly inhibited by HJC0152 treatment. In vivo, HJC0152 administrated either before, concurrent with or after tail vein injection of MDA-MB-231 cells dramatically blocks the development of lung macro- and micro-metastasis in all groups. These results suggest that HJC0152 can specifically reprogram/restore the dysregulated glucose metabolism by inducing specific glycolytic enzyme expression and mitochondrial respiratory chain function, likely via targeting one or more upstream signal molecule(s) that regulates glucose and energy metabolism, thereby suppressing breast cancer progression to metastasis. This work was supported by Grants P50 CA097007, P30DA028821, and R21MH093844 (J.Z.) from the NIH, CPRIT (J.Z.), John Sealy Memorial Endowment Fund (J.Z.), DFI Seed Grants from MD Anderson Cancer Center (Q.S.), and Holden Family Research Grant in Breast Cancer Prevention from the Prevent Cancer Foundation (Q.S).

#330

Development of a highly selective B/CRAF kinase inhibitor that exhibits antitumor activities in RAS and BRAF mutant tumors with minimal paradoxical activation.

Wenlin Shao,1 Yuji Mishina,1 Yun Feng,1 Giordano Caponigro,1 Savithri Ramurthy,2 Vesselina Cooke,1 Lesley Griner,1 Gisele Nishiguchi,2 Alice Rico,3 Ben Taft,3 Matthew Burger,2 Huw Tanner,3 Valery Polyakov,3 Brent Appleton,3 John Tellew,4 Richard Zang,3 Mohammad Hekmat-Nejad,5 Payman Amiri,6 Mallika Singh,6 Darrin Stuart1. 1 _Oncology, Novartis Institutes for BioMedical Research, Cambridge, MA;_ 2 _Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Cambridge, MA;_ 3 _Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, CA;_ 4 _Genomics Institute of the Novartis Research Foundation, San Diego, CA;_ 5 _Infectious Diseases, Novartis Institutes for BioMedical Research, Emeryville, CA;_ 6 _Oncology, Novartis Institutes for BioMedical Research, Emeryville, CA_.

The mitogen-activated protein kinase (MAPK) signaling pathway is frequently activated in human cancers due to genetic alterations that can occur at multiple nodes in the pathway, the most prevalent of which are mutations in RAS or BRAF. While BRAFV600 mutant tumors are effectively treated with existing RAF inhibitors, RAS mutant cancers and tumors expressing atypical BRAF mutants remain an unmet medical need. Emerging biology has demonstrated that the CRAF kinase functions as a critical mediator of mutant KRAS-driven cell proliferation and tumor development. CRAF was also shown to be the mediator of feedback-mediated pathway reactivation following MEK inhibitor treatment in KRAS mutant cancers. Hence selective inhibitors that potently inhibit the activity of CRAF could be both effective in blocking mutant RAS-driven tumorigenesis and in alleviating feedback activation. We have developed a type II ATP-competitive inhibitor that inhibits both B- and CRAF kinase activities at picomolar IC50 values in biochemical assays with high selectivity profile against a panel of 456 human kinases. The inhibitor not only inhibits MAPK signaling activity in tumor models harboring BRAFV600 mutation, but also inhibits mutant N- and KRAS-driven signaling with minimum paradoxical activation, likely due to its activity in inhibiting both RAF monomers and dimers with similar potencies. Correspondingly, profiling data of the inhibitor in a panel of 480 human cancer cell lines shows that it has higher antitumor activities in cell lines harboring BRAF or RAS mutations as compared to those that are wild-type. The inhibitor is orally bioavailable, it demonstrates a direct PK/PD relationship and causes tumor regression in multiple cell line and primary human tumor derived xenograft models that have BRAF, NRAS or KRAS mutations with good tolerability. Thus, we have developed a next generation RAF inhibitor with unique biochemical and cellular properties that enables its antitumor activities in RAS mutant tumors.

#331

Beta-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG.

Kevin J. Lee,1 Ashley S. Lindsey,1 Luciana Madeira da Silva,1 Alisa Trinh,1 Bernard Gary,1 Joel Andrews,1 Veronica Ramirez-Alcantara,1 Adam B. Keeton,1 Wen-Chi Chang,2 Margie Clapper,2 Gary A. Piazza1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Phosphodiesterase 10A (PDE10A) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington's disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibited colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activate PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduced the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibited the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, which resulted in caspase activation and apoptosis. This was determined to be through a PKG mediated pathway through the use of small molecule inhibitors of PKG and PKA. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues in a cDNA array. We also report the increase in PDE10 mRNA in 50% of a small collection of human clinical specimens collected at the Mitchell Cancer Institute (n = 13). In addition, novel PDE10 inhibitor, MCI-030, reduced tumor size and activated PDE10 signaling mechanisms in vivo. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells.

#332

Spliceosome inhibition as novel strategy against diffuse malignant peritoneal mesothelioma.

Rocco Sciarrillo,1 Valentina E. Gomez,1 Marzia Pennati,2 Anna Wojtuszkiewicz,1 Gert-Jan L. Kaspers,1 Carla Molthoff,1 Nadia Zaffaroni,2 Godefridus J. Peters,1 Gerrit Jansen,1 Elisa Giovannetti1. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy_.

Background: Diffuse malignant peritoneal mesothelioma (DMPM) is an aggressive tumor of the lining of the abdomen, characterized by late clinical symptoms and poor prognosis. Systemic chemotherapy together with cytoreductive surgery and intraperitoneal hyperthermic therapy has been introduced as the best treatment option, resulting in an overall 5-year survival rate of approximately 50%. To further increase survival, novel drugs targeting key molecular factors in DMPM are warranted. Analogous to pleural mesothelioma, such a factor may be alternative splicing which can be modulated by inhibiting the SF3B subunit of the spliceosome. This strategy is an emerging therapeutic opportunity for a number of solid tumors and hematological malignancies. In particular, the spliceosome inhibitor Pladienolide B (PB) has low nanomolar IC50 values against a range of cancer cell lines and leukemic cells, but no data are available regarding its antitumor efficacy in DMPM.

Aims: This study investigates (1) the activity of PB (alone or in combination with standard chemotherapeutics) in primary peritoneal mesothelioma cells through in vitro and in vivo assays, and (2) the molecular mechanisms of splicing inhibition through whole-genome RNA-seq and PCR of selected genes involved in apoptosis and invasion.

Methods: The antiproliferative effect of PB was investigated using the SRB assay on two primary mesothelioma cell cultures (MESOII and STO), obtained from resected tumors with well-annotated clinical characteristics. Further in vitro studies were performed to evaluate the pro-apoptotic and anti-invasive activities, while splicing profiles of treated and untreated cells were determined with RNA-seq.

Results: PB impaired DMPM cell growth in a dose-dependent manner, with IC50 values of 1.57 ± 0.30 nM in MESOII and 1.18 ± 0.16 nM in STO (n=3, mean ± standard deviation).

The specific activity on the spliceosome was demonstrated by PB induced time- and dose-dependent alterations of splicing patterns for several apoptotic genes, such as Mcl-1, Bcl-X, Fas, and for the pro-metastatic tyrosine kinase receptor RON, which was shifted to its un-spliced and non-functional variant. In addition, RNA-seq showed several differentially expressed alternatively spliced genes in PB treated samples. The DMPM cells have also been genetically engineered to express Firefly- and Gaussia- luciferases, enabling monitoring of tumor growth inhibition by PB in in vivo orthotopic models.

Conclusions: These data provide evidence that PB has a strong antitumor activity against relevant models of DPMP, associated with modulation of splicing, induction of apoptosis and inhibition of invasion. RNA-seq represents a powerful tool for the identification of alternatively spliced genes that could serve as useful diagnostic markers as well as potential therapeutic targets for DMPM.

#333

Development and validation of a LC-MS/MS method for the quantification of the tropomyosin receptor kinase (Trk) inhibitor pegcantratinib in human skin tumors.

Monique Zangarini, Neil Rajan, Marina Danilenko, Philip Berry, Gareth J. Veal. _Newcastle University, Newcastle Upon Tyne, United Kingdom_.

Background: Pegcantratinib is the first topically applied tropomyosin receptor kinase inhibitor allowing retention in the skin to exert its effects at nanomolar concentrations.

Pegcantratinib is now under early clinical evaluation as an anti-tumour agent in patients with germline mutations in the tumour suppressor gene CYLD, which include multiple clinical presentations of rare, skin appendage tumours. No pharmacological treatment is currently available and patients face repeated and disfiguring surgery to control tumour burden. We report on the development and validation of an analytical method to quantify pegcantratinib in skin tumour biopsies of patients to determine tumour penetration.

Methods: A sensitive and specific HPLC-MS/MS (API 4000) assay coupled with in-source CID was developed and validated according to EMA guidelines, for direct quantitative measurement of pegcantratinib in biopsies of human skin tumours. The sample preparation procedure required a 10 mg sample volume and involved protein precipitation with acetonitrile following tissue homogenization and addition of internal standard. Reversed-phase chromatography under gradient conditions (MP A - 1% formic acid; MP B - 1% formic acid in acetonitrile) was applied with separation on a Kinetex 2.6 μm C18 column (100 Å, 50 x 4.6 mm). Detection was obtained by SRM, following the transitions m/z 419.0 → 376.4. The method is rapid and selective, allowing good resolution of peaks in 2.4 min.

Results: The assay was fully validated and the method exhibited good sensitivity, precision, and accuracy, with overall precision expressed as CV ≤8.5% and accuracy in the range 96-99%. Recovery was high (≥85 %) and consistent with CV ≤13%. No matrix effect was observed in four independent matrix sources including 3 different tumours and normal skin, the calculated CV was 5%. The limit of quantitation was 2.5 ng/ml, with precision and accuracy of 6% and 99%, respectively. The assay developed was linear in the range 1.0-500 ng/ml, with mean precision <8% and accuracy within the range 96-105%. Inter/intra-day precision and accuracy were always <8.5% and within the range 95-99%, respectively. Pegcantratinib is stable in tumour homogenate for at least 4 hours at room temperature, for 10 days at 4°C, before and after extraction, and over 3 freeze-thaw cycles. Preliminary data of an ex-vivo incubation of skin tumours biopsies, from two patients, at 5μM pegcantratinib for 1, 3 and 6h, showed that drug achieved concentrations of 1.5-2.5 μM in the tumours analysed.

Conclusion: We have successfully developed a LC-MS/MS method to measure the anti-tumour agent pegcantratinib in human skin tumour samples. Analysis of samples obtained from the Phase 1b/2a clinical trial is now underway.

Acknowledgements: Work supported by the Department of Health and Wellcome Trust through the Health Innovation Challenge Fund and Cancer Research UK.

#334

Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer.

Jacob Kach, Phillip Selman, Diana C. West, Donald J. Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. _University of Chicago, Chicago, IL_.

(A) Introduction: Because of the central role of androgen receptor (AR) signaling in prostate cancer (PC), AR inhibition is the primary modality of initial PC treatment. While AR-targeted therapies are often initially effective in castration-resistant prostate cancer (CRPC), disease progression is common. AR and glucocorticoid receptor (GR) are structurally similar nuclear receptors, bind the same DNA response elements, and regulate common genes. Previous work from our lab demonstrated that GR expression is upregulated following AR antagonism, GR expression decreased the effectiveness of AR blockade, and inhibiting GR activity reversed these effects. However, the only FDA-approved GR antagonist, mifepristone, is not GR-specific and has significant drug-drug interactions; therefore, we report our initial data with a new generation of selective GR-antagonists (SGRMs) for PC treatment using models of CRPC.

(B) Experimental Procedures: The LAPC4, CWR-22Rv1 and VCAP PC cell lines were utilized. GR target gene expression analysis was performed at various time-points by qRT-PCR and protein immunoblots subsequent to treatment with AR agonist (R1881, 1nM), AR antagonist (enzalutamide, 10 μM), GR agonist (dexamethasone, 100nM) and the new SGRMs CORT108297 and CORT118335(1μM). Quantification of cell numbers in vitro in response to these treatments was determined by trypan blue exclusion/live cell counting and the CellTiter Glo 2.0 ATP-luciferase assay. For in vivo studies, PC cells were injected subcutaneously into male nude mice and grown to a predetermined tumor size, mice were then castrated, and fifteen days later treated with mifepristone (12mg/kg), SGRM (16mg/kg, 20mg/kg), or vehicle by daily intra-peritoneal injection to assess time to CRPC.

(C) Results: AR antagonism with concurrent GR activation resulted in increased expression of canonical AR/GR-target genes and SGRMs (1μM) effectively inhibited expression of glucocorticoid-mediated genes at the RNA and protein levels. SGRMs did not affect AR-mediated gene expression. When AR was antagonized, activated GR promoted cell survival, which was inhibited by SGRM treatment to varying degrees. Preliminary studies support the hypothesis that these novel SGRMs delay CRPC tumor xenograft progression in vivo.

(D) Conclusion: In vitro, new generation SGRMs inhibit GR-mediated, but not AR-selective gene expression and result in decreased tumor cell number. SGRM administration to nude mice bearing PC xenografts can delay castration-resistant tumor growth. Therefore, new SGRMs may be an effective therapy for CRPC treatment worthy of further exploration. Studies are ongoing to determine how SGRMs modulate GR target gene expression globally and whether they decrease expression of tumorigenic pro-survival genes in vivo.

#335

Title: IACS-010759 is a novel clinical candidate that targets AML cells by inducing a metabolic catastrophe through inhibition of oxidative phosphorylation.

Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Jing Han, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Gera, Jay Theroff, Quanyun Xu, Juliana Velez, Carlo Toniatti, Timothy Heffernan, Giulio Draetta, M. Emilia Di Francesco, Philip Jones, Joseph R. Marszalek. _UT MD Anderson Cancer Center, Houston, TX_.

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production leading to robust cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. In an orthotopic xenograft model of primary AML cells derived from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 treatment with 3 and 10 mg/kg orally using a 5 on/2 off schedule extended the median survival by greater than 2-fold. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling and flux analysis revealed that the response to IACS-10759 was associated with induction of a metabolic imbalance that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, and we expect to file an IND at the end of 1Q2016 and initiate a Phase I clinical trial in AML during the second quarter of 2016.

#336

S49076, a kinase inhibitor of AXL, MET and FGFR with strong, selective preclinical activity against tumor cells with acquired resistance to EGFR inhibitors not carrying the T790M mutation.

Jordi Bertran-Alamillo,1 Miguel A. Molina,1 Cattan Valérie,2 Mike Burbridge,2 Cristina Teixido,1 Jordi Codony-Servat,1 Carles Codony-Servat,1 Rafael Rosell3. 1 _Laboratory of Oncology, Pangaea Biotech, Barcelona, Spain;_ 2 _Institut de Recherches Internationales Servier, Suresnes, France;_ 3 _Institut Catala d'Oncologia, Badalona, Spain_.

Background: Aberrant activity of MET, FGFR1 and AXL has been associated with development of resistance to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC) patients. S49076 is a potent ATP-competitive tyrosine kinase inhibitor (TKI) of MET, AXL/MER, FGFR1/2/3 currently in phase I clinical trials.

Methods: We obtained six resistant lines by treating EGFR-mutated, TKI-sensitive PC9 cells with increasing concentrations of gefitinib (GR1-5) or erlotinib (ER). The six resistant cell lines conserved the exon 19 deletion but the T790M resistance mutation only emerged in two (GR1, GR4), which remained sensitive to AZD9291, a third generation EGFR TKI. Six new AZD9291-resistant cell lines were derived from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. Three of the AZD9291 resistant cell lines lost the exon 19 and T790M mutations, one conserved only the sensitizing mutation and two kept both mutations but with the T790M dropping to very low allelic fractions (1% and 0.03%). All resistant cell lines were characterized for AXL, MET, MER, FGFR1 and FGFR2 expression by Q-RT-PCR, immunohistochemistry and Western blotting. The effects of S49076 on the parental and resistant cell lines were analyzed by MTT, colony formation, basal membrane invasion and migration assays. Western blotting of key signal transduction proteins was used to gain insight in the drug's mechanism of action.

Results: AXL overexpression was the most common event related to gefitinib/erlotinib resistance in the panel of cell lines, with T790M mutation, FGFR1 or BCL-2 overexpression and MET activation being less frequent events. Regarding AZD9291 resistance, AXL upregulation was again widespread, with loss of EGFR mutations as the second most frequent event. In proliferation assays, S49076 showed strong antitumor activity against all PC9-derived cell lines with acquired resistance to EGFR TKIs (erlotinib, gefitinib or AZD9291) and not carrying the T790M mutation, with IC50 of 0.2-1.2 µM and less than 10 % surviving cells at 1 µM of drug in most cases. In contrast, S49076 was less active against the parental PC9 cells and the T790M positive resistant lines, with more than 50% of surviving cells at 50 µM of drug. S49076 also inhibited the anchorage-independent growth and migration of the resistant cell lines. A correlation was found between mRNA and protein levels of AXL and sensitivity to S49076. Also, PARP cleavage and moderate but reproducible inhibition of AKT phosphorylation by S49076 were observed exclusively in the non-T790M resistant cell lines.

Conclusions: S49076 shows strong activity in preclinical assays against EGFR mutated cell lines resistant to EGFR TKIs not harboring EGFR T790M mutation and overexpressing AXL. A clinical trial of S49076 in non-T790M patients progressing to EGFR TKI and overexpressing AXL or MET has been initiated.

#337

Genetic analysis of tumors from a phase II trial evaluating AZD1775, carboplatin and paclitaxel in patients with TP53-mutant ovarian cancer.

Naomi Laing,1 Zhongwu Lai,1 J. Carl Barrett,1 Mark J. O'Connor,2 Amit M. Oza,3 David Lawrence2. 1 _AstraZeneca, Waltham, MA;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Background: AZD1775 (formerly MK-1775) is a selective inhibitor of WEE1 kinase, which has been shown to sensitize TP53-mutant cancer cells to genotoxic agents such as platinum-based chemotherapies. Mutation of TP53 abrogates the G1/S checkpoint in cells, which may enhance their dependency on the G2/M checkpoint and WEE1 kinase activity for control of the cell cycle and effective repair of DNA damage. The results from a randomized Phase II trial (NCT01357161) evaluating the effects of adding AZD1775 to carboplatin and paclitaxel in patients with TP53-mutant ovarian cancer showed an increase in progression-free survival (PFS) for the AZD1775 arm versus placebo (enhanced RECIST: median PFS 34.14 vs 31.86 weeks; HR=0.63, 80% CI: 0.45-0.89, P=0.080; RECIST 1.1: median PFS 42.86 vs 34.86 weeks; HR=0.55, 80% CI 0.39-0.79, P=0.030; Oza et al, ASCO 2015). We investigated whether particular genetic factors were associated with an increased response to the combination of AZD1775 and chemotherapy in this trial.

Methods: A retrospective analysis was performed to determine whether any specific subtype of TP53 alteration was associated with a greater response to the AZD1775 combination compared with placebo. In addition, next-generation sequencing (NGS) was performed on archival tumors from a subset of patients who provided consent in an effort to identify additional genetic alterations that may predict increased clinical benefit following the addition of AZD1775 to chemotherapy.

Results: TP53 data were available for 136 patients (15 patients from an initial open-label safety run-in and 121 patients from the randomized trial) and 133 patients were evaluable for response. Fifty-five patients provided additional consent for NGS of tumor samples. The genetic aberrations observed in the NGS subset of tumors from this trial were heterogeneous, and the total mutational load in cancer-related genes was also variable across tumors. Although the small number of patients with a tumor BRCA mutation limited the statistical power of the comparison between randomized arms, the median PFS was longer in those patients treated with AZD1775 (53.86 weeks, 95% CI 24.43-66.57) versus placebo (45.86 weeks, 95% CI 35.71-55.86) in this BRCA-mutated subgroup. TP53 subgroup analyses showed a similar benefit for patients with missense mutations compared to those with splice site, nonsense and frameshift TP53 mutations. The heterogeneity of the G1/S checkpoint gene aberrations limited the statistical power of the subgroup analysis, but specific genes that warrant further investigation were identified.

Conclusions: These results highlight a number of potential candidate genes for increased response to AZD1775 and chemotherapy compared with chemotherapy alone.

#338

A novel small molecule inhibitor of the Notch transcription activation complex.

Rajwinder Lehal,1 Viktoras Frismantas,2 Sylvain Loubéry,3 Viktoria Reinmuller,1 Gerardo Turcatti,4 Marcos Gonzalez-Gaitan,3 Jean-Pierre Bourquin,2 Freddy Radtke1. 1 _ISREC-EPFL, Lausanne, Switzerland;_ 2 _University Children's Hospital, Zurich, Switzerland;_ 3 _University of Geneva, Geneva, Switzerland;_ 4 _Biomolecular Screening Facility, EPFL, Lausanne, Switzerland_.

NOTCH signaling is a developmental pathway known to play critical roles during embryonic development as well as for the regulation of self-renewing tissues. Aberrant activation of NOTCH signaling leads to deregulation of the self-renewal process resulting in sustained proliferation, evasion of cell death, loss of differentiation capacity, invasion and metastasis, all of which are hallmarks of cancer. Over activation of NOTCH in human cancers can be a consequence of over expression of NOTCH ligands/receptors, GOF mutations in NOTCH receptors as well as chromosomal translocations leading to constitutive activation of the pathway. Given the importance of Notch signaling in human cancers, several therapeutic approaches have been utilized to block NOTCH signaling. Two of these strategies are; a) the use of monoclonal blocking antibodies (Mabs) against NOTCH ligands and receptors and b) the use of small molecule γ-secretase inhibitors (GSIs). However, these approaches can only be effective if tumor cells express full-length ligand or receptor molecules. On the contrary, in human cancers harbouring NOTCH gene fusion due to chromosomal translocations, the use of Mabs and GSIs will have very limited clinical benefits. A third, yet not fully explored approach could be the blockage of NOTCH signalling by targeting the most downstream event in the NOTCH cascade i,e NOTCH transcriptional activation complex using small molecule inhibitors.

Here we report discovery and identification of a novel, orally-active small molecule inhibitor, named CB-103, of the NOTCH pathway that blocks NOTCH signaling by targeting the NOTCH transcriptional activation complex in the nucleus. CB-103 has shown the ability to block NOTCH signalling in human cancer cell lines, induce neurogenic phenotype in drosophila, induce muscle cell differentiation and inhibit NOTCH dependent cellular processes in mice. In addition, CB-103 exhibit anti-tumor efficacy in a xenograft model of human triple negative breast cancer resistant to GSIs and Mabs against NOTCH ligands/receptors. Furthermore, CB-103 has shown a remarkable activity in PDX models of human T-ALL harbouring activation of the NOTCH pathway. Additional studies are underway on several analogs of CB-103 to determine ADME/PK/PD profile and nominate a development candidate for further clinical development of this novel inhibitor of the NOTCH pathway.

#339

Identification of novel covalent inhibitors of K-Ras G12C that are efficacious in a xenograft model of NSCLC.

Leena Khare Satyam, Dinesh Chikkanna, Aswani K. G, Vinayak V. Khairnar, Sreekanth Reddy, Vakkapatla Durgaprasad, Kowju Radhakrishna, Sunil K. Panigrahi, Anuradha Ramanathan, Kumari Mahasweta, Anirudha Lakshminarasimhan, Narasimha R. K, Vinutha R, Sreevalsam Gopinath, Suryakant Kumar, Mubarak H. Shah, Raghuveer Ramachandra, Kiran A. B, Chetan Pandit, Murali Ramachandra. _Aurigene Discovery Technologies Ltd., Bangalore, India_.

KRAS is the frequently mutated isoform in RAS driven cancers. The G12C mutation is more predominantly associated with various tumor types over other changes in K-Ras. Although direct targeting of RAS is very challenging, it is possible to selectively target G12C mutant K-Ras using a covalent approach. Mutant specific covalent inhibitors with high selectivity against wild type K-Ras and other GTPases are expected to lead to efficacy with a very high degree of tolerability. Here, we report identification of lead compounds from two distinct chemical series that selectively target K-Ras G12C. Molecular modeling based on the reported crystal structures aided in the identification of these compounds. Covalent binding of the lead compounds to K-Ras G12C was demonstrated by MALDI-TOF. Lead compounds were potent in selectively inhibiting proliferation of cell lines with K-Ras G12C mutation but not with wild type K-Ras. The anti-proliferative activity of the lead compounds correlated well with their potency in a cellular mechanistic assay. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability, permeability lack of CYP inhibition and desired exposure in pharmacokinetic studies. In a xenograft model of NSCLC, the lead compound demonstrated dose-dependent tumor growth inhibition with excellent tolerability upon oral dosing. In summary, we have identified a novel, potent and selective K-Ras G12C inhibitor with optimized drug-like properties including oral bioavailability and efficacy in a NSCLC derived xenograft model. Toxicity evaluation is ongoing towards progressing the lead compound to the clinic.

#340

Talazoparib enhances low-dose temozolomide efficacy in flank glioblastoma models, but intracranial efficacy is constrained by limited brain distribution.

Sani H. Kizilbash,1 Kenneth Chang,1 Shiv K. Gupta,1 Ryo Kawashima,1 Karen Parrish,2 Ann C. Mladek,1 Brett L. Carlson,1 Katrina K. Bakken,1 Mark A. Schroeder,1 Gaspar J. Kitange,1 Paul A. Decker,1 Yuqiao Shen,3 William F. Elmquist,2 Jann N. Sarkaria1. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _BioMarin Pharmaceutical Inc., Novato, CA_.

BACKGROUND: Talazoparib (TAL) is a potent poly (ADP-ribose) polymerase (PARP) inhibitor that robustly enhances in vitro sensitivity to temozolomide (TMZ) in several tumor models. We assessed the in vitro and in vivo efficacy of TAL combined with TMZ in glioblastoma (GBM) models.

METHODS: Established glioma cell lines (T98G, U251) and the GBM12 patient derived xenograft (PDX) line were treated in vitro with TAL (1-10 nM) ± TMZ (2-300 µM). Antitumor efficacy was assessed with CyQUANT, clonogenic and primary neurosphere assays; cell cycle analysis with flow cytometry; DNA damage signaling with phospho-specific western blots for pKap1, pChk1, pChk2 and fluorescent immunocytochemistry for γH2AX and replication protein A foci. Brain and plasma concentrations of TAL in wild-type (WT), Mdr1a/b(-/-), Bcrp(-/-) and Mdr1a/b(-/-)Bcrp(-/-) knockout (KO) mice were assessed with LC-MS/MS. The tolerability of TAL (0.05 - 0.30 mg/kg/day orally, divided twice daily (div. bid)) and TMZ (5-50 mg/kg/day orally) was tested in athymic nude mice. Subsequently, in vivo combination TAL/TMZ efficacy was assessed with survival analyses in intracranial and flank GBM12 PDX models.

RESULTS: TAL 1-3 nM sensitizes the TMZ resistant T98G glioma cell line to TMZ at high TMZ concentrations (30-300 µM) and is associated with G2 cell cycle arrest and enhanced DNA damage signaling. TAL 1-3 nM also enhances the cytotoxic efficacy of lower concentrations of TMZ (10-30 µM) in the TMZ sensitive U251 cell line. At least 3 nM TAL is required to enhance the in vitro efficacy of low TMZ concentrations (2-10 µM) in the TMZ sensitive GBM12 PDX line. PK studies in non-tumor bearing WT FVB mice treated with a single dose of TAL 0.15 mg/kg revealed mean brain and plasma concentrations of 0.49 ng/g (1.3 nM) and 25.5 ng/ml (67.1 nM) respectively at 2 hours. The mean brain/plasma ratio at 2 hours was unchanged in Bcrp(-/-) KO mice compared to WT mice (1.2% vs. 2.0%), but was significantly increased in Mdr1a/b(-/-) KO mice (22.8%) and Mdr1a/b(-/-)Bcrp(-/-) KO mice (23.9%). Standard doses of TMZ (50 mg/kg/day, days 1-5) combined with low doses of TAL (0.05 mg/kg/day, div. bid) were toxic in nude mice. Higher doses of TAL (0.30 mg/kg/day, div. bid) required substantial TMZ dose reductions (5 mg/kg/day, days 1-5) for tolerability. The addition of TAL (0.30 mg/kg/day, div. bid) to low-dose TMZ (5 mg/kg/day) prolonged tumor stasis in flank GBM12 xenografts (median time to endpoint 76 vs. 50 days, p=0.005). However this regimen was ineffective in intracranial GBM12 xenografts (median survival 37 vs. 30 days, p = 0.93).

CONCLUSIONS: TAL is a potent PARP inhibitor that enhances the efficacy of TMZ in both in vitro GBM models and flank GBM12 PDX models, but not in the corresponding GBM12 intracranial xenografts. This lack of intracranial efficacy is associated with limited brain distribution due to active efflux mediated by Mdr1 at the blood-brain barrier.

#341

Preclinical mode of action and anti-tumor efficacy of the selective MKNK1 inhibitor BAY 1143269 in NSCLC models.

Susann Santag, Franziska Siegel, Antje M. Wengner, Claudia Lange, Ulf Boemer, Knut Eis, Florian Puehler, Martin Michels, Franz von Nussbaum, Karl Ziegelbauer, Dominik Mumberg, Kirstin Petersen. _Bayer Pharma AG, Berlin, Germany_.

MKNK1 (MAP kinase-interacting serine/threonine-protein kinase, also known as Mnk1) is activated by the mitogen-activated protein kinases ERK1/2 and p38. Thus, MKNK1 signaling is involved in the cellular response to environmental stress factors and cytokines. Of particular interest, MKNK1 kinase regulates mRNA translation by phosphorylating the translation initiation factor eIF4E (eukaryotic translation initiation factor 4E), known to be critical for malignant transformation but shown to be dispensable for translation in normal cells. Phosphorylated eIF4E levels were found to be elevated in several cancer tissues, including lung cancer. MKNK1 is also involved in resistance mechanisms to cancer therapeutics. Thus, the inhibition of MKNK1 activity may provide an innovative approach for anti-cancer therapy, and in particular for lung cancer, the main cancer-related cause of death worldwide. BAY 1143269 is a potent and selective MKNK1 inhibitor and inhibits eIF4E phosphorylation and reduces MKNK1-regulated translational downstream targets in non-small cell lung cancer (NSCLC) cell lines. In this study, BAY 1143269-mediated effects on molecular mechanisms in lung cancer models were analyzed.

Epithelial-mesenchymal transition (EMT) is associated with the pathogenesis of numerous lung diseases including cancer progression, metastasis and resistance. BAY 1143269 reduced expression of EMT key regulators like Snail1 and cellular junction components, as well as reduced TGFβ1-induced EMT. Accumulating evidence suggests a role for proinflammatory cytokines in the development and progression of cancer; increased serum concentrations of cytokines like interleukin 6 (IL-6) are associated with diminished lung cancer survival rates. BAY 1143269 reduced the secretion of several proinflammatory cytokines, including TNFα and IL-6 in whole blood, and affected IFN-stimulated gene expression in cell lines.

Consistent with the observed effects in vitro, BAY 1143269 showed significant anti-tumor effects in vivo in cell line as well as patient derived NSCLC xenograft models in monotherapy. In combination with chemotherapeutics approved for treatment of NSCLC, BAY 1143269 improved anti-tumor effects in comparison to chemotherapy alone.

In conclusion, BAY 1143269 has the potential to provide therapeutic benefit in NSCLC. A phase I study of BAY 1143269 in combination with docetaxel for subjects with advance solid tumors is ongoing (NCT02439346).

#342

BGB-283, a slow-off inhibitor of RAF dimers, differentiates from vemurafenib in MAPK signaling inhibition.

Ye Liu, Xi Yuan, Zhiyu Tang, Lai Wang, Lusong Luo, Min Wei. _BeiGene, Ltd, Beijing, China_.

Dysregulations of the mitogen-activated protein kinase (MAPK) signaling through abnormal expression or activating mutations of key signaling molecules, including RAS and RAF, have been identified in a wide range of cancers. Targeting these signaling molecules in the pathway represents a promising strategy to target dysregulated MAPK signaling. BGB-283 is a novel pan-RAF inhibitor and currently under clinical development to treat BRAF and KRAS/NRAS mutated tumors. In this study, we show that BGB-283 inhibits WT B-RAF dimer much more potently than vemurafenib at millimolar ATP concentration that is representative of the cellular ATP level, although BGB-283 and vemurafenib display similar inhibitory activity at the Km ATP concentration. BGB-283 has very slow off-rate and the dissociation half-life (t1/2) on WT B-RAF enzyme is well above 24 hrs. In comparison, vemurafenib has much faster off rates with t1/2 in the range of minutes. The crystal structure of BGB-283/B-RAF protein complex shows the benzimidazole and trifluoro group of BGB-283 binds significantly deeper into pocket formed by α-C helix and activation loop than vemurafenib. BGB-283 exhibits time-dependent inhibition both in biochemical assay and in B-RAF or K-RAS mutated cells. BGB-283 potently inhibits the p-ERK signaling in splicing isoform p61B-RAF(V600E) cells which were reported to be resistant to vemurafenib through a dimer mechanism. In vivo, BGB-283 significantly suppressed the tumor growth in A375-p61B-RAF(V600E) melanoma xenograft model. In addition, BGB-283 inhibits vemurafenib induced p-ERK activation and selumetinib induced p-MEK feedback activation in K-RAS mutated cancer cells. In both cases, activation is reported to be mediated by RAF dimers. Taken together, we propose that BGB-283 is a novel, slow-off RAF dimer inhibitor that targets the MAPK signaling differently from the first generation BRAF inhibitor vemurafenib. This feature of BGB-283 may help to address the drug resistance issues in BRAF mutated tumors and further expand its utility as mono- or combination therapy into RAS mutated patient populations.

#343

ERG oncogene-specific inhibitors for prostate cancer.

Ahmed Mohamed,1 Gauthaman Sukumar,2 Charles Xavier,1 Shilpa Katta,1 Lakshmi Ravindranath,1 Muhammad Jamal,1 Taduru Sreenath,1 Gyorgy Petrovics,1 Albert Dobi,1 Meera Srivastava,2 Clifton Dalgard,2 Shiv Srivastava1. 1 _Uniformed Services University of the Health Sci., Rockville, MD;_ 2 _Uniformed Services University of the Health Sci., Bethesda, MD_.

Introduction: Treatment for advanced prostate cancer (CaP) remains challenging due to accumulation of numerous genomic alterations or treatment-related mutations during disease continuum. Therefore, targeting early CaP driver genes may introduce new approaches in complementing current therapeutic paradigms. ERG oncogenic activation by recurrent gene fusions is the most validated driver gene alteration in CaP and represents a high priority therapeutic target. However, the transcription factor nature of ERG poses challenges. Current ERG-targeted strategies include direct or indirect inhibition of ERG/ETS transcription factor activity. Due to high homology of ERG to a large number of ETS-related proteins the specificity of these strategies remains to be established. In order to screen for ERG-specific inhibitors, we developed a new approach for screening of small molecules by assessing inhibition of ERG oncoprotein expression using an In-cell-Western based assay employing a specific anti-ERG monoclonal antibody (9FY). Here we report the characterization of a highly selective small molecule ERG inhibitor (ERGi-USU).

Methods: A small molecule library from the National Cancer Institute was screened for ERG protein inhibition in TMPRSS2-ERG harboring CaP cells (VCaP) using an In-Cell-Western assay employing ERG-MAb (9FY). Among the promising candidates NSC139021 (ERGi-USU) was selected for further characterization in cell culture-based (VCaP, COLO320, MOLT4, KG1, LNCaP, LAPC4, MDAPCa2b, BPH1, RWPE and HUVEC) and VCaP nude mouse xenograft assays. Effect of ERGi-USU was determined using cell growth, cell cycle and apoptosis assays and whole transcriptome sequencing.

Result: Among the cell lines evaluated, ERGi-USU exhibited exclusive inhibitory effects on ERG protein as well as cell growth including TMPRSS2-ERG harboring CaP (VCaP) and ERG positive colon cancer and leukemia cells (COLO320, MOLT4, KG1) (IC50, 50-400 nM). There was no inhibitory effect on cell growth of ERG negative CaP cells or normal prostate-derived epithelial cells. Importantly, ERGi-USU did not inhibit the growth of primary endothelial cells with endogenous ERG. Molecular and cellular mechanisms of ERGi-USU included inhibition of G2 phase of cell cycle, induction of apoptosis. RNA-Seq analyses revealed activation of the ER stress pathway. Importantly, significant inhibitory effect of ERGi-USU was shown on VCaP xenograft growth in nude mice without any significant toxicity.

Conclusion: ERG inhibition through ERGi-USU showed high specificity for ERG expressing tumor cells in experimental models and has potential in further developing ERG-targeted therapeutic strategies for CaP and other ERG positive cancers.

#344

Antimitotic effect and complex of nonmitotic effect on tumor biology of eribulin mesilate in soft tissue sarcoma model.

Satoshi Kawano, Makoto Asano, Yusuke Adachi, Junji Matsui. _Biology Tsukuba, Oncology PCU, Eisai Co., Ltd., Tokodai, Tsukuba-shi, Ibaraki, Japan_.

Background and objectives: Eribulin mesilate (Eribulin), a first-in-class halichondrin B-based microtubule dynamics inhibitor, has been shown to have novel mechanism of actions (reversion of epithelial-mesenchymal transition of tumor cell and promotion of vascular remodeling) beyond its established antimitotic activity in breast cancer models, leading to decrease of tumor metastatic potency. Recently, eribulin showed trends towards greater overall survival compared to Dacarbazine in subjects with leiomyosarcoma and liposarcoma patients in phase 3 trial. The objective of this study was to investigate the effect of eribulin on tumor cell proliferation of soft tissue sarcoma (STS). Furthermore, the effects of eribulin on morphology and gene expression of differentiation markers against STS and tumor blood perfusion in a leiomyosarcoma xenograft model were examined.

Methods: Anti-proliferation activity of eribulin was examined in 6 human STS cell lines. Anti-tumor activity of eribulin was examined in xenografts of A-673 (Ewing's sarcoma), SK-LMS-1 (leiomyosarcoma), SW 872 (liposarcoma), and HT-1080 (fibrosarcoma) at several doses. To analyze the effect of eribulin on in vitro morphological change, 3 human STS cell lines were treated with eribulin at 1, 3, and 10 nmol/L for 7 days. Gene expression change was analyzed by microarray and differentiation marker genes in 2 cell lines were confirmed by qPCR analysis. For tumor blood perfusion analysis, eribulin was administered at doses of 0.38, 0.75, and 1.5 mg/kg one shot in SK-LMS-1 xenografts, and tumor blood perfusion was calculated as Hoechst-positive areas in tumor cryosections 5 days after the administration.

Results: Eribulin showed anti-proliferation activity in vitro against all 6 cell lines in a dose dependent manner with 50% inhibitory concentration values of around 1 nmol/L. Eribulin showed significant antitumor activity against 4 xenografts in a dose dependent manner. Eribulin treatment for 7 days induced apparent morphological change in 3 cell lines. Of these, expression level of smooth muscle differentiation marker gene CNN1 was 7.1-fold upregulated in SK-UT-1 and adipocyte differentiation marker genes MYLK, C/EBPβ, and KIF23 were 1.9-, 1.4-, and 1.3-fold upregulated in SW 872, respectively, by 10 nmol/L treatment compared to vehicle control. In addition, eribulin significantly enhanced tumor blood perfusion compared to vehicle control 5 days after the administration in SK-LMS-1 xenografts at all doses.Conclusions: Eribulin showed anti-proliferation activity against all of STS cell lines we tested in vitro and in vivo. Furthermore, eribulin might cause differentiation of STS cells and remodeling of tumor vasculature to enhance tumor blood perfusion. These data supported that eribulin showed mitotic effect and non-mitotic effect, which may decrease tumor metastatic potency in STS, as well in breast cancer.

#345

Olive oil-based oleocanthal and bioisosteres as c-Met inhibitors for the control of triple negative breast cancer.

Hassan Y. Ebrahim, Mohamed M. Mohyeldin, Abu Bakar Siddique, Mohamed R. Akl, Mostafa Ibrahim, Khalid A. El Sayed. _School of Pharmacy, Monroe, LA_.

Breast cancer is the most commonly diagnosed cancer in women, claiming the lives of hundreds of thousands of women each year. Dysregulation of c Met correlates with aggressive proliferation, invasive character, and pathological motility, especially in triple negative breast cancer (TNBC). The Mediterranean diet is known to reduce the incidence of breast and colon malignancies. Extra-virgin olive oil (EVOO) represents a main ingredient in this diet. The EVOO-derived (-)-oleocanthal was shown to target the RTK c-Met. Oleocanthal selectively inhibited the proliferation, migration and invasion of several c-Met dependent breast and prostate tumor cell lines. Oleocanthal exerted potent in-vivo efficacy in an orthotopic athymic nude mouse breast cancer xenograft model much greater than its modest in-vitro potency. Several oleocanthal bioisosteres have been semisynthized of which the analog HVS have shown significant activity improvement against breast cancer in vitro and in vivo models. Selectivity profiling against a TKs panel, structurally related to c-Met, suggested the HVS has high selectivity to both Met and ABL1 kinases. Overexpression of ABL1 kinase is implicated in the progression of several solid tumors concomitantly with c-Met activation, including breast, colon, lung and other cancers. ABL1 interconnects oncogenic Met and p53 pathways and hence dual blockage of c-Met and ABL1 activation adds significant advantages over currently approved c-Met inhibitors and may justify its potent in vitro and in vivo activities. Therefore, the dual c-Met/ABL1 inhibitor HVS could not only be effective in slowing TNBC progression but also could prevent drug resistance. Detailed molecular and cellular mechanistic studies of oleocanthal and its bioisosteres indicate their potential for future use to control TNBC and other c-Met-dependent malignancies. This study was supported by NIH/NCI project 1R15CA167475-01.

#346

Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies.

Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. _Rigel Pharmaceuticals, Inc., S. San Francisco, CA_.

Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies.

#347

Developing a novel immunotoxin that targets cells overexpressing ErbB2.

Luqun Shen, Kelsey Weigel, Clayton Thomas, Lauren Drapalik, Zachary T. Schafer, Shaun Lee. _University of Notre Dame, Notre Dame, IN_.

Immunotoxins are chimeric proteins comprising a specific cellular targeting domain linked to a cytotoxic factor. Here we describe the design and use of a novel, peptide-based immunotoxin that can initiate selective cytotoxicity on ErbB2-positive cells. ErbB2 is a receptor tyrosine kinase that is overexpressed in the tumor cells of approximately 30% of breast cancer patients. Immunotoxin candidates were designed to incorporate a targeting ligand with affinity for ErbB2 along with a membrane lysin-based toxin domain. The use of a membrane lysin as the toxin domain distinguishes our immunotoxins from numerous others in that internalization of the toxin is not required to induce cell death. One particular peptide candidate, NL1.1-PSA, demonstrated selective cytotoxicity towards ErbB2-overexpressing cell lines. We utilized a bioengineering strategy to show that recombinant NL1.1-PSA immunotoxin expression by Escherichia coli also conferred selective cytotoxicity towards ErbB2-overexpressing cells. Our findings hold significant promise for the use of effective immunotoxins in cancer therapeutics. While Herceptin treatment has been effective in patients with ErbB2 positive breast cancer, our novel immunotoxin may ultimately prove to be a novel and efficacious addition to the arsenal of approaches used to eliminate Herceptin-resistant breast cancer cells.

#348

Small molecule Axl Ig1-Gas6 inhibitors block Gas6-inducible Axl signaling and suppress tumorigenicity.

Nitu Bansal,1 Stanley Kimani,2 Kamlendra Singh,3 Thomas Comollo,2 Sushil Kumar,2 Vladyslav Kholodovych,2 Youyi Peng,4 William Welsh,4 Bertino Joseph,1 Raymond B. Birge2. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Biomedical and Health Sciences Center, Rutgers, Newark, NJ;_ 3 _Bond Life Sciences Center, University of Missouri, Columbia, MO;_ 4 _Department of Pharmacology, Rutgers, New Brunswick, NJ_.

TAM receptors (Tyro-3, Axl, and Mer) are a family of three homologous receptor tyrosine kinases (RTKs) expressed predominantly on myeloid-derived hematopoietic cells and epithelial cells which function as inhibitory receptors that dampen inflammatory responses and maintain tissue tolerance. Additionally, all three TAMs have been implicated in various human malignancies, where level of expression is often associated with more aggressive staging of the cancers, poorer predicted patient survival, as well as drug-resistant cancers. Over the past several years, considerable effort has been made to generate small molecule tyrosine kinase inhibitors and biological therapeutics (such as monoclonal antibodies and soluble extracellular receptor traps) targeting TAM receptors in human cancers. Early preclinical studies show promising anti-tumor activities using either kinase inhibitors or monoclonal antibodies against Axl and Mer, although cross-reactivity with other tyrosine kinase inhibitors is associated with off-target specificities. Here, we report the development and characterization of a series of small molecule inhibitors that target the interface between the Ig1 domain of Axl and Gas6, and inhibit native receptors and Axl reporter lines with sub-micro-molar affinities. Additionally, these compounds inhibit Gas6 inducible motility and invasion in Axl-expressing cell lines, and suppress tumor growth in xenograft models of non small cell lung cancer. Together, these observations demonstrate that Axl can be targeted by small molecules that bind to the Ig1 domain/Gas6 interaction, and validate Ig1 inhibitors as novel agents for treatment of cancer.

#349

Structure-activity relationships and mechanistic analysis of analogues of the clinical-stage anti-cancer small molecule ONC201.

Jessica Wagner,1 Gary Olson,2 Nallaganchu Rao Bhaskara,2 Richard S. Pottorf,2 Garnett J. Mathew,3 Cyril H. Benes,4 Rohinton Tarapore,5 Martin Stogniew,5 Lee Schalop,5 Wolfgang Oster,5 Josh E. Allen,5 Wafik S. El-Deiry1. 1 _Fox Chase Cancer Center: Temple College of Medicine, Philadelphia, PA;_ 2 _Provid Pharmaceuticals, Monmouth Junction, NJ;_ 3 _Welcome Trust Sanger Institute, United Kingdom;_ 4 _Massachusetts General Hospital, Boston, MA;_ 5 _Oncoceutics, Inc., Philadelphia, PA_.

TRAIL is an endogenous protein that initiates apoptosis selectively in cancer without toxic side effects, prompting interest in therapeutic modulation. We previously screened for small molecules that could upregulate the endogenous TRAIL gene to trigger apoptosis and restore anti-tumor immunity within tumor cells in a p53-independent manner. We showed that ONC201 (previously referred to as TIC10) is a dual inactivator of Akt and ERK, leading to nuclear translocation of Foxo3a and TRAIL gene activation (Allen et al., Science Translational Medicine, 2013). We recently found that ONC201 causes an early-stage upregulation of the integrated stress response through ATF4/CHOP/DR5 (Kline et al., Science Signaling, in press) and can inhibit cancer stem cell self-renewal (Prabhu et al., Cancer Research, 2015). ONC201 recently completed its first-in-man phase I clinical trial and several other trials in select advanced cancers are ongoing (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). Leveraging the unique pharmacophore of ONC201, we synthesized ONC201 analogues in search for compounds with distinct therapeutic properties. After establishing the importance of the pyrido[3,4-e]pyrimidinone core structure of ONC201 in its anti-tumor activity (Wagner et al., Oncotarget, 2014), we performed detailed structure activity relationship (SAR) studies. We evaluated several ONC201 analogues with differing N-substituents around the core structure. Certain ONC201 analogues exhibit more rapid kinetics of activity and lowered IC50 values in some human cancer cell lines in vitro. Interim results of ONC212 sensitivity profiling in >100 genetically annotated cell lines from the Genomic of Drug Sensitivity in Cancer collection have corroborated this improvement in potency. One analogue, ONC212, has demonstrated compelling efficacy against several tumor types in vivo with no evidence of toxicity at therapeutic doses. Furthermore, in vitro mechanism studies have demonstrated overlap between ONC201- and ONC212-mediated signaling in tumor cells that includes activation of the integrated stress response. With a wide safety margin, distinct pharmacokinetics (PK), and robust potency, ONC212 is being developed as the next drug candidate from the new class of compounds defined by the novel pharmacophore of ONC201 in indications that complement the parent compound's use spectrum.

### Novel Assays

#350

Visualizing alectinib distribution in mice brain by using MALDI mass spectrometry imaging technique.

Hiroaki Aikawa,1 Mitsuhiro Hayashi,1 Shoraku Ryu,1 Makiko Yamashita,1 Naoto Ohtsuka,2 Masanobu Nishidate,3 Akinobu Hamada1. 1 _National Cancer Center, Tokyo, Japan;_ 2 _Shimadzu Techno-Research Inc., Tokyo, Japan;_ 3 _Chugai Pharmaceutical, Kanagawa, Japan_.

[Introduction]

In the development of anticancer drug, measurement of drug concentration in target tissue has been thought to be crucial for predicting its efficacy and safety. However, measuring homogenized tissue by using quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) completely loss the spatial information of the compound in a target tissue. Mass spectrometry imaging (MSI) has been recently applied as innovative tools to detect pharmaceutical distributions in heterogeneous targets; however, it still needs to be improved in quantitative capability or reproducibility.

[Purpose]

We propose quantitative approach for MSI (qMSI) that administere matrix-assisted laser desorption ionization (MALDI)-MSI, concurrently with LC-MS/MS by using serial sections. We applied qMSI for visualizing spatial distribution of alectinib, novel ALK inhibitor, in mouse brain and examined the effect of MDR1 (p-glycoprotein) on the pharmacokinetic profiles in FVB and Mdr1a/b knockout mice.

[Method]

Pharmacokinetic study of alectinib was conducted in FVB and Mdr1a/b KO mice. Plasma, cerebrospinal fluid (CSF) and brain tissue were collected at 1, 1.5, 2, and 4 hours after a single oral administration dose of 4 and 20 mg/kg. The concentration of alectinib in plasma, CSF and brain tissue sections was measured by LC-MS/MS. The distribution of alectinib in mice brain tissue section was visualized by qMSI that we established.

[Results]

Developing qMSI showed the brain distribution of alectinib in the range from 1.2 to 952.6 pg/mm2 per each section, and the results made with qMSI were in excellent agreement with the complementary measurements by using laser-micro dissection to verify the exposure level. While plasma alectinib concentrations had no difference between both mice, the diffuse alectinib distributions in brain were observed in Mdr1a/b knockout compared with that in FVB mice. The dominant localization of alectinib in choroid plexus and brain blood vessels of FVB mice was observed by 20 μm resolution MSI analysis.

[Conclusion]

Our qMSI technique is expected to contribute to the development of anticancer drugs and translational research by visualizing drug distribution in target organs and tumors.

#351

Uptake of antibody-drug conjugates by cultured Kupffer cells can predict pharmacokinetics.

David Meyer, Sara Shum, Mechthild Jonas, Martha Anderson, Joshua Hunter, Nagendra Chemuturi, Nicole Okeley, Robert Lyon. _Seattle Genetics, Bothell, WA_.

Interest in antibody-drug conjugates (ADCs) has increased rapidly in the oncology field over the past several years. Recent advances in the ADC field have been achieved in part by an improved understanding of how conjugation with drug-linkers impacts the biophysical, pharmacokinetic (PK), and biodistribution properties of a monoclonal antibody. Most of these advances have been driven by in vivo preclinical observations, an inevitably low throughput endeavor. A higher throughput in vitro method that would allow some level of predictive power for ADC disposition in vivo would be highly advantageous, requiring less test article and allowing for much faster turnaround. Thus far no such methods for ADCs have been reported, in contrast to the situation for the prediction of the metabolic clearance of small molecule therapeutics, for which cultured hepatocytes are widely used as an in vitro tool. It has been generally assumed that clearance of ADCs is driven by cells of the monocyte phagocytic system, and we recently published immunohistochemistry (IHC) data implicating hepatic sinusoidal endothelium and the resident liver macrophages, Kupffer cells (KCs), in the accelerated clearance of highly-loaded ADCs. We sought to use this observation to develop a convenient assay that might correlate ADC uptake in vitro with in vivo PK. Initial fluorescent microscopy experiments revealed that cultured rat KCs stain intensely when incubated with fluorescently labeled ADCs, but much less so with unconjugated antibody, similar to the observed IHC results. In an effort to further improve the throughput of this method to allow the rapid comparison of many ADCs, we moved to a FACS-based platform to quantify the amount of KC-associated fluorescent conjugate. To quantify the effect of different drug-linkers, we prepared homogeneous ADCs with 8 drugs per antibody and observed that the KC staining intensity varied widely depending upon the characteristics of the drug-linker that was conjugated. KCs incubated with ADCs loaded with vc-PAB-MMAE had a mean fluorescent intensity (MFI) >20 fold higher than unconjugated Antibody. Additionally, ADCs loaded with MMAE that contain a polyethylene glycol (PEG) masking moiety in the linker had a much lower MFI than those without PEG. This FACS method allowed us to compare fluorescent uptake by KCs with ADC clearance determined in vivo. We have observed that ADC uptake by cultured KCs in vitro correlates well with ADC PK, allowing for more rapid assessment of the PK impact of new drug classes, drug-linker designs, and conjugation methodologies.

#352

HER2 protein isoform heterogeneity investigated by single-cell western blotting.

Ginny (Chi-Chih) Kang,1 Toby M. Ward,2 Jessica Bockhorn,2 Mark D. Pegram,2 Amy E. Herr1. 1 _UC Berkeley, Berkeley, CA;_ 2 _Stanford University, Stanford, CA_.

Accounting for ~20% of breast cancers, half of metastatic HER2-positive breast cancer patients treated with trastuzumab plus chemotherapy will experience progression of disease within one year. Trastuzumab treatment, though associated with improvement in median overall survival, still sees drug resistance as an unmet challenge. Differential response to therapy may arise from tumor heterogeneity. Further complicating the situation is the fact that HER2/erbB2 biology is complex. HER2 is not simply one full-length protein (p185) overexpressed on tumor cell surfaces, but also includes truncated isoforms, t-erbB2 (e.g., 95 and 110 kDa isoforms) lacking the extracellular domain targeted by trastuzumab (and pertuzumab and T-DM1). The t-erbB2 isoforms are implicated in tumorigenesis, metastasis, and therapeutic response. Given existing pathology and life sciences tools, the high heterogeneity and need for single cell resolution presents a tremendous challenge.

We introduce here a novel microfluidic device to quantitatively assess HER2 protein isoforms in heterogeneous tumor biopsies with single-cell resolution. The single-cell western blot (scWB) provides both the sensitivity and specificity needed to detect t-erbB2 isoforms owing to use of a single-cell electrophoretic protein separation followed by immunoprobing. The assay further provides enough sensitivity to obviate the need for cell pooling, which obscures cell-to-cell heterogeneity. We assay ~1000's of single cells on one microscope slide in a 4-6 hr workflow.

First, we validated that the scWB can separate full length (p185) from truncated (p95) HER2 in single p185- and p95-genetically engineered CHO cells. The p95 was resolved from p185 protein in a short ~1 mm separation lane (resolution = 0.97±0.03, n>500 cells). Next, we assessed a HER2-positive breast cancer cell line (BT474) and found <3% t-erbB2 expressing cells. To further investigate the effect of t-erbB2 in HER2-related signaling, we examined phospho-proteins including p-HER2, p-HER3, p-ERK Thr187/Tyr185, p-ERK Thr202/Tyr204, p-rs6 from each t-erbB2 expressing BT474 cell. Protein activation in t-erbB2 expressing cells was similar to cells expressing full length HER2.

This first-in-kind study demonstrates a powerful single-cell resolution multiplexing capability including for phospho-proteins. We are now applying the novel scWB to measure t-erbB2 in HER2-positive and triple positive (ER/PR/HER2) breast tumor biopsies and will report our results at AACR.

Taken together, the microfluidic single-cell resolution western provides a sensitive,

quantitative, and multiplexed tool to assay clinically-relevant oncoprotein isoforms that are currently difficult or impossible to assay. Correlating isoform expression ratios with clinical outcomes may confer a more comprehensive understanding of drug resistance responses derived from oncoprotein isoform profiles within heterogeneous cancer cell populations.

#353

**Development of an** in vitro **screening platform for high capacity evaluation of immunomodulatory drug candidates.**

Sujatha Kumar,1 Felicia Zhao,1 Anatoly Myaskovsky,1 Lydia Kifle,1 Nicola McCarthy,2 Jon Moore,2 Dennis France,1 Janine Steiger,1 W. Frank An1. 1 _Horizon Discovery, Cambridge, MA;_ 2 _Horizon Discovery, Cambridge, United Kingdom_.

Cancer immunotherapy has greatly expanded the therapeutic options against cancer. Recently approved antibody immunotherapies targeting immune-checkpoint molecules expressed on active T cells have shown promise in providing durable clinical anti-tumor responses in a substantial subset of melanoma and lung cancer patients. These clinical successes have prompted renewed efforts to identify efficacious approaches to harness the anti-tumor functions of the immune system. Combination immunotherapy that targets multiple immune-checkpoint molecules and/or combines immunotherapy and chemotherapy will likely lead to improved efficacy and reduced toxicity.

A key challenge in immuno-oncology drug discovery is the lack of high throughput assay technologies to address the complexities of immune cell biology and drug screening. To address this, we have developed a robust and versatile in vitro T cell activation assay that leverages our well-established and validated high throughput combinatorial screening and data analysis platform. Our platform utilizes primary human peripheral T cells and measures a suite of T cell activation parameters including cell surface markers by high-throughput FACS, cytokines (e.g. IL-2, TNFα, IFNγ by HTRF or bead-based multiplexed technology, and proliferation by ATP, BrdU, or dye-based assays. Miniaturized to a 384-well format and supported by automation at each experimental step, including acoustic no-contact compound/antibody delivery, our assays are robust, exhibiting high Z' values, high reproducibility between various donors and days of operation, and high capacity of up to twenty thousand data points per day. The platform is highly customizable in terms of testing agents that either enhance or inhibit CD3 or CD3/CD28-mediated T cell activation, including antibody drug candidates and/or small molecules while examining single agent dose responses and/or combination interactions, as well as accommodating varied dose schedules. Screening data is tracked through a proprietary laboratory information management system (LIMS) and analyzed by the state-of-the-art Horizon Chalice Analyzer software that applies the most appropriate synergy analysis for combination effects. Effect of reference immunomodulatory agents tested in this platform will be discussed. Finally, our high capacity technology platform is compatible with other cellular immunoassays such as the mixed lymphocytes reaction (MLR), NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) assays, and T cell-mediated tumor cell lysis assays aided by engineered bispecific antibodies. We believe that this versatile high throughput immuno-oncology platform will enable large scale interrogation of prospective immunomodulatory agents, either alone or in combination, and thus contribute substantially to the discovery and development of future immunotherapies.

#354

Physiologically relevant target engagement using CETSA.

Jakob Karén,1 Catrine Sioberg,1 Pawel Niekrasz,2 Magnus Neumuller,2 Daniel Martinez Molina1. 1 _Pelago Bioscience, Stockholm, Sweden;_ 2 _Biovica, Uppsala, Sweden_.

Targeted therapies are dependent on adequate occupancy and target engagement to reach their efficacy measure. Yet, more than half of novel candidate drugs entering clinical trial stage fail before proof of concept is achieved. Inadequate target engagement is often at the root cause of these failures. Therefore we have developed a versatile label-free method to quantify the target engagement of drugs on their native physiological relevant targets. The method is validated in multiple sample matrices from bench to bedside: in living cells and tissue from rodents and man.

CETSA is a patented biophysical method measuring the thermal stability of drug target proteins and the shift induced by their ligands. By quantifying the melting temperature and shift induced by the ligand we can quantify the potency of target engagement. This potency determination then allows filtering of candidate drugs by their ability to engage the target in its physiologically relevant form, in relevant sample formats such as intact cells or tissue.

We present a number of applications and readouts of the CETSA method quantifying target engagement of drugs used in the clinic and in development. Furthermore, we show that the target engagement of cyclin dependent kinase inhibitors correlated both to viability and to a dose dependent decrease of a cell cycle activity marker in cellular sample matrices. The biomarker, thymidine kinase activity, was analyzed with the commercially available test DiviTum®. By combining CETSA with DiviTum® in the same samples, it was thus possible to correlate CETSA outcomes to pharmacodynamic effects of a drug.

The utility and value of the CETSA method enabling preclinical development of novel drugs is substantiated by the examples included here. Combining quantifications of efficacy with the molecular target engagement of the drug yields an orthogonal dataset, which has a potential to validate the target-mediated efficacy. Such datasets can be generated from early in vitro systems to clinical studies and may reduce the high failure rate in drug development by facilitating the discovery, development and evaluation of novel drugs.

#355

HT flow cytometry platform enabling measurement of immune signaling in whole blood and T-cells for drug discovery.

mark fereshteh, Litao Zhang, Xin Li, Sha Li, Yi Fan, Gary Schieven. _bristol myers squibb, Princeton, NJ_.

Oral agents targeting Janus-associated kinases (JAKs) are among the most promising new agents in clinical development. To better understand the relationship between JAK inhibition and clinical outcome, compounds targeting JAKs are required to be analyzed in peripheral whole blood. To date, this type of analysis has been low throughput and is associated with high cost. Here, we developed a robust 384-well flow based assay approach to screen small molecules for inhibition of JAK/STAT signaling in whole blood using high-throughput flow cytometry. The platform provides a highly sensitive analysis of signaling events in low volumes of blood facilitating a robust target engagement assay to measure inhibition of proximal signaling by selective JAK inhibitors. Further, automation technologies and processes optimization were established to overcome bottlenecks in sample integrity, handling and multiparametric data analysis without affecting assay performance. The result was dramatically increased sample throughput when compared to conventional manual flow cytometric approaches enabling the identification of novel selective JAK/STAT inhibitors enabling the investigation of IO and Immunology targets earlier in drug discovery.

#356

Development and characterization of an in vitro co-culture angiogenesis assay system using hTERT immortalized cells for high throughput drug screening.

Chaozhong Zou,1 Chia-Wen Hsu,2 Chengkang Zhang,1 Isabela Oliva,1 Metewo S. Enuameh1. 1 _ATCC, Gaithersburg, MD;_ 2 _Division for Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD_.

Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide very useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays were significantly more heterogeneous and more closely resembled capillaries than Matrigel® tubules. Current co-culture models using primary cells have donor variability, and inconsistent results due to lot to lot variation. In this study, we established an in vitro co-culture model system consisting of an assay ready mixture of an aortic endothelial cell line TeloHAEC-GFP (hTERT immortalized human aortic endothelial cell line) and a hTERT immortalized adipose-derived mesenchymal stem cell line hTERT-MSC in a common specially formulated medium, the AngioReady™angiogenesis medium with VEGF supplement. Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts. The new co-culture system forms functional tubular structures in less than 7 days, and in addition, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining (a marker of smooth muscle cells),indicating that the system has physiological relevance. Next, we tested the new system with compounds that impact angiogenesis, results demonstrated that the angiogenesis system responds positively to elevated doses of VEGF and negatively to increasing concentrations of suramin; more importantly, the tubular formation efficiency is reduced or blocked by well-known cancer drugs such as Sunitinib (SUTENT®) and Bevacizumab (Avastin®), both of which target the VEGF pathway. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots of the Angio-ready™ system with the cancer drug, Ramucirumab (Cyramza®), which also targets the VEGF pathway. Finally, we used the Angio-ready™ system validated 4 HIF-1(hypoxia inducible factors-1) inhibitors which have anti-angiogenic properties identified by high-throughput screening methods; data showed the results of the new system match with other screening methods, what's more, Angio-ready™ system screening time can be as short as 3 days. Therefore, the co-culture models developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying cardiovascular biology, drug screening and tissue engineering.

#357

Rapid harvesting of highly concentrated circulating tumor cells from bulk human blood with buoyant immuno-microbubbles.

Guankui Wang,1 Sandeep Pingle,2 Santosh Kesari,2 Yu-Tsueng Liu,3 Dmitri Simberg1. 1 _University of Colorado, Aurora, CO;_ 2 _John Wayne Cancer Institute, Santa Monica, CA;_ 3 _University of California San Diego, La Jolla, CA_.

Circulating tumor cells (CTCs) are the extremely rare cells shed from primary tumor during metastatic disease. A technology to quickly and inexpensively isolate viable CTCs will enable widespread clinical utilization of CTCs for cancer prognosis and drug testing. The lipid-shelled microbubble is a biocompatible material that has been used as ultrasound contrast media. We have previously shown that MB is a promising vehicle to isolate rare cells from biological fluids by flotation. Here we investigated the role of lipid composition, MB/cell ratio, and type of tumor surface marker [epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erbB-2 or human epidermal growth factor receptor 2 (HER2/neu) and epithelial growth factor receptor (EGFR)] on MB stability and binding efficiency to breast cancer cells. Optimized MBs coated with anti-EpCAM antibody showed 95% binding efficiency to human breast carcinoma SKBR-3 cells. Cells were able to proliferate in culture with MBs attached to them. We further developed an inverted setup in order to harvest (skim) highly concentrated tumor cells in a microliter volume on a nitrocellulose membrane. Using vacuum-assisted harvesting we demonstrated a high efficiency (~80%) of isolation of rare tumor cells spiked in 7 ml blood by anti-EpCAM MBs. A blinded study using anti-EpCAM MBs detected epithelial CTCs in samples of metastatic breast and ovarian cancers with brain metastases, but not in samples from non-epithelial brain malignancies, including primary glioblastoma multiforme, hemangioblastoma and metastatic melanoma. The simple and robust MB approach can be developed into an inexpensive tool for batch CTC isolation in basic research and clinical applications.

#358

In vitro assessment of tumor relapse in a 3D tumor microtissue model.

Sumeer Dhar. _InSphero AG, Schlieren, Switzerland_.

One of the most pressing problems in the treatment of cancer is tumor relapse. Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. Current in vitro assays based on monolayer cultures do not provide the required time frame to study tumor recurrence.

We developed a new method to study tumor relapse in a 3D in vitro tumor microtissue model. HCT116 colorectal tumor microtissues were produced and dosed with 3 reference compounds, Taxol (cell division inhibitor), Staurosporine (protein kinase inhibitor) and Doxorubicin (DNA synthesis inhibitor). Based on an initial range finding to determine IC20 and IC50 values, tumor growth was monitored via size profiling and viability (intra-tissue ATP content) over 10 days. Three different conditions were tested: (i) continuous treated tumor microtissues, (ii) microtissues which were supplemented with the drug for 5 days and (iii) non-treated tumor microtissues. Growth differences couldn't be observed comparing IC20 concentrations, however growth profiles significantly varied comparing IC50 concentrations. Whereas a 5-day treatment of Taxol was leading to a complete growth inhibition after removing the drug, Staurosporine treated tumor microtissues relapsed even with a similar growth kinetic as the untreated control group. Pulsed treatment of Doxorubicin was leading to a slower growth kinetic as compared to the control but not to a complete reminiscence. Based on the individual growth profiles relative to the continuous treated and non-treated groups we calculated a relapse index (ReI, patent pending) to enable classification of compounds and compound combinations according to their risk for tumor relapse.

Here we present a novel in vitro tumor relapse assay method based on a 3-dimensional tumor microtissue model, to enable compound classification based on their efficiency to prevent tumor relapse.

#359

A high-throughput PPI assay to quantify inhibition of androgen receptor variant 7 (AR-V7) binding to canonical AR full-length (AR-FL) binding partners.

Jonathan Branch, Michael Quigley, Marco Gottardis, Ian Hickson. _Janssen Research and Development, Spring House, PA_.

Background: Prostate Cancer is the most common non-skin cancer in the United States, affecting 1 in 7 men over their lifetimes [1]. It is the second most frequent cause of cancer-related deaths for men. Despite significant advancements in the treatment of Prostate Cancer within the past several years, numerous resistance mechanisms have been described for patients administered androgen-deprivation therapy (ADT) [2]. Recently, Antonarakis et al. reported that in patients treated with enzaluatmide or abiraterone acetate plus prednisone, high levels of Androgen Receptor Variant 7 (AR-V7) mRNA in circulating tumor cells (CTC's) were strongly correlated with a lack of clinical response to these drugs [3]. AR-V7 is upregulated following suppression of full-length Androgen Receptor (AR-FL) in vitro [4] and its expression is sufficient to induce canonical and distinct AR-FL downstream pathways [4, 5, 6]. Therefore, development of an inhibitor of AR-V7 activity has become a high-priority in pharmaceutical research. In this abstract, we describe a high-throughput protein-protein interaction PPI FRET-like assay, based on Promega's NanoBiT platform, which is amenable to screening potential inhibitors of AR-V7 binding to AR-FL canonical binding partners. Additionally, we report compounds that antagonize these interactions.

Methods: We developed a set of AR full-length, truncated variants, and binding partners (SRC-1, RAP74) to interrogate PPI interactions. These constructs were tethered to one of two components of Promega's NanoBiT system, in which the binding interaction and kinetics are monitored by alterations in the NanoLuciferase (Oplophorus gracilirostris) signal [7]. This platform was validated using described agonists (R1881) and antagonists (enzalutamide and bicalutamide) of N-terminal and C-terminal AR dimerization. Subsequent experiments evaluated the effectiveness of AR-V7/AR-V7 or AR-V7/SRC1 NanoBiT binding and potential for disruption by small molecules.

Results: We demonstrate that our system is capable of detecting activation and inhibition of AR-NTD and AR-CTD dimerization with either R1881 or R1881 in the presence of enzalutamide or bicaluatmide, respectively. Additionally, we have evaluated inhibition of AR-V7 binding to canonical AR-FL binding partners, SRC-1 and RAP74. These results demonstrate the utility of this platform to screen molecules for their ability to inhibit both AR-FL and splice variant dimerization and the resulting nuclear translocation and activation of downstream pathways.

[1] Prostate Cancer Foundation website.

[2] Sprenger and Plymate, Horm Cancer 2014.

[3] Antonarakis et al., NEJM, 2014.

[4] Hu R, Lu C, Mostaghel EA, et al., Cancer Research, 2014.

[5] Hörnberg et al., PLoS ONE, 2011.

[6] Zhang et al., PLoS ONE, 2011.

[7] Hall et al., ACS Chem Biol., 2012.

#360

Detecting intracellular bRAF/cRAF dimerization using a novel luminescent complementation system.

Richard L. Somberg, Marie K. Schwinn, Michael R. Slater, Thomas Machleidt, Mei Cong, Keith V. Wood. _Promega Corp., Madison, WI_.

BRAF mutations can promote constitutive activation of MEK/ERK signaling, leading to unregulated cell proliferation and tumorigenesis. Although inhibitors targeting oncogenic BRAF have shown promise in preventing cell growth, these same inhibitors can paradoxically activate signaling by inducing BRAF interaction with CRAF. Methods for monitoring this undesired outcome are typically low throughput or use kinase binding domains modified with a membrane targeting sequence. We sought to develop a cell-based assay that could reliably detect dimerization of full-length BRAF/CRAF and could potentially be used to rapidly screen inhibitors for modulation of this interaction. To achieve this objective, we utilized a NanoLuc luciferase-based complementation reporter consisting of 1.3 kD (SmBiT) and 18 kD (LgBiT) fusion tags that reconstitute an active luciferase when brought into proximity. In this optimally configured BRAF/CRAF assay, BRAF-LgBiT and CRAF-SmBiT fusions are stably expressed in mammalian cells, and luminescence, indicative of protein interaction, is monitored using a live cell detection reagent. With this assay, we were able to observe rapid and dose-dependent induction of BRAF/CRAF dimerization in response to a panel of kinase inhibitors. The sensitivity and robustness of the assay permitted reliable screening in both 96- and 384-well formats (Z' factors > 0.9 and 0.6, respectively). Furthermore, the brightness of the reporter allowed the assay to be monitored in individual cells using bioluminescence imaging. These results suggest this BRAF/CRAF complementation assay can enable rapid, high-throughput profiling of kinase inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway.

#361

**Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and** in situ **hybridization in breast cancer cells.**

Lisa J. Ma,1 Richard T. Frank,1 Xiaoxiao Li,1 Steve Lai,2 Jeanette Schmidt,2 Peggy Just,1 Sara Becker-Catania1. 1 _Affymetrix eBioscience, San Diego, CA;_ 2 _Affymetrix, Santa Clara, CA_.

Immunocytochemistry (ICC) utilizes antibodies to visualize the localization of specific proteins in cells while in situ hybridization (ISH) is a useful research tool for examining the presence and changes in levels of gene expression in cells. The ability to simultaneously visualize the co-localization of protein and RNA species in individual cells is necessary to address important biological questions. Additionally, studying both protein and RNA can be useful in cases where expression levels vary between mRNA and protein and to detect post-translational modifications. However, a combined technique has proved challenging due to the incompatibility of ICC and ISH protocols. Furthermore, traditional ISH methods have lacked sensitivity to detect low abundance RNA species and have limited multiplexing capability. Here, we describe a novel technique combining ICC staining with RNA ISH (based on the ViewRNA Assay), which utilizes branched DNA signal amplification technology allowing for single copy mRNA sensitivity. We demonstrate the utility and versatility of this technique using a variety of antibody and probe combinations relevant for cancer research. We compared expression of protein and mRNA of HER2, estrogen receptor alpha, and progesterone receptor in several breast cancer cell lines (MCF7, SKBR3, MDA-MB-175, MDA-MB-231, and BT474) known to express differential levels of these targets. We also compared expression of several microRNAs (let-7a, miR-17, miR-30d, miR-107, and miR-93) known to play a role in cancer development in combination with staining for proteins involved in proliferation (Ki-67), anti-apoptosis (survivin), and cell structure (tubulin). We show successful detection of low abundance RNA species together with various protein markers. This novel technique is a valuable tool not only for cancer researchers, but also for investigators across numerous fields in biological research.

#362

A homogeneous bioluminescent succinate assay for detection of jumonjiC domain-containing histone demethylase activities.

Juliano Alves, Said Goueli, Hicham Zegzouti. _Promega Corporation, Madison, WI_.

JumonjiC domain-containing histone lysine demethylases (JMJCs) play a pivotal role in determining the epigenetic status of the genome, leading to either transcriptional repression or activation of target genes by counteracting the activities of histone lysine methyltransferases. Because of their implication in cancer, they have become validated drug targets. Therefore, assays for this enzyme subfamily are desirable in order to facilitate the identification of selective and potent inhibitors for drug discovery and as basic research tools. Since succinate is one of the products of the demethylation reaction catalyzed by these enzymes, an assay that detects succinate would be suitable for monitoring all JMJC activities as well as other succinate-forming enzymes (i.e.: dioxygenases). Thus, a bioluminescent and homogenous succinate detection assay for measuring JMJC activity was developed, and it is performed in a two-step format that relies on converting the succinate product to ATP, and then to light in a robust luciferase reaction. The light output is proportional to succinate concentration from low nM to 15µM, and the assay is highly sensitive and robust, two features that are highly desirable and essential for measuring the activity of the majority of JMJC demethylase subfamilies. Therefore, the succinate detection assay is a simple-to-use method that does not require antibodies or modified substrates. Examples of various applications of this succinate detection assay will be presented, including studies on specificity of different substrates by diverse JMJCs, as well as mode of action studies using specific inhibitors. The development of this succinate detection assay will make it possible to investigate a large number of JMJC demethylases and could have significant impact on diverse areas of Epigenetics research.

#363

Establishment of multicellular tumor spheroids-based assay for screening of novel therapeutics.

Yeonhwa Song, Haengran Seo. _Institut Pasteur Korea, Seoul, Republic of Korea_.

3D cell culture system is highlighted as new method to screen for new cancer therapies. The multicellular tumor spheroid cultures are closely similar to pathophysiological gradients of in vivo tumors and subsequently an ideal screening model for new drugs. Hepatocellular carcinomas (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Hence, we focused in developing tumor spheroids-based screening assay to identify new drugs to treat HCC.

Tumor spheroid cultures were formed in hydrogel coated multi-well plates within 3 days followed by 3 days treatment with reference compounds (Doxorubicin, Etoposide, Methotrexate, Taxol, etc.). Bright images of tumor spheroids were visualized by Operetta imaging system in the presence and absence of reference compounds. Cell viability of spheroids were measured by resazurin assay.

In the results, assay was developed and validated using multicellular tumor spheroids (MCTS) cultures composed of Huh7 (HCC cells), WI38 (fibroblasts), LX2 (hepatic stellate cells) and HUVEC (endothelial cells) to recapitulate HCC tumor microenvironments. We also addressed the effect of sorafenib on the growth of spheroids composed of Huh7 alone or MCTS. Results from this study indicate that sorafenib was less efficacious (IC50=8.0μM) in the MCTS compared to its effect on Huh7 spheroid (IC50=3.8 μM).

In summary, we have established and validated a highly reproducible MCTS-based assay. Based on this results, MCTS based-HTS system can offer a new model for drug screening and significantly improve the efficiency of identifying new drugs for liver cancer therapy.

#364

Validation of human osteoclast cultures for studying the mode-of-action and identification of new compounds with the potential of inhibiting the vicious cycle in osteolytic bone metastases.

Jenni Bernoulli,1 Jussi M. Halleen,1 Mari I. Suominen,1 Johanna Tuomela,2 Jukka Rissanen,1 Katja M. Fagerlund1. 1 _Pharmatest Services Ltd., Turku, Finland;_ 2 _University of Turku, Turku, Finland_.

When tumor cells home to bone microenvironment they secrete factors that stimulate osteoclast formation, which results in increased bone resorption. This in turn increases the release of factors from bone matrix that stimulate the growth of tumor cells, leading to a vicious cycle characterized by extensive bone loss and enhanced tumor growth in bone. Thus, factors that inhibit osteoclast formation or bone resorption activity of mature osteoclasts may have the potential to inhibit the vicious cycle. The RANKL inhibitor denosumab is approved for treatment of bone metastases from solid tumors, and the cathepsin K inhibitor odanacatib has been shown to suppress bone resorption in breast cancer patients with bone metastases. Human osteoclasts can be generated from bone marrow-derived CD34+ mesenchymal stem cells in the presence of M-CSF and RANKL. In this study we report optimization of separate in vitro culture systems for determining osteoclast differentiation and activity, and validation of denosumab and odanacatib as reference inhibitors of osteoclast differentiation and activity, respectively. CD34+ human osteoclast precursor cells were cultured on bovine bone slices for 7 days. Different concentrations of denosumab (0.01 - 10 μg/ml) were added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b activity (TRACP 5b) was measured from the culture medium collected at day 7 as an index of the number of formed osteoclasts. Osteoclast activity was studied by allowing the formed mature osteoclasts to resorb bone during an additional 3-day culture period. The culture medium was changed and different concentrations of odanacatib (0.001 - 1.0 μM) were added into the cultures at day 7, and the amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate bone resorption during days 7-10. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively, with EC50 values of 0.124 μg/ml for denosumab and 0.0433 μM for odanacatib. We conclude that we have validated denosumab as a reference compound of osteoclast differentiation and odanacatib as a reference compound of osteoclast activity in a human in vitro osteoclast culture system. The culture system is a clinically reliable tool for identifying new compounds affecting the vicious cycle of osteolytic bone metastases, and clarifying if these active compounds target directly osteoclast differentiation or activity.

#365

High throughput screen to evaluate combinations with ibrutinib in various B-cell malignancies.

Shalini Chaturvedi,1 Michael Schaffer,1 Cuc Davis,1 Regina Aquino,1 Emily Stepanchick,1 Matthias Versele,2 Sriram Balasubramanian1. 1 _Janssen R &D, Spring House, PA; _2 _Janssen R &D, Beerse, Belgium_.

The primary objective of this study was to discover synergies leading to mechanistic insights and novel combinations for ibrutinib, a small-molecule inhibitor of Bruton's tyrosine kinase (BTK). Ibrutinib has been approved for relapsed/refractory (R/R) and del(17p) chronic lymphocytic leukemia, and R/R mantle-cell lymphoma (MCL). BTK is part of the B-cell receptor (BCR) signaling pathway, so it is of interest to examine synergy between ibrutinib and agents that target other aspects of the BCR pathway. These include PI3K and IRAK inhibitors and apoptosis inhibitors. Combinations were evaluated in a high-throughput, tumor microenvironment-directed format.

Ibrutinib was combined with inhibitors of the BCR pathway and apoptosis, whose targets included MCL-1, BCL2, XPO1, and isoforms of PI3K, IRAK, and BRD. Histologies examined included follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), MCL, acute myeloid leukemia (AML), acute B-lymphoblastic leukemia, and Burkitt's lymphoma. Cell lines were screened in the presence of human marrow stromal-cell-conditioned media and B-cell receptor stimulation via anti-IgG/anti-IgM antibodies in a 72-hour cell viability ATP lite assay (384-well plate, 9×9 optimized matrix, 4 replicates). Dose response matrix screening was used to measure combination effects, which manifest as potency shifts or efficacy boosts. Combination effects can be characterized by comparing each data point to a combination reference model derived from single-agent curves using the Loewe additivity model.

Ibrutinib demonstrated varying activity across cell lines. Combination activity was classified based on synergy score raw values. The best combination among tested compounds was with ABT-199, a BCL2i. This combination showed high or medium synergy in 2/5 DLBCL, 3/4 FL, and 2/5 MCL cell lines; this was also the only agent to show good synergy in AML (3/5 lines). Interestingly, ABT-737, a BCL2i that also targets BCL-XL, has previously been shown to be synergistic with ibrutinib in DLBCL lines. Synergy was also seen with other apoptotic agents such as MCL-1i and the epigenetic BETi JQ-1 across B-NHL, but in fewer lines. Among the PI3Ki, PI3Kα/δi did not show much activity, but the PI3Kδ/γi IPI-145 was synergistic in 2/5 DLBCL, 3/4 FL cell lines, and 1/5 MCL cell lines, a pattern very similar to the PI3Kδi CAL-101 (idelalisib). Ibrutinib also combined well with IRAK1/4i and XPO1i selinexor, with high activity in 1/5 DLBCL cell lines each, and medium activity in 1/5 DLBCL, 1/4 FL, and 2/5 MCL (XPO1i) and 1/5 DLBCL, 2/4 FL, and 1/5 MCL cell lines (IRAK1/4i).

Synergy was mostly observed in B-cell malignancies, but interesting synergy was observed with ABT-199 in AML. In B-NHL, ibrutinib combined with BCL2i and PI3Ki showed the best combination activity. Ibrutinib was also synergistic with other agents in selected B-NHL lines with no observed antagonism, suggesting that further study in specific histologies is warranted.

#367

Assessing the therapeutic efficacy of Cyst(e)inase to induce oxidative stress mediated cytotoxicity in pancreatic cancer cells.

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

Pancreatic ductal adenocarcinoma is the only cancer in the US with a dismal one-digit 5-year survival rate (7%). Its oncogenic program relies on upregulated ROS antioxidant mechanisms to maintain oxidative balance. Hence, perturbation of this balance might be an effective therapeutic strategy for pancreatic cancer. A major intracellular antioxidant is the tripeptide glutathione (GSH). Cysteine (Cys), which has the functional moiety of GSH, can either be synthesized de novo or imported [predominantly as cystine (CSSC) that is reduced intracellularly to Cys]. In cancer, de novo synthesis of Cys has to be supplemented with extracellular import in order to fulfil the excessive metabolic demand of proliferation. This intricate balance between ROS and antioxidants, and the excessive requirement for Cys/CSSC import in tumor cells, in part to maintain oxidative balance through GSH synthesis, potentially provides a therapeutic window. We believe that prolonged depletion of the serum Cys and CSSC pool by a genetically engineered human enzyme called Cyst(e)inase may provide a pancreatic cancer therapeutic avenue either as a monotherapy or in combination with other agents with low or minimal toxicity.

In this study, Cyst(e)inase treatment (3-500 nM) of cultured pancreatic cancer cell lines (MIA-PaCa2, BxPC3, Panc1) caused a dose-dependent decrease in survival and intracellular GSH content. Further mechanistic investigation showed an increase in intracellular ROS, activation of AMP kinase and autophagy signaling, as well as inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway. There was also a concentration dependent reduction in cyclin D1 after treatment with the enzyme. Cyst(e)inase displayed synergistic cytotoxicity when combined with the natural compound curcumin. Notably, a single intraperitoneal 100 mg/kg dose of PEGylated Cysteinase in mice was able to deplete serum CSSC below 5 μM (normal is ~ 70 μM) for over 3 days with a half-life of ~4 days. Longer treatment with Cyst(e)inase at this dose given 4 times/week for 6 weeks did not produce significant signs of toxicity. This in vivo dose also inhibits the growth of prostate cancer cells in xenograft tumor models. Ongoing experiments to evaluate the in vivo efficacy of Cyst(e)inase for inhibition of pancreatic tumor growth as well as further mechanistic studies will be presented. Collectively, the current data suggest that depletion of extracellular Cys/CSSC using Cyst(e)inase may have utility either as a monotherapy or a combination therapy for several cancers including pancreatic cancer.

#368

Specific chromosomic alterations confer therapy resistance in a cohort of 49 patients with newly diagnosed acute myeloid leukemia treated with intensive chemotherapy.

Cristina Papayannidis, Maria Chiara Fontana, Giovanni Marconi, Viviana Guadagnuolo, Giorgia Simonetti, Antonella Padella, Simona Soverini, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Silvia Lo Monaco, Marco Manfrini, Elisa Zuffa, Eugenia Franchini, Claudia Venturi, Maria Teresa Bochicchio, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Giovanni Martinelli. _Inst. of Hematology 'Seragnoli', Bologna, Italy_.

Introduction. Intensive induction chemotherapy in non-M3 young Acute Myeloid Leukemia (AML) patients is represented by the association of an antracycline and Cytarabine. Some treatment regimens including fludarabine or the addition of Gemtuzumab Ozogamicin (GO) as a third or fourth drug, proved to give a benefit in terms of CR rates.

Aims of the study. In a group of 49 patients treated with intensive chemotherapy, we evaluated chromosomal abnormalities with SNP 6.0 and Cytoscan HD (Affymetrix) in order to improve conventional cytogenetic analysis and discover novel chromosomic aberrations related to clinical data and therapy response.

Patients and Methods. From 2001 to 2014, 489 patients were treated in our Institution. Among those, in 49 newly diagnosed AML patients (median age 54 (range 19-71)), SNP microarray based-genotyping were performed and then analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team.

According to karyotype, FLT3 and NPM1 mutational status, 55.9% of the patients were considered at High Risk (HR) and 4.1% at low risk (LR). Ten patients had secondary AML. Patients were treated with induction schemes including MyFLAI, MyAIE, FLAI, FLAN, FLAG, 3+7 or DAE.

Results. The CR rate after induction was 87.8% (43/49 patients). Deaths during induction (DDI), occurring in the first 50 days from 1st line therapy, were 1/49. The median OS was 135 months, the 5-years OS in our patients was 55,1%. Patients treated with GO showed a non-statistical trend toward a better OS than patients treated with other regimens (median OS not reached vs 133 months, respectively).

We explored the alterations found by SNP array in our patients searching for novel markers of therapy resistance. We found a median of 192,5 total copy number aberrations (range 72- 1071): a median of 145,5 total copy number aberrations in responding patients group (RPG), and a median of 361 total copy number aberrations (p=ns) in non-responding patients group (NRPG).

We compared the frequency of detected aberrations in RPG and in NRPG with Fisher's exact test. We found that PIK3CA, Gain chr3:178,927,088-178,929,550 (p=0,0016), SMAD4, Gain chr18:48,573,154-48,573,255 (p=0,0166) and several other gene's loci (CASC18, TCF12, UTY, GRB10, ZFY) are significant aberrations in NRPG compared with RPG.

Conclusions. We identified a number of genes with significant aberrations in NRPG, particularly PIK3CA, a protein-coding gene involved in cell proliferation and metabolic pathway with interaction with HRAS/KRAS and EGF, and SMAD4, a transcription factor activated by TGF-beta. Those 2 genes were found overexpressed in other solid tumors.

We suppose that those genes may be involved in a hyper-proliferative pathway that underlies a mechanism of chemo-resistance.

Acknowledgments Work supported by ELN, AIL, AIRC, Progetto Regione-Universit^ 2010-12 (L.Bolondi), FP7 NGS-PTL project.

#369

Automating recovery of spiked tumor cells from blood.

Mark Sarinana, Ky Truong, Keith Cannon, Antonio Guia. _Aviva Biosciences, San Diego, CA_.

The isolation and study of circulating tumor cells (CTC) enables personalized medicine solutions, monitoring of therapeutics (theragnostics), and monitoring of minimal residual disease (MRD); culturing these rare cells is a frequent goal in cancer research, however the common methods for their isolation (gradient centrifugation, lysis buffer) are damaging to these cells, and many emerging technologies leave the cells bound in a consumable, bound to a biomarker or in a state that is difficult to use in cell culture. Further, the anuclear cells in blood greatly outnumber nucleated cells and present significant challenges to the study of the nucleated cells, for example, erythrocytes can absorb excitation or emission wavelengths in fluorescence studies, and their shape promotes rouleaux formation which can sequester the cells of interest; platelets also have a tendency to be activated by physical handling and cause aggregation that can also sequester and damage the cells of interest, and these all contain the greatest content of RNA and other markers that increase background noise in many assays. We demonstrate a new technology that automates the depletion of erythrocytes, thrombocytes, and plasma markers from, initially, small volumes of blood, using a micromachined membrane filter. We spiked small counts (10 to 1000 estimated from dilution) of tumor cell lines BT474 (breast cancer), DLD1 (colorectal cancer), and K562 (myelogenous leukemia) and other cell types into aliquots of blood. Using calcein-AM-loading to distinguish the spiked cells from blood cells, the recovery rates were determined in a flow cytometer, indicating an average of 80% ±4% (SE, N=27) recovery of the rare cells after depletion of anuclear cells and plasma. The technology is amenable to use of beads for initial negative depletion of common nucleated cells by magnetic beads and has a recovery capacity of a few to as much as 3 million cells per specimen. Tumor cell lines alone that were run through this automation process then seeded for cell culture demonstrated growth rates indistinguishable from those seeded in similar counts in a regular cell passage. In further studies we demonstrate depletion of leukocytes with magnetic beads and of anuclear cells by filtration to handle larger blood volumes. The filtration system may be used for automated recovery of nucleated cells or cell clusters from blood or other fluid samples (liquid biopsies) without use of harsh chemicals or centrifugation, removing a common barrier for emerging analytical approaches.

### PI3K/AKT Inhibitors

#370

The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action.

Kyle A. Edgar, Kyung Song, Stephen Schmidt, Don Kirkpatrick, Lilian Phu, Michelle Nannini, Rebecca Hong, Eric Cheng, Amy Young, Deepak Sampath, Lori S. Friedman. _Genentech, South San Francisco, CA_.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha sparing inhibition of p110beta, is investigated in these preclinical studies.

Taselisib demonstrates greater potency in cancer cell lines harboring activating mutations in PIK3CA vs. wild-type lines, and induces regressions at tolerated doses in both PIK3CA mutant xenograft and patient-derived xenograft (PDX) models. When comparing taselisib to other clinical-stage PI3K inhibitors at Maximum Tolerated Dose (MTD) in vivo, taselisib confers greater activity in PIK3CA mutant models, which may indicate a larger therapeutic index. Unlike other PI3K inhibitors, taselisib has a gain of potency in PI3K alpha mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PI3K alpha mutant cells. Other clinical PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not have improved potency in PI3K alpha mutant cells due to their inability to maintain pathway suppression after alleviation of negative feedback. The unique mechanism of action of taselisib is most notable when comparing signaling suppression at 24 hours vs. 1 hour of drug exposure. In mutant cells, Taselisib displays greater pathway suppression at 24 hours and is more effective at maintaining pathway suppression upon re-activation of growth factor RTK signaling. This mechanism is specific to PIK3CA mutant cells and not observed in wildtype cells. Ongoing studies to further elucidate this mechanism of action will be presented.

#372

Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines.

Heidi M. Savage, Carol O'Brien, Heather Moore, Mark R. Lackner, Robert Yauch, Jeffrey Settleman, Timothy R. Wilson. _Genentech, Inc., South San Francisco, CA_.

The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC 0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013).

To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to taselisib, we profiled over 550 cell lines, encompassing 13 of the main tissue types, to increasing concentrations of taselisib. As expected, a small percentage of all tumor types responded to taselisib and correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 0.5uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Interestingly, the majority of HNSCC cell lines displayed an activated PI3K pathway as evidenced by phosphorylation of key proteins, such as AKT, S6 and PRAS40 implicating that downstream phospho-protein analysis of the PI3K pathway may not predict for sensitivity to taselisib. Mutational profiling identified that only 3 out of the tested 31 HNSCC cell lines harbored a mutation within PIK3CA, suggesting that additional genotypes may explain the sensitivity to taselisib. Additional molecular analysis assessing both gene expression and copy number levels of genes relevant to the PI3K pathway found that many of the taselisib sensitive cell lines had an activated ERBB signaling node through low level amplification of ERBB receptors (e.g. EGFR, FGFR1) or via a NRG1:ERBB3 autocrine mechanism. As many ERBB receptors heterodimerize with ERBB3, which contains p85 binding sites and potentially activates the PI3K pathway, we next tested whether taselisib would enhance the potency of ERBB receptor inhibitors in biomarker-defined subset of HNSCC cell lines. Combination screens were performed in cell lines with amplified EGFR, ERBB2, and NRG1:ERBB3 autocrine signaling with taselisib + tarceva or lapatinib. We found that combination effects assessed using the BLISS excess method showed a synergistic interaction. These results suggest that taselisib may have therapeutic potential for the treatment of HNSCC.

#373

Discovery of selective PI3Kb inhibitors for PTEN-deficient tumors and other indications.

Arnab R. Chowdhury, Jeyaraj D. Athisayamani, Sayan Mitra, Srinivas Maddi, Ram Sudheer Adluri, Sridhar R. Bethi, Hemant Joshi, Vijaya G. Tirunagaru, Jang B. Gupta. _GVK Biosciencies, Hyderabad, India_.

PTEN somatic mutations occur in a large percentage of human cancers, from breast to prostate. Deletions of PTEN e.g. in prostate cancers are associated with tumor aggression and poor outcome, and up to 70 percent of prostate cancer patients have lost one copy of the PTEN gene at the time of diagnosis. Recent pharmacologic and genetic studies have identified the β isoform of PI3K to be required for growth of PTEN-deficient tumors in vivo and highly potent compounds are currently being explored in the clinic. These clinical candidates, however, are either limited by poor bioavailability or moderate selectivity against the delta isoform, thus warranting more selective compounds with better exposure. We have undertaken a comprehensive knowledge-based discovery of selective inhibitors of PI3Kβ with admissible pharmacokinetic and drug like properties. We have successfully identified a chemical series with potency ranging as low as 2.5 nM for PI3Kβ with >100 - fold selectivity for PI3Kδ. The α and γ isoform selectivity was around 1000 and 500 fold respectively. Compounds did not decrease insulin sensitivity and T cell proliferation thus mitigating the PI3Kα and PI3Kδ mediated side effects of nonselective inhibitors. In vitro biochemical potency translated into cell survival assay and pAKT target engagement in recombinant and wild type mammalian cell lines. In vivo tumor regression was observed in xenograft models of prostate cancer. The series did not show any observable hERG or CYP liabilities. The considerable potency, high selectivity and good bioavailability of this novel class of compounds not only makes them attractive for prostate cancer, but also for rare disease indications like hamartoma and other drug resistant cancers that are dependent on PTEN deletions.

#374

In vitro and in vivo anti-tumor activity of ARQ 751, a potent and selective AKT inhibitor.

Yi Yu, Ronald E. Savage, Sudharshan Eathiraj, Terence Hall, Brian Schwartz, Giovanni Abbadessa. _ArQule, Inc., Burlington, MA_.

Dysregulation of the PI3K-AKT signaling pathway is associated with a number of cancers. AKT can be activated through activated receptor tyrosine kinases, gain-of-function mutations of PIK3CA, PTEN deficiency, and AKT amplification or activating mutations such as AKT1-E17K. We present here the preclinical characterization of ARQ 751, which has distinct physico-chemical properties compared to our first generation inhibitor, ARQ 092.

ARQ 751 has IC50 values of 0.55 nM, 0.81 nM and 1.31 nM for AKT1, 2 and 3, respectively, and does not inhibit any other kinase (out of the 245 tested) by greater than 50% at 5 µM, nor does it inhibit AKT lacking the PH domain. ARQ 751 strongly binds to wild-type AKT1 and mutant AKT1-E17K with Kd of 1.2 nM and 8.6 nM, respectively, and suppresses pAKT(S473) in 293T cells transiently transfected with AKT1-E17K. Additionally, membrane translocation of both wild-type and AKT1-E17K is inhibited in NIH 3T3 cells transiently transfected with wild-type AKT1 or mutant AKT1-E17K. Tests on 240 cancer cell lines (Oncopanel) showed the best antiproliferative effects on esophageal (100%, 3/3), breast (87.5%, 14/16), and head and neck cancer cells (67%, 4/6), with GI50 values GI50 < 1 µM. Cancer cell lines with PIK3CA/PIK3R1 mutations (73%; 33/45) are more sensitive to ARQ 751 (GI50<1µM) compared to wild-type (42%, 74/175). Interestingly, among PIK3CA/PIK3R1 wild type cell lines, the PTEN mutant (55%, 11/20) exhibited a similar sensitivity to PTEN wild-type cell lines (48%, 95/197). Of 17 tested breast cancer cell lines, all 8 with PIK3CA mutations are sensitive to ARQ 751. An in vivo efficacy study in AN3CA endometrial cancer xenograft model shows that ARQ 751 inhibits tumor growth (by up to 92% at the well tolerated continuous daily dose of 120 mg/kg) in a dose-dependent manner. Additionally, ARQ 751 exerted dose-dependent anti-tumor activity (by up to 98% at the 5 days on, 2 days off dose of 75mg/kg) in an AKT1-E17K mutant endometrial patient-derived xenograft (PDX) model. ARQ 751 causes significant pathway inhibition in vitro (at the concentrations of 3 nM on pAKT[S473] and 70 nM on pPRAS40 [T246]) and in vivo (on both pAKT[S473] and pPRAS40[T246] after 6 hours of dosing as low as 10 mg/kg) using AN3CA models. Pharmacokinetic data from repeat doses of ARQ 751 show a plasma half-life of 4 to 5 hours and no tissue accumulation.

In conclusion, ARQ 751 is a potent and selective allosteric AKT inhibitor. PIK3CA/PIK3R1 and AKT mutations but not PTEN may predict ARQ 751 sensitivity. Our analysis suggests that endometrial, breast, esophageal, and head and neck cancers may represent viable indications for ARQ 751. Both PIK3CA/PIK3R1 and AKT1 mutations could be predictive biomarkers for patient selection, regardless of tumor type.

#375

Combination of MEK and PI3K inhibition in BRAF wild-type and mutant melanoma.

Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. _St. Louis University, St. Louis, MO_.

Introduction: Targeted therapy against BRAF-mutated melanoma with BRAF and MEK inhibitors has shown clinical efficacy. However, resistance to therapy occurred and lead to therapy failure. We evaluated the effect of combining downstream inhibition of RAS/RAF pathways with MEK (AZD6244) and PI3K (XL765) inhibition on BRAF wild-type and mutant melanoma with variable expression of NRAS as possible therapeutic option.

Methods: Fifteen melanoma cell lines were screened for BRAF mutation status and NRAS expression. BRAF wildtype (Mel628, Mel1098) and BRAF-mutated (Mel1861, Mel1890) cell lines were selected. Drug concentration study with AZD6244 (A, 2.5-40 uM) and XL765 (X, 2.5-40 uM) were performed on Mel628 cells. Subsequently, cells were treated with A (5 uM), X (5 uM), or combination. Cell proliferation assay was performed using Cell Titer Blue assay. Western blotting was performed to for expression of PARP, caspase-9, LC3A/B , Beclin1, pMEK/MEK, pERK/ERK, p-P70S6/P70S6, and p4EBP1/4EBP1. GAPDH was used as internal control. Apoptosis was analyzed using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Cell migration assay was performed by artificial wounding of melanoma cell monolayer and observing migration of cells into the wound up to 72 hours. Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p< 0.05 was considered to be statically significant.

Results: There was dose-dependent inhibition of melanoma cell proliferation with both A and X. Combination A and X had synergistic effect on the anti-melanoma proliferative effect of BRAF mutated cells regardless of expression level of NRAS. Significantly increased apoptosis by Annnexin V assay was detected in BRAF mutated cells (Mel 1860 and Mel1890). Significantly increased expression (p<0.05) of apoptosis markers (PARP and caspases-9) were observed in cells treated with A+X compared with A or X alone. Enhanced phosphorylation of ERK and P70S6 were observed in BRAF mutated cells. No significant difference in autophagy markers (LC3A/B and Beclin1) were observed between A, X, or combination. Variable to minimal additive effect of the combination treatment was observed in BRAF wild-type cells in terms of proliferation, apoptosis, and autophagy. The combination of A and X significantly inhibited melanoma migration compared with either drug alone regardless of BRAF mutation status in the wounding assay model.

Conclusion: We have observed a synergistic effect on suppressing BRAF-mutated melanoma proliferation with combined inhibition of MEK and PI3K. The effect is not dependent on NRAS expression levels of melanoma.

#376

Combination of duvelisib with either ibrutinib or dexamethasone prevents mTOR-dependent feedback in aggressive B-cell lymphoma cell lines.

Kerry White, Erin Murphy, Kerrie Faia, Jennifer Proctor, Nicole Kosmider, Melissa Pink, Stanley Goldstein, Karen McGovern, Jeffery L. Kutok. _Infinity Pharmaceuticals, Inc, Cambridge, MA_.

Background

Duvelisib (IPI-145), an oral dual inhibitor of PI3K-δ and PI3K-γ, is in clinical development for the treatment of hematological malignancies. We previously reported on a high throughput combination screen evaluating duvelisib in combination with standard of care and emerging agents in lymphoma cell lines. Results showed dexamethasone and ibrutinib have significant synergy with duvelisib in selected diffuse large B-cell and follicular lymphoma cell lines. Based on these results we sought to better understand the mechanism of this synergy.

Methods

Duvelisib alone and in combination with dexamethasone or ibrutinib was evaluated in DoHH2 and SU-DHL4 lymphoma cell lines and xenograft models. Cellular signaling was measured by western blot and flow cytometry using phospho-specific antibodies. In vitro, specific inhibitors of ERK (SCH-772984), pan-PI3K (GDC-0941), PI3K-mTOR (PF-04691502), mTORC1 (Rapamycin), and mTORC 1/2 (AZD-8055) were used to evaluate the contribution of these pathways to potential feedback loops. Apoptosis was measured by caspase 3/7 activation, PARP cleavage, and by western blotting and qRT-PCR for pro- and anti-apoptotic Bcl-2 family members after duvelisib or combination treatment.

Results

Western blotting revealed that cell lines treated with duvelisib alone showed inhibition of phosphorylated (p)AKT at serine 473 only out to 12 hours, with mTORC2 dependent re-phosphorylation of AKT evident at 24 hours. Combination with dexamethasone or ibrutinib prevented this reactivation and also inhibited downstream signaling effectors pPRAS40 and pS6. Combination of duvelisib with dexamethasone also significantly reduced p-4EBP1, a regulator of cap dependent translation initiation, leading to the decreased presence of c-MYC 6 hours after treatment. Enhanced caspase 3/7 activation was seen in both cell lines 4-6 hours post combination treatment compared to monotherapies and was accompanied by a decrease in anti-apoptotic MCL-1 protein and an increase in pro-apoptotic BIM protein. These changes correlated to the appearance of cleaved PARP and eventual cell death. In support of the in vitro studies, in vivo studies with xenografts generated from these cell lines revealed that duvelisib in combination with either dexamethasone or ibrutinib led to greater tumor growth inhibition compared to single agent administration.

Conclusions

Synergy between duvelisib and dexamethasone or ibrutinib was observed in aggressive lymphoma cell lines. Mechanistic studies revealed a combined effect on apoptotic pathway family members and inhibition of mTOR signaling, which blocked both reactivation of pAKT and translation initiation. These data provide a rationale for exploring these combinations clinically and suggest that suppression of mTOR-driven survival signaling may be an important mechanism for the observed combination synergy.

#377

Novel mTOR inhibitor MLN0128 inhibits imatinib-resistant GIST more potently than rapalogues by abrogating AKT and 4EBP1 activation.

Thomas Mühlenberg,1 Julia Ketzer,1 Jonathan A. Fletcher,2 Sebastian Bauer1. 1 _Universitätsklinikum Essen, Essen, Germany;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Introduction:

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of the KIT or PDGFRA receptor tyrosine kinases. Most patients respond to the KIT/PDGFRA inhibitor imatinib (IM) but eventually progress due to secondary resistance mutations in KIT. Development of salvage treatments is hampered by the genomic heterogeneity of resistance mutations as single KIT-inhibitory drugs do not inhibit all mutants. The PI3K pathway is strongly activated in GIST regardless of secondary KIT mutations and thus provides a rational target for therapeutic combinations. Rapamycin-analogues have yet shown limited clinical benefit in GIST but next-generation mTOR inhibitors have not been tested in GIST. Here we evaluated the effects of the novel mTOR inhibitor MLN0128 alone and in combination with other kinase inhibitors in GIST.

Methods:

Three IM-sensitive (IM-S; GIST-T1, GIST882, GIST430) and 2 IM-resistant (IM-R; GIST430/654, GIST48B) cell lines were studied. Cells were treated with MLN0128 alone and in combinations with KIT inhibitors (IM, sunitinib, regorafenib), MEK1/2 inhibitor trametinib, and everolimus. Cell viability was evaluated by Sulforhodamin B (SRB) assay after 3 or 6 days of treatment. Biological consequences of on KIT, KIT-dependent signaling intermediates and apoptosis were evaluated by immunoblotting.

Results:

In cell viability assays MLN0128 displayed IC50 values between 15nM (GIST-T1) and 30nM (GIST48B). Immunoblots revealed inhibition of phosphorylation of ribosomal protein S6 (as marker for mTOR activation) at 1nM with complete inhibition at 50-100nM in all cell lines. In contrast to everolimus, MLN0128 did also abrogate phosphorylation of 4E-BP1 at 50-100nM. At these concentrations we also observed strong inhibition of AKT phosphorylation (except in GIST48B), upstream of mTOR, while phosphorylation of KIT remained unchanged. A notable induction of ERK1/2 phosphorylation in response to MLN0128 treatment suggested a feedback loop activating the MEK/ERK signaling. Combinations of MLN0128 with the clinical MEK1/2 inhibitor trametinib as well as with different KIT inhibitors exhibited additive effects.

Conclusions:

MLN0128 has strong antiproliferative effects in IM-sensitive and IM-resistant cell lines in the low nanomolar range, including KIT-negative GIST. The distinct inhibitory profile, inhibition of AKT as well as 4E-BP1, suggests superior clinical efficacy compared to first- and second-generation rapalogues. Combinations with approved KIT and MEK inhibitors display additive effects and may represent a feasible clinical strategy, which warrants further investigation.

#378

Exploring intermittent dosing schedules for the Pan PI3K/mTor inhibitor PQR309.

Karin Jorga,1 Debora Schmitz,2 Natasa Cmiljanovic,2 Doriano Fabbro,2 Sasa Dimitrijevic2. 1 _KarinJorga GmbH, Basel, Switzerland;_ 2 _PIQUR Therapeutics AG, Basel, Switzerland_.

Introduction

PQR309 is a novel oral balanced dual pan-PI3K and mTOR inhibitor currently in clinical development for the treatment of solid tumors and hematological malignancies. Eighty mg PQR309 given once daily (q.d.) has been established as the MTD in patients with advanced solid cancers. This dose and regimen was associated with plasma concentrations that were pharmacologically active in pre-clinical experiments. However, there is emerging evidence that continuous inhibition of PI3K/mTOR may not be needed to achieve the full effect of PQR309 on tumor growth inhibition and therefore further intermittent dosing regimens will be explored.

Methods

In vivo xenograft studies in nude male rats bearing PC3-derived tumors were conducted with continuous and intermittent dosing schedules to estimate effective plasma concentrations. Pharmacokinetic (PK) data from cancer patients were used to establish a 2-compartment PK model with first order absorption. The model was applied to simulate PK profiles after various intermittent dosing schedules including 2 daily administrations followed by a 5 day wash-out period in a 7 day cycle (2 days ON/5 days OFF) and a regimen with administrations on days 1 and 4 in a 7 day cycle (eg MON/THUR).

Results

A maximum plasma concentration (Cmax) of 800-1200ng/mL PQR309 and an impulse interval of 72 hours or less was associated with maximum effect in the rat xenograft model. In rats PQR309 is rapidly eliminated from plasma but in humans the compound has an elimination half-life of about 40 hours and accumulates upon multiple dosing. The applied model predicts that PQR309 administration given 2 days ON/5 days OFF will build up drug exposure to reach levels that strongly inhibit the targeted signalling pathways. During the 5 day wash-out period, PQR309 levels will decrease significantly, thereby reducing the probability of adverse events. For the alternative dosing regimen MON/THUR, the model predicts a sharp Cmax lasting for a few hours followed by a decrease in drug levels over the following 3 or 4 days, thus minimizing accumulation and drug exposure that could lead to adverse events.

Conclusions

Both intermittent regimens are expected to strongly inhibit the targeted signalling pathways with high transient concentrations around Cmax followed by wash-out periods for healthy tissue recovery to optimize the benefit/risk of the compound. These regimens are currently being explored in clinical studies.

#379

Allosteric AKT1/2-inhibitor BAY 1125976 as potent inhibitor in luminal breast cancer resistant to antihormone therapy.

Oliver Politz, Lars Baerfacker, Stuart Ince, Andrea Haegebarth, Ningshu Liu, Roland Neuhaus, Ulf Boemer, Martin Michels, Karl Ziegelbauer, Dominik Mumberg. _Bayer Pharma AG, Berlin, Germany_.

The PI3K-AKT-mTOR signaling cascade is one of the major drivers in the development of cancer. It is constitutively activated in many types of cancers and is one of the prominent pathways that promote tumor cell survival and confers resistance to antihormonal therapies for patients with breast cancer.

Breast cancer has been classified into at least four distinct subtypes, based on molecular profiling. Luminal-B breast cancer, although still expressing the hormone receptor, has been identified as relatively insensitive to endocrine therapy and is an entity with highest need for novel treatments and combination approaches. Despite the notable improvements in endocrine therapy, the invariable appearance of endocrine resistance, either primary or secondary, remains an important issue in this type of tumor. Main cancer signaling pathways, including PI3K/Akt/mTOR and CCND1/CDK4-6, are thought to play an important role in development of this resistance.

Therefore AKT is considered an attractive drug target for the treatment of breast cancer. BAY 1125976, an orally active, potent, highly selective, allosteric AKT1/2 inhibitor is currently in phase I clinical development (NCT01915576). BAY 1125976 is particularly effective in preclinical models with PI3K-AKT pathway aberrations and luminal B status as shown by profiling in a panel of tumor cell lines as well as respective in vivo studies. The efficacy of BAY 1125976 in inhibition of cell proliferation is correlated with luminal status of the tumor as shown in several cell line panels. In vitro combination with anti-hormonal therapeutics showed synergistic anti-proliferative effects and rendered resistant cell lines sensitive towards tamoxifen or fulvestrant treatment. In the MCF-7 cell line tamoxifen combined with BAY 1125976 resulted in a 14 fold reduction of the IC50 for inhibition of cell proliferation compared to monotherapy. This translated into additive to synergistic activity in combination with tamoxifen in a ER+ MCF7 (PIK3CAE545K) BC model and enabled the use of alternative dosing schedules with improved efficacy versus monotherapy.

BAY 1125976 also showed potent inhibition of tumor cell growth in a tamoxifen- and fulvestrant-resistant derivate of MCF-7 enabling a reduction of the therapeutic dose of BAY 1125976 and thereby improving tolerability while keeping efficacy.

Combination of the allosteric AKT inhibitor BAY 1125976 therefore provides an interesting opportunity in improving efficacy of antihormonal therapy in luminal B type breast cancer.

#380

The dual PI3K/MTOR inhibitor PQR309 is active in mature B cell lymphoma cell lines bearing resistance to the PI3K-delta inhibitor idelalisib and specific gene expression features.

Tarantelli Chiara,1 Eugenio Gaudio,1 Ivo Kwee,1 Alberto Arribas,1 Andrea Rinaldi,1 Elena Bernasconi,1 Luciano Cascione,1 Petra Hillmann,2 Anastasios Stathis,3 Laura Carrassa,4 Massimo Broggini,4 Georg Stussi,3 Doriano Fabbro,2 Emanuele Zucca,3 Vladimir Cmiljanovic,2 Francesco Bertoni1. 1 _Institute of Oncology Research - IOR, Bellinzona, Switzerland;_ 2 _PIQUR Therapeutics AG, Basel, Switzerland;_ 3 _IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland;_ 4 _IRCCS-Instituto di Ricerche Farmacologiche Mario Negri, Milan, Italy_.

Introduction The PI3K-delta idelalisib is among the most promising novel agents for lymphomas, but certain patients present a primary resistant disease or relapse after an initial response. Here, we studied the dual PI3K/MTOR inhibitor PQR309 in lymphoma cell lines that were resistant to idelalisib.

Methods IC50 values were calculated with the MTT assay (72h) for 40 lymphoma cell lines. Gene expression profiling was done with Illumina HumanHT12 Expression BeadChips, data mining with Limma, GSEA and LINCSCLOUD.

Results PQR309 and idelalisib showed only a partially overlapping pattern of activity (R=0.6). While no cell line was sensitive to idelalisib and not to PQR309, 10 cell lines were sensitive to both compounds and 7 were sensitive only to PQR309. Idelalisib median IC50 was 28 nM (95%CI; 13-147) in the PQR309/idelalisib group and 13.4 μM (7.2-16.7 μM) in the PQR309-only (P 0.0006). PQR309 median IC50 was 113 nM (33-166 nM) in PQR309/idelalisib sensitive and 193 nM (111-372 nM) in the PQR309-only sensitive cells (P 0.03). No differences between the two groups were observed in terms of histology, diffuse large B cell lymphoma cell of origin, MYC deregulation, TP53 inactivation, BCL6 or BCL2 translocations. The gene expression signature of the PQR309/idelalisib sensitive cell lines was enriched of genes involved in IFN signaling, IL6/Jak/Stat3 signaling, oxidative phosphorylation, PI3K/MTOR signaling, chemokines signaling and proteasome (GSEA FDR <0.05; core enrichment genes including among others: PIK3CD, PTPN6, CCL3, CXCR4, CXCL10, JAK2, MYD88, STAT3, BAX, CDKN1A, AKT1, CDK2, PRKCB, PSMC4, PSMC2, MX1, MX2). Conversely, gene expression profiles of the PQR309-only sensitive cell lines were enriched of genes involved in hypoxia, IL2/STAT5 and glycolysis (GSEA FDR <0.05; core enrichment genes including, among others, ENO2, AKAP12, PRKCA, VEGFA, XBP1). The gene expression signature derived from genetic silencing of SOX4 in different cancer cells overlapped with the differences observed between our groups (connectivity score=-0.5) and the transcription factor itself, a positive-regulator of the PI3K/AKT pathway, was more expressed in the PQR309-only cells (log ratio 0.99 P 0.036). The cell surface marker CD24, involved in PI3K signaling, was the most up-regulated gene in the PQR309/idelalisib sensitive group (log ratio -2.1 P 0.02). Transcripts more expressed in the PQR309-only sensitive cells overlapped with genes down-regulated by PI3K and MTOR inhibitors in cancer cell lines (connectivity scores < -0.55).

Conclusions. The dual PI3K/MTOR inhibitor PQR309 was active in a subset of mature B cell lymphoma cell lines bearing a primary resistance to the PI3K-delta inhibitor idelalisib and specific gene expression features. Expression of SOX4 and CD24 appear worth of validation on clinical specimens derived from prospectively treated patients.

#381

PI3K inhibitor BAY 1082439 and radium-223 dichloride decrease tumor burden and tumor-induced bone formation in an established bone metastatic prostate cancer model in mice.

Mari I. Suominen,1 Jukka Morko,1 Katja M. Fagerlund,1 Esa Alhoniemi,1 Dominik Mumberg,2 Karl Ziegelbauer,2 Jussi M. Halleen,1 Arne Scholz,2 Ningshu Liu2. 1 _Pharmatest Services Ltd., Turku, Finland;_ 2 _Bayer Healthcare, Global Drug Discovery, TRG-Onc/GT, Berlin, Germany_.

Background and objective: Bone is the most common site of metastasis in prostate cancer (PCa) and results in significant morbidity and poor prognosis. Disseminated PCa cells conquer hematopoietic stem cell niches, and during outgrowth they induce osteoblasts to form new, disorganized bone. Also osteoclasts are activated at a varying degree. PI3K activation has important role in survival and proliferation of PCa cells, mediating critical signaling pathways involving tumor and stromal cell interaction in the bone microenvironment. BAY 1082439 is a highly selective and potent PI3K inhibitor currently in Phase I, with equipotent activity against PI3Kα and PI3Kβ isoforms. Radium-223 dichloride (ra-223/Xofigo) is alpha-emitting calcium mimetic that via efficient osteoaffinity provides targeted radiation therapy against bone metastases. We have previously reported that BAY 1082439 and ra-223 showed synergistic anti-proliferative and apoptosis-inductive effects in vitro in LNCaP PCa cells (Suominen et al, AACR Annual Meeting 2014). Here, we investigated the effects of BAY 1082439 and ra-223 as single agents and in combination in the osteoblastic LNCaP tumors in tibia of male nod.scid mice.

Methods: Tumors were established 6 weeks after inoculation of LNCaP cells. Mice were randomized into four treatment groups (vehicle, ra-223; BAY 1082439 and ra-223 + BAY 1082439) based on serum PSA and bone lesion score. Treatments were continued for six weeks and the efficacy was assessed by the following endpoints biweekly and/or at the end of the study: PSA, bone formation marker PINP, bone lesions and bone volume (measured by microCT), and tumor and bone area by histological analysis.

Results: Both ra-223 and BAY 1082439 monotherapies inhibited tumor growth (reduction 67.9%, p=0.029 and 67.4%, p=0.009, respectively). Furthermore, BAY 1082439 monotherapy treatment group displayed 68.8% of necrotic tumor, compared to 6.5% in the control group (p=0.009). In the combination group, tumor growth was further suppressed (tumor reduction 89%, p=0.005) and it is noteworthy that 60% of animals had no detectable tumors in histology. All treatments inhibited the tumor-induced bone formation compared to vehicle, and the combination treatment inhibited progression of bone lesions nearly completely. In conclusion, BAY 1082439 and ra-223 as monotherapies decreased tumor burden and tumor-induced bone reaction, and the observed effects were further enhanced by the combination treatment.

#382

Concomitant administration of acid reducing agents can impact the pharmacokinetics of the balanced pan-PI3K and mTOR inhibitor PQR309.

Karin Jorga,1 Eva Huehn,2 Grace Fraczkiewicz,2 Debora Schmitz,3 Doriano Fabbro,3 Sasa Dimitrijevic3. 1 _KarinJorga GmbH, Basel, Switzerland;_ 2 _SimulationsPlus Inc, Lancaster, CA;_ 3 _PIQUR Therapeutics AG, Basel, Switzerland_.

Introduction

Acid reducing agents (ARAs) - such as proton pump inhibitors, H2-antagonists or antacids - are commonly prescribed medications to reduce stomach acidity in cancer patients. Increased gastric pH can significantly impair the dissolution of agents that exhibit pH-dependent solubility, thus possibly reducing their absorption and influence their pharmacokinetics (PK).

PQR309 is a novel oral balanced dual pan-PI3K and mTOR inhibitor currently in clinical development for treatment of solid tumors and hematological malignancies. PQR309 is a weak base and exhibits pH-dependent solubility. Therefore, the assessment of the potential for drug-drug interactions with ARAs is essential to avoid treatment failure in cancer patients.

Methods

GastroPlusTM v.9.0 (Simulations Plus, Inc.) was used to build a physiologically-based absorption and pharmacokinetic (PBPK) model of PQR309. A baseline model was built with the physicochemical properties of the compound and pre-clinical PK data after iv and po administration. The model was then extended using PK data from cancer patients. Volume of distribution and renal clearance were calculated using the Lukacova and fup*GFR methods, respectively. Hepatic clearance was estimated from in vitro clearance obtained in hepatocytes and the intestinal absorption was calculated by the Advanced Compartmental Absorption and Transit (ACATTM) Model.

Results

The parameter sensitivity analysis showed that at fasted stomach pH of ~1.5, PQR309 is fully dissolved and rapidly absorbed in the upper part of gastrointestinal tract leading to a distinct maximum plasma concentration (Cmax) and a predicted bioavailability of almost 100%. At a stomach pH of 4.0 dissolution is delayed and the absorption shifted towards distal parts of the gastrointestinal tract. The predicted bioavailability is still estimated to be good (> 75%), but the observed plasma concentration time profile is much shallower and the expected Cmax considerably reduced.

Conclusions

Our model predicts that concomitant administration of ARAs can impact PQR309 absorption and change its pharmacokinetic profile. The clinical relevance of this potential drug-drug interaction is unknown. In currently ongoing clinical studies co-medication of PQR309 with ARAs should be avoided until further clinical investigations confirm the model predictions and mitigation strategies are explored.

#383

In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma.

Alex A. Pyuen, Douglas H. Thamm. _Colorado State University, Fort Collins, CO_.

While extremely rare in humans, hemangiosarcoma (HSA) accounts for nearly 2% of canine neoplasia, and is characterized by both aggressive local growth/invasion and a high rate of metastasis. Both canine and human HSA exhibit sustained aberrant PI3K/Akt/mTOR pathway signaling. The purpose of this study was to examine the in vitro effects of a novel dual PI3K/mTOR inhibitor, VDC-597, in canine HSA cells. Three canine HSA cell lines (DEN-HSA, CIN-HSA, and SB-HSA) were employed in multiple in vitro assays. Western analysis evaluated activation (phosphorylation) of key downstream pro-survival proteins in the PI3K/mTOR pathway. Changes in tumor cell growth/apoptosis were assessed using both bioreductive assays (Alamar Blue) and in vitro live-cell imaging (IncuCyte), in the presence and absence of doxorubicin, a standard-of-care cytotoxic drug for canine and human sarcomas, as well as in the presence and absence of U-0126, an inhibitor of the MEK pathway. Migration was assessed using both Boyden chamber and scratch assays, via live-cell imaging (IncuCyte). Matrigel invasion was assessed using traditional Boyden chambers. Finally, ELISA was utilized to quantify relative expression of vascular endothelial growth factor (VEGF). VDC-597 suppressed activation of both Akt and 4eBP1 in canine HSA cells in a dose- and time-dependent fashion, with an IC50 of approximately 0.3 uM, a concentration predicted to be clinically achievable based on preliminary early-phase canine and human studies. VDC-597 dose-dependently reduced proliferation, migration, invasion, and VEGF expression in HSA cells, while promoting tumor cell apoptosis. VDC-597 demonstrated additive antiproliferative effects when combined with doxorubicin and U-0126. Together, these results suggest that inhibitors of the PI3K/Akt/mTOR pathway may act against multiple components of the neoplastic process, including proliferation/apoptosis, chemosensitivity, invasion/migration and angiogenesis, and justify the evaluation of PI3K/mTOR inhibitors in canine, and eventually human, HSA. Experiments to examine the effect of VDC-597 in reducing tumor burden and metastasis in a rodent model are ongoing.

#384

Differential response to a dual PI3K/mTOR inhibitor in PIK3CA mutant urothelial cancer patient derived xenografts.

May F. Elbanna,1 Eric Ciamporcero,2 Remi Adelaiye,1 Ashley Orillion,1 Sreenivasulu Chintala,1 Roberto Pili1. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _Department of Clinical and Biological Sciences, University of Turin, Turin, Italy_.

Background: PI3K pathway has been reported to be deregulated in up to 30% of bladder cancer patients. We have recently established two patient derived xenografts (PDX) models in our lab that carry unique helical domain mutations in the PIK3CA gene that is known to be deregulated in bladder cancer. This sets our models as unique tools for understanding the molecular background that would dictate an optimal response to targeting the PI3K pathway in vivo as well as the potential mechanisms of resistance. Methods: RNA-seq was carried out to understand the molecular make up of our PDX models (RP-B-01 and RP-B-02) and to identify clinically relevant drug targets. In-vivo drug testing was done using a dual PI3K-mTOR inhibitor to test the response to this class of drugs. Cell lines derived from the PDX models were used for in-vitro studies to validate our in-vivo data and to calculate the IC-50 for this class of agents in our cell lines. Western blot was used to assess the molecular effects of these agents using both in-vivo specimens as well as cell lines that were treated in vitro. Results: Our RNA-seq data have shown that our models (RP-B-01 and RP-B-02) are molecularly distinct where RP-B-01 has a basal-like phenotype while RP-B-02 is has a luminal like phenotype. Additionally, these models carry two unique helical domain mutations in the PIK3CA gene (E542K and E545K mutations respectively). Despite the molecular similarity between these two mutations, the two models responded differently to a dual PI3K-mTOR inhibiter in vivo. The RP-B-02 tumor model significantly responded to the drug compared to vehicle treated mice (P Value = 0.03) while the RP-B-01 model was resistant. Cell lines derived from these models were able to recapitulate the same phenotype in-vitro and IC-50 for each cell line was significantly different (IC50 for RP-B-01 cells = 204.1 nM; IC50 for RP-B-02 cells = 73.21 nM). Western blot analysis has shown that despite the ability of the dual PI3K-mTOR inhibitor to hit the target in RP-B-02 PDX, the treatment was not able to have the same biological effect in the other model (RP-B-01). Interestingly, supplementing media with insulin and nutrients switched the response phenotype and rendered cells derived from the RP-B-02 model more resistant to the PI3K-mTOR inhibitor. Conclusion: Response to PI3K pathway inhibitors in bladder cancer is not only dictated by the presence of targetable mutations but more importantly by the molecular make up of individual tumors which should be taken into account when using these agents in the clinic.

#385

mTORC1/2 inhibition as a treatment strategy for subtypes of colorectal cancer.

Stephanie Fricke, Cheri Pasch, Susan Payne, Alexander Yueh, Tyler Foley, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. _University of Wisconsin Carbone Cancer Center, Madison, WI_.

BACKGROUND Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the United States. Several key mutations in CRC include APC (80%), TP53 (50%), and PIK3CA (20-30%). Mutations in the PIK3CA gene, resulting in a constitutively active PI3K, often occur concomitantly with loss of the APC gene in human CRCs. Our lab has developed a murine model system where a constitutively active PI3K and loss of APC occur simultaneously in the colon (AK3K) as well as concomitant loss of p53 (AK3KTO). Colon tumors from these models are cultured as three-dimensional spheroids and treatment studies. METHODS AK3K and AK3KTO spheroids were treated with a dual PI3K/mTOR inhibitor, NVP-BEZ235, or a mTORC1/2 inhibitor, MLN0128. Images were taken both pre- and post-treatment and changes in spheroid diameter were measured. Parallel treatment studies were performed on a primary human colon cancer tumor cell line, SW48, which carries a mutant FBXW7 and also on a cell line transfectedwith a PIK3CA mutation (SW48PK). RESULTS Treatment with NVP-BEZ235 and MLN0128 resulted in a significant treatment response as measured by marked change in spheroid diameter in the AK3K spheroids at 100, 200 and 400nM (p<0.01), with MLN0128 eliciting a greater treatment response. There were no significant differences between drugs in the AK3KTO spheroids. Comparing across cell lines, there were highly significant differences when treated with NVP-BEZ235, as the treatment was much more effective in the AK3KTO cell line at 100, 200, and 400nM (p<0.01). There were no significant differences between cell lines for MLN0128. Immunoblotting further demonstrated a dramatic inhibition of the PI3K/mTOR pathway in both spheroids following treatment with 200nM MLN0128 and 200nM NVP-BEZ235. Treatment of SW48 and SW48PK cells with NVP-BEZ235 and MLN0128 showed highly significant differences between the drugs in both cell lines, with MLN0128 being more effective at 1, 10, and 50nM (p<0.01). At higher drug concentrations (100, 200, and 400nM), there were highly significant differences between drugs for the SW48PK cell line (p<0.01) but no significant differences in the SW48 cells. Comparing across cell lines, there were highly significant differences when treated with MLN0128 at 100, 200, and 400nM (p<0.01), with MLN0128 being more effective in the SW48PK cell line. Immunoblotting demonstrated a reduction in activation of the PI3K/mTOR pathway following treatment with 10nM MLN0128 but this inhibition was not seen when treated with 10nM NVP-BEZ235. CONCLUSIONS mTOR inhibition is sufficient to induce responses in PIK3CA and APC mutated CRCs and concomitant loss of TP53 does not lead to resistance and potentially increases sensitivity. In a cell line predisposed to PI3K/mTOR signaling sensitivity, mTOR inhibition remains sufficient and the addition of a PIK3CA mutation further increases sensitivity

#386

Addition of mTOR inhibition to HER2 blockade results in improved antitumor activity in a preclinical model of uterine serous carcinoma with HER2 gene amplification and a gain of function mutation in PIK3CA.

Silvia F. Hernandez, Sarah Chisholm, Darrell Borger, Rosemary Foster, Bo R. Rueda, Whitfield B. Growdon. _Massachusetts General Hospital, Boston, MA_.

Subsets of uterine serous carcinomas (USC) harbor simultaneous HER2 (ERBB2) over-expression and gain of function mutations in PIK3CA that may play a role in resistance to single agent anti-HER2 therapies. Targeting the mammalian target of rapamycin (mTOR) may be a promising option to heighten anti-tumor response and restore sensitivity to anti-HER2 therapies.

HER2 gene amplified cell lines with (ARK1, E542K) and without (ARK2) PIK3CA gene mutations were divided into four arm cohorts with equivalent tumor volumes and treated with either vehicle, ridaforolimus (1 mg/kg, lapatinib (150 mg/kg) and trastuzumab (10mg/kg) or the combination of lapatinib/trastuzumab and ridaforolimus. At day 22, tumors were collected to measure downstream target proteins and calculate the proliferation and apoptosis index.

In the ARK1 USC xenograft cohort, inhibition of tumor growth was observed following treatment with ridaforolimus compared to vehicle (P<0.05) and this was not statistically different from the lapatinib/trastuzumab group. However, when lapatinib/trastuzumab was combined with ridaforolimus, improved anti-tumor activity was observed compared to all other arms (P<0.01). Similarly, the treatment of ARK2 mice with ridaforolimus slowed tumor growth (P<0.05) compared to the vehicle. Both the lapatinib/trastuzumab and lapatinib/trastuzumab/ridaforolimus arms induced reductions in tumor sizes compared to the vehicle and ridaforolimus arms (P<0.01), but ridaforolimus failed to add any additional anti-tumor activity.

Analysis of the treatment effects revealed the lapatinib/trastuzumab/ridaforolimus therapy was required to decrease Ki67 positive cells in ARK1 xenografts compared to the vehicle (P<0.01) however in ARK2 xenografts, lapatinib/trastuzumab was sufficient to decrease Ki67 positive cells with or without ridaforolimus. Similarly, differential TUNEL staining was observed in the two cell lines with ridaforolimus being required to increase the percentage of apoptotic cells in ARK1, while the apoptotic index was solely dependent of lapatinib/trastuzumab administration in ARK2. In ARK1, we observed that pS6 expression was reduced (p<0.01) only in the arms where ridaforalimus was administered but in ARK2, its expression was again independent of mTOR inhibition.

These data suggest that USCs harboring gain of function PIK3CA mutations are relatively insensitive to anti-HER2 therapies. The addition of downstream mTOR inhibition to HER2 blockade increased the anti-tumor activity only in the xenograft model with PIK3CA mutation and provides rationale for clinical trials in HER2 positive endometrial cancers based on molecular genotype.

#387

Inhaled buformin for lymphangioleiomyomatosis and early (airway confined) lung cancer.

Steven Lehrer,1 Peter H. Rheinstein,1 James L. Mulshine2. 1 _Fermata Pharma, Inc., New York, NY;_ 2 _Rush Medical University, Chicago, IL_.

Pulmonary lymphangioleiomyomatosis (LAM) is a low grade neoplasm related to tuberous sclerosis (TSC) occurring in a sporadic form affecting only females, usually of childbearing age, with an incidence of less than one case per 100,000 population per year. LAM is associated with over-proliferation of bronchial smooth muscle, infiltration and cystic destruction of the lung. Clinically, LAM is characterized by progressive dyspnea on exertion, recurrent pneumothorax, abdominal and thoracic lymphadenopathy, and abdominal tumors. Oral sirolimus (rapamycin) has recently been reported to be an effective therapy that halts progression of LAM. Rapamycin works by inhibiting mTOR, the mammalian target of rapamycin, a serine/threonine protein kinase that regulates cell growth. mTOR integrates the input from upstream pathways, including insulin, growth factors (such as IGF-1 and IGF-2), and amino acids. mTOR also senses cellular nutrient and energy levels. The mTOR pathway is dysregulated in human diseases, especially cancers. Because LAM is predominantly a pulmonary neoplasm, an inhaled therapy, targeting only the lung, could be highly desirable. Rapamycin (sirolimus) cannot be safely inhaled because of its well-documented lung toxicity, interstitial pneumonitis. We propose to test an inhaled version of the biguanide anti-diabetic buformin for LAM. Biguanides also inhibit mTOR, but have no known lung toxicity after decades of use in millions of patients. Buformin would be used rather than metformin because buformin requires one eighth the inhaled dose of powder, and can be given in inhaled doses that may be one tenth or less of the toxic dose of buformin required to produce lactic acidosis, its principal side-effect. Buformin has an octanol/water partition coefficient (log P) of -1.2 and is hydrophilic. Hydrophilic small molecules with a log P less than 0 have a mean lung half life (t½) of about one hour. Long lung residence time of buformin could be increased even further with an appropriate formulation. Pulmonary buformin would likely be useful only for sporadic LAM, because TSC-LAM has been shown to represent clones of the smooth muscle in those patients' renal angiomyolipomas, believed to represent metastases of this "benign" tumor. Buformin has a known systemic safety profile, well established chemistry, manufacturing and control profile, an inexpensive supply with Drug Master Files on file, is non-toxic compared to the vast majority of anti-cancer agents, and readily administered by inhalation aerosol. Inhaled buformin, which is a radiation sensitizer, could also be used to treat early lung cancer detected on spiral CT lung cancer screening. Lung cancer screening will be covered by Medicare. There will be huge numbers of airway-confined lung cancers to treat. In addition, inhaled buformin could prevent lung cancer in heavy smokers.

#388

Pathway-directed high throughput drug screen identifies PI3K inhibitors that synergistically potentiate anti-tumor activity of HDAC inhibitors in mycosis fungoides and Sézary syndrome.

Chen-Yen Yang, Razan Faraj, Taha Rakhshandaroo, Shervin Afghani, Laura Pincus, Sourav Bandyopadhyay, Frank McCormick, Weiyun Ai. _UCSF, San Francisco, CA_.

Introduction and Purposes: Mycosis fungoides and Sézary syndrome (MF/SS) represent a group of heterogeneous diseases. Recent studies demonstrated dysregulation of several signaling pathways in MF/SS, including PI3K/AKT, JAK/STAT, RAS and NFκB pathways. We performed a high throughput drug screen to determine the potential of novel agents targeting these pathways for the treatment of MF/SS.

Experimental Procedures: We compiled a library of 94 compounds targeting pathways known to be relevant in cancer biology. These included kinases involved in growth factor receptor signaling, HDACs, proteasome, DNA repair and regulators of apoptosis. The compounds were screened for anti-proliferative activity against four MF/SS cell lines in high throughput proliferation assays. Selected hits were further studied in xenograft models of MF/SS and in primary T cell lymphomas. Promising candidates from different classes were also tested in combination therapy assays using a matrix block method across dose gradients of each compound designed to detect synergistic activities.

Results: From the high throughput screen, we identified 14 compounds with anti-proliferative activity in MF/SS, including multiple inhibitors of the PI3K pathway. PI3K inhibitors emerged as preliminary hits in this screen and secondary validation assays confirmed the class effect of PI3K inhibitors. From this class, the PI3K inhibitor BKM120 was selected for in vivo studies. In a xenograft model of MF, BKM120 exhibited striking anti-tumor activity measured by a marked suppression of tumor growth and prolonged survival of tumor-bearing mice compared with vehicle control. In a search for even more effective combination therapies, we identified that BKM120 and the HDAC inhibitor class of compounds exhibit synergistic anti-proliferative effects in MF/SS tumor cells. Each of three HDAC inhibitors including LBH, Romidepsin and Vorinostat showed synergistic activity with BKM120, most evident at the GI50 concentrations of each drug, and apparent in both growth inhibition and apoptotic assays.

Conclusion: BKM120 is highly active in preclinical models of MF/SS. Furthermore, it synergistically potentiates the effect of HDAC inhibitors against MF/SS tumor cells. These are highly promising approaches for the treatment of MF/SS and warrant clinical investigation.

#389

The dual mTOR inhibitor, AZD2014, and castration increase intra-tumoral immune cell infiltration and anti-tumour activity in a genetically engineered mouse model of prostate cancer.

Chiranjeevi Sandi,1 Antonio Ramos-Montoya,2 Sergio L. Filisbino,3 Adina Hughes,2 Suzanne Mosely,4 Michelle Morrow,4 Robert W. Wilkinson,4 Sarah Jurmeister,1 Karan Wadhwa,1 Frances M. Richards,1 Duncan I. Jodrell,1 Sabina Cosulich,2 Barry R. Davies,2 Simon Pacey1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _Sao Paulo State University (UNESP), Brazil, Brazil;_ 4 _MedImmune, Cambridge, United Kingdom_.

Background: Medical therapy for men with prostate cancer (PCa) is evolving, including recent evidence to support the use of chemotherapy for men with hormone sensitive disease. PI3K/AKT/mTOR pathway aberrations are common in patients with primary PCa and almost universal in metastatic tumours. The mTOR pathway acts as a central regulator of tumour cell metabolism, proliferation, cell cycle progression and immune regulation. The aim of this study was to investigate the effects of mTOR inhibitor AZD2014 on tumour growth and immune infiltration in a genetically engineered mouse model of PCa.

Methods: PtenL/L;PB-Cre4 mice, which developed invasive hormone-sensitive prostate tumours between 10-14 months of age, were enrolled (n=13-15 per group) and treated with AZD2014 (15mg/Kg) or vehicle orally for 14 doses (QD 5/7) with or without castration. Tumour volumes were assessed by ultrasound imaging. Tumour samples were collected within 4 hours post 14th dose for histological and molecular analysis.

Results: AZD2014 was well tolerated, no overt toxicity was observed. Mean plasma concentrations of AZD2014 were 4.4±2.1µM at time of tumour sampling. AZD2014 alone or AZD2014+castration inhibited mTORC1 and mTORC2 activity as demonstrated by reduced p4EBP1(Thr37/46) 48%±27% (P<0.001) and 37%±11% (P<0.001), pS6(Ser235/236) 74%±43% (P<0.001) and 44%±13% (P<0.001), pAKT(Ser473) 36%±8% (P<0.001) and 20%±3% (P<0.01), respectively compared to vehicle-treated mice. Proliferation (Ki67+) was reduced in AZD2014-treated tumours by 70%±45% (P<0.001) and AZD2014+castration by 42%±16% (P<0.001). Apoptosis (CC3+) was increased in AZD2014 and AZD2014+castration groups by 3.3-fold (both, P<0.001) or castration only group by 2-fold (P<0.001). Tumour volumes were significantly reduced (54%, p<0.05) in AZD2014+castration group. Castration induced increased infiltration of T cells (CD3+, 2-fold, P<0.001) and macrophages (F4/80+, 1.6-fold, P<0.001) in the tumour tissues. These effects were more pronounced when combined with AZD2014 (CD3+, 2.8-fold, P<0.001; F4/80+, 2.3-fold, P<0.001). Multi-channel flow cytometry has confirmed increased proliferation of CD45+, CD3+ and CD8+ cells in tumours from the AZD2014+castration group.

Conclusion: These data confirm the anti cancer effect of AZD2014 in this PCa model. Furthermore, AZD2014+castration was more effective than either treatment as a single agent. The increased intra-tumoral T cell infiltration observed may have contributed to the anti-tumour effect. Further studies are ongoing to maximise this therapeutic effect and will inform current clinical studies in men with hormone sensitive, high risk prostate cancer.

#390

Colorectal cancer pulmonary metastasis treatment with lung-selective delivery of PAN-class I PI3K inhibitors.

Piotr Rychahou,1 Younsoo Bae,2 Yekaterina Zaytseva,3 Eun Y. Lee,4 Heidi L. Weiss,3 B. Mark Evers1. 1 _University of Kentucky Markey Cancer Center and Department of Surgery, Lexington, KY;_ 2 _University of Kentucky Markey Cancer Center and Department of Pharmaceutical Sciences, Lexington, KY;_ 3 _University of Kentucky Markey Cancer Center, Lexington, KY;_ 4 _University of Kentucky Markey Cancer Center and Department of Pathology and Laboratory Medicine, Lexington, KY_.

Colorectal cancer (CRC) is the second leading cause of cancer death in the US, and its frequency is increasing worldwide. Phosphatidylinositol 3-kinase (PI3K) signaling is an important therapeutic target that is currently being evaluated in multiple clinical trials. The purpose of this study was to: i) determine expression of pAkt (Ser473), Akt1 and Akt2 in primary and metastatic CRCs, and ii) evaluate lung-selective delivery of PI3K inhibitors as a novel therapeutic approach against CRC lung metastasis. METHODS. i) To determine the expression of PI3K/Akt pathway components, we obtained primary CRCs (n=12) and CRC lung metastases (n=10). Akt1, Akt2 and pAkt (S473) expression was analyzed by immunohistochemistry (IHC) and blindly scored by a pathologist. ii) To confirm lung-selective nanoparticle accumulation, we used two methods. First, polymeric nanoparticles were constructed and loaded with fluorescent dye (Alexa547). IVIS Spectrum imaging and confocal imaging of frozen tissue sections was used to determine distribution of fluorescently-labeled nanoparticles after intravenous administration (50 µg/g; 300 µl). Second, polymeric nanoparticles were loaded with either wortmannin, PX866, BEZ235, PIK-90, ZSTK474 or GDC-094 and administered intravenously. Western blot analysis of protein extracts from lung, liver, spleen and kidney for pAkt (Ser473) expression was used to determine PI3K pathway inhibition. iii) The anti-metastatic potential of PI3K drug-loaded lung-selective nanoparticles was evaluated in a preclinical model of CRC lung metastasis. RESULTS. i) We found strong Akt2 and pAkt (S473) expression in all primary and metastatic lung CRC patient samples. ii) We confirmed lung-selective accumulation of fluorescent nanoparticles and absence of fluorescent nanoparticle accumulation in liver, spleen and kidney. Lung-selective drug delivery was confirmed by western blot analysis of PI3K pathway inhibition in protein extracts from lung, liver, spleen and kidney after a single (10 µg/g; 300 µl) intravenous administration of drug-loaded nanoparticles. PI3K pathway inhibition was observed only in lung tissue samples. iii) Mice with established lung metastases were treated with PI3K drug-loaded nanoparticles daily (10 µg/g; 300 µl). Treatment in vivo with wortmannin and PX866 showed marked suppression of CRC lung metastatic growth. BEZ235, PIK-90, ZSTK474 or GDC-0941 had no significant effect on growth in metastatic CRC to the lung when delivered by nanoparticles. CONCLUSIONS. We developed a safe and efficient lung selective drug nanocarrier and evaluated lung selective PI3K inhibition as a viable treatment strategy. Treatment of CRC lung metastasis with pan-PI3K-loaded nanoparticles demonstrated a marked suppression of metastatic lung growth, and suggests that lung selective PI3K inhibition is a viable treatment strategy for CRC lung metastasis.

#391

Implications of Akt inhibition for neoadjuvant radiotherapy: improving the rectal cancer treatment.

Fernanda C. Koyama,1 Camila Ramos,1 Angelita Habr-Gama,2 Venâncio Avancini Ferreira Alves,3 Rodrigo O. Perez,2 Anamaria Aranha Camargo1. 1 _Hospital Sirio-Libanes, Sao Paulo, Brazil;_ 2 _Angelita & Joaquim Gama Institute, Sao Paulo, Brazil; _3 _University of Sao Paulo, Sao Paulo, Brazil_.

Resistance to therapy is a major obstacle to a favorable outcome in cancer treatment. For rectal cancer, neoadjuvant chemoradiotherapy (nCRT) can lead to complete tumor regression in a significant proportion of patients (up to 40%) but approximately 60% of patients have only partial or no tumor remission after nCRT. A number of works searching for a differential gene expression signature has failed. As those gene signatures relies on the sample set, there is an increasing agreement that biological understanding is necessary to produce a signature in terms of pathways and biological processes. In this regard we applied pathway enrichment analysis to RNA-Seq data in order to identify biological pathways involved in tumor resistance and discriminate rectal cancer patients that is refractory to neoadjuvant treatment. Mitochondrial oxidative phosphorylation and phosphatidylinositol signaling were identified as the main pathways involved in resistance to nCRT in patients with rectal cancer. Both pathways have been reported as involved in resistance to radiotherapy and chemotherapy in solid tumors but according to the status of gene regulation observed and the literature, there is evidence that Akt pathway could be central in promoting resistance.Therefore, we evaluated the status of Akt activation by immunohistochemistry of pre-treatment biopsies of rectal tumors as well as by immunoblot of colorectal cancer cell lines utilizing antibodies against phosphorylated Akt (pS473). As a result, we observed an increased proportion of Akt phosphorylation in the resistant cell line, SW480, and in patients with TRG 0-2 in comparison to those with TRG 3-4 and in the sensitive cell line, SW48. To evaluate the implication of Akt in promoting nCRT resistance, we performed colony formation assays using Akt inhibitors as radiosensitizers. We observed an additive effect using MK2206 at sub-doses in combination to radiotherapy (2 Gy) in the resistant cell line. In fact, Akt inhibition improved in approximately 40% the tumoricidal effects of radiotherapy. Moreover, at IC50 dose this effect is even higher, reaching 60% the improvement of radiotherapy. Furthermore, the treatment of MK2206 with radiotherapy alone or in combination with 5-FU have similar effectiveness. In this setting, as Akt is involved in a range of cellular functions that promote tumor progression and metastasis, we propose that combination of Akt inhibitor and radiotherapy as neoadjuvant treatment would maximize tumor remission and improve the rectal cancer treatment.

#392

Combined targeting of MET and PI3K improves efficacy in breast cancer models with concurrent MET/PI3K aberrations.

Shuying Liu,1 Naoto T. Ueno,1 Bailiang Wang,1 Mihai Gagea,1 Prahlad T. Ram,1 Mark Merchant,2 John Mendelsohn,1 Debasish Tripathy,1 Gordon B. Mills,1 Ana M. Gonzalez-Angulo1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Genentech, South San Francisco, CA_.

Introduction: Detailed understanding of the genetic abnormalities that drive subsets of cancer has led to the development of highly specific inhibitors targeting key oncogenic pathways. Growing evidence implicates aberrations of both mesenchymal epithelial transition receptor (MET) and its key downstream phosphatidylinositol 3-kinase (PI3K) signaling with poorer prognosis in breast cancer. Co-targeting MET/PI3K and identifying mechanisms of response and resistance could ultimately inform a novel therapeutic strategy for breast cancer treatment. Based on these finding, we hypothesized that concurrent aberrations in PI3K and MET will render breast cancers resistant to therapies targeting each pathway, and that combination therapy targeting the PI3K and MET pathway will improve the efficacy by preventing the acquisition of resistance.

Methods: We established cell models that stably express human wild type (WT) MET or MET-T1010I, a functional germline single nucleotide polymorphism, or mutant PI3K-H1047R alone, or variable concurrent aberrations in PI3K and MET using MCF-10A, a non-transformed mammary epithelial cell line, and HCC1954, a breast cancer cell line with an endogenous PI3KCA-H1047R mutation. Cell behaviors, including survival, invasion and response to MET one arm monoclonal antibody onartuzumab (MetMAb, Genentech Inc) and/or pan class I PI3K inhibitor GDC 941 (pictilisib, Genentech Inc), were detected. HCC1954 expressing WT MET or MET-T1010I along with PI3K-H1047R were transplanted into the mammary fat pad of hepatocyte growth factor-transgenic mice on a severe combined immunodeficiency background. Statistical analyses were carried out using the ANOVA and the Student t test.

Result: Expression WT MET or MET-T1010I act coordinately with PI3K-H1047R to increase colony formation (P < 0.01, respectively). Concurrent MET-T1010I, but not WT MET, acts coordinately with PI3K-H1047R to increase cell invasion (P < 0.0001). Expression MET-T1010I or WT MET induced cells resistance to GDC941, with MET-T1010I stronger than WT MET. Combined targeting MET with onartuzumab and targeting PI3K with GDC941 significantly inhibited cell growth in matrigel, dose dependently than each monotherapy (P < 0.0001, respectively). Invasion assay showed similar pattern. Combined onartuzumab and GDC941 significantly inhibited invasion, dose dependently (P < 0.001). The effects were confirmed using PI3K/mTOR inhibitor, GDC980. Consistently, combination of onartuzumab/GDC941 significantly inhibited tumor growth (P < 0.01 versus GDC941; P < 0.0001 versus onartuzumab alone).

Conclusions: Concurrent aberrations of MET and PI3K render breast cancer cells resistant to therapies blocking either pathway. Targeting both MET and PI3K increases inhibitory efficacy.

#393

NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma.

Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Pan Tong, Tuhina Mazumdar, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson, Mitchell Frederick. _UT MD Anderson Cancer Center, Houston, TX_.

Recently published whole exome sequencing studies in head and neck squamous cell carcinoma (HNSCC) tumors revealed that few had therapeutically targetable alterations using current strategies. This finding defines translational gap between genomics and HNSCC treatment. One potential targetable alteration is PIK3CA mutations. However, clinical trials testing PI3K/mTOR pathway inhibitors have had limited success and these inhibitors only lead to cell cycle arrest in PIK3CA mutant HNSCC cell lines. Thus, there is a critical need to identify therapeutic vulnerabilities for common mutation groups, including tumor suppressors, in HNSCC. One of these molecular subgroups is NOTCH1 which is the second most frequently mutated gene in HNSCC, with a 10-15% prevalence of inactivating mutations. Although there are several studies underscoring the importance of NOTCH1 as a tumor suppressor in HNSCC, none has identified a therapy that targets NOTCH1 mutant (mut) HNSCC. Our objective was to identify predictive biomarkers of sensitivity to PI3K/mTOR inhibitors by integrating drug and multiple-omics data.

Cell viability with six PI3K/mTOR inhibitors in 68 HNSCC lines was measured by the CellTiter Glo assay. The peak plasma concentration of each drug was used as the cut-off to determine sensitivity. We observed a striking correlation between NOTCH1mut and sensitivity to PI3K/mTOR pathway inhibitors. When fisher's exact test was performed, NOTCH1mut lines were more sensitive to GSK2126458 (P<0.027), BYL719 (P<0.004) and PQR309 (P<0.014) than NOTCH1 wild type cell lines. NOTCH1 was also identified as an upstream regulator in sensitive cell lines by Ingenuity® Pathway Analysis. Basal NOTCH1 protein expression was higher in HNSCC lines resistant to PI3K/mTOR inhibition using unsupervised hierarchical clustering of Reverse Phase Protein Array data.

NOTCH1mut lines underwent more apoptosis after GSK2126458 treatment compared to NOTCH1wt lines (PCI15B- 48.1 fold; P<0.05, HN31- 46.9 fold; P<0.05). There was also increased accumulation of cells in G1 after GSK2126458 treatment in NOTCH1mut lines (PCI15B-1.3 fold, P<0.05; HN31- 1.4 fold, P<0.05). To check if inhibition of NOTCH1 pathway inhibition sensitizes NOTCH1wt lines to PI3K/mTOR inhibition, resistant NOTCH1wt lines were treated with Gamma secretase inhibitors and GSK2126458. The combination led to significantly decreased cell viability (DAPT- 1.5 fold and YO010227- 1.7 fold). The combination studies will be further expanded to 38 NOTCH1wt lines. On-going studies include assessment of drug sensitivity in vivo, mechanistic studies and the effect of genetic manipulation of NOTCH1 signaling on sensitivity to PI3K/mTOR inhibitors.

Our data suggests that loss of active NOTCH1 signaling confers sensitivity to PI3K/mTOR inhibition. If the combination of NOTCH1 and PI3K/mTOR inhibition leads to apoptosis, this combination could be translated into the clinic.

#393A

Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620.

Florent Beaufils,1 Denise Rageot,2 Anna Melone,2 Alexander Sele,2 Marc Lang,1 Juergen Mestan,1 Robert A. Ettlin,1 Petra Hillmann,1 Vladimir Cmiljanovic,1 Carolin Walter,3 Elisabeth Singer,3 Hoa HP Nguyen,3 Paul Hebeisen,1 Doriano Fabbro,1 Matthias P. Wymann2. 1 _Piqur Therapeutics AG, Basel, Switzerland;_ 2 _University of Basel, Basel, Switzerland;_ 3 _University of Tuebingen, Tuebingen, Germany_.

Introduction: The mammalian target of rapamycin (mTOR) signaling pathway is an integrating factor in cell physiology that influences many processes like growth, metabolism and proliferation. mTOR signaling is constitutively activated in many cancers. Rapamycin is an allosteric inhibitor of mTOR that targets a subset of mTOR functions via inhibition of the mTORC1 complex. An ATP site-directed mTORC1/2 inhibitor that fully blocks all mTOR functions is desirable as cancer therapeutic. PQR620 is a novel, ATP site directed inhibitor of mTOR that is currently in pre-clinical development. PQR620 potently binds to its target (Kd = 6 nM) and shows excellent selectivity versus related and unrelated kinases [1].

Results: PQR620 inhibits mTOR signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been observed in mice (MTD=150 mg/kg). A 14 day GLP toxicological study in rats showed very good tolerability (MTD=30 mg/kg). Only minor toxicities such as dose-related changes in body weight and blood count were observed. PQR620 was administered to male C57BL/6J mice for a pharmacokinetic (PK) and pharmacodynamics (PD) evaluation. After oral application PQR620 exhibited dose-proportional PK, a maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (4.8 μg/ml and 7.7 μg/ml, respectively). In muscle, Cmax (7.6 μg/ml) was reached after 2 hours. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC0-tz (area under the curve)) was 20.5 μg*h/ml in plasma, while it was approximately 30% higher in both, brain and thigh muscle (30.6 and 32.3 μg*h/ml, respectively). PQR620 potently inhibited mTOR signaling in vivo after administration of a single oral dose of 50 mg/kg. Importantly, no effect on plasma insulin levels was observed. In an OVCAR-3, ovarian carcinoma mouse xenograft, PQR620 effectively attenuated tumor growth using daily, oral dosing.

Conclusion: PQR620 potently inhibits mTORC1/2 in vitro and in vivo. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound.

[1] Beaufils F, Rageot D, et al., Structure-Activity Relationship Studies, Synthesis and Biological Evaluation of PQR620, a Highly Potent and Selective mTORC1/2 Inhibitor, AACR annual meeting 2016

#394

Novel pan-PIM/pan-PI3K/mTOR inhibitors are highly active in preclinical models of pancreatic ductal adenocarcinoma.

Kevin O'Hayer,1 John Barbe,2 Avinoam Nevler,3 Michael O'Neill,4 Darren Cunningham,4 Jordan Winter,1 Jonathan Brody1. 1 _Thomas Jefferson University Hospital, Philadelphia, PA;_ 2 _University of Notre Dame, South Bend, IN;_ 3 _Sheba Medical Center, Tel-Hashomer, Israel;_ 4 _Inflection Biosciences, Dublin, Ireland_.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal and morbid malignancies today. Although newer chemotherapy regimens such as FOLFIRINOX and gemcitabine/nab-paclitaxel have made modest improvements in patient survival, more effective therapies are needed. With the exception of the EGFR inhibitor erlotinib, there are no approved, targeted therapies for metastatic PDA. Recent work demonstrated the importance of PIM kinases, particularly PIM1 and PIM3, in PDA. PIM kinases are involved in apoptosis, cell cycle progression, DNA damage repair mechanisms, and drug resistance mechanisms. Our work showed that hypoxic stress causes the RNA binding protein HuR to translocate to the cytoplasm where it stabilizes PIM1 mRNA leading to increased levels and activity of PIM1. Activation of PIM1 led to oxaliplatin resistance in a hypoxia specific manner and validated PIM1's upregulation upon drug exposure as a potential target for therapeutic inhibition in PDA. The KRas/PI3K/mTOR signaling pathway has been well described in PDA and PI3K as well as dual PI3K/mTOR inhibitors are currently in clinical development. Inflection Biosciences is currently developing IBL-202, a pan-PIM/pan-class I PI3K inhibitor and IBL-301, a pan-PIM/pan-class I PI3K/mTOR inhibitor. These compounds show good oral bioavailability in preclinical PK studies. Unlike prior attempts at PIM inhibition, these compounds do not interact with hERG channels or CYP enzymes, decreasing the likelihood of drug-drug interactions or prolonged QTc. Furthermore, toxicity studies in rodent models demonstrate that the compounds are well tolerated at 200mg/kg and 400mg/kg doses. Our work shows that IBL-202 and IBL-301 have significant preclinical activity in PDA cell lines with diverse genetic backgrounds, including PL5, Panc-1, and HS776t cells. Using PICOgreen assays, we show that the IC50 for IBL-202 ranges from 400nM (PL5) to 800nM (HS776t). The IC50 of IBL-301 ranges from 80nM (PL5) to 400nM (HS776t). As the PIM and PI3K pathways are important for inhibition of apoptosis, we tested the ability of these compounds to induce an apoptotic response. Cells treated for 48 hours at IC50 levels of IBL-202 showed positive cleaved caspase 3/7 staining in 31% (Panc-1) to 90% (HS776t) as examined by flow cytometry. IBL-301 elicited a positive cleaved caspase 3/7 in 28% (Panc-1) to 91% (PL5) of cells. Mechanistically, PIM and PI3K are known to lead to G1/S cell cycle progression through phosphorylation of p21 and p27. PDA cell lines dosed at IC50 levels show a potent G1 to S arrest at 48 hours in all cell lines tested. Overall, this work provides the rationale for the further preclinical development of IBL-202 and IBL-301 in PDA pre-clinical models in an effort to move these compounds towards early phase trials.

#394A

Phosphatidylcholine-34:2 and -36:4 have tumor suppressive function for gastric cancer.

Nobuya Kurabe, Masako Suzuki, Yusuke Inoue, Tomoaki Kahyo, Moriya Iwaizumi, Hiroyuki Konno, Mitsutoshi Setou, Haruhiko Sugimura. _Hamamatsu University School of Medicine, Hamamatsu, Japan_.

Imaging mass spectrometry (IMS) is an emerging technique that enables us the quantitation and visualization of a number of biomolecules including phospholipids. Because antibodies against lipid molecules are hard to generate, IMS is a useful approach for lipid biology. Our colleagues and we used IMS apparatus named "iMScope™ (Shimadzu)" to detect the cancer specific phospholipids in liver, colorectal, oral, and breast cancer. Here, to identify possible anti-cancer drug candidate(s) for GC with no side effects, we performed an imaging mass spectrometry screening using a panel of non-neoplastic and neoplastic gastric tissue. Two species of phosphatidylcholine (PC), PC-36:4 and PC-34:2, were highly downregulated in GC. PC-36:4 and PC-34:2 suppressed the transformation of NIH3T3 cells driven by K-rasV12 and growth of 4 out of 8 GC cell lines, while the growth of non-transformed NIH3T3 was not affected by these lipids. Tumor growth of GC cells was also inhibited in nude mice on administration (i.v.) of these lipids without any side effects in vivo. Phosphorylation of Akt was downregulated by these lipids in some GC cell lines. In an immunohistochemical analysis, the expression of a PC-36:4 and PC-34:2 producing enzyme, LPCAT3, was diminished in GC tissue, and the low expression level of LPCAT3 was a predictor of poor survival of GC patients. Considering that PCs generally have no toxic effect for human, these data indicate the usefulness of PC-36:4 and PC-34:2 as potential anti-cancer drugs of GC.

Stating the biochemical background of this observation, phospholipids are constituents of lipid bilayer and consist of a glycerol backbone, two esterified acyl chains, and a polar head group; phospholipids are classified into five subclass by the head group: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), or phosphatidylglycerol (PG). The Lands' cycle produces the diversity of acyl chains' compositions in phospholipids by phospholipase A2 and lysophospholipid acyltransferases. Recently, novel regulatory mechanisms of Akt activation via phospholipids were reported. For example, PS can bind Akt to induce conformational change, which facilitates the activation of Akt. On the other hand, a specific species of PC suppresses the interaction of Akt to phosphatidylinositol-3,4,5-trisphosphate to inhibit the downstream signaling. Because other specific phospholipids show the physiological functions in lipogenesis, fatty acid use, hepatic steatosis, and diabetes, the combination of the acyl chains are thought to define the functional properties of PCs.

In these contexts, our data first disclosed that a specific PC plays a tumor suppressing role and possibly be a anti-cancer agent with minimal side-effect. Furthermore, our in vitro analysis was mechanistically rational in terms of the Akt-centered pathway of carcinogenesis.

## CLINICAL RESEARCH:

### Biomarkers

#395

Patient stratification and drug combination strategy based on drug response and genomic information from PDX clinical trials (PCTs).

Jingjing Jiang,1 Tengfei Yu,2 Ying Yan,2 Wei Du,2 Tingting Tan,2 Xuqin Yang,2 Jiali Gu,2 Xin K. Ye,2 Zhenyu Gu2. 1 _GenenDesign USA, Menlo Park, CA;_ 2 _GenenDesign, Shanghai, China_.

Cancer is a heterogeneous disease with various molecular lesions and drug response profiles within the same tumor type. Patient stratification in clinical trials based on molecular features has contributed to recent success of several targeted cancer drugs on molecular aberrations such as BCR-ABL translocation in CML, Her2 amplification in breast and gastric cancers, EGFR mutations and ALK fusions in lung cancer and BRAF mutation in melanoma. However, in most cancers, the molecular features which can be used for patient stratification are not as simple as a single genetic aberration. Multiple drug resistance mechanisms caused by various mutations in cancer signaling pathways can also increase the uncertainty of clinical outcomes.

To increase the chance of success in human clinical studies, patient-derived xenograft (PDX) clinical trials (PCTs) have increasingly been used for predictive biomarker validation, resistance mechanism investigation and combination therapy selection. PDX tumor models have been demonstrated to have high correlations with human patients in tumor pathology, molecular characteristics and drug responses. Large scale PCTs have also shown consistency in results when compared to related human clinical trials.

At GenenDesign, we have established over 1000 PDX tumor models and more than 100 resistance models against various cancer drugs. Many of these PDX models have been characterized at RNA/Exome sequence, gene expression, gene copy number and hot-spot mutation levels. We carried out our PDX clinical trials by testing multiple approved drugs and clinical drug candidates such as targeted inhibitors against FGFRs, c-Met/ALK, HER2, EGFR, cell cycle regulators, Ras/Raf pathway, PI3K/Akt pathway, as well as chemotherapy drugs in biomarker-driven multi-drug multi-arm expanded PDX clinical trials. So far, we have accumulated more than 3000 efficacy data sets and associated PD samples.

Analysis of drug response and associated genomic information from PDX clinical trials yielded rich information for predictive biomarker identification and validation. At the same time, many potential resistance mechanisms were also revealed. These information can make human clinical trial better prepared, more efficient and focused. More importantly, testing of a targeted drug with multiple chemotherapies in the same models can also provide guidance on future combination selection strategy.

#396

Analysis of MET-amplified solid tumors using chromogenic in situ hybridization (CISH).

David Arguello,1 Zoran Gatalica,1 Semir Vranic,2 Sandeep Reddy,1 Ari VanderWalde3. 1 _Caris Life Sciences, Phoenix, AZ;_ 2 _University Clinical Center, Sarajevo, Bosnia and Herzegovina;_ 3 _West Clinic, University of Tennessee Health Science Center, Memphis, TN_.

MET amplification has been implicated in signaling pathways that promote cell proliferation, invasion, and survival. It has been identified as an oncogenic driver in various malignancies and is currently being investigated as a potential therapeutic target. To date, MET exon 14 skipping by sequencing and MET amplification by FISH have been found to have potential clinical utility in predicting those patients who may derive benefit from MET-targeted therapy. However, little research has been conducted on alternative technologies to FISH such as CISH, which does not require a dark room and can be interpreted by a board-certified pathologist. The purpose of this study is to report our experience with MET amplification across solid tumors using CISH.

A retrospective analysis was done on 26,619 specimens analyzed for MET amplification by CISH at a CLIA-certified lab (Caris Life Sciences). The validated CISH assay, previously validated against a FISH assay, utilized a gene copy number > 5 to assess amplification. Concordance and correlative studies were done in MET-amplified, non-small cell lung cancer (NSCLC) specimens analyzed using a cMET IHC (SP44, 2+ or 3+ staining intensity in 50% or more tumor cell membrane) analyzing protein expression. Correlative studies involving co-existing aberrations, including PD-L1 (SP142, any intensity in at least 50% of tumor cells), in this MET-amplified, NSCLC cohort were also performed.

MET amplification utilizing CISH was 0.7% (188/26,619) overall. MET-amplified solid tumors included carcinomas such as NSCLC (3.1%, 87/2767), gastric adenocarcinoma (3.8%, 11/293), esophageal and esophagogastric junction adenocarcinoma (3.3%, 11/338), and endometrial carcinoma (0.4%, 9/2020) along with non-carcinomas, including glioblastoma multiforme (1.0%, 5/510), uterine sarcoma (1.3%, 5/400), melanoma (0.4%, 2/538), and rare tumors such as placental-site trophoblastic tumor (100%, 1/1) and prostatic neuroendocrine tumor (100%, 1/1). A sub-analysis of MET-amplified, NSCLC specimens demonstrated co-occurring protein overexpression in 92.6% (75/81) of cases. These same MET-amplified, NSCLC specimens were found to have EGFR pathogenic/presumed pathogenic mutations (19.7%, 15/76), ALK rearrangements (2.5%, 2/80), and PD-L1 overexpression (27.5%, 14/51). ROS1 rearrangements were not detected in this NSCLC cohort (0%, 0/76).

Our data suggest MET amplification detection utilizing CISH is a viable option for identifying MET-driven cancers. The presence of MET across various solid tumors contrasts with biomarkers like HER2, which are exclusive to carcinomas. A sub-analysis of our NSCLC population shows MET-amplified tumors contains a similar molecular distribution to the general NSCLC population. Future studies should incorporate MET CISH in clinical trials utilizing MET-targeted agents to determine its potential as a predictive test for evaluating who may derive the most benefit.

#397

Brain natriuretic peptide (BNP) as molecular marker for bladder, ovarian, and glioblastoma cancers.

Cynthia Lee,1 William Yeh,2 Dongwoon Lee,1 Samuel Dixon,1 Vuong Trieu1. 1 _Autotelic Inc, Fountain Valley, CA;_ 2 _LipoMedics Inc, Fort Worth, TX_.

Background: BNP has been reported as molecular marker for ovarian cancer. This study evaluated BNP as a molecular marker for bladder, ovarian, glioblastoma, and pancreatic cancers. Methods: Serum samples collected at the time of diagnosis were tested using rapid and quantitative point-of-care (POC) devices for BNP blood biomarkers and the data was evaluated using JMP® 11.2.1 statistical analysis software. Samples were collected from bladder cancer patients (48 transitional cell carcinoma of the bladder), glioblastoma patients (51), pancreatic cancer patients (39 adenocarcinoma, 1 mucinous adenocarcinoma), ovarian cancer patients (24 serous adenocarcinoma, 20 serous papillary adenocarcinoma, 7 endometrioid adenocarcinoma, 3 clear cell carcinoma, 1 endometrial adenocarcinoma, 1 endometrioid papillary carcinoma, 1 serous papillary carcinoma), and normal subjects (n = 60). Results: Compared to normal subjects, bladder patients are more likely to be smokers (p = 0.0064). All four patient groups were more likely to be hypertensive (bladder, p = 0.0165; glioblastoma, p<0.0001, ovarian, p = 0.0002; pancreatic, p < 0.0001). No significant differences among the groups versus normal for weight were observed. Significant differences between the groups versus normal for BMI (ovarian, p = 0.0061), age (bladder, p = 0.0006; pancreatic = 0.0085), BNP (ovarian, p = 0.0012; glioblastoma, p = 0.0016; bladder, p = 0.0112) were noted. There was positive correlation between age and BNP level among all four cancer groups (bladder, p = 0.0133; glioblastoma, p = 0.0629; ovarian, p = 0.0304; pancreatic, p = 0.0172); whereas there was a negative correlation between age and BNP level among normal controls (p = 0.0109). Age-adjusted BNP level was highly significant different versus normal (bladder, p <0.0001; ovarian, p=0.0013; glioblastoma, p = 0.0047). Neither BNP nor age-adjusted BNP level was significantly different from normal for pancreatic cancer patients. Hypertensive condition had no impact on BNP or age-adjusted BNP. Conclusions: Bladder, ovarian, and glioblastoma cancers are associated with significant reduction in BNP and age-adjusted BNP blood levels. Pancreatic cancer was an exception. BNP level could be useful in the monitoring of treatment efficacy in which the BNP POC device described here would be highly appropriate due to its ease in deployment at the patient's home or doctor's office.

#398

Real-life example of biobanking: results of the PATH Biobank.

Tobias Anzeneder, Ulla Ohlms, Heinz Bodenmüller. _Stiftung PATH, München, Germany_.

Introduction: Since 2002, PATH Foundation has kept a biobank collecting high-quality fresh frozen breast cancer specimens adhering to uniform SOPs at seven certified breast cancer centres in Germany. Research groups from academia and industry can obtain samples after application and review. PATH Biobank is a not-for-profit organisation and asks for a cost recovery fee in exchange for sample allocation to sustainably finance the expenses it incurs.

Material and methods: The PATH Biobank consists of a centralized database and a decentralized bio repository. The samples are collected and stored at seven institutes for pathology at certified German breast cancer centres. Tumour tissue, along with normal (benign) adjacent tissue and blood serum aliquots are processed, labelled and stored according to uniform SOPs. Informed consent to biobanking and the use of the samples and data for research is obtained from the donors individually during pre-operation discussions.

Results: Since 2004, more than 8,600 breast cancer patients have given their informed consent to the storage and analysis of their tissue and blood serum for research purposes. Breast cancer tissue samples from 59% of donors could be stored due to size of the surgically removed tissue specimen. In addition, normal adjacent tissue is available from 62% of donors and blood serum aliquots can be derived from 92% of patients.

Using the annotating data, 96% of donors can be classified into the intrinsic molecular breast cancer subtypes in accordance with the St. Gallen Criteria. In 2008, PATH Biobank started to support research groups by providing breast cancer samples and data as well as informing the public about the projects on-line. In 2015 three co operations resulted in scientific publications.

One landmark collaborations started in 2012 and has been studying the frequency and background of defined mutations in breast cancer. A number of 701 tissue samples have been used for this study. All samples were subject to quality testing, only 11% failed due to less than 5% tumour content. Tumour content depended on clinical conditions and staging (ranging from 34% failure in samples derived from neo adjuvantly treated patients to 2% in cases with staging UICC IV). Additional results concerning the quality of samples from PATH Biobank will be presented.

Conclusions: For breast cancer research and biomarker studies, PATH Biobank can be a valuable resource.

#399

A new rabbit monoclonal antibody to FOXM1 for immuohistochemical assessment.

Jackie K. Chan, Aihua Li, Jason K. Law, Asish V. Nand, Taiying Chen. _Epitomics - an Abcam company, Burlingame, CA_.

Purpose and Background: Forkhead box protein M1 (FOXM1) is a major oncogenic transcription factor regulating genes involved in G1-S and G2-M cell cycle transitions. FOXM1 has been implicated in tumorigenesis, facilitating tumor proliferation, angiogenesis and migration. Genome wide microarray studies consistently demonstrated FOXM1 upregulation in many solid malignancies. FOXM1 overexpression is associated with poor patient prognosis through blocking cytostatic and cytotoxic activity in taxane, platinum-based, and anthracycline anti-cancer drugs thereby increasing chemotherapeutic resistance. FOXM1 is broadly distributed during embryonic development, but restricted to progenitor and proliferating cells in mature tissues. However, broad tissue immunohistochemistry (IHC) based distribution studies are currently lacking. Furthermore, commercially available FOXM1 IHC antibodies are limited to rabbit polyclonals which are susceptible to batch inconsistencies. The need for a consistent monoclonal antibody validated in IHC is currently unmet. Herein, we report on FOXM1 distribution on a wide ranging collection of normal and tumor tissues using a newly developed rabbit monoclonal antibody (RabMAb), Clone EP372.

Design: Rabbits were immunized with a recombinant N-terminal FOXM1 protein fragment, allowing detection of the three isoforms: FOXM1A, FOXM1B, and FOXM1C. Sera collected from immunized rabbits were screened by ELISA, Western Blot (WB) and IHC. After fusion, antibody from final hybridoma cells were characterized by ELISA and WB. This antibody, designated as Clone EP372 was further characterized through IHC testing using formalin-fixed, paraffin embedded (FFPE) human normal and tumor tissue microarrays (TMA).

Results: A single band at approximately 100 kDa was detected by WB in Hela cell lysates. IHC analysis on 31 cases of human normal TMA comprising 15 different tissue types showed that FOXM1 only labeled cells in regenerative tissues, with nuclear expression in germinal center cells in the tonsil, proliferating colonic crypt cells, stromal cells in the endometrium, spleen and testis. Fifteen of 30 tumor cases were FOXM1 positive, expressed in astrocytoma, melanoma, and carcinomas of the bladder, thyroid, endometrium, ovarian, colon, hepatocellular, lung, cervical, stomach, and breast.

Conclusion: The RabMAb FOXM1 antibody, Clone EP372 specifically labels proliferating cells in regenerative tissues and in a broad spectrum of human tumors. This expression pattern is concordant with results from the literature. To our knowledge, this is the first report that systematically evaluated FOXM1 expression across a broad number of normal human tissues and a variety of human tumors via IHC. Additional research in the role of FOXM1 in tumorigenesis and drug resistance across tissue types will help bolster the prognostic and therapeutic significance of this key oncogenic regulator.

#400

Immunofluorescence chromatographic assay of tumor cells in dried blood spot.

Ming Zhao,1 Dakang Ma,2 Quanxu Shen,3 Bin Hong1. 1 _TeloVISION LLC, West Lafayette, IN;_ 2 _University of Maryland, College Park, MD;_ 3 _The Fifth Central Hospital of Tianjin, Tianjin, China_.

Blood droplet, when desiccated on a surface, forms a ring-like deposit, akin to the coffee ring left after a coffee spill. When blood, coffee, or other aqueous suspensions of the colloidal particles, evaporate with contract line pinned on the underlying hydrophilic surface, a capillary force is generated by the evaporative flux, which drags nearly all the particles to the edge. As the evaporation extends, the volume of the droplet decreases, and the sol-gel transition occurs. The gelation gradually moves inward from the edge to the center, eventually immobilizes all particles, and develops the ring effect. Because smaller sized particles diffuse much more quickly and efficiently than the larger ones, smaller particles tend to preferentially accumulate on the rim, forming multi-layered self-assembly. Contrarily, the majority of the larger particles congregate in the central area. This natural phenomenon simply presents a size-dependent chromatography of variously sized particles in solution. Separation of normal blood cells or break-free tumor cells has been widely utilized for blood disease diagnosis and cancer prognosis. Traditional preparation via blood smear and advanced separation by the external driving forces, e.g. centrifugation and powered microfluidics, could be limited by the technical difficulty, assay complexity, and cell integrity. Coffee-ring based blood cell separation and detection, namely dried spot biopsy, is however a hand-free, cell-harmless, spontaneous process. To perform a dried spot biopsy, 3x1 inch microscope slides were first cleaned to remove surface impurities and residues. 200 uL of freshly collected, anti-coagulated whole blood, bearing fluorescent microbeads (1 and 10 um sized) or pre-stained breast tumor cells (MDA-MB-231, ~15 um sized), was dispensed onto the slide and allowed to dry statically or acceleratedly on a moving stage at controlled temperature and humidity. Three hydrophilic coatings and five surfactants were examined to balance the outward capillary flow with the inward Marangoni flow for optimal coffee ring effect. It is demonstrated that two sizes of microbeads formed separate rings, a few hundreds of microns in average apart from each other, while tumor cells, acting differently, concentrated in the central area with random dispersion, probably because of the diversified cell sizes. The dried spot biopsy could have a potential for detecting blood borne biomarkers in the form of dried blood specimens that poses longer shelf life, less biohazardous risk, and easier transportation protocol than the liquid form. Additionally, specific cells of interest from microscopic regions of dried blood film can be retrieved by laser microdissection for downstream analyses. Particularly, this method could be valuable for use in low-resourced regions and countries.

#401

Preservation of cells in blood and culture.

Cecille Browne, Dan Lu, Daniela Roth, Vasco Liberal. _Biomatrica, Inc., San Diego, CA_.

Introduction: Liquid biopsies and cell-based therapies are emerging approaches for the treatment of cancer. Accuracy of diagnosis using liquid biopsies and effectiveness of treatment using cell-based therapies will rely upon the preservation cells for both viability and the information contained in them.

Experimental procedures: Biomatrica's proprietary chemical library was used to develop formulations that could preserve cells in multiple formats. Cell preservation was assessed by a variety of cell-based and molecular techniques such as microscopy, flow cytometry and real-time PCR.

Results: Blood samples spiked with circulating tumor cells (CTCs) were preserved for several days at room temperature to allow for downstream morphological and transcriptomic analyses. CTC recoveries in preserved samples were as much as 4 times those in non-protected samples over 4 days. Changes in RNA expression of CTC-specific genes in preserved samples ranged from 0 to +/- 2 delta Ct values, while in non-protected samples, changes ranged from 0 to +/-5 delta Ct values over 4 days. In some formulations, CTC viabilities were as much as twice the viabilities of CTCs in non-protected samples. Supporting these viability results was a demonstrated culturability of isolated CTCs. Furthermore, cell-free DNA in plasma of preserved blood samples changed by less than 2 Ct values over 4 days, whereas in control samples, plasma DNA changed by as much as 10 Ct values. Hemolysis in preserved blood samples was also lower by as much as 69% compared to non-protected samples.

In a separate set of experiments, activated T-cells were stored in the presence of common buffers (RPMI, PBS) or formulations at room temperature. T-cells cultured with formulations displayed greater 40 to 60% greater cell viability after 3 days compared to those stored in common buffers.

Conclusions: Biomatrica has developed a library of cell preservatives that can be used to preserve cells in blood samples and in cell culture. The ability to recover cells in blood samples and determine their genetic information would enable monitoring of a patient's cancer status and personalizing drug treatments. In addition, cell preservation may allow further advances in cell-based therapies as cells could be more reliably recovered from patients, modified (e.g. CAR-T, stem cells), and administered to patients.

#402

Multiplex TaqMan assays for rare mutation analysis using digital PCR.

Marion -. Laig, Frances Chan, Le Lac, Ted Straub, Kamini Varma, David Keys. _Thermo Fisher Scientific, South San Francisco, CA_.

Introduction

Detection of rare mutations for research purposes in tumor tissue and cell free DNA (cfDNA) allows for monitoring of tumor progression and regression. cfDNA isolated from plasma combined with a sensitive detection method like digital PCR is non-invasive and enables earlier detection compared to conventional imaging techniques.

Building on the TaqMan based Rare Mutation assay set for detection of rare mutations using digital PCR on the QuantStudio 3D Digital PCR System, we are now developing multiplex assays for simultaneous detection of several mutations. We selected relevant mutations in the EGFR and KRAS genes for our initial multiplex application: EGFR G719, EGFR exon 19 deletions, and KRAS G12/G13. These mutations may have implications for potential future targeted therapy.

Methods

Primers and probes of singleplex Rare Mutation Assays were reformulated to generate multiplex assays detecting the EGFR and KRAS mutations. All multiplex assays were tested on template composed of wild-type genomic DNA background mixed with mutant plasmid reflecting each of the mutations detected by the multiplex assays.

Summary

Initial experimental results were successful and showed excellent signal intensity and clear cluster separation when analyzed with the QuantStudio 3D AnalysisSuite™ Cloud Software. The EGFR G719 mutations (COSM6239, COSM6253, COSM6252) were detected using a 3plex assay, EGFR exon 19 deletions (COSM12383, COSM12422, COSM12678, COSM6223, COSM6254, COSM6255) were detected using a 6plex assay, and KRAS G12/G13 mutations (COSM516, COSM517, COSM518, COSM520, COSM521, COSM522, COSM527, COSM532) were detected using an 8plex.

Conclusion

Multiplexing assays for three relevant mutation loci proved feasible and presents an efficient way to assess the presence and the percentage of mutations at these loci.

#403

Performance assessment of highly sensitive NGS assay for TP53.

Johannes Fredebohm,1 Daniel Mehnert,1 Ann-Kathrin Löber,1 Hiroyuki Shimizu,2 Frank Holtrup,1 Frank Diehl1. 1 _Sysmex Inostics GmbH, Hamburg, Germany;_ 2 _Sysmex Corporation, Kobe, Japan_.

We have designed a highly sensitive assay based on the Safe-Sequencing technology(1) to detect de novo mutations in the TP53 gene. A custom panel was designed to cover 95% of all reported mutations in TP53. To demonstrate assay performance, we assessed LoD, LoB, reproducibility, and repeatability and evaluated concordance with BEAMing(2). A customized data analysis pipeline for ultra-deep sequencing runs was developed that includes extensive quality control and advanced mutation calling. This highly sensitive NGS-based workflow can be applied to monitor recurrent or minimal residual disease in cancer patients after surgery or chemotherapy.

References:

(1) Kinde I, Wu J, Papadopoulos N, Kinzler KW, Vogelstein B. Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A. 2011 Jun 7; 108(23): 9530-9535

(2) Diehl F, Li M, He Y, Kinzler KW, Vogelstein B, Dressman D. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat Methods. 2006 Jul;3(7):551-9.

#404

Development of a RSPO3 CLIA-validated assay as a predictive biomarker for response to anti-RSPO3 antibody treatment in patients with solid tumors.

Chun Zhang,1 Yuwang Liu,1 Min Wang,1 Gilbert OYoung,1 Joy Kavanagh,2 Cheryl McFarlane,2 Fiore Cattaruzza,1 Pete Yeung,1 Jennifer Cain,1 Wan-Ching Yen,1 Marcus Fischer,1 Belinda Cancilla,1 Edwina Dobbin,2 Michelle McCarthy,2 Austin Gurney,1 Leonardo Faoro,1 John Lewicki,1 Tim Hoey,1 Ann M. Kapoun1. 1 _OncoMed Pharmaceuticals Inc., Redwood city, CA;_ 2 _Almac Diagnostics LLC, Craigavon, United Kingdom_.

R-Spondin (RSPO) proteins bind to LGR receptors and potentiate Wnt/β-catenin signaling. We have identified a therapeutic anti-RSPO3 antibody targeting the RSPO-LGR pathway. In preclinical studies, RSPO3 gene expression has shown correlation with anti-RSPO3 antibody efficacy in multiple solid tumor types. A qPCR-based RSPO3 assay has been developed as a predictive biomarker for response to the anti-RSPO3 antibody. In addition, RSPO gene fusions may play a role in the activation of Wnt signaling. A gene fusion detection workflow consisting of a RSPO3 CLIA assay, a RSPO3 RUO assay and next generation sequencing (NGS) has also been developed.

We designed 6 qPCR-based assays for the RSPO3 CLIA assay development and 2 assays for the RUO assay. These assays were designed to span exon-exon junctions or target microarray probe set sequences. Amplification sensitivity and specificity were assessed for assay selection. The analytic performance of the candidate RSPO3 CLIA assay and quality control measures were established in a validation study. The validation study included: 1) performance specifications of the RSPO3 assay including analytical sensitivity, linearity, and precision, 2) determination of a reportable range, 3) establishment of a cut-off for the RSPO3 CLIA assay for patient selection, and 4) establishment of quality control procedures. 104 human cancer tissues and 24 independent patient-derived tumor xenografts (PDX) were used in these studies. To evaluate the fusion detection workflow, the RUO assay was performed on samples that tested above the CLIA assay cut-off. The delta Ct difference between the CLIA and RUO assays was calculated to identify potential fusions.

The limit of quantification was established for the RSPO3 CLIA assay. The 95% reference interval was estimated to be (-2.44, 16.02) with 90% confidence interval for the lower bound (-3.45, -2.12) and upper bound (15.26, 16.57). The delta Ct cut-off for the RSPO3 CLIA assay was set based on sensitivity, specificity and prevalence. No statistically significant difference in the total variance across the tested samples was observed. A549 and OV56 were identified to be cell line controls with established acceptable delta Ct limits. Using NGS, RSPO3 fusions were identified in 6 PDX tumors with delta Ct RUO - delta Ct CLIA>7, including a novel fusion. This cut-off was further refined with NGS of 9 clinical samples. Prevalence of the RSPO3 expression and fusions will be presented.

A qPCR based RSPO3 assay was developed and CLIA-validated for use as a potential predictive biomarker for response to anti-RSPO3 therapy. This RSPO3 CLIA assay, together with the fusion detection workflow, will be evaluated in a Phase 1a/b dose escalation study of anti-RSPO3 (OMP-131R10) in advanced solid tumors and in combination with FOLFIRI in metastatic colorectal cancer (NCT02482441).

#405

Caveolin-1 expression mediates response to albumin-bound paclitaxel.

Terence M. Williams,1 Moumita Chatterjee,1 Ryan Robb,1 Marally Vedaie,1 Star Seum,1 Thirumoorthy Krishnan,1 Palanichamy Kamalakanan,1 Edgar Ben-Josef,2 Arnab Chakravarti1. 1 _James Cancer Hospital & Solove Research Institute, Columbus, OH; _2 _University of Pennsylvania, Philadelphia, PA_.

BACKGROUND:

Albumin-bound chemotherapies such as nab-paclitaxel are approved to treat pancreatic cancer, non-small cell lung cancer (NSCLC), and breast cancer. Predictive biomarkers to select patients who may benefit most from nab-paclitaxel are lacking. Nab-paclitaxel is thought to enter cells through a caveolae-gp60 endocytic mechanism. Caveolin-1 (Cav-1) is a principal structural component of caveolae, and Cav-1 has been shown to be important for albumin uptake in endothelial cells. Cav-1 is known to be up-regulated in multiple tumor types, including pancreatic cancer, and certain subtypes of non-small cell lung cancer, and breast cancer. We hypothesize that Cav-1 expression may predict response to nab-paclitaxel therapy.

METHODS: We correlated Cav-1 expression with nab-paclitaxel sensitivity in a panel of NSCLC and pancreatic cancer cell lines. We also assessed albumin uptake in tumor and non-tumor cell lines. We genetically depleted Cav-1 by shRNA in cells and performed cytotoxicity assays. In addition, we measured how Cav-1 levels affected albumin and nab-paclitaxel uptake into tumor cells by immunofluorescence, immunoblotting, and mass spectrometry. Annexin V flow cytometry analysis and immunoblotting for apoptosis pathway intermediates were also performed. Nab-paclitaxel resistant cell lines were created by culturing cells with increasing doses of nab-paclitaxel for extended periods of time. The role of Cav-1 expression in mediating response to nab-paclitaxel in vivo was assessed using xenograft models.

RESULTS:

H23 and MIA-PaCa2 tumor cells uptake more albumin compared to FHs74Int and HBEC3KT non-tumor cell lines. Higher Cav-1 expression in a panel of pancreatic cancer and NSCLC cell lines was correlated with lower IC50 for nab-paclitaxel. Cav-1 depletion resulted in reduced albumin and nab-paclitaxel uptake by tumor cells as measured by immunofluorescence, immunoblotting, and mass spectrometry. Loss of Cav-1 resulted in resistance to nab-paclitaxel but no change in sensitivity to free paclitaxel in vitro. Cav-1 down-regulation resulted in protection from nab-paclitaxel-induced apoptosis. Conversely, re-expression of Cav-1 in low-Cav-1 endogenously expressing cell lines AsPC-1 and HPAFII resulted in increased nab-paclitaxel uptake and sensitization through apoptosis. Furthermore, nab-paclitaxel resistant cells generated by prolonged exposure demonstrated downregulation of Cav-1 levels and reduced albumin uptake. Finally, genetic depletion of Cav-1 rendered tumor cells resistant to nab-paclitaxel in xenograft models, with concomitant reduced albumin uptake and activation of apoptosis.

CONCLUSIONS: Our data suggest that Cav-1 expression and caveolae are critical determinants of response to nab-paclitaxel. This data supports further testing of Cav-1 as a potential predictive biomarker of response to nab-paclitaxel and potentially other albumin-bound chemotherapies.

#406

Enhancer templated RNAs as predictors of therapeutic response to epigenetic therapy.

Ron Firestein,1 Mark McCleland,2 Kathryn Mesh,2 Florian Gnad2. 1 _Hudson Institute of Medical Research, Clayton, Australia;_ 2 _Genentech, South San Francisco, CA_.

Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we utilized a CRISPR loss of function screen and identified a number of essential genes, including the Bromodomain and Extraterminal (BET) protein, BRD4. We find BRD4 is critical for colon cancer proliferation and its loss leads to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers, characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct super-enhancer in CIMP(+) colon cancers that regulates cMYC transcription. We find that the CCAT1 long non-coding RNA (lncRNA) is transcribed from this super-enhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose CCAT1 as a clinically tractable biomarker for identifying patients likely to benefit from BET inhibitors.

#407

Functional analysis of discoidin domain receptor 2 mutation and expression in squamous cell lung cancer.

Naomi Kobayashi-Watanabe. _Saga University, Saga, Japan_.

The treatment strategy for lung cancer has been rapidly progressed, especially in molecular targeted therapy. However, most treatments are established for adenocarcinoma of lung, but not squamous cell carcinoma (SQCC). SQCC occupies approximately 30% among entire lung cancer patients, but the prognosis is worse than that of adenocarcinoma patients. Discoidin domain receptor (DDR) 2 mutations have been recently reported as one of the candidates of molecular targets for lung SQCC, and they are associated with sensitivity to dasatinib, which is already applicable to treatment of chronic myeloid leukemia. However, precise roles of DDR2 on SQCCs of lung have not been clarified. We performed Sanger sequencing of DDR2 gene in a set of 44 primary lung SQCC samples and 7 lung SQCC cell lines, and identified a novel mutation (T681I) in a lung SQCC patient and a lung SQCC cell line, EBC-1. Endogenous protein expression level of DDR2 measured by Western blot technique was high in a lung SQCC cell line, PC-1. Overexpression of wild type and T681I of DDR2 promoted cancer cell invasion of a human lung SQCC cell line, H226B. In animal model, lung metastasis was enhanced and survival was shortened after transplantation of DDR2 wild type overexpressing cells into NOD/SCID/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and dendritic cell function, and complement activation, as well as T and B cell deficiencies. Using the cDNA microarray and protein phosphorylation array assay, we found that ectopic expression of DDR2 induced TGF-β1 protein and mRNA expression accompanied with phosphorylation of c-Jun, p38 MAP kinase, and MMP1 elevation. During the process of metastasis, TGF-β pathway plays pivotal roles especially on disruption of basement membrane through induction of MMPs from fibroblasts mediated through both Smad and non-Smad pathways. Since phosphorylations of c-Jun and p38 MAPK were reported to be involved in non-Smad pathway, DDR2 would induce TGF-β mainly through non-Smad pathway, resulting in cancer invasion and metastasis. Taken together, elevation of DDR2 protein might contribute on tumor progression as well as some of DDR2 mutations in lung SQCC.

#408

ATM function and mutation in CLL 11q deletion samples.

Yingjun Jiang, Xiaojun Liu, Hsiang-Chun Chen, Kim-Anh Do, Xiaoping Su, William Wierda, Michael Keating, William Plunkett. _UT MD Anderson Cancer Center, Houston, TX_.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of abnormal B-cell development in which cell death mechanisms have been altered. As the second most common type of leukemia in adults, it progresses more slowly than other types of leukemia and is considered incurable. Approximately 10% of previously untreated patients with CLL exhibit a substantial deletion in the q arm of chromosome 11(22-23), the site of the ATM gene, the product of which regulates homologous recombination repair of double strand DNA breaks (DSB). This lesion occurs at increased incidence, about 50% in patients relapsing on fludarabine-cyclophosphamide-rituximab (FCR) therapy. The residual ATM allele is often mutated, suggesting that the region containing ATM is important for response to chemoimmunotherapy. Although some mutations that cause non-synonymous substitution of amino acids associated with loss of ATM function are known, because the ATM protein contains more than 3,000 amino acids, it is unlikely that all most or all non-synonymous changes would cause loss of function. To address this issue, we developed an immunoblot assay to determine ATM function in del(11q22-23) samples from CLL patients. We confirmed that SMC1 and KAP1 are unique substrates of ATM. Their phosphorylation levels after irradiation (IR) were linearly correlated with ATM activity. This was demonstrated using cell lysates mixtures from cells derived from an individual with ataxia telangiectasia that are deficient in ATM, and those cells repleted with ATM. Consequently, the ratios of phospho to total proteins of SMC1 and KAP1 were employed as indicators of ATM function in response to IR treatment. Using a pool made up of lysates from FISH-negative CLL samples (n=8) as a positive standard, we validated this assay in 46 del(11q22-23) CLL samples. The phosphorylation ratios of SMC1 and KAP1 from 46 CLL samples were analyzed simultaneously by the normal mixture model-based clustering method, and a formula was generated to determine ATM function. Using this formula, we identified 13 ATM function-deficient samples. To determine whether there is a correlation between ATM gene mutation and the function of the protein, we conducted targeted sequencing of the ATM gene in the 46 samples. Genomic DNA of T-cells isolated and expanded from each of these samples was extracted to serve as a germline reference for the CLL cells. Through a series of bioinformatics analyses, 12 different ATM somatic mutations were identified. Fifteen other ATM mutations were recognized as germ line mutations. No strong correlation was observed between ATM mutation and function. Therefore, alternative assays of ATM function, such as that we have described, are needed to identify ATM deficient tumors in order to develop selective therapies, e.g. agents that cause DSB or inhibit redundant repair pathways.

#409

Immunofluorescent digital slide technology to evaluate correlation between Glypican-3 expression and response to codrituzumab.

Takayoshi Tanaka,1 Yasuo Sugitani,1 Tokiya Abe,2 Miho Kawaida,2 Ken Yamazaki,2 Akinori Hashiguchi,2 Michiie Sakamoto,2 Toshihiko Ohtomo1. 1 _Chugai pharmaceutical Co., Ltd, Tokyo, Japan;_ 2 _Department of Pathology, Keio University School of Medicine, Tokyo, Japan_.

Background: Codrituzumab/GC33/RO5137382 is a recombinant humanized monoclonal antibody against Glypican-3 (GPC3) which is highly expressed in HCC cells. As part of phase I clinical trial, GC-002US study, evaluating escalating doses of codrituzumab plus sorafenib in HCC, immunofluorescent staining of GPC3 were applied in addition to GPC3 immunohistochemistry (IHC) with conventional DAB staining. The objectives of this work were to compare GPC3 expression levels by different staining methods and evaluate the correlation between these GPC3 scores and codrituzumab activities.

Methods: Biopsies specimens (n = 34) collected at baseline in the phase I trial were evaluated by GPC3-IHC and immunofluorescent quantification digital slide (IQD) methods. GPC3-IHC were conducted using mouse anti-human GPC3 antibody, mouse GC33 (Ventana Medical Systems) as reported previously (Ikeda M. et al., Cancer Sci. 2014: 105; 455-462) and H scores were derived by two pathologists. For IQD, biopsy specimens were stained with same primary antibody, and were incubated with Qdot 655-conjugated secondary antibody. Fluorescent images of whole slides were obtained with the Nano-Zoomer (Hamamatsu Photonics K. K.). The IQD intensity score (immunofluorescent intensity per pixel) and cell score (immunofluorescent intensity per cell) were calculated respectively using MatLab (MathWorks Inc.).

Results: There was a statistically significant correlation between GPC3 H scores and IQD scores. Among GPC3 scores, only the IQD cell score was correlated with serum GPC3 C-terminus increase by treatment (p = 0.032), whereas in the patients treated with higher dose of GC33 (≥ 10 mg/kg/week) the cytoplasmic H and the IQD intensity score were also correlated with serum GPC3 C-terminus increase by treatment. Interestingly, increasing cut-off values of IQD cell score levels led to lower HR and longer PFS. Best HR with lowest p value was observed at a cut-off value of 66,183 (HR = 0.24, p = 0.038).

Conclusion: This study suggested that higher GPC3 expression on HCC cells quantified by IQD method was superior to other scores to predict codrituzumab activity, though this analysis was conducted only with limited number of subjects assessed. Further assessment and validation in clinical study of codrituzumab would be necessary to make a final conclusion on the clinical application of GPC3 IQD.

#410

Exhaustive scanning of mutation hotspot regions using a new tool: The MHS-ddPCR.

Charles Decraene,1 Francois-Clement Bidard,1 Samia Belaabi,1 Etienne Rouleau,1 Adrien Saliou,1 Alexandre Houy,1 Maud Milder,1 Olivier Lantz,1 Marc Ychou,2 Jean-Yves Pierga,1 Marc-Henri Stern,1 Charlotte Proudhon1. 1 _Institut Curie, Paris, France;_ 2 _Institut de Cancerologie de Montpellier, Montpellier, France_.

Background: Recent progress in the 'liquid biopsy' field in combination with the development of the droplet digital PCR (ddPCR) technology now enables non-invasive monitoring of cancer-related genomic alterations with high detection accuracy. Current ddPCR techniques represent the gold standard for the detection of point mutations that have been previously characterized on tumor tissues. However, ddPCR screen for only one mutation (or a few mutations, with multicolor ddPCR) per reaction. This is a clear limitation to use it as a discovery tool, to detect resistance-associated mutations that may appear during therapy (e.g. ESR1 activating mutations in breast cancer) or when tumor tissue is not available (e.g. EGFR activating mutations in lung cancers).

Methods:

We developed the Multiple Hotspot mutations detection by Single droplet digital PCR (MHS-ddPCR) method, a variant of the conventional ddPCR technique, which detects all genomic alterations within a hotspot region, using a unique couple of Taqman oligo-probes. We first established two specific assays covering KRAS and EGFR mutation hotspot regions, which are of clinical importance in the context of colorectal cancer. The assay for KRAS scans for the 7 most common mutations in codons 12 and 13 of the gene as well as all other mutations with lower frequency (<1%). The EGFR assay screens for all in-frame deletions of exon 19, which are frequent activating events in EGFR.

Results:

Sensitivity and specificity of MHS-ddPCR were assessed on decreasing fractions of tumor DNA mutated for KRAS. The KRAS assay reaches a sensitivity of 0.02% and was validated on plasma and tumor samples harboring a panel of different KRAS mutations. Similarly, the EGFR assay can detect numerous exon 19 deletions from patient samples and with limited amounts of tumor DNA.

Conclusions:

The MHS-ddPCR is extremely sensitive, works for numerous hotspot regions with different types of alterations (SNV, deletion) and is cost-effective as it combines multiple assays in one reaction.

#411

Sensitive detection of BRAF V600E mutation by bridged nucleic acid (BNA)-mediate real-time PCR assay.

Xiaoyun Liu,1 Leticia Loredo,1 Houquan Dai,1 Aaron Castro,1 Yuewei Zhao,1 Sung Kun Kim,2 Austin Dinkel,2 Miguel Castro1. 1 _Biosynthesis Inc., Lewisville, TX;_ 2 _Northeastern State University, Tahlequah, OK_.

BRAF is a human gene encoding a serine/threonine protein kinase in the RAS-RAF-MEK-ERK signal pathway, which is important in regulating cellular responses to extracellular signals. Frequent mutations on BRAF gene have been detected in various types of human cancer, including malignant melanoma, non-small cell lung cancer, colorectal cancer, papillary carcinoma of the thyroid, ovarian cancer and hairy cell leukemia. Among all, a single point 1799T>A (V600E) transversion, the most common mutation on BRAF, results in increased kinase activity. It serves as a driver mutation in several types of cancer and confers increased sensitivity to tyrosine kinase inhibitors. Thus, detecting the BRAF V600E mutation may offer valuable information for cancer diagnosis, treatment selection, and prognosis. Here we report a rapid, highly sensitive method for the detection of BRAF V600 mutation using a bridged nucleic acid (BNA) clamp technology associated with quantitative PCR (qPCR). Due to the feature of enhanced affinity for hybridization template, BNA clamp is able to selectively block PCR reactions on the perfect match DNA sequence, while leaves the mismatch template unaffected. In this study several BNA clamps that bind to BRAF V600 wild-type (WT) template was designed, synthesized, and characterized. As compared to DNA clamp counterparts, these BNA clamps increased Tm values for the BRAF V600 WT by greater than 30°C. In addition, BNA clamps efficiently distinguished BRAF WT (perfect match) from V600E (mismatch) templates (deltaTm >12°C). In qPCR assays BNA clamps could suppress amplification of WT by >1,000-fold, while had little or no effects on the V600E mutant. Such BNA clamps allowed us to detect 1.0% or lower levels for the BRAF V600E mutant in a wild-type background. These results indicate that BNA clamp-based qPCR technology offers a new tool for rapid, sensitive detection of BRAF V600E mutation. The technology may be adapted for detection of mutations of a variety of biomarker genes.

#412

RNA-Seq provides cost-effective alternative for typing self-identifying antigens in immunotherapy patients.

Kimberly Robasky, Jason Powers, Donald Trapolsi, Jeff Jasper, Chad Brown. _Q2 Solutions|EA, Chapel Hill, NC_.

The excitement surrounding immunotherapy is being driven by results in the clinic. Currently, autoimmunity is an unfortunate side-effect for a large fraction of those treated. Consequently, understanding how self-antigens are recognized is nearly as important as characterizing the immune repertoire for efficacious delivery of these promising new therapies. One approach to measuring the tumor immune environment is with RNA-Seq assays. Here we show that the genes responsible for presenting self-antigens (HLA Class I and Class II) can also be ascertained from RNA-Seq on total RNA. We present the detailed results from standard RNA-Seq pipeline analysis for 40 lymphoblastoid cell lines to achieve 91.3% overall concordance with "gold standard" typings. These samples were sequenced with 50bp paired-ends and approximately 30M reads. We additionally present RNA-Seq analysis results from calling HLA alleles on 15 replicate pairs of FFPE hepatic tumor samples, finding 88.33% overall replicate concordance with 2-digit precision, and 90% for Class I alleles. Finally, we present HLA-types on triplicates from 2 common breast cancer cell lines, MCF7 and T47D for loci HLA-A,-B,-C,-DRB1,-DQB1, with 90% overall replicate concordance. Emerging targeted DNA-Seq assays aimed at high-throughput clinical trials require high quality whole-blood samples that yield larger amounts of assay input material. Alternatively, the analyses presented here do not require HLA-region enrichment and thus can also be performed on legacy RNA-Seq data. Notably, the data here are generated from sample types and amounts that are more typical to a clinical oncology setting, and because it does not rely on targeted capture as do DNA-Seq assays, these analyses hold greater promise for assembling rare alleles and fusions.

#413

Validation of an antibody independent tool for patient selection: RNA in situ hybridization detects Met expression levels predictive for response to Met inhibition by Bay 853474.

Oliver von Ahsen, Thomas Krahn, Christoph Schatz. _Bayer Pharma AG, Berlin, Germany_.

This study was performed in order to validate RNA in situ hybridization (RNA ISH) as tool for patient selection. Met expression was analyzed in a matched sample set of FFPE versus fresh frozen tumor samples comprising 20 cases of gastric cancer. Classical immunohistochemistry using the antibody SP44 and RNA ISH (RNAscope by ACD) were used to detect c-met expression in FFPE material. The results were confirmed by sandwich-immunoassays on Met and its phosphorylation on tyrosine 1349 (MSD) as well as mass spectrometry. The level of functional relevance was determined by testing a set of cell lines comprising some with genomic amplification of the met gene as well as some non-amplified lines showing different expression levels. Among gastric cancer cell lines only those with met amplification respond to treatment with the small molecule Met inhibitor Bay 853474. The cell line result generates a responder hypothesis that can be used to define a cutoff for clinical samples. 2 of the 20 investigated clinical samples were shown to have high level Met expression by RNA ISH and IHC that could be confirmed by sandwich-immunoassays also showing high level of functional activity by phosphotyrosine 1349. Met expression in these cases was also confirmed by mass spectrometry. Expression levels and functional activity in these 2 cases were in the range that predicts response to treatment as established with gastric cancer cell lines. Determination of predictive biomarkers by immunohistochemistry can be limited due to lack of high quality antibodies of sufficient specificity. Due to its high specificity, RNA in situ hybridization is a technique that can be used to confirm the findings obtained by immunohistochemistry and may potentially even replace immunohistochemistry it if no suitable antibodies are available or not specific enough e.g. to discriminate between closely related protein isoforms. We show the biological relevance of RNA in situ hybridization on FFPE samples by correlation with immunohistochemistry, ELISA based approaches and mass spectrometry. RNA ISH is shown to be specific and sensitive enough to identify cases of functionally relevant MET overexpression levels in gastric cancers and can be used to select patients for treatment.

#414

Biochemical and cellular monitoring of the activity of the ecto-5'-nucleotidase (CD73), a key cancer modulator using HTS-formatted bioluminescent technology.

Said A. Goueli, Kevin hsiao. _Promega Corp., Madison, WI_.

The ecto-5'-nucleotidase (CD73) is glycosylphosphatidylinositol (GPI) anchored cell surface protein that is involved in switching on adenosinergic signaling. Its enzymatic and nonenzymatic activities (via its interaction with extracellular matrix components) are involved in cancer associated processes and not completely independent of each other. It catalyzes the hydrolysis of AMP into adenosine and phosphate, where adenosine plays important role in tumor immune escape. It is overexpressed in many types of cancer cell lines and patient's biopsies including breast, colon, ovarian, gastric, ovarian, etc. In addition to being important as clinical and prognostic marker in cancer patients, overexpression of CD73 is associated with resistance to antitumor agents, and inhibition of CD73 activity or knocking it down by SiRNA reversed chemoresistant phenotype of glioblastoma multiforme cells. Because of the significant role of CD73, it is of importance to develop an assay that monitor the activity of CD73 in order to develop modulators of its activity. Towards this goal, we have developed a bioluminescent assay to monitor the activity of the enzyme in biochemically pure as well cellular anchored enzyme forms. The assay is homogenous and formatted for HTS screening research, very sensitive, and robust as indicated by the high Z'. We also show that the use of inhibitors such as adenosine 5'-(α-β-methylene) diphosphate (APCP) generates data with biochemically pure enzyme similar to the cellular bound form. We also demonstrate that cells that are enriched in CD73 can be easily identified from those that have low CD73. Thus, the current assay is robust, sensitive, and easy to use making it an ideal assay for HTS screening for new modulators of CD73 and generation of potential novel cancer therapeutics

#415

A new, highly sensitive ALK antibody improves the screening of rearranged-ALK by IHC.

Hsiangmin Lu,1 Rachel Gonzalez,1 Yi Shen,1 Mu-lan Jin,2 Yipan Wu,1 Yungang Zhang,2 Kehu Yuan,1 Boyang Chu,1 Lili Qi,1 Huibo Liu,1 Chenlin Wang,1 Guangli Wang,1 Youmin Shu,1 Julie McDowell,1 Donghui Ma,1 Wei-wu He,1 Jian Chen,3 Ray Lin1. 1 _Origene Technologies, Rockville, MD;_ 2 _Department of Pathology, Beijing Chaoyang Hospital,Capital Medical University, China;_ 3 _Institute of Functional Nano and Soft Materials (FUNSOM),Soochow University, Suzhou, MD_.

All non-small cell lung cancer (NSCLC) patients are recommended to be screened for anaplastic lymphoma kinase (ALK)-rearrangement despite its low occurrence (< 7%). This is due to recent advances in treatment for patients with ALK-positive NSCLC with receptor tyrosine kinase inhibitors Crizotinib and Ceritini. Current rearranged-ALK testing includes fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). Despite the high sensitivities and specificities, the limitations of the first two assays include time-consuming, infeasible, and specialized techniques that make them unsuitable for the routine screening for which IHC has been proposed. However, the biggest obstacle to detecting rearranged-ALK in lung cancer patients by IHC is the lack of highly sensitive ALK antibodies that detect the extremely low abundance of ALK in patients. To overcome this limitation, we have developed an ALK mouse monoclonal antibody (clone OTI1A4) with higher sensitivity in comparison to a rabbit ALK antibody (clone D5F3). Moreover, OTI1A4 identified ALK-positive NSCLC samples (16/16 cases) and showed negative staining for ALK-negative samples (11/11 cases) previously validated by FISH (10/16 ALK-positive cases) or qPCR (10/16 ALK-positive cases). This indicated the potential advantage of using clone OTI1A4 over FISH or qPCR to detect ALK rearrangements. Lastly, there was 100% concordance between OTI1A4 and D5F3 in detecting ALK-rearranged NSCLC samples previously confirmed by FISH (76 ALK-positive and 438 ALK-negative samples). These results support the possibility that ALK clone OTI1A4 could be used for routine screening of patients with ALK-positive NSCLC by IHC.

#416

Tumor cell isolation using antibody/aptamer-based multivalent binding.

Z. Hugh Fan, Jinling Zhang, Weian Sheng. _University of Florida, Gainesville, FL_.

Isolation and enumeration of circulating tumor cells (CTCs) in peripheral blood have been used for cancer diagnosis, prognosis, and theragnosis. The majority of the CTC isolation methods, including the FDA-approved CellSearch®, employ one antibody to capture tumor cells. Using such a monovalent-binding capture agent likely results in lower cell capture efficiency than multivalent-binding methods, which tend to have stronger binding force (or lower dissociation constant).

We have investigated in a multivalent-binding-based tumor cell isolation method by immobilizing a mixture of aptamers and antibodies on the surfaces of microfluidic devices. Since aptamers are much smaller in the size (8-15 kDa) than antibodies (150 kDa), aptamers and antibodies could interact with different receptors on cell surfaces, resulting in a configuration that one cell simultaneously interacts with multiple different types of capture agents. The microfluidic devices we used are in the size of a microscope slide and each of them consists of an inlet, eight parallel channels connected via consecutive bifurcation, and an outlet. There are micromixers inside the channels for enhanced interactions. Biotinylated aptamers and biotinylated antibodies were coated onto microchannel surfaces using avidin-biotin chemistry.

We tested the devices by isolating human leukemia cells (CCRF-CEM) in devices immobilized with a mixture of sgc8 aptamers and protein tyrosine kinase-7 (PTK7) antibodies. Both sgc8 aptamers and anti-PTK7 exhibited strong binding with CCRF-CEM cells according to flow cytometry. An antibody-to-aptamer ratio of 1:300 showed the highest capture efficiency among the range we studied (from 1:30 to 1:3000). We found that the antibody-aptamer surfaces had higher capture efficiency than antibody-alone or aptamer-alone surfaces; the degree of enhancement increased with the flow rate. Control experiments using non-specific aptamer (TD05) and non-specific antibody (anti-EpCAM) were carried out to verify the benefits of multivalent binding. We also found that the multivalent-binding surfaces have higher specificity than monovalent-binding surface, especially at a high flow rate. A calibration curve was obtained by spiking leukemia cells into human whole blood.

In summary, antibody/aptamer-coated multivalent-binding surfaces in microfluidic devices showed higher capture efficiency and specificity than monovalent-binding surfaces, suggesting their potential for the isolation and enumeration of CTCs for clinical applications.

#417

A novel predictive biomarker model for MEK sensitivity.

Marie Wagle, Christiaan Klijn, Bonnie Liu, Shilpi Mahajan, Peter Haverty, John Moffat, Mark Merchant, Bob Yauch, Garret Hampton, Lukas Amler, Mark Lackner, Shih-Min A. Huang. _Genentech, Inc., South San francisco, CA_.

KRAS mutations occur in approximately 25% of NSCLC (1). Tumors with these mutations are predicted to be sensitive to MEK inhibition due to activation of MAPK signaling. However, MEK inhibitors in multiple clinical trials, either as a monotherapy or in combination with chemotherapies, have not shown superior efficacy in the KRAS mutant subgroup when compared to the KRAS wild-type subgroup, indicating a limitation of utilizing KRAS mutation status as a predictive biomarker of efficacy to MEK inhibition (2, 3). Furthermore, stratification based on KRAS mutation status may inadvertently miss wild-type KRAS tumors that could be addicted to MAPK signaling regardless of KRAS mutation status, thus denying patients potential benefit from MEK inhibitors. Here we describe a novel predictive model that more accurately forecasts the sensitivity of the KRAS wild-type NSCLC subpopulation to MEK inhibitors such as cobimetinib and trametinib. Cell viability data from cobimetinib or trametinib-treated cells, with concomitant gene expression data (RNAseq), from 46 colon, 106 lung, and 37 pancreatic cell lines were used to create an elastic net regression model trained on gene expression features (alpha = 0.5 and optimal lambda chosen by 5-fold cross validation) (4). From the model, we established two distinct predictive gene lists: (1) a longer low cross-validation list, and (2) a shorter low error list. Initial analysis of the model demonstrated that predicted mean viabilities of the cell lines used to create the model correlated well with their actual mean viabilities (R: 0.65-0.7 for trametinib and cobimetinib respectively). Predicted mean viabilities of 40 previously unscreened NSCLC cell lines were then generated by the model based on their expression features (RNAseq). The 40 cell lines were categorized either as sensitive or resistant by the median of predicted mean viabilities derived from the model. Subsequently, the actual experimental GI50 values of cobimetinib were obtained for each of these cell lines. We found that KRAS mutational status predicted that only 8 of the 40 cell lines screened would be sensitive to MEK inhibition. In contrast, our model predicted that 15 of the 40 cell lines screened would be sensitive. Experimentally, we demonstrated that 24 of the cell lines were sensitive to MEK inhibition with a measured GI50<1uM. In conclusion, we describe a novel predictive model that more accurately predicts sensitivity to MEK inhibition, provides a potentially larger patient segment, and could be translatable to the clinic.

References

(1) Blumenschein GR. et al., (2015) Annal Oncol. 26(5):894-901.

(2) Gandara DL et al., (2013) J Clin Oncol 31, (suppl; abstr 8028).

(3) Laethem JLV et al, (2014) J Clin Oncol 32:5s, (suppl; abstr 4025).

(4) Barretina J. et al. (2012) Nature 483, 603-607.

#418

Mitochondrial perturbations as a novel approach to personalized medicine.

shruti bhatt, david weinstock, Anthony Letai. _Dana-Farber Cancer Institute, boston, MA_.

Recent progress in cancer research has been marked by the continued development of exciting drugs that selectively target vulnerabilities present only in cancer cells, so-called targeted therapies. Too often, however, we are lacking the proper predictive biomarkers to identify which patients will benefit most from individual targeted therapies. Since most chemotherapeutic agents ultimately function by inducing the mitochondrial apoptotic pathway, we hypothesized that a tool that measures mitochondrial sensitivity may serve as a broadly predictable biomarker. Hence to address the unmet need for predictive biomarkers, we have developed a tool called Dynamic BH3 Profiling (DBP). In this approach, we briefly treat cancer cells (14h) from patients with targeted drugs and measure the apoptotic threshold of a cell "priming" by treatment with peptides derived from BH3 domains of pro-apoptotic proteins. Previous studies from our laboratory demonstrated that when a patient's cells exhibit robust death signaling in the DBP assay in response to a particular drug, it predicts a good clinical response to that drug in patients.

Using xenotransplantation of primary AML samples into NSG mice (PDX models), we validated the utility of DBP to personalize acute myeloid leukemia therapy. We established 12 independent AML PDX models and determined the mitochondrial response of splenic myeloblasts from each PDX to 25 targeted agents. Ex-vivo DBP measurement demonstrated heterogeneous responses; while some drugs induced priming in all PDXs, others increased priming in only selective PDX models. For instance, JQ-1 (BRD-4 inhibitor) and birinapant (SMAC mimetic) demonstrated inverse correlation of their activity among 3 different PDXs. These suggested that mitochondrial priming can stratify AML PDX models according to predicted sensitivity to targeted agents. When we compared DBP results with cell death assay, we found that myeloblasts from most of the PDXs failed to sustain long-term culture (72h), a requirement for cytotoxicity measurements. However, myeloblasts from 3 of the PDXs with high starting viability showed strong correlation between mitochondrial priming and cell death, implying that cells with increased mitochondrial response are destined to commit apoptosis. By testing functional state of the apoptotic pathway in myeloblasts from spleen, bone-marrow and peripheral blood with BH3 profiling, we found that splenic blasts were relatively more primed for apoptosis in 8 out of 10 PDXs. Correlatively, drug induced priming also varied between myeloblasts from these 3 different sites. Finally, we are directly testing the performance of DBP as a predictive biomarker by treating a variety of PDX models with drugs, including drugs predicated to be active and drugs predicated to be inactive.

Collectively, mitochondria based measurements can predict good and bad response to individual targeted agents and may serve as a powerful biomarker to choose patient therapy.

#420

Translational strategy for targeting MCL1 amplified tumors with CDK9 inhibitor.

Ziping Yang, Ricky Bellin, Paul Hessler, Xin Lu, Tamar Uziel, Lloyd T. Lam. _AbbVie, North Chicago, IL_.

BCL-2, BCL-XL, and MCL-1 are members of the prosurvival family of proteins that regulate the mitochondrial apoptotic pathway. These proteins are often amplified or overexpressed in multiple tumor types. Previously, we showed that non-small cell lung cancer (NSCLC) cell lines with low BCL-XL expression (high MCL-1 / BCL-XL ratio) are MCL-1-dependent. TCGA data shows MCL1 amplification is one of the frequent genetic events in NSCLC adenocarcinoma (20%) and breast cancer (15%), while BCL2 is frequently amplified in activated B-cell like diffuse large B cell lymphoma (11%) and BCL2L1 in colorectal cancer (15%). We hypothesize that cancer cell lines with MCL1 amplification and/or high MCL-1 / BCL-XL ratio depend on MCL-1 for survival and are sensitive to inhibition of CDK9, a component of the transcriptional elongation complex that regulates MCL-1 expression. In this study, we demonstrate that NSCLC, triple negative breast cancer (TNBC) and ovarian cell lines with amplified MCL1 or low BCL-XL expression (high MCL-1 / BCL-XL expression ratio) are MCL-1-dependent, and are sensitive to dinaciclib, a CDK9 inhibitor. Cell lines with high BCL-XL expression could be re-sensitized to dinaciclib when co-treated with BCL2/BCL-XL inhibitor navitoclax (ABT-263) or BCL-XL-selective inhibitor A-1155463, suggesting BCL-XL as a resistance factor. Indeed, exogenous expression of BCL-XL rescues sensitive cell lines from dinaciclib. As reported in TCGA, ovarian, NSCLC and TNBC patients with MCL1 amplification also have high MCL-1 expression, and we hypothesized that these patients to be sensitive to CDK9 inhibitors. We developed a fluorescence in situ hybridization (FISH) assay to detect MCL1 amplification which can potentially select patients that may benefit from dinaciclib treatment. We show that fifteen percent of NSCLC patients have high MCL1 amplification, similar to published data. In addition, we have identified other predictive biomarkers associated with sensitivity in TNBC cell lines from gene expression analysis. In conclusion, we have developed a translational strategy for identifying MCL-1-dependent cancers that may be sensitive to CDK9 inhibitors in the clinic.

Disclosures:

All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication

#421

Non reportable results - maternal neoplasm can alter results from non-invasive prenatal testing (NIPT).

Daniel Grosu, Nilesh G. Dharajiya, Ron M. McCullough, Youting Sun, Juan-Sebastian Saldivar, Dirk van den Boom, Mathias Ehrich. _Sequenom, Inc., San Diego, CA_.

Cell free DNA is a powerful new analyte for the detection of chromosomal abnormalities of fetuses from the plasma of pregnant women. In non-invasive prenatal testing (NIPT), cfDNA is contributed into the plasma by the placenta. In rare cases, these results are distorted by additional sources of cfDNA in the maternal bloodstream. For example, organ transplantation can severely affect the cfDNA levels and lead to uninterpretable results. Another source of cfDNA that can lead to similar difficulties and ultimately to a non-reportable NIPT results, can be a maternal neoplasm. In our clinical laboratory, we have processed samples from over 450,000 pregnant patients. In cases of uninterpretable findings due to aberrant genomic profiles, potential reasons for non-reportable results were discussed with the caregiving physician. When feedback about these cases was available, it was collected in a database. In summary, we observed non-reportable NIPT results with aberrant genomic profile for 55 cases. A set of 43 had sufficient information available to allow inclusion in this summary. In 40 cases, a maternal neoplasm was confirmed (18 malignant, 20 benign, and two with imaging but without pathological confirmation). These observational results support further investigation by prospective controlled trials.

#422

Functional analysis of family with sequence similarity 83, member B in lung adenocarcinoma.

Takumi Yamaura, Naoyuki Okabe, Hironori Takagi, Yuki Owada, Takuya Inoue, Yuzuru Watanabe, Mitsuro Fukuhara, Satoshi Muto, Yuki Matsumura, Takeo Hasegawa, Atsushi Yonechi, Mika Hoshino, Jun Osugi, Yutaka Shio, Mitsunori Higuchi, Junji Ezaki, Satoshi Waguri, Mitsukazu Gotoh, Hiroyuki Suzuki. _Fukushima Medical University, Fukushima, Japan_.

Background:

We have reported the usefulness of Family with sequence similarity 83, member B (FAM83B) as a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma. FAM83B expresses not only in lung cancer but also in several types of solid cancer such as breast, esophageal, and so on. More recently, novel finding showing FAM83B involve to the resistance of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in breast cancer cell lines has been shown. EGFR signaling pathway also plays an important role in lung adenocarcinoma, therefore we hypothesize that FAM83B associates to the tumor proliferation in lung adenocarcinoma, and its results in the resistance to EGFR TKI and in a novel therapeutic target in lung adenocarcinoma.

Materials and methods:

Sixty-four paired tumor and corresponding normal tissue samples obtained from patients with lung adenocarcinoma who underwent surgical resection since 2008 to 2011 were subjected. FAM83B mRNA expression were examined and analyzed relationship between the level of FAM83B and clinicopathological variables. Then, FAM83B knock down study was performed in several types of cancer cell lines (PC-14, H820, H1650, H1975, HLC-1, H2347) to understand whether FAM83B involve to cell proliferation.

Results:

Multivariate analysis show that smaller cancer(≦3cm vs >3cm, p=0.044), and wild type EGFR(wild type vs. mutant, p=0.004) tend to express higher FAM83B mRNA. There is no significant difference between FAM83B expression and progression-free survival. FAM83B knock down results in suppression of proliferation in HLC-1, H1650, which express high level of FAM83B protein.

Conclusions:

FAM83B could involve to tumor proliferation of lung adenocarcinoma in part, and could be the novel therapeutic target.

#423

Toxicity and efficacy probability intervals design for phase I dose-finding in oncology trials.

Daniel H. Li,1 Jim Whitmore,1 Yuan Ji2. 1 _Juno Therapeutics, Seattle, WA;_ 2 _NorthShore University HealthSystem, Chicago, IL_.

We propose a toxicity and efficacy probability interval (TEPI) design for dose-finding in phase I oncology studies. This approach incorporates efficacy outcomes to inform dosing decisions to optimize both efficacy and safety. Decision rules can be prespecified, allowing for transparency to clinicians and non-statisticians. The TEPI design is an extension over the modified toxicity probability interval (mTPI) design, which uses only dose-limiting toxicity (DLT) data for dose escalation. The TEPI method is motivated by recent promising data from cancer immunotherapies, for which response or a biomarker for response can be observed in the same interval as a DLT.

The TEPI method is based on a two-way decision table constructed by combining the target probability intervals of DLT and efficacy. It uses a beta/binomial hierarchical model to compute the posterior probabilities of intervals for toxicity and efficacy. The dosing decision depends on the joint unit probability mass of toxicity and efficacy data, which follows the Bayes rule under independent beta prior distributions. TEPI can pre-calculate all dosing actions for any possible combination of toxicity and efficacy outcome at a certain time point during a trial. For example, dose escalation or de-escalation decisions at time of 6, 9, and 12 treated patients are provided in Table 1.

In the current era of potent novel agents in oncology, phase I trials that simultaneously optimize antitumor activity and safety may accelerate development of these therapies. The proposed TEPI method recognize this needs, and is appealing to clinicians because all dose escalation or de-escalation can be prespecified. Simulation studies have been performed to evaluate the performance of TEPI, and compared to other designs.  | |  | |

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

|  | No. of Responders

No. of Treated Patients at Current Dose Level | No. of DLTs | 0 | 1-4 | 5-6

6 | 0-1 | EU | E | S

|

2-3 | S | S | S

|

4-6 | DU | DU | DU

|  | |

|

|  | 0 | 1-6 | 7-9

9 | 0-2 | EU | E | S

|

3-4 | S | S | S

|

5-9 | DU | DU | DU

|  | |

|

|  | 0-1 | 2-7 | 8-12

12 | 0-3 | EU | E | S

|

4-6 | S | S | S

|

7-12 | DU | DU | DU

Table 1. The letters in cells are computed based on the decision rules under the TEPI method and represent different dose-finding actions. EU: Unacceptable efficacy; DU: Unacceptable safety. Target toxicity probability=0.3 and target response rate probability=0.5.

#423A

A tumor-associated mRNA localizing in circulating exosomes as a novel serological marker for pancreatic cancer: the retrospective clinical study.

Keisuke Taniuchi. _Kochi University, Nankoku, Japan_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in the Western world. Early diagnosis of PDAC is difficult, and no biomarkers in blood can identify patients with pancreatic cancer at an early stage. Serum cancer antigen 19-9 (CA19-9) remains the gold standard serum marker for patients with PDAC; however, inadequate sensitivity and specificity limit the use of CA19-9 in the early diagnosis of PDAC. Therefore, the discovery of biomarkers derived from blood that facilitate the distinction of PDAC would greatly affect patient management. Exosomes are extracellular lipid microvesicles, secreted by nearly all cells in body fluids such as peripheral blood. The exosome-associated RNA is called exosomal shuttle RNA that includes microRNAs and messenger RNAs (mRNAs).

Methods: The aims of the present study are to assess if the mRNA that we found in the intracellular exosomes of PDAC cells is secreted from PDAC cells, and it is useful as a serum diagnostic marker for differentiating PDAC from individuals without pancreatic disorders compared with CA19-9. 20 PDAC patients (stage III: n = 5, stage IVa: n = 7 and stage IVb: n = 8 according to the classification of pancreatic carcinoma of the Japan Pancreas Society) and 30 control individuals without pancreatic diseases were analyzed in a retrospective cohort study. Circulating exosomes were isolated from serum, and then the mRNA localizing in exosomes was measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Results: We confirmed that the mRNA was localized in secreted exosomes from cultured PDAC cells: a moderately differentiated PDAC cell line (S2-013) by the use of condition media real-time RT-PCR. Furthermore, the retrospective clinical study showed that the area under the curve (AUC) of reciever operating characteristic curves (ROC curves) was 0.805 (95%CI 0.691-0.9199) for this RNA and 0.8 (95%CI 0.675-0.926) for CA19-9. If the mRNA was combined with serum CA19-9, the AUC increased (0.921 [95%CI 0.851-0.991]).

Conclusions: Our data suggest that measuring the level of this exosome-associated mRNA has the potential to improve detection of PDAC. Further research is necessary to understand whether this mRNA has clinical implications for early detection of PDAC (stage I and II) and how much this information adds to serum CA19-9.

### Biomarkers for Genitourinary and Gynecological Cancers

#424

Urine cf-DNA SNPs as early biomarkers of prostate cancer.

Amrita Datta, Hogyoung Kim, Allison Feibus, Melody Badoo, Adedoyin Johnson, Ahmed Moustafa, Jonathan Silberstein, Krishnarao Moparty, Raju Thomas, Asim B. Abdel-Mageed. _Tulane University, New Orleans, LA_.

Background: The current strategies of prostate cancer (PC) detection, such as serum PSA, digital rectal examination (DRE) and prostate biopsy have been hampered by many limitations, including the degree of aggressiveness, invasiveness, non-specificity to PCa, over diagnosis and over treatment. Recent studies have proposed the use of cell-free circulating DNA (cf-DNA) as non-invasive biomarkers for cancer diagnosis, prediction of tumor burden and prognosis. We hypothesize that a selective subset of PC associated Single Nucleotide Polymorphisms (SNPs) in urine cf-DNA detects PCa with high specificity and sensitivity.

Aim: To identify PC specific SNPs in urine cf-DNA from prostate cancer (PC) patients and further validate their clinical utilities as non-invasive biomarkers for early detection of PC in comparison to other genitourinary conditions, such as benign prostate hyperplasia (BPH) and prostatitis.

Method and Results: A protocol was optimized for collection, processing and storage of urine samples and isolation of urine cf-DNA. Our discovery dataset was constructed by comparative Illumina HiSeq next generation sequencing (NGS) of urine cf-DNA from three different cohorts of patients presenting with PC (Gleason score 6), benign prostatic hyperplasia (BPH) or prostatitis. The data was analyzed by SNAPE-pooled and annotated using ANNOVAR; revealing 24 PC-specific non-synonymous SNPs. The selective detection of urine cf-DNAs in PC patients was verified by conventional PCR using primers flanking their genomic sites. Our validation analysis was performed in patients (n=50/group) diagnosed with PC (Gleason score ≤ 3+4), Gleason score ≥ than 4 + 3, or patients deemed as "no cancer seen". This was performed by Droplet Digital PCR (ddPCR) targeting wild-type versus variant (SNP) DNA in each patient sample. The results have revealed detection of two PC specific SNPs in urine cf-DNA (>60% for SNP1 and >75% SNP2 in 91 patients; patent pending).

Conclusion: Our study brings forth the potential clinical utility for two novel urine cf-DNA SNPs as early biomarkers for prostate cancer.

#425

Nuclear morphometry predicts prostate cancer progression.

Guangjing Zhu,1 Aniq ur rehman Gajdhar,2 Jonathan I. Epstein,3 Neil Carleton,4 Christine Davis,1 Luciane Tsukamoto Kagohara,1 Robert W. Veltri1. 1 _The Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _SAIMS Hospital, India;_ 3 _The Johns Hopkins University Hospital, Baltimore, MD;_ 4 _Carnegie Mellon University, PA_.

Introduction:

The progression of prostate cancer (PCa) involves tissue morphometric changes, which laid the base for current pathological diagnosis of prostate cancer with the Gleason Scoring system. Nuclear morphometry (NM) changes contribute significantly to these tissue morphometric changes, but accurate and autonomous quantification of NM changes are challenging. Here we created a macro to quantify the changes of size, shape, DNA content, etc., and used these parameters to predict PCa progression in 80 men that underwent radical prostatectomy (RP).

Materials & Methods:

Two tissue microarrays (TMAs) with 80 RP PCa cases, stratified by Gleason scores (GS) were used for this study. The two continuous sections of TMAs were stained with H&E and Feulgen reagents and the H&E slides were used for pathological diagnosis of PCa. Both kinds of slides were scanned with Aperio scanner and image of each core were separated using Aperio ImageScope software. The H&E or Feulgen stained nuclei of cancer core were quantified using Smart Segmentation of ImagePro® Premier 9.1 software. For each core, data of all ROIs were pooled and the covariance of each parameter were generated. Data were first analyzed alone and then in combination using multivariate logistic regression (MLR) to predict the aggressive RP cases or biochemical recurrence (BCR). Decision curve analysis (DCA) was used for the MLR models to evaluate their power and effectiveness for clinical decision making. For all analysis, a p<0.05 is considered as statistically significant.

Results:

Using multivariate logistic regression (MLR), to differentiate aggressive PCa (Gleason score 4+3 & >=8) from less aggressive PCa (Gleason score 3+3 & 3+4) on the TMAs of RP cases, our Feulgen NM model generated a receiver operating characteristic curve-area under the curve (ROC-AUC) of 0.90 with a sensitivity of 75.51% and specificity of 86.21% while our H&E NM model yield an ROC-AUC of 0.96 with a sensitivity of 82.00% and specificity of 90.00%. DCA analysis both showed better decision using both models compared with considering the patients as all or none of them as aggressive. Both models also show strength in differentiating two Gleason score 7 (3+4 vs 4+3). Feulgen NM model showed moderate power in the prediction of BCR based on the MLR (ROC-AUC = 0.79) and DCA analysis, while H&E MLR model performed better with a ROC-AUC of 0.86. Further Kaplan-Meier analysis of BCR with GS and NM (H&E and Feulgen) showed that the combination of GS with either Feulgen or H&E NM showed significant power in the differentiation patients with BCR survival time (HR = 3.14 & 3.89, respectively).

Conclusions:

Our accurate quantification of tissue morphometry demonstrated its translational clinical relevance since it can predict PCa aggressiveness in men that have undergone RP. Future application of this tool in active surveillance biopsies may provide an early diagnosis and prognosis prediction of PCa patients effectively.

#426

Identifying a predictive biomarker for chemotherapy (taxane) in patients with metastatic castration resistance prostate cancer.

Greg A. Rothchild,1 George Sandusky,1 Constance J. Temm,1 Costantine Albany2. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _IU Simon Cancer Center, Indianapolis, IN_.

In 2015, American Cancer Society predicts that 220,800 men will be diagnosed with prostate cancer (PC). Patients diagnosed with metastatic PC are first treated with androgen deprivation therapies (ADT) in order to lower testosterone to castration levels. Unfortunately, it is only a matter of time before becoming metastatic castration resistant prostate cancer (mCRPC). Subsequent treatments include combined androgen deprivation therapies such as (abiraterone, enzalutamide), immunotherapies, investigational drugs, and/or chemotherapy (docetaxel and cabazitaxel). Individualizing therapy for prostate cancer patients is an overarching goal of research. Currently, no valid practical biomarkers exist that can predict resistance to the taxane in PC. Identifying a marker that predicts taxane sensitivity would delineate patients who would benefit from taxane regimens from those who would suffer from added side-effects without any improved response. Previous studies have confirmed TLE3 is associated with a response to taxane therapy in breast and ovarian cancers. Preclinical studies have also shown that TLR4, RAGE, and HMGB1 confers chemo resistance to docetaxel on PC-3 human PC cells. After obtaining IRB approval (protocol IUCRO-0499), 8 metastatic castration resistant prostate cancer samples and 6 control samples were pulled for this study after scrutinous review of the H&E slides from clinical cases. This study then consisted of investigating the proteins TLE3, TLR4, RAGE, and HMBG1. Immunostaining was performed using the automated immunostainer, Dako FLEX System, and the slides were scanned into the whole slide Aperio Scanscope CS digital imaging system. Computer-assisted morphometric analysis of digital images was then performed using the Aperio ImageScope software. Review of the TLE3 and TLR4 expression in metastatic castration resistance prostate cancer samples showed a difference between the expressions of these two proteins. TLR4 positivity was found to be 43.88% higher than the expression of TLE3. This is suggestive of resistance to docetaxel treatments. Review of the RAGE and HMGB1 expression in metastatic castration resistance prostate cancer samples compared to normal prostate epithelium showed a 32.9% increase in RAGE and 36.6% increase in HMGB1 expression. The findings of this study are suggestive that there is a possible correlation between the overexpression of these protein biomarkers and the patient's response to a specific drug treatment.

#427

Integrated PDEF and Twist1 expression distinguishes lethal prostate cancer from an indolent disease.

Fengtian Wang, Sweaty Koul, Parveen K. Jaiswal, Runhua Shi, Glenn Mills, Hari K. Koul. _Louisiana State University Health Sciences Center/FWCC, Shreveport, LA_.

Introduction: Prostate cancer (PCa) is the third most commonly diagnosed cancer in the world. Advanced genomic studies have characterized it as a molecularly heterogeneous disease with a multiple clinical spectrum ranging from indolent to highly aggressive. Conventional therapies produce a high rate of cure for patients with localized PCa, but there is no effective treatment for metastatic PCa. These facts underline the urgent, yet unmet, need for identification and characterization of new targets that can help distinguish metastatic PCa from an indolent disease, and pave a way for novel mechanism based anti-metastasis therapies against PCa. Our previous studies have shown that loss of PDEF, a putative tumor suppressor, could lead to more invasive phenotype in PCa cell lines through promoting Epithelial to Mesenchymal transition (EMT). In this study, we evaluate the role of PDEF and Twist1 in PCa through NCBI omnibus and The Cancer Genome Atlas. We also evaluated the mechanism by which PDEF is regulated epigenetically.

Methods: PDEF expression was monitored by immunohistochemistry using tissue microarray. Western blot was employed to measure the PDEF level in PCa cell lines. PDEF and Twist1 gene expression was measured by real time PCR. The PDEF promoter was cloned and methylation specific PCR was performed. PDEF methylation in clinical samples was extracted from TCGA database and PDEF/Twist1 microarray data was extracted from GSE16560 in NCBI gene expression omnibus with R language. GraphPad prism was used to generate figure and statistical analysis. P < 0.05 is considered significant

Results: IHC staining of PDEF on PCa patient samples and in commonly used PCa cell lines suggested an inverse correlation between the level of PDEF and Twist1. Over-expression of PDEF in PC3 cells inhibits Twist1 expression. DNA Methylation status near the promoter region of PDEF was negatively associated with the expression levels of PDEF in established PCa cells. These results suggest that PDEF levels are regulated in part by promoter methylation. Using TCGA data we identified 12 methylation site and the methylation level of multiple site correlates with the increased of Gleason Score (GS). Analysis of gene expression data from GSE16560 revealed that low expression of PDEF and high expression of Twist1 were independently associated with poor survival in PCa patients. Integrated PDEF and Twist1 expression can distinguished a high-risk group of people for hormone refractory PCa along with short median survival time.

Conclusions: Loss of PDEF combined with gain of Twist1 expression can serve as a potential biomarker set for distinguishing aggressive PCa from an indolent disease. PDEF expression is epigenetically regulated in part by promoter hyper-methylation.

Acknowledgement: Carroll W. Feist Endowed Chair Funds .

#428

Hypermethylation of GATA2 is validated as a marker of progression in non-muscle invasive bladder cancer.

Kim E.M. van Kessel,1 Kirstin A. van der Keur,1 Lars Dyrskjøt,2 Ferran Algaba,3 Naeromy Welvaart,1 Willemien Beukers,1 Ulrika Segersten,4 Bastian Keck,5 Tobias Maurer,6 Tatjana Simic,7 Marcus Horstmann,8 Joost L. Boormans,1 Francisco X. Real,9 Nuria Malats,9 Per-Uno Malmström,4 Torben F. Ørntoft,2 Ellen C. Zwarthoff1. 1 _Erasmus MC, Rotterdam, Netherlands;_ 2 _Aarhus University Hospital, Aarhus, Denmark;_ 3 _Universitat Autónoma de Barcelona-Spain, Barcelona, Spain;_ 4 _Uppsala University, Uppsala, Sweden;_ 5 _Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany;_ 6 _Klinikum rechts der Isar der Technischen Universität München, Munich, Germany;_ 7 _University of Belgrade, Belgrade, Serbia;_ 8 _Friedrich-Schiller-University Jena, Jena, Germany;_ 9 _Spanish National Cancer Research Centre (CNIO), Madrid, Spain_.

Objective

Hypermethylation patterns of a set of four genes (GATA2, TBX2, TBX3, and ZIC4) were previously identified to predict progression of non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). In this prospective study we aimed to validate these four genes as progression markers in NMIBC.

Materials and Methods

A total of 856 patients in follow-up for stage Ta urothelial carcinoma of the bladder were included in hospitals in Denmark, Germany, Serbia, Spain, Sweden and the Netherlands. Available pathology sections were reviewed by a single expert uropathologist. According to the WHO 2003 grading, 633 patients were diagnosed with a Ta low-grade tumor and 223 with a Ta high-grade tumor. Progression to muscle-invasive disease was seen in 18 (3%) of the low-grade tumors and in 12 (5%) of the high-grade tumors. Mean follow up was 22 months (range 0 to 76 months). DNA was isolated from fresh frozen tumor tissue and analyzed for mutations in FGFR3, TERT, RAS and PIK3CA. Methylation assays were done using a single-nucleotide probe extension assay for GATA2, TBX2, TBX3 and ZIC4. Methylation percentages were calculated and cut-off values were determined for dichotomization. All results were combined and the optimal model for predicting progression was selected by logistic regression analysis. Survival analysis was done using the Kaplan-Meier method.

Results

Multivariate logistic regression analysis using backward selection resulted in an optimal prediction model including GATA2, TBX2, age and gender for Ta low-grade tumors. The predictive capacity of the model represented by the AUC was 0.896. For Ta high-grade tumors, multivariate regression analysis resulted in a predictive model that included only GATA2 and smoking status with an AUC of 0.783. Mutation analysis did not add to these models. KM curves were plotted of the best performing methylation marker GATA2 and progression-free survival. Progression-free survival was significantly worse in patients having a GATA2-positive Ta high-grade tumor (P=0.025).

Conclusion

Hypermethylation of the GATA2 gene can predict progression of NMIBC to MIBC in low- as well as high-grade Ta tumors. Further, survival is significantly worse in Ta high grade patients with a GATA2 positive primary tumor. Hypermethylation of GATA2 is therefore a validated progression marker for NMIBC.

#429

Establishment of the classifier for a response to vascular endothelial growth factor (VEGF)-tyrosine kinase inhibitor (TKI) in metastatic renal cell carcinoma.

Heounjeong Go,1 Mun Jung Kang,1 Pil-Jong Kim,2 Ja-Min Park,1 Jae-Lyun Lee,1 Ji Young Park,3 Yong Mee Cho1. 1 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Biomedical Knowledge Engineering Laboratory, Seoul National University College of Dental Medicine, Seoul, Republic of Korea;_ 3 _Daegu Catholic University Medical Center, Daegu, Republic of Korea_.

Vascular endothelial growth factor (VEGF)-targeted therapy has been demonstrated to improve the outcome of metastatic renal cell carcinoma (mRCC) patients. However, validated predictors that predict response and prognosis to VEGF-tyrosine kinase inhibitors (VEGF-TKIs) remain to be elucidated. We aimed to define a classifier for VEGF-TKI response in mRCC patients. Among 101 mRCC patients treated with VEGF-TKIs, 73 patients were responder defined as patients showing complete or partial response, or ≥24 weeks stable disease to VEGF-TKI; and 28 patients were nonresponder defined as <24 weeks stable or progressive disease. Clinical and laboratory data were obtained from the medical records. Histologic features of all tumors were reviewed. Twenty-one protein expressions such as VEGF, pAKT, PD-L1, PD-L2, HIF-1α and HIF-2α were evaluated on immunohistochemical staining. Mutation of various cancer-related genes was investigated on OncoMap ver. 4.4 core. Microarray and quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) were also performed to compare the expression patterns of miRNAs between responders and nonresponders in tissues samples. Patient's age, time from diagnosis to TKI initiation and thrombodytosis were different between responder and nonresponder (all, p <0.05). Histologically, tumor size, T stage, ISUP grade, sarcomatous change, necrosis and lymph node metastasis of responder were significantly lower or smaller than nonresponder (all, p <0.05). pAKT, PD-L1, PD-L2, FGFR2, pS6 and PDGFRβ showed more expression in nonresponder than in responder; on the other hands, HIF-1α, IL-8 and CA9 showed more expression in responder than in nonresponder (all, p <0.05). Expression of miR-421 was higher in responder than nonresponder (p=0.008). A classifier to VEGF-TKI response was developed with various clinicopathological features, protein expression and miRNA expression. Tumor size, T stage, ISUP grade, necrosis, sarcomatoid change, pAKT, PD-L1, CA9, pS6, HIF-1α and miR-421 were selected by chi-square test with p <0.01. Using 10-fold cross validation (CV) by support vector machine, 3 features, i.e., necrosis, sarcomatoid change and HIF-1α, were finally selected as features for classifier and accuracy of 10-fold CV was 0.87. When the classifier was checked with all patients, apparent accuracy was 0.875 (95% CI, 0.782-0.938). In addition, the classifier could be presented by a simple decision tree for clinical use. In conclusion, we built a VEGF-TKI response classifier by comprehensive inclusion of clinical, laboratorical, histological and immunihistochemical features, mutation of cancer-related genes and miRNA expression using machine learning method and it may be helpful to receive a proper treatment in mRCC patients.

#430

Novel biomarkers for VEGFR inhibitors in metastatic renal cell carcinoma: BIM expression, and germline polymorphisms of BIM and PIK3R1.

Jee Hung Kim,1 Woo Sun Kwon,2 Won Suk Lee,2 Tae Soo Kim,2 Kyu Hyun Park,2 Jae Kyung Roh,2 Joong Bae Ahn,3 Hyun Cheol Chung,4 Sun Young Rha4. 1 _Division of Medical Oncology, Yonsei Cancer Center, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea, Republic of; College of Medicine, Yonsei University Graduate School, Seoul, Korea, Republic of, Seoul, Republic of Korea;_ 2 _Song-dang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Division of Medical Oncology, Yonsei Cancer Center, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 4 _Song-dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

Molecular targeted therapy, especially VEGFR tyrosine-kinase inhibitors (TKIs) including sunitinib, pazopanib and sorafenib has been the most efficacious therapy for metastatic renal cell carcinoma (mRCC). However, approximately 35% of mRCC patients do not get the benefit from TKIs treatment, due to resistance and/or drug induced toxicities.

Pro-apoptotic Bcl-2 family member (BIM) is a particularly critical mediator of targeted therapy-induced apoptosis in solid tumors. In previous, it had known that TKIs induced to BIM upregulation and then accelerated to apoptosis of tumor. However, according to recent several studies, BIM germline deletion polymorphism was identified as one of the primary resistant mechanisms of various TKIs in EGFR mutated NSCLC or CML. In addition, the alteration of phosphoinositide 3 kinase(PIK3)/protein kinase B(AKT) pathway has also been found as important mechanism in various cancer cell growth, proliferation and survival. PI3KR1 encodes the p85α subunit, regulatory subunits of Class IA PIK3s. Germline PIK3R1 variant M326I(c.978G>A) might affect the sensitivity of receptor tyrosine kinase (RTK) inhibitors and its downstream pathway inhibitors.

We evaluated the possibility of BIM, PIK3R1 as a potential biomarker for VEGFR TKIs in mRCC. BIM deletion polymorphism status of gDNA from formalin fixed paraffin embedded (FFPE) tumor tissues and peripheral blood mononucleated cells (PBMC) of mRCC patients was determined by separated PCR. And we evaluated BIM protein expression by immunohistochemistry. We also evaluated PIK3R1 germline mutation by pyrosequencing in PBMC of mRCC patients.Germline BIM deletion accounted for 17.8% of total 124 mRCC patients. Unlike with previous other studies, our data suggested that BIM wild type showed tendency to poor prognosis with sunitinib or sorafenib treatments for clear cell type RCC (median OS of BIM wild and deleted type: 44.9 vs 67.3 months, p=0.279), and high expression of BIM (median OS of BIM low(<10%), median(10-50%) and high(>50%) respectively: 76.5 vs 60.0 vs 21.3 months, p=0.005). In addition, we found 26% (19/71) of the germline PIK3R1 variant in mRCC. Patients with PIK3R1 germline variant showed a tendency of poor prognosis to 1st line VEGFR TKI treatment than wild type (median PFS: 7.78 vs 15.87months, p=0.342). Especially PFS of 1st line sunitinib treatment was significantly shorter in PIK3R1 variant compared to wild type (median PFS 7.78 vs 16.33 months, p=0.004). But, there is no difference in mTOR inhibitor sensitivity both with PIK3R1 or BIM polymorphism.In conclusion, BIM expression and germline variants of BIM and PIK3R1 might be candidate biomarkers for VEGFR TKIs in mRCC.

#431

Circulating fibroblast growth factor 21 (FGF21) as diagnostic and prognostic biomarker in renal cancer.

Maria Elena Knott,1 Jose Nicolas Minatta,2 Lucia Roulet,2 Guillermo Gueglio,2 Leonardo Pasik,1 Stella Maris Ranuncolo,1 Myriam Nuñez,3 Lydia Ines Puricelli,1 Mariana Silvia De Lorenzo4. 1 _Instituto de Oncologia "Angel H. Roffo", Buenos Aires, Argentina;_ 2 _Hospital Italiano, Buenos Aires, Argentina;_ 3 _Facultad de Farmacia y Bioquimica, University of Buenos Aires (UBA), Argentina;_ 4 _Rutgers, The State University of New Jersey, Newark, NJ_.

Clear-cell renal cell carcinoma (ccRCC), the most frequent renal parenchyma malignant neoplasm, is considered a cell metabolic disease. The finding of new biomarkers is needed for better sub-classification of renal cell tumors as well as reliable predictors of outcome and therapy response. Fibroblast Growth Factor 21 (FGF21) is a hepatokine that regulates glucose, energy and lipid metabolism during stress-induced pathologies. Despite the beneficial effects of FGF21 in diabetes and obesity; up to date, the clinical implication of FGF21 as a cancer biomarker was not investigated. Our main goal was to evaluate the role of circulating FGF21 as a diagnostic and prognostic biomarker for ccRCC. Initially, we measured the levels of circulating FGF21 in human healthy controls (HC, n=51) using a quantitative ELISA test (R&D Systems, Inc.). No associations were observed between FGF21 serum concentration and gender or age. FGF21 levels significantly correlated only with triglycerides levels (Spearman's test: p <0.01). Interestingly, we found that ccRCC patients (n=98) have higher levels of serum FGF21 compared to HC (KW test: p<0.0001). FGF21 serum levels were not associated with BMI or other metabolic parameters. Using 130.64 pg/ml of serum FGF21 as a cut-off value, the sensitivity was 80.61% and the specificity was 64.61%. Serum FGF21 was increased since the earliest stages of the disease without differences among the various ccRCC stages. To analyze the associations between FGF21 and clinico-pathological parameters; FGF21 values were dichotomized into "low" or "high" using 219.57 pg/ml (50th percentile) as cut-off point. No significant association was observed with age, sex, obesity, triglycerides and known risk factors (Chi square test, NS). The prognostic value of FGF21 was analyzed in terms of disease-free survival (DFS) and overall survival (OS). No significant association was found between serum FGF21 levels and 5- years OS. Kaplan-Meier plots of DFS showed that high levels of serum FGF21 were associated with worse prognosis with a borderline significance. However, multivariate analysis showed that FGF21 expression is a significant independent prognostic factor when adding the variables Fuhrman grade and stage (Cox Regression test). We also collected a second serum sample in 30 patients after successful surgery and we observed that the levels of serum FGF21 decreased in 41.4 % of ccRCC patients. In addition, we showed that serum FGF21 levels were significantly increased in 14 patients with chromophobe renal cancer respect to HC (MW test: p<0.0001), although similar to RCC patients. Our results indicated that serum FGF21 is useful as a diagnostic ccRCC biomarker in combination with other clinical or molecular parameters. Moreover, in our cohort of ccRCC patients, high levels of FGF21 showed to be an independent prognostic biomarker, associated with worse disease-free survival.

#432

Loss of MGMT promoter methylation and resistance to cisplatin in non-seminomatous germ cell tumors.

Cátia Moutinho,1 Xavier Garcia-del-Muro,2 Elisabet Guino,2 August Vidal,2 Sara Puertas,1 Clara Muñoz,1 Josep Piulats,2 Alberto Villanueva,2 Manel Esteller1. 1 _Inst. d'Investigació Biomèdica de Bellvitge (IDIBELL), Barcelona, Spain;_ 2 _Catalan Institute of Oncology (ICO), Barcelona, Spain_.

To explore if MGMT promoter methylation changes have a role in cisplatin chemoresistance, first we study its methylation status in cisplatin sensitive and resistant paired human non-seminomatous germ cancer cell lines. Secondly in xenograft paired tumors and after in human non-seminomatous germ cell primary tumors, from patients treated with cisplatin based chemotherapy. In general we found that cisplatin sensitive samples are related with MGMT promoter hypermethylation associated with its loss of expression. Resistance is present when MGMT promoter is not methylated and expressed. Clinically, the presence of MGMT promoter methylation is related with better overall survival (p=0.025) in patients with testicular germ cell cancer. Inhibition of MGMT with O6-benzylguanine in vitro or in vivo increases the sensitivity to cisplatin and temozolomide, being this a possible chemotherapeutic approach to re-sensibilize human non-seminomatous germ cell refractory tumors.

#433

Utility of p16 immunohistochemistry in evaluating negative cervical biopsies following high-risk pap test results.

Alana F. Shain, Shirley Kwok, Ann K. Folkins, Christina S. Kong. _Stanford University, Stanford, CA_.

The Lower Anogenital Squamous Terminology (LAST) Standardization Project for HPV-associated Lesions recommends the use of p16 immunohistochemistry as an adjunct to morphologic assessment of cervical biopsies interpreted as negative from patients that are at high risk for missed high-grade disease (defined as a prior cytologic interpretation of HSIL, ASC-H, ASC-US/HPV-16+, or AGC-NOS) (Darragh et al., 2012). However, few studies have specifically evaluated the utility of performing p16 on negative cervical biopsies and endocervical curettage specimens following high-risk Pap test results.

A search of the Stanford Cytopathology database from 7/1/2002-6/30/2012 for cervical cytology cases with diagnoses of ASC-H, HSIL, AGC-NOS, and atypical endocervical cells NOS (AGC-EC) yielded 1517 cases. Of these, 703 cases had histologic follow-up. Biopsies were excluded if diagnosed as atypical, low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesion (HSIL), or insufficient, or if the time to follow up was greater than 1 year. Immunostain for p16 (CINtec) was performed on 350 cases from 339 patients (age 16-85 yrs, average 42+/-14) and scored as positive (diffuse strong), negative or equivocal. 6 cases were excluded due to insufficient tissue and 3 cases with equivocal staining were excluded from further analysis. For p16(+) and equivocal cases, HPV in situ hybridization (ISH) (Ventana HPV III Family 16 probe) was performed and the corresponding H&E section reviewed. Based on the H&E review, the diagnosis was revised. Follow-up data was also obtained.

12/341 cases (3.5%) were p16(+) corresponding to missed diagnoses of LSIL (1 cases), HSIL-CIN2 (2 cases), and SIL-indeterminate grade (6 cases). Two biopsies from one patient at different time points exhibited p16 staining of bland metaplastic cells undermining endocervical glands that morphologically did not meet criteria for SIL and were interpreted as atypical metaplasia. Follow-up biopsies showed HSIL.

p16 immunostain increases the detection rate of SIL by 3.5% in benign appearing cervical biopsies and endocervical curettages from patients with a prior high-risk Pap test result. The benefit of p16 immunostain is highest for cases with a prior Pap diagnosis of HSIL. Cytoplasmic only staining and staining of surface mucosa, endocervical glands, and tubal metaplasia may lead to over-interpretation of p16 as positive.

Reference:

Darragh, T. M. et al. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch. Pathol. Lab. Med. 136, 1266-1297 (2012).

#434

Rational molecular assessment and innovative drug selection (RAIDs): exome data from cervical cancer.

Suzy Scholl,1 Maud Kamal,1 Els Berns,2 Balzs Balint,3 Attila Kereszt,3 Leanne de Koning,1 Emmanuelle Jeannot,1 Windy Luscap-rondof,1 Vonick Sibut,1 Philippe Hupe,1 Gemma Kenter,4 Sanne Samuels,4 Katja Jordanova,4 Sandrine Blanchet,5 Laurence Lafanachere,5 Marc Billaud,5 RAIDs consortium1. 1 _Institut Curie, Paris, France;_ 2 _ERASMUS, Rotterdam, Netherlands;_ 3 _SeqOmics, Hungary;_ 4 _NKI-AVL, Amsterdam, Netherlands;_ 5 _INSERM, Grenoble, France_.

Background Recent retrospective data1,2 identified major molecular alterations in cervical cancer (CC), but so far there has been no prospective assessment on patient outcome using a complete molecular profiling with quality control evaluation of treatment. The Cetuxicol (phase 2) clinical trial showed that the addition of Cetuximab over a 6 week period, did not improve DFS. PI3K pathway mutations in the tumor in the Cetuximab treatment arm led to a worse DFS3. We are lacking prognostic and predictive biomarkers for CC treatment and there is a growing need for the development of biomarkers to follow up the course of the disease.

Methodology RAIDs (http://www.raids-fp7.eu) is a multidisciplinary co-operation between academic clinical centers, SMEs and translational research platforms in seven European countries. It includes: 1) a cognitive cohort study (BioRAIDs)4, one of the first prospective trials intended to define patient stratification for targeted therapies, 2) a targeted clinical trial using an HPV directed vaccine and 3) preclinical studies aiming at assessing new treatment strategies. Molecular analysis on quality controlled tumor and sera samples from 500 patients enrolled in BioRAIDs combine Next Generation Sequencing at SeqOmics (Hungary), PIK3CA mutations detection in circulating tumor (ct) DNA at ERASMUS (The Netherlands), Reverse Phase Protein array and HPV insertion sites analyses at Institut Curie (France). Bioinformatics pipelines to detect somatic mutations and clustering methods were developed in order to stratify the patients into different subtypes. Following quality control, the pharmacological profiling of a panel of 20 CC cell lines using a panel of drugs which may potentially synergize with "standard treatment" has been completed. Drugs were chosen so as to interfere with different signaling pathways.

Results Molecular profiles including exome sequencing and ctDNA analyses on 48 quality controlled samples from the BioRAIDs patients and 20 CC cell lines will be presented. Preliminary results on HPV insertion sites in tumor and serum will be reported. Somatic and DNA copy number alterations from exome sequencing profiles confirm PI3K pathway mutations to be a dominant feature in CC. Stratifications of patients' tumors based on their mutation profiles show that some cell lines cluster similarly to patients' tumors, suggesting that they will be helpful for the prediction of response to drugs for patients within the same cluster.

Conclusions The identification of predictive tumor/blood based biomarkers will permit the definition of new strategies for precision medicine in CC. A bioinformatics analysis which will assess drug responsiveness in CC cell lines in relation to specific genomic alterations is ongoing. Its results should permit to select treatments according to genetic "constellations" of the tumors.

#435

Prognostic biomarkers as molecular targets for individualized neoadjuvant treatment for cervical cancer.

Pablo Moreno-Acosta,1 Oscar Gamboa,1 Diana Mayorga,1 Alfredo Romero,1 Jinneth Acosta,2 Maria Carolina Sanabria,1 Martha Cotes Mestre,1 Nicolas Magne3. 1 _National Cancer Institute, Bogotá, D.C., Colombia;_ 2 _National University of Colombia, Bogotá, D.C., Colombia;_ 3 _Institut de Cancérologie Lucien Neuwirth, Saint-Priest en Jarez cedex, France_.

Cervical cancer is one of the most prevalent malignancy and of higher mortality in the world, and is considered a marker of underdevelopment. Conventional radiotherapy is one of the treatments used for this type of cancer. 30 to 40% of patients with similar prognosis factors not respond equally to a comparable standard treatment. The poor response to radiotherapy leads to the development of innovative and effective therapies for cervical cancer locally advanced, metastatic and refractory. A comparative analysis of cervical cancer in the context of other cancers may reveal that it is relatively smaller number of targeted molecular agents that have been tested. Accordingly, a number of biological agents are currently in clinical development for the purpose of, inhibiting angiogenesis, molecularly address EGFR and IGF-1R, modulation of cell cycle, of histone deacetylases, COX-2, mTOR and tumor microenvironment (hypoxia and glycolysis). Within work that we have been developing, reported that gene expression of IGF1R is a strong predictive marker for lack of response to radiotherapy, patients have 28.6 times higher risk of failure treatment; Objective: To determine whether expression of IGF-IR, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, HKII and clinicopathological parameters can be used as prognostic biomarkers to treatment outcome and as possible molecular targets. Patients & Methods: This prospective cohort study included 149 patients with squamous cell carcinomas of the uterine cervix in FIGO stages IIB and IIIB between 2008 and 2011. The mean age was 46 years. Of the 149 patients, 61 were treated with radiotherapy and 88 with concurrent radiochemotherapy. Expression of the proteins CAIX, GLUT-1, HIF1α, HKII, IGF-IRα, IGF-IRβ and Survivin was determined by immunohistochemistry in biopsies taken before treatment. Results: The highest increase was found in expression of GAPDH (100%), Survivin (87%), followed of, IGF-IRα (76.5%), IGF-IRβ (74.5%), IGF-IRα and IGF-IRβ concordance in the expression(73%), HIF1α (74.1%); strong expression was observed with low frequency for GLUT-1 (31.1%), CAIX (16.2%), HKII (10.6%). We found that patients who do not express IGF-1Rß, GLUT1 and having hemoglobin levels > 11g/dl have improved overall survival compared to those that express IGF-1Rß, GLUT1 and having hemoglobin ≤ 11g/dl (P= 0.0158). Conclusions: Using the expression of GLUT1, IGF-1Rß and Hb levels (≤ 11g/dl) as therapeutic molecular targets could contribute to an appropriate therapeutic management as individualized neoadjuvant treatment for cervical cancer

#436

Glycodelin expression in endometrial carcinoma.

Hannu Koistinen,1 Annukka Pasanen,2 Laura Hautala1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Department of pathology, University of Helsinki, Helsinki, Finland_.

Glycodelin is a lipocalin protein mainly expressed in well-differentiated epithelial cells in reproductive tissues, including endometrium and seminal vesicles. Previously, glycodelin has been shown to induce differentiation of endometrial carcinoma cells, which was associated with reduced xenograft tumor growth. Glycodelin is highly expressed in secretory endometrium, but not in proliferative or postmenopausal endometrium. While glycodelin is weakly, if at all, expressed in cancer tissues and, when expressed, associated with better prognosis, results regarding the expression of glycodelin in endometrial carcinoma are discordant. Therefore, we studied the expression of glycodelin in endometrial carcinoma and hyperplasia by immunohistochemical staining using highly validated antibody developed in house. Furthermore, the levels of mRNA were addressed utilizing in silico transcriptomics databases.

Glycodelin transcripts were more abundant in normal uterus than in uterine carcinomas. Glycodelin staining in endometrial carcinoma sections was also much lower than that in secretory phase endometrium. Interestingly, in some endometrial hyperplasia samples glycodelin levels were much lower in hyperplastic regions of the sample as compared to those having normal histology.

In conclusion, our data show that glycodelin is not highly expressed, if at all, in endometrial carcinoma tissues. This is in keeping with the reduced expression of glycodelin in postmenopausal endometrium and association of glycodelin in epithelial differentiation.

#437

**Lumipulse** G **HE4 assay for monitoring of ovarian cancer recurrence and progression.**

Rachel Radwan,1 Anders Öhrvik,2 Katherine Falcone,1 Sara Gannon,1 Candice Felman,1 Natalya Benina,1 Natascha Svensson,2 Savitha Raju,1 Sharee Jones,1 Catherine Peacock,1 Julianna Young,1 Zhong-Qian Li,1 Christian Fermer,2 Diana Dickson1. 1 _Fujirebio Diagnostics, Inc., Malvern, PA;_ 2 _Fujirebio Diagnostics, AB, Gothenburg, Sweden_.

Human epididymis protein 4 (HE4), a member of Whey acidic four-disulfide core protein (WFDC) family, was demonstrated as one of the most useful biomarkers for ovarian cancer (HellstrÖm I, et al. 2003; Drapkin R, et al. 2005; Moore RG, et al. 2007).This study was to evaluate the use of Lumipulse G HE4 assay with patient serum for monitoring recurrence and progression of epithelial ovarian cancer. Recently, Lumipulse G HE4 assay was cleared by US FDA as an aid in monitoring recurrence or progressive disease in patients with epithelial ovarian cancer. Methods: Lumipulse G HE4 is a Chemiluminescent Enzyme Immunoassay (CLEIA) for the quantitative determination of HE4 in human serum and plasma on the Lumipulse G System by a two-step sandwich immunoassay method. In the assay, serum was added to and incubated with an anti-HE4 monoclonal antibody (MAb)-linked magnetic particles. The particles were then washed and rinsed to remove unbound materials. Alkaline phosphatase-labeled 2nd anti-HE4 MAb was added to and incubated with the HE4-bound particles. The particles were then washed and rinsed again to remove unbound materials. Substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane disodium salt (AMPPD) solution was then added to and mixed with the particles. Luminescence signals were thus generated by the cleavage of dephosphorylated AMPPD and converted into the amount of HE4 in the serum. Results: In the monitoring study, changes in HE4 levels in serial serum samples collected in SST tubes from 72 subjects with epithelial ovarian cancer were compared to changes in disease status, that is, progression or no progression. A total of 330 observations were undertaken with an average number of 5.6 observations per subject. A positive change in the HE4 value was defined as an increase in the observation value that was at least 18% greater than the previous observation value. Of the 61 samples with a positive change, 49% of them correlated with the progression of epithelial ovarian cancer while 80% of the 269 subject serial samples with no significant change in the HE4 value correlated with no progression. The total concordance was 74%, positive predictive values (PPV) 35%, and negative predictive value (NPV) 87%. In addition, a comparison of Lumipulse G HE4 with the predicate device, HE4 EIA, was carried out using specimens consistent with CLSI Protocol EP09-A3 and weighted Deming regression analysis. The slope and correlation coefficient (r) obtained were 1.03 and 0.9891, respectively, for the tested specimens (n = 143) which ranged from 33.4 - 969.5 pmol/L, and slope and r of 1.03 and 0.9917, respectively, for the tested specimens (n = 168) ranged from 33.4 - 4602.0 pmol/L. Conclusion: The Lumipulse G HE4 assay has demonstrated to be useful in monitoring the course of disease in women with epithelial ovarian cancer and well correlated with the predicate device HE4 EIA.

#438

Development of an autoantibody panel that reflects disease pathogenesis in ovarian cancer.

Makoto Kobayashi,1 Clayton R. Boldt,1 Wei-Lei Yang,2 Clemente Aguilar-Bonavides,3 Hong Wang,1 Zhen Lu,2 Robert C. Bast,2 Samir M. Hanash1. 1 _Department of Clinical Cancer Prevention,The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction

The serous subtype of ovarian cancer is responsible for most ovarian cancer deaths. When patients are diagnosed with early stage disease localized to the ovary or pelvis, current therapies can achieve survival rates up to 75-90%. However, approximately 70% of cases are diagnosed at an advanced stage, with a 5-year survival rate of less than 20%. A strategy based on autoantibodies which occur early during tumor development has merit for early cancer detection. We have applied a combined approach of mass spectrometry of circulating antigen-antibody complexes and with high-density protein microarrays to develop ovarian cancer serum markers based on autoantibody reactivity.

Materials and methods

Serum samples were obtained from women diagnosed with ovarian cancer (cases) and healthy controls undergoing ovarian cancer screening, women undergoing gynecologic surgery for various conditions but with normal ovarian pathology (surgical controls), and women without malignancy but with benign ovarian disease (benign controls). All samples were obtained following Institutional Review Board approval and informed consent. For autoantibody analysis, we used recombinant protein arrays containing 5,005 recombinants arrayed in duplicate and Alexa 647-labeled anti-human IgG were purchased from Invitrogen. Circulating Immunoglobulin (Ig) bound antigen analysis was performed using LC-MS/MS.

Results

Autoantibodies against 20 targets were identified in ovarian cancer patients compared to healthy, surgical and benign controls by protein array analysis with P<0.01. Ingenuity Pathway Analysis of these 20 antigens, exhibited major nodes of p53, MYC, and ESR1 associated networks reflecting ovarian cancer pathogenesis. Concordant findings were observed with LC-MS/MS analysis of Ig-antigen complexes.

Conclusion

We have uncovered a set of 20 protein antigens associated with the development of autoantibodies in ovarian cancer that reflect disease pathogenesis and that have potential relevance for early detection.

#439

Detection of aurora kinase A (AURKA) focal amplification in plasma samples of patients with recurrent ovarian cancer.

Gloria Juan, Katherine Paweletz, Abraham Anderson, Erick Gamelin, Gregory Friberg, Robert Loberg, Florian Vogl. _Amgen, Inc, Thousand Oaks, CA_.

Purpose: Aurora kinases are associated with acquired resistance to therapy in human cancers. We hypothesized that AURKA amplification is a late event in ovarian cancer (OC) progression and that frequency of amplification might be underestimated if assessed in tumor samples collected at the time of initial diagnosis. To test this hypothesis, we determined AURKA amplification levels in patients with recurrent OC and

compared those to AURKA amplification levels in the same patients at the time of diagnosis.

Methods: Samples were collected from 33 patients with taxane- and platinum-resistant OC enrolled in a first-in-human (FIH) study that evaluated monotherapy with AMG 900, an orally administered pan-aurora kinase inhibitor (NCT00858377). We performed a targeted analysis of

the AURKA locus and surrounding regions using next-generation paired-end sequencing (Illumina HiSeq2500 and 100 bp paired-end reads). By using extremely high sequence coverage (average coverage of > 2,000-fold), we were able to detect and quantify circulating tumor DNA (ctDNA) in plasma and to identify chromosomal alterations characteristic of tumor DNA. Sequencing was performed at Personal Genome Diagnostics (PGDx, Baltimore, MD). In addition, we performed AURKA (20q13) / 20q11 FISH in archival FFPE sections from the same patients. The AURKA (20q13) / 20q11 dual color FISH probe kit was purchased from Kreatech (now Leica Biosystems, Buffalo Grove, IL). Amplification states were defined as follows: amplified: AURKA (20q13) / 20q11 signal > 2.0; borderline: AURKA (20q13) / 20q11 signal < 2.0 and > 1.5; non-amplified: AURKA (20q13) / 20q11 signal < 1.5. FISH analyses were performed at Mosaic Laboratories (Lake Forest, CA). Survival analysis was performed using the Kaplan-Meier method.

Results: Plasma samples were available from all patients. Analysis of rearrangements surrounding the AURKA locus in plasma samples identified 11 of the 33 patients as AURKA amplified. Archival FFPE

sections were available from 27 of the 33 patients, and 21 of these 27 patients were evaluable by FISH for AURKA amplification. FISH only identified 1 patient as AURKA amplified and 1 additional patient as borderline amplified. Patients with AURKA amplification in plasma had received a median (min, max) of 6 (2,18) prior lines of chemotherapy, while patients without AURKA amplification had received a median (min, max) of 3 (1, 14) lines of prior chemotherapy. AURKA amplification as determined in plasma ctDNA at the time of AMG 900 study entry was an indicator of shorter PFS (median 9.4 vs 19.9 weeks; P = 0.04) and OS (median 9.4 vs 27.6 weeks; P = 0.013).

Conclusions: Our results suggest that AURKA amplification is a late event in ovarian cancer progression,and that monitoring AURKA focal amplification in plasma ctDNA allows the non-invasive assessment of acquired resistance to cancer therapy.

#440

Quantification of somatic chromosomal rearrangements in circulating cell-free DNA from ovarian cancers.

Faye R. Harris, Irina Kovtun, James Smadbeck, Francesco Multinu, Aminah Jatoi, Kimberly R. Kalli, Stephen J. Murphy, Geoffrey C. Halling, Farhad Kosari, Sarah H. Johnson, Andrea Mariani, George Vasmatzis. _Mayo Clinic, Rochester, MN_.

Circulating tumor DNA (ctDNA) provides great potential for the non-invasive clinical assessment of tumor burden. We developed a monitoring protocol using next generation mate-pair sequencing to identify chromosomal rearrangement signatures in primary tumors to follow ctDNA in blood. Primary tumors of 10 patients with ovarian cancer were sequenced, and individualized tumor-specific panels were designed to detect the junctions in ctDNA. A novel algorithm was developed and applied to data generated by quantitative PCR to calculate the percent of ctDNA. Selected junctions, some with potential as therapeutic targets, were detected in pre-surgically drawn blood in eight out of ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA post-surgery, and five had undetectable levels, consistent with their documented presence or absence of disease. The detection of somatic junctions derived from ctDNA could be an effective way to monitor ovarian cancer patients for relapse and therapy response.

#441

Association of interferon inducible genes with tumor immune microenvironment and chemotherapy resistance in high-grade serous epithelial ovarian cancer.

Katrina K. Au,1 Liliane Meunier,2 Cécile Le Page,2 Charles H. Graham,1 Andrew WB Craig,1 Timothy Childs,3 Julie-Ann Francis,4 Jeremy Squire,5 Anne-Marie Mes-Masson,2 Madhuri Koti1. 1 _Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada;_ 2 _Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Institut du Cancer de Montréal, Montreal, Quebec, Canada;_ 3 _Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada;_ 4 _Department of Obstetrics and Gynaecology, Kingston General Hospital, Kingston, Ontario, Canada;_ 5 _Department of Genetics and Pathology, Faculdade de Medicina, Ribeirão Preto, São Paulo, Brazil_.

Chemotherapy resistance is a major hurdle in high-grade serous epithelial ovarian cancer (HGSC) management. We previously reported differential expression of interferon inducible genes in pre-treatment tumours from chemoresistant and sensitive HGSC tumors. STAT1 expression was evaluated as a prognostic and predictive biomarker via immunohistochemistry in Phase I (n=183) and Phase II (n=550) biomarker validation studies conducted on HGSC tumours accrued from the Terry Fox Research Institute- Canadian Ovarian Experimental Unified Resource (TFRI-COEUR). Tumor STAT1 expression levels significantly correlated with the density of tumor infiltrating CD8+ T lymphocytes in both Phase I and Phase II cohorts. STAT1 expression significantly associated with progression free survival and response to chemotherapy in both Phase I and Phase II validation studies. Significant positive correlation between STAT1 expression levels and intratumoral CD8+ T cell density was observed. Intratumoral CD8+ T cell infiltration did not associate with progression free survival or response to chemotherapy in both cohorts. Interestingly, the prognostic relevance of CD8+ T cell was enhanced in combination with STAT1 in both cohorts. These findings provide evidence that STAT1 as an independent biomarker and a combination of CD8+ T cell infiltration with STAT1 could be novel immune-based prognostic and predictive biomarkers in HGSC. Findings from the current study will aid in patient stratification for novel immunomodulatory therapies for the management of chemotherapy resistance in HGSC.

#442

RASSF1A gene promoter methylation in primary tumors, adjacent morphologically normal tissues and plasma samples of patients with high grade serous ovarian cancer.

Lydia Giannopoulou,1 Issam Chebouti,2 Kitty Pavlakis,3 Sabine Kasimir-Bauer,2 Evi S. Lianidou4. 1 _Analysis of Circulating Tumor Cells Laboratory, Dept of Chemistry, Univ. of Athens, Athens, Greece;_ 2 _Department of Gynecology and Obstetrics, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany;_ 3 _Pathology Department, IASO Women's Hospital, Athens, Greece;_ 4 _Analysis of Circulating Tumor Cells Laboratory Dept of Chemistry, Univ. of Athens, Athens, Greece_.

Introduction: RASSF1A promoter methylation is frequent in high grade serous ovarian cancer (HGSC), the most common histological subtype. We examined RASSF1A promoter methylation in primary tumors, adjacent normal tissues and corresponding plasma samples of patients with HGSC, using real-time methylation specific PCR (real-time MSP) and methylation-sensitive high-resolution melting analysis (MS-HRMA).

Materials and methods: A training group of 78 primary HGSC FFPEs was first analyzed using both real-time MSP and MS-HRMA. Our validation group consisted of 61 primary HGSC FFPEs, 58 adjacent tissues (analyzed using both real-time MSP and MS-HRMA), and 59 corresponding plasma samples (analyzed using real-time MSP). The specificity of both assays was evaluated by analyzing a group of 16 fallopian tube samples from healthy individuals. OVCAR29 and IGROV1 cell lines were used as positive controls.

Results: In the training group, RASSF1A promoter methylation was detected in 33/78 (42.3%) by real-time MSP and in 33/78 (42.3%) by MS-HRMA (Agreement=94.9%, P=0.001, k= 0.895). The validation group results are shown in Table 1. According to the semi-quantitative MS-HRMA, RASSF1A promoter methylation was detected at significantly lower percentages in the adjacent morphologically normal tissues, compared to the paired primary tumors. In corresponding plasma samples, methylation was detected in 15/59 (25.4%) (Agreement with paired tumors=60.7%, P=0.259, k=0.143). Both real-time MSP and MS-HRMA revealed no RASSF1A promoter methylation in the small group of normal fallopian tubes (n=16).

Conclusion: RASSF1A promoter is highly methylated in primary tumors and at lower percentages in the adjacent normal tissues. In all cases, MS-HRMA gave comparable results with real-time MSP. Evaluation of the clinical significance of RASSF1A promoter methylation in corresponding plasma requires further investigation.

Table 1  | |

|

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

RASSF1A promoter methylation

Method | Primary tumor samples (validation group) N=61  | Adjacent tissues N=58 | Agreement (paired samples, N=57)

Real time MSP | 25/61 (41%) | 17/58 (29.3%) | 48/57(84.2%), P=0.001, k=0.652

MS-HRMA | 28/61 (45.9%) | 21/58 (36.2%) | 51/57(89.5%), P=0.001, k=0.782

Agreement (methods) | 58/61(95.1%), P=0.001, Cohen's kappa=0.900 | 50/58(86.2%),P=0.001, Cohen's kappa= 0.689

|

#443

Prognostic significance of CXCL12 protein levels in high-grade serous ovarian carcinoma.

Laurence Buisseret, Vincent Delisle, Laudine Communal, Sandra Pommey, John Stagg. _CR CHUM, Montréal, Quebec, Canada_.

Signals mediated by the C-X-C chemokine ligand 12 (CXCL12; stromal-derived factor 1) and its receptor CXCR4 have been implicated in cancer progression through multiple tumorigenesis processes, including cancer cell proliferation, survival, invasion and tumor angiogenesis. In vitro and murine models recently demonstrated that this axis is also involved in the mechanism of tumor immune evasion and resistance to immune checkpoint inhibitors. In human ovarian cancer, this axis has been associated with ascites, nodes and peritoneal metastasis.

We investigated the prognostic value of CXCL12 protein expression and association with CD8+ T cell infiltration in a cohort of 203 patients diagnosed with a high-grade serous ovarian carcinoma (HGSC). Quantitative immunofluorescence allowed us to evaluate the frequency and level of expression of CXCL12 and CD8+ cell density in the epithelial and stromal tumor compartments. CXCL12 expression in the epithelial tumor compartment was significantly higher than in the stromal compartment (p<0.0001). Strikingly, high levels of tumoral CXCL12 expression were significantly associated with a worse progression-free survival (PFS) and overall survival (OS) (log-rank test, p=0.007 & p= 0.006, respectively). The Kaplan-Meier median PFS and OS were respectively of 17±1.5 and 42±6.9 months in patients with high levels and of 18±3.6 and 61±7.5 months in patients with low levels of CXCL12. Notably, multivariate analysis showed that CXCL12 levels were associated with a worse outcome independent of FIGO stage and residual disease. No correlation was observed between CXCL12 expression levels and CD8+ T cell density. Intriguingly, in CD8+ subgroup analyses, the prognostic value of CXCL12 levels was restricted to tumors with low intraepithelial CD8+ T cell densities.

Flow cytometry and quantitative real-time PCR results suggest that CXCL12 promotes a mesenchymal phenotype and increased expression of cancer stem cells markers on human ovarian cancer cells.

In conclusion, modulation of the CXCL12/CXCR4 axis is a promising approach in HGSC that may potentiate immunotherapies and may act on epithelial-to-mesenchymal transition. Our study highlights the rationale to evaluate in the clinical setting treatments targeting the CXCL12/CXCR4 in HGSC, specifically in patients lacking CD8+ T cell tumor infiltration.

#444

Hyaluronan-mediated motility receptor (RHAMM) in ovarian cancer.

Stephanie Buttermore, Patricia Kruk. _University of South Florida, Tampa, FL_.

This study focuses on Hyaluronan Mediated Motility Receptor (RHAMM) protein expression in ovarian carcinoma (OC). RHAMM protein increases migration, invasion and is associated with poor prognosis when overexpressed. It is a dualistic oncogenic protein located within and on the cell surface acting as a receptor for Hyaluronan (HA). RHAMM is not generally overexpressed in normal tissues, but is upregulated in a number of cancers and is associated with poor clinical outcome. However, the role of RHAMM in OC progression is unknown.

Hypothesis: Elevated RHAMM levels promote OC progression and represent a novel urinary biomarker for OC.

Procedures: Measuring RHAMM expression in OC cell protein lysate and conditioned media (CM) was done by western blot (WB) and densitometry. With institution approval, patient cohort urine from male and female healthy controls and patients with cancer of the ovary, lung, breast, prostate, cervix, colorectum, skin, endometrium, head and neck, and brain were acquired from the H. Lee Moffit Cancer Center at the University of South Florida. Enzyme-linked immunosorbent assay (ELISA) designed for RHAMM detection, was used to measure patient urine expression levels.

Results: Cultured OC cells overexpress and secrete RHAMM. Using OC, SV-40 large T-antigen transfected human ovarian surface epithelial (IOSE) cells (HIO118, IOSE-121) and human dermal fibroblasts (HDF), we found >2x the expression of RHAMM protein in BC (MCF7) and OC (OVCAR5, OV2008, C13) cells by WB. WB of CM indicated >3x RHAMM levels secreted by OC compared to IOSE cells.

Urinary levels of RHAMM are elevated in OC patients. Since RHAMM is not an integral membrane protein, but rather loosely tethered to the plasma membrane, we determined whether RHAMM could be detected in bodily fluids of OC patients. ELISA showed detection of urinary RHAMM protein levels averaged 0.140 ng/ml (n=26) in healthy patient controls whereas OC patients averaged 0.411 ng/ml (n=132).

Conclusions: Prior to this study, overexpression and secretion of RHAMM by OC cells had not been reported. This has expanded our knowledge of OC etiology by delineating a functional role for RHAMM-driven OC progression. Validating elevated urinary levels of RHAMM suggests it is a potential diagnostic marker alone or in combination with other markers for OC detection. Further, the ability to monitor OC through the course of disease using urinary RHAMM levels could indicate recurrent disease or therapeutic efficacy. Understanding RHAMM expression in OC may lead to a possible diagnostic/prognostic indicator or therapeutic target for OC.

#445

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

Jeffrey D. Krimmel, Michael W. Schmitt, Scott R. Kennedy, Maribel I. Harrell, Kathy J. Agnew, Larry A. Loeb, Elizabeth M. Swisher, Rosa Ana Risques. _University of Washington, Seattle, WA_.

The detection of subclonal tumor-specific somatic mutations in clinical samples could revolutionize cancer diagnostics, but is limited by insufficiently sensitive sequencing methods. Duplex Sequencing is a novel next-generation sequencing (NGS) technology that implements single-molecule barcoding of both strands of DNA to allow internal error correction. This modification reduces the error rate of NGS from 1 in 1000 to less than 1 in 10 million nucleotides, an unprecedented sensitivity that enables accurate ultra-deep sequencing for clinical applications. In this study we sought to determine whether Duplex Sequencing could detect extremely rare TP53 mutated cancer cells disseminated into the peritoneal cavity of women with high-grade serous ovarian carcinoma (HGSOC). HGSOC is the most common and most aggressive type of ovarian cancer, shows early transperitoneal dissemination, and is characterized by near-universal prevalence of driver TP53 mutations. We analyzed 17 peritoneal fluid samples from women with HGSOC and known TP53 tumor mutation (cases) and 20 peritoneal fluid samples from women without cancer (controls). The tumor-specific TP53 mutation was detected in matched peritoneal fluid from 94% of cases (16/17), including 2 patients with occult tubal intraepithelial neoplasia, 7 patients with early stage HGSOC, and 8 with negative peritoneal fluid cytopathology. Tumor-specific alleles were detected as low as <1/25,000. Surprisingly, we also detected additional extremely low-frequency somatic TP53 mutations in peritoneal fluid from nearly all (35/37) cases and controls. These mutations were more abundant in cases than in controls, and in the latter correlated with age. The total burden of somatic TP53 mutations (tumor specific and non-specific) in a peritoneal fluid sample could distinguish cases from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Duplex Sequencing also revealed low-frequency, age-associated somatic TP53 mutations in 100% (15/15) of matched peripheral blood samples. Our results demonstrate the ability of Duplex Sequencing to detect very rare cancer cells, but also provide evidence of widespread, low frequency somatic TP53 mutation in non-cancerous tissue. This compromises the specificity of TP53 mutation tests for cancer detection in liquid biopsies.

#446

Systematic discovery of biomarkers for ovarian cancer by using the Human Protein Atlas database.

Jianxiang Shi,1 Hao Sun,2 Hongfei Zhang,1 Mengtao Xing,3 Jitian Li,3 Pei Li,3 Liping Dai,3 Chenglin Luo,3 Jian-Ying Zhang3. 1 _Zhengzhou University, Zhengzhou, China;_ 2 _Henan Cancer Hospital, Zhengzhou, China;_ 3 _University of Texas at El Paso, El Paso, TX_.

Purpose: The extensive accumulation of human protein expression datasets in normal and cancer tissues provides unique opportunities to perform systematic selection of tumor biomarkers. This study aims to discover biomarkers for ovarian cancer (OC) by data mining the Human Protein Atlas databases in a systematic manner.

Design methods: The Human Protein Atlas website (HPA, www.proteinatlas.org) is a publicly available database with spatial distribution of 20,356 proteins in 44 different normal human tissues and 20 different types of cancers. It is ideal for biomarker discovery. SAS software version 9.4 was used to data mine HPA database. All the potential biomarkers should meet the following criteria: the protein have ≥ 75% high or medium expression in OC but have low or no expression in other types of cancers, and the protein have low or no expression in at least one type of tissues of ovary included in the HPA database, the overall expression in normal tissues is ≤ 40%. Then ELISA was used to test the diagnostic performance of some of the selected biomarkers.

Results: Nineteen out of twenty different types of cancers included in the HPA database were studied in our study, carcinoid was excluded in the selection process due to the lack of proper normal control tissues. In general, PLAT might be a universal biomarker for 19 cancers. In total, 2,593 potential biomarkers for OC were selected. Ninety seven proteins out of 2,593 proteins might be OC-specific according to the available data. After an intensive manual search from the website, twelve proteins (STC1, MSLN, LSM3, WFDC2/HE4, ZNF2, CRIP3, XAF1, SLC35E2B, IFT88, ATP13A3, ZNF787 and TROAP) were selected for further research. Among them, WFDC2/HE4, MSLN have already been reported to have good diagnostic performance in OC by other researchers. ELISA was further used to test whether serum autoantibodies against PLAT or MSLN can distinguish 44 OC patients from 48 normal controls. The AUC of anti-PLAT and anti-MSLN were 0.783 (P < 0.001) and 0.666 (P = 0.006), respectively.

Conclusions: HPA database have made systematic discovery of cancer biomarkers possible. PLAT and MSLN can distinguish OC from normal controls. Another 11 proteins have great potential to serve as OC biomarkers and further validation studies are warranted to test their diagnostic performance and whether these biomarkers are OC specific.

Keywords: ovarian cancer, tumor associated antigen, big data, data mining, systematic discovery

This study was supported by grants from the General Program of National Natural Science Foundation of China (No. 81172086 and No.81372371). We would also like to acknowledge the Border Biomedical Research Center (BBRC) Core facilities at The University of Texas at El Paso (UTEP) for their help, which were funded by NIH Grant (5G12MD007592).

#447

The presence and persistence of ERCC1 positive circulating tumor cells predicts worse prognosis in primary ovarian cancer patients.

Issam Chebouti,1 Paul Buderath,1 Jan Dominik Kuhlmann,2 Yvonne Bokeloh,3 Siggi Hauch,3 Rainer Kimmig,1 Sabine Kasimir-Bauer1. 1 _Department of Gynecology & Obstetrics, Essen, Germany; _2 _Department of Gynecology & Obstetrics, Dresden, Germany; _3 _Qiagen Hannover GmbH, Hannover, Germany_.

Background: Resistance to platinum based chemotherapy in ovarian cancer can only be assessed retrospectively during post-chemotherapy follow-up. We recently showed that excision-repair cross-complementing rodent repair deficiency complementation group 1 nuclease (ERCC1)-positive (+) circulating tumor cells (CTCs) were an independent predictor for progression free survival (PFS), overall survival (OS) and for platinum-resistance in our ovarian cancer patients (pts). We now analyzed whether the negative prognostic impact of CTCs was related to the presence and persistence of ERCC1+ CTCs after platinum based chemotherapy.

Methods: 10 ml blood of 65 primary ovarian cancer pts before and after chemotherapy were studied for CTCs by immunomagnetic enrichment followed by multiplex RT-PCR to detect the transcripts EpCAM, MUC-1 and Ca 125 (AdnaTest OvarianCancer; QIAGEN Hannover GmbH, Germany). A patient was considered CTC+, if at least one of the three markers was detectable. ERCC1 was analyzed in a separate approach by single plex PCR, ß-actin served as an internal control and PCR-products were quantified on the Agilent Bioanalyzer 2100.

Results: CTCs were detected in 18/65 pts (28%) before and in 13/65 pts (20%) after chemotherapy. A persistence of CTCs was found in four pts, 14 pts were only positive before and nine pts only after therapy whereas in 38 pts, no CTCs were detected at any time. Pts with persisting CTCs had a significantly shorter PFS (p=3.32E-05) and OS (0.00018) while pts initially CTC-negative before but CTC+ after therapy showed a reduced OS (p=0.0039). ERCC1+CTCs were observed in 12/65 (18%) pts before and in 8/65 pts (12%) after therapy. A persistence of ERCC1+CTCs was found in three pts, nine pts were only positive before, five pts only after therapy whereas in 48 pts, no ERCC1+CTCs were detected at any time. Pts with persisting ERCC1+CTCs had a significantly shorter PFS (p=0.032) and OS (0.0058) while pts initially ERCC1-negative before but ERCC1+ after therapy showed a reduced OS (p=0.039). Up to now, no association between clinically defined platinum resistance and ERCC1-positivity was observed.

Conclusion: The negative prognostic impact of CTCs was related to the presence and persistence of ERCC1+CTCs after therapy supporting the idea to implement ERCC1 expression in CTC analysis of ovarian cancer pts as a biomarker for monitoring the disease to possibly change treatment in case of ERCC1 persistent CTCs.

#448

Expression of Galectins in high grade serous ovarian cancer.

Marilyne Labrie,1 Laudine Communal,2 Maria-Claudia Vladoiu,1 Pascal Dupont,1 Anne-Marie Mes-Masson,2 Yves St-Pierre1. 1 _INRS - Institut Armand-Frappier, Laval, Quebec, Canada;_ 2 _Institut du cancer de Montréal et centre de recherche du CHUM, Montreal, Quebec, Canada_.

Ovarian cancer (OC) is the 5th leading cause of cancer-related deaths in the Western world. Despite improvements in various treatments, the five-year survival of patients with advanced stage disease remains less than 40%. Approximately 70% of malignant OCs are attributed to the high-grade serous carcinoma (HGSC) histological subtype, which is typically treated by a surgical tumor debulking followed by platinum/taxane chemotherapy. Unfortunately, although approximately 80% of patients respond well to the initial chemotherapy treatment, a large portion of patients develop drug resistance during subsequent cycles of chemotherapy. The non-uniformity of response to therapy complicates the clinical decision-making process and reflects the genetic complexity that gives rise to intra-tumor heterogeneity. Clearly, there is an urgent need to develop reliable clinical biomarkers to identify patients at different levels of risk for specific outcomes. Here, we have investigated the expression of galectins (gal) in HGSC and examined their potential as biomarkers. Galectins have been shown to play a dual role in cancer progression. When released in the extracellular space by cancer cells, they are particularly efficient in creating an immunosuppressive tumor microenvironment, both locally and systemically. Inside cancer cells, they provide cancer cells with increased resistance to apoptosis induced by chemotherapeutic agents. Using tissue microarrays constructed from formalin fixed paraffin embedded tissues from 209 patients with HGSC, we have identified a new protein signature that takes into account the expression of galectins in both cancer and stromal cells. Our study showed that increased protein expression of stromal gal-1 and epithelial gal-8/9 in primary tumors was associated with a poor response to treatment in patients with HGSC. This signature also increased the predictive value of CA-125 on five-year disease-free survival, chemotherapy treatment response and five-year overall survival. Univariate and multivariate analyses revealed that gal-8 and gal-9 were both independent predictors of chemoresistance and overall survival, respectively. From a fundamental point of view, our project also contributes to better define the heterogeneity of the disease. Overall, these data provide a rational for further studies of Galectins in OC.

#449

Targeted proteomic analysis for personalized treatment of muscle invasive bladder cancer.

Fabiola Cecchi,1 Sheeno Thyparambil,1 Adele Blackler,1 Todd Hembrough,1 Shahrooz Rabizadeh,2 Patrick Soon-Shiong,2 Henry Frierson,3 Daniel Theodorescu4. 1 _NantOmics, Rockville, MD;_ 2 _NantOmics, CA;_ 3 _University of Virginia Health System, VA;_ 4 _UC Denver, CO_.

Background: Standard treatment for muscle-invasive bladder cancer (MIBC) includes chemotherapy with either gemcitabine-cisplatin (GC) or methotrexate, vinblastine, adriamycin and cisplatin (MVAC). These regimens have similar clinical complete response rates of approximately 30%. While no targeted treatment has been approved for bladder cancer, clinical trials have identified biochemical markers that predict the chemoresponsiveness of MIBC tumors to specific chemotherapeutic agents. For example, patients with high hENT1 and low RRM1 expression responded better to GC-based chemotherapy than their counterparts, and HER2 overexpression predicted resistance to cisplatin-based therapy. To quantify targets that are known indicators of tumor behavior, we used targeted proteomic analysis to assess 30 different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) MIBC tumor tissue.

Methods: Twelve FFPE MIBC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area from 1 or 2 slides. Following laser microdissection of the marked areas, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins in each patient sample.

Results: Of the 12 patient samples, 7 (58%) expressed high levels of hENT1 and 11 (92%) expressed low levels of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. A single patient expressed high levels of RRM1, an indication for non-gemcitabine based therapy. All 12 patients expressed TUBB3, a marker of resistance to taxane (vinblastine) and 10 patients (83%) lacked expression of FR-alpha, a marker of sensitivity to methotrexate. The majority of patients expressed a marker of sensitivity to anthracycline (TOPO2A) and did not express a resistance biomarker for platinum therapy (ERCC1).

Of 3 patients whose tumors expressed HER2, 2 had overexpression (>750 amol/ug) and would thus be eligible for HER2 basket trials. Further, multiplex-targeted proteomics discovered patients expressing FGFR1 (17%), cMET (33%), Axl (17%) and IDO1 (25%), which would make them eligible for clinical trials of targeted or immunotherapies.

Conclusion: MIBC is heterogeneous and expresses a wide range of proteins. Yet, it continues to be treated with only 2 chemotherapeutic regimens. Multiplexed proteomics is currently being used in clinical practice to inform personalized patient care decisions with identification and the relative quantities of actionable proteins known to predict tumor behavior.

#450

Serum biomarkers in patients treated with stereotactic body radiation therapy (SBRT) for prostate cancer.

Einsley Janowski,1 Simeng Suy,1 Rency S. Varghese,1 Olga Timofeeva,1 Stuart G. Field,2 Rachel Ostroff,2 Steve Williams,2 Anatoly Dritschilo,1 Sean P. Collins1. 1 _Georgetown University, Washington, DC;_ 2 _Somalogic, Boulder, CO_.

Purpose: The goal of this study was to determine the feasibility for developing prognostic protein biomarkers in serum samples from patients undergoing Stereotactic Body Radiation Therapy (SBRT) for organ confined prostate cancers.

Methods: 130 patients presenting with organ confined prostate cancers were treated with SBRT to doses of 35-36.25 Gy in 5 fractions. Peripheral blood samples were collected prospectively from patients at time 0 prior to radiation and serially after the first treatment (24 hours), follow-up months 1, 3, 6, 9, and 12 during the first year, and every 6 months thereafter, for up to 3 years. Processed study samples were analyzed by SomaLogic, Inc., using the SOMAscan Version 3 proteomic assay. Statistical analysis was performed on log-transformed data, with statistically significant differences identified by evaluating false discovery rate corrected p-values. Protein correlations were discovered with the Jonckheere-Terpstra (JT) test. Ingenuity Pathway Analysis (IPA) software was used to analyze cellular signaling pathways. PSA levels and clinical recurrence information were prospectively obtained at each follow-up visit and maintained in an institutional database.

Results:

Patient stratification by risk assessment identified 27 low-, 71 intermediate- and 32 high-risk patients in the study cohort. Proteins that function in cell proliferation, angiogenesis, protein signaling, gonad development, and cell migration correlated with Gleason's grade. CGA.LHB, KLK3, and CNTFR correlated both with tumor stage and Gleason's grade. With a median follow up of 3 years, ten patients experienced biochemical failures. While no proteins identified as differentially expressed in recurrent patients achieved significance, IPA pathway analysis of the top differentially expressed proteins converged on the IL-6 canonical pathway.

183 proteins were attributed to radiation effects on differential expression in patients by comparing pre-treatment to the 3 months post-treatment specimens. IPA network pathway analyses of these paired samples revealed changes in cell activation and signaling pathways, with the primary regulatory networks associated with ERK1/2, NF-κB, IFNG, ADIPOQ, histone 3, and histone 4. Of particular interest, high adiponectin levels in patients presenting with higher tumor stage decreased after radiation exposure, underscoring the potential for this molecule as a signal for determining response to radiation treatment.

Conclusion:

These hypothesis-generating experiments show differential serum protein levels in prostate cancer patient cohorts that have been stratified by risk factors and in longitudinal studies of patients following treatment with SBRT. Candidate biomarker proteins and molecular pathways have been identified for validation as potential predictors of prostate cancer response to radiation treatment.

#451

Large-scale evaluation of SLC18A2 in prostate cancer reveals diagnostic and prognostic biomarker potential at three molecular levels.

Christa Haldrup,1 Anne-Sofie Lynnerup,1 Tine M. Storebjerg,1 Søren Vang,1 Peter Wild,2 Tapio Visakorpi,3 Christian Arsov,4 Wolfgang A. Schulz,4 Johan Lindberg,5 Henrik Grönberg,5 Lars Egevad,5 Michael Borre,6 Torben F. Ørntoft,6 Søren Høyer,7 Karina D. Sørensen6. 1 _Aarhus University Hospital Skejby, Aarhus, Denmark;_ 2 _University Hospital Zürich, Zürich, Switzerland;_ 3 _Tampere University Hospital, Tampere, Finland;_ 4 _Heinrich Heine University, Düsseldorf, Germany;_ 5 _Karolinska Institute, Stockholm, Sweden;_ 6 _Aarhus University Hospital Skejby, Aarhus N, Denmark;_ 7 _Aarhus University Hospital, Aarhus, Denmark_.

Limitations of current diagnostic and prognostic tools for prostate cancer (PC) contribute to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using Mann-Whitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni- and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923-0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P<0.05). Likewise, SLC18A2 protein was down-regulated in PC (AUC 0.898) and had independent prognostic value for BCR (multivariate HR 0.51, P<0.05). Reduced SLC18A2 protein expression was also associated with poor overall survival in univariate analysis (HR 0.29, P<0.05).

Our results highlight SLC18A2 methylation as a new promising biomarker candidate for PC diagnosis. Furthermore, both SLC18A2 RNA and protein expression showed promising prognostic potential beyond routine clinicopathological variables. Thus, novel SLC18A2-based molecular tests could have useful future applications for PC detection and identification of high-risk patients.

#452

MIC-1 and Endoglin are protein serum biomarkers capable of increasing the clinical diagnostic specificity of the PSA test.

Luciane T. Kagohara,1 Ji Li,2 Christian P. Pavlovich,1 Christine Davis,1 Leslie Mangold,1 Guangjing Zhu,1 Colm Morrissey,3 Alan W. Partin,1 Wlodeck Mandecki,2 Robert W. Veltri1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _PharmaSeq, Inc., Monmouth Junction, NJ;_ 3 _University of Washington, Seattle, WA_.

PSA test and digital rectal examination have been routinely used to screen for prostate cancer (PCa). PSA screening revolutionized the management of PCa, especially with regards to early detection. Before the PSA test was available, ~20% of men were diagnosed with PCa that had already spread to the bone. Today, that number is around 4%. However, it is estimated that 56% of the cases are overdiagnosed resulting in overtreatment and exposing the patients to serious unnecessary treatment morbidities. Also the rate of false-positives is incredibly high (60-80%), since PSA is not cancer specific. It is critical to discriminate clinically significant PCa patients requiring definitive treatment from those who would safely undergo active surveillance monitoring.

The aim of this project is to identify and incorporate multiple serum protein biomarkers into an assay that will enhance the clinical diagnostic specificity of the PSA screening test. Using ELISA, we analyzed protein serum levels of Endoglin, IL-8 and MIC-1 in samples obtained from 50 PCa patients and 50 non-cancer individuals. ROC curve analysis was used to determine if these biomarkers were capable of separating the 2 groups. Multivariate logistic regression (MLR) was used to identify a panel of biomarkers that combined to PSA would increase the test specificity.

MIC-1 was the only biomarker capable of discriminating PCa from normal cases (AUC=0.76; p<0.0001). Endoglin protein levels were significantly different (p=0.0010) between the groups, although the AUC was borderline (AUC=0.69) to our cut-off (AUC>0.7) it suggests a good potential for this protein as a biomarker in PCa diagnosis. IL-8 did not show significant differences (p=0.1828) in the levels detected between cancer and normal samples. The expression of these proteins were also evaluated for their prognostic association with clinical-pathological features: Gleason Score (GS), T and N stage. MIC-1 protein levels was capable to separate GS 3+3 and 3+4 from those 4+3 or higher (AUC=0.73), suggesting that this biomarker might be associated with more aggressive cases. Using MLR, we identified Endoglin, MIC-1 and PSA as a multiple biomarker panel that enhanced specificity of the PSA screening test. The panel resulted in an assay with 88% specificity and 86% sensitivity that is slightly higher than PSA alone (82% specificity; 84% sensitivity). Our results suggest that MIC-1 is a strong candidate as a biomarker for cancer screening and disease aggressiveness. We also showed that when PSA test is combined with the presence of MIC-1 and Endoglin in serum, it resulted in an assay with higher specificity and sensitivity than when these biomarkers are evaluated alone. To validate these results, protein serum levels of Endoglin, MIC-1 and IL-8 will be determined in a bigger cohort of normal and PCa patients. Other biomarkers are also being evaluated.

#453

MEK activation is associated with a molecular subgroup in high grade serous ovarian cancer.

Nuala McCabe,1 Charlie Gourley,2 Andrena McGavigan,1 Caroline O. Michie,2 Niamh McGivern,3 Michael Churchman,2 Eamonn J. O'Brien,1 Laura Hill,1 Timothy S. Davison,1 Alistair Williams,4 Glenn McCluggage,5 Karen E. Keating,1 Denis P. Harkin,1 Richard D. Kennedy1. 1 _Almac Diagnostics, Craigavon, United Kingdom;_ 2 _University of Edinburgh Cancer Research UK Center, Edinburgh, United Kingdom;_ 3 _Centre for Cancer Research and Cell Biology, Craigavon, United Kingdom;_ 4 _Division of Pathology, University of Edinburgh, Edinburgh, UK, Edinburgh, United Kingdom;_ 5 _Department of Pathology, Royal Group of Hospitals Trust, Belfast, United Kingdom_.

Introduction

We have previously defined 3 molecular subgroups of High grade serous ovarian cancer (HGSOC), 'Angio', 'Immune' and 'Angio_immune' subgroups using gene expression data from 265 FFPE HGSOC samples obtained from treatment naive patients and who were subsequently treated with platinum-based standard of care (SoC) chemotherapy (carboplatin +/- paclitaxel) (Gourley, et al. J Clin Oncol 32:5s, 2014). Patients within these 3 molecular subgroups respond differently to SOC treatment. The immune subgroup has the best outcome compared to the Angio and Angio_immune subgroups (HR of 0.63 and 0.66 respectively on multivariate analysis). Since it has previously been shown that the MAPK pathway is an important mediator of cisplatin resistance in ovarian cancer, we wanted to investigate if the MAPK pathway was associated with one of the poor prognosis subgroups.

Methods

A gene signature that could detect each of the subgroups Angio, Immune and Angio_immune was generated from the clinical samples. Data from the Cancer Genome Atlas (TCGA) Project was used to test for correlation between each subgroup and phospho-MEK as measured by Reverse Phase Proteomic Array (RPPA). Sensitivity to the MEK inhibitor trametinib (GSK1120212) and cisplatin was determined by 10-day colony formation assay.

Results

We found a statistically significant association between the Angio_immune subgroup signature and Phospho-MEK (serine 217/221) expression (p=0.047) indicating activation of the MAPK pathway in this subgroup. Additionally we have demonstrated that the Angio_immune subgroup signature is suppressed by MEK inhibition (p=0.0055) and elevated by KRAS, NRAS and MEK1 overexpression in cell line models (0.0072, 0.0004 and <0.0001). These effects were specific to the Angio_immune subgroup signature as there was no association with the Angio or immune subgroup signatures. Additionally the Angio_immune gene signature could predict resistance to cisplatin and sensitivity to MEK inhibitors in a panel of breast and ovarian cancer cell line models (p=0.0017 and p=0.0091). Acquired resistance to cisplatin in cell line models was also associated with MEK activation and an elevated Angio_immune gene signature score, which could be reversed by a MEK inhibitor.

Conclusion

We have identified a molecular subgroup in HGSOC that is associated with MAPK signalling. A gene signature to detect this subgroup from formalin fixed paraffin embedded samples has been developed and predicts sensitivity to MEK inhibitors in pre-clinical model systems. Further work aims to validate the signature in clinical samples from patients treated with a MEK inhibitor. 

### Biomarkers for Melanoma and Uncommon Cancers

#454

Identification and characterization of tumor-associated antigens (TAAs) and anti-TAAs autoantibodies as biomarkers in immunodiagnosis of human osteosarcoma by serological proteome analysis.

Jitian Li,1 Manyu Huang,2 Manli Luo,2 Pei Li,1 Wen Xie,2 Zongchang Han,2 Wuyin Li,2 Jianying Zhang1. 1 _The University of Texas at El Paso, El Paso, TX;_ 2 _Luoyang Orthopedic-Traumatological Hospital, Luoyang Institute of Orthopedic and Traumatology, Luoyang, China_.

Osteosarcoma (OS) is the most common highly malignant primary solid bone-tumor. Despite its relatively low incidence rate among overall cancers, it remains one of the most harmful primary malignant tumors in childhood and adolescence. Although some tumor markers like mutant p53 can be potentially used as biomarker to detect OS, the sensitivity and specificity are not optimal, especially for early diagnosis. The establishment of a methodology to identify patient with early stage of OS remains to be investigated. There has been a growing interest in using serum autoantibodies against tumor-associated antigens (TAAs) as serological cancer biomarkers, which stems from the notion that anti-TAA antibodies are "sensors" or "reporters" of molecular events associated with tumorigenesis. The objective of this study is to identify and characterize the targeted TAAs as biomarkers in OS, and further to analyze the frequency and specificity of autoantibodies in OS patients. In this initial study, we have tested 35 sera from OS patients, 12 sera from Osteochondroma (OC) patients and 32 age-matched normal human sera (NHS), for the presence of autoantibodies to the TAAs from extracted protein antigens from U2-OS culture cells in 1-D Western blot and by indirect immunofluorescence (IIF). Autoantibodies were detected in 34 of 35 (97.1%) sera from patients with OS, which were significantly higher than that in NHS (12.5%, 4/32). In contrast, there is no significate association between OC and NHS group, which implies that the OS sera may encompass more specific autoantibodies than OC sera. Interestingly, 35% (7/20), 25% (5/20), 20% (4/20) and 35% (7/20) OS sera were identified by 1D Western blotting analysis containing antibodies against unknown cellular protein antigens around 35 kD, 45 kD, 55 kD and 60 kD respectively. Additionally, no reactivity with the 45 kD and 55 kD protein was detected in 32 NHS. In the further study, these cellular proteins targeted by serum antibodies in OS will be identified by using serological proteome analysis (SERPA) approach. This preliminary data supports that autoimmune responses to certain cellular proteins maybe a by-product in the transformation to OS, and further studies of novel targeted proteins in OS may provide insights into how these proteins might be involved in malignancy.

Key Words: Osteosarcoma (OS), Tumor-associated antigens (TAAs), Serological proteome analysis (SERPA), Cancer early detection, Immunodiagnosis

#455

Identification of exosomal surface marker derived from Ewing sarcoma cells.

Aki Yoshida,1 Tomohiro Fujiwara,1 Koji Uotani,1 Yusuke Yoshioka,2 Koji Ueda,3 Takuya Morita,1 Ken Takeda,1 Toshiyuki Kunisada,1 Yutaka Nezu,2 Hiroyuki Kawashima,4 Takahiro Ochiya,2 Toshifumi Ozaki1. 1 _Dept. of Orthopaedic Surgery, Okayama University Graduate School, Okayama, Japan;_ 2 _Div. of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan;_ 3 _Genome Center, Japanese Foundation for Cancer Research, Tokyo, Japan;_ 4 _Dept. of Orthopaedic Surgery, Niigata University Graduate School, Niigata, Japan_.

Introduction: Exosomes are small vesicles that are secreted by a multitude cell types. Recent studies have identified that circulating exosomes play an important role in tumor develompment and provide perspectives in novel diagnostic and therapeutic challenge. In this study, we investigated the surface protein on the tumor-derived exosomes by proteome analysis to utilize for a new biomarker of Ewing sarcoma.

Methods: Exosomes was collected from the culture medium of two Ewing sarcoma cells (SKES1, RDES) and human mesenchymal stem cells (hMSC) by gel filtration (EVsecond®) and ultracentrifugation. Exosomes extracted from each cell line were checked by transmission electron microscopy (TEM) and Nanosight®, a nanoparticle-tracking system. Global analysis of exosomal surface proteins were performed by liquid chromatography and mass spectrometry (LC/MS), then ES specific protein candidates were validated by immunoblot and ExoScreen for exosomes extracted by ultracentrifugation, and enzyme-linked immunosorbent assay (ELISA) for exosomes extracted by EVsecond®.

Results: Extracellular vesicles from ES cells were obtained by ultracentrifugation and EVSecond®. TEM and Nanosight® showed that the size of extracted exosomes derived from ES cells were 40-100 nm in diameter. Proteomic analysis detected 287 proteins from SKES-derived exosomes, 256 proteins from RDES-derived exosomes, and 133 proteins from hMSC-derived exosomes by LC/MS. We identified CD99 as entrapped protein on the exosomes derived from ES cells. For validation study, we performed immunoblot analysis that CD9, CD63, CD81 and CD99 were detected in ES-derived exosomes. Furthermore, immunogold TEM detected CD81 and CD99 on the exosomal surface. Next, we performed ELISA to detect the CD9 and CD99 on the surface of ES-derived exosomes purified by EVsecond®. Finally, we identified that ExoScreen, that has been clinically useful, was able to detect ES-derived exosomes, which expressed CD9, CD63 and CD99 on their surface.

Discussion and conclusion: Although the EWS-FLI1 oncogenic activity is increased by CD99 in ES, no study, to date, has reported that CD99 is on the tumor-derived exosomes and circulating in the patients' serum. This study demonstrates that exosomes derived from ES cells can be detected by the CD99 surface marker, providing the possibility of the exosome as a novel biomarker for liquid biopsy.

#456

Identification of circulating tumor-derived microRNA signatures in osteosarcoma.

Tomohiro Fujiwara,1 Koji Uotani,1 Aki Yoshida,1 Takuya Morita,1 Tadashi Komatsubara,1 Kazuhisa Sugiu,1 Toshinori Omori,1 Ken Takeda,1 Toshiyuki Kunisada,1 Yutaka Nezu,2 Akira Kawai,3 Hirotaka Kanzaki,4 Takahiro Ochiya,2 Toshifumi Ozaki1. 1 _Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan;_ 3 _Department of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan;_ 4 _Department of Pharmacy, Okayama University Hospital, Okayama, Japan_.

Introduction:

The lack of useful biomarkers is one of the most important clinical problems of bone and soft tissue sarcomas, although early detection of recurrent or metastatic disease and early decision making according to tumor response to chemotherapy is important to improve patient prognosis. The recent discovery of circulating cell-free microRNAs (miRNAs) in human blood has represented a new approach for the diagnostic screening for malignant diseases. In this study, we investigated whether the serum miRNA levels could be used as novel biomarkers for osteosarcoma (OS) patients.

Methods:

Global miRNA profiling was performed using the serum samples collected from OS patients, age-matched non-OS patients and healthy volunteers. The expression levels of extracted miRNA were confirmed in human OS cell lines (SaOS2, U2OS, HOS, 143B), human mesenchymal stem cell (hMSC), and their culture media. The miRNA candidates were evaluated using the serum samples of the validation set.

Results:

The miRNA expression profile of the serum samples of 10 OS patients, 10 age-matched non-OS patients, and 10 healthy controls identified 236 serum miRNAs to be highly expressed in the OS compared to the non-OS patients and healthy controls. Among these miRNAs, 8 miRNAs were overlapped with the secretory miRNAs in the culture medium of 7 osteosarcoma cell lines.

Quantitative RT-PCR revealed that two candidates were significantly upregulated in the OS cells and culture medium, compared to MSCs. The expression of these miRNAs in the culture media from all OS cell lines increased along with culture time and tumor cell numbers, indicating that it is an important secretory miRNA derived from OS cells.

The serum concentration of these miRNAs in OS patients was significantly higher than those in healthy volunteers and non-OS patients. Our ROC analyses revealed that an AUC value based on the miRNA expression was 0.868 (95% confidence interval = 0.743 to 0.993), which was was higher than that of alkaline phosphatase (ALP). Finally, the detected serum miRNA expression levels markedly decreased at the postoperative status in operative cases, while gradually decreased during neoadjuvant chemotherapy, which revealed that serum miRNA expression levels correlated with the tumor burden and drug sensitivity.

Conclusions:

We identified circulating tumor-derived miRNA signatures in the serum obtained from the OS patients. Furthermore, the definitive miRNA reflected tumor burden in OS patients. Evaluating the serum miRNA expression may thus have important clinical implications for risk stratification and the planning of post-therapeutic surveillance.

#457

FGFR1 overexpression is frequent in adult soft tissue sarcoma and predicts sensitivity to FGFR inhibitors.

Priya Chudasama,1 Marcus Renner,2 Katja Specht,3 Sadaf Mughal,4 Barbara Hutter,4 Ingo Alldinger,5 Simon Schimmack,5 Dirk Jäger,6 Christof von Kalle,7 Stefan Gröschel,8 Stephan Wolf,4 Benedikt Brors,9 Wilko Weichert,3 Hanno Glimm,8 Gunhild Mechtersheimer,2 Claudia Scholl,9 Stefan Fröhling10. 1 _German Cancer Research Center; National Center for Tumor Diseases Heidelberg, Heidelberg, Germany;_ 2 _Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany;_ 3 _Institute of Pathology, Technische Universität München, Munich, Germany;_ 4 _German Cancer Research Center, Heidelberg, Germany;_ 5 _Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany;_ 6 _National Center for Tumor Diseases Heidelberg; Department of Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany;_ 7 _German Cancer Research Center; National Center for Tumor Diseases Heidelberg; Section for Personalized Oncology, Heidelberg University Hospital; DKFZ-Heidelberg Center for Personalized Oncology, Heidelberg, Germany;_ 8 _German Cancer Research Center; National Center for Tumor Diseases Heidelberg; Section for Personalized Oncology, Heidelberg University Hospital, Heidelberg, Germany;_ 9 _German Cancer Research Center; National Center for Tumor Diseases Heidelberg; German Cancer Consortium, Heidelberg, Germany;_ 10 _German Cancer Research Center; National Center for Tumor Dieases Heidelberg; Section for Personalized Oncology, Heidelberg University Hospital; German Cancer Consortium, Heidelberg, Germany_.

Purpose: Soft-tissue sarcomas (STS) are highly diverse, devastating and clinically challenging malignancies. In advanced-stage STS, conventional chemotherapy provides symptom palliation but not prolonged survival, which typically ranges from 11-15 months after development of distant metastases. Thus, there is an urgent need for more effective, well-tolerated and targeted STS therapies. Alterations of the FGFR1 receptor tyrosine kinase are under investigation as biomarker of FGFR inhibitor sensitivity in several epithelial cancers. Prompted by the detection of high-level FGFR1 amplification in a patient with metastatic leiomyosarcoma through clinical exome sequencing, we investigated the role of FGFR1 as oncogenic driver and potential drug target in STS. Specific aims were to (i) determine the frequency of FGFR1 amplification and overexpression in adult STS, (ii) evaluate the sensitivity of FGFR1-altered STS cells to genetic and pharmacologic FGFR1 inhibition, and (iii) delineate the signaling pathways engaged by FGFR1 in these cells.

Experimental design: The frequency of FGFR1 amplification and overexpression was established in a cohort of patients with treatment-näive high-grade STS using array-based comparative genomic hybridization and mRNA expression profiling, respectively, and results were validated using data from The Cancer Genome Atlas (TCGA). FGFR1 dependence was assessed in STS cell lines with and without FGFR1 alteration through RNA interference (RNAi)-mediated knockdown and selective small-molecule FGFR inhibitors. FGFR ligand and pharmacologic inhibitors were employed to dissect FGFR signaling in FGFR1-altered STS cells.

Results: FGFR1 amplification and overexpression were present in 31% and 34% of cases in the untreated high-grade STS cohort (n=176) and in 16% and 15% of cases in the TCGA cohort (n=256). Interestingly, FGFR1 overexpression was detected in a substantial proportion of cases

with no amplification (high-grade sarcoma cohort, 26%; TCGA cohort, 6.6%) comprising seven STS subtypes. Functional studies employing RNAi and different FGFR inhibitors (PD173074, AZD4547, BGJ398) demonstrated that the degree of dependence on FGFR1 for proliferation, survival, and anchorage-independent growth was primarily determined by FGFR1 expression levels. Furthermore, we identified the MAPK-ERK1/2 signaling axis as critical FGFR1 effector pathway whose suppression dictates the sensitivity of FGFR1-driven STS cells to FGFR-targeted therapy.

Conclusions: FGFR1 amplification and overexpression are frequent events in multiple STS subtypes, and high FGFR1-expressing STS cells are sensitive to FGFR inhibition. These findings support FGFR1 as novel therapeutic target in STS and point to FGFR1 overexpression as a more reliable biomarker of sensitivity to FGFR pathway inhibition than genomic amplification, which might be of value for enrichment of future clinical trial populations.

#459

The use of circulating SAA and CXCL4 to predict outcome of osteosarcoma at diagnosis.

Ricardo J. Flores. _Texas Children's Cancer and Hematology Centers, Houston, TX_.

Background: Osteosarcoma is the most common malignant bone tumor in children. The survival rate of osteosarcoma is 60-70%, which has remained stagnant for the past four decades. Identifying biomarkers that can better prognosticate osteosarcoma patients and guide therapies would improve survival of this disease.

Material and Methods: In this study, we used ELISA to evaluate the expression of two previously identified circulating biomarkers, namely Serum Amyloid A (SAA) and CXC Chemokine Ligand 4 (CXCL4), in a large cohort of serum samples (n=233) obtained at from the Children's Oncology Group at initial diagnosis. The biomarkers' expression was correlated with survival and evaluated for prognostic significance in the OS patients.

Results: Analysis showed that patients with high SAA and low CXCL4 levels had significantly poorer outcome (p = 0.014, HR = 1.68), which was independent of initial metastasis status. The 5-year overall survival rates for the "high-risk" and "low-risk" groups were 47% (95% CI; 36% - 62%) and 64% (95% CI; 57% - 72%), respectively. In addition, low tumor expression of CXCL4 correlated with poor survival (p = 0.017) in an osteosarcoma tissue microarray. In conclusion, this study reports a novel prognostic model consisting of high SAA and low CXCL4 levels in sera that prognosticates osteosarcoma patients at initial diagnosis. This model could serve as the basis for future biomarker-guided clinical trials by incorporating targeted therapy for the poor prognostic population.

#460

Diagnostic and prognostic value of expression of CCND1 splice variant cyclin D1b in papillary thyroid carcinoma.

Chan Kwon Jung,1 Sora Jeon,2 Yourha Kim,2 Ahwon Lee,1 Gyeong Sin Park1. 1 _Department of Hospital Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea;_ 2 _Department of Biomedicine & Health Science, Graduate School, The Catholic University of Korea, Seoul, Republic of Korea_.

Background: An active cyclin D1 isoform, cyclin D1b, arises as a result of a failure to splice the fourth intron of the CCND1 pre-mRNA transcript. The expression of cyclin D1b has been associated with tumorigenesis and tumor progression in many types of human cancer. However, no studies with cyclin D1b have yet been reported in the thyroid cancer.

Methods: We investigated the CCND1 rs9344 (cDNA 870) polymorphism by TaqMan SNP genotyping assay and expression profiles of mRNA and protein of full-length cyclin D1 (cyclin D1a) and cyclin D1b by quantative real-time PCR and immunohistochemistry in 286 thyroid tumors including 16 nodular hyperplasias, 33 follicular adenomas (FA), 179 papillary thyroid carcinomas (PTC), 32 follicular thyroid carcinomas (FTC), 5 poorly differentiated carcinomas (PDC), 4 anaplastic thyroid carcinomas (ATC), and 17 medullary thyroid carcinomas (MTC). The relationships between cyclin D1 expression levels and clinicopathological features were analyzed.

Results: The cyclinD1b mRNA expression level in all thyroid tumors was significantly higher in the AA and GA genotype groups compared with the GG genotype group (p<0.001 and p=0.006, respectively). The mRNA expression levels of cyclin D1b was significantly higher in PTCs when compared to benign or other malignant tumors (p<0.001) while no significant difference in cyclin D1a mRNA expression was observed according to the genotypes or tumor types. PTCs showed similar expression levels of cyclin D1b mRNA regardless of histologic subtypes, even when compared with follicular variant. In immunohistochemistry analysis, cyclin D1b overexpression was observed in PTC, PDC, ATC, and MTC, but not in FA and FTC. Cytoplasmic overexpression of cyclin D1b was significantly associated with advanced tumor stage in all PTCs (p=0.025). Nuclear overexpression of cyclin D1b was associated with an increased incidence of lymph node metastases (p=0.014) in classic PTCs.

Conclusion: The A allele of the rs9344 polymorphism predisposes for cyclin D1b production in thyroid cancer. Cyclin D1b overexpression has a diagnostic biomarker utility to differentiate PTC from benign follicular nodules and may be functionally involved in the progression of the disease.

#461

Combined analysis of circulating epithelial cell count and serum thyroglobulin for differentiating disease status of the patients with papillary thyroid carcinoma.

Ching-Ping Tseng,1 Jen-Der Lin,2 Hung-Chih Lin,1 Ju-Chien Cheng3. 1 _Chang Gung University, Taoyuan, Taiwan;_ 2 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 3 _China Medical University, Taichung, Taiwan_.

Papillary thyroid carcinoma (PTC) is one type of thyroid cancer and accounts for about 80% of the cases. Routine surveillance by serum thyroglobulin (Tg) and medical imaging is the current practice to monitor disease progression of the patients. However, the presence of anti-Tg antibody or other influence factors may discount the value of serum Tg in disease monitoring. Whether enumeration of circulating epithelial cells (CECs) and its combined analyses with serum Tg help to define disease status of PTC patients was investigated. CECs were first enriched from the peripheral blood of the healthy control subjects (G1, n=17) and the patients at disease-free status (G2, n=26) or with distant metastasis (G3, n=22). The number of CECs expressing epithelial cell adhesion molecule (EpCAM) was determined by immunofluorescence microscopy analyses. The medium number of EpCAM+-CECs was 6 (intequartile range 1-11), 12 (interquartile range 7-16) and 91 (interquartile range 31-206) cells/ml of blood for G1, G2 and G3, respectively. EpCAM+-CEC counts were significantly higher in G3 than in G1 (p<0.05) and G2 (p<0.05). Our data further revealed that combined analysis of serum Tg with EpCAM+-CEC defined the disease status (disease-free vs. distant metastasis) in 46 (95.8%) of the 48 patients with the AUC equivalent of 0.962 (p<0.0001) in the ROC analysis. The sensitivity and specificity of the assay was 100% and 92.3%, respectively. Moreover, all 22 patients (100%) with metastatic status were defined accordingly. These data demonstrate that combined analysis of serum Tg with EpCAM+-CECs is suitable to distinguish the patients at disease-free status from the patients with distant metastasis. CEC testing thereby can supplement the current standard methods for monitoring disease status of PTC.

#462

The role of Her2 overexpression in Middle Eastern papillary thyroid cancer.

Abdul K. Siraj, Shaham Beg, Rong Bu, Saif AlSobhi, Fouad Al-Dayel, Khawla Al-Kuraya. _King Faisal Specialist Hospital & Res. Ctr., Riyadh, Saudi Arabia_.

Thyroid cancer is the second most common malignancy among females in Saudi Arabia accounting for 7.4% of all cancers and 10.6% of all female malignant cancer. Among different subtypes, papillary thyroid cancer (PTC) is the most common subtype of thyroid cancer. Although the overall cancer related ten year survival rate for PTC has reached 90%, local, regional and distant metastasis does occur in 10% of cases. Therefore, in search for new therapeutic targets for PTC, we investigated the role of Her2 oncogene in PTC as this gene is known to be involved in other cancers such as breast cancer. The aim of this study was to determine the prevalence and prognostic role of Her2 overexpression in Middle Eastern papillary thyroid carcinoma by immunohistochemistry (IHC), Fluorescent In-Situ Hybridization (FISH) and clinical data. A tissue microarray (TMA) containing >1000 PTC cases with follow-up data was used. TMA sections were analysed at protein and DNA level using IHC and FISH. FISH analyses were performed to look for the gain or amplifications in Her2 gene. IHC analysis showed Her2 overexpression in 19.7% of our cases of PTC. Elevated expression was almost exclusively 2+ (194 tumors) and only rarely 3+ (1 tumor). Interestingly, no amplification of Her2 gene was visualized by FISH. Only 3% of our cases showed mildly elevated HER2 gene copy numbers not reaching the threshold for amplification. HER2 overexpression and copy number gains were unrelated to tumor stage, metastasis, patient survival and other clinical and pathological parameters. Our results demonstrated that Her2 overexpression occurs at relevant frequency in papillary thyroid cancer and in the absence of gene amplification. Additionally, expression of Her2 seems to hold no clinical value as prognostic factor in PTC

#463

Immunohistochemical study of TROP-2 protein expression in thyroid lesions.

Zhiming Liao. _Spring Bioscience, Pleasanton, CA_.

Introduction

Tumor-associated calcium signal transducer 2 (TROP-2) is a transmembrane glycoprotein overexpressed in various types of epithelial carcinomas from lung, breast, stomach, ovary, cervix, colon and pancreas. A recent immunohistochemical (IHC) study indicated that TROP-2 is also overexpressed in thyroid papillary carcinoma, but the sensitivity is only 82% in formalin-fixed and paraffin-embedded thyroid (FFPE) tissues when a polyclonal anti-TROP-2 antibody was used. A high quality of TROP-2 antibody for IHC applications is needed to improve the sensitivity of the detection of thyroid papillary carcinoma in FFPE tissues.

Methodology

A rabbit monoclonal antibody (clone SP295) against human TROP-2 protein was developed. Western blot was performed using SK-BR-3 cell lysates to verify the specificity of the TROP-2 antibody. An IHC study of TROP-2 was performed on normal tissue and tumor arrays from the thyroid and other organs. TROP-2 staining intensity was semi-quantitatively scored using a 0-3 scale. The sensitivity and specificity of the TROP-2 IHC assay for detecting papillary carcinoma were determined using 65 cases of thyroid papillary carcinoma, 29 cases of follicular carcinoma, and 31 cases of other thyroid conditions (normal, adenoma, Grave's disease, thyroiditis, Goiter, medullary carcinoma, clear cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma).

Results

A single band at 33 kDa was detected by SP295 on a western blot. In normal tissues, TROP-2 is detectable by IHC mostly in squamous epithelia of many normal organs (skin, pharynx, esophagus, and cervix) and cuboidal and columnar epithelium (breast, kidney, liver, uterus, pancreas, prostate and transitional epithelium of the bladder). In thyroid lesions, the TROP-2 positive rate is 100% (65/65) in papillary adenocarcinoma and it is negative in all other thyroid conditions (normal, adenoma, Grave's disease, thyroiditis, Goiter, medullary carcinoma, clear cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma). The sensitivity and specificity of TROP-2 IHC for detecting papillary carcinoma were both 100% in this study.

Conclusions

Rabbit monoclonal TROP-2 antibody (clone SP295) is highly sensitive and specific for detecting TROP-2 in FFPE tissues and it can be used to identify thyroid papillary carcinoma.

#464

Methylation of a single CpG site in the CD95-ligand promoter is a biomarker predicting the response to therapy with APG101 in glioblastoma.

Christian Gieffers,1 Claudia Kunz,1 Jaromir Sykora,1 Christian Merz,1 Meinolf Thiemann,1 Harald Fricke,1 Benedikt Wiestler,2 Wolfgang Wick3. 1 _Apogenix GmbH, Heidelberg, Germany;_ 2 _Department of Neurooncology, Heidelberg, Germany;_ 3 _Depertment of Neurooncology, Heidelberg, Germany_.

CD95 (APO-1/Fas) is a member of the Tumor Necrosis Factor Receptor Super Family. Binding of CD95-ligand (CD95L) to CD95 triggers intracellular signal transduction that is critically involved in the invasive growth of glioblastoma cells. Invasion of malignant glioblastoma cells into the brain parenchyma is responsible for poor therapeutic outcome of currently available treatments. The inhibition of CD95/CD95L mediated invasive growth of glioblastoma cells represents an attractive novel therapeutic concept. Apogenix has developed APG101, a fully human fusion protein consisting of the extracellular domain of CD95 and the Fc-domain of an IgG. APG101 has been confirmed as a potent inhibitor of CD95L induced invasion of glioblastoma cells in vitro.

In a randomized phase 2 study in glioblastoma patients with 1st or 2nd relapse the combined therapy of APG101 plus radiotherapy (RT) was found to be superior to RT alone in a clinically relevant order of magnitude in all efficacy endpoints (i.e. PFS-6, PFS and OS). At the same time APG101 exhibited an excellent safety profile and was well tolerated.

To identify potential biomarkers we used available tissue sections originating from archived primary tumor of the study patients and analyzed them for the expression of CD95L as well as for the DNA methylation status. A genome-wide assessment of DNA methylation identified a single CpG-site (CpG2) upstream of the CD95L-promotor that showed differential methylation between APG101 responders (PFS>5 months) and non-responders (PFS < 2 months). Available patient DNAs were in addition analyzed by MassARRAY and Pyro-sequencing to confirm differential CpG2 methylation. Based on this data we used the median of the CpG2 methylation level as a threshold to analyze for a correlation of CpG2 methylation and response to APG101 therapy. Patients showing a low level of CpG2 methylation responded best to therapy with APG101 whereas patients with a high level of CpG2 methylation did not show a relevant benefit when treated with APG101 compared to the control RT-group. The analysis shows a significant survival benefit achieved in patients with low CpG2 methylation (median OS: 16.1 vs 7.3 months, p = 0.029).

Hence, the level of CpG2 methylation in the CD95L promoter in the patients` glioblastoma tissue is a prognostic biomarker predicting response to therapy with APG101. Based on the observations described, Apogenix currently develops a qPCR-based assay to quantify CpG2 methylation. This assay is intended as companion diagnostic to identify patients that may respond best to APG101 treatment.

#465

CD44 as a potential therapeutic target and prognosis marker for glioblastoma.

Fengfei Wang, Xin Shen, Jessica Pruszynski, Jason Huang, Batool Kirmani, Erxi Wu, Ekokobe Fonkem. _Baylor Scott & White Health, Temple, TX_.

Glioblastoma (GBM) is a common and very aggressive primary brain tumor with no cure. The current diagnosis relying on magnetic resonance imagining, computed tomography, and biopsy is a very expensive and aggressive process. More effective therapeutic strategies such as target therapies and simplified early diagnosis tools are urgently needed for GBM management and prognosis. As recent reports suggest that GBM expresses CD44, a type-I transmembrane glycoprotein, is a marker of cancer stem cell and therapy resistance, with a goal of identifying a therapeutic target and a prognosis maker for GBM, we analyzed several data sets previously detected by microarrays using an online R2 Genomics analysis and visualization platform (http://r2.amc.nl). Through examining of the expression levels of CD44 in the tissues of normal cerebellum (without brain tumor) and in GBM tissues, we find that the expression level of CD44 in GBM is higher than that in non-brain tumor tissues. Comparing the expression levels of CD44 in Primary and recurrent GBMs, GBM cell lines, neuron stem cell lines, and normal cortex tissues, we observe that the expression levels of CD44 in recurrent GBMs are higher than primary GBMs; the CD44 levels in GBM cell lines are similar to neuron stem cell lines which are significantly higher than that in the normal cortex tissues. We further evaluated the correlation of CD44 levels with patients' overall survival in 4 available complete data sets in the R2 database, including 2 from The Cancer Genome Atlas (TCGA) database. We show that an elevated level of CD44 in GBM is associated with a short overall survival time although variations exist from cohort to cohort. In addition, we studied the effect of p53 status on CD44 expression, we reveal that CD44 level is higher in the GBMs carrying p53 mutation compared to those with wild-type p53. Taken together, these results indicate that CD44 could be a potential therapeutic target of GBM and the expression level of CD44 in the GBM may be an indicator of poor outcome for patients with GBM.

#466

MR prediction of tumor burden in Patient-Derived Mouse Xenografts model of glioblastoma using an adaptive model.

Hassan Bagher-Ebadian,1 Ana deCarvalho,1 Azimeh NV Dehkordi,2 Tavarekere Nagaraja,1 Susan Irtenkauf,1 Swayamparva Panda,1 Meser M. Ali,1 Arbab S. Ali,1 Hamid Soltanian-Zadeh,1 Robert Knight,1 James R. Ewing1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Shahid Beheshti University, Tehran, Islamic Republic of Iran_.

Introduction: In human glioblastoma multiforme (GBM), infiltrating cells are found in remote locations, even in the hemisphere contralateral to the primary lesion. Magnetic Resonance Imaging (MRI) allows approximation of the extent of tumor cell infiltration. However, the actual extent of infiltration may be greater or less than the edema, and there is no standard MRI practice that allow exploring the infiltrating tumor burden. This pilot study investigates the feasibility of using a set of MR modalities for the development of an MRI estimate of infiltrating tumor burden in Patient-Derived Mouse Xenografts model of GBM using an adaptive model.

Material and Methods: 8 mice implanted with GBM CSC HF2927 were studied. MRI studies were performed in a Direct Drive Varian 7 Tesla. The following image sets were acquired: high-res. T1-weighted; T2-weighted (TE/TR = (20, 40, 60, 80)/3000 ms); MT-weighted fast spin-echo. Magnevist (0.25 mmol/kg i.p) was injected about 5 minutes before the post-contrast T1-weighted image set was acquired. The animal was sacrificed immediately after MRI and stained for the presence of human GBM cells. We used the following MRI sequences to establish a basis set for training the AM: pre- and post-contrast (Magnevist, I.P.) T1-weighted, 4-echo T2, MT, 3-direction, 3 b-value diffusion-weighted. Maps of T2 and proton density (T2-PD) were produced by fitting the T2 data. The histology image was warped and co-registered to the T2-PD image using mouse brain anatomical landmarks. MR image sets were normalized to the white matter area of the brain, and each voxel profile extracted from the 9 image set was normalized to the summation of two normalized T2 images (echo 2 and 3), following which the normalized profile along with the co-registered histology was used for training and testing of an artificial neural network (ANN) with Multi-Layer perceptron architecture to predict the presence of local tumor burden in form of tumorous cell density.

Results and Conclusions: The ANN was successfully trained and validated using K-Fold Cross Validation technique (KFCV) and the 9 MR modalities as its input set. Results imply that the chosen MRI feature set contains adequate information content for training the ANN. The predictive power of the ANN was ~ 0.81. The correlation coefficient for the association between the predicted map and histology was ~ 0.85. Given the success of training an ANN to predict infiltrating tumor burden, it may be possible to identify similar or different basis set of MRI images that can robustly predict both infiltrating and solid tumor burden in human GBM.

#467

Immunohistochemical multiplex staining strategies with CD8, CD103, PD-1, FOXP3 and pan melanoma cocktail in tumor infiltrating lymphocytes in melanoma.

David E. Tacha, Wei Yuan, Jillian Terrell. _Biocare Medical, Concord, CA_.

Introduction

Tumor-associated immune suppression can lead to defective T-cell mediated antitumor immunity. CD8 T-cells play a critical role in the host defense against cancers and infectious diseases. However, the presence of antigen-specific CD8 T-cells does not always imply that cancers and/or pathogens are efficiently eliminated in the body. In tumor infiltrating lymphocytes (TILS), other markers including CD103, PD-1 and FOXP3 are broadly expressed and have shown a wider range of immunoregulatory and important roles in T-cell activation, T- cell regulatory and programmed cell-death checkpoints. Existing methods can either deliver phenotypic information on homogenous samples such as flow cell cytometry or give immunohistochemical information on single biomarker. However, it would be of advantage to be able to visualize the distributions of multiple biomarkers in TILs that would discriminate stromal cells versus solid tumor. Therefore, a multiplex IHC stain utilizing a melanoma marker as a staining mask that separates the malignant tumor cells from stromal cells maybe a good strategy to facilitate better interpretation.

Materials and Methods

Monoclonal mouse antibodies to CD8, FOXP3 and PD-1 and rabbit monoclonal antibodies CD8, CD103 and PD-1 were optimally tittered for single, double and triple stains and visualized with HRP DAB/Black chromogens and/or alkaline phosphatase fast red/blue chromogens. A mouse monoclonal pan melanoma cocktail was visualized with a fast red chromogen and was used as a staining mask for melanoma tumors cells, but not stromal cells.

Results

Single, double and triple stains that included pan melanoma cocktail (mask) and CD8, CD103 FOXP3 and PD-1 were successfully established. All double and triple stains gave comparable results to single stains. The co-expression of TILs including CD8 and CD103 or CD8 or FOXP3 and PD-1 was easily identified with contrasting chromogens. TILs in stroma tissues and in malignant tumors cells were easily discriminated with the pan melanoma (mask) that stained only tumor cells with fast red chromogen, but did not stain TILS or stromal tissues.

Conclusion

The evaluation of TILS in malignant melanoma was enhanced with multiplex stains. TILs in stromal tissues and melanoma tumors cells were easily distinguished with the Pan Melanoma staining mask. These finding may help facilitate important prognostic information and thus may support the rationale for using immunotherapy strategies.

#468

BRAF mutation analysis in cell free tumoral DNA (cfDNA) of melanoma patients: results from the prospective study GEM1304 (Spanish Melanoma Group).

Maria Gonzalez Cao,1 Jose Luis Manzano,2 Virtudes Soriano,3 Teresa Puertolas,4 Ainara Soria,5 Clara Mayo,6 Margarita Magem,7 Miguel Angel Molina,6 Clara Montagut,8 Eva Muñoz,9 Delvys Rodriguez,10 Elizabeth Perez,11 Almudena Garcia,12 Javier Cortes,9 Nuria Jordana,6 Jordi Rodon,9 Niki Karachaliou,1 Rafael Rosell1. 1 _IOR, Dexeus, Barcelona, Spain;_ 2 _Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Barcelona, Spain;_ 3 _Fundación IVO, Valencia, Spain;_ 4 _Hospital Miguel Servet, Zaragoza, Spain;_ 5 _Hospital Ramon y Cajal, Madrid, Spain;_ 6 _Breakthrough Cancer Research Unit, Pangaea Biotech, Dexeus University Institute, Barcelona, Spain;_ 7 _Hospital Sant Pau, Barcelona, Spain;_ 8 _Hospital del Mar, Barcelona, Spain;_ 9 _Hospital Vall d'Hebron, Barcelona, Spain;_ 10 _Hospital Insular de Gran Canaria, Gran Canaria, Spain;_ 11 _Hospital Costa del Sol, Malaga, Spain;_ 12 _Hospital Marques de Valdecilla, Santander, Spain_.

Backgroud: Tumor-derived circulating cell-free DNA (cfDNA) is a dynamic source for determination of tumor mutation status. We have previously demonstrated the prognostic value of BRAFV600 mutation status in pretreatment cfDNA (BRAF pre-cfDNA) in advanced melanoma patients (p) treated with BRAF inhibitors (median overall survival [OS] 7 months [m] vs 22m for BRAF pre-cfDNA positive and negative p, respectively p=0.017)1. Based on these results, the Spanish Melanoma Group conducted a prospective study in 13 centers to determine the prognostic value of BRAFV600 mutation in pre-cfDNA, the change in mutation status at time of first evaluation (BRAF early-cfDNA), and the correlation of BRAF cfDNA dynamics with clinical evolution (GEM1304) (ClinicalTrials.gov Identifier: NCT01960634).

Methods: One hundred and fifty nine plasma and serum samples from 66 stage IV BRAF mutant melanoma p were collected before and during treatment, until disease progression.

A quantitative 5'-nuclease PCR based assay was used to determine BRAFV600 mutation status in cfDNA.

Results: Most p were stage M1c (62%), treated with BRAF inhibitors (53%), and not previously treated (67%). BRAF pre-cfDNA was positive in 42 p (64%). Median OS was 6.4 m (95% CI: 10.9-23.6) and 17 m (95% CI: 3.5-9.2) for p with positive and negative BRAF pre-cfDNA, respectively (p=0.06). Significant differences in OS were observed according to BRAF early-cfDNA negativization: 4.7 m (95%CI: 1.2-8.1) in those with persistence of BRAF in cfDNA (12 p), not reached (NR) in p with BRAF early-cfDNA negativization (11 p), and 22 m (95%CI:0.6-43.9) in those who continued to be negative (17 p) (p<0.001). Median progression free survival (PFS) was 3.4 m (95% CI: 2.1-4.6), 16.8 m (95% CI: 6.9-26.8) and 15.3 (95%CI: 1.1-29.6), respectively (p<0.001). There were also significant differences according to BRAF early-cfDNA among p treated with BRAF inhibitors: in p with persistence of BRAF in cfDNA (8p), median OS was 4.7 m (95% CI:16-7.8), NR for those with BRAF early-cfDNA negativization (9p), and 22 m (95%CI: 7.9-36.6) for those who continued to be negative (8 p) (p<0.001). Median PFS was 3.6 m (95% CI: 2.2-5.1), 16.9 m (95% CI: 6.9-26.9) and 18 m (95% CI: 0.6-35.3), respectively (p=0.001). In those p with serial samples taken during treatment, BRAF mutation disappeared from cfDNA in all cases who responded (18). Those with persistence of mutation during follow up had rapid progression and death (10). BRAF mutation had relapsed in cfDNA at time of progression in 6/15 cases.

Conclusions: Patients with early negativization of BRAFV600 in cfDNA have excellent prognosis, at least as good as those with negative BRAF in pre-cfDNA.

González-Cao et al. Mel Res 2015; 25:486

#469

Systematic analysis of circulating tumor DNA in melanoma patients to uncover mechanisms of resistance and disease clonality.

Gabriela Gremel,1 Rebecca J. Lee,1 Maria R. Girotti,1 Grace Garner,1 Amit K. Mandal,1 Sally Wood,1 Alberto Fusi,1 Sara Valpione,2 Patricio Serra-Bellver,2 Nathalie Dhomen,1 Paul Lorigan,2 Richard Marais1. 1 _Cancer Research UK Manchester Institute, Manchester, United Kingdom;_ 2 _The Christie NHS Foundation Trust, Manchester, United Kingdom_.

Recent advances in targeted and immunotherapies have unlocked potent treatment options for malignant melanoma patients. However, targeted therapies are associated with limited response durations and only a fraction of patients benefit from immunotherapies. The analysis of circulating tumour DNA (ctDNA) provides a powerful tool for the continuous assessment of treatment responses in melanoma patients.

We have established a robust pipeline for the serial, prospective analysis of ctDNA in melanoma patients. By combining next generation sequencing (NGS)- and droplet digital PCR (ddPCR)-based approaches we make ideal use of currently available technologies. To date, plasma samples from over 120 individuals have been collected, with follow-up times surpassing one year in many cases.

For routine monitoring of patients commencing BRAF-mutant targeted therapies, pre-treatment DNA, derived from tumour biopsy or plasma, is tested to confirm the exact BRAF mutation and rule out pre-existing resistance-associated mutations using a custom NGS sequencing panel. Timely follow-up throughout treatment is achieved by measuring mutant BRAF fractions in ctDNA using ddPCR. When increasing BRAF variant allele frequencies are detected, additional ddPCR-based profiling for NRAS mutations and NGS-based re-sequencing for other resistance-associated mutations is initiated. We demonstrate superiority of ctDNA monitoring over LDH measurements and present a strategy to detect emerging resistant disease ahead of clinical scans in a cost-effective, timesaving and low technology way.

In addition to the basic characterization of BRAF-mutant cases undergoing targeted therapy, we have used the analysis of ctDNA to uncover clonally distinct tumour sub-populations resulting in complex responses to treatment. For instance, we monitored a case of metastatic vaginal mucosal melanoma undergoing sequential targeted, immuno- and chemotherapy. Despite the presence of a KIT mutation in the primary tumour, response to KIT inhibitor imatinib was mixed. In the absence of tumour biopsies, we used whole exome-wide NGS of ctDNA to study the underlying mechanisms. This strategy revealed a KIT-mutant tumour sub-clone that responded to imatinib and a second sub-clone including an SF3B1 mutation that did not respond. The sub-clones also responded differentially to immunotherapies, but both responded to chemotherapy.

The analysis of ctDNA is a powerful approach to monitor responses to systemic therapies in melanoma. By providing early warning of resistance and crucial information of clonal composition/evolution of the disease, ctDNA analysis has the potential to revolutionize the implementation of precision medicine.

#470

Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma.

Maria R. Girotti,1 Gabriela Gremel,1 Rebecca Lee,1 Elena Galvani,1 Dominic Rothwell,1 Amaya Viros,1 Amit Kumar Mandal,1 Kok Haw Jonathan Lim,1 Grazia Saturno,1 Simon J Furney,1 Franziska Baenke,1 Malin Pedersen,2 Jane Rogan,1 Jacqueline Swan,1 Matthew Smith,1 Alberto Fusi,3 Deemesh Oudit,3 Nathalie Dhomen,1 Ged Brady,1 Caroline Dive,1 Richard Marais1. 1 _Cancer Research UK Manchester Institute, Manchester, United Kingdom;_ 2 _The Institute of Cancer Research, London, United Kingdom;_ 3 _The University of Manchester, The Christie NHS Foundation Trust,, Manchester, United Kingdom_.

BRAF/MEK inhibitors and immunotherapies have revolutionized care for patients with advanced melanoma, improving expected median survival from 9 months to 25-30 months, but the majority of patients still die of their disease. Personalized medicine strives to individualize and improve patient care. To individualize treatment decisions in advanced melanoma we analyzed 364 samples from 214 patients. We performed whole exome sequencing (WES) and circulating tumor DNA (ctDNA) analysis, and we developed patient derived xenografts (PDX). WES and targeted sequencing of ctDNA allowed us to predict responses to therapy and to identify and monitor mechanisms of resistance. WES of tumors revealed new therapeutic strategies in BRAF V600 wild-type and BRAF inhibitor-resistant melanoma and we validated these in patient derived xenografts (PDX). Thus, we describe a powerful combination of techniques for personalized medicine to improve the management of melanoma patients.

#471

Detection of circulating tumor cells in high-risk primary uveal melanoma by the photoacoustic method.

Ryan M. Weight,1 Shingo Sato,1 Masahiro Ohara,1 Mizue Terai,1 Michael Mastrangelo,1 Marlana Orloff,1 Benjamin Goldschmidt,2 John Viator,2 Takami Sato1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Duquesne University, Pittsburgh, PA_.

Circulating tumor cells (CTCs) have been shown to be a prognostic marker in breast cancer1. We hypothesize that circulating melanoma cell (CMC) detection could be utilized in the management of uveal melanoma, including early intervention. Prior methodologies for circulating uveal melanoma cell (CUMC) detection have been fraught with poor sensitivity, limiting their clinical utility2. Development of an improved method is necessary to establish the clinical utility of CUMC monitoring. Photoacoustics, also referred to as laser-induced ultrasound, is a novel platform for the detection and capture of CMCs. Photoacoustics uses short duration pulsed light to create ultrasonic acoustic waves in an optically absorbing medium, in this case melanin within melanoma3. As light is absorbed by irradiated chromophores, the optical energy gets converted into kinetic thermal energy trapped within the chromophore and subsequent thermal expansion ensues. Transient thermoelastic expansion of the absorbent cell results in the propagation of ultrasonic acoustic waves which can be detected and analyzed using a piezoelectric response mechanism. In addition, detected CMCs can be isolated by a two-phase flow cell separation technique4. Due to the low cost and melanoma specific capabilities of photoacoustics, we evaluated this technology for the purpose of CUMC detection. Methods: Cells from uveal melanoma cell line UM002B, established at Thomas Jefferson, were titrated to various cell concentrations and analyzed in a neutral density solution utilizing the photoacoustic method. Uveal melanoma cells of differing concentrations were spiked into isolated healthy donor peripheral blood mononuclear cells (PBMCs) and healthy whole blood samples. PBMC isolates were analyzed for CUMCs. Results: CUMCs were successfully quantified by the photoacoustic method including single cell detection. Recovery rates of cultured cells in a neutral density solution approached 25%. Recovery rates for CUMCs in whole blood averaged 10% of expected cell yield (56/540 cells detected) with a higher detection rate at lower cell concentrations. Photoacoustics offers a viable method for the detection of CUMCs with an accuracy that meets or exceeds previously reported CUMC yields. Studies analyzing CUMCs from patients with metastatic disease are ongoing.

1. Cristofanilli, M, et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med , 2004. 351(8): 781-91.

2. Bidard FC, et al., Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma. Int J Cancer. 2014 Mar 1;134(5):1207-13.

3. Weight RM, et al. Photoacoustic detection of metastatic melanoma cells in the human circulatory system. Opt Lett . 2006 Oct 15;31(20):2998-3000.

4. O'Brien CM, et al. Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow. J Biomed Opt . 2012 Jun;17(6):061221.

#472

A digital sorting-based detection and isolation assay for single disseminated cancer cells from lymph nodes of melanoma patients.

Sebastian Scheitler,1 Kathrin Weidele,1 Barbara Alberter,1 Christian Werno,1 Christoph A. Klein,2 Bernhard Polzer1. 1 _Project Group Personalized Tumor Therapy, Fraunhofer ITEM, Regensburg, Germany;_ 2 _Experimental Medicine and Therapy Research, University of Regensburg and Project Group Personalized Tumor Therapy, Fraunhofer ITEM, Regensburg, Germany_.

We recently showed that sentinel lymph node (SLN) disaggregation followed by immunocytology enables precise quantification of disseminated cancer cells (DCC). We demonstrated that this approach has a 20-fold higher sensitivity to detect melanoma DCCs than routine histopathology and that the DCC density (DCCD, defined as the number of DCCs per million lymph node cells) together with ulceration state and tumor thickness enables individual risk prediction superior to current AJCC guidelines (Ulmer et al., PLoS Medicine, 2014). Here, we present the adaptation of this method to a semi-automated workflow, including automated single cell detection and isolation by DEPArrayTM technology for subsequent molecular analysis of DCCs from SLN of melanoma patients.

The developed workflow includes a mechanical disaggregation of lymph node tissue and collection of the mononuclear cells after Percoll density centrifugation. Several tumor cell enrichment methods were tested (CellSearch® and size-based enrichment technologies), however, failed to process cell suspensions generated from lymph nodes due to clumping of cells during the enrichment procedure. Tumor cell enrichment using MACS depletion of CD45+ cells delivered approx. 50% of spiked-in cell line cells with a mean depletion rate of lymphocytes and erythrocytes > 98 %. To prevent limitations in DCC detection caused by phenotypic heterogeneity of tumor cells, we established a double staining against two melanoma-associated markers gp100 and MCSP. Individual melanoma cells are then detected and isolated by DEPArrayTM technology enabling single cell whole genome amplification (Ampli1TM) for subsequent molecular analysis as assessment of copy number alterations (e.g. by arrayCGH) or sequence analysis for melanoma specific point mutations (e.g. BRAF or NRAS). We successfully applied the workflow to first SLN samples from melanoma patients.

The established workflow enables a standardized detection of single DCCs from lymph nodes of melanoma patients applicable in clinical diagnostic labs. Automated single cell isolation and subsequent molecular analysis of DCCs is feasible within short time after SLN biopsy. In the future, this approach could help to select individualized therapies for melanoma patients.

#473

A tumor and immune related miRNA signature predicts progression-free survival of melanoma patients treated with ipilimumab.

Ahmad Tarhini, Priyanka Vallabhaneni, Theofanis Floros, William A. LaFramboise, Panayiotis V. Benos, Lucas Santana dos Santos. _University of Pittsburgh Cancer Institute, Pittsburgh, PA_.

Background

Patients with regionally advanced melanoma were treated with neoadjuvant ipilimumab in a previously reported study (Tarhini et al, PLOS One 2014). MicroRNA (miRNA) expression profiles of tumors of treated patients were investigated for their therapeutic predictive value.

Methods

Patients were treated with ipilimumab (10 mg/kg IV every 3 weeks x2 doses) bracketing surgery. Tumor specimens were obtained at baseline and following ipilimumab at definitive surgery (week 6-8). MiRNA expression profiling was performed on the tumor biopsies of 30 patients using Affymetrix miRNA array (v.4). Significance Analysis of Microarrays (SAM) was performed to test the association of each differentially expressed miRNA molecule with outcome. Targets of the selected miRNAs were obtained from miRTarBase (http://mirtarbase.mbc.nctu.edu.tw). Functional annotation analysis of the list of miRNA target genes were performed using DAVID (https://david.ncifcrf.gov). The FDR method was used to adjust for multiple testing in SAM and the functional analysis.

Results

An expression profile consisting of a 4-miRNA signature was associated with improved progression free survival. The signature consisted of miR-34c (previously reported to suppress cancer growth and invasion), miR-711 (reported as a prognostic marker in cutaneous T-cell lymphomas and to target and suppress Heat Shock Protein 70 highly expressed in melanoma), miR-641 (activates MAPK by targeting NF1 and cooperates with its host gene AKT2 in human cancer) and miR-22 (reported to function as a tumour suppressor). Functional annotation analysis of target genes for the 4-miRNA signature was statistically significantly enriched for various cancer-related pathways including regulation of cell proliferation (GO:0042147), regulation of apoptosis (GO:0042981), MAPK signaling pathway (hsa04010) and positive regulation of T cell activation (GO:0050870).

Conclusions

MiRNA expression profiling identified a 4-miRNA signature that is significantly associated with PFS in advanced melanoma patients treated with neoadjuvant ipilimumab. Preliminary results show that targets of these 4 miRNAs are biologically relevant and important. These findings warrant further investigation in relation to ipilimumab and other immunotherapeutics.

#475

NTF2 regulates nuclear size in mammalian cells and may contribute to altered nuclear size in melanoma.

Lidija D. Vukovic,1 Bradley A. Stohr,2 Dan L. Levy1. 1 _University of Wyoming, Laramie, WY;_ 2 _University of California, San Francisco, CA_.

Nuclear size and morphology are distinct cancer features that pathologists use for diagnostic purposes. Proteins that regulate nuclear size have been identified in Xenopus, where nuclear import rates correlate with nuclear size. The levels of two nuclear import factors, importin α and NTF2, were shown to be particularly important in nuclear size regulation. It has also been shown that importin α2 is a potential biomarker in non-small cell lung cancer, as well as importin α1 in breast cancer. Our data implicate that NTF2 is a possible cancer biomarker.

Our main goal is to analyze how these nuclear scaling factors affect nuclear size during carcinogenesis and to test how nuclear size impacts cancer growth characteristics. We find that manipulating the levels of importin α2 and NTF2 in HeLa and MRC-5 cells leads to altered nuclear size, demonstrating a conserved function for these factors in nuclear size regulation. To place these results in a cancer context, we are focusing on melanoma cell lines, including radial and vertical growth primary melanomas and metastatic melanomas. We find that, compared to normal melanocytes, the nuclear-to-cytoplasmic (N/C) volume ratio is increased in the cancer cell lines. Western blot analysis revealed an inverse correlation between NTF2 protein levels and nuclear size. We confirmed these results using a melanoma tissue microarray, finding similar differences in nuclear size and NTF2 expression levels between benign nevi and melanomas.

We next performed transient transfection experiments with our melanoma cell lines. Importin α2 knock down led to smaller nuclear size, while knocking down the levels of NTF2 increased nuclear size, consistent with our Xenopus results. We also observed reduced nuclear size when NTF2 was ectopically expressed. Currently we are generating stable, inducible melanoma cell lines where we can precisely alter NTF2 and importin α expression levels using doxycycline. We will use these inducible cell lines to examine how altering nuclear size impacts cell proliferation rates, invasiveness, and apoptosis. We will also investigate how nuclear size affects gene positioning using FISH, focusing on genes involved in melanoma formation and metastasis. In the long term, we plan to move these studies into a mouse model where we can test how nuclear size affects in vivo metastatic capacity.

#476

Differential prognostic value of MYC immunohistochemistry in papillary RCC subtypes.

Julia Bellut,1 Simone Bertz,2 Elke Nolte,1 Christine Stoehr,2 Iris Polifka,2 Edwin Herrmann,3 Rudolf Jung,2 Arndt Hartmann,2 Bernd Wullich,1 Helge Taubert,1 Sven Wach1. 1 _Dept. of Urology, Univ. Hospital Elangen, Erlangen, Germany;_ 2 _Institute of Pathology, Univ. Hospital Elangen, Erlangen, Germany;_ 3 _Dept. of Urology, Univ. Hospital Muenster, Muenster, Germany_.

Introduction

The histomorphological classification of type 1 and type 2 papillary renal cell carcinomas (pRCCs) has substantially improved the prognostication of patients' long-term outcome. We previously established microRNA (miRNA) expression profiles of type1 and type 2 pRCCs and, based upon the results, hypothesize a differential expression of the MYC proto-oncogene in pRCCs.

Material and methods

An multi-institutional tissue microarray with more than 200 histomorphological classified pRCCs was acquired from the PANZAR consortium. Immunohistochemistry for MYC, the MYC-induced nuclear antigen MINA53 and Ki67 was performed. The prognostic value for patients' long-term survival was examined using uni- and multivariate survival models.

Results

As expected, a pRCC type 2 morphology was associated with reduced overall and cancer-specific survival. Interestingly, pRCCs with a mixed type 1/2 morphology showed an even worse clinical outcome. When combining the pRCC morphology and immunohistochemical staining, only the combination of morphology and MYC staining patterns was able to further sub-stratify the group of pRCC type 1 tumors in a good and worse prognosis group. None of the patients with pRCC1 tumors and the favorable MYC staining pattern (>44% of all pRCC type 1 tumors) suffered a tumor-related death. Patients with pRCC type 1 tumors and the adverse MYC staining pattern had an 4.5-fold elevated risk of death (multivariate corrected for pathological tumor stage).

Conclusions

Although a pRCC type 1 histomorphology is regarded as a favorable prognostic factor, associated with good clinical outcome, still patients die of tumor-related causes. We conclude, that in particular for patients with pRCC type 1 tumors, MYC immunohistochemistry is able to provide additional prognostic information for a sub-stratification of this patient population. This knowledge could be used for the establishment of risk-stratified follow-up schemes for patients after surgical treatment.

#477

Correlation of PD-L1 expression and HPV status among primary and metastasized HNSCC tumors.

Chenglu C. Quon, Xiaoling Xia, Lupe Manriquez, Crystal Schemp, Dawn Sloane, Shahad Alabagi, Mohammed G. Abdelwahab, Pengfei Gu, Lizhen Pang, Khalid Hanif, Nicole Schechter. _Ventana Medical Systems, Inc., a member of the Roche Group, Tucson, AZ_.

Dysregulation of the immune checkpoints has been identified as one of the mechanisms tumor cells employ for immune escape. Human papillomavirus (HPV) plays an important role in the etiology of one subset of Head and Neck Squamous Cell Carcinomas (HNSCC). Recent studies reported that PD-1/PD-L1 pathway may be correlated with HPV-associated HNSCC. The degree of PD-L1 expression has been reported to be increased in those patients with HPV-positive disease. Also, PD-L1 expression in HNSCC was observed in the primary, recurrent, and metastatic disease setting. However, the clinicopathological implications associated with PD-L1 in HNSCC, as well as in disease metastasis, remain largely unclear.

Using immunohistochemistry (IHC) and In situ hybridization (ISH) on VENTANA BenchMark ULTRA automated stainers, 40 cases of HNSCC, with matching primary and metastatic cancer stages, were evaluated for expression of PD-L1, p16, a surrogate marker of transforming HPV infections, and presence of HPV. Formalin-fixed paraffin-embedded tumor samples were subjected to PD-L1- and p16-IHC as well as HPV-ISH. A potential association between PD-L1 expression and HPV status among primary tumors and matched metastases was analyzed.

Our data show that expression of PD-L1 or p16 is concordant in the majority of HNSCC primary vs metastatic tumor cases tested. Agreement rate of p16 expression among 40 case pairs of primary vs metastatic tumor was 88.2% with a 95% Confidence Interval (CI) of (73.4-95.3). PD-L1 expression was 76.9% (61.7-87.4) concordant among the 40 case pairs. Agreement rate between PD-L1 and p16 was 77.1% (61.0-87.9) in primary cases and 51.3% (35.9-66.6) in metastatic cases. No obvious association between PD-L1 and p16 expression was observed.

#478

A mouse model for highly metastatic, nonfunctioning pancreatic neuroendocrine tumors.

Tanupriya Contractor,1 Shinta Kobayashi,2 Richard Clausen,1 Edaise da Silva,3 Laura Tang,3 Chang Chan,4 Chris R. Harris1. 1 _Raymond and Beverly Sackler Foundation, New Brunswick, NJ;_ 2 _Chugai Pharmaceutical Co., LTD, Japan;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Pancreatic neuroendocrine tumors are generally classified as functioning or nonfunctioning. Patients with functioning tumors can have severe hypoglycemia because their tumors express high amounts of insulin as well as chromogranin A; fortunately, these tumors are seldom metastatic. Patients with nonfunctioning tumors express high amounts of chromogranin A but little insulin; unfortunately these tumors are highly metastatic to the liver. Nonfunctioning tumors are more common, and a more severe health issue. The highly studied RT2 B6 mouse, in which the SV40 T antigen is expressed from an insulin promoter in an inbred C57Bl/6 genetic background, is a model for functioning pancreatic neuroendocrine tumors: liver metastasis is uncommon in RT2 B6 mice, which are very hypoglycemic and often die when tumors are very small. We have changed the genetic background and are studying RT2 AB6F1 hybrid mice, which have a C57Bl/6 father and a mother from an A/J inbred background. RT2 AB6F1 mice are much less hypoglycemic, and these mice develop liver metastasis with high frequency. We have mated the hybrid AB6F1 mice to their littermates in order to map metastasis genes by F2 analysis, and have discovered that a locus at mouse chromosome 2qG1 links tightly to liver metastasis. One gene within this locus, InsM1, is less expressed in the primary tumors of mice with liver metastatic disease. InsM1 is also less expressed in primary tumors from humans with liver metastatic disease. Previous studies of InsM1 have shown that this protein maintains the differentiated status of pancreatic beta cells. We find that tumors from mice with low expression of InsM1 also express lower amounts of several distinctive markers of differentiated beta cells, but higher amounts of several markers of liver cells. These data suggest that dedifferentiation to stem cells, which can then redifferentiate to other cell types such as liver cells, promotes metastasis of pancreatic beta cell tumors. Intratumoral expression of liver markers may be a useful predictor of metastatic disease in these patients. Dedifferentiation of mature beta cells is also a mechanism by which type 2 diabetes occurs, so there may be similarities between these two diseases.

#479

Detection and clinical significance of circulating tumor cells in squamous cell carcinoma of the head and neck.

Kazuaki Chikamatsu, Hideyuki Takahashi, Koichi Sakakura, Minoru Toyoda. _Gunma Univ Graduate School of Med, Maebashi, Japan_.

Background: With a developing tumor, tumor cells shed a variety of substances into the blood stream, such as tumor cells, tumor DNA, or exosomes, and these are considered to be as attractive circulating biomarkers to monitor disease progression in cancer patients. However, impact of circulating tumor cells (CTCs) in squamous cell carcinoma of the head and neck (SCCHN) is still not fully understood. In this study, we show that CTCs are present in the peripheral blood of patients with SCCHN, and then investigate the correlation with clinicopathological factors. Materials and Methods: 7.5mL of peripheral blood was obtained from 32 patients with pathologically and clinically confirmed SCCHN. Samples were run with a CellSieve™ Microfiltration Assay (Creatv MicroTech, Inc., Rockville, MD) using a low-pressure vacuum system. The CTCs captured by the filter were stained with an antibody mixture of FITC-conjugated anti-CK 8, 18, and 19, PE-conjugated anti-EpCAM, and Cyanine 5-conjugated anti-CD45. Cells that stained positive for DAPI and CK but negative for CD45, were identified as CTCs. Results: The CTCs were found in 29 (90.6%) of 32 patients. Although there was no association between the number of CTCs and tumor sites, the number of CTCs in patients with advanced disease (stage III/IV) was significantly increased, as compared with those with early disease (stage I/II) (p<0.05). Furthermore, patients with T3/4 had higher number of CTCs compared to patients with T1/2 (p<0.01). Meanwhile, nodal status was not significantly associated with the number of CTCs. In 12 of 32 patients tested, the number of CTCs after treatments was also evaluated. As expected, the number of CTCs was significantly decreased after treatments, regardless of the treatment modalities. Conclusion: Our results suggest that microfiltration is very efficient in capturing CTCs from SCCHN patients. Moreover, CTCs analysis in SCCHN may provide useful information for assessment of treatment efficacy.

#480

Analysis of the expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in a cohort of 178 primary locally advanced oral squamous cell carcinoma cases: the prognostic implication.

Monica C. Solomon,1 Mammadipuli Vidyasagar,2 Donald Fernandes,2 Vasudeva Guddattu3. 1 _Manipal College of Dental Sciences, Manipal, Manipal University, Manipal, India;_ 2 _Kasturba Medical Hospital, Manipal, Manipal University, Manipal, India;_ 3 _Department of Biostatistic, Manipal University, Manipal, India_.

Background: Oral Squamous Cell Carcinomas comprise of a heterogeneous tumor cell population with of varied molecular characteristics; which makes prognostication of these tumors a complex and challenging issue. Thus, molecular profiling of these tumors is advantageous for an accurate prognostication and treatment planning.

Study design: A retrospective study on a cohort of primary locally advanced oral squamous cell carcinomas (n=178) of an Indian rural population

Aim:

• To evaluate the expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in a cohort of primary locally advanced oral squamous cell carcinomas.

• To assess the reliability of this panel of biomarkers for prognostication and treatment planning of locally advanced oral squamous cell carcinomas

• To identify a potential biomarker that can predict the tumor response to treatment.

Patient Selection: All patients who were diagnosed with locally advanced oral squamous cell carcinomas cases between the year 2009 and 2013 were reviewed. These patients were treated with radiotherapy and adjuvant chemotherapy. The maximum duration of follow-up was 5 years and the minimum follow-up time is 1 ½ years.

Material and Methods: Formalin fixed paraffin embedded tumor blocks of (n=178) of histopathologically diagnosed cases locally advanced oral squamous cell carcinomas were selected. The areas where the tumor cells appeared most aggressive were identified in the Hematoxylin and Eosin stained tissue sections of these cases. The corresponding areas were identified in the tumor blocks and two cores of 2 mm diameter for each case were obtained and tissue microarray blocks were constructed. Four microns thick sections were cut from these tissue microarray blocks. These tissue microarray sections were immunohistochemically stained for EGFR, p53 and Bcl-2 and cyclin D1 and p16. The expression of these markers by the tumor cells was evaluated in a semi quantitatively. The dat

Results:

In this cohort, EGFR was the most expressed in 150/178 (84%) of the cases. The expression of EGFR was significantly associated with p53 (p=.012), and with cyclin D1 (p=.011). Kaplan Meier analysis showed a significant association (p=.038) between expression of p53 and a poor prognosis. A Poisson regression analysis showed that tumors that expressed p53 had a two times greater chance of recurrence (Unadjusted IRR -95% CI 2.08 (1.03, 4.5), Adjusted IRR- 2.29 (1.08, 4.8) compared with the tumors that did not express this biomarker.

Conclusion:

Molecular profiling of oral squamous cell carcinomas will enable us to categorize our patients into more realistic risk groups. With biologically guided tumor characterization, personalized treatment protocols can be designed for individual patients, which will improve the quality of life of these patients.

#481

Low-molecular-mass secretome profiling identifies prognostic biomarkers for oral cavity squamous cell carcinomas.

Kai-Ping Chang,1 Jau-Song Yu2. 1 _Chang Gung Memorial Hospital, Taipei, Taiwan;_ 2 _Chang Gung University, Taipei, Taiwan_.

The profiling of cancer cell secretomes is considered to be a good strategy for identifying cancer- related biomarkers, but few studies have focused on identifying low-molecular-mass (LMr) proteins (<15 kDa) in cancer cell secretomes. Here, we used tricine-SDS-gel-assisted fractionation and LC-MS/MS to systemically identify LMr proteins in the secretomes of five oral cavity squamous cell carcinoma (OSCC) cell lines. Cross-matching of these results with nine OSCC tissue transcriptome datasets allowed us to identify 33 LMr genes/ proteins that were highly upregulated in OSCC tissues and secreted/released from OSCC cells. Immunohistochemistry and quantitative real-time PCR were used to verify the overexpression of two candidates, HMGA2 and MIF, in OSCC tissues. The overexpressions of both proteins were associated with cervical metastasis, perineural invasion, deeper tumor invasion, higher overall stage, and a poorer prognosis for post-treatment survival. Functional assays further revealed that both proteins promoted the migration and invasion of OSCC cell lines in vitro. Collectively, our data indicate that the tricine-SDS-gel/LC-MS/MS approach can be used to efficiently identify LMr proteins from OSCC cell secretomes, and suggest that HMGA2 and MIF could be potential tissue biomarkers for OSCC.

#482

Atypical expression of βV-tubulin in secretory cells of the fallopian tube as a biomarker for early dysplasia.

Deepti Mathew,1 Yanhua Wang,2 Anne Van Arsdale,2 Susan Band Horwitz,1 Hayley McDaid1. 1 _Albert Einstein College of Medicine, NY;_ 2 _Montefiore Medical Center, NY_.

Class V βeta tubulin isotype (βV-tubulin), was recently found to have a tissue-specific expression pattern in epithelial tissues with secretory function, and deregulated expression in certain tumors. The overall aim of this study was to define the localization of βV-tubulin in the fallopian tube and its expression in pre-malignant dysplasia. Fallopian tube epithelium (FTE) was obtained from patients undergoing salpingectomy for various reasons including familial history that comprised BRCA mutant cases. Additionally, βV-tubulin expression was also examined in serous ovarian neoplasms. Immunohistochemistry, using a novel antibody developed in our lab against human βV-tubulin, was used to evaluate expression in paraffin-embedded sections of the distal fallopian tube (n=55) and tumors (n=13), from prospectively selected cases, categorized according to reasons for salpingectomy. Staining with standard markers, Pax-8 and acetylated α-tubulin was done to demarcate the secretory and ciliated cells of the FTE, respectively. Pilot data indicate that in the FTE, βV-tubulin is present in secretory cells and essentially never in ciliated cells. Morphologically "normal" FTE had only rare, scattered βV-tubulin positive cells; whereas, percentage positivity was increased in cases with serous ovarian neoplasms, and BRCA-wildtype familial breast cancer. The highest expression was observed in FTE from patients with BRCA-mutant breast cancers. Four distinct types of FTE atypia were observed in patients with known BRCA mutations. βV-tubulin was highly expressed in serous ovarian neoplasms, with the extent and intensity of staining elevated in high-grade serous carcinomas, compared to serous borderline tumors. In summary, βV-tubulin was localized to secretory cells of the distal FTE and its expression varied according to the clinical diagnosis. The frequency of these cells, and thus expression of βV-tubulin was dramatically enriched in tissue obtained from BRCA mutant cases. BRCA mutants also exhibited pronounced histologic atypia indicative of early dysplasia. Furthermore, elevated expression of βV-tubulin correlated with poor differentiation status in serous ovarian neoplasms.

#483

Detection of circulating tumor DNA in oral rinses and plasma from head and neck cancer cases in Argentina.

Sandra Perdomo,1 Patrice Avogbe,1 Marta Vilensky,2 James Mckay,1 Paul Brennan1. 1 _IARC, Lyon, France;_ 2 _Instituto de Oncologia Angel H Roffo, Buenos Aires, Argentina_.

In 2012, almost 700,000 new head and neck cancer (HNSCC) cases were estimated to occur worldwide with two thirds of those cases occurring in developing countries. Within the geographic areas in Latin America, characterized by high incidence rates of head and neck cancer, the region comprising Argentina, southern Brazil and Uruguay has the highest levels of incidence. Despite current therapy approaches, the survival rate is around 50%, and the main reason for treatment failure is the frequent development of regional recurrences, affecting around 30% of patients. In this context the use of ctDNA (specific biomarker for tumors) using techniques of mass sequencing (high sensitivity and specificity) is presented as a promising tool for noninvasive diagnosis and monitoring of HNSCC cases.

Our primary goal was to provide a comprehensive evaluation of the presence of ctDNA in both plasma and oral rinses from a series of HNSCC cases without previous knowledge of tumor mutational status. 43 cases diagnosed as head and neck squamous cell carcinoma were selected from a multi center case-control study conducted in Argentina between 1998 and 2002. Subsite and stage distribution was as follows: 14 oral cavity, 6 oropharynx, 1 hypopharynx, 15 larynx and 7 overlapping lesions. 6 cases were stage III and 17 stage IV. Additionally, 50 controls matched by center, age, sex, smoking and alcohol status were included to assess a threshold of the background noise after sequencing.

Targeted sequencing was performed independently in duplicate on plasma, oral rinses, germline DNA and the tumor material using the Ion Proton™ System. Sequencing included the entire coding region of TP53 as the most frequently mutated gene in HNSCC. Both Plasma and oral rinses samples were sequenced at an average depth of 10000X. For somatic variant calling we used a recently developed new statistical model and a bioinformatics pipeline based on Poisson distribution for estimation of sequencing errors distribution to accurately identify variants above the sequencing background. Statistical tests were carried out in R statistical programming environment.

74% of tumor samples sequenced harboured at least one TP53 somatic mutation. 10 TP53 variants were identified in the corresponding plasma samples and 1 of these variants matched the tumor mutation, possibly reflecting high tumor heterogeneity. In contrast, 90% of the variants detected in the tumor were also found in in the oral rinses with allelic frequencies ranging from 0.002 to 0.05 showing higher specificity of oral rinses as a surrogate sample for mutation detection.

The detection and analysis of ctDNA in noninvasive samples might be included as a potential biomarker to improve early diagnosis and patient monitoring in HNSCC cases. 

### Circulating Biomarkers 1

#484

Pre-treatment platelet counts as a prognostic factor in stage II and III rectal adenocarcinoma.

Ioannis Voutsadakis, Morgan Steele. _Sault Area Hospital, Sault Ste Marie, Ontario, Canada_.

Background. Thrombocytosis is often associated with malignancies and has been confirmed as an adverse prognostic factor in various cancers. We investigated if pre-treatment elevated platelet counts could provide prognostic information in patients with stage II and III rectal adenocarcinoma that received neo-adjuvant treatment before surgery.

Patients and Methods. Platelet number on diagnosis of stage II and III rectal cancer was evaluated in 51 patients receiving neo-adjuvant treatment and for whom there were complete follow-up data on progression and survival, as well as pathologic outcome at time of surgery. Pathologic response after neo-adjuvant treatment of patients with lower platelet counts (150-300× 109/L) was compared with that of patients with higher platelet counts (>300× 109/L) by the χ2 test. Overall and progression free survival Kaplan-Meier curves of the two groups were constructed and compared with the LogRank test.

Results. A significant difference was present between the two groups in regards to pathologic response with patients with lower platelet counts being more likely to exhibit a good or complete response to neo-adjuvant treatment than patients with higher platelet counts (P=0.015). There was also a significant difference between the CEA at presentation of patients that exhibited a good or complete response and those that had no response or a minimal to moderate response. Patients with a good or complete response were more likely to present with a CEA of less than 5 (P=0.00066). There was no significant difference in overall and progression free survival between the two groups (LogRank tests p=0.42 and p=0.35, respectively).

Conclusion. In this retrospective analysis of stage II and III rectal cancer patients, platelet counts at the time of diagnosis had prognostic value for neo-adjuvant treatment pathologic effect. Pre-treatment CEA also held prognostic value in regards to treatment effect.

#485

Blood-based profiling of patients with NSCLC using CLIA certified cell-free DNA tests for EGFR, KRAS and BRAF point mutations and test use in clinical practice.

Hestia Mellert, Scott Thurston, Trudi Foreman, Westen Hahn, Nicholas Dupuis, Gary Pestano. _Biodesix, Inc., Boulder, CO_.

Nearly 80% of patients will not have mutation results available at their initial oncology consult and as many as 1 in 4 patients will begin treatment in advance of receiving their tumor mutation results. These factors hinder diagnosis at the molecular level, which is an important component in the decision to commence treatment with specific targeted therapies. The goal of this study was to measure the utility of a CLIA-certified Laboratory Developed Test (LDT) in the absence of a tissue diagnosis. EGFR, KRAS and BRAF point mutations within the cell-free DNA (cfDNA) isolated from the plasma of patients with advanced stages of cancer were analyzed using Droplet Digital™ PCR (ddPCR) technology. The specific tests detected the EGFR sensitizing mutations L858R and exon 19 deletion (E746 - A750) and EGFR resistance mutation, T790M, the KRAS mutations G12C, G12V and G12D, as well as the BRAF V600E mutation. The tests were comprised of three components: (i) a whole-blood collection kit that ships at ambient temperature, (ii) cfDNA isolated from plasma for analysis using ddPCR, and (iii) a secure laboratory information management system (LIMS) for sample accessioning and report generation. Test mutation status results were reported within 72 hours of blood shipment from the physicians' office. To date we have processed greater than 2000 individual variant tests for patients with NSCLC. The predominant tumor type for EGFR sensitizing mutations requested was NSCLC (95%). Interestingly, even BRAF test orders were > 90% from NSCLC patients. Additionally, the percentage of tests requested that were positive for each mutation category were 12% for EGFR sensitizing, 20% for EGFR resistance, 14% for KRAS and 1.8% for BRAF. Significantly, T790M test orders were observed to have increased five-fold over the last three months of testing. We have developed highly sensitive, 72 hour test to results blood-based assays as a part of the GeneStratTM panel, that expand the utility of laboratory testing for patients previously diagnosed with cancer.

#486

A comparison of cfDNA isolation kits: isolation and quantification of cell-free DNA in plasma.

Laure Sorber,1 Karen Zwaenepoel,2 Vanessa Deschoolmeester,1 Geert Roeyen,3 Sofie Goethals,4 Christian Rolfo,5 Patrick Pauwels6. 1 _CORE Antwerp University (UA) - Antwerp University Hospital (UZA), Antwerp, Belgium;_ 2 _Department of Pathology - Antwerp University Hospital (UZA), Antwerp, Belgium;_ 3 _Hepatobiliairy transplantation and endocrine surgery - Antwerp University Hospital (UZA), Antwerp, Belgium;_ 4 _UZA Tumor bank - Antwerp University Hospital, Antwerp, Belgium;_ 5 _Oncology & Phase I - Early Clinical Trials - University Hospital of Antwerp (UZA), Antwerp, Belgium; _6 _Department of Pathology - University Hospital of Antwerp (UZA) - CORE Antwerp University (UA) - UZA Tumor Bank, Antwerp, Belgium_.

Introduction: In oncology, circulating cell-free DNA (cfDNA) has emerged as a new and sensitive biomarker. However, the low concentrations of cfDNA, especially in early stages of disease, make it a challenging analyte for extraction, consequently high isolation efficiency is essential.

In this study we compared the isolation efficiency of one of the most commonly used DNA isolation kits, namely the QIAamp® circulating nucleic acid kit (QIA - Qiagen), with four newly launched cfDNA isolation kits.

Material & Methods: The QIA kit was compared with the PME free-circulating DNA Extraction Kit (PME - Analytik Jenna), the Maxwell® RSC ccfDNA Plasma Kit (RSC - Promega), the NEXTprep-MagTM cfDNA Isolation Kit (NpM - BiooScientific), and the EpiQuickTM Circulating Cell-Free DNA Isolation Kit (EQ - Epigentek).

CfDNA was extracted from 10 plasma samples of patients treated at the Antwerp University Hospital (UZA); including two patients with benign pancreatic cysts, three early stage pancreatic cancer patients, and four pancreatic cancer patients with metastasized disease, of which one had a second blood sample taken during follow up. Only the patients with metastasized disease were KRAS mutated.

To determine the isolation efficiency of the different kits, the QX200TM Droplet DigitalTM PCR system (ddPCR - Bio Rad) was used. All samples were screened with the ddPCRTM KRAS Screening Multiplex kit (Bio Rad) for the presence of seven KRAS mutations and a specific wild type sequence, which results in the KRAS mutated cfDNA fraction and the quantification of the total cfDNA.

Results: The QIA and the RSC kits displayed similar isolation efficiencies of both KRAS mutated and non-mutated cfDNA; with the detection of the KRAS mutated fraction ranging from approximately 1% to 40% and on average a yield of 76,7 and 83,1 copies/µl, respectively. On the other hand, the PME kit could not detect any KRAS mutations and in four cases there was not enough cfDNA isolated to be detected with ddPCR. DdPCR of cfDNA isolated with the NpM and EQ kits did not generate any positive signals. qPCR was performed on cfDNA isolated with both kits with control primers of the NpM kit. All samples of the NpM kit and three of the samples of the EQ kit were shown to contain cfDNA of at least 180 bp; which leads to the conclusion that ddPCR can't be used as a downstream procedure for the NpM and EQ kits.

Conclusion: The RSC kit enables the isolation of a sufficient quantity of cfDNA similar to the yield generated with the QIA kit, and both kits have a higher isolation efficiency than the PME kit. This study presents two highly efficient isolation kits, of which the RSC kit has the advantage of a fully automated, magnetic beads based protocol over the labor intensive and time consuming QIA kit. We also concluded that ddPCR as a downstream procedure is not feasible with the NpM and EQ kits. However, an improved version of the NpM kit has recently been produced and will also be included in this comparison study.

#487

Usefulness of exosome quantification by cholesteryl ester transfer protein activity as a biomarker for human esophageal squamous cell carcinoma (ESCC).

Yasunori Matsumoto, Masayuki Kano, Yasunori Akutsu, Naoyuki Hanari, Isamu Hoshino, Kentaro Murakami, Akihiro Usui, Hiroshi Suito, Masahiko Takahashi, Ryota Otsuka, Hisahiro Matsubara. _Chiba University, Chiba, Japan_.

Exosomes are small membrane vesicles (30-100 nm) and are thought to play an important role in tumor progression, chemoresistance, dormancy etc. However, the exosome dynamics and transportation between cancer cells and the other cells are still unclear. In addition, although it is common method to quantify exosomes using standard protein quantification methods, it is actually incorrect.

In this study, we assessed the exosome dynamics with mouse model and quantify plasma exosome by cholesteryl ester transfer protein activity, which is known to be enriched within exosomes. We analyzed the relationship between exosome quantification and clinical characters in ESCC patients.

In order to image and analyze the movement of cancer cell-derived exosomes, green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes, was expressed in human esophageal squamous cancer cell line TE2. Mouse model of human ESCC were then established by s.c. injection of TE2-CD63-GFP or TE2 cell lines. Eight weeks after injection, the tumor, blood and other organs were harvested and imaged with fluorescence. Our data indicates tumor derived exosome can emitted from the tumor and circulate in the blood flow.

Quantification of plasma exosome in ESCC patients (n=64) and no malignant patient (n=10) were performed by measurement of cholesteryl ester protein activity, and then analyzed the relationship between the exosome number and the patients' clinical characteristics, such as age, sex, TNM classification, prognosis, tumor marker. Our data indicates the exosome number was higher in ESCC patients than no malignant patients (P=0.0014). Althogh there were no difference in tumor depth, lymph node status, distant metastasis, the exosome number can be a prognostic marker for ESCC (P=0.04).

#488

Detection of somatic mutations in plasma allows for non-invasive real-time therapy response monitoring of lung cancer patients.

Jose L. Costa,1 Gabriela Fernandes,2 Joana Reis,1 Miguel Silva,1 Venceslau Hespanhol,2 José C. Machado1. 1 _Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal;_ 2 _São João Hospital Center, Porto, Portugal_.

Tumor-specific (somatic) mutations in plasma can serve as biomarkers for tumor detection, monitor tumor response to specific therapies, detect residual disease after surgery, and long-term follow-up. The intrinsic low abundance of circulating cell-free tumor DNA (cfDNA) makes the detection and quantification of such mutations in plasma a challenging task. This study aimed to establish a comprehensive strategy to be used in the detection of clinically relevant somatic mutations in plasma of lung cancer patients both at diagnosis and during follow-up of disease.

Plasma samples from healthy controls were used to isolate cfDNA and to optimize the experimental approach. A custom multiplex PCR panel was designed to target 420 unique genetic variants. To cover a broad range (0.0 -100%) of variant allele fractions (VAF), as expected for somatic mutations in the plasma of cancer patients, the cfDNA samples from the controls were mixed at five different ratios and sequenced using five different levels of coverage. In order to evaluate our strategy, plasma samples obtained at different stages of disease were collected from a group of 100 lung cancer patients and used to isolate cfDNA. The mean treatment follow up time was 6 months with a minimum of 1 and a maximum of 23 months. Different enrichment strategies where tested. All amplified products were used to prepare libraries and sequenced using the Ion PGM system. The QuantStudio 3D Digital PCR System was used to confirm selected results.

Our controlled data set enabled us to define VAF, allele depth and allele quality score cutoffs for a reliable detection of low VAF in cfDNA. For variants present at VAF ≥5%, a sensitivity of 100% was achieved at ≥500x coverage. At ≥1000x coverage, more than 94% of the variants present at VAF <5% were successfully detected with an average sequencing quality score >Q30. In the plasma isolated from lung cancer patients, tumor derived genetic mutations could be identified in as little as 10ng of cfDNA. Mutations identified in cfDNA mirrored the mutations identified in tumor biopsies. Mutations with VAF as low as 0.1% could be detected in cfDNA and confirmed using Digital PCR. The changes in tumor-specific mutations in cfDNA was then correlated with the clinical follow up of the patients. Response to therapy, including resistance, could be observed by tracking specific mutations in cfDNA and the detection of tumor relapse could be anticipated up to two months using cfDNA when compared to standard methodologies.

In conclusion, the comprehensiveness and speed of the NGS methodology, combined with the high sensitivity of detection, delivered an experimental protocol for the detection of somatic mutations in cfDNA for non-invasive real time therapy response monitoring of cancer patients. This strategy can be implemented in any molecular pathology laboratory with clear benefits for the management of cancer patients.

#489

NGS analysis of the single CTC or DTC isolated and subtyped by the integrated subtraction enrichment and immunostaining-FISH.

Peter Lin. _Cytelligen, San Diego, CA_.

Majority of the circulating or disseminated tumor cell (CTC or DTC) detection methodologies rely on existence of epithelial markers (such as EpCAM and/or cytokeratins) on tumor cells. However, highly heterogeneous expression or even absence of EpCAM, and down-regulation of both EpCAM and cytokeratins in CTCs/DTCs during epithelial-mesenchymal transition (EMT) has been reported elsewhere, resulting in significant false negative detection of those uncatchable and invisible CTCs/DTCs. A novel strategy integrating subtraction enrichment which efficiently enriches non-hematopoietic tumor cells shed from various types of solid tumor into biofluid including peripheral blood, bone marrow, ascites, pleural effusion, and cerebrospinal fluid (CSF), with immunostaining-FISH (iFISH), which enables in situ phenotypic immunofluorescent staining of tumor biomarker and chromosomal FISH on the identical tumor cell, has shown its unique advantage in terms of detecting CTCs/DTCs with higher sensitivity and specificity. Diversified CTCs/DTCs could be identified and subtyped upon specific chromosome ploidy and tumor biomarker expression, and each subtype of CTCs/DTCs respectively correlate with therapeutic drug sensitivity or resistance, tumor metastasis or relapse. Whole genome amplification and subsequent next generation sequencing (NGS) analysis performed on the single subtyped breast, pancreatic, gastric and NSCLC cancer CTC or DTC targeted by iFISH, and collected by means of a microscopic cell manipulator is able to reveal ploidy of all the chromosomes and status of either unknown genes or a series of known oncogenes including HER2, Kras, BRAF, etc. It is anticipated that SE-iFISH could guide and promote more significant single cell based genomic and functional analyses of CTCs and/or DTCs.

#490

High-throughput mutational analysis in cell-free DNA by targeted next-generation sequencing.

Nga wan Rachel Tam, David Shames, Walter Darbonne. _Genentech, South San francisco, CA_.

Circulating cell-free DNA (cfDNA) in plasma offers a non-invasive approach to monitor tumor molecular profiling in real-time at multiple time-points, detection of emerging genomic alterations associated with drug resistance and clarifying cancer prognosis and diagnosis of cancer recurrence or progression. We developed a robust, multiplexed and PCR-based targeted next-generation sequencing approach to detect actionable mutations in cfDNA with low DNA input (1-5 ng). We employed the latest Ion TorrentTM S5/XL bench-top sequencer with Ion ChefTM Automation System and successfully automated the entire 2-day workflow from libraries preparation to semi-conducting chip sequencing. The hands-on time has been reduced to only 30 min.

We will present data showing validation of this NGS platform using commercially available DNA control, archival FFPE tissue and cfDNA matched samples and demonstrate robust sensitivity and specificity by using the off-the-shelf Ion AmpliSeq Cancer Hotspot Panel v2 that covers 50 oncogenes. We also compared this approach to other orthogonal technologies, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR) and achieved 100% concordance across these platforms. NGS can detect mutations down to 1% allelic frequency with 100% sensitivity and specificity.

This robust and automated NGS platform is being routinely used in our lab to analyze cell-free DNA samples from multiple clinical trials. These efforts enable the development of a non-invasive method to overcome existing challenges to provide molecular understanding of patient's tumor evolution in real time, and aid in the development of personalized therapies for cancer patients.

#491

Salvage MET amplification detection and therapy through cell-free DNA NGS in a progressing lung cancer patient.

Nir Peled,1 Anna Belilovski,1 Lior Soussan-Gutman,2 Richard B. Lanman,3 AmirAli Talasaz3. 1 _Tel Aviv University, Tel Aviv, Israel;_ 2 _Oncotest Teva, Teva Pharmaceuticals, Shoham, Israel;_ 3 _Guardant Health, Inc., Redwood City, CA_.

Background: Mutational analysis has become standard of care in lung cancer. However, many patients do not have tissue available due to lack of tissue, or more commonly due to tissue exhaustion by previous tissue analysis. Next generation sequencing of circulating cell-free DNA (cfDNA) provides a non-invasive alternative. EGFR point mutations and indels are well reported in this manner, however gene copy number amplification is more challenging. Here we present a cases where MET amplification has been detected and guided highly efficient therapy in advanced lung cancer patient.

Methods: Guardant360TM is a targeted cfDNA NGS panel using hybrid capture and complete exon sequencing exons for single nucleotide variant detection in 68 genes, copy number amplifications (CNA) in 16 genes, and selected indels and fusions. CNA has been validated against cell lines with known amplifications.

Results: A 70 y old former light smoker (15 PY) with pulmonary fibrosis and moderate pulmonary hypertension was diagnosed with a 30 mm RMB lung adenocarcinoma that was treated by stereotactic beam radiotherapy due to poor pulmonary reserve. After 5 months, mediastinal, liver and multiple bone metastases were diagnosed. Genomic analysis of the primary tissue biopsy yielded a rare EGFR mutation (I744F) and afatinib therapy was started to cover uncommon EGFR mutations. However, 2 months later, a significant progression has occurred and the patient had severe local progression with respiratory near-failure. The patient was not a candidate for chemotherapy and there was no further tissue available for molecular testing. Therefore cfDNA testing was performed finding no residual EGFR mutation in the blood but a MET amplification reported above the 90th percentile for the Guardant Health laboratory with copy number of 53.6 in the blood.

Upon this result, crizotinib 250 mg BID was started with immediate clinical improvement, followed with a dramatic imaging response on CT/PET scans. Currently, the patient is in PS-0 and free of any symptoms, 3 months since treatment has been started.

Conclusion: cfDNA detection of MET amplification as a key resistance mechanism after EGFR TKI therapy is feasible in patients where tissue is not accessible or was undergenotyped and may be accompanied by a fast and dramatic clinical improvement.

#492

Automated circulating cell-free DNA purification from large volume draws.

Robert Ray, Mark Bratz, Doug Wieczorek, Douglas White, Douglas Horejsh, Eric Vincent, Trista Schagat. _Promega Corporation, Madison, WI_.

Circulating, cell-free DNA (ccfDNA) has emerged as an important tool in molecular oncology research. Because it is highly fragmented, present in small quantities, and highly susceptible to degradation, purification of ccfDNA poses unique challenges to researchers. While plasma is the main focus of many researchers for ccfDNA, recent work has shown that it is present in other biological fluids.

Promega has developed a unique chemistry to selectively purify ccfDNA from plasma. The Maxwell RSC ccfDNA Plasma system is a completely automated system that allows purification of ccfDNA from 1ml of plasma. To add flexibility, we adapted the chemistry to automated platforms in 24-well configurations. These platforms include the Hamilton Microlab STAR series and the KingFisher Flex Processor. Because the biomarkers in ccfDNA can be of very low frequency, many researchers prefer to process volumes of samples >1ml which can exceed the capacity of many purification systems. Using a sequential bind strategy, we demonstrate that ccf DNA can be purified from at least 8ml sample draw using a fully automated method. Liquid draws tested include plasma and urine. DNA quantity was assessed by qPCR using an autosomal target, and quality was assessed using an internal PCR control.

This study shows the flexibility and robustness of the Maxwell RSC ccfDNA chemistry. We successfully adapted it for purification from relatively large volume liquids in a fully automated manner making this a convenient option for early biomarker research.

#493

Quantity of KRAS mutations in cell-free DNA is associated with survival of patients with advanced cancers.

Kiran Madwani,1 Helen J. Huang,1 Dawne N. Shelton,2 Siqing Fu,1 Apostolia M. Tsimberidou,1 Sarina A. Piha-Paul,1 Aung Naing,1 David S. Hong,1 Daniel D. Karp,1 Debra L. Andrews,1 Goran Cabrilo,1 E. Scott Kopetz,1 Rajyalakshmi Luthra,1 Bryan K. Kee,1 Cathy Eng,1 Van K. Morris,1 George A. Karlin-Neumann,2 Funda Meric-Bernstam,1 Filip Janku1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA_.

Background: Cell-free (cf) DNA from plasma offers an easily obtainable material for KRAS mutation analysis for diagnostics and monitoring. There is emerging evidence that the percentage of mutant cfDNA in the wild-type background (mutant allele fraction, MAF) and/or absolute quantity of mutant cfDNA can be associated with survival of patients with advanced cancers.

Methods: Plasma-derived cfDNA from patients with progressing advanced cancers was purified and 16 ng of DNA was tested with a KRAS multiplex assay to distinguish the wild-type allele from 7 of the most common mutations in the G12 and G13 hotspot of exon 2 using the QX200 Droplet Digital PCR™ platform (Bio-Rad). Results were compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory and clinical outcomes including survival.

Results: Of the 117 patients (colorectal cancer, 71; non-small cell lung cancer, 12; melanoma, 10; pancreatic cancer, 5; ovarian cancer, 5; appendiceal cancer, 5; other cancers, 9), KRAS mutations were detected in 85 (73%) archival FFPE tumor samples and 85 (73%) plasma cfDNA samples. The two methods had overall agreement in 109 patients (93%; kappa, 0.83, standard error, 0.06; 95% confidence interval [CI], 0.71-0.94), sensitivity of 95% (95% CI, 0.88-0.99), specificity of 88% (95% CI, 0.71-0.96), even though median time from tissue to blood sampling was 18.5 months (1.1-134.4 months). A higher MAF (>7%) of KRAS in cfDNA as determined by 5% trimmed mean value was associated with shorter survival compared to lower (<7%) MAF (5.4 vs. 7.6 months; P=0.001), which was confirmed on multivariate analysis. A total of 20 patients with KRAS mutations in cfDNA had longitudinal testing of cfDNA from at least two time points obtained before and on experimental therapy. In these patients, changes in MAF demonstrated a trend towards positive correlation with changes in measurement of target tumor lesions on imaging per RECIST (r=0.42, P=0.07).

Conclusions: A higher percentage of KRAS mutation in plasma cfDNA is an independent predictive factor for shorter survival in patients with advanced cancers.

#494

Evaluation of soluble FLT3 ligand as a marker of bone marrow stress and recovery.

Christine Sastri, Mercedesz Balazs, Priya Koppikar, James R. Lipford, Angela Coxon, Tara L. Arvedson. _Amgen, Thousand Oaks, CA_.

Depletion of bone marrow (BM)-resident myeloid progenitor cells is a consequence of various anti-cancer treatments, including bispecific T-cell engager (BiTE®) antibodies targeting myeloid cell antigens. Currently, aspirates are required to determine the extent of myeloid cell depletion in the BM. This procedure is painful and potentially invasive for patients. As a surrogate marker for BM myeloid cell depletion, we evaluated soluble FLT3 ligand (FLT3L) in non-human primates treated with myeloid cell-depleting BiTE® antibodies. While FLT3L is not detectable in serum during steady-state hematopoiesis, it is detectable in response to hematopoietic deficiency. The goal of this study was to evaluate the relationship of serum FLT3L with depletion and recovery of BM myeloid cells in non-human primates treated with BiTE® antibodies against CD33.

Serum FLT3L levels were determined using an anti-human FLT3L ELISA kit. Validation studies demonstrated that both the capture and detection antibodies recognized cynomolgus FLT3L. Serum FLT3L concentrations were determined using a standard curve with rhesus FLT3L (99.6% identical to cynomolgus FLT3L). Serum samples were taken from cynomolgus monkeys before, during and after treatment with anti-CD33 BiTE® antibodies and compared with the number of BM-resident

target cells.

In the first study, cynomolgus monkeys were dosed once with different dose levels of anti-CD33 BiTE® antibody ranging from 1- 15 µg/kg. BM-resident myeloid cells were transiently reduced in some, but not all, cohorts. Soluble FLT3L was detected only when there was a reduction in BM myeloid cells. When FLT3L was detectable, the serum concentration correlated with the extent of target cell reduction and FLT3L levels decreased as the myeloid cells recovered.

In the second study, cynomolgus monkeys were dosed three times with anti-CD33 BiTE® antibody. In these studies target cell reduction was greater and persisted longer compared to the single dose studies. Similarly, the magnitude and duration of increased FLT3L in the multidose studies was also greater. Within the multi-dose study, FLT3L levels continued to increase as more BM-resident myeloid cells were depleted, suggesting that it was possible to detect cumulative stress to the BM.

In summary, serum FLT3L levels are correlated with the extent and duration of myeloid cell depletion in the BM suggesting that serum FLT3L levels reflect depletion and subsequent myeloid cell replenishment. All studies to date have involved treatment with myeloid antigen-specific BiTE® antibodies; future studies will evaluate FLT3L levels following treatment with anti-lymphoid or solid tumor antigen-specific BiTE® antibodies to determine if FLT3L increases are associated with myeloid cell depletion or indicative of general BM stress or inflammation. The potential benefit of this marker is that it could allow for repeat non-invasive BM monitoring in patients on myelosuppressive therapies.

#495

The impact of preoperative hydroxyprogesterone on serial levels of circulating tumor cells [CTCs] in patients undergoing surgery for operable breast cancer.

Ujjwala M. Warawdekar,1 Vani Parmar,2 Akshita Singh,2 Swapnil Aher,1 Abhay Kulkarni,1 Meenal Chaudhari,1 Sudeep Gupta,1 Rajendra A. Badwe3. 1 _Advanced Centre for Treatment,Research and Education in Cancer [ACTREC], Navi Mumbai, India;_ 2 _Tata Memorial Hospital, Mumbai, India;_ 3 _Tata Memorial Hospital , Tata Memorial Centre, Mumbai, India_.

Introduction: Assessment of Circulating Tumour Cells [CTCs] from the peripheral blood of patients diagnosed with cancer is emerging as a valuable biomarker for prognostication and tailoring therapy. A previous study demonstrated that a single depot injection of hydroxyprogesterone prior to surgery improved the DFS of patients with large tumours or positive nodes, forming the basis for this investigation, designed to assess effects of this drug on serial levels of CTCs on patients with operable breast cancer.

Methods: Seventy patients were enrolled, randomized, and as previously described a single depot injection of hydroxyprogesterone was administered prior to surgery in patients in the experimental arm. Peripheral blood samples were obtained at three time points during surgery; prior to induction

of anaesthesia, intra operative and at completion. Peripheral Blood Mononuclear Cell [PBMC] fractions separated on Ficoll hypaque were enriched for tumour cells with double positive immunomagnetic selection for EpCAM [Epithelial Cell Adhesion Molecule]. Half the enriched fraction was routed for multistep sequential labelling of cell-surface markers; EpCAM and CD45, cytoplasmic Cytokeratin and staining with DAPI for the nucleus, facilitating acquisition with multi parametric flow cytometry and imaging using confocal microscopy. With pre-amplification of other half of the enriched fraction, a quantitative expression analysis for a panel of epithelial genes was performed.

Results:With multi parametric flow analysis, events that were CD45-/ EpCAM+/ CK+ with DAPI positivity were

defined as CTCs, reported as events/ml blood and in all samples were in the range of 0 to 155 /ml of blood with no significant difference observed between the two arms. Detailed analysis with pooled data of all time points, in premenopausal patients, using the Mann Whitney test, showed that the mean number of CTCs is significantly lower[p=0.005] in the experimental arm[n=30] as compared to the control arm[n=36] .Analysis within the hydroxyprogesterone group using the Wilcoxon signed rank test showed that the mean number of CTCs is lower in the post-surgery time point when compared to the pre surgery time point, in patients with positive nodes[n=16][ p=0.044], large tumours [n=18] [NS; p=0.085] and post-menopausal node positive group [n=7] [NS; p=0.063] The expression analysis for the selected markers and confocal images validated the identity of tumour cells.

Conclusions: Patient follow-up will ascertain the benefits of reduced numbers observed and establish CTCs as a surrogate marker for prognostication.

#496

Androgen receptor expression on circulating tumor cells in metastatic breast cancer.

Takeo Fujii,1 Yipeng Wang,2 James M. Reuben,1 Rachel Krupa,2 Ryon Graf,2 Lyndsey Dugan,2 Jessica Kouw,2 Dena Marrinucci,2 Bora Lim,1 Carlos H. Barcenas,1 Angela N. Marx,1 Debu Tripathy,1 Ryan Dittamore,2 Naoto T. Ueno1. 1 _The University of Texas, MD Anderson Cancer Center, Houston, TX;_ 2 _Epic Sciences, San Diego, CA_.

Background: Androgen receptor (AR) is a nuclear transcription factor and a member of the steroid hormone receptor superfamily that can be detected by immunohistochemical staining in all three subtypes of invasive breast cancer with frequency of >70% of estrogen receptor-positive tumors (ER+), >60% of HER2-positive tumors (HER2+), and 40-50% of triple-negative breast cancers (TNBC) respectively. AR-target therapies are showing early activity in AR+ breast cancer. Given early evidence of activity with agents targeting AR, it is desirable to improve our understanding of the AR biology in breast cancer. Although circulating tumor cells (CTCs) have been proposed as a liquid biopsy for biomarker detection in multiple cancer types, the rate and heterogeneity of AR expression in CTCs in breast cancer is still unclear. In this study, we determine the rate and heterogeneity of AR expression in CTCs of patients with metastatic breast cancer.

Methods: Blood samples from patients with metastatic breast cancer were smeared onto glass slides and subjected to nuclear staining with DAPI and reacted with fluorescent-labeled antibodies to detect CD45 on leukocytes and cytokeratin and AR on epithelial cells; the slides were scanned on the Epic Sciences CTC platform and the data analyzed using established algorithms.

Results: Among the 59 patients analyzed, 25 (42.4%) had ER+/HER2-, 12 (20.3%) had ER+/HER2+, 6 (10.2%) had ER-/HER2+, and 16 (27.1%) had TNBC based on the most recent staining. CTCs were detected in 50 of 59 (84.7%) patients. Characterization of CTCs identified CK+ CTCs (45 of 59, 76.3%), CK-negative CTCs (16 of 59, 27.1%), CK+ CTC clusters (16 of 59, 27.1%), and apoptotic CTCs (45 of 59, 76.3%), respectively. Number of CTCs per patient ranged from 0 to 282.1 per mL (median=4.4). Six of the 50 patients (12%) with CTCs had AR+ CTCs. The number of AR+ CTCs per patient ranged from 0.3 to 52.6 per mL (median=1.15). Among the patients with AR+ CTCs, the percentage of CTCs that were AR+ ranged from 5% to 50% (median=21%). Among the six patients with AR+ CTCs, five patients had ER+ breast cancer and one had TNBC.

Conclusions: We demonstrated that the Epic Sciences non-enriching or selecting platform can detect AR expression in CTCs in breast cancer. These preliminary results suggest the need for clinical validation of CTC AR expression as a potential test to stratify and identify patients who might benefit from AR therapy. The heterogeneity of intra-patient CTC AR expression leads us to another hypothesis that patients with AR+ CTCs might have heterogeneous disease with multiple drivers. Further studies are warranted to allow serial monitoring of changes in AR and to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

#497

PIK3CA mutational status in circulating tumor cells (CTCs) and corresponding circulating tumor DNA in breast cancer patients.

Elena Tzanikou,1 Athina Markou,1 Nikos Malamos,2 Vasilis Georgoulias,3 Evi S. Lianidou1. 1 _Analysis of Circulating Tumor Cells lab, Lab of Analytical Chemistry, Univ. of Athens, Athens, Greece;_ 2 _Oncology Unit , Helena Venizelou Hospital, Athens, Greece;_ 3 _Medical School, University of Crete, Heraklion, Greece_.

AIMS: Molecular characterization of Circulating Tumor Cells (CTCs) and analysis of circulating tumor DNA (ctDNA) in cancer patients holds promise as an extremely powerful and reliable non-invasive clinical tool for the individual molecular profiling of each patient in real time. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is implicated in human cancers, and somatic mutations in the p110α catalytic subunit of PI3K, are very frequent and play a crucial role in response to molecular targeted therapies in breast cancer. In this study, we analyzed PIK3CA hotspot mutations (1633 G>A, 3140 A>G) in CTCs and corresponding ctDNA of early and metastatic breast cancer patients. We also examined whether there is a correlation between the presence of PIK3CA mutations in CTCs and ctDNA.

METHODS: We used our highly sensitive methodology for the detection of PIK3CA hotspot mutations in exons 9 (1633 G>A) and 20 (3140 A>G), based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis (HRMA) (Markou et al, CCR 2014). We analyzed PIK3CA hotspot mutations in the EpCAM-positive CTCs fraction and the corresponding plasma samples of: i) a group of 19 patients with operable breast cancer, ii) a group of 40 breast cancer patients with verified metastasis and iii) 30 healthy female volunteers. All ctDNA samples (extracted from 2 ml plasma) were examined for their DNA quality; to verify DNA quality, primers specific for the wild type in exactly the same PIK3CA gene region for exon 9 were used to assess for hotspots mutations. The mutation status of PIK3CA gene in ctDNA samples was detected by the developed methodology exactly as previously described. All samples were analyzed in triplicate.

RESULTS: The assay is highly sensitive as it can detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons (9 and 20) and highly specific (0/30 healthy donors). PIK3CA hotspot mutations were identified in ctDNA in 12/40 (30%) of metastasis-verified breast cancer patients and 4/19 (21%) of operable breast cancer patients. In metastasis-verified breast cancer patients, the concordance between EpCAM- positive CTCs and ctDNA for 1633 G>A mutation was 72.5%, whereas the corresponding concordance for the 3140 A>G mutation was 97.5%. In operable breast cancer patients, the concordance between EpCAM-positive CTCs and ctDNA for the 1633 G>A mutation was 80%, whereas the corresponding concordance for the 3140 A>G mutation was 100%.

CONCLUSIONS: Detection of PIK3CA hotspot mutations in EpCAM-positive CTCs and corresponding ctDNA has shown a correlation both in metastasis-verified and operable breast cancer patients. We will further evaluate our findings in a large cohort of patients before and after treatment, to evaluate response to molecular targeted therapies in breast cancer.

#498

Meta-analysis of genomic aberrations identified in CTCs andctDNA in triple negative breast cancer.

Kellie Howard,1 Sharon Austin,1 Fang Yin Lo,1 Arturo Ramirez,2 Debbie Boles,3 John Pruitt,3 Elisabeth Mahen,4 Heather Collins,1 Amanda Leonti,1 Lindsey Maassel,1 Christopher Subia,1 Tuuli Saloranta,1 Nicole Christopherson,1 Kerry Deutsch,1 Jackie Stilwell,2 Eric Kaldjian,2 Michael Dorschner,4 Sibel Blau,5 Anthony Blau,4 Marcia Eisenberg,3 Steven Anderson,6 Anup Madan1. 1 _Covance, Seattle, WA;_ 2 _RareCyte, Inc, Seattle, WA;_ 3 _Laboratory Corporation of America® Holdings, Research Triangle Park, NC;_ 4 _Center for Cancer Innovation, University of Washington, Seattle, WA;_ 5 _Northwest Medical Specialties, Puyallup, WA;_ 6 _Covance, Durham, NC_.

Technological innovation and scientific advances in understanding cancer at the molecular level have accelerated the discovery and development of both diagnostics and therapeutics. Circulating tumor cells (CTCs) and plasma circulating tumor DNA (ctDNA) are non-invasive prognostic markers that have been associated with metastatic and aggressive disease. Both CTCs and ctDNA allow molecular characterization of a tumor that is inaccessible or too risky to biopsy. The analysis of genomic aberrations in both sample types provides insights into drug resistance and can help determine appropriate, targeted cancer treatments. Mutations found in the primary or metastatic tumor can be identified in both CTCs and ctDNA as well as novel mutations that may reflect intratumoral and intermetastatic heterogeneity. When collected and evaluated over an extended period of time, changes in the CTC and/or ctDNA mutational profile can offer guidance into the effectiveness of a treatment, indicate the progression of disease, and detect recurrence of disease earlier.

We have performed whole exome sequencing of CTCs and ctDNA from a metastatic triple negative breast cancer (TNBC) patient to better understand the evolution of tumor heterogeneity during therapy. The patient was enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001) and initially received weekly cisplatin infusions followed by additional targeted therapy. Longitudinal peripheral blood samples were collected over a period of 272 days following enrollment in the clinical trial. CTCs were identified using the AccuCyte-CyteFinder system (RareCyte, Seattle WA).

We used next generation sequencing, and computational biology tools to analyze genomic DNA from multiple CTCs, white blood cells (WBCs) and ctDNA from various time points. We observed similar genomic aberrations in both CTCs and ctDNA that could be classified into three groups: a) a static group that remains unchanged during the course of therapy, b) a sample-specific group that is unique to each time point and c) an intermediate group that has variants that are short-lived but are present across multiple time points. Variants identified in the liquid biopsy samples were compared with variants observed in primary breast tumor, metastatic bone marrow tumor and publically available pan-cancer datasets. We then performed meta-analysis on somatic variants to identify changes in affected networks in response to therapy over time. Several key nodes were identified that could rationally have been targeted for therapy using compounds currently in clinical trials. We then compared and combined the perturbed networks obtained from the CTCs and ctDNA to better understand the etiology of TNBC. These studies represent the first step of a synergistic partnership between the genetic information obtained from the analysis of CTCs and ctDNA with innovative health care for patients with metastatic breast cancer.

#499

ESR1 methylation in circulating tumor cells of patients with breast cancer.

Sofia Mastoraki,1 Areti Strati,1 Maria Chimonidou,1 Nikos S. Malamos,2 Vasilis Georgoulias,3 Evi S. Lianidou1. 1 _Analysis of Circulating Tumor Cells lab, Lab of Analytical Chemistry, Univ. of Athens, Athens, Greece;_ 2 _Helena Venizelou Hospital, Athens, Greece;_ 3 _School of Medicine, University of Crete, Heraklion, Athens, Greece_.

AIMS: DNA methylation is an epigenetic alteration which plays a decisive role in the regulation of signal translation processes. In our lab, we have demonstrated for the first time the epigenetic silencing of tumor and metastasis suppressor genes in CTCs through their promoter methylation. Estrogen receptor (ER) is an important prognostic biomarker and is predictive of response to endocrine therapy in breast cancer. In this study, we evaluated for the first time ESR1 methylation in CTCs of breast cancer patients.

METHODS: We developed and validated a novel highly sensitive and specific qMSP assay for ESR1 methylation using commercially available DNA methylation controls and the MDA-MB-231 cell line. We further examined its performance in EpCAM-positive immune-magnetically isolated CTC fractions, followed by DNA isolation and sodium bisulfite (SB) treatment from: a) 74 operable, b) 48 metastasis- verified breast cancer patients and c) 30 healthy donors (control group).

RESULTS: The developed assay is highly specific and sensitive since it can detect 0.1% methylated ESR1 sequences in the presence of 99.9% un-methylated. ESR1 was found to be methylated in 16/74 (21.6%) operable breast cancer patients, in 10/48 (20.8%) patients with verified metastasis, but only in 1/30 (3.3%) healthy donors (EpCAM-positive CTC fraction).

CONCLUSIONS: The EpCAM-positive CTC fraction was found to be methylated for ESR1 in about 20% of patients with breast cancer. We will further evaluate these findings in respect to the clinical outcome of these patients, since the epigenetic silencing of ESR1 could be of important clinical significance especially for its impact on the efficacy of treatment.

#500

Prognostic impact of CTC detected using a novel fluidic cell microarray chip CTC detection system in patients with breast cancer.

Takeshi Sawada,1 Jungo Araki,2 Toshinari Yamashita,1 Manami Masubuchi,2 Tsuneko Chiyoda,2 Mayu Yunokawa,3 Kumiko Hoshi,2 Shoichi Tao,2 Shohei Yamamura,4 Shouki Yatsushiro,4 Kaori Abe,4 Masatoshi Kataoka,4 Tatsu Shimoyama,1 Yoshiharu Maeda,1 Katsumasa Kuroi,1 Kenji Tamura,3 Tsuneo Sawazumi,2 Hironobu Minami,5 Yoshihiko Suda,2 Fumiaki Koizumi1. 1 _Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;_ 2 _Konica Minolta, Inc., Tokyo, Japan;_ 3 _National Cancer Center Hospital, Tokyo, Japan;_ 4 _Health Research Institute, National Institute of Advanced Industrial Science and Technology, Kagawa, Japan;_ 5 _Kobe University Graduate School of Medicine, Kobe, Japan_.

No current circulating tumor cell (CTC) enumeration system provides better prognostic value than CellSearch. We have developed a novel semi-automated CTC enumeration system (fluidic cell microarray chip system, FCMC) that captures CTC independently of tumor-specific markers or physical properties. This system was validated in preclinical studies and in blood samples from 20 healthy donors and 22 breast cancer patients in this study. Using spike-in samples, a statistically higher detection rate (p = 0.010) of MDA-MB-231 cells and an equivalent detection rate (p = 0.497) of MCF-7 cells were obtained with FCMC in comparison with CellSearch. The number of CTC detected in samples from patients that was above a threshold value as determined from healthy donors was evaluated. The CTC number detected using FCMC was significantly higher than that using CellSearch (p = 0.00037). CTC numbers obtained using either FCMC or CellSearch had prognostic value, as assessed by time-to-treatment-failure. The hazard ratio between CTC+ and CTC- was 4.043 in CellSearch (95% CI, 1.248 to 13.10; p = 0.01985); in contrast, it was 10.97 in FCMC (95% CI, 2.18 to 55.21; p = 0.003662). CTC detected using FCMC have the potential to be a better prognostic factor than CTC detected using CellSearch.

#501

PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer.

Martha Zavridou,1 Areti Strati,1 Nikos Malamos,2 Vasilis Georgoulias,3 Evi S. Lianidou1. 1 _Analysis of Circulating Tumor Cells Lab, Dept of Chemistry, Univ. of Athens, Athens, Greece;_ 2 _Oncology Unit, Helena Venizelou Hospital, Athens, Greece, Athens, Greece;_ 3 _Medical School, University of Crete, Heraklion, Crete, Greece_.

Background: Programmed cell Death receptor Ligand 1 (PD-L1) is a very promising biomarker for the selection of patients for cancer immunotherapy. We recently developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA. The aim of the present study was to study the expression of PD-L1 in CTCs from breast cancer patients with verified metastasis.

Methods: We quantified the expression of PD-L1 mRNA transcripts in EpCAM-positive CTCs, by using our recently developed RT-qPCR assay, based on the following procedure: i) immunomagnetic enrichment of EpCAM-positive CTCs from 20mL of peripheral blood, ii) total RNA isolation iii) cDNA synthesis and iv) RT-qPCR for PD-L1. PD-L1 expression in respect to the expression of B2M as a reference gene, was normalized using the 2-ΔΔCt approach. Peripheral blood samples (20mL) were obtained from 22 breast cancer patients with verified metastasis and 14 healthy donors.

Results: According to our results, 11/22 (50%) of metastasis-verified breast cancer patients were found to be positive for PD-L1 overexpression in CTCs. These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Our results are in concordance with a recent study by Mazel et al (Mol Oncol 2015), that by using the CellSearch(®) system found PD-L1((+)) CTCs in 11/16(68.8%) patients with metastatic breast cancer.

Conclusion: This is the first time that a quantitative RT-qPCR molecular assay is used for the evaluation of PD-L1expression levels in EpCAM-positive CTCs in metastatic breast cancer patients. The assay is closed-tube, quantitative, highly specific and sensitive, and high-throughput. We are currently evaluating the assay in a large number of clinical samples.

#502

Gene expression signatures in circulating tumor cells are prognostic for metastatic lesions in breast cancer patients and correlate with response to therapy.

Maren Bredemeier,1 Philippos Edimiris,1 Pawel Mach,1 Mikael Kubista,2 Robert Sjoback,2 Marie Jindrichova,3 Eva Rohlova,2 Vendula Novosadova,2 Katarina Kolostova,4 Siegfried Hauch,5 Bahriye Aktas,1 Mitra Tewes,6 Rainer Kimmig,1 Sabine Kasimir-Bauer1. 1 _University Hospital Essen, Department of Gynecology and Obstetrics, Essen, Germany;_ 2 _TATAA Biocenter, Goetheburg, Sweden;_ 3 _Institute of Biotechnology CAS, Prague, Czech Republic;_ 4 _University Hospital Kralovske Vinohrady, Department of Laboratory Genetics, Prague, Czech Republic;_ 5 _QIAGEN Hannover GmbH, Langenhagen, Germany;_ 6 _University Hospital Essen, Department of Medical Oncology, Essen, Germany_.

Background: Circulating tumor cells (CTC) are discussed to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC). Besides CTC characterization for targeted therapies, it would also be desirable to know where these cells derive from or to which organ site they are going to. Here we investigated whether it is possible to predict the origin of metastatic lesion based on the expression of 46 genes in CTC of MBC patients (pts).

Materials and Methods: 2x5 ml blood of 45 MBC pts and 20 healthy controls was collected at the time of disease progression (T0) and at two consecutive clinical staging (T1 and T2) for the detection of CTC applying immunomagnetic enrichment using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Pts were grouped a) into overall responders (OR) and overall non-responders (ONR), thus responding or not responding at T1 and T2 and b) according to sites of metastasis. PCR assays, targeting 46 transcripts and reference markers were used for a workflow based on pre-amplification and high throughput profiling (each samples in duplicates) with the full set of markers including also ValidPrime to correct for genomic background and InterPlate Calibrator to even out variations between runs. Data were analyzed using GenEx (MultiD, Sweden) and SAS. qPCR as well as technical reads were normalized using several normalization strategies.

Results: The multidrug resistant protein gene MRP1 was significantly differently expressed if OR and ONR groups were compared. In the following order of significance, VEGFR1, Keratin (KRT) 19, EGFR, MET1, ALDH, progesterone receptor (PR), UPA, Cathepsin D, KIT1 and Ki67 were differentially expressed in CTC of pts who had already developed liver metastasis as compared to pts without liver metastasis. Interestingly, a small group of pts, developing liver metastases in the course of disease, showed the estrogen receptor (ER), PR, HER2, mammaglobin, KRT19 on a significantly lower level as compared to the other pts. Similarly, once CTC were ER and PR positive, the probability of bone metastasis development decreased.

Conclusion: Our preliminary results indicate that the development of metastatic lesions is associated with site-specific CTC. Thus, besides using CTC as a monitoring tool to guide therapy, they might also indicate the site of metastasis which will allow a more precise decision concerning treatment strategy.

#503

**Molecular characterization of** in vivo **isolated EpCAM-positive circulating tumor cells in breast cancer.**

Areti D. Strati,1 Martha Zavridou,1 Galateia Kallergi,2 Eleni Politaki,2 Tobias Gorges,3 Andra Kuske,3 Anna-Lena Bohnen,3 George Koutsodontis,4 Amanda Psyrri,4 Klaus Lucke,5 Vasilis Georgoulias,2 Klaus Pantel,3 Evi Lianidou1. 1 _Univ. of Athens, Athens, Greece;_ 2 _Univ. of Crete, Heraklion, Greece;_ 3 _University Medical Center Hamburg-Eppendorf, Hamburg, Germany;_ 4 _Attikon University Hospital, Athens, Greece;_ 5 _GILUPI GmbH, Potsdam, Germany_.

Introduction: In the early stages of cancer, the chance to detect rare CTCs is increasing by increasing the sample volume. The aim of our study was to evaluate the diagnostic sensitivity of a novel clinical device for the in-vivo isolation of EpCAM-positive CTCs (CellCollectorTM, GILUPI, GmBH), by using highly sensitive RT-qPCR molecular assays.

Patients and methods: 29 breast cancer patients without overt metastases before the beginning of adjuvant chemotherapy (M0), 26 breast cancer patients with overt metastases before starting of therapy (M1) and 12/26 of them before the second cycle of therapy (M2), as well as 18 healthy donors participated in the study. After in-vivo isolation, total RNA was extracted from captured cells, lysed in Trizol, followed by cDNA synthesis. RT-qPCR was used for the molecular characterization of captured cells, for: CK-19, HER-2, TWIST1, VEGF, ER, PR, EGFR, CD44, CD24, and ALDH1, while B2M was used as a reference gene. Peripheral blood was also collected for CTC analysis by the FDA cleared CellSearchTM system. In addition, immunofluorescence staining of cytospins was performed and screened for CTCs using the ARIOL system, using ER, HER2, CK (8, 18, 19) and CD45 for CTC identification.

Results: Results are shown in Table 1. At least one gene was expressed in 10(34.5%) of M0, 15(57.7%) of M1 and 4(33.3%) of M2 patient groups, but in none of healthy donors 0/18(0%). CellSearchTM gave positive results in 5(17.2%) of M0, 10(38.5%) of M1 and 0(0%) of M2. Immunofluorescence (Ariol system) was positive for ER, HER2, CK (8, 18, 19) in 5/15(33.3%) M0, in 4/12(33.3%) M1 and in 1/7(14.3%) M2 groups.

Table 1.  | |  | |

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

Gene expression in CTC  | Healthy

N=18 | M0

N=29 | M1

N=26 | M2

N=12

CK-19 | 0 (0%) | 6(20.7%) | 6 (23.1%) | 2 (16.7%)

HER2 | 0 (0%) | 2 (6.9%) | 0 (0%) | 0 (0%)

ER | 0 (0%) | 2 (6.9%) | 0 (0%) | 0 (0%)

PR | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%)

EGFR | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%)

TWIST1 | 0 (0%) | 1 (3.4%) | 0 (0%) | 2 (16.7%)

VEGF | 0 (0%) | 3 (10.3%) | 5 (19.2%) | 1 (8.3%)

CD44+/CD24-, | 0 (0%) | 4 (13.8%) | 3 (11.5%) | 1 (8.3%)

ALDH1high/CD24-, | 0 (0%) | 2 (6.9%) | 8 (30.8%) | 1(8.3%)

Conclusions: In-vivo isolation of CTC is minimally invasive, and in combination with high specific and sensitive RT-qPCR assays for CTC detection and molecular characterization seems promising. Comparison studies with the CellSearch and immunofluorescence have shown poor agreement. These results should be validated in large patient cohorts, and in respect to the clinical outcome.

#504

Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are superior to CA 15-3 in predicting tumor burden, patients response to treatment and overall survival (OS) rates in metastatic breast cancer patients from Egypt.

Abdel-Rahman N. Zekri, Abeer A. Bahnassy. _National Cancer Inst. Cairo Univ., Cairo, Egypt_.

Background: Monitoring of tumor burden in patients with metastatic breast cancer (mBC) is crucial to determine response to treatment. In addition to radiological procedures and serological biomarkers, circulating, tumor cells (CTCs) are now used to assess the tumor burden and patients' response to treatment. The circulating cell-free tumor DNA (ctDNA) harboring tumor-specific aberrations has not been properly assessed yet.

Methods: 100 Egyptian patients with locally mBC were assessed for CA 15-3 levels, CTCs number by flowcytometry (FCM) confirmed by RT-PCR (CK and mammaglobin expression), and for ctDNA. The results of the three techniques were compared to the radiographic imaging of tumors, patients response to treatment and overall survival (OS) rates. Paraffin blocks for 40 tumor samples, obtained from the studied patients (20 non-responders and 20 responders) were used to detect p53 gene mutations exons 5-9. CA 15-3 levels and CTC numbers were measured at the same time intervals.

Results: Somatic mutations of p53 were detected in 23/40 (57.5%) sequenced tumors. ctDNA showing the identified mutations in tumor samples, was detected in 78 cases (78%) with high dynamic range. CTCs>4/7.5ml blood were present in 57 out of the 78 (73.1%) ctDNA positive cases, and CA 15-3 was detected in 30 (38.5%) cases. Changes in ctDNA levels and CTCs>4 correlated significantly with the tumor burden (p=0.034), patients response to treatment (p<0.01), and lower OS rates (p=0.034&p=0.01; respectively). CTCs number and ctDNA levels showed higher correlation with changes in tumor burden, compared to CA 15-3 (p<0.001 versus p=0.046). However, ctDNA provided the earliest measure of treatment response in most of the patients (53%).

Conclusions: ctDNA is an informative, highly sensitive and specific biomarker that could be used to monitor tumor burden in mBC. Together with enumeration of CTCs they can predict tumor response and OS in mBC patients with high accuracy.

#505

Optimized 7-Plex cell-free DNA assay for clinically relevant KRAS mutations in colorectal cancer.

Rajeswari Avula, Benjamin R. Kipp, Jesse S. Voss, Keegan E. Haselkorn, W. Edward Highsmith, Kevin C. Halling, Jeremy C. Jones, Steven R. Alberts, Michael B. Campion, Cassandra J. Nelson, Jin Jen, Eric D. Wieben, Julie M. Cunningham, Minetta C. Liu. _Mayo Clinic, Rochester, MN_.

Background: KRAS gene mutations are present in approximately 40% of colorectal adenocarcinomas and predict for nonresponse to the anti-epidermal growth factor receptor antibodies used in routine clinical practice. The most common activating mutations in this malignancy occur in codons 12 and 13 of exon 2. Determination of KRAS mutational status from tumor samples is an essential tool for managing patients with colorectal cancer (CRC) but requires tissue obtained by invasive percutaneous or surgical biopsies. Recent advances allow for the isolation and analysis of cell-free DNA (cfDNA) from the peripheral blood. A highly sensitive multiplex technology that uses low amounts of input DNA is needed to establish cfDNA as a liquid biopsy to personalize care.

Methods: Digital droplet PCR (ddPCR) is a particularly sensitive method for detecting rare and low copy targets. Related work led to 7 individual assays, and to companion 4-plex and 3-plex assays, to accommodate the 7 common KRAS mutations and internal controls. A single 7-plex assay is favored to maximize efficiency and minimize cost. We optimized the RainDrop Digital PCR System to collectively detect the G12/G13 KRAS mutations, given that documentation of a mutation (not the specific mutation) is the factor of clinical relevance. Commercially available cell lines and genomic DNA aliquots were used as reference standards and for determinations of sensitivity and limit of blank (LOB). Initial clinical validation samples included tumor DNA derived from FFPE tissue (n=10), cfDNA from healthy donors (n=10), and cfDNA from metastatic CRC patients with tumors of known KRAS mutation status (n=4). All assays were run in triplicate to confirm reproducibility.

Results: Custom Taqman MGB probes were selected for KRAS wild type (WT), G12A, G12C, G12D, G12R, G12S, G12V, and G13D to allow for testing in a single reaction. LOB was 9 per 1x10(6) droplets on the basis of WT controls, including cfDNA samples from healthy donors. Evaluation of cell line DNA, genomic DNA aliquots, and FFPE samples demonstrates that the assay detects mutant KRAS with 100% accuracy and 0.1% mutant DNA in a total input of 10 ng WT/mutant DNA. With the clinical samples, concordant KRAS findings between cfDNA and tissue were obtained for two patients with WT tumors and one patient with a KRAS G12S mutant tumor. cfDNA analysis did not detect the G12V mutation identified in the colon primary of the other patient. Evaluation of additional clinical samples is in progress.

Conclusions: Our 7-plex G12/G13 KRAS assay has excellent accuracy and is able to detect the KRAS mutations of interest at low (0.1%) mutation frequencies. Additional studies are being performed on prospectively collected plasma samples from patients with metastatic CRC of known KRAS mutation status. Large scale correlation between tumor mutation status and cfDNA findings will be performed to validate our assay for clinical implementation.

#506

Post-surgical resection monitoring in early stage colorectal carcinoma patients using a circulating cell-free DNA assay with ultra-high accuracy and specificity.

Stefanie A. Mortimer,1 Katharine Dilger,1 Stephen Fairclough,1 Diana Abdueva,1 Darya Chudova,1 Ankit Sarin,2 Jim Leng,2 Jeeyun Lee,3 Helmy Eltoukhy,1 AmirAli Talasaz1. 1 _Guardant Health, Redwood City, CA;_ 2 _University of California, San Francisco, CA;_ 3 _Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) allows non-invasive real-time profiling of actionable genomic alterations. Liquid biopsy provides an option for disease monitoring in early stage cancer patients post surgical resection, with a potential to aid in adjuvant decision making. However, to be applicable, tests must cover a broad enough genomic footprint to not require a priori knowledge of mutations, have high specificity, and sensitivity higher than conventional methods. NGS is necessary, since inactivating mutations are the most common alteration type in many common cancer types such as colorectal carcinomas (CRC).

Here we present a highly efficient and specific NGS assay for detection of ctDNA in early stage cancer patients, capable of detecting single molecule variants across a 12 kb gene panel with an analytical sensitivity of >0.02% for single nucleotide variants (SNVs) and indels. This panel was applied to a clinical study involving 14 early stage (II/III) CRC patients with both pre- and post-op blood draws (up to 7 days post surgery). A subset (6 patients) also had tumor samples collected at the time of the surgical resection of the tumor.

Overall, the detection rate of ctDNA in pre-op blood draws was 93%. In the post-op blood draws ctDNA was detectable in 43% of cases. The estimated average minor allele frequency (MAF) is 0.58% (± 0.82%) in pre-op, 0.18% (±0.21%) in post-op, and 40% (±18%) in tumor samples. When tumor tissue was available and used as a reference, the clinical sensitivity, specificity, and accuracy in pre-op blood samples were 83%, 99.995%, and 99.99%, respectively. SNVs with MAF as low as 0.04% were confirmed in tissue data. The clinical specificity of variants detected in post-op blood samples using pre-op samples as the reference is 99.996%. Cohort expansion to 50 patients and follow-up for clinical recurrence in both cohorts is ongoing.

In conclusion, we have developed an assay with ultra-high accuracy and specificity, for the detection of ctDNA in early stage CRC patients that is capable of detecting alterations present in the tumor post-surgical resection. This technology allows for a promising non-invasive route for molecular monitoring of residual disease post surgery and for early detection of relapse compared to traditional methodologies.

#507

Prediction of colorectal cancer recurrence with plasma miRNAs in the ColoCare Study.

Brandon Dickinson,1 Scott V. Adams,2 Cornelia Ulrich,3 Kathy Vickers,2 Michelle Wurscher,2 William M. Grady,2 Karen W. Makar4. 1 _University of Washington School of Medicine, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Huntsman Cancer Institute, Salt Lake City, UT;_ 4 _Bill and Melinda Gates Foundation, Seattle, WA_.

Background

Colorectal cancer is the third most common cancer and third most common cause of cancer-related death in the US. Recurrence of primary tumors occurs in 10-40% of patients depending on tumor stage. Early detection of recurrence can increase the treatment options for those eligible for curative therapy, which may lead to better survival. We determined the levels of multiple different plasma miRNAs and assessed their association with clinical outcomes to identify specific miRNAs that may serve as predictors of CRC recurrence.

Methods

Colorectal cancer patients (age 18-80, newly diagnosed, stage I-IV) were recruited as part of the ColoCare Study in Seattle, Washington, between 2007 and 2011. Patients who had a blood sample taken >30 and <365 days after surgery and prior to any recurrence were included in the study (n=83).

Candidate miRNAs (miRNA 31, 141, 200b, 203, 16, 17-3p, 29a, 92a, 125b) were quantified using pre-designed TaqMan microRNA assays in triplicate assays. Plasma miRNA expression was normalized and expressed as copy number. Cox proportional hazards regression with robust standard errors was used to estimate hazard ratios of recurrence associated with level of each miRNA, adjusted for age, sex and tumor site and stratified by stage. No recurrences were observed in stage I patients. Statistical analyses were conducted separately for stages II-III and II-IV colorectal cancer.

Results

During a median of 1,067 days (33-2,254 days) follow-up after surgery, 20 recurrences were observed. In stage II-III patients, the presence of detectable circulating levels of miR-141 (HR=4.30 [1.10-16.84]) or miR-203 (HR=8.04 [1.39-46.4]) were associated with risk of recurrence. In stage II-IV patients, detectable miR-203 (HR=3.52 [1.13-10.96]) was associated with recurrence, and elevated miR-29a levels were associated with greater risk of recurrence (HR: 1.80 per natural log unit, 95% CI: 1.06-3.04).

Conclusion

The present study identified multiple plasma miRNAs that may help to predict those patients at greatest risk of colorectal cancer recurrence. Our results require validation in order to determine the role for such plasma markers in clinical practice.

#508

Prognostic value of circulating tumor DNA in advanced colorectal cancer patients: quantification of hypermethylated or mutant sequences using picoliter-droplet digital PCR.

Fanny Garlan,1 Pierre Laurent-Puig,2 Nathalie Siauve,3 Audrey Didelot,4 Geraldine Perkins,5 Hélène Blons,6 Julien Taieb,7 Valerie Taly,8 Aziz Zaanan9. 1 _Université Paris Sorbonne Cité; INSERM UMR-S1147; CNRS SNC5014, Paris, France;_ 2 _Université Paris Sorbonne Cité; INSERM UMR-S1147/ Department of Biology, European Georges Pompidou Hospital, AP-HP, Paris, France;_ 3 _Department of Medical Imaging, European Georges Pompidou Hospital, AP-HP, Paris, France;_ 4 _Université Paris Sorbonne Cité; INSERM UMR-S1147, Paris, France;_ 5 _Université Paris Sorbonne Cité; INSERM UMR-S1147/ Department of Digestive Oncology, European Georges Pompidou Hospital, AP-HP, Paris, France;_ 6 _UMR 1147 - Univ. of Paris-Descartes/ Department of Biology, European Georges Pompidou Hospital, AP-HP, Paris, France;_ 7 _Hepatogastroenterology and Digestive Oncology Department, Hôpital Européen Georges Pompidou, Université Paris Descartes, Paris, France;_ 8 _UMR 1147 - Univ. of Paris-Descartes, Paris, France;_ 9 _UMR 1147 - Univ. of Paris-Descartes/Department of Digestive Oncology, European Georges Pompidou Hospital, AP-HP, Paris, France_.

Background: Circulating tumor DNA (ctDNA) has been suggested as a new marker in different cancer types including colorectal cancer (CRC). Its prognostic role needs to be validated in prospective clinical studies using precise and robust procedures. In this context, picoliter-droplet digital PCR has arisen as a highly sensitive and quantitative approach offering a broad dynamic range of detection.

Patients and methods: All consecutive advanced CRC patients receiving first-line chemotherapy were included in this monocentric prospective study. Plasma samples were collected before the 1st cycle of chemotherapy, then at 14 and 28 days after the beginning of the chemotherapy. Both, carcinoembryonic antigen (CEA) dosages and computed tomography (CT) were performed at baseline and every 8 weeks. For each patient, tumor DNA from biopsies was tested for the presence of KRAS, BRAF and TP53 mutations either by conventional qPCR or Next-Generation Sequencing. When no mutation was identified in the tumor, ctDNA was screened for hypermethylated sequences of WIF1 and NPY genes. CtDNA sequences (mutated or hypermethylated) were quantified (concentration, ng/mL) using picoliter-droplet digital PCR coupled to Taqman probes. Associations of ctDNA concentration with progression-free survival (PFS) and overall survival (OS) were analysed using a Cox proportional hazards model. Multivariate models were adjusted on age, gender, Kohne's score, CEA and type of genetic alteration.

Results: Up to now 43 patients were included (mean age: 66±1.8years [62.6-69.6], gender ratio: M/F 1.15). At baseline ctDNA was detectable in 38/43 (88%) patients based on KRAS, BRAF or TP53 mutation (n=27), or hypermethylation of the WIF1 or NPY genes (n=16). Values ranged from 0 to 208 ng/mL, mean and median ctDNA concentration were 12.7 ng/mL and 2.05 ng/mL, respectively. After adjustment on prognostic covariates, the concentration of baseline ctDNA was significantly positively associated with a worse PFS (HR: 1.01 CI95% [1-1.02]; padj=0.03) and OS (HR: 1.02 CI95% [1.01-1.035] padj=0.004). The median PFS were 8.6 and 2.3 months in patients with less or more than 5 ng/mL of baseline ctDNA, respectively (HR: 6.7, CI95% 2-22; padj=0.001). When ctDNA concentration was considered at 14 or 28 days, patients with a ctDNA ≥ 0.2 ng /mL have a significantly worse PFS (HR: 4.12 CI95% [1.41-12.0]; padj=0.009) than the others independently of the baseline concentration of ctDNA.

Conclusion: This study suggests that ctDNA is a strong prognostic factor highlighting the clinical relevance of quantifying circulating tumor DNA by picoliter-droplet digital PCR in first-line advanced colorectal cancer treatment.

#509

HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant colorectal cancer.

NAOKI TAKEGAWA. _Department of Medical Oncology Kinki University Hospital Cancer Center, Osaka-shi, Japan_.

Background: Patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS benefit from epidermal growth factor receptor (EGFR)-targeted therapy. However, patients who are treated with anti-EGFR antibodies will eventually develop the resistance to those agents. We previously found that HER2 amplification was one of the mechanisms conferring resistance to anti-EGFR antibody therapy and could therefore be a potential therapeutic target (Yonesaka et al, STM 2011). The aim of this study was to detect HER2 amplification in circulating tumor DNA (ctDNA) from patients with CRC and acquired resistance to anti-EGFR antibody therapy.

Methods: We analyzed plasma ctDNA using digital polymerase chain reaction (PCR) from 18 patients with CRC, who had been treated with anti-EGFR antibody-based therapy (cetuximab) and subsequently acquired resistant cetuximab. We aimed to obtain at least 100 droplets for HER2 and EFTUD2 to accurately assess the ratio. Digital PCR data were analyzed using the QuantaSoft analytical software package (Bio-Rad). The copy number concentration of each gene (HER2 and EFTUD2) was estimated from the Poisson distribution. HER2 amplification with digital PCR was defined as a HER2 ratio (HER2/EFTUD2 copy number ratio) of 1.25 accordingly (Gevensleven H et al, CCR 2013). HER2 gene copy number was analyzed using fluorescence in situ hybridization also in tumor samples before and after acquisition of resistance to cetuximab-based therapy.

Results: Our data showed various HER2 copy number in plasma ctDNA from CRC patients (median: 1.09; range: 0.94-5.18). 22% (4/18) of patients in the cohort exhibited HER2 amplification. One of these patients was found to be positive for HER2 amplification in matched tumor specimens collected after cetuximab therapy, at which point the

patient had acquired cetuximab resistance, despite being negative for HER2 amplification prior to therapy ( pre: 1.14, post: 2.78).

Conclusion: Analysis of plasma ctDNA by digital PCR could be useful for detecting HER2 amplification in patients with CRC who were resistant to anti-EGFR antibody therapy. We are now conducting another, large-scale study for evaluating HER2 amplification by ctDNA in CRC patients using digital PCR.

#510

A novel circulating cell free DNA-based assay in colorectal cancer patients during treatment with systematic chemotherapy.

Toshiaki Toshima,1 Takeshi Nagasaka,1 Yoshiko Mori,1 Takashi Kawai,1 Tomokazu Fuji,1 Fumitaka Taniguchi,1 Keisuke Kimura,1 Kazuya Yasui,1 Hiroyuki Kishimoto,1 Yuzo Umeda,1 Hiroshi Tazawa,1 Ajay Goel,2 Toshiyoshi Fujiwara1. 1 _Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayamashi, Japan;_ 2 _Center for Gastrointestinal Cancer Research; Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, TX_.

Introduction: Although circulating cell-free DNA (cfDNA) in blood plasma is being touted as a frontier noninvasive approaches, its clinical utility still remains questionable. The purpose of this study was to compare the efficacy of cfDNA by comparing blood CEA levels and radiological evaluation in patients with unresectable metastatic colorectal cancer (mCRC) who received systemic chemotherapy.

Experimental Procedures: In this study, we measured aberrant cancer-specific methylation in cfDNA and the concentration of cfDNA in plasma obtained following each treatment cycle of systemic chemotherapy in three patients with mCRC. To analyze aberrant cancer-specific methylation, we used a modified highly sensitive assay for bisulfite DNA followed by fluorescence-based PCR, as reported previously (JNCI 2009). This methodology can detect methylation status in eight regions, therefore both recovery score (RS) and methylation score (MS), ranged from 0-8 at a given time. We measured RS and MS two-times in each plasma specimen obtained before administration of systemic chemotherapies.

Results: In this pilot study, we examined a series of blood plasma obtained from three patients who received oxaliplatin-based chemotherapy together with molecularly-targeted agents. Despite initial tumor shrinkage in the metastases, all patients ultimately developed progressive disease (PD). Patient1 had wild-type KRAS, but had developed a sigmoid colon cancer with synchronous multiple liver and lung metastases. In contrast, Patient2 had mutant KRAS with sigmoid colon cancer and synchronous multiple liver metastasis. Both patients 1 and 2, demonstrated decreasing levels of CEA after the first-line chemotherapy, along with low methylation scores and concentration of cfDNA. Interestingly, in both patients, MS and concentration level of cfDNA increased prior to radiographic documentation of PD. Patient3 harbored BRAF V600E mutation, and a cancer in the ascending colon with systemic lymph node metastasis. Although, in this case, the tumor development progressed rapidly, similar to patients 1 and 2, MS and the concentration levels of cfDNA also increased prior to radiographic documentation of rapid PD.

Conclusions: Our novel DNA methylation and concentration-based monitoring assay is a novel methodology for capturing DNA methylation in circulating cell-free DNA in plasma, and is useful for the early identification of colorectal cancer patients who are at risk of developing PD prior to radiographic documentation.

#511

Unbiased enrichment of circulating tumor cells directly from whole blood.

Carrie E. Peters, Danay Maestre-Battle, Steven M. Woodside, Terry E. Thomas, Allen C. Eaves. _STEMCELL Technologies Inc., Vancouver, British Columbia, Canada_.

Enrichment of rare circulating tumor cells (CTC) in peripheral blood is required prior to most analytic procedures. The ideal enrichment method would be rapid (<30 minutes (min)), permit a high recovery of viable CTC, and would be independent of surface marker expression, since CTC in the peripheral blood may be undergoing epithelial-mesenchymal transition and may not express epithelial markers. We have developed a new immunomagnetic method (EasySep™ Direct) that fulfills these criteria and can be used directly with whole blood without any preparatory steps. The method was tested using whole blood samples seeded with the adenocarcinoma cell line CAMA to a final concentration of approximately 1%. The CAMA cells were then enriched using either EasySep™ Direct CTC Enrichment (immunomagnetic separation) or RosetteSep™ (immunodensity based control). For immunomagnetic enrichment, the samples were incubated with EasySep™ Direct Enrichment Cocktail for 5 min, then EasySep™ Direct RapidSpheres™ were added and the sample placed in a magnet for 10 min. Magnetically labeled cells were retained in the magnet and the unlabeled cells poured or pipetted off. Rapidspheres™ were added again and the magnetic incubation and pour-off steps repeated. To perform RosetteSep™ enrichment, the samples were incubated with either the CD45+ cell depletion cocktail or a more extensive hematopoietic cell depletion cocktail for 10 min. Samples were then pipetted onto Lymphoprep in SepMate™ tubes, centrifuged for 10 min at 1200 x g with the brake on, and poured off. The number of cells recovered with each procedure was counted and the depletion of hematopoietic cells (CD45+/EPCAM-), enrichment of CAMA cells (CD45-/EpCAM+), and depletion of red blood cells (RBCs, Glycophorin A+/CD45-) in each sample were evaluated by flow cytometry. EasySep™ Direct was evaluated on five different samples. Each sample was separated on five different EasySep™ magnet formats (5, 14, and 50 mL single tube magnets and 5 and 14 mL multi-tube magnets) with each condition in duplicate; the two RosetteSep™ cocktails were tested on three of the samples, each in duplicate. There was no significant difference in CD45 depletion, CAMA cell recovery, or residual RBC contamination between samples enriched using the different magnets (Least Squares Fit; Prob F>0.05) or pooled and compared to the pooled RosetteSep™ controls (Least Squares Fit; Prob F>0.05). Over all magnet separation conditions and samples, the mean log depletion of CD45+ cells was 2.9±0.4, the mean recovery of CAMA cells was 42±23%, and the residual RBC contamination was ~9000 RBC/mL of start sample. Rare cells were enriched in 25 minutes using EasySep™ Direct and in less than 40 minutes using RosetteSep™ with SepMate™. CTC can be enriched directly from whole blood with either EasySep™ Direct or RosetteSep™. Since enriched cells are unlabeled there is nothing to interfere with subsequent further enrichment, culture, or evaluation.

#512

KRAS mutation in cell-free DNA was correlated with treatment response to gemcitabine/cisplatin chemotherapy in patients with pancreatic cancer.

Min Kyeong Kim,1 Yoon Kyong-Ah,2 Woo Sang Myung,3 Kim Yun Hee,4 Park Sang Jae,3 Kong Sun-Young1. 1 _Department of System Cancer Science, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Republic of Korea;_ 2 _Cancer Genomics Branch, Division of Convergence Technology, National Cancer Center, Goyang, Republic of Korea;_ 3 _Liver and Pancreatobiliary Cancer Branch, Division of Translational & Clinical Research I, National Cancer Center, Goyang, Republic of Korea; _4 _Molecular Imaging & Therapy Branch, Division of Convergence Technology, National Cancer Center, Goyang, Republic of Korea_.

Cell-free DNA (cfDNA) of cancer patients has been known to be a useful marker for minimal residual disease detection and treatment monitoring. However, there are a few studies which showed correlation of cfDNA with pancreatic cancer treatment. Here we investigated applicability of cfDNA as a response and prognosis biomarker through comparing the qualitative and quantitative changes of the KRAS mutant in the patients with pancreatic cancer before and after treatment.

Total of 44 pancreatic cancer patients who treated by gemcitabine and cisplatin included in the study. Serum from the blood samples was separated by centrifugation through the method established. cfDNA was extracted through the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany) from 0.8 ml of serum. Extracted cfDNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). QX200 Droplet Digital PCR System (Biorad, USA) was applied to measure frequency of KRAS mutation. We performed digital PCR with KRAS screening multiplex droplet digital PCR kit which covers G12A, G12C, G12D, G12R, G12S, G12V and G13D sites (Biorad, USA). Mutant concentration and fractional abundance were analyzed by QuantaSoft software (Biorad, USA).

The positive rate of KRAS mutation was 61% (27/44) and 47% (16/34) in before and after treatment. When we analyzed the treatment response according to the positive presence of KRAS mutation before treatment, 50% and 61% of KRAS mutations were observed in patients who showed either partial response (PR) or stable disease (SD) after chemotherapy, respectively. However, all the 5 patients (100%) with progressive disease (PD) showed KRAS mutation implicating significant correlation (p=0.046) with treatment response.

The fraction change of KRAS mutation was showed decrease in 18 (58.1 %) patients and increase in 13 (41.9%) patients comparing before and after treatment status. When we observed the association the fraction change direction (decrease vs. increase) with the survival, there was no significant difference.

These results implicate that KRAS mutation in cell-free DNA might be associated with treatment response to gemcitabine/cisplatin chemotherapy. In further studies, we will conduct in prospective study for pancreatic cancer patients. (This study was supported by National cancer center, Korea, Grant no. 1510203-1)

#513

Standardization of technologies for CTC, ctDNA and miRNA enrichment, isolation and analysis for liquid biopsies during the first year of IMI's CANCER-ID.

Thomas Schlange,1 Nikolas Stoecklein,2 Rui P. Neves,2 Sabrina Pleier,1 Sebastian Bender,1 Nora Brychta,3 Merlin V. Luetke-Eversloh,3 Kiki Andree,4 Leon Terstappen,4 Thomas Krahn,1 Thomas Krahn1. 1 _Bayer Pharma AG, Wuppertal, Germany;_ 2 _University Hospital and Medical Faculty of the Heinrich-Heine University, Dusseldorf, Germany;_ 3 _Bayer Pharma AG, Berlin, Germany;_ 4 _Faculty of Science and Technology, Medical Cell BioPhysics, Enschede, Netherlands_.

Within the European Innovative Medicines Initiative (IMI) consortium CANCER-ID (www.cancer-id.eu), scientists at academic, clinical and industrial sites across Europe and in the US joined forces to evaluate innovative technologies in the field of liquid cancer biopsies. This project aims at implementing standard operating procedures (SOPs) for pre-analytical sample handling, enrichment, isolation and analysis of Circulating Tumor Cells (CTCs), circulating free tumor DNA (ctDNA) and microRNAs (miRNAs) as novel blood-based biomarkers, with a focus on Non-Small Cell Lung Cancer (NSCLC) and HER2-treatment refractory breast cancer. In order to determine sensitivity and specificity of different technologies for CTC isolation and analysis (e.g. detection of mutations, amplifications, protein phosphorylation), complex samples comprising a mixture of NSCLC or breast cancer cell lines spiked in healthy donor blood were distributed to different CANCER-ID partner sites. These cell lines have been selected based on their molecular/genetic properties to reflect clinically relevant subtypes of the disease and have been further characterized in terms of cell-surface marker expression and cell size distribution. The use of complex spiked samples better models the heterogeneity of real-life patient material. Furthermore, healthy donor and patient derived plasma samples are investigated using different technology platforms to validate tumor-specific miRNA or ctDNA profiles that might characterize molecular tumor subtypes. To this end, differences in exosome-derived versus free circulating miRNAs are of special interest. As for CTCs the development of ctDNA and miRNA standards that can be used to compare and validate different technologies are in the focus of this effort. In summary, our results pave the way for the next phase of CANCER-ID, which includes the analysis of cancer patient samples in clinical studies using different technologies and thereby advance the concept of liquid biopsy particularly in indications in which conventional tissue biopsies are difficult to obtain.

### Radiation Oncology

#514

Effect of radiation therapy on breast epithelial cells in BRCA1/2 mutation carriers.

Huai-Chin Chiang,1 Richard Elledge,1 Paula Larson,2 Ismail Jatoi,1 Rong Li,1 Yanfen Hu1. 1 _UT Health Science Center at San Antonio, San Antonio, TX;_ 2 _Methodist Healthcare System, San Antonio, TX_.

Women carrying BRCA1 and BRCA2 mutations have significantly elevated risk of developing breast and ovarian cancers. BRCA1-associated breast cancer likely originates from progenitors of the luminal epithelial lineage. Recent studies indicate that radiation therapy (RT) for BRCA1 cancer patients is associated with lower incidence of developing subsequent ipsilateral breast cancer. In the current study, we analyzed tumor-free breast tissue procured via prophylactic bilateral mastectomy from three BRCA1 and one BRCA2 mutation carriers, who had been previously treated with RT for unilateral breast cancers. Freshly isolated breast cells from the irradiated and non-irradiated breasts of the same individuals were subjected to flow cytometry, using established cell-surface markers. Two out of the three BRCA1 carriers and the BRCA2 carrier exhibited significantly diminished luminal cell population in the irradiated breast versus the non-irradiated side. The remaining BRCA1 carrier showed similar cell abundance between the two sides. Our finding suggests that prior RT could result in preferential depletion of the luminal epithelial compartment and thus reduced incidence of BRCA1-associated breast cancer. Further investigation may inform development of RT-based preventive approaches aimed at specifically decimating the source of BRCA1-associated tumors.

#515

Survivin contributes to radiation resistance in head and neck cancer through regulation of DNA damage repair.

Xu Wang, Jonathan J. Beitler, Wen Huang, Guo Chen, Sreenivas Nannapaneni, Nabil F. Saba, Xingming Deng, Zhuo G. Chen, Dong M. Shin. _Emory University Winship Cancer Institute, Atlanta, GA_.

Background: A major challenge affecting outcomes of patients with squamous cell carcinomas of the head and neck (SCCHN) is radiation resistance. Overexpression of survivin is associated with radiation resistance and poor survival. Our study objectives were to: 1) evaluate the effects of survivin expression level on radiosensitivity in SCCHN cell lines; 2) identify the effect of survivin on DNA damage repair following radiation exposure in SCCHN cells; 3) evaluate the feasibility and efficacy of the combination therapy of survivin inhibitor YM155 and radiation in SCCHN. Methods: SCCHN cell lines, including JHU-022, PCI-15A and Tu686 were used to evaluate the role of survivin in DNA double strand break (DSB) repair following radiation exposure. To determine whether survivin specifically targets the sites of DNA damage, we performed ChIP assays using PCI-15A cells carrying the DR-GFP homologous recombination reporter. The in vitro effects of YM155 on sensitizing resistant cells to radiation were also evaluated using these cell lines. Results: The fraction surviving radiation was about 2.5-fold greater in PCI-15A and Tu686 cells compared with that in JHU-022 cells. As expected, survivin protein levels were elevated in PCI-15A and Tu686 cells compared with JHU-022 cells, suggesting an inverse correlation between survivin expression and radiosensitivity. Clonogenic survival following radiation exposure was inhibited by depletion of survivin in PCI-15A cells by siRNA, and increased by overexpression of survivin in JHU-022 cells. Inhibition of survivin by YM155 enhances the efficacy of radiation in PCI-15A cells. Immunoblotting revealed that radiation induced nuclear accumulation of survivin and its complex with γ-H2X, and DNA-PKCs in PCI-15A cells, suggesting survivin was involved in DNA damage response and repair induced by radiation. ChIP assay further confirmed that survivin specifically targeted and bound to the DNA DSB site, which is consistent with results obtained from immunofluorescence analysis. Conclusions: Survivin is involved in DNA damage response and repair induced by radiation exposure in SCCHN cells. Inhibition of survivin by YM155 enhances the efficacy of radiation, and may provide a novel therapeutic approach to improve the efficacy of radiotherapy in SCCHN. (This research was supported by the National Cancer Institute award P50 CA128613, and GCC Distinguished Cancer Scholar to Dong M. Shin, Zhuo (Georgia) Chen, and Jonathan J Beitler)

#516

The mechanism associated with radio-resistant renal cell carcinoma and its targeting strategy.

Eun-Jin Yun, Chun-Jung Lin, Debabrata Saha, Jer-Tsong Hsieh. _UTSW medical center, Dallas, TX_.

Introduction and Purpose: Although ionizing radiation (IR) represents an effective regimen for most solid tumors, renal cell carcinoma (RCC) is known to be highly radio-resistant and the underlying mechanisms associated with IR-resistance remained elusive. In this study, we unveiled the factors associated with IR-resistance and the potential targeting strategy.

Experimental procedures: Several RCC sublines were generated with different genetic manipulation and cells were irradiated with different doses and the surviving fractions were evaluated in 7 days. To investigate the underlying mechanism, various biochemical and molecular biologic methods, such as quantitative real-time PCR, mass-spectroscopy (MS), immunoprecipitation (IP), western blot, and ELISA were performed.

Results: DAB2IP (DOC-2/DAB2 interactive protein) known as a tumor suppressor that functions as a scaffold protein modulating many signal pathways associated cell growth/survival, apoptosis/autophage, and cell migration/invasiveness. In RCC, the status of DAB2IP has not been characterized. We found DAB2IP was frequently lost in RCC; more than 50% in every subtype of RCC. We observed that DAB2IP-deficient RCC cells exhibited IR-resistance whereas re-introduction of DAB2IP could sensitize RCC cells to IR. From MS and IP, it appeared that DAB2IP could directly interact with poly (ADP-ribose) polymerase-1 (PARP-1). In the presence of DAB2IP, PARP-1 activities were suppressed because DAB2IP was able to induce PARP-1 protein degradation via unique proteasome pathway. Since PARP-1 is commonly involved in many cellular functions such as DNA damage repair or transcription modulation, which is also considered as a therapeutic target particularly IR therapy. In this study, elevated PARP-1 led to rapid DNA repairing and IR resistance in DAB2IP-expressing RCC cells. In contrast, by knocking-down PARP-1 in DAB2IP-deficient RCC cells can sensitize cells to IR, suggesting that PARP-1 inhibitors can be a potential radio-sensitizer.

Conclusion: In summary, this study demonstrates a new functional role of DAB2IP in IR-resistant RCC cells via modulating PARP-1 expression and activities. Therefore, targeting PARP-1 by small molecular inhibitor or gene therapy becomes a potential therapeutic strategy to overcome IR resistant RCC.

#517

An MC1R targeted 225Ac radiopharmaceutical agent for treatment of uveal melanoma.

Narges K. Tafreshi,1 Michael L. Doligalski,2 Darpan N. Pandya,3 Hyun Joo Kil,2 Mikalai Budzevich,1 Thaddeus J. Wadas,3 Mark L. McLaughlin,1 David Morse1. 1 _H.Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _University of South Florida, Tampa, FL;_ 3 _Wake Forest University, Health Sciences, Winston-Salem, NC_.

Prognosis in metastatic uveal melanoma is poor with median survival being less than one year. There is no approved therapy for metastatic disease. Hence, new targeted therapies are needed. The melanocortin 1 receptor (MC1R) is expressed in 94% of uveal melanomas but is not expressed in normal tissues of concern for toxicity. We have developed a MC1R specific ligand (MC1RL), conjugated imaging contrast agents to it, demonstrated high selectivity for MC1R expressing tumors in mice, and demonstrated rapid systemic clearance, without retention in tissues of concern for toxicity. The aim of this study is to use MC1RL as a targeting scaffold for development of a radiopharmaceutical by conjugation of 225Ac chelate. 225Ac is a therapeutic alpha emitting radionuclide. A targeted approach to deliver 225Ac to uveal melanoma without causing toxicity in normal tissues would provide clinicians with a novel and powerful therapeutic option for treatment of metastatic disease.

Here, we conjugated 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) to the MC1R specific scaffold (DOTA-MC1RL) and chelated the nonradioactive surrogate 139La (substitute for 225Ac) and demonstrated high binding affinity to MC1R (0.2 nM Ki). We anticipate that the radioactive conjugates will have comparable affinities. We also synthesized 225Ac-DOTA-MC1RL and showed a radiochemical yield greater than 95% and high radiochemical purity (≥99.8%) as determined by radio-TLC, radio-HPLC, and gamma-counter quantification. Moreover, 225Ac -DOTA-MC1RL showed excellent in vitro stability, i.e. 90% after 10 days in human serum at 37ºC. A maximum tolerated dosage (MTD) study was performed by administration of 0, 9, 18, 28, 37, 56, 74 and 148 kBq of 225Ac-DOTA-MC1RL. Animals were followed for 120 days and there were no signs of altered behavior among the groups. The group that received the highest dosage had a slightly but significantly lower increase in body weight over the course of the study, suggesting that 225Ac-DOTA-MC1RL is tolerated extremely well. Blood work and organ pathology did not show any significant deleterious effects even at the highest dose. In vitro cytotoxicity assays showed significant cell death in uveal and cutaneous cell lines in an MC1R dependent manner. Biodistribution studies showed tumor selectivity and a combination of renal and hepatic clearance with minimal retention in other normal tissues. In vivo efficacy studies in SCID mice bearing MC1R expressing human A375 subcutaneous xenograft tumors showed complete loss of tumor within one week of intravenous administration of 225Ac-DOTA-MC1RL relative to controls that had continued tumor growth.

In conclusion, we have demonstrated radiosynthesis of 225Ac-DOTA-MC1RLwith high yield, purity and stability. In vitro studies demonstrated MC1R dependent cytotoxicity in uveal melanoma cells. In vivo studies demonstrated favorable biodistribution and significant antitumor efficacy.

#518

Ionizing radiation-induced DNA damage response decreases during induced pluripotent and embryonic stem cell differentiation.

Kalpana Mujoo,1 Raj K. Pandita,1 Anjana Tiwari,1 Vijay Charaka,1 Walter N. Hittelman,2 Murlidhar Hegde,1 Haibo Wang,1 Clayton R. Hunt,1 Bhuvanesh Dave,1 Jenny Chang,1 E Brian Butler,1 Tej K. Pandita1. 1 _THMRI, Houston, TX;_ 2 _MDACC, Houston, TX_.

Embryonic as well as induced pluripotent stem cells (ESC/iPSC) have a robust DNA damage response to facilitate efficient repair of DNA strand breaks as well as interstrand cross-links (ICLs). Stem cell abundance in tumors can impact the efficacy of the therapeutic agents, and such agents also have an impact on the normal tissue toxicity. Therefore, it is important to compare DNA damage repair in progenitor stem cells and their differentiated lineages since the normal tissues surrounding the tumor predominantly contain differentiated cells. We therefore asked whether the DNA damage response is altered during differentiation of embryonic as well as induced pluripotent stem cells and found that sensing of ionizing radiations, interstrand cross-linking or hydroxyurea induced DNA damage was minimally altered by cellular differentiation. Furthermore, DNA DSB repair by non-homologous end joining was minimally impacted. However, the homologous recombination of DSB repair or ICL repair was significantly reduced in differentiated cells. In addition, differentiated cells showed a reduction in the frequency of new origins of replication and increased stalled replication forks. Interestingly, similar to ESC or iPSC lines, breast tumor stem cells also exhibited a decrease in DNA damage repair by homologous recombination in differentiated cells. The defect in DNA damage repair by homologous recombination was reversed in de-differentiated cells, supporting the argument that differentiation can influence homologous recombination repair of DSBs as well as repair of ICLs. These studies have implications in modulating the therapeutic efficacy of DNA damage inducing tumor-killing agents.

#519

Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair.

Leslie A. Parsels, David Karnak, Joshua D. Parsels, Zachery Reichert, Jonathan Maybaum, Theodore S. Lawrence, Meredith Ann Morgan. _University of Michigan, Ann Arbor, MI_.

Mutant KRAS is found in approximately 30% of non-small cell lung cancers (NSCLC) and is associated with poor prognosis. Despite the use of radiation (RT) therapy for the treatment of locally advanced disease, local recurrence remains an issue. The observation that mutations in KRAS lead to both replication stress and DNA double-strand breaks (DSBs) suggests these cancers may have a greater reliance on DNA damage response pathways and therefore may be preferentially radiosensitized by therapies targeting DNA repair. In this study, we investigated the combined inhibition of WEE1 and PARP1 using the small molecule inhibitors, AZD1775 and olaparib, respectively as a radiosensitizing strategy in KRAS mutant NSCLC. We began by comparing radiosensitization by AZD1775+olaparib, in KRAS isogenic H1703 lung cancer cells and found that KRAS mutant cells were preferentially radiosensitized (ER, enhancement ratio 1.8) compared to KRAS wild type cells (ER 1.4). Combined WEE1 and PARP1 inhibition also radiosensitized KRAS mutant Calu-6 and NCI-H23 lung cancer cells (ER 1.9 and 1.5, respectively). These findings were further confirmed in vivo: Calu-6 tumor xenografts were significantly radiosensitized by AZD1775+olaparib, as evidenced by an 11 day delay in tumor volume doubling time relative to RT treatment. Given that WEE1 and PARP1 function to prevent and manage replication stress, respectively, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent replication stress. While replication stress did contribute to AZD1775-mediated radiosensitization in Calu-6 cells, as evidenced by pan-nuclear γH2AX staining, and the ability of exogenous nucleosides to protect cells from radiosensitization by AZD1775 alone, nucleosides had little effect on radiosensitization by AZD1775+olaparib. These results suggest that replication stress is not required for radiosensitization by AZD1775+olaparib. As WEE1 and PARP1 both promote repair of radiation-induced DNA damage, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent, unrepaired DSBs. Assessment of the kinetics of DSB repair by γH2AX flow cytometry demonstrated that while total γH2AX levels in cells treated with RT alone had returned to control levels within 24 h, AZD1775+olaparib treatment significantly delayed the resolution of γH2AX following RT, with 44.8% or 46.2% of Calu-6 or KRAS mutant H1703 cells, respectively remaining γH2AX-positive 24 h post-RT. This delay corresponded with the inhibition of radiation-induced RAD51 foci by AZD1775, indicating inhibition of homologous recombination repair. Taken together these data demonstrate the efficacy of combined inhibition of WEE1 and PARP1 with radiation in KRAS mutant lung cancer. Furthermore, these results suggest that although replication stress occurs in response to AZD1775 and olaparib, persistent DSBs are the cause of radiosensitization.

#520

The role of the calpain pathway in early response and acquired resistance to radiation in glioblastoma.

Emily A. Bassett, Arnab Chakravarti. _Ohio State University, Columbus, OH_.

Glioblastoma (GBM) is the most aggressive, malignant brain tumor that is highly heterogeneous and often develops resistance to conventional chemotherapy and radiation treatments. The median survival time of GBM patients is 12-15 months despite aggressive treatment modalities, and there is an urgent need for the development of novel treatment strategies. The goal of this study is to identify novel mechanisms of radioresistance in GBM through mass spectrometry-based phosphoproteomic profiling, with the objective that more effective therapies can be rationally developed in the future. We and others have previously reported that GBMs are characterized by deregulated signaling pathways that control critical cellular processes including survival, migration, and proliferation. Very little is known about acquired resistance mechanisms to radiation, specifically how radiation alters protein phosphorylation patterns in tumors on a global level, or how radiation-induced changes in protein phosphorylation contribute to treatment resistance. In order to identify radiation-induced changes in protein phosphorylation, we performed phosphoproteomic profiling of 6 GBM cell lines and 1 normal human astrocyte cell line both before and after radiation treatment in 3 independent experiments. Specifically, cells were collected before treatment and at 2 time-points following a 10 Gy radiation treatment. Following protein isolation and phosphopeptide enrichment, global phosphoproteomic analysis was performed by mass spectrometry. Relative quantitation and statistical analysis were performed to determine fold changes in phosphorylation at both post-radiation time-points. An average of 3,971 phosphopeptides mapping to 1,649 phosphoproteins were quantified for each independent experiment, and a total of 621 phosphoproteins were quantified across all 3 datasets. We identified a major component of the calpain signaling pathway that consistently shows an increase in phosphorylation following radiation treatment in GBM cell lines. The post-radiation increase in phosphorylation was confirmed in 2 independent experiments and observed across all 6 GBM cell lines. This is the first study linking the calpain signaling pathway to radiation response in GBM. Mechanistic studies using phosphomimetic and nonphosphorylatable mutants of the protein of interest will determine the effect of phosphorylation on calpain activity and radiation resistance. Results of this work will contribute to our understanding of how GBM tumors become resistant to radiation therapy, and may identify a novel target to overcome radiation resistance in GBM.

#521

c-MYC as a differentiating marker between angiosarcoma and atypical vascular lesion.

Ipshita Kak,1 Bekim Sadikovic,2 Guillaume Pare,3 Tom Corbett,4 Snezana Popovic1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _Schulich School of Medicine and Dentistry, London, Ontario, Canada;_ 3 _David Braley Cardiac, Vascular and Stroke Research Institute, Hamilton, Ontario, Canada;_ 4 _Juravinski Cancer Center, Hamilton, Ontario, Canada_.

Introduction: Angiosarcoma is a rare, aggressive malignancy that accounts for less than 1% of all sarcomas, characterized by a dismal 5 year survival of 20-30% at best. In striking contrast, atypical vascular lesion (AVL), which typically occurs secondary to radiation, follows for the most part, a benign course. However, debate rages over the true nature of AVL with reports describing both benign and malignant behavior. Further compounding the issue is the fact that overlapping histological features make the important differentiation between AVL and angiosarcoma difficult, especially on limited biopsy specimens. There have been a number of recent studies of c-MYC expression in vascular tumors in relation to this question, yielding varied results.

Objectives: This pilot study aimed to investigate c-MYC expression in atypical vascular lesions and angiosarcomas (primary and secondary) to evaluate the clinical utility of c-MYC testing as an adjunct to the histological diagnosis.

Methods: A retrospective search for biopsy, resection specimens and internal consult cases with diagnosis of angiosarcoma and/or atypical vascular lesion from January 2008- February 2014 was performed. A total of 32 cases (including controls) obtained after review were stained by dual colour c-MYC copy number probe set. The expression of c-MYC was read by two independent evaluators and final data was collated along with histology findings, follow-up and survival data.

Results: c-MYC amplification was found to be a major differentiating factor (p value: 0.00002, 68% sensitivity, 96% specificity) between AVL and angiosarcoma(median c-MYC expression 1.0 vs. 12.14, 95% confidence interval: 0.8-1.9 vs. 9.2-22.3 respectively). The amplification levels of primary (median c-MYC expression:10.9, 95% confidence interval: 3.8-16.6) and secondary angiosarcoma (median c-MYC expression:13.2, 95% confidence interval: 9.0-25.5) were statistically not found to be significantly different (p=0.07). Although no correlation was found between level of c-MYC amplification and outcome, the study was underpowered to accurately evaluate this relation.

Conclusion: Amplification of c-MYC can be used as a reliable ancillary test to differentiate between diagnostically challenging cases of angiosarcoma and AVL.

#522

Role and regulation of Aurora A in radiation resistance mechanisms in glioblastoma.

Brinda Ramasubramanian, Kamalakannan Palanichamy, Disha Patel, Saikh Jaharul Haque, Arnab Chakravarti. _The Ohio State University, Columbus, OH_.

Glioblastoma (GBM) is the most aggressive tumor of the human brain, which inevitably escapes the standard treatment modalities that include surgery, radiotherapy and chemotherapy. Consequently, the median survival of GBM patients remains 12-15 months despite these aggressive treatments. Therefore, to develop effective therapies, it is important to understand how GBM cells develop therapeutic resistance. Aurora A is a ubiquitously expressed serine/threonine kinase, which plays essential roles in mitotic regulation. This protein kinase, which phosphorylates p53, PP1, CEBP, and histone H3 among other regulators of cell division, is overexpressed in various neoplasms including GBM and invariably associated with poor prognosis. Recent studies have demonstrated that Aurora A contributes to radio-resistance in various solid neoplasm including GBM. Importantly, Aurora A inhibitors are currently in phase I/II clinical trials for solid cancer. Notably, Aurora A inhibitor MLN8237 that crosses the blood brain barrier specifically inhibits Aurora A at concentrations ≤ maximally tolerated dose in animal models and in phase I clinical trials. We found that Aurora A protein level is upregulated by ionizing radiation in GBM cells. This was, in part, mediated by radiation-induced inhibition of 26S-proteosomal degradation of Aurora A. Aurora A was silenced using both lentiviral mediated shRNA expression and a specific Aurora A inhibitor in three different established cell lines, and were treated with 6 gy radiation. We found that pharmacological inhibition and RNAi-mediated knockdown of Aurora A sensitized the GBM cells to radiation therapy resulting in reduced cell proliferation and increased apoptosis. Interestingly, we observed that RNAi-mediated down-regulation of Aurora A was associated with a decrease in X-linked inhibitor of apoptosis (XIAP) expression in GBM cells. We also found that Aurora A and XIAP were co-localized in GBM cells. Taken, together our results reveal a mechanism by which Aurora A confers radio-resistance in GBM cells and further rationalize the use of Aurora A inhibitors as radio-sensitizer in solid neoplasms including GBM.

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

#523

Activating mutations in EGFR abrogate hypoxia-associated radiation resistance in non-small cell lung cancer.

Mohammad Saki,1 Haruhiko Makino,2 Nozomi Tomimatsu,3 Lianghao Ding,3 Michael D. Story,3 Sandeep Burma,3 Chaitanya Nirodi1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _Tottori University Hospital Cancer Center, Tottori, Japan;_ 3 _U.T. Southwestern Medical Center, Dallas, TX_.

Hypoxic tumors tend to be 3-5 times more resistant to ionizing radiation relative to well-oxygenated tumors in part because oxygen is required to stabilize radiation-induced DNA damage. Tumor-adaptive responses also contribute to hypoxia-associated radioresistance but the precise mechanisms are not fully understood. Here we test our evidence-based hypothesis that the epidermal growth factor receptor (EGFR), which is frequently over-expressed in non-small cell lung carcinomas (NSCLCs), has critical roles in a tumor adaptive DNA damage response (DDR) pathway in hypoxic tumors. We find that, despite elevated EGFR activity and pro-survival signaling, hypoxia-associated radioresistance is dramatically reduced in a biologically distinct class of NSCLCs harboring activating mutations, L858R and ΔE746-E750 in EGFR. Hypoxic NSCLCs with EGFR mutations exhibit a unique spectrum of basal and hypoxia-induced DSBs not found in hypoxic wild type EGFR-expressing NSCLCs. These lesions dominate the S-phase of the cell cycle and are linked to collapsed replication forks in hypoxic cells. Replication fork-associated DSBs are repaired in wild type, but not in mutant, EGFR expressing NSCLCs. Microarray gene expression analysis revealed that mutant, but not wild type, EGFR expression results in an altered DDR characterized by dramatic down-regulation of several key components of the HR pathway within 24 hours of exposure to 0.1% oxygen. Our model predicts that NHEJ, not HR, dominates an altered DDR in hypoxic tumors. Elevated EGFR signaling may contribute to this altered DDR state where the EGFR-NHEJ axis becomes critical for hypoxia associated radioresistance and could be a potential target for hypoxic tumor radiosensitization.

## IMMUNOLOGY:

### Genetic Determinants and Regulators of Cancer Immunity

#524

Novel and shared neoantigen for glioma T-cell therapy derived from histone 3 variant H3.3 K27M mutation.

Hideho Okada,1 Gary Kohanbash,1 Kaori Okada,1 Shuming Liu,1 Yi Lin,1 Sabine Mueller,1 Ian F. Pollack,2 Angel M. Carcaboso,3 Yafei Hou1. 1 _UCSF, San Francisco, CA;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _Hospital Sant Joan de Déu Barcelona, Barcelona, Spain_.

Malignant gliomas, such as glioblastoma (GBM) and diffuse intrinsic pontine gliomas (DIPG), are lethal brain tumors in both adults and children. Indeed, brain tumors are the leading cause of cancer-related mortality and morbidity in children. Children with DIPG have median overall survival of 9 to 10 months with current treatment. Recent genetic studies have revealed that malignant gliomas in children and young adults often show shared missense mutations, which encodes the replication-independent histone 3 variant H3.3. Approximately 30 % of overall GBM and over 70% of DIPG cases harbor the amino-acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3. We evaluated whether H3.3-derived peptides that encompass the H3.3 K27M mutation can induce specific cytotoxic T lymphocyte (CTL) responses in human leukocyte antigen (HLA)-A2+ CD8+ T-cells.

Four candidate peptides encompassing different amino-acid positions around the H3.3 K27M mutation were synthesized, and peptide-specific CTL lines and clones were generated from peripheral blood mononuclear cells of HLA-A2+ donors by in vitro stimulation with each of the synthetic peptides. One of the 4 peptides (the H3.3.K27M epitope, hereafter) induced CTL lines which recognized not only the synthetic peptide loaded on T2 cells but also lysed HLA-A2+ DIPG cell lines which endogenously harbor the H3.3.K27M mutation. On the other hand, CTL lines did not react to HLA-A2+, H3.3 K27M mutation-negative cells or HLA-A2-negative, H3.3 K27M mutation+ cells. Furthermore, CTL clones with high and specific affinities to HLA-A2-H3.3.K27M-tetramer were successfully obtained, and α- and β-chain cDNAs from a high-affinity T cell receptor (TCR) were cloned into a lentiviral vector. Human T-cells transduced with the TCR demonstrated an antigen-specific but CD8-independent activation, which has been often observed with high-avidity TCRs. Furthermore, alanine scanning revealed the key amino-acid sequence motif in the epitope which was responsible for the TCR reactivity. Available human protein data bases indicate that the motif is not shared by any known human protein.

These data provide us with a strong basis for developing peptide-based vaccines as well as adoptive transfer therapy using autologous T-cells transduced with the TCR.

#525

Higher mutation rate is associated with more frequent expression of cancer/testis antigens in human tumors.

Junping Jing,1 Patrick Mayes,1 Yan Degenhardt,1 James R. Brown,1 Philippe Sanseau2. 1 _GlaxoSmithKline, Collegeville, PA;_ 2 _GlaxoSmithKline, Stevenage, United Kingdom_.

Tumors with high mutation rate such as those of melanoma and non-small cell lung cancers (NSCLC) have shown greatest response to the emerging immune therapies. These tumors harbor a large number of somatic mutations, some of which could be translated into neoantigens, potentially evoking host immune response. To what degree self antigens, many of which are cancer testis (CT) antigens, contribute to enhanced immune response in these tumors has never been systematically studied. Here we show that cancers with higher mutation rate frequently express a larger number of CT antigens than cancers with fewer mutations. In NSCLC, tumors with more than 300 non-synonymous mutations (75th percentile, n=158) express more than 3 times as many CT antigens as those tumors with fewer than 100 mutations (25th percentile, n=162), with a P-value of 1.6e-13. In head and neck cancers, tumors with more than 140 non-synonymous mutations (75th percentile, n=79) express nearly twice as many CT antigens as those tumors with fewer than 60 mutations (25th percentile, n=80), with a P-value of 0.004. Additionally, in NSCLC, tumors with high mutation rate are associated with higher expression of CD8B and granzyme B, markers representing cytotoxic lymphocytes. Tumors with more expressed CT antigens are also associated with higher expression of these markers. These data suggest that CT antigens, along with neoantigens, may contribute to a stronger host immune response in these hypermutative tumors.

#526

Identification of novel cancer immunotherapy targets using Sleeping Beauty mutagenesis.

Laura M. Rogers, Adam J. Dupuy, George J. Weiner. _University of Iowa, Iowa City, IA_.

Immune checkpoint blockade antibodies (eg. anti-CTLA-4 and anti-PD-1) enhance T cell anti-tumor activity and have produced exciting and durable results in treatment of a number of cancers including melanoma. Unfortunately, response to first generation checkpoint blockade therapy is limited to a subset of patients. A more comprehensive understanding of the genes and molecules involved in T cell checkpoint control and other aspects of anti-tumor T cell activity may allow a wider patient population to benefit from this exciting and new approach to cancer therapy.

To this end, we performed a forward genetic screen to identify gene pathways that influence intratumoral T cell infiltration using Sleeping Beauty (SB) mutagenesis. More specifically, this genetic T cell screen was designed to identify additional genes and molecules responsible for selection and expansion of intratumoral T cells via a variety of T cell processes including T cell receptor (TCR)-mediated activation, clonal expansion of tumor-specific T cells, T cell trafficking into the tumor, and maintenance of prolonged viability once there. Advantages of using SB in a genetic screen include the ability to cause both gain- and loss-of-function mutations, as well as mutagenesis of the entire genome followed by easy identification of insertion sites. In addition, SB screens are performed in vivo, and thus preserve the complexity of the tumor microenvironment.

We generated a pilot cohort of mice (n=12) with SB-mutagenized endogenous T cells. These mice were challenged with syngeneic B16F0 melanoma cells subcutaneously. After tumor development (21 days after tumor challenge), tumor-infiltrating CD4+ and CD8+ T cells were harvested. Splenic T cells representing unselected CD4+ and CD8+ populations were also isolated from the same animals at time of tumor harvest. Harvested T cells were evaluated by high throughput sequencing to identify SB insertion sites. T cells harvested from tumors demonstrated a decrease in clonal insertion sites compared to splenic T cells suggesting intratumoral clonal selection. Moreover, clonal insertions in intratumoral T cells were significantly enriched in or near genes, signifying likely selection for insertions impacting gene function (p<0.000003, Fisher's exact test). Clonal insertion sites in T cells from tumors that were absent from spleens, representing potential immunotherapy targets and including insertion sites in genes known to be associated with the T cell response, were identified. We are currently in the process of expanding this experimental cohort using a larger number of mice.

We conclude T cell specific SB mutagenesis has the potential to identify novel molecules that influence intratumoral T cell infiltration and can be used to identify additional genes that may contribute to the anti-tumor T cell response.

#527

Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition.

Mario Kuttke,1 Emine Sahin,1 Julia Pisoni,1 Sophie Percig,1 Andrea Vogel,1 Daniel Kraemmer,1 Leslie Hanzl,1 Julia Stefanie Brunner,1 Hannah Paar,1 Klara Soukup,2 Angela Halfmann,2 Alexander Dohnal,2 Carl-Walter Steiner,1 Stephan Blüml,1 Jose Basilio,1 Bernhard Hochreiter,1 Manuel Salzmann,1 Bastian Hoesel,1 Günther Lametschwandtner,3 Robert Eferl,1 Johannes Schmid,1 Gernot Schabbauer1. 1 _Medical University of Vienna, Vienna, Austria;_ 2 _St. Anna Children's Cancer Research Institute, Vienna, Austria;_ 3 _Apeiron Biologics AG, Vienna, Austria_.

In the current study we are investigating the effects of PTEN-deficient myeloid cells on tumor immune surveillance. We could previously show that hyper-activation of the PI3K signaling cascade by genetic knock-out of the counteracting phosphatase PTEN induced an anti-inflammatory phenotype in myeloid cells. This resulted in protection of conditional knock-out mice in models of acute infection and inflammation.

A reduction in pro-inflammatory responses could however increase tumor burden. To address this question we induced colitis associated colon cancer in conditional PTEN-KO mice and found an increase in tumor burden and a reduction in survival in male KO mice. This was accompanied by increased numbers of splenic antigen-presenting cells (APC) expressing the immune checkpoint regulators PD-L1 and PD-L2. Furthermore, transcriptome analysis in these cells revealed a shift towards gene expression profiles found in professional APCs capable of cross-presentation. As expected, ex-vivo stimulated T-cells from KO-mice showed a reduction in proliferative capacity. These findings were further substantiated by findings in a second tumor model using implanted B16 melanoma cells. In this model myeloid PTEN-deficient mice showed a decrease in T-cell activation and a reduction in melanoma cell killing capacity.

Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin increases splenic APCs expressing immune checkpoint regulators resulting in a decrease in tumor immune surveillance. Our study shows that PI3K-inhibitors which are currently tested as anti-cancer drugs might have additional beneficial effects on immune cells by shifting their inflammatory phenotype.

#528

Identify and prioritize candidate neoantigens from cancer exome sequencing with unmatched accuracy.

James White, Sam Angiuoli, Mark Sausen, Sian Jones, Lisa Kann, Manish Shukla, Maria Sevdali, Victor Velculescu, Luis Diaz, Theresa Zhang. _Personal Genome Diagnostic, Baltimore, MD_.

Somatic, nonsynonymous genetic alterations present in cancer can lead to the formation of novel protein sequences and thus production of immunogenic "non-self" neoantigens. Some of those neoantigens will be processed, presented on MHC molecules, and induce tumor-specific T cell responses. Because neoantigens play central roles in the cancer-immunity cycle, it is critical to identify the most potent immunogenic neoantigens effectively and accurately.

Combining PGDx's highly accurate cancer exome analyses (CancerXome™ ) with in silico neoantigen prediction, we have launched ImmunoSelect-R™ that identifies and prioritizes the most relevant mutation-derived neoantigens. To ensure detection of true somatic mutations and prevent false positive mutations from confounding neoantigen identification, ImmunoSelect utilizes CancerXome that delivers unparalleled cancer whole exome sequencing accuracy, achieving 95% sensitivity and 97% positive predictive value at 10% mutant allele frequency with 150x coverage. ImmunoSelect also provides accurate HLA typing from whole exome sequencing with >99.9% sensitivity and specificity. Once exome-based mutations and novel open-reading-frames are identified and HLA genotypes defined, ImmunoSelect utilizes state of art bioinformatics pipelines for prediction and prioritization of the most relevant neoantigens. When applied to a set of experimentally validated neoantigens, ImmunoSelect identified 18 out of 19 of them as being strong neoantigen candidates, suggesting a sensitivity of greater than 90%. Moreover, ImmunoSelect consistently ranked experimentally validated neoantigens within top 20% of all neoantigen candidates derived from whole exome sequencing.

In summary, ImmunoSelect is able to identify and prioritize candidate neoantigens from cancer exome sequencing results effectively and accurately, enabling personalized cancer vaccine development, adoptive T-cell transfer, and prediction of response to checkpoint inhibitors

#529

A novel kinase connection between oncogenic signaling and myeloid-derived suppressor cell functions in tumor evasion.

Jingying Zhou,1 Man Liu,1 Hanyong Sun,2 Zhiwei Chen,3 Alfred Sze Lok Cheng1. 1 _School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 3 _AIDS Institute and Department of Microbiology, Research Center for Infection and Immunity, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong_.

Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of immature myeloid cells that suppress anti-tumor immune responses. The accumulation of CD33+HLA-DR- MDSCs correlates with tumor stage and metastatic burden in various cancers including hepatocellular carcinoma (HCC) (1). Cancer cells can secrete a variety of cytokines and chemokines to facilitate the peripheral expansion and tumor infiltration of MDSCs. However, the cancer cell-specific signaling cascades that promote MDSC expansion remain poorly understood. We previously demonstrated that cell cycle-related kinase (CCRK) acts as a new oncogenic signaling hub in hepatocellular proliferation and transformation (2-4). To investigate whether CCRK regulates tumor microenvironment in HCC, we determined the role of CCRK signaling in the crosstalk between cancer cells and MDSCs.

Our results showed that treatment of human peripheral blood mononuclear cells (PBMCs) with conditional medium of CCRK-over-expressing immortalized hepatocytes and HCC cells resulted in expansion of CD33+ HLA-DR-MDSCs with increased inhibitory effects on T cell responses. In contrast, knockdown of CCRK in hepatic cells downregulated MDSCs. Cytokine profiling analysis revealed that CCRK significantly induced hepatocellular interferon-6 (IL-6) expression and release, which mediated MDSC expansion as shown by IL-6 interfering experiments. Mechanistic studies demonstrated that CCRK triggered nuclear factor-kappa B (NF-κB) signaling in an enhancer of zeste homolog 2 (EZH2)-dependent manner. Simultaneously, the phosphorylation of p65 by CCRK facilitated the co-occupancy of IL-6 promoter by NF-κB-EZH2 complex for transcriptional activation. Using a Hepa1-6 orthotopic HCC model in immune-competent C57BL/6J mice, we showed that knockdown of Ccrk significantly decreased hepatic tumorigenicity and the levels of circulating and tumor-infiltrating MDSCs as well as related T cell responses. Notably, adoptive transfer of MDSCs rescued the effects of Ccrk knockdown. In a complementary experiment, we found that MDSC depletion by anti-Gr-1 antibody significantly reduced CCRK-induced tumorigenicity. Furthermore, transgenic over-expression of CCRK in mouse liver led to activation of EZH2/NF-κB signaling and elevated circulating levels of MDSC. As we also showed concordant over-expression of CCRK, IL-6 and MDSC markers (CD33, Arg-I) in human HCCs, our results uncover CCRK to be a critical immune regulator to promote MDSC, thereby providing a new mechanistic link between aberrant oncogenic signaling and tumor evasion for therapeutic exploitation.

Acknowledgement: This project was supported by the University Grants Committee through the Collaborative Research Fund C4017-14G.

References

1) Marigo I., et al., 2010. Immunity.

2) Feng H., et al., 2011. J Clin Invest.

3) Yu Z., et al., 2014. Gut.

4) Feng H., et al., 2015. J Hepatol.

#530

Location of oncogene expression within a stratified squamous epithelium drives distinct B and CD4 T-cell crosstalk to dictate the tumor immune response.

Michael A. Podolsky,1 Carrie J. Oakes,1 Andrew Gunderson,2 Kyle Breech,1 Jacob Bailey,1 Adam B. Glick1. 1 _The Pennsylvania State University, State College, PA;_ 2 _Oregon Health & Science University, Portland, OR_.

Tumors from stratified epithelium contain proliferating and differentiating compartments, but it is not clear how tumor cells in different layers alternatively engage the immune system. We have established two doxycycline inducible Ras models in which oncogenic RasV12G is targeted to either the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or suprabasal differentiating cells with an Involucrin-tTA transgene (InvRas). On threshold doxycycline levels yielding similar tumor numbers and Ras expression over 30 days, mice with basal cell targeted Ras developed focal squamous cell carcinoma while suprabasal targeting caused benign squamous papilloma formation. On a Rag1-/- background InvRas mice developed fewer tumors that regressed over time while K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Depletion and adoptive transfer studies revealed that naïve B and CD4 T cells together, but not alone, suppressed tumor formation in K14Ras mice but restored tumor numbers in InvRas mice. Tumors developing in K14Ras mice showed a loss of proinflammatory CD4 T and B cells and increased percentages of regulatory cells infiltrating the tumor tissue. In vivo cotransfers show that B and CD4 T cells reciprocally prime each other towards a regulatory phenotype in the K14Ras tumor microenvironment, but in the InvRas tumor microenvironment proinflammatory CD4 T and B cell phenotypes result. Coculture of tumor-conditioned B cells with stimulated naïve CD4 T cells showed an importance of direct contact and CD40/CD40L interactions for the generation of regulatory T cells (Tregs) by B cells in K14Ras mice. In contrast, tumor-conditioned B cells from InvRas mice support generation of proinflammatory CD4 T cells, and antagonize Treg development. This function is restricted to tumor-conditioned B cells, as splenic B cells from tumor-bearing mice had no effect on CD4 T cell phenotype. In vivo blockade of CD40L in K14Ras mice resulted in significantly increased tumor counts as well as reduced B and Treg prevalence. Blockade of CD40L in InvRas mice significantly reduced tumor number, increasing Treg cell count and decreasing neutrophil infiltration into the tumor tissue. Time-course experiments suggest a protective role of Tregs in late stages of K14Ras tumors, and a tumor-promoting role of proinflammatory CD4 T cells in InvRas tumors. Thus, basal/stem cell expression of Ras provokes a regulatory cell inducing microenvironment, suppressing tumor-promoting inflammation in late-stage tumors. Ras expression in differentiating cells activates a tumor promoting proinflammatory phenotype in B and CD4 T cells without provoking immunosurveillance. Taken together, these data show that tumor cell position within a stratified epithelium differentiation hierarchy provokes distinct B and CD4 T cell interactions with opposing effects on tumor development.

#531

The landscape of immunostimulatory RNA transcription.

Alexander Solovyov,1 Antoine Tanne,1 David Ting,2 Arnold Levine,3 Nina Bhardwaj,1 Benjamin Greenbaum1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Institute for Advanced Study, Princeton, NJ_.

Recent studies have demonstrated an unexpected connection between aberrant transcription of noncoding RNA (ncRNA) in tumors and innate immune system activation in the tumor microenvironment (TME). Such ncRNA are often of unknown function and may consist of typically silenced interspersed elements, satellite repeats, and endogenous retroviruses (ERVs). For instance, satellite RNA from the pericentromere (particularly HSATII) has been shown to be abundantly transcribed in several solid tumors, including the often lethal pancreatic ductal adenocarcinoma, yet is virtually silent in normal tissue. The genomic DNA repetitive regions this RNA is transcribed from frequently expand during tumerigenesis. Using a novel computational approach, we have shown that, unlike ncRNA expressed under normal conditions, a set of such repetitive elements, abundantly expressed in tumors, display patterns typically associated with viruses. We therefore predicted they are immunogenic, particularly HSATII. In a novel, theory-experiment collaboration between the laboratories of Professors Benjamin Greenbaum and Nina Bhardwaj, the most significant set of these ncRNA have been validated as immunogenic (HSATII and murine GSAT), capable of activating antigen presenting cells - HSATII stimulated production of IL-6, IL-12 and TNFalpha (Tanne, et al., PNAS, 2015).

At the same time a set of recent papers has shown that ERV transcription may be a predictor of immunotherapy response in melanoma. Hence, it is critical to profile key "immunogenic" ncRNA (i-ncRNA) in the tumor microenviroment, understand which immune pathways different sets of i-ncRNA activate, and assess the link between the specific pathways activated, prognosis, and immunotherapy. Our preliminary work on melanoma transcriptomes has shown that activation of endogenous elements implies better prognosis. We further profile the landscape of activation for i-ncRNA candidates. We have currently demonstrated a key set of ncRNAs preferentially expressed in cancer cells have sequence features that are immunostimulatory in humans. Quantitatively, our analysis uses a new, unique set of computational methods to study the innate immune system in the TME, originally designed to find motifs in viruses targeted by innate immune receptors (Greenbaum, et al., PNAS, 2014). Our methods for motif characterization utilize a novel mathematical approach we created based on transfer matrix approaches in statistical physics, and are here utilized for the purpose of discovering immunogenic nucleic acids in the tumor microenvironment. They offer several advantages over previous approaches. In particular they are far more computationally efficient, allowing the discovery of anomalous, potentially immunostimulatory patterns of motif usage in far larger sequence datasets than was previously practical.

#532

Impact of APOBEC mutagenesis on the immune microenvironment of lung adenocarcinoma (LUAC).

Edwin R. Parra,1 Pan Tong,1 Humam Kadara,1 Vassiliki Papadimitrakopoulou,1 Julie Izzo,1 Jaime Rodriguez-Canales,1 Carmen Behrens,1 John D. Minna,2 Jing Wang,1 Ignacio I. Wistuba,1 John V. Heymach,1 Ferdinandos Skoulidis1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _UT Southwestern Medical Center, Dallas, TX_.

Introduction: It is currently unknown how specific mutational processes that sculpt the genome of non-small-cell lung cancer (NSCLC) impact the tumor immune micro-environment. APOBEC-mediated cytidine deamination has emerged as a major source of somatic mutation across several epithelial malignancies, including NSCLC. Among the 11 APOBEC family members, APOBEC3B (A3B) is overexpressed in several cancers and has been considered the dominant mutator deaminase. However, recent data have also implicated APOBEC3A (A3A) as an important contributor to tumor somatic hypermutation. In this study, we evaluated the relationship(s) between A3B/A3A expression and the immune contexture of LUAC.

Methods: Our datasets include two large cohorts of chemotherapy-naïve, early stage LUAC: a) the TCGA collection of 431 LUACs with available RNASeq data and b) the PROSPECT dataset of 183 LUACs with available array-based mRNA expression data (Illumina platform) and automated IF-based quantitative analysis of immune cell sub-populations for 107 tumors included in a tissue micro-array (TMA). We have further extended our studies to include two datasets of metastatic, platinum-refractory LUACs from the BATTLE-1 and BATTLE-2 clinical trials of targeted therapy. Finally, we have analyzed an in-house panel of 78 comprehensively annotated NSCLC cell lines.

Results: Expression of A3B correlated significantly with the expression of CD274 (PD-L1) in both the TCGA (r=0.25, P=1.49e-07) and PROSPECT (r=0.168, P=0.023) cohorts, as well as in our panel of NSCLC cell lines (r=0.283, P=0.012). The correlation was stronger among KRAS-mutant LUACs (r=0.462, P=7.64e-08, TCGA). Within co-mutation defined subgroups of KRAS-mutant LUAC, KP (KRAS;TP53) tumors expressed significantly higher levels of A3B mRNA (P=4.41e-08, ANOVA). When data were dichotomized into top and bottom tertiles for A3B mRNA expression, A3B-high tumors expressed significantly higher levels of CD274 mRNA in both the TCGA (P=3.32e-08, t-test) and PROSPECT (P=0.0096, t-test) cohorts. Importantly, A3B-high tumors displayed increased density of intra-tumoral CD8+ T-lymphocytes (P= 0.03, Mann-Whitney test). Analysis of chemo-refractory tumors from the BATTLE trials and assessment of the impact of the A3B deletion polymorphism are underway and updated results will be presented at the meeting.

Conclusions: Elevated expression of A3B is associated with a "hot" tumor immune micro-environment with engagement of PD-1/PD-L1 axis immune checkpoints. Distinct mutagenic processes may shape the tumor immune microenvironment and present opportunities for targeted intervention with immunotherapy.

#533

Accurately identifying expressed somatic variants for neoantigen detection and immuno-oncology.

Sean M. Boyle, Michael J. Clark, Ravi Alla, Shujun Luo, Deanna M. Church, Elena Helman, Parin Sripakdeevong, John West, Rich Chen. _Personalis, Menlo Park, CA_.

Accurate detection of somatic variants is a staple of both research and clinical cancer analysis, with applications ranging from detecting new common driver mutations in large patient cohorts to selecting therapeutic small molecule treatment courses for an individual patient. Recent research into neoantigens and immunotherapy has shown great promise as a precision therapeutic, and somatic variant detection by next-generation sequencing represents an ideal method of identifying candidate neoantigens. Somatic variant detection typically involves assaying the DNA for changes in gene sequences without assessing whether those variants are actually expressed in RNA. However, the expression of small variants is key because only expressed peptides will be displayed as neoantigens on the cell surface.

From a technical standpoint, detection of somatic variants in the RNA represents additional challenges above and beyond those of somatic detection in DNA. The widely varying expression levels of cancer genes, alternative splicing, and RNA editing are all features that make somatic variant calling in RNA uniquely challenging. However, accurately detecting variants directly from expressed transcripts is beneficial to neoantigen prediction, and therefore we sought to create and validate a method for somatic variant calling in RNA.

We have designed a highly accurate expression-based somatic variant detection pipeline utilizing extensive discovery and filtering methods to overcome the challenges inherent in RNA somatic variant calling. We validated our pipeline using a combination of well-characterized cell lines, commercially available reference standards, and real world FFPE patient samples. To our knowledge, this is the most extensive validation of its kind to date, representing over 29,158 small variants across 39 samples. In testing, we measured our detection method at >99% sensitivity and >99% PPV using a combination of gold set small variants and orthogonal validation. This method, in combination with our validated DNA somatic variant calling pipeline (>99% sensitivity and >99% PPV), enables precise detection of variant expression levels in a given sample, even at low allele frequency (5%).

After validating our RNA somatic variant calling method, we applied it to detect candidate neoantigens in patient tumor samples. We performed HLA typing for each sample using HLAssign software and predicted MHC presentation of the expressed somatic variants. In ongoing studies, we are validating our most promising putative neoantigens using orthogonal technologies and demonstrating our ability to detect the most promising clinically effective peptides for therapy.

#534

Trastuzumab increases uptake and cross-presentation of HER2-derived antigens by dendritic cells.

Victor A. Gall, Anne V. Philips, Na Qiao, Karen C. Dwyer, Alexander A. Perakis, Mao Zhang, Pariya Sukhumalchandra, Jeffrey J. Molldrem, Gheath Alatrash, Elizabeth A. Mittendorf. _MD Anderson Cancer Center, Houston, TX_.

Introduction:

The purpose of this study was to identify the effect of trastuzumab on the uptake and presentation of HER2-derived peptides by dendritic cells (DCs). Clinical trials for HER2-derived peptide vaccines have shown improved outcomes for patients receiving a combination of vaccine and trastuzumab suggesting potential synergy between the two. We hypothesized that antigen processing and cross-presentation would be enhanced via trastuzumab facilitating antigen presenting cell (APC) uptake of HER2 shed from breast cancer cells.

Methods:

An ELISA assay was used to evaluate HER2 shedding in multiple cancer cell lines (SKBR3, SKOV3, BT474, MDA-MB-231). To look for uptake of HER2 shed from breast cancer cells into culture media by APCs, DCs were generated by incubating healthy donor monocytes in media supplemented with GM-CSF, IL-4, and TNF-α. These DCs were co-incubated with SKBR3, BT474, and SKOV3 (high HER2 expressers) as well as MDA-MB-231 (low HER2 expresser) in the presence of trastuzumab. Rituximab was used as an isotype control to determine trastuzumab specificity and an Fc blocker was used to evaluate the importance of the Fc receptor in the uptake mechanism. Cells were then stained for DC markers, including CD11c and HLA-DR, permeabilized and stained for HER2 to detect uptake, and then analyzed using flow cytometry. To evaluate antigen cross-presentation, antigen-specific T cells were expanded by co-culturing healthy donor PBMCs with DCs that have been pulsed with SKBR3 cells or trastuzumab-treated SKBR3 cells. DCs pulsed with E75 peptide (HER2, aa: 369-377) were used as a control. After T cell activation and expansion, the number of E75-specfic CD8+ T cells was enumerated using an E75-dextramer assay.

Results:

When compared to SKOV3 and MBA-MB-231 cells, SKBR3 and BT474 cells shed a significantly higher concentration of HER2 into the growth media. DCs took up HER2 when co-cultured with cell lines with higher levels of HER2 shedding (SKBR3, BT474). Trastuzumab treatment of SKBR3 resulted in a 50% increase in HER2 uptake by DCs at 24 hours (p= 0.0014). This finding persisted among all trastuzmab doses. Treatment with rituximab showed no significant increase in HER2 uptake when compared to untreated SKBR3. The effect of trastuzumab on uptake was abrogated by adding an Fc blocking agent. DCs treated with SKBR3 and trastuzumab also led to an increase in the generation of E75-CTLs as measured using a dextramer assay.

Conclusions:

Trastuzumab treatment leads to increased uptake of soluble HER2 by DCs in vitro. This effect is specific to trastuzumab and is Fc receptor mediated. Increased HER2 uptake led to increased E75-CTL generation secondary to increased cross-presentation of E75 by DCs. These data have global implications for HER2-targeting vaccine approaches. Specifically, by enhancing antigen presentation, trastuzumab can augment the immune response initiated by a peptide vaccine.

#535

Deregulated Myc is an immunosuppressive switch.

Roderik M. Kortlever,1 Nicole M. Sodir,1 Catherine H. Wilson,1 Deborah L. Burkhart,1 Lamorna Swigart,2 Trevor D. Littlewood,1 Gerard I. Evan1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _University of California San Francisco, San Francisco, CA_.

Early tumor evolution through sustained oncogene activity selectively bypasses the engagement of cell-intrinsic tumor-suppressor signaling and cell-extrinsic microenvironmental restrictions. How the immune system may be involved is virtually unknown. We describe here the contributions of a conditional and reversible low-level expression of Myc in a mouse model of KrasG12D-driven non-small cell lung cancer. Deregulated Myc activity results in highly expansive tumors that appear embedded in inflamed regions and leads to a rapid reduction of mouse survival. Myc activation imposes an immediate switch to an immunosuppressive and pro-angiogenic microenvironment, facilitated through IL23- and CCL9-associated recruitment of PD-L1 loaded macrophages and local exclusion of T-lymphocytes. Reversibly, blocking the activity of Myc-driven IL23 and CCL9 expression or withdrawal of deregulated Myc activity in tumors established by oncogene cooperation results in tumor cell death and regression, associated with a collapse of the established microenvironmental changes and re-engagement of cytotoxic T-cells. During oncogene cooperation with Ras, deregulated Myc directs a sufficient and necessary switch to a microenvironment that shields tumor growth and expansion from immune suppression.

#536

Excessive HCK kinase activity in the tumor stroma promotes colorectal cancer progression.

Matthias Ernst,1 Ashleigh Poh,2 Frederic Masson,1 Tracy Putoczki,2 Robert O'Donoghue1. 1 _Olivia Newton-John Cancer Research Institute, Heidelberg, Australia;_ 2 _Walter and Eliza Hall Institute, Parkville, Australia_.

Excessive activation of the myeloid-specific Src-family kinase Hematopoietic Cell Kinase (HCK) acts as a tumor intrinsic oncogene by promoting proliferation and survival of immune cells, and confers a poor prognosis for leukemia. However, the role of HCK in the tumor stroma of solid malignancies remains unexplored.

We analyzed the expression level of HCK in matched biopsies from sporadic colorectal cancer patients and observed elevated HCK phosphorylation in tumors compared to unaffected colons. Analysis of corresponding RNAseq data revealed a striking correlation between tumors with high HCK gene expression and a gene signature indicative of a tumor-promoting alternatively-activated macrophage (AAM) endotype. To functionally assess this observation, we exploited HckCA mice [Ernst et al., J Exp Med 2002] that express a constitutively active form of Hck as a knockin mutation, and subjected these mice to a chemically-induced model of sporadic colorectal cancer. HckCA mice developed more and larger tumors compared to wild-type (WT) animals, and this was associated with a significant bias towards CD206+ AAMs in tumors of HckCA mice without affecting the total number of tumor-associated leukocyte and lymphocytes. Likewise, adoptive bone-marrow transfer experiments revealed enhanced tumor formation and AAM differentiation in WT mice reconstituted with HckCA bone marrow, with a reciprocal decrease of these parameters in HckCA mice reconstituted with WT marrow. Finally, pharmacologic targeting of the catalytic activity of Hck with a small molecule inhibitor significantly reduced tumor progression and impaired AAM polarisation.

Collectively, our findings suggest that excessive Hck activity in the tumor stroma promotes the progression of solid cancers by modulating the endotype of tumor-associated myeloid cells. Therefore, Hck represents a rational therapeutic target for macrophage re-education in solid cancers by limiting polarization of tumor-promoting AAM.

#537

**Evaluation of immune function in the tumor microenvironment by RNA** in situ **hybridization to reveal spatial information on immune cell infiltration and the expression of functional mRNA biomarkers.**

Ming-Xiao He, Courtney Anderson, Na Li, Xiao-Jun Ma, Emily Park. _Advanced Cell Diagnostics, Hayward, CA_.

Growing evidence for the antitumor activity of the immune system has supported recent success in cancer immunotherapy. Therapeutic approaches such as anti-cancer vaccines and suppression of immune checkpoints have been successfully applied in various cancers to raise specific immune responses and to enhance antitumor immune responses. In particular, blocking immune checkpoint pathways such as CTLA-4 and PD-1/PD-L1 is transforming cancer therapeutics. Based on these successes, clinical trials of combinatorial strategies are in progress to target multiple pathways. While many biomarker analysis technologies are employed to guide specific therapeutic approaches, spatially mapped biomarker expression data at the single-cell level is crucial to understanding the composition and activities of various immune cells in the tumor microenvironment.

In this study, in order to visualize gene expression of key immune biomarkers in the tumor microenvironment, we have applied the RNAscope® technology, an RNA in situ hybridization (ISH) method that provides single molecule RNA detection with spatial and morphological context. This highly sensitive ISH presents a unique advantage to the visualization of mRNA biomarkers for secreted proteins such as cytokines and chemokines in individual cells. Specific probes for key cytokines ( e.g., IL6, IL12A, TGF-β, IFN-γ, TNF-α), chemokines ( e.g., CCL5, CXCL9, CXCL10, CXCL13), and immune subset markers ( e.g., CD3, CD4, CD8A, FOXP3) were designed in addition to key receptor/ligand pairs in immune check points ( e.g., PD1/PD-L1, OX40/OX40L). Expression of each target and combinations of multiple targets were assessed on formalin-fixed paraffin embedded (FFPE) tissue sections derived from multiple tumor types. Using this approach, the various functional mRNA biomarker profiles of the immune cell types infiltrated in the tumor are directly visualized as punctate dots. The visible mRNA dots are readily quantifiable, so that enumeration of immune cells is feasible.

RNAscope® based ISH is well-suited to study immune function in the tumor microenvironment by visualizing gene expression. By applying this method, it is feasible to evaluate the relationship of specific biomarkers in different cell types. With the emerging effort towards combinatorial approaches to target multiple pathways, the method presented here can be applied as a universal platform to study both cancer specific molecular pathways as well as immune function.

#538

A role for mutated hydrophobic amino acids in patients with triple-negative breast cancer.

Vassiliki Kotoula,1 Sotiris Lakis,1 Kyriaki Papadopoulou,1 Ioannis Vlachos,1 Eleni Giannoulatou,2 Zoi Alexopoulou,3 Eleni Timotheadou,1 Ioannis Efstratiou,1 Flora Zagouri,1 George Pentheroudakis,1 Helen Gogas,1 Spyros Miliaras,1 Maria Sotiropoulou,1 George Zografos,1 Dimitrios Pectasides,1 George Fountzilas1. 1 _Hellenic Cooperative Oncology Group (HeCOG), Athens, Greece;_ 2 _Victor Chang Cardiac Research Institute, Darlinghurst and The University of New South Wales, NSW, Australia;_ 3 _Health Data Specialists Ltd, Athens, Greece_.

Background - Aim: Nodal status and stromal tumor infiltrating lymphocyte (TILs) density are independently associated with patient outcome in triple-negative breast cancer (TNBC). Herein we examined whether the nature of tumoral mutated amino acids, in association with TILs and nodal status, interferes with the outcome of patients with operable TNBC treated with adjuvant chemotherapy.Methods: Two independent cohorts of TNBC patients and tumors were examined: (a) 133 patients treated upon clinical service (training set; TS), and (b) 190 patients treated in the context of phase III adjuvant trials (validation set; VS). All 323 patients had received adjuvant anthracycline-taxanes chemotherapy. All 323 tumors yielded informative data upon massively parallel sequencing for breast cancer related targets in 47 genes. Amino acid changing variants were considered as mutations (MUT) for minor allele frequencies 50% TILs) and nonLP. Disease-free survival (DFS) was the clinical endpoint.Results: The 2 cohorts did not differ regarding MUT tumor incidence (TS, 71%; VS, 67%); TILs (10% LP in each set); nodal status (TS, 0-3 positive nodes in 68%; VS, 64%); and, MUT a.a. classes (TS, charged 11%, polar 25%, hydrophobic 13%, mixed 15%; VS, 12%, 17%, 9%, and 13%, respectively). No statistically significant difference was obtained in the rates of MUT tumors or MUT a.a. classes with respect to TILs and nodal status. In the TS, patients with tumors bearing hydrophobic MUT a.a. fared worse than those with other MUT types (HR 2.43, 95%CI 1.17-5.03, p=0.017) or than those without MUT (HR 5.13, 95%CI 1.95-13.52, p<0.001). Similar trends were observed in VS. When classifying tumors for nodal status, TILs and the presence of hydrophobic MUT a.a., the latter independently conferred worse DFS in patients with >3 positive nodes and non-LP tumors (TS, HR 3.03, 95%CI 1.11-8.29, p=0.031; VS, HR 2.90, 95%CI 0.97-8.70, p=0.057). Patients with >3 positive nodes, non-LP tumors with hydrophobic MUT a.a. were revealed as the only subset with significantly worse DFS when compared to patients with LP tumors in both TS (p=0.003) and VS (p=0.015). Charged and polar MUT a.a., as well as the presence of MUT in the most frequently affected genes (TP53 and PIK3CA) did not have any impact on DFS in either TS or VS.Conclusions: In the adjuvant setting, the presence of hydrophobic MUT a.a. seems to be associated with worst prognosis in nonLP TNBC patients with high nodal burden. The finding was obtained in 2 independent patient series, which makes it worthy validating in further studies for this most difficult to treat breast cancer patient subset.

#539

Analysis of genetic alternations in multiple tissue specimens from each patient: Implications for field-effect colon cancerization.

Chansu Lee,1 Sung Noh Hong,1 Sungjin Kim,1 Sunghoon Cho,2 Sohyun Youn,2 Kwang-Sung Ahn,2 Young-Ho Kim1. 1 _Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Functional Genome Institute, PDXen Biosystems Inc., Seoul, Republic of Korea_.

Colorectal cancer (CRC) develops through a polyp-to-cancer progression sequence, in which normal colorectal epithelium transforms into an adenoma, which then progresses to cancer via the accumulation of progressive molecular changes, including both genetic and epigenetic alterations. Given that Colon epithelial cells can acquire pro-tumorigenic mutations that are insufficient to cause morphological change, genetic alternations in adjacent tumor tissues are primed to become neoplastic cells. It is known that individuals with a personal history of colon adenomas or cancer are at increased risk for metachronous colon neoplasms. To examine the genetic alternations involving in a polyp-to-cancer progression sequence, whole-transcriptome expression profiling of resected normal tissues (N1 and N3), adenoma (A1 and A2), and colorectal cancer (C1) obtained from individuals was performed. Differentially expressed genes among groups were identified using Subio platform. GATK and haplotypecaller was used to compare the somatic mutations among three different tissues. Tophat, Cufflink, Mev, R was used for determining the expression levels. Splicegrapher and Genomon-fusion was used for identifying the fusion genes. Ordinal regression analysis was performed to characterize site-dependent expression profiles. Gene interaction was examined using String (Open database). All statistical tests were two-sided, except where noted. Expression profile showed that highly expressed genes associated with inflammation were detected in adenoma. Our results indicated that highly expressed genes associated with inflammation were initially found in adenoma tissues and its levels were consistent in carcinoma tissues. Number of somatic mutation was extremely increased in carcinoma tissues when compared to adenoma tissues. The same mutations in KRAS, CDKN2A(p16) and TP53 that were observed in carcinoma tissues. They were also present in normal tissues and adenoma tissues. Disruption of TP53, CDKN2A, and KRAS were all seen as possible initial events in tumorigenesis; the sequence of mutations (the tumor development pathway) differed among lesions. Also, unique somatic mutations were found 17 genes which were not exist in normal tissues. Those somatic mutations were found in carcinoma tissues. The results of fusion gene analysis showed that 9 fusion genes were detected. Four fusion genes (FIIR-RBM2292, ST7-AS1-ST7, MMP11-IGLL5, EXOC4-LOC101928861) was detected in all of tissues. Five fusion genes were found in normal tissues and adenoma tissues. Unique fusion gene in carcinoma tissue was not detected. One explanation for this increased risk could be field cancerization, which is a phenomenon in which the histologically normal tissue in an organ is primed to undergo transformation. Our analysis of genetic alterations in a single patient appears to be promising markers for field cancerization. 

### Immune Modulating Agents 1

#540

Adjuvant effect of anti-PD-L1 in boosting HER2-targeted T cell adoptive immunotherapy.

Yang Xia,1 Yangyang Hu,1 Yi Wang,2 Yangyi Bao,2 Fu Dai,2 Shiang Huang,3 Elaine Hurt,4 Robert E. Hollingsworth,4 Archana Thakur,5 Lawrence Lum,5 Alfred E. Chang,1 Max S. Wicha,1 Qiao Li1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _The No.1 People's Hospital of Hefei, Hefei, China;_ 3 _Hubei Province Stem Cell Research & Appling Center, Wuhan Union Hospital, Wuhan, China; _4 _MedImmune, LLC., Gaithersburg, MD;_ 5 _Wayne State University, Detroit, MI_.

In a phase I clinical trial, we used anti-CD3 x anti-HER2 bispecific antibody (HER2Bi) armed T cells. Utilizing HER2-directed T cells benefitted both HER2+ patients and patients with 1 or 2+ HER2 expression, ones that would be considered "HER2-negative" by classical criteria. We have also shown that the level of cancer stem cell (CSC) marker ALDH in HER2+ breast cancer cells (ALDHhighHER2+) is much higher than that in HER2- breast cancer cells (ALDHlowHER2-), and that in luminal breast cancers that are considered HER2-, HER2 is actually selectively expressed in the ALDHhigh CSC population. These observations might account for the surprising result that HER2Bi-armed T cells, while intended to target HER2, seemed to benefit HER2- patients after adoptive transfer. In this study, we found that the HER2Bi-armed T cells used in the clinical trial killed ALDHhigh human breast CSCs isolated from MCF7 (HER2-) tumor significantly more than ALDHlow MCF7 cells in vitro, while the same HER2Bi-armed T cells killed ALDHhigh human breast CSCs (ALDHhighHER2+) isolated from BT474 (HER2+) tumor equally to ALDHlow BT474 cells (ALDHlowHER2+). We then tested the "mouse HER2" (neu) expression on ALDHhigh vs. ALDHlow 4T1 cells (mouse TNBC), and found that mHER2 was selectively expressed in the ALDHhigh 4T1 CSC population. These results replicated our findings in human breast cancers that HER2 is selectively expressed on CSCs, even in HER2- murine tumors, such as 4T1. For mHER2 targeting in animal models, we generated anti-mouse HER2-CD3 bispecific (mHER2Bi) that binds to mouse HER2 and mouse CD3. In vitro, the mHER2Bi-armed T cells killed ALDHhigh 4T1 CSCs significantly more than ALDHlow 4T1 cells. In vivo, adoptive transfer of mHER2Bi-armed T cells for HER2 targeted therapy showed antitumor effect in mHER2- 4T1-bearing host. To boost the therapeutic efficacy of mHER2-targeted T cells, we administrated anti-mouse PD-L1 during mHER2Bi-armed T cell adoptive transfer. This combination decreased metastases significantly more than the use of either strategy alone. Collectively, these studies have generated evidence providing proofs of principle that due to the selective expression of HER2 on CSCs, HER2-targeted T cell therapy could benefit HER2- host as well as HER2+ hosts via immune destruction of HER2+ CSCs, and use of anti-PD-L1 as an adjuvant could significantly boost the efficacy of HER2-targeted T cell therapy.

#541

Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance.

Chandini M. Thirukkumaran,1 Zhong Qiao Shi,1 Joanne Luider,2 Karen Kopciuk,1 Marta Chesi,3 Leif Bergsagel,3 Yuan Dong,4 Chunfen Zhang,4 Ahmed Mostafa,1 Kathy Gratton,1 Satbir Thakur,1 Don Morris1. 1 _Tom Baker Cancer Ctr., Calgary, Alberta, Canada;_ 2 _Calgary Laboratory Services, Calgary, Alberta, Canada;_ 3 _Mayo Clinic, Scottsdale, AZ;_ 4 _University of Calgary, Calgary, Alberta, Canada_.

Introduction: Multiple myeloma (MM) is a malignancy of plasma cells that is still considered incurable due to the 90% relapse rate in patients. Despite the advent of new agents the majority of MM patients relapse secondary to therapy resistance. The potential of reovirus (RV) as a novel therapeutic agent for MM under in vitro, in vivo and ex vivo conditions has been demonstrated by us and a phase I clinical trial of MM with RV therapy has shown indications of efficacy. Recently we have shown that RV could synergize with several standard of care MM drugs such as bortezomib (BTZ), carfilzomib (CFZ) and dexamethasone and importantly, enhance its therapeutic potential in therapy resistant human MM cell lines in vitro. Utilizing the syngeneic Vk*MYC BTZ resistant transplantable MM mouse model, we demonstrate that mice harbouring BTZ insensitive MM tumors significantly respond to RV treatment. Our data indicate that this RV treatment sensitivity is manifested via, direct oncolysis in conjunction with immune modulatory effects.

Methods: Vk*MYC myeloma cells (Vk12598) were injected via tail vein (IV) into 3 groups of C57BL/6 wt recipient mice. Seven days post tumor injection, mice were treated with PBS (vehicle) or live (LV) or dead reovirus (DV) (5x10^8 PFU) administered IV every 6 days in a total of 5 doses. Serum gamma globulins (M-spike) were assessed weekly by serum protein electrophoresis. Mice were sacrificed on day 35 post tumour injection and their spleens and bone marrow (BM) were harvested. Splenic and BM cells were stained for CD45+/CD45R-/CD138+ (MM tumors), CD4+/CD8+ (T cells), CD3+/NKG2D+ (NKT cells), CD11b+Ly6C+ (monocytic myeloid derived suppressor cells (Mo-MDSC) and CD11b+Ly6G+ (polymorphonuclear (PMN) - MDSCs) and analyzed by flow cytometry.

Results: Mice treated with RV demonstrated highly significant (P<0.001) reductions in their M-spikes at 4 and 5 weeks post treatment. RV treatment led to a significant NKT cell accumulation in splenic and BM of RV treated mice in comparison to vehicle treated mice suggestive of immune activation due to RV. Further, RV treatment led to a significant increase in BM Mo-MDSC number but not PMN-MDSC numbers. Interestingly, significant down regulation of splenic PMN-MDSCs was seen with RV treatment.

Conclusions: Our results indicate reovirotherapy is successful in a validated syngeneic BTZ resistant MM model. These results have important implications for future clinical trials. Currently we are conducting further experiments to assess the immune modulatory (such as T - memory and T - regulatory cells) contributions of RV in this model.

#542

Assessing and augmenting the immune response to glioblastoma using repurposed pharmaceuticals.

Breanna R. Brenneman, Desiree H. Floyd, Tajie Harris, Benjamin Purow. _University of Virginia, Charlottesville, VA_.

In order to improve the therapeutic responses to immunotherapies in glioblastoma (GBM) patients, a known pharmaceutical, ritanserin, was repurposed to augment cytotoxic T cell responses and suppress tumor growth. To determine the kinetics of the immune response to GBM, the transplantable syngeneic glioma cell line GL261 was used in C57BL/6 mice to assess the tumor immune response in the brain, spleen, and cervical lymph node via flow cytometry weekly for one month. After identifying the optimal T cell response temporally, mice with established tumors were treated with 100mg/kg of Ritanserin for one week and harvested post treatment. Ritanserin is able to inhibit the activity of diacylglycerol kinase alpha (DGKA), an enzyme that converts diacylglycerol to phosphatidic acid, and can inhibit tumor growth in mice. In addition, DGKA is expressed in T cells, and is more highly expressed in those that have entered an unresponsive (anergic) state, characteristic of dysfunctional T cell responses to tumors. To determine if ritanserin can counteract T cell anergy, T cell responses were determined by assessment of T cell number by subset (CD8+, CD4+, CD4+FoxP3+), by activation (CD62L-CD44+), and by tumor-specific response to ovalbumin-expressing GL261 tumor cells in the brain, spleen, and cervical lymph nodes. In comparison to mock-injected mice, tumor-bearing mice showed a robust T cell response to tumors in the brain, peaking at days 21 and 28 post injection. Treatment with ritanserin resulted in a trend toward increased number and activation of T cells in the brain compared to vehicle-treated mice. In addition, there was a significant increase in OVA-specific T cells in the brain with treatment. In conclusion, T cells respond robustly to brain tumors in mice, and ritanserin treatment appears to increase this response by increased activation and priming of tumor-specific T cells. This indicates that ritanserin may have combinatorial effects on T cells when combined with checkpoint inhibitors such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1). Future experiments will focus on combining ritanserin and other repurposed drugs with these checkpoint inhibitors and further characterizing the effect of ritanserin on T cells.

#543

Preclinical combination strategies to enhance the efficacy of the anti-PD-1 antibody pembrolizumab.

Elaine M. Pinheiro,1 Marlene Hinton,1 Ruban Mangadu,1 Mingmei Cai,1 Uyen Phan,1 Michael Nebozhyn,1 Heather Hirsch,1 Andrey Loboda,1 Sarah Javaid,1 Yaolin Wang,2 Venkataraman Sriram,3 Joseph H. Phillips,1 Terri McClanahan,1 Brian Long1. 1 _Merck & Co., Inc., Kenilworth, NJ; _2 _SUNY Stony Brook, Stony Brook, NY;_ 3 _University of Madras, Palo Alto, CA_.

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), has shown activity in multiple types of cancer. Although pembrolizumab is showing a dramatic impact in patients that respond, a large fraction of patients do not respond to single agent therapy. Combination approaches may be the key to improving response rates in these patients. One of the challenges is how best to tailor combination strategies to provide maximal benefit to patients. How we prioritize new combinations will require an understanding of which mechanism is best for which tumor. To elucidate mechanisms that may create an immunogeneic tumor microenvironment and enhance the antitumor activity of PD-1 blockade, we have tested combination strategies with muDX400, a murine anti-PD-1 antibody. The murine preclinical syngeneic tumor models selected for these studies had varied tumor microenvironment compositions and displayed a range of responses to therapy with single agent anti-PD-1. Here we show enhanced anti-tumor activity when PD-1 blockade is combined with conventional chemotherapies, small molecule therapies and additional immunotherapy approaches that target multiple steps in the immune-oncology pathway. Pre and post treatment tumors from these models were extensively characterized by gene expression profiling, whole exome sequencing, flow cytometry and immunohistochemistry analysis. Molecular and cellular correlates of response to anti-PD-1 as a single agent and in combination with these agents were evaluated. These data support potential combination strategies that could either increase efficacy or expand the patient population in which pembrolizumab will demonstrate benefit.

#544

Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) promotes tumor growth and metastasis via inhibition of protein phosphatase 1 in Ewing sarcoma.

Wen Luo,1 Changxin Xu,2 Janet Ayello,1 Filemon Dela Cruz,3 Jeremy Rosenblum,1 Stephen Lessnick,4 Mitchell S. Cairo1. 1 _New York Medical College, Valhalla, NY;_ 2 _University of Utah, Salt Lake City, UT;_ 3 _Columbia University, New York, NY;_ 4 _Nationwide Children's Hospital, Columbus, OH_.

Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. Children with metastatic Ewing sarcoma have a cure rate of less than 30% (De loris MA et al, 2013). The major hurdle of curing Ewing sarcoma is the lack of specific and druggable targets. EWS/FLI is the master regulator of Ewing sarcoma and functions as an aberrant transcription factor to deregulate downstream targets which eventually leads to cancer phenotypes. By comparing genes deregulated by EWS/FLI in RNA-sequencing analyses across multiple model systems, we identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A), a potent protein phosphatase 1 (PP1) inhibitor, as one of the core targets of EWS/FLI (Niedan S et al, 2014; Sankar S et al, 2013; Tirode F et al, 2007). Public data mining and western blotting analysis has demonstrated that PPP1R1A is highly expressed at both RNA and protein levels in Ewing sarcoma, but not the putative cell of origin, mesenchymal stem cells, or other sarcomas. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that PPP1R1A is up-regulated by EWS/FLI. Further data mining with published chromatin immunoprecipitation (ChIP)-Seq, Encyclopedia of DNA Elements (ENCODE), and formaldehyde-assisted isolation of regulatory elements (FAIRE) data suggested, and subsequent luciferase reporter assay validated, that PPP1R1A is directly targeted by EWS/FLI via a GGAA microsatellite enhancer element. We next studied the role of PPP1R1A in Ewing sarcoma by functional assays in vitro and in vivo. Intriguingly, depletion of PPP1R1A caused a loss of oncogenic transformation of patient-derived Ewing sarcoma cells in soft agar, and limited xenograft tumor growth in NOD-SCID mice (p=0.0001). Furthermore, reduction of PPP1R1A resulted in decreased cell migration in wound healing and Boyden chamber assays, and limited lung metastasis in an orthotopic mouse model. Further studies demonstrated that activation and phosphorylation at Thr35 of PPP1R1A by protein kinase A (PKA), is required for PPP1R1A to promote tumorigenesis and metastasis, as evidenced by rescuing of PPP1R1A-knockdown induced phenotypes by constitutively active (T35D) but not wild-type PPP1R1A. Consistently, we found that Ewing sarcoma but not HEK293 cells were sensitive to the treatment of PKA inhibitors (H89 and PKI 5-24). Finally, we found that further mutating of the PP1 binding site (R8A/K9A) in T35D PPP1R1A rendered this constitutively active mutant unable to rescue the knockdown phenotype, indicating that PP1 binding and inhibition by activated PPP1R1A is indispensable for PPP1R1A function. Collectively, our data suggest that PPP1R1A and related pathway plays a positive role in tumorigenesis and metastasis in Ewing sarcoma. Therefore, targeting PKA/PPP1R1A/PP1 pathway has potential therapeutic value in the treatment of both primary and metastatic Ewing sarcoma.

#545

Differential regulation of human T-cells by TGR-1202, a novel PI3Kδ inhibitor.

Kamira K. Maharaj,1 John Powers,1 Renee Fonseca,1 Hari Miskin,2 Dave Maryanski,2 Eva Sahakian,1 Javier Pinilla-Ibarz1. 1 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _TG Therapeutics, Inc., New York, NY_.

INTRODUCTION: TGR-1202 is a novel, next-generation PI3Kδ inhibitor presenting significant structural and pharmacological differences from prior small-molecule PI3Kδ inhibitors. TGR-1202 has high clinical efficacy in treatment of B cell malignancies with a substantially differentiated adverse event profile compared to previous PI3Kδ inhibitors, specifically concerning hepatotoxicity or colitis which have been minimal or non-existent. It has been postulated that these effects may be due to T cell immune-mediated mechanisms. We hypothesized that TGR-1202 preserves the function of the regulatory T cell (Treg) population, translating to decreased immune-mediated side effects after treatment. Here, we aimed to compare effects of clinically available PI3Kδ inhibitors on T cells with an emphasis on Tregs.

METHODS: We compared activity of idelalisib, duvelisib, and TGR-1202 as single agents in vitro in isolated human T cells. Viability, apoptosis, and proliferation were determined using CellTiter Blue®, annexin V/PI and CFSE staining. CBA and qRT PCR were used to measure cytokines and transcription factors. Flow cytometry was used to detect subset ratios and surface marker expression.

RESULTS: First, we observed comparable dose-dependent increases in cytotoxicity beginning at 25uM following treatment of isolated T cell populations with idelalisib, duvelisib, or TGR-1202. At this dose, apoptosis was induced between 48 and 72h. Second, all inhibitors reduced cytokine production in CD3+ T cells upon stimulation. Particularly, IFN-γ, IL-10 and IL-17a reduction was less pronounced after TGR-1202 treatment, indicating relative conservation of T cell response. All inhibitors lowered mRNA expression of T-bet (Th1), GATA-3 (Th2) and FoxP3 (Treg), however FoxP3 levels were consistently higher in TGR-1202 treated T cells. Third, we detected normal CD4:CD8 ratio and unaffected proliferative capacity of CD4+ and CD8+ subsets after drug treatment. Finally, all inhibitors decreased total percent of Tregs following stimulation (CD4+ CD25HI FoxP3+) accompanied by decreased expression of co-inhibitory molecules CTLA-4 and PD-1 on the Tregs. Interestingly, TGR-1202 significantly preserved the percent of Tregs closer to normal as well as surface expression of CTLA-4 and PD-1 on Tregs, indicating greater retention of immune checkpoint blockade and suppressive phenotype.

CONCLUSIONS: We report herein that TGR-1202 affects human T cells differently than idelalisib and duvelisib. TGR-1202 sustains IL-10 production, FoxP3 mRNA expression, and maintains Treg percentage and expression of immune checkpoint molecules, suggesting relative preservation of numbers and function of Tregs. Data presented begin to provide novel insight into immune mediated cellular mechanisms responsible for lack of side effects in clinical trials of TGR-1202. In vivo models to further characterize effects on the T cell compartment are ongoing.

#546

Impact of therapeutic EGFR inhibition on immune checkpoint blockade in head and neck squamous cell carcinoma.

Laura M. Rogers, Madelyn M. Espinosa-Cotton, Andrean L. Simons, George J. Weiner. _University of Iowa, Iowa City, IA_.

Patients with advanced recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) have few effective treatment options and a poor median survival of 6-8 months. Limited improvement in overall survival has been observed in HNSCC patients treated with EGFR inhibitors despite high expression of epidermal growth factor receptor (EGFR) in these tumors. Remarkable clinical results are emerging from immunotherapy targeting programmed death-1 (PD1) and its ligand programmed death ligand-1 (PDL1). Blockade of the PD1/PDL1 interaction is believed to enhance endogenous anti-tumor immunity by restoring T cell cytotoxic action. PDL1 is highly expressed in primary, recurrent, and metastatic HNSCC and phase 1 clinical trials are ongoing for monoclonal antibodies targeting the PD1/PDL1 axis in HNSCC.

We found that activation of EGFR in HNSCC cells by epidermal growth factor (EGF) significantly increases PDL1 expression in a dose dependent manner. EGFR inhibition using tyrosine kinase inhibitors (TKI) or cetuximab suppresses this EGF-induced PDL1 expression in cells with both wild type and constitutively active EGFR. This is consistent with published data that constitutively active EGFR in lung cancer cells correlates with higher PDL1 expression, and this can be reversed by EGFR inhibition. These results suggest there may be interactions between EGFR-targeted therapy and immunotherapy involving the PD1/PDL1 axis.

To evaluate potential therapeutic interactions, we used an immunocompetent, syngeneic mouse model of human EGFR-expressing TUBO cancer cells on a BALB/c background. The murine TUBO-EGFR cells are sensitive to human EGFR inhibitors both in vitro and in vivo. Initial studies show that treatment of tumors with EGFR inhibitor (erlotinib) results in increased intratumoral CD8+ T cell infiltration and tumor regression, suggesting that EGFR inhibition can impact the anti-tumor T cell response. We are currently evaluating the effects of EGFR inhibition on PDL1 expression and tumor response to PD1/PDL1 blockade using this model. Further, a clinical trial investigating the impact of cetuximab treatment on intratumoral expression of PDL1 in HNSCC patients is planned to open shortly. We conclude that signaling mediated by EGFR can impact expression of PDL1 by HNSCC. Understanding the interactions between EGFR inhibitors and immune checkpoint blockade could lead to new approaches to combining TKI and cancer immunotherapy.

#547

Radio- and chemotherapy causes up-regulation of immunoinhibitory ligands pd-l1 and galectin-9 in gastric cancer.

Sven H. Petersen. _National University of Singapore, Singapore, Singapore_.

Apart from the cytotoxic effect of radiation- or chemotherapy it has become evident that these treatments are able to induce an immune response which can play an

additional and pivotal role in clearing tumours. This treatment-induced immunogenic tumour cell death (ICD) enables the host's immune system to recruit to the tumour and recognise and kill tumour cells which can even extend to yet untreated metastases, a phenomenon called "abscopal effect". For the first time, we found evidence that radiation and/or chemo-treatment stimulates a pronounced up-regulation of both, PD-L1 and Galectin-9 on pre-apoptotic and apoptotic gastric tumour cells. Both these proteins exert immuno-modulatory signals which in turn may dampen the aimed immunogenicity of the treatment. PD-L1, which is the ligand to programmed cell death protein 1 (PD1) on T cells, and its upregulation is commonly used by cancer cells to evade immunity. PD-L1 causes T cell apoptosis and hence an inhibition of T cell driven anti-tumour immunity. Galectin-9 exerts immunoinhibitory functions and is, together with the immunostimulatory molecule HMGB1, a competitive binding partner for the receptor T-cell immunoglobulin domain and mucin domain 3 (Tim-3). Once engaged by its ligand, Tim-3 either exerts stimulatory or inhibitory effects, depending on ligand, cell type, maturation state and Tim-3 expression rate. Together with PD-L1, Galectin-9 enables a complex interplay between cancer cells and cells of the adaptive immune response expressing PD1 and Tim-3. Based on these results, we are investigating the mechanism responsible for PD-L1 and Galectin-9 upregulation and the effect on the efficacy of radio- as well as chemotherapy on tumour eradication. Additionally, due to their potentially enhancing effect on anti-tumour immunity, we are studying the therapeutic value of blocking Abs specific for human PD1 and/or Tim-3 in cancer immunotherapy.

#548

Characterization of myeloid cells in bladder cancer.

Neelam Mukherjee, Xiang Ru Zhao, Niannian Ji, Robert S. Svatek. _UT Health Science Center at San Antonio, San antonio, TX_.

Both cancer-associated inflammation and tumor-mediated immune suppression have been linked with expansion of myeloid cells in multiple tumor types, including bladder [1]. We previously observed a predominance of M2-like macrophages in mouse bladder tumors [2]. However, little is known about the characteristics of these myeloid cells in human bladder cancer. In this study, we analyzed myeloid cells from tumor tissues collected from patients with muscle-invasive bladder cancer. We used flow cytometry to characterize the different subsets of these myeloid cells.Myeloid cells were identified in the bladder tissue. Our results showed that bladder cancer patients have two major CD11b+CD33\+ myeloid derived suppressor cell subsets: granulocyte-type HLADR-CD15+ cells and monocyte-type HLADR+CD14+ cells. Analysis of CD45+ CD11b+cells demonstrated that bladder tumors are dominated by phenotypical M2-like (CD163+ CD80lo) CD163+ M2 macrophages.

The primary treatment modality for bladder cancer is Bacillus Calmete-Guerin (BCG), a live attenuated mycobacterium that is instilled into the bladder via a urethral catheter [3]. This agent remains one of the most effective immune therapies to date. However, though it is effective in prevention of tumor recurrence, response is heterogeneous and tumor recurrence is observed in up to 50% of patients over time. The predominance of immunosuppressive myeloid cells subsets among TILs in human bladder cancer suggests myeloid cells could be potential targets for therapeutic intervention in bladder cancer. We investigated the role of macrophages in BCG therapy in the MB49 bladder cancer model. Depletion of macrophages by liposomal clodronate (LC) during MB49 tumor challenge resulted in decreased tumor growth compared to BCG monotherapy suggesting that macrophages have a critical role in BCG immunotherapy. This study characterizes these myeloid populations in bladder cancer patients and also investigates their potential role in bladder cancer development and therapeutic response.

References:

1.

Eruslanov, E., et al., Circulating and tumor-infiltrating myeloid cell subsets in patients with bladder cancer. International journal of cancer. Journal international du cancer, 2012. 130(5): p. 1109-1119.

2.

Svatek, R.S. et al., Sequential intravesical mitomycin plus Bacillus Calmette-Guérin for non-muscle-invasive urothelial bladder carcinoma: translational and phase I clinical trial. Clinical Cancer Research, 2015. 21(2): p. 303-11

3.

Askeland, E.J., et al., Bladder Cancer Immunotherapy: BCG and Beyond. Advances in urology, 2012. 2012: p. 181987.

#549

Immune profiling uncovers neo-antigens and TCR repertoire responding to WX-mPD-1 and WX-mPD-L1 immune checkpoint inhibitors in syngeneic mouse xenograft models.

Hua Dong,1 Yan Lu,1 Qiyao Zhang,1 Lingling Zhang,1 Xiaoyue Chen,2 Guizhu Yang,1 Shuqun Yang,1 Miao He,1 Xuzhen Tang,1 Yuxin Qin,1 Yong Zheng,2 Jiexing Cai,2 Yong Cang,1 Shaoyu Yan,1 Jing Li,2 Qunsheng Ji1. 1 _Oncology BU, WuXi AppTec, Shanghai, China;_ 2 _WuXi Biologics, Shanghai, China_.

Introduction: PD-1 and PD-L1antibodies have shown impressive clinical benefits to cancer patients. However, still a significant number of patients fail to respond to those checkpoint inhibitors. To understand the underlying mechanism, we performed genomic and immune profiling of both tumor tissues and cytotoxic T cells isolated from syngeneic mouse xenograft models treated with in-house WuXi anti-mouse PD-1(WX-mPD-1) and anti-mouse PD-L1 (WX-mPD-L1) antibodies.

Material and methods: Syngeneic mouse xenograft models from melanoma cell lines, CloudmanS91 and B16-F10, were used to evaluate the anticancer efficacy of WX-mPD-1 and WX-mPD-L1 antibodies. Cancer cells and xenograft tumors before and after treatment were collected for RNASeq profiling, from which mRNA expression and DNA mutations were called and neo-antigens were predicted. FACS analysis and TCR sequencing were also performed to examine the tumor infiltrating lymphocytes (TILs) population and TCR repertoire diversity, respectively.

Results: Among forty-eight established syngeneic models, nine had been tested with WX-mPD-1 and WX-mPD-L1 antibodies as single agent or in combination. More importantly, we have investigated the expression, mutation loads, neo-antigens for responsive model from CloudmanS91 and partially responsive model from B16-F10. CloudmanS91 model had 8 fold increases in mutation loads compared with B16-F10 model. Neo-antigens identified in CloudmanS91 cells were further knocked out to dissect their roles in activating immune response to WX-mPD-1 and WX-mPD-L1 antibodies in vivo.

Conclusions: WX-mPD-1 and WX-mPD-L1 antibodies as well as syngeneic tumor models are useful tools for pre-clinical combination and MoA studies for cancer immunotherapy. Analysis of Neo-antigen and TCR repertoire helps us better understand immune response to these antibodies in preclinical settings, and suggest novel approaches to enhance the current immunotherapeutic interventions.

#550

Evaluating changes in T cell activation by measuring expression of IL-2, CTLA-4, NFAT, and NF-kB in Jurkat cells.

Jacquelyn M. Bement, Yuko J. Miyamoto. _Elon University, Elon, NC_.

Insufficient stimulation of T cells due to activated checkpoint pathways can lead to ineffective clearance of tumor cells and thus, tumor growth and cancer. Developing methods to measure changes in the activation of such pathways are needed to evaluate the efficacy of immunotherapy approaches to treating cancer by altering T cell activation. Current methods that use measurments of IL-2 expression are limited by the fact that IL-2 is not specific to T cell activation and that it provides little insight into the mechanisms by which activation is affected. This project aims to address such limitations by determining whether changes in T cell activation can be effectively evaluated by measuring expression of CTLA-4, NFAT, and NF-kB, in addition to IL-2. Jurkat cells were cultured and activated with PMA (100 ng/mL) and ionomycin (500 ng/mL) for five hours. Staining for IL-2 and CTLA-4 with antibodies and transfection of the cells with genetic constructs that link NFAT and NF-kB to fluorescent proteins provided the means to link expression of fluorescence to expression of the molecules of interest. FACS analysis was then used to measure changes in the expression of the four signaling molecules in response to the induced changes in activation. Activation of the cells resulted in increased expression of all four molecules. Following activation, fluorescence due to CTLA-4, NFAT, and NF-kB expression changed by average fold increases of 2.58, 1.10 and 1.13, respectively. By measuring these key elements of T cell signaling pathways in addition to IL-2, these methods provide a potentially more effective alternative to current methods for evaluating the efficacy of immunotherapeutic treatments for cancer.

#551

In situ vaccination with TLR9 agonist and anti-Ox40 antibody is sufficient to induce abscopal responses even in mice with spontaneous oncogene-driven tumors.

Idit Sagiv-Barfi, Debra K. Czerwinski, Ronald Levy. _Stanford University, Stanford, CA_.

Background: Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns on bacterial, fungal, or viral pathogens. Intratumoral (IT) injection of unmethylated CG-enriched oligodeoxynucleotides (CpG), a TLR9 agonist, results in local tumor eradication but on its own is not able to induce a systemic anti-tumor immune response. OX40 is a potent costimulatory receptor that can potentiate the action of conventional T cells leading to their proliferation, effector function and survival, but can also inhibit or kill T regulatory cells by ADCC. In preclinical studies OX40 agonists increased antitumor immunity and improve tumor-free survival.

Scientific question: Does local injection of a CpG with anti-OX40 agonistic antibody trigger a systemic anti-tumor immune response?

Results: Using transplantable syngeneic tumor models, we implanted the tumor bilaterally in opposite sides of the abdomen. After tumors were established we administered CpG and anti-OX40 antibody into the tumor on one side and monitored both the injected and the uninjected sites. Mice bearing the A20 lymphoma tumors were cured and were protected from a second challenge with A20 cells. To examine the potential to treat spontaneous, non-transplanted cancers we chose the mouse mammary gland tumor model- FVB/N-Tg(MMTV-PyVT)634Mul/J. Injections of CpG and anti-OX40 antibody to the first arising tumor significantly reduced the incidence and outgrowth of subsequent tumors at un-injected susceptible mammary glands and the number of lung metastases. This anti tumor effect was T cell dependent, since depletion of either CD4+ or CD8+ T cells abrogated the therapeutic effect of in situ vaccination.

Significance: TLR9 agonists and anti-OX40 antibodies are currently under clinical development for cancer treatment. We show here that combining anti-OX40 antibody with a TLR9 agonist at a single established tumor is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at distant sites in both transplantable and spontaneously occurring oncogene-driven murine tumors. This anti-tumor effect was long lasting, specific and required T cells.

Impact: We recently published positive results showing that the combination of CpG with antibodies against CTLA4 and OX40 induced a therapeutic immune response against transplantable syngeneic tumors (Marabelle, Levy, et al. JCI, 2013). Our current results suggest that CpG and anti-OX40 are sufficient to induce fully protective and curative anti tumor immune responses, even in spontaneously arising cancer. Anti-OX40 and CpG are both currently in phase-I clinical trials as single agents. Our results provide the rationale for testing these agents in combination in the form of locally injected inducers of therapeutic anti-cancer immunity.

#552

Immuno-oncology agent CB-1158 is a potent and selective arginase inhibitor and causes an immune-mediated anti-tumor response.

Melissa Works, Mark Bennett, Jason Chen, Ethan Emberley, Tony Huang, Julie Janes, Weiqun Li, Andy Mackinnon, Gisele Marguier, Silinda Neou, Alison Pan, Francesco Parlati, Mirna Rodriguez, Susanne Steggerda, Tracy Wang, Jing Zhang, Winter Zhang, Matthew Gross. _Calithera Biosciences, South San Francisco, CA_.

L-arginine is a critical metabolite for T-cell receptor signaling and subsequent T-cell proliferation, and depletion of arginine arrests T-cell growth. In the tumor microenvironment, infiltrating myeloid-derived suppressor cells (MDSCs), macrophages, and neutrophils produce arginase, which depletes local arginine concentrations and dampens T cell-mediated immune surveillance. Pharmacological inhibition of arginase is expected to restore arginine levels and allow T-cells to proliferate, thereby leading to an immune-mediated anti-tumor response. CB-1158 is a potent inhibitor of human arginase (IC50 = 98 nM). In culture, human granulocytes release arginase and deplete media arginine to levels that inhibit T-cell proliferation. In a co-culture system of human granulocytes and T-cells, CB-1158 potently blocks granulocyte-derived arginase activity, maintains extracellular arginine levels, and restores proliferation of T-cells.

CB-1158 has high oral bioavailability in rodents and is very well tolerated. BID oral dosing of CB-1158 leads to dose-dependent pharmacodynamic increases in plasma and tumor arginine levels resulting in single agent anti-tumor efficacy in mouse syngeneic tumor models including Lewis Lung carcinoma (LLC) and Madison 109. The anti-tumor effects of CB-1158 are consistent with promoting a proinflammatory tumor microenvironment. Following CB-1158 treatment, multiple Th1 T-cell, NK-cell, and M1 macrophage-associated chemokines, cytokines, and activation markers are elevated in the LLC tumor microenvironment. The anti-tumor efficacy of CB-1158 requires an intact tumor microenvironment since CB-1158 has no effect on LLC cell growth in vitro. Furthermore, CB-1158 treatment of immunocompromised C57/SCID mice bearing LLC tumors has no anti-tumor effect, supporting an immune-mediated anti-tumor mechanism.

Immunosuppression in the tumor microenvironment can occur via multiple mechanisms, including arginine depletion, and our data support the combination of checkpoint inhibitors and arginase inhibition by CB-1158. In mice bearing LLC tumors, CB-1158 in combination with checkpoint inhibitors reduced tumor growth, increased the number of tumor infiltrating CD8+ T-cells, and increased the level of Th1/NK/M1-associated chemokines, cytokines, and activation markers in the tumor microenvironment. In mice bearing 4T1 tumors, a tumor type that is highly refractory to checkpoint inhibition, the combination of CB-1158 with anti-PD-1 and anti-CTLA-4 reduces tumor growth and lung metastases. These results support the development of CB-1158, a first-in-class arginase inhibitor, as a novel immuno-oncology agent targeting the immunosuppressive effects of tumor-infiltrating myeloid cells.

#553

Ibrutinib, a BTK inhibitor, impairs the generation and function of myeloid derived suppressor cells.

Andrew R. Stiff, Prashant Trikha, Robert Wesolowski, Kari Kendra, Sarvani Uppati, David Abood, Elizabeth McMichael, Megan Duggan, Amanda Campbell, Natarajan Muthusamy, Susheela Tridandapani, Michael Caliguiri, John C. Byrd, William E. Carson. _The Ohio State University, Columbus, OH_.

Myeloid derived suppressor cells (MDSC) interfere with anti tumor immune responses. MDSC have also been shown to antagonize the effectiveness of immune based therapies including immune checkpoint blockade. As a result, MDSC have received attention as potential targets for immune based combination therapies. There has been limited success in the identification of clinically active agents with the ability to inhibit the function or generation of MDSC. Ibrutinib is an orally available irreversible inhibitor of Bruton's tyrosine kinase (BTK) that is FDA approved for the treatment of B cell malignancies. In addition to B cells, cells of the myeloid lineage including monocytes and macrophages express BTK, and treatment with ibrutinib has been shown to alter their function and differentiation. As a result, it was hypothesized that ibrutinib would interfere with the function or generation of MDSC in the setting of cancer. MDSC isolated from the spleens of multiple murine tumor models (EMT6, 4T1, and C26) as well as MDSC from patients with metastatic melanoma expressed BTK. Treatment with ibrutinib at doses ranging from 0.1-5 μM inhibited the phosphorylation of BTK in both murine and human MDSC. Ibrutinib treatment of murine and human MDSC resulted in a significant reduction in nitric oxide (NO) production (p< 0.05), but had only modest effects on MDSC levels of IDO and arginase. Ibrutinib was also able to inhibit murine MDSC migration in response to EMT6 cell line conditioned media and the chemokine CXCL12 (p< 0.05). In addition, ibrutinib inhibited human MDSC migration in response to GM CSF (p< 0.05). Ibrutinib reduced the expression of the myeloid adhesion molecules CD11a (p< 0.05) and CD49D (p< 0.01) by MDSC, which could explain the reduction in migration. Importantly, ibrutinib significantly reduced the ability of MDSC to suppress CD8+ T cell proliferation compared to DMSO (21.98% vs. 12.49% proliferation, p< 0.05). Daily treatment with ibrutinib effectively inhibited the in vitro generation of human MDSC from monocytes by promoting HLA DR expression (p< 0.05). Using the EMT6 mammary carcinoma model in vivo, ibrutinib treatment resulted in a significant reduction of MDSC in both the spleen and tumor (p< 0.05). Ibrutinib also reduced the frequency of splenic MDSC in wild type B16F10 tumor bearing mice, but not in BTK mutant XID mice. In addition, both murine and human MDSC did not express significant levels of alternative ibrutinib targets including ITK, Bmx, and Blk. These results suggest that inhibition of BTK is the primary driver behind the observed effects of ibrutinib on MDSC function and generation. Finally, the combination of ibrutinib and anti PDL1 therapy was significantly more effective than either agent alone (p< 0.01 and p< 0.05) producing complete tumor regression in 50% of EMT6 tumor being mice. The results support further investigation of ibrutinib in combination with immune based therapies for solid tumors.

#554

Checkpoint blockade therapy is improved by altering the immune suppressive microenvironment with IPI-549, a potent and selective inhibitor of PI3K-gamma, in preclinical models.

Olivier De Henau,1 Taha Merghoub,1 David Winkler,2 Sujata Sharma,2 Melissa Pink,2 Jeremy Tchaicha,2 Matthew Rausch,2 Jennifer Proctor,2 Nicole Kosmider,2 John Soglia,2 Vito Palombella,2 Jeffery L. Kutok,2 Jedd D. Wolchok,1 Karen McGovern2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Infinity Pharmaceuticals, Inc, Cambridge, MA_.

The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is predominantly expressed in leukocytes and not expressed in most epithelial tumors or sarcomas. Genetic studies highlight an important role for PI3K-γ in myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Schmid et al. Canc Cell 2011). Targeting PI3K-γ could therefore alter the immune tumor microenvironment, enabling the immune system to attack tumor cells more effectively. We are developing IPI-549, an investigational small molecule inhibitor of PI3K-γ, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition.

IPI-549 is a potent and selective inhibitor of PI3K-γ with favorable pharmacological properties. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization, but did not affect M1 polarization. Oral administration of IPI-549 to tumor-bearing mice resulted in significant tumor growth inhibition in multiple syngeneic solid tumor models at PI3K-γ selective doses. Analysis of the tumor-associated immune cells demonstrated that IPI-549 treatment results in decreased immune suppressive myeloid cells and increased CD8+ T cells, suggesting enhanced anti-tumor immunity. To address the requirement for targeting myeloid cells by IPI-549, CD11b+ cells were depleted from a transplanted whole tumor Lewis Lung Carcinoma model and the effect of IPI-549 on limiting tumor growth was abrogated. In addition, a myeloid-infiltrated B16-GMCSF model, but not the isogenic B16 model without GMCSF, was responsive to IPI-549. Studies in immune-deficient mice or CD8 T-cell depleted tumor bearing mice demonstrated the T-cell dependence of IPI-549-mediated tumor growth inhibition. IPI-549 treatment also led to a significant reduction in lung metastases in the 4T1 and B16-GMCSF models. Importantly, in vivo studies with IPI-549 in combination with the immune checkpoint inhibitors anti-PD-1, anti-PDL-1 and anti-CTLA-4 showed increased tumor growth inhibition in multiple models compared to monotherapies alone. These data can inform combinations for future clinical trials.

Our studies support a role for PI3K-γ in immune suppressive myeloid cells in the tumor microenvironment and provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit tumor growth in preclinical models. A Phase 1 study evaluating IPI-549 as an orally administered therapeutic, as a single agent and in combination with an anti-PD-1 antibody therapy, in patients with selected solid tumors is expected to begin in early 2016.

#555

**Prostate cancer cell-induced differentiation of human monocytes into MDSCs** ex vivo **is inhibited by targeting STAT3.**

Rebecka Hellsten,1 Karin Leandersson,1 Anders Bjartell,1 Martin E. Johansson2. 1 _Department of Translational Medicine, Lund University, Malmo, Sweden;_ 2 _Glactone Pharma, Helsingborg, Sweden_.

Background: The transcription factor Signal Transducer and Activator of Transcription Factor 3 (STAT3) is implicated in acquired drug resistance, metastases and immune suppression in prostate cancer and is a potential target in drug-resistant prostate cancer. Today, the treatment options for metastatic drug-resistant prostate cancer are very limited and existing immunotherapies have so far shown low efficacy. Cancer cells may avoid immune responses by expressing immunosuppressive markers or activating immunosuppressive cells. Myeloid-derived suppressor cells (MDSC) play a major role in the suppression of antitumor immunity and active STAT3 signaling has been shown to be involved in the immunosuppressive functions of these cells. Elevated levels of MDSC have been found in the peripheral blood and at the tumor site of prostate cancer patients and to correlate with disease progression.

Aims: In this study we aimed to investigate if human monocytes could be induced to differentiate into immunosuppressive cells in the presence of prostate cancer cells and if this effect could be blocked by the STAT3 inhibitor GPA500 (galiellalactone).

Methods: Monocytes were isolated from peripheral blood mononuclear cells from healthy human donors using a magnetic monocyte enrichment kit. Monocytes were co-cultured with the prostate cancer cell lines DU145, LNCaP or longterm IL6 treated LNCaP cells (LNCaP-IL6+) in transwells allowing cancer cells and monocytes to share culture medium. The co-cultures were grown for three days with or without the STAT3 inhibitor GPA500 (5 uM or 10 uM) with subsequent analysis by flow cytometry for cell surface markers of MDSC differentiation (CD14+ HLA-DRlo). The presence of active STAT3 (pSTAT3) in the prostate cancer cell lines was determined by Western blot.

Results: Monocytes in the presence of DU145 and LNCaP-IL6+ cells showed a significantly increased population of CD14+ HLA-DRlo cells. In the presence of GPA500 the induction of this population was inhibited in a dose dependent manner compared to untreated cells. Co-culture with LNCaP cells that lack pSTAT3 did not induce this population in monocytes after three days in culture. Furthermore, GPA500 induced a dendritic cell-like population in monocytes in the absence of prostate cancer cell co-culture. This population was also observed in monocytes when co-cultured with LNCaP-IL6+ cells, but not in DU145 co-cultures, with GPA500 present.

Conclusion: The prostate cancer cell lines DU145 and LNCaP-IL6+ were able to induce a monocyte population displaying MDSC markers and this induction could be inhibited by the STAT3 inhibitor GPA500.

Impact: This study demonstrates that inhibiting the transcription factor STAT3 in prostate cancer could potentially reverse the immunosuppressive mechanisms caused by MDSC activation and that STAT3 inhibitors, alone or in combination with other therapies, could be a new treatment option for prostate cancer.

#556

Costimulatory T-cell engagement by the HER2/CD137 bispecific PRS-343 leads to strong antitumor effect in humanized mouse model.

Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Alexander Wiedenmann, Andrea Allersdorfer, Gabriele Matschiner, Sven Berger, Ulrich Moebius, Christine Rothe, Shane A. Olwill. _Pieris Pharmaceuticals, Inc., Freising, Germany_.

Background. CD137 (4-1BB) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. While multiple lines of evidence show that CD137 is a highly promising therapeutic target in cancer, current mAb-based approaches are not designed to achieve a tumor-target driven activation and may display toxicity and a limited therapeutic window due to peripheral T cell and NK cell activation. To overcome this limitation, we generated PRS-343, a HER2/CD137 bispecific that is designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells.

Methods. Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. PRS-343 was obtained by genetic fusion of the CD137-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone.

Results. The bispecific fusion PRS-343 targets CD137 and HER2 with nearly identical affinities compared to the parental building blocks, and is capable of binding both targets simultaneously. We show ex vivo that T cells are efficiently activated when incubated with PRS-343 and HER2-positive cells, and that the activation is HER2-dependent. The in vivo activity of PRS-343 was investigated utilizing a humanized mouse model with a tumor cell-line-derived, HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC via an intravenous route and weekly intraperitoneal injections of PRS-343 or controls for three weeks. We found that PRS-343 led to strong tumor growth inhibition and a significantly better response compared to either isotype control or anti-CD137 benchmark mAbs. The data, which include phenotyping of peripheral and intra-tumoral lymphocytes, support the envisaged mode of action of tumor-localized costimulatory T cell activation.

Conclusion. We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the HER2/CD137 bispecific PRS-343. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The positive functional ex vivo and in vivo data of PRS-343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials.

#557

Taking the sweet out of Th17 cells to potentiate immuno-oncology drugs.

Sucheta Telang, Kavitha Yaddanadupi, Gilles Tapolsky, Rebecca Redman, Jason Chesney. _University of Louisville, Louisville, KY_.

The major subsets of T cells that produce IL-17 include adaptive CD4+ Th17 cells and innate γδ T17 cells. IL-17 and both cellular sources are elevated in multiple human cancers and have been found to correlate with decreased patient survival. IL-17 produced by these cells promotes tumor growth by increasing the tumor infiltration and function of myeloid-derived suppressor cells (MDSCs), which in turn stimulate angiogenesis and suppress CD4+ and CD8+ T cell tumor homing and activation. Recently, two independent groups discovered that Th17 cell differentiation requires the transcription factor, hypoxia inducible factor 1α (HIF-1α), which promotes glycolytic enzymes and increases glucose metabolism. An established transcriptional target of HIF-1α and stimulator of glucose metabolism is 6-phosphofructo-2-kinase (PFKFB3) which synthesizes fructose 2,6-bisphosphate (F2,6BP), a potent allosteric activator of the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK-1). In unpublished studies, we have found that human Th17 cells generated ex vivo produce increased PFKFB3 and F2,6BP relative to total T cells. We postulate that Th17 cells may selectively require the activity of PFKFB3 for their differentiation and tumor-promoting functions. We examined the immunomodulatory effects of a first-in-class PFKFB3 inhibitor, (E)-1-(pyridyn-4-yl)-3-(7-(trifluoromethyl)quinolin-2-yl)-prop-2-en-1-one (PFK-158) on Th17 cells in vitro, in B16 melanoma-bearing mice and in cancer patients participating in a phase 1 multi-center clinical trial (clinicaltrials.gov # NCT02044861) and found that PFK-158: (i) suppresses human Th17 cell differentiation in vitro (200 nM); (ii) decreases splenic and tumor-infiltrating Th17 cells, γδ T17 cells and MDSCs, and increases CD4+ and CD8+ T cells in the tumors of B16-F10 melanoma-bearing mice (0.06 mg/gm QD x 3 days); and (iii) decreases peripheral blood Th17 cells, γδ T cells and MDSCs and increases activated effector CD4+ and CD8+ T cells in cancer patients. Furthermore, we observed that homozygous genomic deletion of Pfkfb3 in C57Bl/6 mice (but not in implanted B16 melanoma cells) reduces B16 tumor growth, decreases splenic and tumor-infiltrating Th17 cells, γδ T17 cells and MDSCs, and increases tumor-filtrating CD4+ and CD8+ T cells. Based on these immunological effects, we predicted that PFK-158 would improve the anti-tumor activity of an immune checkpoint inhibitor, anti-CTLA4, in the B16-F10 model - we observed a marked increase in tumor growth inhibition by anti-CTLA4 when combined with PFK-158 in vivo. Taken together, these studies provide the first pre-clinical and clinical rationale for the conduct of phase 1/2 trials to examine the anti-cancer efficacy of PFKFB3 inhibitors in combination with FDA-approved immune checkpoint inhibitors and other immunostimulatory agents such as the GM-CSF-producing oncolytic herpes virus talimogene laherparepvec.

#558

Durable antitumor activity of the CD122-biased immunostimulatory cytokine NKTR-214 combined with immune checkpoint blockade.

John L. Langowski, Rhoneil Pena, Yolanda M. Kirksey, Murali K. Addepalli, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych. _Nektar Therapeutics, San Francisco, CA_.

Background: While immune checkpoint blockade is a promising therapeutic approach, combination with agents that modulate complementary pathways may improve responses. Interleukin-2 (IL-2) immunotherapy leads to long-term responses in a small percentage of cancer patients, but systemic toxicity limits its use. In addition, IL-2 expands T regulatory cells, antagonizing antitumor immunity. NKTR-214 is a novel CD122-biased immunostimulatory cytokine which combines biased activation of the IL-2R beta receptor, greatly favoring activation of effector over regulatory T cells in the tumor microenvironment, with improved pharmacokinetics and tolerability compared to Proleukin® (aldesleukin) in non-clinical models. Here we examine the efficacy and mechanism of NKTR-214 combined with checkpoint blocking antibodies in murine tumor models.

Methods: Mice bearing subcutaneous B16, LLC, CT26 or EMT6 tumors were treated with single agent NKTR-214 (q9d), aldesleukin (bid or qd), murine anti-CTLA-4 or anti-PD-1 (twice-weekly), or combinations of these agents. Tumor volume was measured before, during and after treatment. Immune cells were profiled by flow cytometry; CD8 or NK requirements for efficacy were assessed by in vivo depletion through serial anti-CD8 or anti-asialo-GM1 antibody injections, respectively. Antitumor memory and specificity was assessed in complete responders by challenging with EMT6 or CT26 followed by observation with no additional test article treatments.

Results: In the B16 model, superior single-agent efficacy was achieved by NKTR-214 with a 10-fold lower dose than aldesleukin, yet significantly increased CD8/Treg ratio in the tumor (>400). In the EMT6 model, while both aldesleukin and NKTR-214 synergized with anti-CTLA-4, a greater percentage of complete responders were consistently observed with NKTR-214 (73% versus 44%). Antitumor immunity induced by the combination required both NK and CD8 T cell activity, and was durable and specific with mice remaining tumor-free after re-challenge with an EMT6 but not CT26 implant. Finally, NKTR-214 combined with anti-PD-1 provided superior activity in the CT26 model compared with the combination of anti-CTLA-4 and anti-PD-1 (90% versus 60% tumor-free, respectively).

Conclusions: NKTR-214 enables access to the potent IL-2 pathway, providing a mechanism of action complementary to checkpoint inhibition. Favorable pharmacokinetics of NKTR-214 allows sustained tumor exposure and dosing schedules commensurate with antibody therapies. The nonclinical results support the ongoing Phase 1 trial of NKTR-214 in patients with solid tumors.

#559

Small molecule T-reg inhibitors for cancer immunotherapy.

Suresh Kumar,1 Jian Wu,1 Liquing Wang,2 Feng Wang,1 Matthew P. Kodrasov,1 Saket Agarwal,1 Ivan Sokirniy,1 Thomas Yeckley,1 Joseph Weinstock,1 Michael R. Mattern,1 Wayne W. Hancock2. 1 _Progenra, Inc., Malvern, PA;_ 2 _Children's Hospital, University of Pennsylvania, Philadelphia, PA_.

Immune evasion is a hallmark feature of tumors as they employ various strategies to suppress the immune system's ability to recognize and destroy cancer cells. T cell checkpoint inhibitors such as anti-PD1 and anti-CTLA-4 antibodies spearhead the immune response against a variety of tumors. Numerous studies suggest that the complex immunosuppressive milieux require the development of additional therapeutic agents to potentiate active drugs and thereby broaden the utility and increase the therapeutic indices of revolutionary immune-oncology treatment modalities. The presence of immunosuppressive regulatory T cells (Tregs) in tumors, keeping tumoricidal Teffector cells in check, signals poor prognosis. Thus, depletion of Tregs or impairment of Treg function is an attractive therapeutic approach for cancer. USP7, a deubiquitylase enzyme implicated as a critical node in several cancer signaling pathways, has recently emerged as an essential factor in maintaining Treg functions. Progenra identified small molecule USP7 inhibitors and employed them to show that Treg specific inhibition of USP7 results in impairment of Treg function leading to immune activation, commensurate with the ablation of Foxp3, a transcription factor that is a target of USP7 and is essential to Treg activation. This USP7 inhibitor class was subsequently lead optimized, and selected USP7 inhibitors were evaluated in ADME/PK studies and shown to impair Treg functions and to exhibit powerful anti-tumor activity against syngeneic lung tumor models in immunocompetent mice. In addition, Progenra's USP7 inhibitors significantly augmented the antitumor activity of anti-PD1 antibody, CAR T cell therapy, and cancer vaccines. These studies provide a strong rationale for the use of USP7 inhibitors in combination therapy protocols to improve the efficacy of currently approved cancer immunotherapy agents.

#560

Development of a novel class of traps that potently block transforming growth factor-beta (TGF-beta) thereby counteracting TGF-beta mediated immunosuppression and promoting T-cell infiltration into tumors.

Maureen D. O'Connor-McCourt,1 Anne E.G. Lenferink,2 John Zwaagstra,2 Traian Sulea,2 Jason Baardsnes,2 Catherine Collins,2 Christiane Cantin,2 Yves Durocher,2 Renu Singh,2 James Koropatnick3. 1 _Formation Biologics, Montreal, Quebec, Canada;_ 2 _National Research Council Canada, Montreal, Quebec, Canada;_ 3 _Lawson Health Research Institute, London, Ontario, Canada_.

Introduction: Elevated TGF-β ligand markedly augments cancer progression primarily by suppressing the immune system in the tumor microenvironment, in particular by suppressing T-cell recruitment and/or activation. We developed a novel class of decoy receptor traps to potently block TGF- β and induce T-cell infiltration into tumors. This promotes the "T-cell-inflamed" tumor state, which is expected to render tumors sensitive to immune checkpoint inhibitors and other immunotherapeutics.

Experimental Procedures: We have computationally designed a class of avidity-enhanced receptor-ectodomain-based traps which bind and neutralize TGF-β. Several trap formats have been produced and tested, with each format exhibiting varying characteristics, including differing circulating half-lives and in vitro blocking potencies (from nM to pM). Representative therapeutic candidates from the different trap formats were evaluated for efficacy in in vivo studies using the syngeneic 4T1 triple negative breast cancer (TNBC) tumor model. Additionally, ex vivo studies were performed on CD4+ and CD8+ T-cells harvested from the draining lymph nodes of treated animals.

Results: In efficacy studies using the syngeneic 4T1 TNBC model, novel TGF-β traps were shown to promote significant T-cell infiltration into tumors. This infiltration resulted in reduced primary tumor growth as well as significant reductions in metastatic lesions. Additionally, ex vivo studies revealed that trap treatment decreased T-cell apoptosis, promoted T-cell proliferation in response to tumor cell lysates in the presence of dendritic cells, as well as increased the capacity of T-cells to specifically lyse 4T1 tumor cells.

Conclusion: Novel computationally-designed TGF-β traps are capable of promoting the "T-cell-inflamed" tumor state. Combination studies in which this novel class of anti-TGF-β immunotherapy is combined with immune checkpoint inhibitors are ongoing.

#561

MEDI1873: A novel hexameric GITRL fusion protein with potent agonsitic and immunomodulatory activities in preclinical systems.

Ross A. Stewart,1 Natalie Tigue,1 Samantha Ireland,1 James Hair,1 Lisa Bamber,1 Michael Oberst,2 Rebecca Leyland,1 Amanda Watkins,1 Maureen Kennedy,2 Cann Jennifer,2 Lesley Young,1 Robert W. Wilkinson1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _MedImmune, Gaithersburg, MD_.

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the tumor necrosis factor receptor (TNFR) superfamily. GITR is expressed constitutively on regulatory T cells (Tregs) and is up-regulated on other T cells following activation. Agonistic antibodies to GITR have demonstrated significant activity in preclinical models of cancer. Here we describe the generation and characterisation of a GITR ligand (GITRL) fusion protein (FP) (MEDI1873), currently in phase 1 clinical trials.

Protein engineering was used to generate a series of GITRL FPs, which were screened using a high throughput reporter gene assay for GITR signalling. The most potent fusion protein resulted in a 20 times greater maximal signal and a 5 times higher EC50 when compared to a GITR targeting antibody. This increased potency was considered to be a result of the enhanced valency achieved by the hexameric format.

Two versions of GITRL FP, MEDI1873 and MEDI5607, bearing an IgG1 and IgG4 Fc respectively, both demonstrated equivalent potency in a reporter assay and were able to enhance T-cell activation, with respect to proliferation and cytokine release, and to overcome the suppressive effect of Tregs, in primary human cell based assays.

Assessment of two surrogate mouse GITRL FPs in the CT26 model of colorectal cancer indicated that the version with increased binding to Fc gamma receptors resulted in increased activity, coincident with an increased depletion of intratumoral Tregs, likely through Fc mediated effector functions.

A comparison of GITR expression on Tregs and effector T cells in mouse and human, via flow cytometry, indicated a similar pattern of expression across species, with significantly higher expression observed on Tregs. Immunohistochemical analysis indicated the presence of high levels of both GITR and FoxP3 in sections from human tumors; suggesting that the intratumoral Treg depletion observed in mice could also occur in humans.

Both MEDI1873 and MEDI5607 demonstrated enhanced binding to Fc gamma receptors when compared to antibody controls of the same isotype, again considered to be a result of their increased valency. However, only MEDI1873 was able to mediate ADCC against activated T cells in vitro; resulting in an increase in the CD8:CD4 ratio within the culture.

As a result of these studies, MEDI1873 was selected as an optimal GITR targeting agent that possessed the ability to both agonise GITR and to modulate Tregs through suppression and/or depletion. MEDI1873 is currently being assessed in a phase 1 clinical study (NCT02583165) in patients with solid tumors.

#562

Dinaciclib induces immunogenic cell death and enhances anti-PD-1 mediated tumor suppression.

Dewan Mohammed Sakib Hossain,1 Fernando Ugarte,1 Anandi Sawant,1 Mingmei Cai,2 Venkataraman Sriram,1 Elaine Pinheiro,2 Svetlana Sadekova,1 Alissa Chackerian1. 1 _Merck Research Laboratories, Palo Alto, CA;_ 2 _Merck Research Laboratories, Boston, MA_.

Blockade of the checkpoint inhibitor PD-1 has demonstrated remarkable success in the clinic for the treatment of a growing list of different cancers. However, several tumor types are resistant to anti-PD-1 monotherapy. This observation has spurred numerous combination studies to reveal what additional therapeutic interventions may complement anti-PD1 blockade. Recently it has been shown that immunogenic cell death (ICD), induced by radiation and/or chemotherapy, improves T cell responses against different tumor types. ICD is characterized by damage-associated molecular patterns (DAMPs) including surface expression of calreticulin, and release of ATP and HMGB1. Recognition of DAMPs triggers dendritic cell maturation and functions that are critical for tumor antigen-specific T cell activation. Thus therapies that evoke ICD may further augment anti-tumor immunity elicited by anti-PD-1.

In mouse syngeneic tumor models, we observed combinatorial anti-tumor activity of anti-PD1 and the cyclin-dependent kinase inhibitor, dinaciclib. We hypothesized that dinaciclib potentiates the effects of anti-PD-1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib express the hallmarks of ICD including HMGB1 and ATP release and surface expression of calreticulin. Dinaciclib treatment also increases tumor cell phagocytosis and induces dendritic cell maturation. Furthermore, mice immunized with dinaciclib-treated tumor cells are resistant to subsequent tumor challenge. Tumors from mice receiving anti-PD1 and dinaciclib have increased T cell infiltration and dendritic cell activation, indicating the overall quality of the immune response generated may be improved with the combo. Taken together, these findings suggest a potential mechanism for the observed synergy between dinaciclib and anti-PD1. Dinaciclib induces immunogenic cell death, converting the tumor cell into an endogenous vaccine and thereby boosting the effects of anti-PD-1.

#563

Combined treatment with PD-L1 blockade and a TLR7/8 agonist dramatically enhances antitumor immunity.

Naoto Nishii,1 Yuta Kondo,1 Lixin Li,2 Walter Lau,2 Hiroyuki Harada,3 Miyuki Azuma1. 1 _Department of Molecular Immunology, Tokyo Medical and Dental University, Tokyo, Japan;_ 2 _Birdie Biopharmaceuticals, Inc., China;_ 3 _Department of Oral and Maxillofacial Surgery, Tokyo Medical and Dental University, Tokyo, Japan_.

[Aim] Toll like receptor (TLRs) are expressed in many types of immune cells and function as a key sensor of microbial products. Ligand binding to TLRs leads to activation of signaling pathways in innate immune cells, particularly dendritic cells and subsequent induction of adaptive immune responses. TLR7/8 agonists have been shown to enhance antiviral and antitumor immune responses via multiple cytokine production such as IFN-α, IL-1β, IL-6 and TNFα. Resiquimod (R-848) is a synthetic imidazoquinoline-like molecule that binds to TLR7 and TLR8. Presently, resiquimod has been used as a topical drug for skin cancer and viral infection, because of severe side effects of systemic administration. Thus far, the clinical results of anti-PD-1 or anti-PD-L1 therapy have showed great benefits by inducing tumor regression and improving survival. However, the patients obtained the benefits of this treatment are around one third. An additional approach is required to enhance the efficacy of PD-1 blockade therapy. In this study, we examined the effects of combined treatment with anti-PD-L1 antibody and a low dose of resiquimod administration in two murine tumor models (a squamous cell carcinoma cell line, SCCVII and a colon carcinoma cell line, Colon26). [Methods] Anti-PD-L1 mAb (MIH5, 200 μg/mouse) and/or resiquimod (1.73 μg/mouse) were intraperitonealy administrated at three times a week and tumor growth was evaluated. [Results and Discussion] In a Colon26 model, either anti-PD-L1 (MIH5) mAb or resiquimod treatment alone slightly decreased tumor growth, but the combined treatment dramatically reduced the tumor growth and completely eradicated the tumor in some of mice. In a SCCVII model, monotherapy with resiquimod markedly reduced the tumor growth and completely eradicated the tumor in some of mice. The combined treatment further enhanced the efficacy. The delayed combined treatment at day 7 also showed the reduction of tumor size. Functional analyses at 3-4 wks after the tumor inoculation revealed that monotherapy with resiquimod in the SCCVII model and the combined treatment in both models significantly enhanced the percentages of IFN-γ+-expressing CD8+ T cells, but did not clearly affect the percentages of Foxp3+ regulatory T cells. Our results suggest that TLR7/8 agonist administration alone markedly enhanced antitumor responses in a selected tumor model and the combined treatment with PD-L1 blockade and a TLR7/8 agonist further amplify their effects.

#564

Combination therapy of chemokine receptor inhibition plus PD-L1 blockade potentiates antitumor effects in a murine model of breast cancer.

Heiyoun Jung, Linda Ertl, Karen Ebsworth, Christine Janson, Penglie Zhang, Punna Sreenivas, Thomas Schall, Israel Charo. _ChemoCentryx, Mountain View, CA_.

Trafficking and expansion of myeloid derived suppressor cells (MDSCs) plays a major role in the immune suppression of tumors. MDSCs express chemokine receptors which likely mediate their recruitment to the tumor microenvironment. Suppression of T cells is also mediated by the interaction of programmed death-1 (PD-1) and its ligands which are abundantly expressed in cancer cells and immune infiltrates, including MDSCs. Here, we show that targeting both pathways through administration of a small molecule chemokine receptor antagonist (CCX9588, which blocks CCR1) and a PD-L1 monoclonal antibody (mAb) significantly reduced tumor burden in mice who received orthotopic transplants of 4T1 cells, a cell line used to model triple negative breast cancer. Primary tumor growth was modestly reduced by either agent (PD-L1 mAb or CCR1 inhibitor) alone, but the combination of CCR1 inhibitor plus PD-L1 mAb resulted in significantly reduced tumor progression as compared to either of the single agents. Furthermore, lung metastasis was also significantly reduced by CCR1 antagonist and combination treatment. Orthotropic 4T1 cell engraftment induced robust expansion of CD11b+Ly6Ghi Ly6Chi MDSCs, a subpopulation of which express CCR1. Analysis of the tumor infiltrating cells revealed that CCX9588 significantly reduced the number of MDSCs and increased CD8 T cells infiltration in primary tumors, suggesting that CCR1 blockade of MDSC trafficking in combination with PD-L1 mAb translates into reduced tumor burden, possibly through increased CD8 T cell response against the tumor. Analysis of human breast cancer patient samples from The Cancer Genome Atlas (TCGA) database revealed that the CCR1 ligands, MCP-7 (CCL7) and RANTES (CCL5) are present at significantly higher levels in breast cancers as compared to normal breast tissue. Interestingly, ligands for CCR1 and PD-1 are significantly higher in triple negative breast cancer samples than in the other breast cancer subtypes. These data are the first to show that CCR1 chemokine receptor antagonists can act synergistically with PDL-1 inhibitors, and suggest a novel approach for potentiating the activity of immune cell checkpoint inhibitors in one of the most aggressive forms of breast cancer.

#565

RORγ agonists regulate immune checkpoint receptors to enhance anti-tumor immunity.

Xiao Hu,1 Xikui Liu,1 Jacques Moisan,1 Chrystal Paulos,2 Yahong Wang,1 Chauncey Spooner,1 Charles Lesch,1 Rodney Morgan,1 David Mertz,1 Dick Bousley,1 Clarke Taylor,1 Chad Van Huis,1 Don Skalitzky,1 Thomas Aicher,1 Peter Toogood,1 Laura Carter1. 1 _Lycera Corp, Ann Arbor, MI;_ 2 _Medical University of South Carolina, Charleston, SC_.

As the master transcription factor for Type 17 T cells, RORγt activates a program of gene expression associated with enhancing effector function and overcoming immune suppression. Lycera is developing synthetic, small molecule RORγ agonists for immunotherapy of cancer.

We have previously reported that RORγγ agonists increase type 17 cytokine and chemokine production, enhance cell survival and have single agent anti-tumor activity in syngeneic tumor models. Interestingly, transcriptional profiling and cytometry studies revealed that treatment of murine and human Th17 or Tc17 cells with RORγ agonists increases the expression of costimulatory receptors such as CD137 and decreased expression of coinhibitory receptors like PD-1.

Given the importance of PD-1 in anti-tumor immunity, we further characterized the effects of RORγ agonist on this pathway. In vitro treatment with RORγ agonists significantly decreases mean fluorescent intensity and percent PD-1+ cells after resting and repetitive restimulation of Type 17 T cells. RORγ agonists do not modulate PD-1 expression in RORγ-/- T cells. In co-cultures of wild type and RORγ-/- T cells in the presence of the agonist, RORγ-/- T cells have reduced PD-1 expression suggesting that RORγ agonists induce a transmissible effect on RORγ-/- T cells. However, ChIP-seq data indicates that RORγt does not directly bind to the promoter or enhancer element of PD-1. Transcriptional and epigenetic profiling experiments have identified several pathways modulated by RORγ agonists that may regulate PD-1 expression. Reduced PD-1 expression following RORγ agonist treatment has a functional impact as treated cells also resist PD-L1-mediated inhibition of cytokine production and proliferation.

Importantly, the decreased PD-1 expression observed after in vitro treatment with RORγ agonists is maintained following adoptive transfer of tumor specific T cells. These cells are highly effective at controlling tumor growth without further agonist treatment in vivo, suggesting that in vitro RORγ agonist treatment results in durable epigenetic changes.

In summary, RORγ agonists have been shown to decrease checkpoint receptor expression while enhancing cytokine production and promoting long term survival and self-renewal of T cells. These results provide rationales for combining an RORγ agonist with checkpoint inhibitor such as anti-CTLA4 or anti-PD-1. By integrating effects on different effector pathways, RORγ agonists represent a promising immunotherapy approach for the treatment of cancer.

#566

BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy.

Gro Gausdal,1 Kjersti Davidsen,2 Katarzyna Wnuk-Lipinska,1 Kathleen Wiertel,3 Jing Kang,2 Agnete Engelsen,2 Sébastien Bougnaud,1 Monica Hellesøy,1 Magnus Blø,1 Lavina Ahmed,1 Linn Hodneland,1 Sergej Kiprijanov,1 Oddbjørn Straume,4 Rolf A. Brekken,3 James B. Lorens2. 1 _BerGenBio AS, Bergen, Norway;_ 2 _Department of Biomedicine, Center for Cancer Biomarkers, University of Bergen, Bergen, Norway;_ 3 _Division of Surgical Oncology Department of Surgery, Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, TX;_ 4 _Department of Oncology, Haukeland University Hospital, Bergen, Norway_.

Signaling via the AXL receptor tyrosine kinase is a key suppressor of the anti-tumor innate immune response. AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer cells, dendritic cells and tumor-associated macrophages. AXL is also an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) that drives tumor immune evasion and resistance to cytotoxic T cell-mediated cell killing. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms. We therefore evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in syngeneic cancer mouse models that display limited immunogenicity.

We conducted studies in the aggressive mammary adenocarcinoma (4T1) syngeneic (Balb/C) mouse model. We found that AXL expression increased in 4T1 tumors treated with anti-CTLA-4/anti-PD-1 and correlated with lack of response to immune therapy. Combination with BGB324 (50 mg/kg bid) significantly enhanced responsiveness to anti-CTLA-4/anti-PD-1 treatment (10 mg/kg of each, 4 doses) in Balb/C mice bearing established 4T1 tumors. The combination of BGB324 + anti-CTLA-4/anti-PD-1 resulted in durable primary tumor clearance in 23 % of treated mice versus 5.6% obtained with anti-CTLA-4/anti-PD-1 alone (p=0.0157). In a separate study, BGB324 + anti-CTLA-4 treated resulted in 22% long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of this model were concomitantly abrogated in the animals responding to the combination treatment. In addition, BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity.

In conclusion, targeting AXL signaling represents a unique opportunity to address multiple tumor immune suppression mechanisms. Our results support combining the clinical-stage AXL inhibitor, BGB324, with immune checkpoint inhibitors to improve treatment of human cancers.

#567

In vivo **intra-tumoral electroporation of Gp96-Ig/Fc-OX40L stimulates CD8+ T cell cross-priming to tumor specific neoantigens.**

Suresh de Silva,1 George Fromm,1 Jamil Haque,2 Jean S. Campbell,2 Robert H. Pierce,2 Taylor H. Schreiber1. 1 _Heat Biologics, Durham, NC;_ 2 _Oncosec Medical Inc., San Diego, CA_.

Cancer immunotherapy relies on presentation of shared- and neo- antigens from a patient's tumor cells for recognition and clearance by the immune system. However, the tumor microenvironment deploys multiple strategies to evade immune recognition and often remains non-immunogenic, which is one of the challenges that need to be addressed when designing new therapies. Among the strategies to increase a tumor's immunogenicity is their genetic manipulation in situ via expression of molecular chaperones, T cell costimulators and/or pro-inflammatory genes using DNA/RNA vectors packaged in oncolytic viruses, lipid based components or through electroporation. In vivo electroporation-mediated gene transfer of IL-12 triggers tumor regression and systemic anti-tumor immune responses in experimental mouse models and in patients, demonstrating the feasibility of this intratumoral (IT) gene-transfer technology. We set out to test whether intratumoral electroporation of Gp96-Ig/Fc-OX40L, a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression, would generate a robust anti-neoantigen CD8+ T cell response. Gp96-Ig/Fc-OX40L is a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression. To assess antigen-specific CD8+ expansion, mice were adoptively transferred with OT-I cells after B16.F10-ovalbumin cells were injected to generate primary and contralateral melanotic tumors. Contralateral tumors were monitored to assess whether a systemic CD8+ T cell response could be elicited following primary tumor electroporation. IT electroporation of DNA expressing Gp96-Ig/Fc-OX40L in the primary tumor triggered a significant expansion of antigen-specific OT-I cells, which was absent in control mice. Remarkably, increases in antigen-specific OT-I cells correlated with regression of both the treated primary and untreated contralateral tumors. We further validated our findings in a CT26 mouse colorectal cancer tumor model, in which the expression of Gp96-Ig/Fc-OX40L from electroporated DNA stimulated an expansion of antigen-specific CD8+ T cells and again led to regression of both the treated primary and untreated contralateral tumor. Our findings demonstrate that in situ manipulation of intratumoral cells to express Gp96-Ig/Fc-OX40L stimulates potent antigen-specific cross priming to tumor specific neoantigens that culminates in robust systemic anti-tumor response. These findings provide exciting proof-of-principal and warrant further investigation into the direct delivery of molecular chaperones such as Gp96-Ig/Fc-OX40L and/or pro-inflammatory molecules for elevating the immunogenicity of tumors for a potent anti-tumor CD8+ T cell response.

#568

FAK/PYK2 inhibition enhances immune checkpoint inhibitor efficacy.

Yan Wang, Jennifer E. Ring, Kam Sprott, David T. Weaver, Jonathan A. Pachter. _Verastem, Needham, MA_.

Although durable responses to single agent immune checkpoint inhibitors have been reported, additional approaches are needed to improve upon this therapeutic benefit. Combinations of immunotherapy agents with tumor microenvironment modulators have the potential to overcome barriers that tumor cells develop to evade the immune system, and provide benefit to a greater proportion of patients. Focal Adhesion Kinase (FAK) and its family member, PYK2, are potentially valuable targets due to their roles in regulating key cellular populations in the tumor microenvironment. In addition to targeting cancer stem cells, the FAK/PYK2 dual inhibitors, VS-6063 and VS-4718, have been shown to inhibit monocyte-derived macrophages, reduce tumor-associated macrophages in xenograft models, and promote a CD8+ T cell-mediated anti-tumor response in squamous cell carcinoma models.

We now report that the combination of VS-4718 with an anti-PD-1 mAb shows improved efficacy over anti-PD-1 mAb alone and extends survival of MC38 syngeneic tumor bearing animals. Analysis of MC38 tumors at day 12 of treatment revealed a significant increase in the CD8+ T cells/Treg ratios in tumors in the VS-4718 + anti-PD-1 combination group, providing a mechanistic understanding for the enhanced efficacy of this combination.

To explore additional combination options, we tested the combination of VS-4718 with anti-4-1BB in the MC38 model. Consistent with what was observed with the anti-PD-1 combination, VS-4718 also enhanced the efficacy of an anti-4-1BB mAb.

To further delineate direct effect of FAK inhibition on human T cells, in vitro T cell proliferation assays were conducted. VS-6063 and VS-4718 dose-dependently stimulated proliferation of CD8+ cytotoxic T cells isolated from healthy donors. This is in distinct contrast to other protein kinase inhibitors, such as the SRC inhibitor dasatinib which impaired the proliferation of CD8+ cytotoxic T cells. In addition, both VS-4718 and VS-6063 decreased CD8+ T cell exhaustion markers, and increased T cell-mediated tumor cell killing in vitro.

These data provide a rationale for clinical trials in cancer patients to test whether a FAK/PYK2 inhibitor in combination with an immune checkpoint inhibitor could increase the breadth of responsive tumor types, increase the number of responders, and confer more durable anti-tumor responses.

#569

MicroRNA-mediated resistance to anti-PD1 therapy in lung cancer.

Maria A. Cortez, Xiaohong Wang, Cristina Ivan, Jonathan E. Schoenhals, Sharareh Niknam, Ailin Li, David Valdecanas, James P. Allison, Padmanee Sharma, Willem W. Overwijk, Daniel Gomez, Joey Y. Chang, Stephen Hahn, George A. Calin, James W. Welsh. _UT MD Anderson Cancer Center, Houston, TX_.

Immunotherapies targeting programmed cell death receptor-1 (PD-1) have shown some clinical success, validating the role of immune modulation in the treatment of cancer. However, only 18% of non-small cell lung cancer patients have responded to anti-PD1 treatment thus far, the remaining 82% have not. This raises fundamental questions about mechanisms of resistance to anti-PD1 therapy and potential strategies to overcome resistance. Previously, our group generated an anti-PD-1-resistant preclinical tumor model after sequential in vivo passage of the murine lung cancer cell line 344SQ (p53R172HΔg/+KrasLA1/+) into syngeneic mice, previously treated with anti-mouse PD-1 antibodies. In this study, we analyzed global gene expression of microRNAs and correlated with our previous mRNA microarray data to validate potential target candidates in order to identify mechanisms of resistance to anti-PD1 therapy. For the microRNA microarray studies, 344SQ_P (parental) or 344SQ_R (resistant) cells were inoculated into the right flank of 129Sv/ev mice (female mice, 12-14 weeks old, 5 mice per group). Anti-PD1 or control IgG (10mg/kg) antibodies were given on days 4 and 7 after tumor inoculation. On day 11, tumor tissues were collected and immediately frozen in liquid nitrogen. RNAs from three independent biologic replicates per group were used for GeneChip miRNA 4.0 Array (Affymetrix). Significant differences in microRNA gene expression levels between groups were defined as an adjusted P value <0.05 and absolute fold change of > 2. Heatmaps were created by using the heatmap.2 function in the gplots R package. Our previous studies demonstrated that major histocompatibility complex (MHC) class I and II and antigen presentation pathway, were significantly downregulated in the anti-PD-1-resistant tumors compared with parental tumors. Here, we found that microRNAs significantly upregulated in the anti-PD1 resistant model directly regulates MHC class I and II and antigen presentation machinery genes, including B2M, CIITA, PSMB8, PSMB8, TAP2 and TAPBP, promoting resistance to anti-PD1 therapy. Because activated CD8+ tumor-infiltrating lymphocytes must bind to MHC I proteins to kill target tumor cells, one way in which tumors can evade destruction by the immune system is through downregulating expression of MHC I on their surfaces. Therefore, our study identified upregulation of microRNAs that modulates the antigen presentation pathway as an underlying mechanism by which some tumors do not respond to anti-PD1 therapy. Our future goal is to validate these findings on our ongoing clinical studies.

### Therapeutic Antibodies

#570

Long-term efficacy and downstream mechanism of anti-Annexin A2 monoclonal antibody in a pre-clinical model of aggressive human breast cancer.

Mahesh C. Sharma,1 George P. Tuszynski,2 Marc R. Blackman,1 Meena Sharma3. 1 _VA Medical Center Washington DC, Washington, DC;_ 2 _Temple University School of Medicine, Philadelphia, PA;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Abstract:

There are considerable direct evidences to support that calcium binding protein ANX A2 is a potential target for aggressive breast cancer. The most compelling evidence is based on the fact that ANX A2 overexpress specifically in aggressive triple negative human breast cancer (TNBC) cell lines as well as in human breast cancer tissues. Previously, we and others have demonstrated a unique role of ANX A2 in cancer invasion including breast cancer. Moreover we demonstrated that anti-ANX A2 monoclonal antibody (anti-ANX A2mAb)-mediated immunoneutralization of ANX A2 inhibited invasive human breast growth in a xenograft model. We further evaluated long-term effect of multiple treatments of anti-ANX A2mAb and its mechanism of inhibition of human breast tumor growth. We now show that three treatments of anti-ANX A2mAb showed significant inhibition of breast tumor growth in immunodeficient mice and anti-tumor response was started from day 94. After treatments, we followed tumor growth for 172 days and our results demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated anti-ANX A2mAb treatment caused significant inhibition of tissue plasminogen activator (tPA) synthesis in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathway in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for the treatment of aggressive breast cancer.

#571

Pregnancy associated plasma protein A (PAPP-A) is a potential novel therapeutic target in Ewing sarcoma.

Sabine Heitzeneder, John F. Shern, Javed Khan, Crystal L. Mackall. _NIH, NCI, POB, Bethesda, MD_.

Background: Ewing sarcoma (EWS) is the second most common bone malignancy in the pediatric population. Despite intensive treatment regimens, 5-year overall survival for patients with metastatic or relapsed disease is less than 20%, indicating a clear need for novel targeted therapies. The importance of IGF signaling in EWS is illustrated by activity of antagonistic targeting of IGF-1R in clinical trials, although this approach is plagued by rapid development of resistance. Pregnancy Associated Plasma Protein A (PAPP-A) anchors to cell surface proteoglycans and mediates metalloproteinase activity, that cleaves IGFs from IGFBP-4, IGFBP-2 and IGFBP-5, thus increasing local bioavailability of bioactive, free IGF. PAPP-A is expressed at high levels by placental trophoblasts, where it induces high levels of IGFs, which are required for fetal growth.

Methods: In an attempt to identify novel targetable cell surface antigens in EWS, we performed ribosomal depleted RNA sequencing in 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal samples of variable tissues.

Results: PAPP-A was identified as one of the top 5 membrane associated proteins overexpressed in EWS (> than 4-fold) compared to normal tissue (p = 3.89E-30). PAPP-A showed substantial expression in tumors (median log2FPKM = 3.933, n=122) and minimal expression in normal tissue (median log2FPKM = -1.564, n=96). We confirmed cell surface expression of PAPP-A by flow cytometry on 10 EWS cell lines tested, and identified PAPP-A in the supernatant of 12 EWS cell lines, measured by ELISA. Targeting the PAPP-A locus in the EWS cell line EW8 utilizing CRISPR/Cas9 completely abrogated PAPP-A secretion in single cell clones and significantly diminished bioactive IGF-1 levels in EWS cultures, while increasing IGF-1 bound to IGFBP-4, results which confirm absent PAPP-A metalloproteinase activity. Gene knockout of PAPP-A in EW8 resulted in decreased cell growth in vitro.

Conclusions: We have identified PAPP-A as a novel potential cell surface target in Ewing sarcoma, characterized by high level tumor expression, limited normal tissue expression and biological significance in this tumor. Ongoing work is focused on studying growth of PAPP-A KO cell lines, and assessing whether PAPP-A neutralizing antibodies may mediate antitumor effects in EWS.

#572

A novel anti-PDL1 x anti-VEGFR2 bispecific antibody for enhanced antitumor immunity.

Dan Lu, Zhanna Polonskaya, Haifan Zhang, Xenia Luna, Stella Martomo, Zhikai Zhang, Zhaojing Zhong, Yan Wu, Jeegar Patel, James Tonra, Larry Witte, Sam Waksal, Zhenping Zhu. _Kadmon Corp, New York, NY_.

The importance of VEGF/VEGFR2 signaling in cancer growth and metastasis has been clearly highlighted by the therapeutic benefits of bevacizumab (Avastin), a humanized antibody to VEGF, and Cyramza, a fully human antibody to VEGFR2, in multiple cancer treatment modalities. Recently, immune checkpoint antagonistic antibodies to PD1 and PDL1 have shown some success in a variety of clinical settings across multiple cancer types. The overall clinical efficacy of these individual antibody therapies has been, however, rather limited and, only evident in a fraction of patients. To this end, combinations of antibodies against both VEGF/VEGFR2 and PD1/PDL1 may represent promising approaches to further enhance the antitumor efficacy of individual antibody therapies. In this study, we engineered a bispecific anti-PDL1 x anti-VEGFR2 antibody using two fully human antibodies derived from antibody phage display libraries. In this bispecific format, a high affinity single chain antibody to PDL1 was genetically fused to the C-terminus of the heavy chain of a conventional IgG antibody against VEGFR2. The bispecific antibody was efficiently expressed in mammalian cells and could be purified to homogeneity via single step affinity chromatography. The bispecific antibody retained the binding activity of its parental antibodies to PDL1 and VEGFR2, and strongly blocked both VEGF/VEGFR2 and PDL1/PD1 interaction. Further, the bispecific antibody inhibited VEGF-stimulated VEGFR2 phosphorylation and proliferation of endothelial cells, and promoted proliferation of human T cells and secretion of cytokine such as IL2 and INFγ. The bispecific antibody is currently being evaluated in vivo in relevant animal models.

#573

Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody.

Jennifer S. Michaelson,1 Christopher J. Harvey,1 Kutlu G. Elpek,1 Ellen Duong,1 Matthew Wallace,1 Chengyi J. Shu,1 Sriram Sathyanarayanan,1 Robert Mabry,1 Lindsey Shallberg,1 Tong Zi,1 Amit Deshpande,1 Stephen L. Sazinsky,1 Joshua Apgar,2 Deborah Law1. 1 _Jounce Therapeutics, Cambridge, MA;_ 2 _Applied BioMath, Winchester, MA_.

ICOS (inducible co-stimulator molecule) is a co-stimulatory molecule and a member of the CD28 superfamily expressed primarily on T lymphocytes. Analysis of cancer patient samples as well as rodent preclinical data have implicated a role for the ICOS pathway in cancer immunotherapy. We have generated a panel of anti-ICOS monoclonal antibodies with in vitro agonistic properties. The anti-ICOS antibodies are efficacious as monotherapies and in combination with anti-PD1 in multiple syngeneic tumor models. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8:T regulatory (Treg) cells as well as preferential reduction in ICOS-high Tregs in the tumor microenvironment. JTX-2011, a species cross-reactive high affinity humanized agonist monoclonal antibody, has been selected for development. Evaluation of JTX-2011 in nonhuman primate models will be presented, including data informing safety and PK parameters. Our preclinical data provides rational for clinical development of JTX-2011 as a cancer immunotherapeutic to be tested as both a monotherapy as well as in combination with immunotherapies in solid tumor indications.

#574

AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status.

Nandini Rudra-Ganguly, Pia M. Challita-Eid, Christine Lowe, Mike Mattie, Sung-Ju Moon, Brian A. Mendelsohn, Monica Leavitt, Cyrus Virata, Alla Verlinsky, Linnette Capo, Mi Sook Chang, Deanna L. Russell, Baljinder Randhawa, Gao Liu, René Hubert, Mary Brodey, Hector Aviña, Chunying Zhang, Joseph D. Abad, Banmeet Anand, Sher Karki, Zili An, Roland Luethy, Fernando Doñate, Daniel S. Pereira, Kendall Morrison, Ingrid B.J. Joseph, David R. Stover. _Agensys Inc., an Affiliate of Astellas Pharma Inc., Santa Monica, CA_.

FLT3 is a member of the class III receptor tyrosine kinase family that includes C-KIT, C-FMS and platelet derived growth factor receptor (PDGFR). FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human leukemia, FLT3 is expressed on 70-90% acute myeloid leukemia (AML) and most B-acute lymphoblastic leukemia (B-ALL). FLT3 genetic aberrations are commonly detected in patients with AML. The most common aberration is internal tandem duplication (ITD), which occurs in 25-30% of AML patients and causes constitutive activation of FLT3. Point mutation in codon D835 of the FLT3 tyrosine kinase domain is reported in 7-10% of AML patients and also causes constitutive activation of the receptor. FLT3 small molecule inhibitors targeting the kinase domain are predominantly active against FLT3 activated AML. The restricted normal tissue expression profile and higher differential in leukemic specimens makes FLT3 amenable to antibody-based therapeutics, requiring only target expression independent of kinase activation status. Therefore, development of an antibody-drug conjugate (ADC) may provide a therapeutic alternative for AML patients.

Here, we report the development of the first FLT3specific ADC, AGS62P1, employing site-specific conjugation using the non-natural amino acid, p-acetyl phenylalanine (pAF). AGS62P1 comprises a human gamma one antibody including an inserted pAF residue in each of the heavy chains. The antibody was conjugated to a potent cytotoxic payload via an oxime bond at the pAF sites, thus creating a nearly homogeneous drug distribution, with approximately 2 drug molecules per antibody. Strong binding affinity (0.1-0.9 nM) and potent in vitro cytotoxic activity (IC50 = 0.2-12 nM) was achieved in AML cell lines. The anti-FLT3 ADC was highly efficacious in AML tumor xenografts, leading to statistically significant tumor growth inhibition of both FLT3 ITD and non-ITD models. Additional characterization of both the antibody and ADC was performed, including ligand receptor interaction, degradation, internalization, and apoptosis.

In summary, we have developed a site-specific ADC targeting FLT3 that exhibits potent anti-tumor activity in xenograft models regardless of FLT3 activation status. This drug can potentially offer a new and more versatile approach in targeting FLT3-expressing leukemia through a mechanism independent of FLT3 genetic aberration.

#575

Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types.

Ana Alvarez-Cienfuegos,1 Lawrence H. Cheung,1 Khalid A. Mohamedali,1 Yunli Zhao,2 Kathryn E. Ruisaard,1 Jeffrey A. Winkles,3 Michael G. Rosenblum1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Shenyang Pharmaceutical University, Shenyang, China;_ 3 _University of Maryland School of Medicine, Baltimore, MD_.

The cytokine TWEAK, a TNF superfamily member, and its cognate receptor, Fn14, have emerged as potentially valuable targets for cancer therapy since their expression has been frequently identified as independent negative prognostic indicators in a variety of tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This human protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14 targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 containing either WTCYS or EACYS mutants of GrB, and a human scFv targeting Fn14 were expressed in HEK293E and suspension CHO cells, harvested under serum-free conditions and purified to homogeneity. Both constructs display high-level protein production (>50 mg/L), enhanced serum stability and impressive cytotoxic activity (IC50 < 20 nM) against Fn14 positive cells. The specific activity of the GrB moiety, as assessed by cleavage of a synthetic chromogenic GrB substrate, was comparable to that of human GrB for the WTCYS while the EACYS variant showed a 50% reduction in specific activity. On the other hand, in vitro stability studies showed that EACYS was more stable after 48h incubation in human serum, retaining over 60% of its initial specific activity. Against a panel of 20 human cell lines expressing Fn14, both constructs showed high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) and were two to over a hundred times more potent than free GrB. Mechanistic studies demonstrated that GrB-Fc-IT4 constructs activated caspase cascades and cytochrome C related pro-apoptotic mechanisms in keeping with the known intracellular functions of GrB in target cells. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 fusion proteins exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½ α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2 β = 35 hours). Toxicity studies in mice demonstrated that the MTD is above 100 mg/kg total dose. Murine orthotropic tumor model efficacy and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research.

#576

Immunohistochemistry and radioimaging with hJAA-11 antibody to the Thomsen-Friedenreich antigen: Potential theranostic application for breast cancer.

Loukia Karacosta,1 Holly Johnson,2 Julia Abdullah,2 Taylor Chrisikos,1 Rachel Ludwig,1 Bradley Turner,3 Swetha Tati,1 Diala Ghazal,1 Munnawar Sajjad,2 Stephen Koury,2 Susan Morey,2 Julie Adams,2 Kate Rittenhouse-Olson2. 1 _For-Robin, Inc., Buffalo, NY;_ 2 _University at Buffalo, Buffalo, NY;_ 3 _University of Rochester, Rochester, NY_.

Theranostics through the utilization of immunohistochemistry followed by radioimaging to determine if metastatic foci will react with a therapeutic antibody will allow for the selection of the patient population that will most benefit from this immunotherapy. The Thomsen-Friedenreich antigen (TF-Ag) has been shown to be involved in ~90% of carcinomas, specifically breast carcinomas, making it a suitable target for radioimaging and therapy. The anti-TF-Ag antibody, JAA-F11, a mouse monoclonal antibody (mAb), has had success in localization, blocking metastasis, and inhibiting cell proliferation, and binds to ~80% of breast cancer cell lines, without preference to receptor status. This is significant since the triple negative breast cancer (ER-/PR-/HER2-) has no current targeted treatment. Studies are extended to human breast cancer tissue microarray immunohistochemical (IHC) analysis, which was scored blindly by a pathologist on a semi- quantitative scale. JAA-F11 stained approximately 76% of all breast cancer specimens, which included cases of mucinous, medullary, invasive ductal and neuroendocrine carcinomas. The staining observed was irrespective of receptor status, whereas in normal breast tissue, staining was either absent or very weak/weak. In addition, all available matched lymph node metastasis stained, with greater intensity observed in 39% of cases. JAA-F11 IHC studies performed on human normal organ tissue arrays, showed staining that for the most part was not significantly different from staining obtained with isotype control antibody, or was observed in areas that would not be therapeutically accessible. Furthermore, in an additional IHC study, preliminary results suggest that JAA-F11 significantly stained other carcinomas including those of the colon, bladder, ovary and prostate. In vitro studies show that the humanized JAA-F11 (hJAA-F11) has similar chemical specificity and higher affinity towards the TF-Ag. Imaging studies were performed in a BALB/c mouse breast cancer model with the hJAA-F11 to determine biological reactivity and to predict the feasibility of theranostic imaging prior to therapy. After ~10 days of tumor growth, mice pretreated with cold iodine water and rabbit IgG to inhibit binding to Fc receptors, were given tail vein injections of hJAA-F11 conjugated with [124]-I. Clear tumor images were obtained up to 144 hours post injection. Biodistribution analysis has provided further results indicating increased %ID/g (7.0±3.9%) in tumor tissue as compared to healthy tissues (brain %ID/g to be 0.21±.09, stomach 0.80±0.19%, and bone 0.90±2.4%). Results support that the hJAA-F11 antibody can be used in a multi-step theranostic approach, with analysis of tumor binding in IHC, followed by imaging to determine in vivo tumor targeting prior to direct immunotherapy or antibody drug conjugate therapy with hJAA-F11.

#577

Systemic delivery of CBT-15G DCLK1-targeted monoclonal antibody dramatically decreases tumorigenesis in a xenograft model of pancreatic cancer.

Nathaniel Weygant,1 Dongfeng Qu,1 Randal May,1 Parthasarathy Chandrakesan,1 Yang Ge,2 Chelsea D. Ryan,1 Guangyu An,2 Michael J. Schlosser,3 Edwin Bannerman-Menson,3 Courtney W. Houchen1. 1 _University of Oklahoma HSC, Oklahoma City, OK;_ 2 _Beijing Chao-Yang Hospital, Capital Medical University, Dept. of Oncology, Beijing, China;_ 3 _COARE Biotechnology Inc., Oklahoma City, OK_.

Introduction

Pancreatic cancer is the 4th leading cause of cancer mortality in the US. Treatments for pancreatic cancer are desperately needed as current drugs lead to little or no improvement in patient survival. Doublecortin-like kinase 1 (DCLK1) marks putative stem cells in colon and pancreatic cancers and regulates important oncogenic processes including epithelial-mesenchymal transition, which supports metastasis. DCLK1's extracellular domain is a novel target for therapeutic monoclonal antibodies (mABs) that has not previously been pursued.

Materials and Methods

Development of a DCLK1-targeted mAB: Balb/c mice were immunized with proprietary KLH-linked peptides targeting the DCLK1 extracellular domain. After immunization, mice were killed and spleens were taken and used to generate hybridoma cell lines, which were subcloned to the monoclonal level. Hybridoma media containing DCLK1-mAB were screened for their ability to bind immunogen peptide and DCLK1 purified protein, and the best clone was selected for further production (CBT-15G). CBT-15G was produced on a large scale in roller bottles, purified using a MEP column, and dialyzed into PBS.

Xenograft study: 0.5x106 SW1990 human pancreatic cancer cells suspend in matrigel were injected into the flanks of 8-week old athymic nude mice and allowed to grow until reaching an average tumor volume of 100 mm3. Xenografts were treated with intraperitoneal CBT-15G mAB or isotype control at 25 mg/kg twice per week for 4 weeks. Tumor volume measurements were taken every other day using calipers. 30 days from the start of injections mice were killed and tumors excised, measured, weighed, and lysed for downstream analysis. Changes in volume and weight were analyzed statistically using Graphpad Prism 6.0.

Results

CBT-15G dramatically decreases tumorigenesis in SW1990 xenografts via a DCLK1-based mechanism of action: Biweekly injections of CBT-15G mAB significantly inhibited SW1990 pancreatic cancer xenograft growth over a period of 4 weeks (p<0.0001 by ANOVA). Excised tumor volume and weights were also significantly different and confirmed these findings. Furthermore, analysis of cell lines treated with CBT-15G and the xenograft tissues confirmed a DCLK1-based mechanism of action.

Conclusion

These findings for the first time demonstrate the systemic in vivo efficacy of DCLK1-targeted mABs against pancreatic cancer. As viable treatments for pancreatic cancer are currently severely lacking, CBT-15G DCLK1-targeted therapy should be investigated further as a potential treatment.

#578

Potent complement-dependent cytotoxicity of tumor cells by target bound hexamerization of EGFR antibodies.

Annalina Tammen,1 Frank J. Beurskens,2 Stefanie Derer,3 Ralf Schwanbeck,1 Paul W.H.I. Parren,2 Janine Schuurman,2 Thomas Valerius1. 1 _Christian-Albrechts-University, Kiel, Germany;_ 2 _Genmab BV, Utrecht, Netherlands;_ 3 _University Hospital Schleswig Holstein, Lübeck, Germany_.

Introduction: Activation of complement (C) constitutes an important effector mechanism of monoclonal antibodies in cancer therapy. Unmodified IgG1 antibodies against the epidermal growth factor receptor (EGFR), a well-validated tumor target, lack the capacity to induce complement-dependent cytotoxicity (CDC) as single agents. Recently, significantly improved CDC was demonstrated after antigen-bound IgG molecules formed hexameric structures through intermolecular Fc-Fc contacts. Single point mutations (E345K or E430G) that enhance hexamerization of IgG molecules conditional on antigen binding have been identified to represent a platform for enhanced C activation (Diebolder et al Science 343:1260, 2014, de Jong et al, PLOS Biology, in press). Alternative strategies to enhance CDC have focused on enhancing C1q binding via affinity-maturation of the C1q binding site (S239D/H268F/S324T/I332E; DFTE) or by generating IgG1/IgG3 isotype chimeras (113F). The aim of the present study was to investigate whether EGFR antibodies optimized for each of these approaches differed in terms of CDC activity.

Methods: EGFR antibodies comprising engineered Fc domains that carry mutations to enhance hexamerization (E345K or E430G), or increase C1q affinity (DFTE), or that exist of IgG1/IgG3 chimeras (113F) were constructed. CDC activity was assessed by 51Cr release assays in the presence of human serum. Antibody mediated C1q binding or C4b deposition on tumor cells was analyzed by flow cytometry. Apparent C1q binding affinity was determined by C1q specific ELISA.

Results: Here, we demonstrate that affinity maturation of the C1q binding site (DFTE & 113F) increased C1q binding in the absence of antigen, whereas mutations that enhance Fc-Fc interactions (E345K & E430G), and so enhance hexamerization after target binding, did not. However, these latter mutations strongly increased C1q avidity and CDC for EGFR antibodies bound to cells. Notably, the latter strategy translated in superior CDC activity, which was particularly evident for tumor cells with low EGFR expression levels. Furthermore, hexamerization enhanced CDC was particularly sensitive to peptides inhibiting Fc-Fc interactions, supporting their different mode of action compared to other CDC-enhancing strategies. Interestingly, also the EGFR epitope recognized by different antibodies impacted on the CDC activity.

Conclusion: Enhancement of hexamer formation by EGFR antibodies conditional on antigen binding is a promising novel approach to improve their anti-tumor activity. Strategies that enhanced complement binding independent of antigen engagement by the antibody appeared less effective.

#579

Antibody drug conjugates (anti-CD79b-vc-MMAE, Polatuzumab Vedotin) exhibit enhanced cell death targeted to CD79b+ Burkitt lymphoma (BL) and primary mediastinal large B-cell lymphoma (PMBL).

Aradhana Awasthi Tiwari,1 Janet Ayello,1 Carmella van de Ven,1 Lisa Kurien,1 Matthew J. Barth,2 Mitchell S. Cairo1. 1 _New York Medical College, Elsmford, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood, 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al.Blood, 2013). However, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease a dismal prognosis (≤ 10% 5 years, EFS) (Cairo et al. JCO, 2012, Miles/Cairo et al. BJH, 2012). The anti-CD79b-vc-MMAE antibody drug conjugates (Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res. 2009, Dornan et al. Blood, 2009). More recently PV has been safe and well tolerated in adult with CD79b refractory NHL (Palanca-Wessels et al. Lancet oncol, 2015) but its preclinical activity against mature BL/PMBL is unknown.

Objective: To determine the efficacy of ADC (anti-CD79b-vc-MMAE) against RTX sensitive/resistant CD79+ BL and PMBL tumor cell lines in-vitro.

Methods: BL: Raji/Raji4RH (provided by M. Barth, Roswell Park Cancer Institute) and PMBL: Karpas1106p and MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with hu anti- CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and cell proliferation was analyzed by alamar blue by flow-cytometry, n=3.

Results: Hu- anti -CD79b-vc-MMAE compared to hu anti-CD79b Ab or control hu IgG1 Ab alone (10µg/ml, 24hrs), significantly enhanced cell death ( apoptosis) in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p=0.0008 and p=0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p=NS and p=0.03), Karpas1106p, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p=0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p=0.002 and 0.0001), respectively. Hu anti- CD79b-vc-MMAE also significantly reduced cell proliferation compared to hu anti- CD79b Ab or control hu anti -IgG1 Ab alone (20µg/ml, 24hrs). Raji, 56.1±5.1% vs 38.1±0.7% vs. 14.8±0.4% (p=0.001 and p=0.0008), Raji4RH, 53.4±5.4% vs 23.4±5.51% vs. 11.3±0.8% (p=0.004 and 0.006), Karpas1106p, 46.4±0.3 %vs 29.0±1.5% vs. 15.9±0.6% (p=0.005 and 0.00007) and MedB-1, 47.2±7.5% vs 27.7±8.5% vs. 12.3±0.5% (p=0.01 and p=0.0005), respectively.

Conclusion: Our preliminary data indicates that hu anti- CD79b-vc-MMAE significantly enhances cell death and reduced cell proliferation in sensitive/ RTX resistant CD79b+ BL and PMBL compared to CD79b Ab or isotype control IgG1 Ab alone. Hu anti- CD79b-vc-MMAE may be a novel agent to investigate as immunotherapeutic agent in patients with relapsed refractory CD79b+ BL and/or PMBL.

#580

Anti-EGFRvIII TandAbs recruiting either T or NK cells are highly specific and potent therapeutic antibody candidates for the treatment of EGFRvIII+ tumors.

Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Thorsten Gantke, Stefan Knackmuss, Martin Treder. _Affimed GmbH, Heidelberg, Germany_.

EGFRvIII is the most prevalent tumor-specific variant of the wild-type EGFR and represents an attractive tumor target in various solid tumors such as GMB, HNSCC or NSCLC. Despite the clinical successes achieved with EGFR targeting antibodies or small molecule inhibitors little therapeutic progress has been made with EGFRvIII. There is still a high medical need in such cancers and several agents are in development to address this notoriously difficult target: Celldex´ vaccine rindopepimut in combination with bevacizumab in late-stage development, or ADCs in early development by Amgen or Abbvie. However, despite the high tumor specificity of EGFRvIII expression, no potent T- or NK-cell engaging are currently in clinical development.

It has been recognized that there may be more therapeutic potential with bi-specific antibodies recruiting immune effectors for targeted destruction of antigen-positive tumor cells. We developed tetravalent, bi-specific TandAbs that recognize EGFRvIII, and recruiting either T-cells or NK-cells by binding to the activating receptors CD3 and CD16A, respectively. This targeted antibody approach allows the selective destruction of EGFRvIII+ tumor cells employing highly potent and efficacious immune effector cells whilst sparing normal cells that are EGFRvIII- or cells presenting the ubiquitously expressed EGFR.

The selected EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs exhibited exquisite specificity towards EGFRvIII in Western Blot, SPR, ELISA, and flow cytometric assays. No binding was observed to recombinant EGFR or to EGFR+ cells. They also displayed potent cytotoxicity towards EGFRvIII+ cell lines with EC50 values in the low picomolar range. No cytotoxicity was observed on EGFRvIII- target cells or cells expressing EGFR demonstrating the high selectivity of anti-EGFRvIII TandAbs. Importantly, in the absence of EGFRvIII+ target cells our TandAbs did not elicit T- or NK-cell activation and activation-induced immune cell proliferation suggesting an excellent safety profile. In vivo pharmacodynamics for anti-EGFRvIII TandAbs was demonstrated in a mouse xenograft model.

The clinical relevance of EGFRvIII as a tumor marker and the binding of our anti-EGFRvIII variable domains were evaluated by immunohistochemistry. Binding was shown in glioblastoma and other solid tumors, but no expression was detectable on healthy tissue.

In summary, the presented in vitro and in vivo studies qualify EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs as highly attractive therapeutic antibody candidates and provides us with the possibility of employing both T-cell and NK-cell recruiting strategies for selective immunotherapy of EGFRvIII+ tumors. Due to tumor specific expression of EGFRvIII and the absence of off-target activity our TandAbs display an excellent safety profile reducing the risks of side effects associated with other anti-EGFRvIII therapies.

#581

Bone marrow-derived B-cell hybridomas from neuroblastoma patients generate antibodies that bind to patients' own tumors.

Girja S. Shukla,1 Giselle S. Sholler,2 Yujing Sun,1 Stephanie C. Pero,1 Chelsea L. Carman,1 Ping Zhao,2 David N. Krag1. 1 _University of Vermont College of Medicine, Burlington, VT;_ 2 _Helen DeVos Children's Hospital and Michigan State University, Grand Rapids, MI_.

Today antibody therapy is considered to be one of the most important and successful strategies to treat a variety of cancers. For example, the addition of antibodies such as Herceptin and Avastin to a chemotherapy regimen has shown improved survival in the treatment of breast cancer and colorectal cancer, respectively. Main problems with this kind of therapy are that many patients are not candidates because their tumors do not overexpress the drug target and that patient develop resistance to the targeted drug. A method to rapidly develop different sets of therapeutic antibodies would greatly contribute to the field of targeted anticancer therapy. This work evaluated the feasibility of using residual clinical material from pediatric neuroblastoma patients to generate antibodies to autologous tumor. Neuroblastoma is the most common extracranial solid tumor in children, accounting for 8-10% of all childhood cancers. Most patients with neuroblastoma are young and commonly present with metastatic disease. Bone marrow aspirate from neuroblatoma patients was the source material for the mononuclear cells and the tumor cells used in present study. Tumor cells were cultured and xenograft tumors were produced in mice. Hybridomas were generated by electrofusion of stimulated bone marrow mononuclear cells with plasmacytoma P3X63.Ag8.653 under hypo-osmolar condition using Eppendorf Multiporator/Helix chamber. Following hypoxanthine-aminopterin-thymidine (HAT) selection and monoclonal distribution, the culture supernatants were assayed for immunoglobulin secretion by ELISA. The supernatants from the positive clones were evaluated by immunofluorescence microscopy for binding to cultured neuroblastoma cells and neuroblastoma xenograft tissue sections derived from the same patient from which the hybridomas were generated. The results demonstrated that multiple hybridomas of bone marrow mononuclear cells secreted monoclonal antibodies that bound autologous neuroblastoma cells. Further evaluation of the tumor-binding antibodies on a panel of normal human tissues showed no binding to most of the tissues in the panel. Successful outcome of these experiments demonstrate the feasibility of generating human monoclonal antibodies from residual marrow specimens that bind autologous neuroblastoma cells. However, it remains to be determined whether these antibodies are bioactive and whether this approach will be generally applicable in more patients with neuroblastoma. It may be concluded that the strategy described here, which exploits the cancer patient's own immune repertoire, has a great potential for neuroblastoma target discovery and developing antibodies with possible therapeutic and/or diagnostic utility in cancer.

#582

**Novel insights into the antitumor activity of an antibody specific for transferrin receptor 1 (ch128.1) in an** in vivo **model of human multiple myeloma.**

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

The transferrin receptor 1 (TfR1), also known as CD71, is a type II transmembrane homodimeric protein involved in cellular iron uptake and regulation of cell growth. The high levels of TfR1 expression on cancer cells, its extracellular accessibility, and its central role in cancer pathology make it an attractive target for antibody-mediated cancer therapy. We have previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits direct cytotoxic activity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. In addition, we showed that ch128.1 is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) against malignant B cells in vitro. Importantly, ch128.1 shows exceptional antitumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige) in an early disease setting. Intriguingly, this activity is observed even in malignant cells that show no sensitivity to the direct cytotoxic activity of ch128.1 in vitro. In order to study the mechanism of the in vivo antitumor activity, we generated a ch128.1 mutant with abolished binding to FcγR and the complement component C1q (L234A/L235A/P331S). Interestingly, this antibody mutant exhibited a total lack of in vivo protection against MM. This lack of antitumor activity is not due to increased clearance as determined by bioavailability studies. If fact, the two antibodies showed comparable affinity for the mouse FcRn (the neonatal Fc receptor, also known as the Brambell receptor) as determined via surface plasmon resonance analysis. This result suggests a critical role for the antibody Fc fragment in ch128.1-mediated antitumor activity. However, preliminary in vivo studies showed that depletion of complement using cobra venom factor (CVF) does not decrease the antitumor activity of ch128.1, suggesting that CDC is not a relevant mechanism of action, at least in the model used. Furthermore, we recently found that treatment with ch128.1 also significantly increased survival in late MM disease. Further studies aiming to provide a better understanding of the in vivo activity conferred by this antibody are in progress. Our results suggest that ch128.1 can be effective in the therapy of incurable human B-cell malignancies such as MM.

#583

Evaluation of EphA2 as a therapeutic target for redirected T-cell killing by DART® bispecific molecules.

Claudia B. Fieger,1 Kalpana Shah,2 Gurunadh Chichili,2 Jonathan Li,1 Thomas Son,1 Jeffrey Hooley,1 Francine Chen,1 Sergey Gorlatov,2 Steve Burke,2 Valentina Ciccarone,2 Ralph Alderson,2 Deryk Loo,1 Syd Johnson,2 Ezio Bonvini,2 Paul Moore2. 1 _MacroGenics, Inc., South San Francisco, CA;_ 2 _MacroGenics, Inc., Rockville, MD_.

Introduction: EphA2 is a receptor tyrosine kinase that plays a critical role in cancer progression through both ligand-dependent and independent mechanisms. The broad overexpression in tumors but limited normal tissue expression of EphA2 makes it an attractive therapeutic target amenable for redirected T-cell killing via an EphA2 x CD3 Dual-Affinity Re-Targeting (DART®) molecules designed to co-engage cytotoxic T cells with EphA2 expressing tumor cells.

Methods: Anti-EphA2 monoclonal antibodies (mAbs) were identified by target-specific screening of a library generated by whole-cell immunization with proprietary cancer cell lines, including models of cancer stem cells. The binding and signaling properties of the antibodies were characterized by ELISA, SPR, flow cytometry and phosphorylation assays. Receptor binding regions were determined by ELISA based competition assays and by utilizing human-mouse chimeric EphA2 molecules. Immunohistochemistry (IHC) was performed on frozen normal and tumor tissues. In vitro functional studies were performed with various cancer as well as transfected cell lines, and primary human T cells or PBMCs. In vivo activity was evaluated in xenograft models in immune deficient mice.

Results: EphA2 mAbs encompassing diversity in binding kinetics and effects on receptor phosphorylation were classified in 5 discrete binding groups. The majority interacted with the N-terminal ligand-binding domain of EphA2 and most mAbs within that group interfered with ligand binding. The majority of mAbs displayed little IHC reactivity with normal tissue, while strong staining of cancer tissues was observed, including colon, lung, pancreas, ovary, bladder and breast cancers. Seven mAbs recognizing independent epitopes were selected for conversion into EphA2 x CD3 DART molecules that showed a range of potency in redirecting T cells to kill EphA2-expressing target cells. A lead EphA2 x CD3 DART molecule was selected based on potency and cross-reactivity with the cynomolgus monkey ortholog; this lead was engineered with a human Fc domain to confer an extended circulating half-life. The resulting Fc-bearing EphA2 x CD3 DART molecule demonstrated in vivo anti-tumor activity at doses as low as 20 mcg/kg in NOD/SCID/IL-2 gamma chain null mice co-implanted with activated human T cells and MDA-MB231 breast cancer cells.

Conclusion: EphA2 is a potential cancer target for redirected T-cell killing applications independent of ligand mediated mechanisms. Further preclinical assessment of EphA2 x CD3 DART molecules as a strategy for targeting EphA2-expressing malignancies is warranted.

#584

Significant enhancement of efficacy of an anti-Trop-2 antibody-drug conjugate, sacituzumab govitecan (IMMU-132), in experimental triple-negative breast cancer (TNBC) when combined with microtubule or PARP inhibitors.

Thomas M. Cardillo, Serengulam V. Govindan, Maria Zalath, Ali Mostafa, Roberto Arrojo, Robert M. Sharkey, David M. Goldenberg. _Immunomedics, Inc., Morris Plains, NJ_.

Purpose: In current clinical trials (ClinicalTrials.gov, NCT01631552), TNBC patients treated with IMMU-132, which is composed of the active metabolite of irinotecan, SN-38, conjugated to an anti-Trop-2 antibody (drug:Ab ratio = 7.6), shows manageable toxicity and encouraging responses in relapsed/refractory cases. Preclinical studies were performed to determine the utility of combinations of IMMU-132 with either a poly(adenosine diphosphoribose) polymerase (PARP) inhibitor (olaparib) or microtubule inhibitors (paclitaxel or eribulin mesylate) in mice bearing BRCA1/2 defective (HCC1806)and wild-type (MDA-MB-468) TNBC tumor xenografts.

Procedures: In vitro, human TNBC cell lines were incubated with IMMU-132 and olaparib to determine a combination index number and whether the interaction was synergistic, as well as incubating with SN-38 or IMMU-132 ± olaparib with analysis by western blot or flow cytometry (FACS) for double-stranded DNA breaks, as evidenced by increases in phosphorylated histone H2AX (p-H2AX). In vivo, mice bearing MDA-MB-468 or HCC1806 tumors were treated with either paclitaxel (qwklyx5wks) or eribulin mesylate (wks 1, 2, 4, & 5) alone or in combination with IMMU-132 (wks 1, 2, 4, & 5). Additionally, mice bearing TNBC tumors were treated with olaparib (qdx5d) plus IMMU-132 (qwkly) for 4 wks. Study survival endpoint was tumor progression to >1.0 cm3.

Results: Treatment with IMMU-132 plus paclitaxel in HCC1806 or MDA-MB-468 tumor-bearing mice significantly inhibited tumor growth compared to monotherapy (P<0.0195 and <0.0328, respectively). IMMU-132 plus eribulin mesylate also resulted in significant tumor regressions when compared to all other treatments in these two disease models (P<0.0007 and <0.0432, respectively). In vitro, olaparib combined with SN-38 or IMMU-132 increased p-H2AX levels. Cytotoxicity assays revealed this interaction to be synergistic in both BRCA1/2 defective and wild-typeTNBC cell lines. In vivo, IMMU-132 plus olaparib had significant anti-tumor effects in both HCC1806 and MDA-MB-468 tumor-bearing mice when compared to single-agent responses (P<0.0017 and <0.004, respectively). In all studies, the combination of IMMU-132 with either microtubule inhibitors or olaparib was well tolerated, with no observable toxicities (e.g., weight loss).

Conclusions: Combining IMMU-132 with a PARP inhibitor achieves synergistic growth inhibition in TNBC, regardless of BRCA1/2 status. The combination of IMMU-132 therapy with either microtubule or PARP inhibitors results in significant anti-tumor effects in TNBC disease models with no observable toxicity. These data provide the rationale for the clinical evaluation of IMMU-132 in combination with these chemotherapeutics in TNBC patients.

#585

Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy.

Olivier Nolan-Stevaux,1 Flordeliza Fajardo,1 Lily Liu,1 Suzanne Coberly,1 Patricia McElroy,2 Aaron Nazarian,2 Franziska Bott,3 Elisabeth Nahrwold,3 Tobias Raum,3 Roman Kischel,3 Ralf Lutterbuese,3 Patrick Hoffmann,3 Peter Kufer3. 1 _Amgen, Inc., South San Francisco, CA;_ 2 _Amgen, Inc., Thousand Oaks, CA;_ 3 _Amgen, Inc., Munich, Germany_.

BiTE® therapeutics are single chain antibody constructs harboring two binding moieties: one directed at a tumor associated antigen and one directed at the CD3e protein, which trigger T cell dependent cellular cytotoxicity (TDCC) against targeted cancer cells. Here, we evaluated the suitability of ENPP3 as a potential BiTE® target. The ENPP3 mRNA is highly differentially expressed in clear cell Renal Cell Carcinoma (ccRCC) and the ENPP3 protein is detected with high uniformity and intensity in ccRCC tumor samples by immunohistochemistry. We demonstrated the surface expression of this protein in Renal Cancer cell lines and confirmed that the ENPP3 protein was highly restricted to the luminal side of normal epithelial layers in which it was detected (proximal renal tubules, bronchial epithelium, salivary glands). We developed highly potent anti-ENPP3 BiTE® molecules and demonstrated the in vitro TDCC activity of these molecules. Two anti-ENPP3 BiTE® molecules further demonstrated tumor formation inhibition activity in a human T cell / human target cell admixture mouse xenograft model.

#586

Discovery of a novel TIM3 binding partner and a key role for TIM3 on macrophages: Identification of specific antibodies capable of converting immune-suppressive macrophages to immune-enhancing.

Jamie Wong, Ryan Phennicie, Igor Feldman, Sriram Sathyanarayanan, Don Shaffer, Mohammad Zafari, Steve Sazinsky, Kenneth Crook, Debbie Law. _Jounce Therapeutics, Cambridge, MA_.

Our Translational Science Platform uses an unbiased bioinformatics-based approach to interrogate particular cell types within the tumor microenvironment (TME). Given the correlation between high levels of immune-suppressive macrophages within the tumor TME and poor patient prognosis across a number of solid tumor types we focused initially on developing novel immunotherapies to modify this cell type. We identified 10 targets as candidates for converting tumor-associated macrophages from immune-suppressing to immune-enhancing.

One of these targets was TIM3. To date, TIM3 has been pursued mainly as a checkpoint target for T cell-directed immunotherapies based on its expression on exhausted T cells. Anti-TIM3 mAbs, generated by multiple groups, induce responsiveness in T cells and demonstrate anti-tumor benefit in certain mouse models. However, our macrophage-centric approach has identified a previously unrecognized protein-protein interaction between TIM3 and one of our additional macrophage targets. Based on knowledge of this interaction, we were able to generate and select for panels of mAbs to TIM3 and to its binding partner capable of converting macrophages from an "M2" to an "M1" pro-inflammatory phenotype.

In contrast to published anti-TIM3 mAbs, our particular anti-TIM3 mAbs lacked activity in T cell-based assays, but promoted an increase in pro-inflammatory cytokines with a reduction or no effect in anti-inflammatory cytokines in a macrophage activity assay. In this assay, monocytes were prepared from human peripheral blood and cultured in the presence of M-CSF to bias toward an M2 phenotype. Under sub-optimal stimulation with LPS or CD40L or HMGB1, treatment of these cells with the anti-TIM3 mAbs led to increases in pro-inflammatory cytokines including IL-1β and TNFα.

The conversion to an "M1" macrophage by anti-TIM3 mAbs had downstream consequences on T cells as demonstrated by mixed lymphocyte reaction experiments. In these studies, the addition of anti-TIM3 led to a macrophage-dependent increase in IFNγ from the T cells. To assess the impact of our anti-TIM3 mAbs in the tumor setting, tumor histoculture experiments were performed. Tumor tissue slices from ovarian cancer patients treated with anti-TIM3 showed an increase in a range of cytokines and in this tumor setting the initial sub-optimal stimulus was not required.

Specific antibodies to TIM3 and its binding partner that are able to promote a pro-inflammatory macrophage phenotype have been generated. We are developing these as modulators of the TME, to be assessed either as single agents or in combination with other therapies such as checkpoint inhibitors.

#587

Superior anti-tumor effects of an anti-HLA-DR IgG4 antibody, IMMU-114, in chronic and acute lymphocytic leukemia (CLL and ALL): Comparison to anti-CD20 therapy, chemotherapy, or combined with kinase inhibitors.

Thomas M. Cardillo, Maria Zalath, Roberto Arrojo, Robert M. Sharkey, David M. Goldenberg. _Immunomedics, Inc., Morris Plains, NJ_.

Purpose: IMMU-114 is a humanized anti-HLA-DR IgG4 monoclonal antibody currently under investigation for non-Hodgkin's lymphoma and CLL (ClinicalTrials.gov, NCT01728207). This study was undertaken to continue preclinical evaluations in CLL and ALL models, comparing IMMU-114 efficacy to anti-CD20 or doxorubicin therapy, respectively, as well in combination with Bruton's tyrosine kinase (Btk) or phosphoinositide-3-kinase (PI3K) inhibitors in CLL.

Procedures: The human CLL cell line, JVM-3, was grown s.c. in SCID mice. Once tumors reached ~0.2 cm3, they were divided into treatment groups of either IMMU-114 or rituximab (200, 100, or 50 μg, twice weekly for 4 weeks). Study survival endpoint was tumor progression to >1.0 cm3. In vitro, JMV-3 was treated with various concentrations of either a Btk inhibitor (ibrutinib) or PI3K inhibitor (idelalisib) in the presence of a constant amount of IMMU-114. IC50-values were determined, data were normalized, and isobolograms generated for each inhibitor to determine overall effect. For ALL, MN-60 cells were injected i.v. into SCID mice. After 5 days, animals received IMMU-114 (50 or 25 μg, 2 x weekly for 4 weeks) or doxorubicin (3x20 μg qdx3d induction phase, followed by a 60μg bolus injection maintenance phase on week 3). Disease progression was declared upon the onset of hind-limb paralysis.

Results: Mice with JVM-3 tumors had a median survival time (MST) of 14 days for saline controls, while therapy with rituximab significantly improved survival (P<0.0102); the MST was only 19 days for the two highest doses. In contrast, mice treated with IMMU-114 had a MST ≥42 days for all three doses tested (P<0.0001), providing an overall superior tumor growth control over rituximab (P<0.0116). In vitro, an additive effect was observed in JVM-3 when IMMU-114 was combined with either ibrutinib or idelalisib. In the ALL disseminated MN-60 disease model, mice were refractory to the doxorubicin treatment, succumbing to disease at the same rate as saline controls (MST=23 and 21 days, respectively). Importantly, IMMU-114, at both the 50 and 25 μg doses, provided a significant survival benefit compared to both saline control and doxorubicin-treated animals (MST>39 days, P<0.0001). IMMU-114 therapy was well tolerated in all these studies, as evidenced by no significant loss in weight.

Conclusions: In a preclinical model of human CLL, IMMU-114 was superior to anti-CD20 therapy using rituximab, and had an additive effect when combined with Btk or PI3K inhibitors. IMMU-114 also achieved a significant survival benefit in the doxorubicin-refractive MN-60 ALL model. These data demonstrate IMMU-114's overall activity in diverse hematopoietic cancers and show the need for continued clinical and preclinical evaluation.

#588

Inhibition of liver inflammation and tumorigenesis by blocking Ccl2-Ccr2 axis in a mouse model.

Kun-Yu Teng. _The Ohio State University, Columbus, OH_.

Hepatocellular carcinoma (HCC), the second cancer-related deadly disease, commonly arises in the setting of chronic liver inflammation. CC chemokine ligand 2 (CCL2), a chemokine that recruits CCR2-positive immune cells to promote inflammation, is highly upregulated in HCC patients. Here, we examined the therapeutic efficacy of inhibitors of the Ccl2-Ccr2 axis against hepatitis and HCC in the miR-122 knockout (aka KO) mouse model, in which upregulation of Ccl2 promotes liver inflammation that progresses to HCC with age. To determine whether blocking the Ccl2-Ccr2 axis has any therapeutic potential, we treated KO mice with Ccl2 neutralizing antibody (nab) or a small molecule inhibitor of Ccr2 and monitored liver pathology. Both agents were able to reduce the bridging inflammation in these mice by reducing the population of CD11highGr1+ cells, and expression of IL-6 and TNF-alpha in KO livers. Furthermore, treatment of tumor-bearing KO mice with Ccl2 nab for 8 weeks significantly reduced the recruitment of CD11bhighGr1+ monocytes, IL-6 and TNF-alpha production, and HCC incidence as well as tumor burden. The down-regulation of pStat3 and c-Myc (IL-6 downstream) as well as NF-kappaB (TNF-alpha downstream) upon targeting Ccl2 positively correlated with tumor growth suppression. In addition, inhibition of tumorigenesis by Ccl2 nab also correlated with enhanced cytotoxicity and IFN-gamma expression of NK cells isolated from the tumor microenvironment. Collectively, our results demonstrate that blocking the Ccl2-Ccr2 axis can be an effective therapeutic approach against both hepatitis and HCC.

#589

Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model.

Bernhard Robl,1 Sander Martijn Botter,1 Aleksandar Boro,1 Dario Neri,2 Bruno Fuchs1. 1 _Balgrist University Hospital, Zurich, Switzerland;_ 2 _Swiss Federal Institute of Technology (ETH Zürich), Zurich, Switzerland_.

Osteosarcoma (OS) is a malignancy of bone and five-year survival rates are still at a low 20% if metastatic disease is present. Metastatic OSs most frequently metastasize to the lung. TNF-α is a powerful cytokine already shown to be active against many cancers. Nevertheless, TNF-α is highly toxic at clinically relevant concentrations. To overcome this limitation, targeted forms of TNF-α can be used. In this study, targeting is achieved through linking TNF-α to the antibody fragment F8, targeting the EDA-domain of fibronectin. To test the efficacy of F8-TNF-α against OS, we sought to investigate the effects of F8-TNF-α in an orthotopic syngeneic OS model. In addition to evaluating compound efficacy, we also tested whether the route of administration (systemic versus local application) can even further increase treatment efficacy. Ultimately, we aimed at measuring treatment success by assessing different stages of OS progression, especially metastatic disease.

To show clinical relevance, we performed immunofluorescence (IF) of EDA on primary human OS samples. In a second step, we evaluated drug efficacy in a syngeneic OS mouse model. K7M2L2 OS cells, stably infected to express the marker proteins lacZ and mCherry, were orthotopically injected into the tibia of mice. Once primary tumors were detectable, the mice were treated with either PBS or F8-TNF-α. The compound was given either systemically (intravenously (i.v.), tail vein) or locally (intraarterial (i.a.), femoral artery). Four days after the last treatment, all mice were sacrificed, terminal blood sampling was performed and blood samples were analyzed for the presence of circulating tumor cells (CTCs) via FACS/mCherry fluorescence. Following X-gal staining, metastases on the surface of lungs were counted. EDA and infiltration of immune cells (CD4+, CD8+, natural killer (NK) cells, CD19+) were assessed histologically.

Using IF, we detected strong expression of EDA in primary tumors as well as bone and lung metastases from human OS patients. Preliminary results from our treatment study showed a significant decrease in the number of lung metastases as well as the number of CTCs in mice treated with i.a. F8-TNF-α while the treatment was well tolerated by the mice. The reduction of lung metastases could, at least in part, be explained by a strong expression of EDA in the lung metastases. In addition, significant increases in lung-infiltrating immune cells were observed after administration of F8-TNF-α (i.v. F8- TNF-α: increased CD4+ T-cells; i.a. F8-TNF-α: increased NK cells). Although significant reductions in numbers of metastases and CTCs were detected, F8-TNF-α did not significantly impair the growth of the primary tumors.

In conclusion, our study demonstrates a reduction of CTCs as well as lung metastases after administration of F8-TNF-α and thus, provides a rationale for further studying the anti-metastatic effects of this compound in a clinically relevant model of OS.

#590

Co-injection of human monocytes improves the in vivo antitumoral activity of bevacizumab in two NSCLC PDX models.

Cordula Tschuch,1 Kerstin Klingner,1 Anne Löhr,1 Yana Raeva,1 Teppo Haapaniemi,2 Eva Oswald,1 Julia B. Schüler1. 1 _Oncotest GmbH, Freiburg, Germany;_ 2 _BioSiteHisto Oy, Tampere, Finland_.

Nowadays, an increasing number of monoclonal antibodies (mAbs) that specifically target malignant cells or interfere with different compartments of the tumor microenvironment are available for cancer therapy. They take effect via different modes of action including the initiation of a tumor-targeting immune response. Preclinical platforms such as PDX models have to be improved to better recapitulate all possible modes of action for mAbs as well as other immune-modulating agents. In the current study, we evaluated the antitumoral activity of Bevacizumab in two NSCLC PDX growing subcutaneously in NMRI nu/nu mice with and without co-injection of human monocytes.

Two NSCLC PDX, LXFA 2478 and LXFA 677, were subcutaneously implanted into 4-6 weeks old female NMRI nu/nu mice (Harlan, Denmark). Tumor models were chosen based on their VEGFA expression level. Additionally, LXFA 2478 and LXFA 677 show high expression of TLR2 and CD14 respectively, two factors known to be involved in monocyte attraction. When median tumor size reached 150 - 300 mm³, mice were equally distributed to treatment groups (n=4/group). Animals were treated once weekly for 7 cycles with a) control vehicle b) control vehicle + 5x106 human monocytes c) Bevacizumab at 40 mg/kg/d and d) Bevacizumab + 5x106 human monocytes. Tumor volume was determined twice weekly by caliper measurement. At the end of the study, tumor and lymphatic organs of the animals were harvested and subsequent IHC analysis for human CD14, CD68 and CD163 was performed.

In both investigated tumor models, Bevacizumab showed moderate antitumoral activity with a maximal tumor load reduction of 71% (LXFA 2478) and 84 % (LXFA677) as compared to untreated controls on days 39 and 35. The co-injection of human monocytes markedly enhanced the therapeutic effect of Bevacizumab in both NSCLC PDX: maximal tumor load reduction was 89 % in LXFA 2478 and 95 % in LXFA 677 in combination groups. The injection of monocytes alone did not affect tumor growth as compared to untreated control. As predicted by the high VEGFA expression, NSCLC PDX showed high sensitivity against Bevacizumab. This antitumoral activity was increased by 18% and 11% for LXFA 2478 and LXFA 677, respectively, through the additional injection of monocytes. Monocyte recruiting factors (namely CD14 and TLR2) likely contribute to the mechanism of action.

Monocytes and macrophages have been reported to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAbs, like Bevacizumab. Our results confirm these observations in a PDX based NSCLC in vivo model. Therefore, the study highlights the suitability of PDX for immuno-oncology approaches by supplementation of the murine host with human immune cells. This advanced PDX approach will lead to more predictive preclinical data for innovative mAb´s and other compounds acting via the activation of monocytes and related immune cells.

#591

**A novel high energy alpha-pharmaceutical:** In vitro **and** in vivo **potency of a mesothelin-targeted thorium-227 conjugate (TTC) in a model of bone disease.**

Urs B. Hagemann,1 Else-Marie Hagelin,1 Katrine Wickstroem,1 Kristine Sponheim,1 Roger Smeets,1 Jenny Karlsson,1 Roger M. Bjerke,1 Mari I. Suominen,2 Yvonne Konkol,2 Jenni Bernoulli,2 Jukka Rissanen,2 Jussi Halleen,2 Liv-Ingrid Oedegaardstuen,1 Alan Cuthbertson1. 1 _Bayer AS, Oslo, Norway;_ 2 _Pharmatest, Finland_.

Mesothelin (MSLN) is a 40 kDa membrane-anchored glycoprotein, involved in mediating cell-cell adhesion, metastatic spread, promotion of cell proliferation and resistance to apoptosis. Overexpression of MSLN is most prominent in mesothelioma, ovarian, lung, triple-negative breast (TNBC) and pancreatic cancers, while in healthy tissue, MSLN is confined mainly to the mesothelial cells of the peritoneum and pericardium. Several MSLN-targeting approaches are currently being investigated, including antibody drug conjugates. We describe herein a high energy, alpha-particle emitting MSLN Targeted Thorium Conjugate (MSLN-TTC).

Thorium-227 (227Th) has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life 11.4 days), a calcium-mimetic used in the treatment of CRPC [1]. The MSLN-TTC comprises an anti-mesothelin monoclonal antibody covalently linked via an amide bond to a chelator moiety possessing high affinity for thorium-227. We present data from in vitro cytotoxicity assays demonstrating selective cell killing on MSLN positive cell-lines as well as in vivo efficacy in a mouse orthotopic bone xenograft model using NCI-H226 luciferase labeled cells.

Experimental procedures:

MSLN-TTC was prepared in high radiochemical yields and purity. In vitro cytotoxicity experiments were performed on the mesothelin-positive cell lines Ovcar-3 (ovarian), NCI-H226 (lung mesothelioma) and mesothelin-transfected HT29 (HT29MSLN/colorectal) cells. An in vivo model was established by orthotopic intratibial inoculation of luciferase-transfected NCI-H226 cells in athymic mice. Development of bone disease was monitored by luciferase activity and the extent of bone lesions determined by x-ray imaging and microCT.

Results:

MSLN-TTC induced specific in vitro cytotoxicity via induction of DNA double strand breaks as determined by phosphorylated histone protein H2AX . MSLN-TTC demonstrated statistical significant in vivo potency administered as a single dose of either 250 or 500 kBq/ kg in the orthotopic bone xenograft model. Animals treated with MSLN-TTC showed a) significantly reduced disease in the bone metastatic lesions b) decreased metastatic disease in the lungs and c) significant reduction in osteolytic/ osteoblastic bone lesions as evidenced by X-Ray and microCT compared to the vehicle control group. Furthermore, no significant loss in body weight was observed during the course of the study demonstrating that the MSLN-TTC was well tolerated.

The data presented support the further investigation of the MSLN-TTC in bone metastatic disease.

References:

1. Henriksen, G., et al., Targeting of osseous sites with alpha-emitting 223Ra: comparison with the beta-emitter 89Sr in mice. J Nucl Med, 2003. 44(2): p. 252-9.

#592

Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation.

Marije B. Overdijk,1 Kristin Strumane,1 Antonio Ortiz Buijsse,1 Claudine Vermot-Desroches,2 Andreas Lingnau,1 Esther C.W. Breij,1 Janine Schuurman,1 Paul W.H.I. Parren1. 1 _Genmab, Utrecht, Netherlands;_ 2 _iDD Biotech, Lyon, France_.

Death receptor 5 (DR5) is a highly interesting tumor target based on the enhanced sensitivity of cancer cells for DR5-dependent apoptosis. In recent years, multiple therapeutic DR5 antibodies have been evaluated in the clinic for which results however have been disappointing. IgG molecules against membrane-bound targets have shown an ability to form ordered hexameric structures upon antigen binding, a process that is dependent on Fc-Fc interactions between IgG molecules. We identified specific mutations in the human IgG1 Fc domain that enhance such antigen-dependent hexamerization while retaining solution-monomericity and developability characteristics of regular IgG1 molecules (HexaBody technology). We hypothesized that antibody-mediated hexamerization, when applied to DR5-specific antibodies, would enhance DR5 signaling and apoptosis, resulting in strongly improved therapeutic potential.

The technology was applied to two non-crossblocking DR5-specific IgG1 antibodies, IgG1-DR5-01 and IgG1-DR5-05, by mutating a glutamic acid residue at position 430 in the Fc domain to glycine (the HexaBody mutants were designated Hx-DR5-01 and Hx-DR5-05). Cytotoxicity of the DR5 antibodies was explored in vitro using the CellTiter-Glo luminescent cell viability assay and the Caspase-Glo 3/7 assay in a broad panel of cancer cell lines, and in vivo in xenograft models.

Both Hx-DR5-01 and Hx-DR5-05 induced increased cytotoxicity compared to their wild type (WT) IgG1 counterparts. Moreover, the combination of Hx-DR5-01 and Hx-DR5-05 (referred to as Hx-DR5-01/05) was found to be more potent than either Hx-DR5-01 or Hx-DR5-05 alone, or than the combination of the WT antibodies (IC50 in BxPC3 cells 0.5 and 1.5 µg/ml; maximal cytotoxicity 91% and 25% for Hx-DR5-01/05 and WT IgG1-DR5-01/05 respectively). In contrast to wild type agonistic DR5 antibodies, tumor cell killing by Hx-DR5-01/05 was independent of secondary crosslinking. Potent anti-tumor activity was observed in seven xenograft models for multiple indications, with Hx-DR5-01/05 consistently showing significantly better efficacy than the WT DR5 comparator antibody conatumumab.

The cytotoxic activity of DR5 antibodies was significantly enhanced by the introduction of a hexamer-enhancing mutation in the IgG1 Fc domain. Maximal killing activity was obtained by combining two non-crossblocking DR5 antibodies carrying this mutation (Hx-DR5-01 and Hx-DR5-05). The strong cytotoxicity of Hx-DR5-01/05 was completely dependent on target binding but, in contrast to WT antibodies, did not require secondary crosslinking. These promising pre-clinical results support the selection of Hx-DR5-01/05 for clinical development for the treatment of cancer.

#593

Highly cytotoxic EGFR/CD16A TandAbs specifically recruit NK cells to potently kill various types of solid tumors.

Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Erich Rajkovic, Martin Treder. _Affimed GmbH, Heidelberg, Germany_.

Constitutive EGFR activation through amplification or dysregulation plays an important role in the pathophysiology of numerous solid cancers, such as colorectal cancer (CRC), non-small cell lung cancer (NSCLC) or squamous cell carcinomas of the head and neck (SCCHN), thus providing a strong rationale for the development of therapeutic strategies targeting EGFR. Different therapeutic approaches have been approved for treatment of such cancers, including tyrosine kinase inhibitors that interfere with signal transduction by blocking the kinase domain or monoclonal antibodies (mAbs) that prevent EGFR ligand binding, dimerization and activation. However, despite demonstrated clinical efficacy, intrinsic or acquired resistance to such treatments has been described for a larger number of patients.

Natural killer cells (NK-cells) play a central role in the innate immune system and have the capacity to destroy virally-infected or neoplastic cells. To specifically utilize the cytotoxic potential of NK-cells for the elimination of EGFR+ cancer cells, we developed tetravalent bispecific EGFR/CD16A NK-cell TandAbs, with two binding sites for EGFR being expressed on tumor cells, and two binding sites for CD16A expressed on NK-cells.

Using antibody phage display technologies, we identified high affinity scFvs recognizing conformational epitopes in the extracellular domain of EGFR that were different to those targeted by other therapeutic antibodies. We engineered a set of bispecific EGFR/CD16A TandAbs and analysed their binding, thermostability and cytotoxic properties in a panel of in vitro assays. TandAbs containing our EGFR-specific domain 21 were highly potent in cytotoxicity assays towards endogenously EGFR-expressing tumor cell lines or transfected CHO cells with single digit picomolar or subpicomolar EC50 values. In contrast to comparator NK-cell recruiting TandAbs containing the Fv sequences from cetuximab, the TandAbs containing our EGFR-binding domain 21 did not exhibit any signs of temperature-induced instability or aggregation in thermostability studies.

Taken together, our data suggest that EGFR/CD16A TandAbs are novel, highly potent drug candidates suitable for the treatment of EGFR-overexpressing malignancies and overcoming resistance to other therapeutic agents.

#594

Antibodies targeting LAG-3 , TIM-3, KIR cloned from healthy human donors.

Angeles Estelles, Robert Stephenson, Keyi Liu, Evelene Lomongsod, Da Ngyen, Jianzhong Zhzng, Yifan Yang, Manfred Heideker, Lawrence Kauvar, Stefan Ryser, Mikhail L. Gishizky. _Trellis Bioscience, Menlo Park, CA_.

The immunosurveillance/immunoediting theory postulates that tumor growth is repressed in healthy individuals through the activation of adaptive and innate immune mechanisms. The clinical success of antibodies that target inhibitory checkpoint pathways of the immune system (i.e. CTLA-4, PD-1) has provided compelling evidence in support of this theory and raises the question of whether antibodies targeting these, or other inhibitory immune cell modulators (ICM), are present in apparently healthy individuals. The present studies were designed to answer this question by probing the circulating memory B-cell compartment that represents a historic record of an individual's lifelong adaptive immune response.

Methods: Blood from donors, with no known cancer history, was screened for the presence of anti-ICM binding memory B-cells targeting LAG-3, TIM-3, B7-H3, KIR, and VISTA, using Trellis's established CellSpotTM technology that can detect the presence of antigen specific memory B-cells at a frequency of 0.1 per 100,000 memory B-cells.

Results: Circulating memory B-cells targeting ICM's were present in all apparently healthy individuals tested (20 total). There was substantial variation as to which ICM's were recognized within each individual across this sample population. Antibodies cloned from these rare B-cells were of IgG class and exhibited low to sub-nM affinity toward the specific ICM. These high affinity antibodies bound their respective antigens on activated T-cells and were able to augment immune stimulatory cytokine release in cellular assays. Data from ongoing studies will be presented.

Conclusion: These studies demonstrate that apparently healthy individuals produce high affinity, pharmacologically active anti-ICM antibodies. Thus, healthy individuals represent a previously unrecognized source of antibodies that have undergone natural selection and hold particular promise as potential therapeutic agents for the treatment of cancer.

#595

Next-generation engineered toxin bodies: CD38, PD-L1 and HER2 targeted ETBs.

Sangeetha Rajagopalan, Garrett L. Robinson, Brigitte Brieschke, Jennifer Erdman, Jane Neill, Jack P. Higgins, Erin K. Willert. _Molecular Templates Inc., Georgetown, TX_.

Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit to specifically kill target expressing cells. Once delivered to appropriate cells, the Shiga toxin A subunit inhibits protein synthesis and promotes apoptosis of tumor cells. Bacterial or plant derived toxin moieties have the potential to induce an immune response, commonly limiting repeat dosing of immunotoxins. Our next-generation ETB scaffold has been modified to overcome this limitation through genetic engineering to systematically and comprehensively reduce B and CD4+T cell epitopes.

Molecular Templates has developed a proprietary epitope class switching technology designed to both reduce the anti-drug response and promote the anti-tumor response by replacing naturally occurring CD4+ T cell epitopes with CD8+ T cell epitopes. We have found that a combination of surface remodeling and epitope class switching combined in one protein results in powerful reductions in the anti-drug response against ETBs after repeat administration. This de-immunized next-generation scaffold retains the potency and specificity of the unmodified ETB. Additionally, the engineering of CD8+ T cell epitopes on the ETB scaffold can allow for foreign antigen presentation in complex with MHC class I on the tumor cell surface. This antigen presentation may recruit activated cytotoxic T lymphocytes (CTLs) to the tumor microenvironment thereby adding an additional mechanism of action to the direct cell kill activity of the ETBs.

The second generation ETB scaffold has been combined with multiple target binding domains, including scFvs that target CD38, PD-L1 and HER2. CD38 is a validated target for multiple myeloma, and our CD38 targeted ETB has shown potent activity in vitro and in vivo. Pre-clinical studies have shown effective combination of the CD38 targeted ETB with standard of care agents such as IMiDs. Additionally, we have developed a PD-L1 targeted ETB that can be used against various PD-L1 expressing malignancies including Hodgkin's lymphoma and melanoma. HER2 is a well characterized target in breast cancer that persists in many patients after HER2 targeted treatment relapse; a novel MOA to this target may impart additional clinical benefit. The potency, reduced immunogenicity, unique mechanism of action and immune modulating activities of ETBs in this second generation scaffold allows for these agents to fit in well in a refractory setting as well as in combination with other agents in the growing field of immuno-oncology.

#596

Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC).

Natasha Bodyak,1 Alex Yurkovetskiy,1 Dmitry R. Gumerov,1 Dongmei Xiao,1 Joshua D. Thomas D. Thomas,1 Laura L. Poling,1 LiuLiang Qin,1 Mao Yin,1 Michael J. DeVit,1 Peter U. Park,1 Winnie Lee,1 Bianka Prinz,2 Donald A. Bergstrom,1 Timothy B. Lowinger1. 1 _Mersana Therapeutics, Cambridge, MA;_ 2 _Adimab LLC, Lebanon, NH_.

XMT-1522 is an anti-HER2 ADC that incorporates HT-19, a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery. Several parameters such as cell binding, internalization rate, cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) downstream signaling, affinity maturation, NHP cross-reactivity, normal human tissue cross-reactivity and in vivo efficacy were used to screen a wide range of antibodies to select a lead candidate optimized for use in ADC applications. In addition HT-19 was selected to be non-competitive for HER2 binding with existing therapies - trastuzumab or pertuzumab, to allow for potential combinability. In vivo efficacy as an ADC did not directly correlate with typical parameters used in antibody screening cascade such as in vitro cytotoxicity, internalization or binding affinity. The lead antibody underwent affinity maturation, and despite increases in affinity, affinity maturation significantly decreased the in vivo efficacy of the ADC in vivo xenograft models. This phenomenon was observed in all the antibody hits. HT-19 showed antibody-dependent cell-mediated cytotoxicity (ADCC) activity. When administered as a single agent in NCI-N87 gastric cancer xenograft model it had biological activity at 20 mg/kg as well as at 3 mg/kg. Consistent with the hypothesis that a non-competitive ADC is combinable with current anti-HER2 regimens, the combination of XMT-1522 with trastuzumab and/or pertuzumab showed more rapid internalization, more complete HER2 degradation, and significantly great anti-tumor activity in the NCI-N87 gastric cancer xenograft model relative to XMT-1522 alone or the combination of pertuzumab + trastuzumab.

#597

Defining the role of macrophage mediated trogocytosis in the clearance of antibody-opsonized tumor cells.

Ramraj Velmurugan,1 Dilip Challa,1 Sripad Ram,2 Raimund J. Ober,3 E. Sally Ward1. 1 _Texas A &M Health Science Center, College Station, TX; _2 _Carl Zeiss Microscopy, Thornwood, NY;_ 3 _Texas A &M University, College Station, TX_.

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is pro-tumorigenic. However, there is limited information concerning the role of trogocytosis in antibody-based treatment of solid tumors such as breast cancer.

We have used advanced microscopy methods and quantitative flow cytometric assays to study the effect of antibody-mediated trogocytosis on breast cancer cells and whether they can lead to tumor cell death.

Our results show that long-term coculture with macrophages can reduce cancer cell numbers in an antibody-dependent manner even at low effector:target ratios. Quantitation from simultaneous long-term imaging of macrophage:cancer cell interactions reveals that this cell death is caused by both macrophage-mediated phagocytosis and trogocytosis, with the contribution of each process differing by the phenotype of the effector macrophages.

Together, our results add to our understanding of the numerous interactions macrophages can have with cancer cells, and how therapeutic antibodies modulate their effects. Our recent observations in this area will be presented.

#598

Utilization of Meditope Biosciences SnAP technology to enhance antibody internalization.

Calin D. Dumitru,1 Elisabeth Gardiner,2 John Williams,3 Cindy Zer4. 1 _Meditope Biosciences Inc., CA;_ 2 _Meditope Biosciences, San Diego, CA;_ 3 _City of Hope, Duarte, CA;_ 4 _TSRI, San Diego, CA_.

Therapeutic antibodies can bind to cell-surface receptors, prompting receptor internalization and effectively blocking further stimulation by the cognate receptor ligands. Receptor internalization is also exploited by antibody-drug conjugates (ADC) to promote effective delivery of cytotoxic payload to the inside of the cell. Meditope Biosciences has developed a way to use its SnAP (Site-specfi novel Antibody Platform) technology to promote enhanced internalization of antibody receptor complexes. The Meditope's SnAP technology functionally enables antibodies to bind to specific peptides, termed "meditopes', and this property can be used to directly facilitate receptor crosslinking when meditope enabled antibodies are bound to cell surface receptors. Here we show that the affinity of the meditope enabled antibodies (trastuzumab anti-HER2 and gemtuzumab anti-CD33) for cell surface receptors results in the formation of an antibody receptor complex that can be detected by native gel electrophoresis and gel filtration. Binding of the meditope peptide to the enabled antibody does not interfere with normal antigen-antibody interaction and the formation of the peptide bound antibody:receptor complexes results in accelerated internalization of the complex, as shown by decreased surface fluorescence of Alexa 488-labeled SnAP peptides as well as by increased pHrodo-Red increased fluorescence of internalized antibodies when they reach the endosomal compartment. Finally, as a result this increased internalization, the downregulation of specific antibody mediated signaling events are much more effectively downregulated in the presence of the meditope peptide complex as compared to the antibody alone.

#599

MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis.

Runhua Feng,1 Ming Li,1 Yuling Wang,1 Dongqing Zhang,2 Xiaolian Gao,3 Xuehua Chen,4 Joe X. Zhou,5 Victor G. Prieto,1 Jian H. Song,1 Shenying Fang,1 Binya Liu,4 Zhenggang Zhu,4 Xiangcang Ye,1 Lee M. Ellis,1 Mason Lu,1 Jeffery E. Lee1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Shanghai Institute of Immunology, Shanghai, China;_ 3 _University of Houston, Houston, TX;_ 4 _Shanghai Ruijin Hospital, Shanghai, China;_ 5 _LC Sciences, LLC, Houston, TX_.

Background: Gastric cancer is the primary cause of cancer-specific mortality worldwide. PODXL (podocalyxin-like protein, or podocalyxin) is a cell-surface glycoprotein that belongs to CD34 family of sialomucins. PODXL-v1 is widely expressed on vascular endothelium, mesothelial cells, and platelets; in contrast, PODXL-v2, a truncated short form of PODXL is specifically expressed or up-regulated in numerous cancer cell types as well as in small blood vessels in tumor tissues. There remains considerable interest in generating monoclonal antibodies (mAbs) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy.

Methods: Mixed live cells from four human GC cell lines were used as the immunogen in A/J mice. MS17-38 mAb was generated by hybridoma technology and from high throughput screening (HTS), with GC cell lines versus normal human PBMC live cells. Purified MS17-38 mAb was used to confirm the target Ag through a combination of western blot (WB), immunoprecipitation (IP) and mass spectrometry (MS). Peptide microarrays of the identified Ag were layered on chips and double screening was performed to achieve epitope mapping of binding of the mAb its target. Dose-dependent treatment of GC cells with MS17-38 mAb and target siRNA-was followed by staining with CCK-8 and CyQuant GR dyes and read at 450nm and 480/520nm for cell proliferation and transwell migration assays. GC MKN-45 cells were injected subcutaneously (s.c.) into the flank of nu/nu mice treated with MS17-38 mAb or control mAb. In addition, tumor tissues from 42 human GC patients were stained by immunohistochemistry (IHC) and evaluated for PODXL expression using MS17-38, and the survival of the GC patients compared to target antigen expression levels.

Results: MS17-38 mAb was generated by live cells HTS and was demonstrated to bind to a specific conformational epitope of PODXL-v2 on GC cells. PODXL-v2 expression inhibition by PODXL-v2 siRNA or the PODXL-v2-neutralizing antibody MS17-38 resulted in inhibition of GC cell growth and prevention of GC cell migration in vitro. Furthermore, in vivo studies demonstrated that MS17-38 mAb can potently inhibit tumor growth and prevent MKN-45 metastasis to the lungs in nu/nu mouse models. Finally, high expression of PODXL-v2 was associated with advanced GC stage and short survival time (P<0.01). Additional engineering of this mAb into chimeric or humanized forms could permit development of MS17-38 for diagnosis, staging or therapy of human malignancy.

Conclusions: Our findings indicate that PODXL-v2 is specifically expressed in the extracellular matrix of GC, and MS17-38 mAb directed against a conformational epitope of PODXL-v2 identified through HTS can functionally inhibit GC cancer cell growth and migration/metastasis both in vitro and in vivo. MS17-38 is a promising functional antibody directed against GC that could potentially be developed into a therapeutic mAb for clinical application.

## TUMOR BIOLOGY:

### Drug Testing in Cell Lines and 3D Models

#600

Melanoma and ovarian cancer cells tested for drug sensitivity using anchorage-independent growth.

Asaf Rotem, Benjamin Izar, Levi A. Garraway. _Dana Farber Cancer Institute, Boston, MA_.

This study aims to test patient-derived melanoma and ovarian cancer cells for their sensitivity to clinically available drugs and drug combinations. The experiments were performed by using the growth in low attachment (GILA) assay, which is equivalent to the classic soft-agar assay and allows screening of 3D ex vivo cultures, which resemble the tumor and its microenvironment.

To assay tumor cells from malignant lesions we isolated ovarian cancer cells from peritoneal fluid (ascites) derived from patients and melanoma cells from metastatic melanoma patients. The fresh samples were enriched for tumor cells and later cultured in an ex vivo manner on either high- or low-attachment surfaces. These cells were subjected to drug treatments and cellular viability was measured at the end of these experiments.

Our experiments demonstrate that the GILA assay is a highly sensitive method for pharmacological tests, as compared to the traditional 2D method (growth on high-attachment surfaces). By using GILA assay we found that melanoma cells are sensitive to drugs that inhibit driver oncogenes, such as BRAF or MEK. In addition, we found interesting targets in metastatic melanoma lesions that carry an unknown mutational background.

Drug sensitivity assays for ovarian cancer cells using the GILA approach showed that cancer cells from various patients with the same cancer type display differential sensitivity to drugs. Additionally, ovarian cancer cells were found to have a different drug-sensitivity threshold when grown in spheres.

Our studies present an approach that utilizes the unique ability of transformed cells to grow in anchorage-independent conditions and enables ex vivo drug sensitivity testing. This phenotypic approach is complementary to a genetic approach that uses DNA sequencing to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein.

#602

BEZ235 efficacy in 3D-cancer cell line screening.

Macarena Irigoyen, Gonzalo Castillo. _BIOENSIS, Bellevue, WA_.

In vitro efficacy assessment of drugs in panels of tumor-derived cell lines has proven to be a powerful tool for predictive biomarker discovery. However, in the last few years it has become evident that particularly for tumor cell lines derived from solid tumors, the results do not always correlate with what is seen in xenografts as well as clinically. One of the reason is that traditionally these type of studies are done in monolayers (2D), which lack the complexity normally seen in vivo. 3D cultures offer the ability to carry out the same type of studies with the added benefit of incorporating 3-dimensional cellular interactions and physiological gradients, normally seen in a tumor microenvironment. In this study, we evaluated the efficacy of BEZ235, an imidazoquinoline, targeting the phosphatidylinositol 3 kinase (PI3K) and the mammalian target of rapamycin (mTOR). BEZ235 was evaluated in a panel of 60 cell lines grown as spheroids, and this information was used to investigate genes associated with sensitivity. Cell lines grown as spheroids proved to be highly sensitive to the drug, where 45 of 60 cell lines showed an IC50 lower than 1 µM. Biomarker association analysis identified two mutated genes associated to BEZ235 sensitivity. The first biomarker was the phosphatase and tensin homolog (PTEN) a tumor suppressor gene, that when deregulated plays an important role in the development of several cancers. The second biomarker was AXIN1, a negative regulator of the WNT signaling pathway, and its deregulation has been associated with hepatocellular carcinoma. Since both PTEN and AXIN1 are thought to play a role in tissue invasion and metastasis, we studied the ability of BEZ235 to inhibit cellular invasion, and the results are discussed.

#603

A high-throughput image cytometry-based screening method for the cytotoxic effect of drug compounds on 3D tumor spheroid.

Leo Li-Ying Chan, Olivier Déry, Scott Cribbes, Dmitry Kuksin, Sarah Kessel. _Nexcelom Bioscience LLC, Lawrence, MA_.

Cancer is a disease caused by uncontrolled proliferation or division of abnormal cells in an organism. Traditionally, cancer cell proliferation is measured using a two-dimensional (2D) in vitro model by culturing and treating the cells in standard microplates to study inhibition and cytotoxicity effect of the drug compounds. After further testing the potential cancer drug candidates in the 2D environment, the investigation is often transferred to an in vivo model by measuring the change in three-dimensional (3D) tumor sizes in animals. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed in a 3D environment, which physiologically has a closer resemblance to a human body. Therefore, cancer drug discovery research has demonstrated poor translation from in vitro to in vivo.

Previous publications have demonstrated that growing cancer cells in the form of 3D tumor spheroids can be more predictive of the in vivo study outcomes comparing to the 2D cell culture method. Therefore, by investigating the effect of cancer drugs on 3D tumor spheroids, may improve the successful translation rate to in vivo animal study. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids on 384-well ultra-low attachment (ULA) round bottom microplates using the Celigo image cytometer. In order to validate this screening method, five experiments were conducted demonstrating comparable results in both 3D and 2D models. First, optimal cell seeding density for tumor spheroid formation is determined by investigating U87MG (glioblastoma) cell seeding number in respect to the desired spheroid size. Second, the dose response effect of 17-AAG in respect to spheroid size is measured to determine the IC50 value. Third, the effect of 17-AAG on the viability of tumor spheroid is investigated in a time-course study. Next, the developed high-throughput method is used to perform dose response measurement of 4 drugs (17-AAG, Paclitaxel, TMZ, and Doxorubicin) in respect to the spheroid size. Finally, the effect of the 4 drugs on tumor spheroid viability is measured. Each experiment is performed simultaneously in the 2D model, where 384-well flat bottom microplates are used. In the 2D adherent cell model, confluence percentage and direct cell count-based viability results are generated to compare to the 3D model. This proposed method allows an efficient process for more qualified drug candidates, which can reduce time and the financial burden of animal testing, as well as the overall time of the cancer drug discovery project.

#604

A multi-layered, hydrogel system for automated 3D high throughput drug screening of cancer-stromal cell co-cultures.

Pamela E. Constantinou,1 Brian J. Engel,1 Lindsey K. Sablatura,1 Nathaniel J. Doty,2 Daniel D. Carson,1 Mary C. Farach-Carson,1 Daniel A. Harrington,1 Thomas I. Zarembinski2. 1 _Rice University, Houston, TX;_ 2 _BioTime, Inc, Alameda, CA_.

Pre-clinical drug screens, involving culturing well-annotated cancer cell lines on two-dimensional (2D) tissue culture plastic, poorly recapitulate in vivo tumor characteristics and yield candidate drugs which fail clinical trials. We created a multi-layer, hyaluronic acid (HA)-based hydrogel system that incorporates three layers: an acellular cushion layer; an encapsulated cancer cell layer for growth in three dimensions (3D); and a collagen-containing layer that supports the growth of stromal cells on top of the hydrogel (2.5D). Utilizing high-throughput robotic delivery coupled with automated advanced imaging, this formulation provides a highly reproducible system for spheroid culture of prostate (C4-2B) or endometrial (Ishikawa) cancer cell lines in mono- or co-culture with matched stromal cells (HS27a or ESS-1, respectively). Both culture systems provided high cancer cell viability over one week of culture. Cells were treated with drugs from a panel of chemotherapeutic compounds. Cells cultured in our 3D multi-layer HA-based system responded to cytotoxic drugs distinctly from cells grown in 2D and 3D-aliginate, and better predicted the efficacy of chemotherapeutics in clinical trials. We have also successfully incorporated cells derived from patient derived xenograft (PDX) models in our 3D culture system. Wider adoption of 3D automated screening has the potential to speed drug discovery and increase success of drugs in clinical trials.

#605

Building better cell models for screening oncology compounds.

David M. Evans,1 Michael Selby,1 Rene Delosh,1 Julie Laudeman,1 Chad Ogle,1 Russell Reinhart,1 Thomas Silvers,1 Ralph E. Parchment,1 Beverly Teicher2. 1 _FNLCR, Frederick, MD;_ 2 _Division of Cancer Treatment and Diagnosis, NCI, Bethesda, MD_.

The NCI60 screen is a valuable compound screening service offered to researchers for 20+ years. The screen allows the submission of small molecules, biologics and natural products for testing in the NCI60 cell line panel (60 cell lines covering 9 major tumor types). Sophisticated tools such as COMPARE allow stratification of compounds based on pattern of cell line response and these tools elucidated several compounds with differential specificity for a disease type (e.g. breast cancer over melanoma).

Since inception the NCI60 screen has been performed in 96-well plates on monolayer cultures of cells. SulfoRhodamine B was used to measure cell viability after a 48h exposure to the compounds at either a single concentration or 5 point concentration response assay. However, recent work has suggested that cells in 3D culture may respond differently to compounds than in 2D. 3D culture systems produce tumor cell spheroids that exhibit features seen in vivo including greater cell-cell contact, and gradients of nutrients and oxygen between the periphery and the center of the spheroid. We sought to optimize the NCI60 cells for use in a 3D spheroid model that would be suitable for screening compounds. By varying NCI60 cell plating density and incubation times, we optimized the formation of spheroids of a given diameter (500um) in Ultra Low Attachment (ULA) plates. Spheroids were imaged using a high content imaging plate reader and classified into 4 categories based on their apparent morphologies. Comparisons were made in drug sensitivity between 2D and 3D model systems using the NCI60 and cells derived from PDX samples. Data on the optimal cell densities of the NCI60 lines used for spheroid formation as well as concentration response effects of select agents compared in 2D and 3D models are presented. These data will allow others to rapidly utilize these 3D models to determine the differential drug sensitivity of select cell types. Funded by NCI Contract No. HHSN261200800001E.

#606

Similar 3D pharmacodynamic (3D-PD) responses of human tumor spheroids and xenografts to topoisomerase 1 inhibitor-induced DNA damage.

David M. Evans,1 Rene Delosh,1 Julie Laudeman,1 Chad Ogle,1 Russell Reinhart,1 Michael Selby,1 Silvers Thomas,1 Robert Kinders,1 Tony Navas,1 Scott Lawrence,1 Anne Monks,1 Annamaria Rapisarda,1 Ralph E. Parchment,1 James H. Doroshow,2 Beverly Teicher2. 1 _FNLCR, Frederick, MD;_ 2 _Division of Cancer Treatment and Diagnosis, NCI, Bethesda, MD_.

Three-dimensional (3D) cultures have been proposed as higher fidelity models of in vivo tumors than 2D cultures. To determine whether this idea extends to drug pharmacodynamics, we examined whether 3D cultures of the human melanoma line A375 (grown as tumor spheroids) could replicate the timing and magnitude of the nuclear γH2AX response to the topoisomerase 1 inhibitor, topotecan observed in A375 tumor xenografts (Kinders et al, Clin Can Res 2010). The appearance of γH2AX-positive nuclear foci has been used as a biomarker for drug- and radiation-induced double-strand breaks in DNA; we reported previously that treatment of nu/nu mice harboring A375 xenografts with a single-dose of topotecan induced nuclear γH2AX foci in a dose- and time-dependent manner, which peaked 4 hours after drug administration. A375 spheroids were generated by seeding cells into ULA U-bottom plates; 4 hours of exposure to 0.1 μM topotecan elicited a γH2AX signal in these 3D cultures while cell viability and intracellular ATP levels remained unchanged, indicating a DNA damage repair response at the maximally tolerated topotecan concentration for human hematopoietic cells (Erickson-Miller et al, Can Chemo Pharmacol 2009). Extending drug exposure to 24 hours caused substantial loss of viable cells (calcein AM+) and 50% decline in ATP levels but no further increase in γH2AX. In contrast, the HT29 tumor line was refractory to topotecan in vivo, and exposing 3D cultures of HT-29 spheroids to 0.1 μM topotecan for 24 hours elicited a strong nuclear γH2AX biomarker response in only a small fraction of cells at the surface of the spheroids. Longer exposure durations or supra-pharmacological concentrations (1 μM) of topotecan were required to achieve a strong nuclear γH2AX response in HT-29 spheroids. These results support the hypothesis that the 3D pharmacodynamics (PD) of drug response is similar to PD drug response in vivo for the camptothecin class of topoisomerase 1 inhibitors. Funded by NCI Contract No. HHSN261200800001E.

#607

Analyzing cell viability in 3-D tissue models with the ViaLight™ plus cell proliferation and cytotoxicity bioassay.

Stefanie Buesch,1 John Langer,2 Sabine Schaepermeier,1 Lubna Hussain,2 Jeffrey Bergeron,3 Volker Vogel,1 Jenny Schroeder1. 1 _Lonza Cologne GmbH, Cologne, Germany;_ 2 _Lonza Walkersville Inc., Walkersville, MD;_ 3 _Lonza Rockland Inc., Rockland, ME_.

Conventional in vitro assays are based on cells grown on two-dimensional (2D) substrates, which are not representative of the true in vivo cell environment. In tissue environments, cells interact with neighboring cells and the extracellular matrix (ECM). Three-dimensional (3D) cell culture methods allow cells to grow in structures more resembling the in vivo environment. Cells can develop cell-cell and cell-ECM interactions in 3D.

The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant concentrations. Cells and neutralized collagen are mixed and subsequently incubated at 37°C to allow the formation of a cell-seeded hydrogel. Specialized RAFT™ Absorbers are placed on top of the hydrogels. The RAFT™ Absorbers gently remove abundant medium, thus compacting the cell/collagen hydrogel. Additional epithelial or endothelial cells may be added as overlays on top to study co-cultures or more complex cultures.

Another commonly used 3D cell culture model is spheroids. Spheroids are compact aggregates of cells that are generated without the addition of exogenous ECM -- e.g. in so-called ultra-low attachment (ULA) plates or by the hanging-drop method.

While 3D cultures more accurately mimic the in vivo cell environment, it might be difficult to analyze cells in 3D due to the dense, tissue-like structure of these cultures compared to 2D cell monolayers. This presentation explains how to measure cell viability in both RAFT™ 3D cell cultures and in spheroid cultures with the ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay. The ViaLight™ Assay is based on the bioluminescent detection of cellular ATP as a measure of cell viability. Only minor modifications of the standard two-step protocol that is used for 2D cell cultures are required for using the assay for 96-well and 24-well RAFT™ 3D cell cultures as well as for spheroid cultures in 96-well ULA plates.

Two different cell types were used in this study: Normal Human Dermal Fibroblasts (NHDFneo) and the colon cancer cell line HCT-116. Both cell types efficiently form spheroids in ULA plates. Also in RAFT™ 3D cultures HCT-116 cells build compact aggregate-like structures, whereas NHDFneo grow in as individual cells interspersed in the collagen scaffold.

By elongating the first step of the Vialight™ Assay, the lysis step, from 10 minutes to 30 minutes, a linear performance of the assay for cell numbers of up to 96,000 cells in the 96-well format and 480,000 cells in the 24-well format could be obtained for RAFT™ cultures. For spheroid cultures an extension of the lysis time to 60 minutes was required to obtain efficient lysis of spheroids with a diameter of up to 400µm, as confirmed by CalceinAM and propidium iodide staining.

In summary this presentation shows that analyzing cells in 3D cultures can easily and routinely be done by slightly adjusting standard 2D cell culture assays like the ViaLight™ Assay.

#608

Comprehensive drug testing of patient-derived conditionally reprogrammed cells from castration-resistant prostate cancer.

Khalid Saeed,1 Vesa Rahkama,1 Samuli Eldfors,1 Dmitrii Bychkov,1 John Patrick Mpindi,1 Bhagwan Yadav,1 Tero Aittokallio,1 Peter Horvath,1 Donna Peehl,2 Lassi Paavolainen,1 Caroline Heckman,1 Krister Wennerberg,1 Tuomas Mirtti,1 Antti Rannikko,3 Olli Kallioniemia,1 Päivi Östling,1 Taija af Hällström1. 1 _Institute for Molecular Medicine Finland, FIMM, University of Helsinki, Helsinki, Finland;_ 2 _Department of Urology, Stanford University School of Medicine, Stanford, CA;_ 3 _Department of Urology, Helsinki University Hospital, Helsinki, Finland, Helsinki, Finland_.

Culture of human prostate cancer (PCa) progenitor cells would facilitate the discovery and testing of new, potentially curative therapies for PCa. Here, we established and characterized patient-derived conditionally reprogrammed cells (CRCs) from seven patients to determine their biological properties (genomic, transcriptomic, protein expression) and to apply these cells to test efficacies of 306 emerging and clinically approved drugs. The patient samples came from both primary prostate cancer as well as from advanced Castration-Resistant Prostate Cancer (CRPC).

Patients with primary prostate cancer generated six benign CRC cultures which all had an androgen receptor (AR)-negative, basal/transit-amplifying phenotype with few CNAs. In 3D cell culture, these cells could re-express AR. The CRCs from a CRPC patient (HUB.5) displayed multiple CNAs, many of which were shared with the parental tumor. We carried out high-throughput drug-response studies with 306 emerging and clinical cancer drugs. Bcl-2 family inhibitor navitoclax emerged as the most potent cancer-specific drug for the CRCs from CRPC patient. Other drug efficacies observed included taxanes, mepacrine and retinoids, thus covering both existing (and clinically validated) as well as new drug efficacies.

In conclusion, comprehensive cancer-pharmacopeia-wide drug testing of CRCs from a CRPC patient highlighted both known and novel drug sensitivities in PCa, including navitoclax, which is currently tested in clinical trials of CRPC.

#609

An established ex vivo 3D culture platform using patient-derived xenograft (PDX) tumors replicate the anti-tumor efficacy observed in vivo.

Xiaoxi Xu,1 Wenna Zhang,1 Bingying Xie,2 Zheng Zheng Bao,1 Zhu Mei,1 Jean-Pierre Wery,1 Jinying Ning1. 1 _Crown Bioscience, Inc., Beijing, China;_ 2 _Peking University Cancer Hospital & Institute, Beijing, China_.

Patient-derived xenograft (PDX) models closely mimic the pathological phenotypes of the original tumors, thus they are ideal surrogates for preclinical anticancer drugs efficacy testing. However, there is an increasing demand to rapidly and reliably screen out candidate drugs at earlier stages, and in vivo animal studies usually take months to produce efficacy data. Compared to traditional two-dimensional (2D) culture, three-dimensional (3D) culture systems more closely reflect in vivo responses. Therefore, we developed an ex vivo 3D culture platform using freshly isolated PDX-derived tumor cells, cultured on the natural methylcellulose polymer as an ex vivo drug screening system to meet the need for patient relevant, lower cost and high-throughput methods.

Single-cell suspensions freshly isolated from PDX tumors are seeded in semisolid methylcellulose media and the drug efficacy is quantitatively evaluated by CellTiter-Glo® (CTG) luminescent assay. The main advantage of this method is the possibility of testing cytotoxicity within days (7 days on average), rather than months. Also, the process is carried out in mild condition and the scaffold material is inert to avoid unwanted cellular response. Moreover, cell spheroids can be easily retrieved for further analysis.

Dissociated primary tumor cells derived from either fresh or frozen PDX tumors remain highly viable and form colonies when cultured in 3D. The success rate of generating ex vivo 3D culture from PDX tumors is higher than 90%, and over 60 PDX models were validated from cancer of various origins. We monitored tumor colony formation and generated a fitted drug concentration-response curve with small variations in CTG cell viability assays. Most importantly, the ex vivo 3D drug response profiling correlated well with the response obtained in vivo from PDX models (over 80% in average), for anticancer drugs with a variety of mechanisms of action including kinase inhibitor, antineoplastic agent and antibodies. Compared with a commercialized 3D drug testing plates, similar results were obtained in our system, with the advantage of a significant reduction in the cost. These data suggest that our 3D ex vivo cell culture platform from freshly isolated or frozen PDX-derived cells is a feasible and authentic method for testing drug efficacy in a short time period before transitioning to multiple PDX models.

To sum up, our ex vivo 3D culture platform can save time and reduce costs, and is extremely useful for preclinical evaluation of anticancer drug, filling the gap between cell line-based and tumor bearing mice-based drug studies, allowing low cost, high throughput in vitro drug screening and the clinically relevant in vivo drug efficacy studies.

#610

Effect of scaffold and growth factors on anti-cancer drug screening with multicellular spheroids: mimicking in vivo response.

Norio Masuda,1 M. Mamunur Rahman,2 Kazuya Arai,1 Atsushi Mizuno,1 Manabu Itoh1. 1 _SCIVAX Life Sciences, Inc., Kawasaki-shi, Kanagawa, Japan;_ 2 _SCIVAX USA, Inc., Woburn, MA_.

Background:

For in vitro cancer research, cancer microenvironment is being increasingly recognized as a key factor. In the past, several 3D cell culture models have been tested as the powerful method for reproducing cancer microenvironment. However, in some cases, the growth and drug sensitivity of cells grown on anchorage-independent 3D culture models have been different to that of cells grown in vivo. Further, development of the 3D culture model including the physical (the cell-ECM interaction (scaffold)) and chemical factor (cytokine) are essential for mimicking cancer microenvironment in vitro. Therefore, we explored the effect of the growth factor dependent proliferation and drug sensitivity in scaffold/scaffold-free culture models, and try to understand which method is mimicking in vivo response due to stimulation and suitable for anti-cancer drug development.

Methods:

We cultured lung cancer, breast cancer, pancreatic cancer and ovarian cancer cell line on monolayer, Matrigel (ECM-embedded 3D culture, scaffold type), NanoCuluture Plate (NCP, a gel-free scaffold type 3D culture plate) and Low adhesion round-bottom plate (scaffold-free 3D culture plate). For growth curve experiment upon addition of growth factors (EGF, HB-EGF, FGF, HRG), we evaluated cell viabilities by ATP assay in several cancer cell lines. We examined drug sensitivities of cancer cell lines against clinically active anti-cancer agents by addition of growth factors.

Results:

Growth factor promoted the proliferation of cancer cell lines cultured on NCP while it was not enhanced the cell growth when cultured on 2D, Matrigel and Low adhesion round-bottom plate. In drug response, the cells grown on NCP by growth factor stimulation was more sensitive to drug than 2D and other 3D culture models.

Conclusion:

Differences of the cell growth and drug sensitivity were appeared by effect of scaffold and growth factor. Growth factor dependent cell proliferation and drug sensitivity on NCP were similar to in vivo behavior (1). These results demonstrated that scaffold and growth factor are the novel approach to be mimicking the cancer microenvironment in vitro. Therefore, it is obvious that NCP can be used as a suitable 3D culture model for mimicking in vivo response and anti-cancer drug development.

1. J Cell Sci. 2009 Dec 1;122(Pt 23):4277-86.

#611

A complex 3D model of glioblastoma for patient-specific drug response profiling.

Tessa DesRochers,1 Lillia Holmes,1 Lauren O'Donnell,1 Qi Guo,1 David Schammel,2 Jeff Edenfield,3 Charles C. Kanos,3 Hal E. Crosswell1. 1 _KIYATEC, Inc., Greenville, SC;_ 2 _Pathology Consultants, Greenville, SC;_ 3 _Greenville Health System, Greenville, SC_.

Approximately 70,000 new cases of brain tumors will be diagnosed this year with glioblastoma (GBM) accounting for about 17% of those cases. Unfortunately the median survival for patients with GBM is only 14.6 months due to the complexity of the tumor, the pattern of diffuse spread within the brain, and the lack of effective therapeutic options. Complex in vitro tumor models developed in 3D better mimic in vivo biology, and, when utilizing patient-derived cells, may offer robust platforms for new drug development and patient-specific drug response profiling. We have previously shown this to be true with both breast and ovarian cancer and we have adopted this approach to modelling GBM ex vivo. We hypothesize that primary derived GBM tissues and stem cells cultured in complex 3D microenvironments can recapitulate the intra-tumor heterogeneity and drug resistance similar to that found in the clinical. To this end, we have developed 3D models of GBM that incorporate tumor cells, endothelial cells, and macrophages within a complex extracellular matrix and cultured them for up to 2 weeks under perfusion flow. We have created these models using both cell lines and primary patient cells and these 3D tissues have been analyzed for changes in viability (PrestoBlue and PicoGreen), marker expression (flow cytometry, Luminex, and protein arrays), macrophage differentiation and invasion (flow cytometry, multiphoton microscopy, and IHC), and methylation patterns (ChIP on chip arrays and methylation specific PCR). Initial optimization work using cell lines has revealed that culturing with endothelial cells and/or macrophages has an effect upon proliferation dependent upon the differentiation state of the macrophages. Additionally, co-culture conditions affect protein secretion and phosphorylation along with patterns of gene methylation. We have also examined how changes in the tumor microenvironment affect these different metrics by examining the role of hypoxic growth conditions. Work using patient cells isolated from primary glioblastoma has focused upon drug response dependent upon the tumor microenvironment and the presence of cancer stem cells utilizing a neurosphere generation assay. We have demonstrated temozolamide resistance in tumors with early relapse after initial chemo-radiation, and sensitivity to EGFR inhibitor afatanib in tumors with EGFR amplication, suggesting clinical and molecular correlation or our ex vivo 3D drug response profiling using patient derived GBM models. Taken together, our data support the further development and use of complex, 3D co-cultures to better mimic GBM in a patient-specific manner. These models are currently being considered for preclinical assessment of novel therapies, including chimeric antigen receptor T cells, and may be a useful adjunct in precision medicine applications to improve patient outcomes.

#612

Identification of selective inhibitors of diffuse-type gastric cancer cells by screening of annotated compounds.

Shu Shimada, Yoshimitsu Akiyama, Hiroshi Fukamachi, Yasuhito Yuasa, Shinji Tanaka. _Tokyo Medical and Dental University, Tokyo, Japan_.

Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and a poor prognosis. We have reported an E-cadherin/p53 double conditional knockout (DCKO) mouse line as the first genetically engineered one, which morphologically and molecularly recapitulates human DGC. We have also established mouse gastric cancer (GC) cell lines from DGC of the DCKO mice, demonstrating strong resistance to cytotoxic anti-cancer agents and high tumorigenic property when subcutaneously injected to nude mice. Then, we performed a synthetic lethal screening of 1535 annotated chemical compounds, and identified 27 candidates with selective inhibitory activity against the GC cell lines. These candidate drugs were classified into some classes, and members of the most potent class specifically attenuated cell proliferation of the GC cell lines by induction of apoptosis as well as suppressing tumor growth in vivo. Expectedly, E-cadherin-mutant and -low human gastric cancer cell lines were more sensitive to them in contrast to E-cadherin-intact ones. These findings may lead to development of novel drugs and discovery of potential targets in human DGC.

#613

Astrocytes and endothelial colony forming cells (ECFCs) influence the migration and drug response of glioblastoma cells in a 3D culture model.

Marisol Herrera-Perez,1 Melissa Fishel,2 Sherry Harbin,1 Jenna Rickus1. 1 _Purdue University, West Lafayette, IN;_ 2 _Indiana University, Indianapolis, IN_.

Glioblastoma, a brain cancer with low prognosis, is characterized by rapid infiltration and drug resistance that leads to tumor reappearance. Cell-extrinsic factors, such as the development of a supportive microenvironment and synergistic relationships between normal cells and GBM play an important role in the tumor successful progression. Despite the importance of the microenvironment as a regulator of tumor response, there is a lack of predictive in vitro models that represent the dimensionality and characteristics of the glioma microenvironment. To this end, we developed a 3D in vitro model that recapitulates the composition and physical features of the GBM microenvironment by generating a composite matrix of a hyaluronan supported by collagen. Human astrocytes and endothelial colony forming cells (ECFCs) were incorporated into the matrix and co-cultured with GBM cells to evaluate the effect of normal cells on the GBM 3D cell migration and drug response. Time-lapse microscopy was used to evaluate the 3D migration distance, velocity and persistence of GBM cell lines GBM10, GBM43 and GBAM1 (CD133+) during 18h after drug treatment with temozolomide and a STAT3-inhibitor. Survival of GBM cells was also evaluated 18h and 72 h after drug treatment. All results were compared to standard monolayer culture. Presence of astrocytes and ECFCs increased GBM net migration distance and velocity in the 3D model and in 2D monolayer culture. Additionally, viability of GBM after drug treatment was higher when astrocytes and ECFCs were present in the 3D model compared to only GBM 3D culture and standard monolayer culture. Our results support the importance of the 3D-microenvironment as modulator of tumor response and provide a tunable in vitro platform for primary brain tumor studies aimed to compliment in vivo model observations.

#614

Cytotoxic activity of enantia chlorantha, nauclea latifolia, and citrus medica extracts on carcinoma cells.

Chenel M. Alford, Alexandra Onyejiaka, Elbert L. Myles. _Tennessee State University, Nashville, TN_.

Over 562,340 Americans are expected to die of cancer, more than 1500 people a day. Cancer is the second most common cause of death in the US, exceeded only by heart disease. In the US, cancer accounts for nearly 1 of every 4 deaths. Our study focuses primarily on three plants never before investigated for antitumor activity. They were chosen based upon native ethno medicinal usages. Indigenous primarily to the southwestern region of Nigeria; Enantia chlorantha, Nauclea latifolia, and Citrus medica have all been traditionally used and studies have proven their noteworthy antibacterial, antimicrobial, and anti parasitical activities. These important characteristics provide the rationale behind studying these plants further for potential antitumor agents. Experimental: Methanolic leaf and bark extracts in a series of concentrations were taken from the previously stated plants and exposed to two breast and two colorectal cancer cells for 24 hours. Growth analysis is determined using a cell viability indicator alamar blue and a florescent plate reader. Results: Enantia chlorantha and Citrus medica demonstrated the most potential for containing novel antitumor compounds. However, E. chlorantha proved to be exceedingly toxic to the cellular proliferation of all four tumors cell lines at all concentrations. In addition to this, Nauclea latifolia extracts illustrated the least potential for antitumor activity. In most cases this extract showed little or no effect in reducing cellular proliferation. Conclusion: We conclude that leaf extracts from E. chlorantha and C. medica should be studied further to characterize their antitumor potential, investigate their main active compounds, and to better understand their mechanisms of action.

#615

Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine.

Manuele Giuseppe Muraro,1 Simone Muenst,2 Valentina Mele,1 Luca Quagliata,2 Alexandar Tzankov,2 Walter P. Weber,3 Giulio C. Spagnoli,1 Savas D. Soysal3. 1 _Institute of Surgical Research and Department of Biomedicine, University Hospital Basel, Basel, Switzerland;_ 2 _Institute of Pathology, University Hospital Basel, Basel, Switzerland;_ 3 _Department of Surgery, University Hospital Basel, Basel, Switzerland_.

INTRODUCTION Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell responses to drugs but they are not suited for the development of immune-mediate therapies. Here we present an innovative method for in vitro culture primary breast cancer (BrCa) tissues in porous 3D scaffolds by using a perfusion-based bioreactor system (U-CUP).

MATERIALS & METHODS Freshly excised breast cancer specimens were fragmented and cultured in a 3D "sandwich-like format" between collagen porous scaffolds under perfusion flow. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. Malignant and non-malignant cells survival, expansion into the scaffold and the ability to recapitulate features of the original BrCa specimens were histologically assessed. For estrogen receptor (ER) positive tissues we tested the response to hormonal therapy by adding the anti-ER drug Fulvestrant. Furthermore, the maintenance of immune-infiltrating cells allowed testing immune blockade therapy in vitro using anti PD-L1 on PD-L1 positive samples. Response to treatment was evaluated by histology and qRT-PCR for markers of immune-response.

RESULTS By culturing BrCa using the U-CUP we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of anti-ER treatment was associated with decreased expansion of cancer tissue into the scaffold after 21 days. After 7 days of anti PD-L1 antibody treatment we observed a reduced number of tumor cells due to the activation of infiltrating lymphocytes, as shown by increased expression of IFNg and decreased expression of IL10.

CONCLUSIONS The scaffold-based perfusion bioreactor represents a successful organotypic tumor model allowing in vitro long-term culture of breast cancer specimens. Our findings shed the light on a promising system for selecting personalized treatment based on a patient's tumor specific microenvironment.

#616

High throughput robotic drug screening against microprinted tumor spheroids.

Pradip Thakuri,1 Gary Luker,2 Hossein Tavana1. 1 _The University of Akron, Akron, OH;_ 2 _University of Michigan, Ann Arbor, MI_.

Cancer cell spheroids present a biologically relevant model of avascular tumors and a unique tool for anti-cancer drug discovery. Despite being used in research laboratories for several decades, spheroids are not routinely used in the mainstream drug discovery pipeline primarily due to the difficulty of mass-producing uniformly sized spheroids and intense labor involved in handling, drug treatment, and analyzing of spheroids. We overcome this barrier using a novel technology to robotically microprint spheroids in standard 384-microwell plates. An aqueous drop containing cancer cells is dispensed into a bath of a second, immiscible aqueous phase to allow generation of a single spheroid. Using several cancer cells, we establish that this approach results in spheroids of well-defined size with less than 8% deviation from the mean diameter in 384-microwell plates. We demonstrate the feasibility of robotic, high throughput compound screening against tumor spheroids using of a collection of 25 standard chemotherapeutics and molecular inhibitors against three different cancer cells, HT-29 colon cancer cells, U-87 MG brain cancer cells, and MDA-MB-157 breast cancer cells. Dose-dependent experiments are done with each drug at a wide range of concentrations. After six days of incubation, viability of cancer cells in drug-treated spheroids is measured using a PrestoBlue assay that we have optimized for three-dimensional cell cultures. Post-treatment morphological changes are used as a secondary measure for analysis.

This screening identifies specific MEK inhibitors including Trametinib and Selumetinib that potently inhibit growth of HT-29 spheroids at nanomolar concentrations, whereas several PI3K inhibitors such as pictilisib significantly block proliferation of U-87 MG spheroids at micromolar concentrations. Spheroids of MDA-MB-157 cells show resistance to most of the drugs. We identify few compounds such as a Survivin inhibitor, YM155, and doxorubicin that disintegrate spheroids and induce significant cell death in all three cells. To generate a scoring system, we use half-maximum inhibitory concentration (IC50), maximum inhibition (Emax), and area under the dose-response curve (AUC) to present a multi-parametric approach that takes into account both potency and efficacy parameters for evaluating drug responses in tumor spheroids. In conclusion, our robotic technology offers a low cost and convenient platform for high throughput screening of large collections of chemical compounds against tumor spheroids. Incorporation of this technology in drug discovery applications will make drug testing and screening with realistic tumor models a routine laboratory technique prior to expensive and tedious in vivo analyses, dramatically improving testing throughput and efficiency and reducing costs.

#617

Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib.

Belen Hernandez,1 Kathryn Cronise,1 James C. Costello,2 Rodney Page,1 Susan Lana,1 Kenneth L. Jones,2 Dawn L. Duval1. 1 _Colorado State University, Fort Collins, CO;_ 2 _University of Colorado Anschutz Medical Campus, Aurora, CO_.

Transitional cell carcinoma (TCC) is the most common bladder cancer in humans and their canine companions. Canine TCCs are usually papillary infiltrative TCCs of intermediate to high grade. Genetic defects identified in human TCCs aid in the diagnosis and therapy of human bladder cancer. We utilized whole exome sequencing to screen canine TCCs for gene mutations that contribute to pathogenesis. Genomic DNA was isolated from 11 canine TCCs, 3 matched normal tissue samples, and 2 canine TCC cell lines. Whole exome capture was conducted using the Canine Agilent Sure-select in-solution capture system, and captured fragments were sequenced using an Illumina HiSeq2000 platform. The sequences were mapped to the CanFam3.1 canine reference genome and somatic mutations were identified using Freebayes. Somatic mutations were characterized and compared to the Cancer Gene Census (COSMIC). Nonsense, missense, and insertion/deletion mutations were identified in 126 genes shown to be drivers or repressors in human cancer. Missense mutations were further screened using SIFT to identify alterations deleterious to protein function. The genes exhibiting in/dels, nonsense, or missense mutations with SIFT scores < 0.45 in at least 2 sites or 2 samples were: BRAF, RPL5, RANBP2, EWSR1, NONO, PTPRB, LYL1, JAK1, MSH2, PER1, PIM1, and WRN. In fact, an activating BRAF V to E mutation was identified in 5 of the tumors and both cell lines. The RPL5 and RANBP2 mutations were also hotspot mutations identified in 5 and 3 of the samples, respectively. Mutations were confirmed using Sanger sequencing of amplified genomic DNA. Interestingly, deleterious RANBP2 mutations were only observed in non-BRAF mutant samples. Drug sensitivity assays using the BRAF V600E targeting drug, Vemurafenib, were conducted in the BRAF mutant canine cell lines as well as the BRAF mutant human A375 melanoma cell line. As previously described, the IC50 for the sensitive A375 line was approximately 100 nM, while each of the BRAF mutant canine lines had an IC50 value ≥ 10 µM. Reverse transcriptase PCR was used to amplify the coding sequence for BRAF from the Bliley canine TCC cell line. The amplified transcript (2125 bp) was sequenced, confirming the heterozygous expression of the V548 to E mutant form of BRAF in this cell line. The sequence also indicated that the predicted expressed protein (AA 10 - 715 of XP_013975364.1, corresponding to predicted exons 1 through 20 of XM_014119889.1 ) exhibited 99% homology to human BRAF AA53 - 763 (NP_004324.2). BRAF protein expression in these cell lines was confirmed by Western blot analysis. Thus, insensitivity to Vemurafenib is not due to differences in canine BRAF, reduced expression, or alternative splicing of the expressed transcript as previously observed in some Vemurafenib resistant human melanomas. These data indicate that although constitutively active BRAF is expressed in canine TCC, other factors may contribute to pathogenesis.

#618

Dual PI3K/mTOR inhibition as a treatment strategy for PIK3CA and APC mutant colorectal cancers.

Susan Payne, Tyler Foley, Cheri Pasch, Alexander Yueh, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. _University of Wisconsin Carbone Cancer Center, Madison, WI_.

BACKGROUND: Colorectal cancer (CRC) is understood as a diverse group of cancers calling for the personalization of therapeutics to individual subtypes of CRC. Mutations in PIK3CA, which result in a constitutively active phosphoinositide-3-kinase protein (PI3K), are present in 20-30% of CRCs. Aberrations in the Adenomatous Polyposis Coli (APC) gene are found in 86% of PIK3CA mutant CRCs. The potential for treatment of tumors with this specific profile is of great interest. METHODS: Mice expressing a constitutively active PI3K and loss of APC were generated and spheroid cultures were derived from the resulting cancers. PIK3CA and APC mutant spheroids were treated with GDC0941 (GDC), a pan PI3K inhibitor, NVP-BYL719 (BYL), a PI3K alpha isomer specific inhibitor, or NVP-BEZ235 (BEZ), a dual mTOR/PI3K inhibitor. Proliferation was measured by changes in spheroid diameter over time. BEZ treatment of mice with PIK3CA and APC mutant CRCs was performed over 14 days. Tumor response was measured with serial murine colonoscopy and dual hybrid 18F-FDG PET/CT imaging. RESULTS: Persistent PIK3CA and APC mutant CRC spheroid growth was observed with BYL and GDC treatment, while BEZ treatment resulted in a significant reduction in spheroid size. Immunoblotting determined that this correlated with significant suppression of phosphorylated AKT, RPS6 and 4EBP1 in BEZ-treated spheroids and incomplete PI3K pathway inhibition following BYL and GDC treatment. BEZ treatment was then investigated in mice with PIK3CA and APC mutant CRCs. BEZ elicited a dramatic treatment response in these cancers on endoscopy, with a 53% decrease in median lumen occlusion compared to a 60% increase in controls over the 14 day treatment period. PET/CT imaging results confirmed these findings demonstrating a decrease in tumor size and avidity post BEZ treatment. BEZ resulted in inhibition of the PI3K pathway in these tumors. Reactivation of PI3K signaling was observed within 24 hours of the withdrawal of BEZ. BEZ was well-tolerated in these mice and associated with a decrease in the endoscopic anemia score. CONCLUSIONS: PIK3CA and APC mutant CRCs are a potentially targetable subtype of cancer. These cancers are resistant to proximal inhibition of the PI3K pathway, but are responsive to dual PI3K/mTOR inhibition. These results warrant further investigation in clinical studies.

#619

Personalized models to guide precision medicine.

Chantal Pauli, Loredana Puca, Brooke M. Emerling, Benjamin Hopkins, Andrea Sboner, Olivier Elemento, Juan Miguel Mosquera, Himisha Beltran, Mark A. Rubin. _WCM, New York, NY_.

BACKGROUND

Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patient's cancer. Available cell line models alone often do not accurately recapitulate the genetic profile of individual patient tumors and therefore limit preclinical evaluation of newly targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to the use of monolayer cultures as the initial screening method that is associated with highly variable responses and does not predict clinically observed chemoresistance. In our Englander Institute for Precision Medicine we developed a program utilizing patient derived tumor organoids, in combination with individualized genomic sequencing, targeted and/or high throughput drug screenings to nominate drug candidates in a precision patient care setting. Drug candidates are further validated with personalized in vivo models. Utilizing these various genomic and biological platforms for pharmacological screenings, we can more closely recapitulate the in vivo tumor of individual patients and can more accurately model personalized therapeutic response and resistance in vitro and in vivo.

DESIGN

Fresh tissue samples were collected, washed and mechanically or enzymatically dissociated and then plated in a Matrigel (BD) scaffold with primary culture media. Primary spheres were characterized according to our cytology, histology and genomic platforms. Established and characterized tumor organoids were expanded, cryopreserved for banking, used for in vitro studies and implanted in nude mice for patient derived xenografts (PDXs) to further validate potential drug candidates.

RESULTS

Our success rate in generating patient derived pan-cancer tumor organoids is 30%, depending on specimen quality and tumor type (e.g. endometrial cancer 70%, metastatic prostate cancer 15%). Morphology and molecular profiles show good concordance among tumor organoids and native tumor tissues. The success rate in establishing PDXs from organoid cultures is currently at 70-80%. In vitro and in vivo drug screenings show tumor specific drug sensitivity.

CONCLUSION

We have developed protocols for the generation and characterization of individual patient-derived tumor organoids. Cytopathology, histopathology and molecular pathology represent important platforms in our Precisicon Medicine Program. Tumor organoid characterization, pharmacological screenings and drug validation in PDX models are effective models which can be used to tailor standard of care treatment, study drug resistance, and nominate novel therapeutic targets unique to the individual genomic landscape and biology of each tumor.

#620

Microfluidic cancer-on-a-chip platform for assessing anti-cancer drug efficacies.

Shantanu Pradhan,1 Ashley M. Smith,2 Charles J. Garson,2 Iman Hassani,1 Kapil Pant,2 Robert D. Arnold,1 Balabhaskar Prabhakarpandian,2 Elizabeth A. Lipke1. 1 _Auburn University, Auburn, AL;_ 2 _CFD Research Corporation, Huntsville, AL_.

Introduction: The tumor microenvironment is known to play an influential role in the angiogenic and metastatic progression of cancer and is regulated by different factors (stromal fibroblasts, extracellular matrix (ECM) proteins and endothelial cells) present in the complex milieu. Recapitulation of this complexity in three-dimensional (3D) tumor models is critical in understanding the processes involved in cancer progression and to provide clinically relevant efficacy data for cancer drugs. To address this challenge, we developed a microfluidic oncomimetic platform where breast cancer cells are co-cultured with fibroblasts along with a complex, intricate vascular network. We further investigated the effect of mechanical stiffness of the tumor stroma on the growth and morphology of cancer cells, migration of cancer cells into surrounding vasculature and the ability of standard cancer drugs to perfuse through the vasculature and target cancer cells.

Materials and Methods: Poly(dimethyl siloxane) (PDMS)-based microfluidic devices, containing vascular networks in communication with tumor chamber, were fabricated using photolithography as described earlier1. Poly(ethylene glycol)-fibrinogen (PEG-Fb), used for 3D co-culture of cancer cells and fibroblasts, was prepared using established protocols2. Human breast tumor associated endothelial cells (hBTECs) were seeded within fibronectin-coated vascular channels and maintained under flow to develop endothelial networks. To obtain 3D cancer-fibroblast co-culture system, MCF7 or MDA-MB-231 breast cancer cells were co-encapsulated with BJ-5ta human fibroblasts in PEG-Fb hydrogel in the tumor chamber. Immunostaining with standard endothelial and cancer markers was conducted to confirm maturity and functionality of seeded cells. GFP-labelled cancer cells were co-cultured with hBTECs to visualize tumor cell migration. Cancer cells were also maintained in culture over several weeks within the devices to demonstrate applicability of this system to perform long-term drug dosing experiments. Finally, the cytotoxic effects of doxorubicin and paclitaxel at two different concentrations on cancer cells were evaluated by perfusing the drugs through the endothelial channels and cell viability quantified via Live/Dead staining.

Conclusions: We have developed a novel 3D microfluidic, vascularized cancer-fibroblast co-culture platform, with the ability to predict drug efficacy for breast cancer. This platform can also be extended in future for cancer-immunotherapy based investigations and screening of novel nano-carrier based anti-cancer drugs.

Acknowledgements: We gratefully acknowledge financial support from NIH (#HHSN261201400037C) and AURIC.

References: 1. B. Prabhakarpandian et al., J Control Release 2015; 201:49-55

2. L. Almany et al., Biomaterials 2005; 26(15):2467-77

#621

Combined treatment of fisetin and cabazitaxel significantly inhibits tumor growth and metastasis of prostate cancer cells.

Eiman Mukhtar, Vaqar M. Adhami, Imtiaz A. Siddiqui, Hasan Mukhtar. _University of Wisconsin-Madison, Madison, WI_.

Drugs that affect microtubule dynamics are among the most effective anticancer agents in routine clinical use. However, the effectiveness of these drugs has been impaired by severe side effects and subsequent development of drug resistance. Continued investigation into the development and discovery of new drugs, and exploring new treatment strategies that reduce side effects and circumvent drug resistance may provide more effective therapeutic options for cancer patients. Considering these facts we believe that natural agents from dietary sources could offer a safe and effective alternative to existing repertoire of microtubule-targeting agents in combination with standard of care. In line with this we have observed that fisetin is a microtubule stabilizing agent and significantly affects microtubule dynamics. We hypothesized that fisetin will enhance the efficacy of cabazitaxel in advanced and metastatic human PCa cells both in vitro and in vivo. We determined the effect of fisetin alone and in combination with cabazitaxel on in vitro and in vivo in a subcutaneous xenograft mouse model. Human prostate cancer PC-3M-luciferase cells (3x103) were injected into 24 athymic nude mice. Two weeks post cell implantation, mice were divided into four groups of 6 animals, and then treated with fisetin (20 mg/kg; 3 times/week), cabazitaxel (5 mg/kg; once/week), combination of fisetin (20 mg/kg; 3 times/week) and cabazitaxel (5 mg/kg; once/week), or vehicle (control) via intraperitoneal route for 8 weeks. Tumor growth of live mice was monitored manually and by IVIS™ imaging system. Combination treatment with fisetin and cabazitaxel significantly (p = 0.0008) inhibited the growth of tumors compared to control and individual treatment groups. We observed only 2.7 fold increase in tumor volume in the combination group over three weeks, while cabazitaxel or fisetin alone resulted in 5 fold increase, and tumor in the control group increased by 7 folds. In addition, we also observed that treatment with fisetin and cabazitaxel alone resulted in 10% and 12% inhibition in tumor growth respectively. However, combination of fisetin and cabazitaxel resulted in 71% inhibition in tumor growth. We next tested the effect of the combination in vitro using PC-3M-luciferase cells. We observed significant inhibition in the protein expression of Ki-67 and PCNA and metastatic markers MMP9 and MMP2 in the combination group compared with each agent alone. In addition, PC-3M-luciferase cells treated with fisetin (20 μM) for 48 hours and then treated with cabazitaxel (5nM) significantly inhibited the migration, invasion and proliferation of these cells. The combination resulted in significant reduction in the effective dose of cabazitaxel. Taken together, these results suggest that fisetin can be used in combination with cabazitaxel to inhibit tumor growth and metastasis of advanced human prostate cancer.

### Human in Mouse Models

#622

Gastric cancer in the age of targeted agents: identification and validation of novel therapeutic strategies through the generation of a patient-derived xenografts platform.

Silvia Menegon,1 Maria Apicella,1 Cristina Migliore,1 Tania Capeloa,1 Marilisa Cargnelutti,1 Maurizio Degiuli,2 Anna Sapino,1 Paola Cassoni,3 Michele De Simone,1 Paolo M. Comoglio,1 Silvia Marsoni,1 Simona Corso,1 Silvia Giordano1. 1 _IRCCS, Candiolo, Italy;_ 2 _Department of Surgery - Ospedale Molinette, Torino, Italy;_ 3 _Department of Medical Science - University of Turin, Torino, Italy_.

Gastric cancer is the world third leading cause of cancer mortality. In spite of the significant therapeutic advances, the overall clinical outcome for patients with advanced gastric cancer is poor, with 5-20% 5-year survival. The only targeted therapy approved so far are trastuzumab, and Ramucirumab which have given unsatisfactory results. Around 50% of gastric tumors bear genetic alterations affecting tyrosine kinase pathways (mainly EGFR, HER3, FGFR2 and MET pathways, besides HER2) but their clinical validation as tumor drivers is missing. The need for new therapeutic options and the possible presence of 'druggable' targets prompted us to investigate potential targeted therapies for this disease.

Our project aims at identifying and validating targeted therapeutic strategies in gastric cancer, through the generation of a platform of gastric tumor patient-derived xenografts (PDXs), animal models in which tumor surgical specimens are directly transferred into mice. Upon engraftment, the tumor is split and re-implanted in a cohort of mice, allowing the simultaneous testing of different drugs on the same tumor. Thanks to the establishment of a network of 15 Italian centers for samples collection, we generated around 80 gastric PDXs and successfully derived cell lines and organoids from engrafted tumors. Among the tumors collected so far, we found HER2, EGFR, FGFR2, MET and KRAS amplifications. This gastric PDX platform will be exploited for: 1) Validation of candidate oncogenes as relevant targets and identification of efficient therapeutic strategies 2) Identification of novel molecular targets; 3) identification of genetic predictors of response/resistance.

In the PDX platform we identified one tumor bearing a high level of MET gene amplification (26 gene copies). We thus performed a preclinical study on a cohort of patient-derived xenografts generated from the MET-amplified gastroesophageal tumor. Despite the high amplification level, MET inhibitors induced only a partial response, while the combined anti-MET/EGFR treatment led to complete tumor regression. Most important, the combo treatment also prevented resistance onset. This data represent the proof of concept that a combined anti-MET/EGFR therapy can be more effective than anti-MET treatment alone in MET-amplified gastroesophageal tumors, in the absence of EGFR genetic lesions.

#623

Feasibility of surgical orthotopic implantation for establishment of patient-derived liver metastatic uveal melanoma xenograft model in NSG mouse.

Ken Kageyama, Masahiro Ohara, Kengo Saito, Shinji Ozaki, Mizue Terai, Andrew E. Aplin, Michael J. Mastrangelo, Takami Sato. _Thomas Jefferson University, Philadelphia, PA_.

The prognosis of metastatic uveal melanoma (UM) is extremely poor. Approximately 90% of patients with metastatic UM die from hepatic metastasis. Controlling hepatic metastases has been a rational approach to extend survival. This raises an urgent need to develop a novel mouse model for further investigation. Patient-derived tumor xenograft (PDX) mouse model provides potential benefit through a personalized medicine approach. This model may be useful for predictions of clinical outcomes, drug efficacy and biomarker identification. Most PDX models, including UM PDX models, are made by ectopic subcutaneous implantation. Subcutaneous PDX models have some obstacles in translational research due to differences in anatomic microenvironment from tumor origin, low engraftment rate, and slow tumor growth. Our goal was to develop an orthotopic PDX model of liver metastatic UM which would overcome these difficulties.

In developing our model, we first compared the differences in growth in a liver with a subcutaneous site using two UM cell lines (UM001 and UM004) in NSG mice, to ascertain which site is a more suitable tumor microenvironment. Second, we investigated the feasibility of surgical orthotopic implantation (SOI) of tumor masses to develop a practical liver tumor xenograft mouse model with UM cell lines in 20 mice. Finally, we tried to establish an orthotopic PDX mouse model of liver metastatic UM in 5 mice. We also evaluated concordance for tumor characteristics between pre-implant and post-implant tumors.

Our results showed that the liver was a more suitable microenvironment to grow the two UM cell lines compared to the subcutaneous site. UM001 engrafted in the liver, whereas it did not engraft in the subcutaneous site. UM004 displayed more rapid growth in the liver compared to the subcutaneous site. A new SOI of tumor chunk was created to develop a practical liver tumor xenograft model. Donor tumors, which were generated in the liver after injecting UM cell lines, were surgically transplanted into the liver of recipient mice. The new SOI was successfully performed in all 20 mice without adverse events. Recipient mice formed tumors in the liver after SOI. For comparison, the tumor chunks of UM cell lines were also implanted subcutaneously, in which we were able to show that the growth was more rapid in the liver. Both donor and recipient tumors had matching tumor characteristics in histological and proteomic analysis. We established an orthotopic PDX mouse model of liver metastatic UM with a new SOI method. The success rate of engraftment was 80% (4/5) within 6 months. Tumor characteristics of both patient's original tumors and xenograft mouse tumors matched in histological, genomic and proteomic analysis.

This new SOI method can establish a patient-derived liver metastatic uveal melanoma xenograft model in NSG mouse at a highly successful rate.

#624

Humanized mouse models for evaluation of cancer therapies.

Olivier Duchamp,1 Francis Bichat,1 Caroline Mignard,1 François Ghiringhelli,2 Jean-François Mirjolet,1 IMODI Consortium. 1 _Oncodesign S.A., Dijon, France;_ 2 _INSERM 866 / Centre Georges François Leclerc, Dijon, France_.

Mice with a humanized immune system, so called "humanized" mouse models, have been established to study the complex interaction of the human immune system during human disorders. In case of cancer research, the in vivo model ideally should recapitulate the biological characteristics of the human tumor and of the related tumor microenvironment in patient such as immune system.

Human immune system is reconstituted in immunodeficient mice using either human PBMCs or hematopoietic stem cells (HSCs). Humanized mice bearing human target tumor cells constitute relevant models for evaluation of cancer therapeutics such as bispecific antibodies, immune cell targeting antibodies.

In vivo proof of concept studies were performed with humanized mice xenografted with either human disseminated lymphoma or with human solid tumors (tumor cell line and Patient Derived Xenograft model, Her2 positive breast, colon, ovarian and lung cancers) and treated with therapeutic antibodies.

Biomarkers of response to immune cell targeting therapies were evaluated by flow cytometry as well as immunohistochemistry analyses. In case of disseminated B-cell lymphoma grafted into mice reconstituted with human PBMC, efficacy of antibody was evidenced by an increased survival and a high decrease of human B cells in mouse bone marrow. Lung and ovarian PDX developed in HSC reconstituted BRGS mice induced an increase in Tregs and a change in T/B ratio in blood and spleen samples. Furthermore, we evidenced the presence of Tregs within human PDX tumor by immunohistochemistry.

#625

Patient derived xenografts of renal carcinoma with human vasculature.

Jie Cai, Likun Zhang, Jean-Pierre Wery, Henry Li. _Crown Biosciences, Santa Clara, CA_.

Patient derived xenografts (PDXs) mirrors original patients' histo-/molecular pathology, thus considered as predictive models. One limitation is that its tumor microenvironment may differ from that of patients. PDXs tend lose human stroma (fibroblasts, endothelial (vasculature) and immune cells) 1,2 that is replaced by mouse stromal components. Since many murine factors do not cross-react with human tumor cells, PDXs may behave differently from human tumors in some respects. To explore this further, we built the largest and most comprehensive PDX library of ~2000 models of different cancer types with genomic annotations2-7. We tested a subset of our PDXs for the presence of stromal components. We first examined PDX histopathology and compared it to the patient counterparts, confirming morphological similarity, e.g. renal carcinoma has a rich vasculature and pancreatic cancer has a high stromal content (fibrosis)8. Immunohistochemistry (IHC) assay also confirmed most negative staining for human stroma, , even at passage 1. Interestingly, a small subsets of PDXs displayed positive staining for human stromal markers, including human vimentin2. One particular renal clear cell carcinoma, KI0326, not only shows a rich vasculature, but also stained positively for all tested human endothelial markers, including human prostate specific membrane antigen (PSMA), platelet/endothelial cell adhesion molecule 1 (PECAM1/CD31) and von Willebrand factor (VWF). To the best of our knowledge, this is the first report of a renal carcinoma PDX that has fully preserved human vasculature. Although its mechanism remains to be elucidated, this model could be particularly useful to study its role in tumor growth and response to therapy.

References

1. Tentler, JJ, et al. Patient-derived tumour xenografts as models for oncology drug development. Nat Rev Clin Oncol 9, 338-350 (2012).

2. Chen, D. Autocrine c-met/HGF HCC Patient Derived Xenograft (PDX) Models for Evaluating c-Met Inhibitors. AACR Annual Meeting 2012 Poster(2012).

3. Chen, D.,et al. A set of defined oncogenic mutation alleles seems to better predict the response to cetuximab in CRC patient-derived xenograft than KRAS 12/13 mutations Oncotarget (2015).

4. Yang, M., et al. Overcoming erlotinib resistance with tailored treatment regimen in patient-derived xenografts from naive Asian NSCLC patients. Int J Cancer 132, E74-84 (2013).

5. Zhang, L., et al. A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy. Sci Rep 3, 2992 (2013).

6. S. Guo1*, W.Q., J. Cai1, Likun Zhang, JP Wery1, H. Q.X. Li1,2. Molecular pathology of patient derived xenografts EORTC-AACR-NCI Abstract (2015).

7. Akashi, Y., et al. Histological advantages of the tumor graft: a murine model involving transplantation of human pancreatic cancer tissue fragments. Pancreas 42, 1275-1282 (2013).

#626

Application of patient-derived xenograft model in nasopharyngeal carcinoma research.

Cheng-Lung Hsu,1 Yung-Chia Kuo,1 Yenlin Huang,1 Yin-Cheng Huang,1 Kar-Wai Lui,1 Kai-Ping Chang,1 Tung-Liang Lin,1 Hsien-Chi Fan,1 An-Chi Lin,1 Chia-Hsun Hsieh,1 Li-Yu Lee,1 Hung-Ming Wang,1 Hsin-Pai Li,2 Angel Chao,1 Yu-Sun Chang2. 1 _Chang Gung Memorial Hospital, Taipei, Taiwan;_ 2 _Chang Gung University, Taipei, Taiwan_.

Nasopharyngeal carcinoma (NPC) was an Epstein Barr virus (EBV)-related malignancy and tumor microenvironment had a pivotal role in tumor progression. Paucity of good NPC animal models hindered the research in this field. Recently, patient-derived xenograft (PDX) had been shown to be a good preclinical model for drug screening and cancer related research. We had developed two PDX mice lines from engrafting NPC metastatic tumors. Positive EBV-encoded small RNAs staining confirmed these tumors harboring EBV. Further gene expression profile analysis showed higher similarity of PDX to primary parent tumor than NPC cell line xenograft. In vivo drug screening in the PDX system demonstrated gemcitabine had the best antitumor effect among the tested drugs. In this PDX corresponding patient also showed excellent response to gemcitabine treatment. Combination of gemcitabine and valproic acid had synergistic antitumor effect. Further adding ganciclovir in this two combined regimen enhancing cytolytic viral activation had the best antitumor response among the tested regimens. This three combined regimen treated group had lower plasma EBV-DNA load and tumor viral concentration and less viable tumor cells than gemcitabine + valproic acid group. These promising results would open a new era for EBV-targeting therapy in NPC treatment.

#627

Establishment and characterization of a panel of cell-based and patient-derived chordoma tumor models.

Michael J. Wick,1 Melissa Rundle,1 Lizette Gamez,1 Armando Diaz,1 Josh Sommer,2 Patricia Cogswell,2 Byron Hann,3 Joanna Phillips,3 Kyriakos P. Papadopoulos1. 1 _START, San Antonio, TX;_ 2 _Chordoma Foundation, Durham, NC;_ 3 _UC San Francisco, San Francisco, CA_.

Background: Chordoma is a rare cancer (0.08 per 100k/yr) that originates from the notochord and develops in the skull and spine. Treatment options for chordoma include resection and local radiation therapy; however, recurrence rates following treatment are high and the majority of patients ultimately succumb to the disease. Currently there are no approved chemotherapy options for chordoma, and effective treatment options for patients with recurrent or advanced disease are limited. Recent molecular analyses of chordoma have revealed multiple tractable therapeutic targets, providing rationale for new systemic therapies. However, lack of relevant, validated chordoma models has limited preclinical evaluation. To address this need, the Chordoma Foundation and START have collaborated to establish, bank and characterize chordoma tumor models derived from patient samples and established cell lines.

Methods: Chordoma patients undergoing resection or biopsy procedures are identified and consented by the Chordoma Foundation under an IRB-approved protocol; tissue is shipped to START and implanted into immune-deficient mice for patient-derived xenograft (PDX) model establishment and banking. Similarly, models from immortalized chordoma cell lines have been established and banked, along with established PDX chordoma models from START or collaborators. Banked PDX models are anonymously linked to available patient clinical information. All chordoma models are subjected to immunohistochemistry (IHC) to confirm histology and presence of brachyury, a protein highly expressed in chordoma tumors. Following validation, models are used at START for drug sensitivity studies and available to investigators for characterization studies.

Results: To date, clinical samples from five chordoma patients have been implanted and two PDX models designated CF322 and CF345 have been established. In addition, xenograft models from the UCH-1 and UCH-2 chordoma cell lines have been established at START. PDX models previously developed at START (ST087) and UCSF (SF8894) have also been validated and banked. Drug sensitivity studies have been initiated these two and the U-CH1 models evaluating agents selected and prioritized through a peer review process established by the Chordoma Foundation. Nominations for additional test agents from academic and industry collaborators are reviewed on a rolling basis and evaluation of selected therapies is funded by the Chordoma Foundation.

Conclusion: We have established a collaboration to create, bank and characterize a panel of preclinical chordoma tumor models originating from patient samples and established cell lines. To date we have generated three chordoma models available for drug sensitivity studies. This panel of models is intended to serve as a shared resource to the chordoma research community to enable more rapid preclinical evaluation of therapeutic hypotheses.

#628

A method to examine spatial relationships between tumor cells and vasculature using a mouse orthotopic PDX glioblastoma model.

Tavarekere N. Nagaraja, Kelly Keenan, Susan Irtenkauf, Laura Hasselbach, Swayamprava Panda, Glauber Cabral, James R. Ewing, Tom Mikkelsen, Ana deCarvalho. _Henry Ford Hospital, Detroit, MI_.

Introduction: Glioblastoma progression requires angiogenesis and/or vascular co-option. A major pathway for its infiltration is the perivascular space. We present herein a method to visualize glioblastoma cells with respect to tumor-associated vasculature within and beyond the tumor mass.

Experimental procedures: A neurosphere cell line, HF3253, developed from cultures of a human glioblastoma biopsy sample was labeled with PKH26, a red fluorescent dye, using a commercially available kit (Sigma-Aldrich). The labeled cells were implanted into the right striatum in immunocompromised nude mice (n=5) and allowed to grow for 2-4 weeks. Terminally (one mouse at 2, two at 3 and the remaining two at 4 weeks), the mice were intravenously injected with fluorescent isothiocyanate (FITC) dextran of 70 kDa molecular weight. The brains were removed after 5 min of dextran circulation, immersion fixed in 10% neutral buffered formalin for 2 days and then sectioned into 100 µm thick coronal slices using a vibratome. Slices were stained with the nuclear counterstain 4',6-diamidino-2-phenylindole (DAPI) to visualize native mouse brain cells at 405 nm in an Olympus FV1200 laser scanning confocal microscope. Relative distributions of the dextran and PKH26 fluorochromes were also examined using the same microscope. FITC-dextran (green fluorochrome)-labeled microvessels and PKH26-labeled tumor cells (red fluorochrome) were excited by a laser beam at 488 and 568 nm, respectively, and emissions were detected with a photomultiplier tube through 522 and 585 nm emission filters, respectively.

Results: The normal vasculature on the contralateral side appeared well perfused, filled with the green dextran fluorescence. Intricate, fine networks of microvessels were present. The ipsilateral hemisphere showed bright red spots of tumor cells in the striatum in closely distributed groups among the blue DAPI-stained mouse cells. Compared to contralateral side, the vasculature associated with such groups was dilated and irregular, with fewer interconnections. Interestingly, these irregular networks were also observed away from the main tumor cell mass in 3- and 4-week-old tumors, suggesting probable future infiltrative pathways. Red-labeled single cells and groups of tumor cells could be seen along vascular branches penetrating brain tissue adjacent to the tumor. The strong fluorescence of the tumor cell tracer PKH26 persisted over the 4 week experimental period, suggesting its utility in imaging tumor infiltration in this model.

Conclusions: This technique generated high resolution images of tumor cell distribution and associated microvasculature in an orthotopic glioblastoma model. With further quantification, it can help in establishing infiltrative patterns of different PDX tumor models, and may also be useful to measure discrete responses to therapies that affect tumor cells and/or vasculature.

#629

The impact of BMP4 on breast cancer metastasis in an in vivo xenograft mouse model.

Minna Ampuja,1 Emma L. Alarmo,1 Philip Owens,2 Riikka Havunen,1 Agnes E. Gorska,2 Harold L. Moses,2 Anne Kallioniemi1. 1 _University of Tampere, BioMediTech, Tampere, Finland;_ 2 _Department of Cancer Biology, Vanderbilt Ingram Cancer Center, Vanderbilt University, Nashville, TN_.

Background. Breast cancer is the most common cancer in women worldwide. Bone morphogenetic proteins (BMPs), members of the transforming growth factor β superfamily, are known to regulate cell proliferation, differentiation and motility, and have also been shown to be involved in cancer pathogenesis, also in breast cancer. We have previously demonstrated that BMP4 is able to consistently reduce breast cancer cell proliferation through G1 cell cycle arrest and to simultaneously induce migration and invasion in a subset of breast cancer cell lines. Similarly, our clinical data revealed a correlation between elevated BMP4 expression in primary breast tumors and reduced proliferation as well as increased risk of recurrence. The growth inhibitory effects of BMP4 have also been demonstrated in vivo but its possible metastasis promoting functions are less well characterized. Here we set out to investigate this topic using a xenograft mouse model.

Methods. MDA-MB-231 breast cancer cells were transduced with a luciferase-expressing vector to allow monitoring of the metastasis formation using bioluminescence imaging. Cells (2 x 105) were injected into the mice intracardially and BMP4 (100 ng/g, 10 animals) or vehicle control (11 animals) was administered through tail vein three times a week. After seven weeks, the mice were sacrificed and metastases collected for histological analyses.

Results. The overall amount of metastases was similar in both groups (13 in BMP4-treatment group vs. 12 in control group). There was a slight but non-significant trend of metastases developing earlier in the BMP4 group compared to controls. Most of the metastases occurred in bone and adrenal glands. There were somewhat more metastases in bone in the BMP4-treated mice (10 vs. 7) and more adrenal gland metastases in vehicle-treated animals (5 vs. 1). To assess the contribution of BMP4 to the characteristics of the metastases, the tumors were stained for pSMAD1/5/9 (BMP signaling activation), Ki67 (proliferation), MECA32 (blood vessels), mesenchymal marker vimentin, α-SMA (cancer-associated fibroblasts) and basal markers K5 and K14. No major dissimilarities were observed between the BMP4 and vehicle tumor groups in the staining patterns. Interestingly, the osteoclast marker Tartrate-resistant acid phosphatase (TRAP) was expressed in both groups in the cancer cells whereas Toluidine Blue staining revealed that the bone morphology was not detrimentally affected by BMP4 treatment.

Conclusions. Despite its ability to enhance breast cancer cell migration and invasion in vitro, BMP4 does not seem to have a dramatic impact on in vivo metastasis formation, although a small acceleration in appearance of the metastases was observed. However, the limitations of the xenograft model do not allow us to exclude the possible long-term effects of BMP4 that might be more applicable to human situation.

#630

Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice.

Eva Oswald,1 Vitor E. Santo,2 Albin Rudisch,3 Catarina Brito,2 Wolfgang Sommergruber,3 Helmut Dolznig,4 Julia B. Schüler1. 1 _Oncotest GmbH, Freiburg, Germany;_ 2 _Animal Cell Technology Unit, ITQB-UNL, iBET, Lisbon, Portugal;_ 3 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _4 _Institute of Medical Genetics, Vienna, Austria_.

Human tumor xenografts in immunodeficient mice have led to valuable insights into the biology of human cancer. The corresponding limitations and pitfalls of xenograft models have been described extensively. To tackle some limitations, we supplemented the murine tumor microenvironment (TME) with human stromal fibroblasts to mirror tumor-stroma cross-talk in vivo.

Non-small cell lung cancer (NSCLC) cell lines Calu-1 and H1437 were co-injected with different types of fibroblasts into NOG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice in 3 independent experiments (n=4-6/group; 106 in total). Human Dermal Fibroblasts (HDFs), lung cancer associated fibroblasts (CAFs), corresponding normal fibroblasts (NFs) and NSCLC cells were implanted into the lungs of 4-6 week old recipient mice. The influence of extracellular matrix components was evaluated by using either a matrigel/collagen mixture or alginate capsules. Tumor load was determined via overall survival (OS) and flow cytometry (FC). When mice had to be sacrificed, organs were collected and analyzed by FC and patho-histological examination. In a subsequent experiment, mice were sacrificed and tumors analyzed at defined time points and similar analyses performed.

OS and metastatic pattern were similar in both cell lines when injected in mono-culture. OS of Calu-1 injected mice was not influenced when cells were co-injected with HDF but prolonged when co-injected with CAFs or NFs. In H1437 bearing mice, OS was significantly reduced when cells were co-injected with HDF or NF (Log-rank [Mantel-cox] test; p< 0.002 & 0.005), whereas CAFs had no influence on OS. Co-injection of HDF and Calu-1 enhanced the number of tumor cells in bronchoalveolar lavage fluid, whereas CAFs had the opposite effect (1way ANOVA for all following statistics, p< 0.0001). Consistently, the number of circulating Calu-1 cells was highest in co-culture with HDF and significantly lower in mice bearing Calu-1 and CAF or NF (p<0.0021). Co-injection of fibroblasts enhanced the H1437 tumor load in all compartments. Of note, the effect was not statistically significant. The use of alginate capsules was favorable compared to matrigel/collagen, improving fibroblast engraftment in all investigated organs. Furthermore, alginate capsules enhanced the number of circulating H1437 cells when co-injected with HDF as compared to matrigel/collagen (p<0.0001).

Our results demonstrate the major impact of fibroblasts on tumor cell behavior in a preclinical setting. With the successful co-cultivation of human fibroblast and NSCLC cells in vivo, it will be possible to study tumor-stroma interactions in a clinically relevant mouse model. Once validated by a compound screening approach, this model will help to reduce drug failure rates and contribute to a more efficient development of urgently needed novel anti-cancer treatments.

#631

The mouse tumor biology database (MTB): An integrated data resource for mouse and patient derived xenograft (PDX) models of human cancer.

Dale A. Begley, Debbie M. Krupke, Steven B. Neuhauser, Joel E. Richardson, John P. Sundberg, Janan T. Eppig, Carol J. Bult. _The Jackson Laboratory, Bar Harbor, ME_.

The laboratory mouse is the premier model organism for understanding the genetic basis of human cancer and is a powerful platform for investigating novel targets for therapeutic intervention. Research using genetically engineered mouse models has led to key insights into the genetics of cancer susceptibility, the function of tumor suppressors and oncogenes, and therapy responses in pre-clinical and co-clinical studies. Patient Derived Xenografts (PDX) models are another model system for in vivo cancer studies. PDX models are created by implanting patient tumors into immunodeficient or humanized mouse hosts. PDX models are a powerful translational research platform for pre-clinical and co-clinical studies. The number of mouse models and the volume and heterogeneity of data related to the characterization of these models has increased dramatically in recent years, making integrated searches of these data and identifying relevant models a significant barrier to their effective use. The Mouse Tumor Biology database (MTB) (http://tumor.informatics.jax.org) provides on-line query tools to facilitate cohesive searches and visualization of these varied data, thus enabling the identification of novel mouse models of human cancer and potential therapeutic treatments.

The Mouse Tumor Biology database is an expertly curated resource for information and data about genetically modified mouse strains and PDX models of human cancer. Enforcement of standard gene and strain nomenclature and use of controlled vocabularies within MTB enables complete and accurate searching of the published literature for relevant mouse models. MTB contains data from spontaneous or endogenously induced tumors from genetically defined mice including tumor classification, incidence and latency, tumor associated QTLs, pathology reports, images and genetic changes in the tumor (somatic) and background strain (germline) genomes. The PDX resource enables searches based on tumor type, cancer diagnosis, and genomic properties of the engrafted tumors. Information in MTB is obtained from curation of peer-reviewed scientific publications and from direct data submissions from individual investigators and large-scale programs. MTB contains over 71,000 Tumor Frequencies, and over 2,080 Pathology Reports with over 5,800 images from over 3,600 references. MTB also provides access to detailed clinical, pathological, expression and genomics data from over 450 PDX models. Information in MTB is integrated with cancer models data from other bioinformatics resources including PathBase, the Gene Expression Omnibus (GEO), and ArrayExpress. MTB is supported by NCI grant CA089713.

#632

Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma.

WT Lin,1 L Xia,1 Dan Dan Wen,1 CM Tsang,1 KW Lo,2 ML Lung,1 George Sai-Wah Tsao1. 1 _Univ. of Hong Kong, Pokfulam, Hong Kong;_ 2 _Chinese Univ. of Hong Kong, Shatin, Hong Kong_.

Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection.

Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF ); University of Hong Kong (CRCG and SRT cancer)

#633

Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer.

Benjamin G. Cuiffo,1 Olivier Deas,2 Ingrid Rankin,1 Gregory D. Lyng,1 Stephano Cairo,2 Katie Pedrick,1 Alexandra Kury,1 Enora Le Ven,2 Jean-Gabriel Judde,2 Stephen T. Sonis1. 1 _Biomodels, Watertown, MA;_ 2 _XenTech, Paris, France_.

Background: Patient derived xenografts (PDX) are regarded as the gold-standard for translational preclinical cancer research due to their preservation of tumor heterogeneity and propagation in vivo. Similarly, grafting human tumor cells of a particular tissue origin into the corresponding orthotopic context enhances translational accuracy; with recapitulation of tumor::microenvironment biology, as well as more accurate tumor progression kinetics and metastasis, a s well as for systemic delivery of therapies as compared to traditional subcutaneous xenografts. Bioluminescent luciferase-reporters into traditional cancer cell lines has allowed for in-life imaging of tumor growth kinetics in real-time, within obfuscated internal organs, improving experimental precision. We have reported on the development and validation of bioluminescent pancreatic cancer and breast cancer PDX in clinically recapitulative orthotopic models. Here, we characterize these bioluminescent PDX by gene-expression profiling; comparing orthotopic and ectopically propagated bioluminescent PDX to their non-transduced parental PDX counterparts.

Methods: Pancreatic (PANx-005-Luc) or metastatic breast (HBCx-14-Luc) PDXs stably transduced with lentiviral luciferase were dissociated and implanted orthotopically into pancreas or mammary fat pad respectively or subcutaneously into the flank of NOD scid gamma (NSG) mice. Likewise, the parental reporter-free PANx-005 or HBCx-14 PDX were implanted subcutaneously into NSG mice. For luciferase-expressing PDX implanted orthotopically, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). For the PANx-005-Luc, orthotopic tumors were harvested at a mean tumor radiance (TR) of 2.53 x 106 + 1.63 x 106 ph/s, while ectopic luciferase-transduced and parental tumors were harvested at a mean tumor volume of 530 + 337mm3. For the HBCx-14-Luc, orthotopic tumors were harvested individually when TR reached 6.5 x 109 ph/s while ectopic luciferase-transduced or parental tumors were harvested when tumor volumes reached 1500 + 362.5mm3. Tumor samples were fixed in 10% neutral buffered formalin and embedded in paraffin-blocks for histological analysis. RNA was extracted from all tumors and full-coverage human genome gene-expression profiling was performed. Major differences observed in microarray data was validated via quantitative biochemical assays.

Results: Stable expression of lentiviral luciferase did not result in significant gene-expression changes to PDX lines, while site of implantation altered some gene-expression characteristics of the PDX.

Conclusions: The introduction of an integrating stable bioluminescent reporter does not significantly alter the gene-expression characteristics of PANx-005 or HBCx-14 PDX. Site of implantation plays a role in gene-expression of PDX tumor models.

#634

Impact of tumor inoculation site on the development of cancer cachexia.

Takashi Ando, Takeshi Ishikawa, Tastuzo Matsuyama, Tomoyo Yasuda, Toshifumi Doi, Tetsuya Okayama, Naoyuki Sakamoto, Satoshi Kokura, Yuji Naito, Yoshito Itoh. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Background and Aim

The incidence of cancer cachexia varies greatly among tumor type. Although certain tumor types are more commonly associated with cachexia, even for the same tumor type, the degree to which patients exhibit cachexia varies. This is due to variations in tumor phenotype and host genotype that contributes to the development of cancer cachexia. The influence of the site of tumor growth on the development of cancer cachexia remains unclear. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model.

Methods

The same number of colon26 cells (5×10^6 cells/mouse) was inoculated subcutaneously or intraperitoneally in 8-week-old male BALB/c mice. Body weight, food intake, and tumor size were monitored. On day 14 after the tumor inoculation, mice were anesthetized and blood collected, and the weight of peritesticular adipose tissue, gastrocnemius muscle and heart were measured. 23 plasma cytokines were determined by using the Bio-Plex array system and plasma Myostatin and Activin A levels were measured by ELISA. Expression of MuRF-1 and Antrogin-1, which are required for muscle atrophy, in gastrocnemius muscle or heart was assessed by RT-PCR.

Results

The body weight, adipose tissue and gastrocnemius muscle decreased in tumor-bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. The weight loss of the body, adipose tissue and gastrocnemius was more severe in the intraperitoneal group than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor-bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. As for plasma cytokine levels, several cytokine levels including IL-6, TNF-α, and activing A of tumor-bearing mice were significantly higher than those of control mice. Eotaxin and G-CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group.

Conclusion

Based on these data, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment.

#635

Establishment of a large panel of patient-derived tumor xenograft models of prostate, bladder and kidney cancers.

Hervé Lang,1 Claire Béraud,2 Audrey Bethry,2 Sabrina Danilin,3 Véronique Lindner,4 Catherine Coquard,3 Sylvie Rothhut,3 Thierry Massfelder3. 1 _Department of Urology, Strasbourg University Hospital, Strasbourg, France;_ 2 _Urolead, Strasbourg, France;_ 3 _INSERM U1113 Team 3, University of Strasbourg, Strasbourg, France;_ 4 _Department of Pathology, Strasbourg University Hospital, Strasbourg, France_.

Prostate, bladder and kidney cancers represent 1 900 000 new cases and 620 000 deaths per year worldwide with an incidence increasing by 1-10% each year. Surgery is curative at localized stages; however, current therapies are inefficient at advanced stages. One of the major needs in new drugs development is the availability of clinically pertinent models faithfully reproducing the heterogeneity of patient tumors. Patient-derived tumor xenografts (PDX) are thus developed as essential tools for drug testing and identification of predictive biomarkers for a better clinical response, the first step for personalized medicine.

Prostate, bladder and kidney tumors were obtained from patients undergoing surgery, subcutaneously (and some orthopically) xenografted in nude mice and serially passaged up to passage (P) 12. Normal corresponding tissues were also harvested. Tissues were conserved at all passages for characterization and grafting. For all patients, informed consent and clinical history are available. For the 3 cancers, primary tumor and tumors grown in mice were characterized for growth behavior, histopathology, genetic stability (short tandem repeat fingerprinting), mRNA expression profiling and response to current therapies. For each cancer type, we also investigated more specific features: expression of the androgen receptor, PSA and pan-cytokeratin for prostate PDXs, expression and status of hotspot mutations including FGFR3, PIK3CA, HRAS, RXRa and p53 for bladder PDXs, and von Hippel-Lindau gene mutation status for kidney PDXs. Metastatic models were followed by infrared imagery (IR780 dye).

So far and since 9 years, we have collected 230 prostate tumors, 130 bladder tumors and 336 kidney tumors at all stages, and established 5, 25 and 31 models (> P3 in mice) respectively (8.8% success rate). Tumor take rate was positively correlated to advanced stage and high grade and for kidney cancer, to sarcomatoid component. Tumor growth evaluation reveals that PDXs were stable from mouse to mouse and throughout passages. Histopathologic and genetic characteristics were preserved between original tumors and case-matched PDXs. Molecular characteristics were also stable with less than 5% of genes differentially expressed between the primary tumors and the PDXs. In bladder PDXs, this analysis and mutation status of the selected genes allowed to define molecular subtypes. The comparison with patient therapeutic response, when available, showed the clinical predictivity of the models. Orthotopic models developed metastases at classical sites.

In conclusion, we developed here a unique platform of preclinical PDXs models for urologic cancers with stable biological characteristics and clinically predictive. This is an invaluable tool for the clinical design of efficient therapies, the identification of predictive biomarkers and translational research.

#636

An orthotopic xenograft animal model for functional analysis of podoplanin in oral squamous cell carcinoma.

Hsing-Ying Lee,1 Yao-Wen Chang,1 Ju-Chien Cheng,2 Ching-Ping Tseng1. 1 _Chang Gung University, Taoyuan, Taiwan;_ 2 _China Medical University, Taichung, Taiwan_.

Podoplanin (PDPN) is a marker for poor prognosis in human oral squamous cell carcinoma (OSCC). Subcutaneous or intravenous injection of OSCC cells has been used in a number of studies as the animal models to investigate the function of PDPN in OSCC cancer progression. However, these models usually overlook the potential interactions between cancer cells and the microenvironment of oral cavity. In this study, we aimed to establish an animal model for functional and mechanistic study of PDPN in OSCC by taking into account the influence of the microenvironment of oral cavity. Profiling of nine human OSCC cell lines revealed that PDPN was highly expressed in five of the cell lines, including OECM-1, OC2, TW2.6, HSC-3 and CAL27. Notably, OECM-1 cells were shown by immunofluorescence staining to compose of two different cell populations with highly expression (P+) or absence (P-) of PDPN. To preclude the potential artifacts associated with gene overexpression study, the P+ cells were isolated by fluorescence-activated cell sorting followed by establishing the sublines shLacZ or shPDPN that co-expressed luciferase and the short-hairpin RNA targeting on β-galactosidase or pdpn. In vitro analysis revealed no difference for the cell growth of shLacZ and shPDPN cells on day 5 (p = 0.79). Orthotopic xenograft animal model for functional analysis of PDPN in OSCC was established by inoculating shLacZ or shPDPN cells into the anterior neck region of athymic nude mice. Consistent with the in vitro assays, there is no difference in the growth of the tumors derived from shLacZ and shPDPN cells (p > 0.05). Notably, the survival of the mice bearing tumors derived from shPDPN cells was significantly prolonged when compared with the mice bearing shLacZ cells-derived tumors (p < 0.001). At day 50 after orthotopic inoculation of cancer cells, 10% (n = 10, 1/10) and 90% (n =10, 9 /10) of the mice bearing tumors derived from shLacZ and shPDPN cells was alive, respectively. These results were consistent with the notion that PDPN is a poor prognosis factor for the patients with OSCC. In contrast to the orthotopic xenograft model, subcutaneous injection of shLacZ and shPDPN cells into the athymic nude mice did not result in a significant increase in tumor growth and tumor size. These data further demonstrate that the microenvironment of the oral cavity has significant effect on promoting the survival and growth of OSCC cells. The orthotopic xenograft mouse model we established in this study represents a useful model for functional and mechanistic study of PDPN in the cancer progression of OSCC.

#637

Establishment of a realistic patient-derived xenograft (PDX) model for prostate cancer bone metastasis.

Yvonne Konkol,1 Maija Valta,2 Jenni Bernoulli,1 Pekka Taimen,3 Peter Boström,2 Pirkko Härkönen,3 Jussi Halleen,1 Johanna M. Tuomela3. 1 _Pharmatest Services ltd, Turku, Finland;_ 2 _Turku University Hospital, Turku, Finland;_ 3 _Univ. of Turku, Turku, Finland_.

Bone metastases are frequent and fatal outcome of advanced prostate cancer. Many of the currently used preclinical models lack typical characteristics of the heterogenic human disease. We improved existing methodology by using fresh patient-derived material in a xenograft model of bone metastasis.

Clinical prostate tumor specimens were collected from robotic-assisted laparoscopic radical prostatectomy operations in Turku University Hospital (Turku, Finland). Patient-derived tumor tissue of Gleason grade 8-10 tumors (n=5) were cut into small pieces, digested overnight, and then cell suspension was intratibially inoculated into the bone marrow cavity of nude mice (n= 8-10/patient). X-ray pictures were taken and blood samples were collected once a month. Mice were sacrificed 6 months after tumor cell inoculation, and hind limbs and lungs were collected for histological and immunohistochemical analysis. PSA was measured from serum using commercial kit.

X-rays demonstrated osteosclerotic bone lesions. Immunohistochemical analysis showed that 80% of tumors in bone expressed similar characteristics compared with original tumor (androgen receptor AR, prostate specific antigen PSA, proliferation marker Ki67). However, no changes were seen in serum PSA. Lung metastases were detected in 50% of tumor-bearing mice. Interestingly, the most of lung metastases were negative for AR and PSA, which indicates that the cell population that forms metastases may be undifferentiated clone of heterogenic tumor and therefore the most aggressive.

Our platform provides new tools for prostate cancer research.

#638

Clinical characteristics of breast cancer xenograft models.

Wendie D. den Brok,1 Stephen Chia,1 Cherie Bates,2 Steve Kalloger,2 Samuel Aparicio,2 Mar Mar,1 Karen Gelmon,1 Peter Eirew2. 1 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 2 _BC Cancer Research Centre, Vancouver, British Columbia, Canada_.

Purpose: We have expertise in breast cancer xenograft models and have previously published data on clonal dynamics. Our aim was to explore clinical characteristics of those patients (pts) whose breast cancer tumours engrafted versus those that did not.

Methods: Tissue from pts enrolled in a locally advanced/metastatic (MBC) study and a breast tumour tissue repository between Sept. 2008 and July 2014 underwent xenografting using NodScid/IL2rgKO (NSG) mice. Xenografts were passaged when tumour volume reached 1 cm3. Mice were sacrificed if no engraftment by 12 months (mos). Pt charts were reviewed to determine biomarker status (hormone receptor [HR], HER2), grade, LVI, time free from disease/progression, and pathologic complete response (pCR) for pts receiving neoadjuvant therapy.

Results: A total of 64 pts had known xenograft status: 32 engrafters, 32 non-engrafters. Biomarker status did not predict likelihood of engraftment (p=.0695) and is shown in Table 1 along with site/timing of biopsy for tissue engraftment within biomarker groups. When HER2+ cases are excluded from analysis, the HR-/HER2- phenotype yields a 72% probability of engraftment compared to 39% for the HR+/HER2- group (p=.0418). For engrafters, 22/32 (68%) of pts had or went on to have relapsed or de novo MBC. For non-engrafters, 7/32 (22%) had or went on to have relapsed disease (p=.0004). Grade and LVI did not predict engraftment (p=.1806 and p=.8657 respectively). No grade 1 tumours engrafted (n=3). There were 25 pts who received neoadjuvant chemotherapy: 14 engrafters, 11 non-engrafters. None of the engrafters achieved a pCR; 3 non-engrafters achieved a pCR. Median time to engraftment was 5 mos for pts with relapsed/advanced disease vs 9.8 mos for pts who did not relapse however, treatment was variable.

Conclusion: This preliminary study highlights potential differences in clinical characteristics of engrafters vs non-engrafters in breast cancer xenograft models and warrants further exploration. (Funded by CBCRA, BCCF)

TABLE 1. Biomarker status, tissue site/type in attempted xenografts.

---

|

Engrafter (N=32)

N, (%) | Non-engrafter (N=32)

N, (%)

HR+/HER2- | 14 (44) | 22 (69)

Tissue from: | |

Primary tumour | 4 | 19

Recurrence | 2 | 3

Advanced dz on therapy | 8 | 0

HR-/HER2- | 13 (40) | 5 (16)

Tissue from: | |

Primary tumour | 11 | 4

Recurrence | 2 | 1

Advanced dz on therapy | 0 | 0

HER2+/HR+ | 0 (0) | 2 (9)

Tissue from: | |

Primary tumour | 0 | 2

Recurrence | 0 | 0

Advanced dz on therapy | 0 | 0

HER2+/HR- | 5 (16) | 3 (9)

Tissue from: | |

Primary tumour | 4 | 2

Recurrence | 0 | 1

Advanced dz on therapy | 1 | 0

#639

Subtyping of pancreatic cancer patient-derived xenograft tumors and implications for anticancer agent testing.

Peter Bronsert,1 Tim Kees,2 Bruno Zeitouni,3 Anne-Lise Peille,3 Manuel Landesfeind,3 Heinz-Herbert Fiebig,3 Simon Kuesters,4 Vincent Vuaroqueaux3. 1 _Institute of Pathology, University Medical Center Freiburg, Freiburg, Germany;_ 2 _Novartis, Basel, Switzerland;_ 3 _Oncotest GmbH, Freiburg, Germany;_ 4 _Clinics for General and Visceral Surgery, University Medical Center Freiburg, Freiburg, Germany_.

Despite improvements in treatment, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, with a continuous increase in incidence emphasizing the need for further research and therapeutic development. In recent years, we have developed a collection of >40 patient-derived xenograft (PDX) models from PDAC. In order to determine their relevance for anticancer agent testing, we extensively characterized our models for histology features, whole exome mutations (Hiseq 2000), chromosome rearrangements, gene copy number variations (Affymetrix SNP6) and gene expression (Affymetrix U133 Plus2.0).

PDAC from 65 patients were implanted into immuno-compromised mice, resulting in the development of 42 PDX models (success rate 65%). The PDAC from which models were established included moderate and poorly differentiated tumors and were heterogeneous for stroma content. In patient tumors, we showed fibroblast activation but not stroma content predicted poor patient outcome (p<0.0001) and success of PDX establishment (p<0.01). Correlating with those of the parental tumors, the resulting PDAC_PDX also showed heterogeneity for stroma content and for murine fibroblast activation. As seen in patient tumors, the PDAC_PDX were characterized by frequent chromosomal instability, with 38% of the models presenting a moderate hypoploidy while the 62% remaining ones showed a hyperploidy. Two models showed hypermutation due to mismatch repair deficiency while the others had on average a lower mutation load when compared to other histotypes such as colon, lung or melanoma tumors. In total, we identified more than 9000 genes altered by homozygous deletions, high gene amplifications and/or mutations, most of the models showed alterations in KRAS (81%), TP53 (67%), CDKN2A (64%) and TGFBR2/SMAD4 (64%) genes. Next, PDAC_PDX profiles were merged with those of patient tumors to determine PDAC_PDX subtypes and were found to be of the classical (61%), quasi mesenchymal (QM) (34%) and exocrine like subtype (5%) according to the 62 gene expression signature established by Collisson et al. 2011. Preliminary biomarker analysis revealed certain associations between genomic alterations and transcriptome subtypes, such as more frequent alterations in the TGFB pathway in classical PDX (80%) compared to QM (47%). Further detailed biomarkers analyses also addressing questions of molecular determinants of stroma content, of fibroblast activation and of sensitivity toward anticancer agents will be presented.

Extensive characterization of our PDAC_PDX collection revealed similarities with patient tumors with regards of histology features including stroma content and fibroblast activation. At molecular level, the models showed similar genomic and transcriptomic patterns as those reported for patient PDAC, altogether, proving the value of this collection for drug development investigations.

#640

New models of breast and lung cancer bone metastases for preclinical efficacy testing.

Mari I. Suominen,1 Urs B. Hagemann,2 Yvonne Konkol,1 Jenni Bernoulli,1 Katja M. Fagerlund,1 Roger M. Bjerke,2 Jenny Karlsson,2 Jussi M. Halleen,1 Alan Cuthbertson2. 1 _Pharmatest Services Ltd., Turku, Finland;_ 2 _Bayer AS, Oslo, Norway_.

Introduction

Clinically, bone is a very common site of metastatic spread in many cancers. In breast, and in particular cases of advanced estrogen receptor positive (ER+) cancers, the propensity of bone involvement is 85%. Similarly in lung cancer, 30-40% of patients with advanced disease develop bone metastases, and as recent advances in lung cancer therapies improve survival, the number of patients living with bone metastases is expected to increase. At the same time there is a paucity of especially ER+ and osteoblastic animal models available for the nonclinical evaluation of new treatment strategies. We present herein the development of four mouse models of breast and lung cancer suitable for screening of new therapies.

Experimental procedures

Human breast cancer cell lines BT-474 and MFM-223 and non-small cell lung cancer (NSCLC) cell lines NCI-H226luc and NCI-H322 were used. BT-474 is ER+, i.e. luminal B subtype and MFM-223 is basal subtype with androgen receptor (AR) expression. H226 originates from squamous cell carcinoma and H322 from adenocarcinoma of the lung. The different cell lines were inoculated in the tibia of female nude or NOD.scid mice. Half of the BT-474 inoculated mice had a s.c. slow release 17-beta estradiol pellet implanted. The formation of bone lesions was monitored by X-ray imaging. For H226 transfected with luciferase, tumor growth was also followed by bioluminescence imaging (BLI). Finally, tumor growth and type of bone lesion, i.e. ostelytic or oestoblastic, was confirmed by histology.

Results

Development of bone lesions was successful in 100% and 90% of animals, with or without hormonal supplementation respectively, four weeks after inoculation of BT-474 cells. Bone lesions were detected earlier in mice with estradiol pellet and were of lytic type. In contrast, bone lesions in mice without hormonal supplementation were strongly osteoblastic. For MFM-223, bone lesions were observed 4-6 weeks after inoculation and the success rate was 60% in nude mice and 70% in NOD.scid mice. For both lung cancer cell lines, 100% of the mice developed bone lesions and were detectable already two weeks after inoculation. H226luc cells developed osteoblastic-mixed lesions and H322 cells induced lytic lesions. Very interestingly, H226luc cells also formed lung metastases in all animals, as evidenced by BLI. Some lung metastases were also found in H322 inoculated mice.

Conclusions

Two new osteoblastic models are added to the current scarce selection and altogether four new bone lesion models representing different subtypes of breast and lung cancer were successfully established. The different types of bone reaction in these models offer a platform for studying the underlying pathways resulting in response to treatment in osteoblastic vs. osteolytic tumor microenvironment.

#641

Use of conditional reprogramming to develop and characterize cell cultures from patient-derived xenograft (PDX) models of human lung and ovarian cancer.

Alexandra Borodovsky,1 Travis J. McQuiston,2 Brian Dougherty,1 Ambar Ahmed,1 David Whitston,1 Daniel Stetson,1 Gretchen K. Hubbard,2 Sharon S. Challberg,2 Brian A. Pollok,2 Celina M. D'Cruz1. 1 _AstraZeneca Pharmaceuticals, Boston, MA;_ 2 _Propagenix, Inc, Gaithersberg, MD_.

Patient-derived xenografts (PDX) are widely recognized as a more physiologically relevant preclinical model to standard cell line xenografts. PDX models faithfully recapitulate the original patient genetic profile, gene expression patterns and tissue histology. Despite their benefits, PDX models are limited by their inherent variability, lower throughput and lack of growth in vitro. The ability to generate cell lines from PDX models would enable high throughput chemosensitivity screens, ex vivo genetic manipulation and the development of novel orthotopic models. Development of stable PDX cell lines remains a challenge due to murine stromal outgrowth, lineage commitment and limited differentiation potential. Conditional reprogramming (CR) cell technology is a novel cell culture system facilitating the generation of stable cultures without genetic manipulation (Liu, Am J Pathol, 2012). The success of CR cell technology is dependent upon the combination of feeder cell-derived factors and Rho Kinase (ROCK) inhibitor. CR cells, therefore, represent a new class of progenitor-like cells, distinct from the phenotype of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. The purpose of this study was to identify the advantages, limitations and potential applications of CR technology for derivation of PDX explant cell lines. Early passage human lung and ovarian PDX tumors were cultured in CR conditions to create stable explant cell lines. Cell lines were established from 5/8 (63%) PDX tumors and were expanded over 6 months in culture with varying morphologies and growth kinetics. Due to normal outgrowth of murine stromal cells, early CR cultures contained mixed populations and required murine depletion to enrich for human cells. Key oncogenic mutations in a model of ovarian papillary serous adenocarcinoma were preserved in the enriched tumor cell population. While purified CR PDX cell lines were amenable to high throughput chemosensitivity screens, in vitro chemosensitivity did not consistently predict response in in vivo murine models. The CR PDX cell lines were additionally assessed for genetic manipulation and ability to form tumors in vivo. Collectively, these results demonstrate the applications of CR technology for the generation of stable explant cell lines from PDX models for preclinical studies.

#642

Hypoxia-regulated gene expression explains differences between cell line-derived xenografts and patient-derived xenografts.

Joydeep Bhadury, Berglind O. Einarsdottir, Agnieszka Podraza, Roger Olofsson Bagge, Ulrika Stierner, Lars Ny, Marcela Dávila López, Jonas A. Nilsson. _University of Gothenburg, Gothenburg, Sweden_.

Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but resulted in reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors.

#643

Establishment and mutation analysis of a novel malignant peritoneal mesothelioma cell line, TU-MM-1.

Seiya Sato,1 Nao Oumi,2 Hiroaki Itamochi,1 Tetsuro Oishi,2 Tasuku Harada,2 Toru Sugiyama1. 1 _Iwate Medical University School of Medicine, Morioka, Japan;_ 2 _Tottori University School of Medicine, Yonago, Japan_.

Malignant peritoneal mesothelioma (MPM) is a rare aggressive neoplasm of the peritoneum, and its molecular biological characteristics and mechanisms of development are poorly understood. We, therefore, established a new cell line of human MPM, TU-MM-1, to elucidate its biological properties and to develop novel therapeutic strategies. The cells showed polygonal morphology, grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 41 to 44. A low rate of proliferation was observed and the doubling time was 67.9 h. Genomic DNA sequencing revealed that TU-MM-1 cells harbored missense mutations in APC, LATS2, BRCA1/2, and TP53, and mutation of a splice donor site in BAP1 and loss of CDKN2A gene. We observed the absence of BAP1 and p16INK4a proteins, underexpression of LATS2 protein, and overexpression of p53 protein in TU-MM-1 cells in western blot analysis. Heterotransplantation to nude mice produced tumors that had the characteristics of the original tumor. This cell line may be useful for studying biological properties and contribute to novel treatment strategies.

#644

Utilizing a novel luciferase labeling technique to establish and validate preclinical models of pancreatic cancer.

Jenni Bernoulli,1 Matthias Bozza,2 Katja M. Fagerlund,1 Johanna Tuomela,3 Mari I. Suominen,1 Suzanne Dilly,4 George Morris,4 Jussi M. Halleen,1 Richard Harbottle2. 1 _Pharmatest Services Ltd., Turku, Finland;_ 2 _German Cancer Research Center DKFZ, Heidelberg, Germany;_ 3 _University of Turku, Turku, Finland;_ 4 _ValiRx, London, United Kingdom_.

Bioluminescent-labeling imaging (BLI) allows sensitive non-invasive sequential imaging of tumor development and early metastasis. Current methods for the genetic modification of cells typically use integrating genotoxic viruses that can disrupt the molecular behavior of cancer cell lines due to their random nature of integration. A primary aim of the study was to utilize a non-integrating DNA vector that comprises an S/MAR (Scaffold/Matrix Attachment Region) element to stably genetically modify pancreatic cancer cells to persistently express the reporter gene luciferase without altering the molecular behavior of the cell or altering its sensitivity to therapeutic drug treatments. Once a novel isogenic cell line is generated the cells can subsequently be used in xenograft studies. A second aim was to validate these established models with gemcitabine and test the efficacy of VAL401, formulation of risperidone in rumenic acid. Human BxPC3, Capan-1, MiaPaCa-2 and Panc-1 pancreatic cancer cells were stably transfected with a pSMARt-UBC-Luc DNA vector and cultured for 4 weeks under selection. Colonies that formed after this period were isolated and expanded in normal medium and evaluated for luciferase expression and the molecular integrity of the DNA vector. Efficacy of gemcitabine was tested in these new luciferase expressing cell lines and VAL401 was tested in Capan-1-luc cells. For in vivo studies, BxPC3-luc cells were inoculated orthotopically into the pancreas of athymic nude mice and stratified into groups: control, gemcitabine, VAL401 (1mg/kg, p.o. daily) and VAL401 (2mg/kg, p.o. daily). In vitro validation results indicated that the luciferase transfected cells maintained their original properties with stable expression. Gemcitabine inhibited cell proliferation in all established cell lines. VAL401 inhibited cell proliferation of Capan-1-luc cells at 50 μM concentration. BxPC3-luc cells inoculated orthotopically into the pancreas were followed for 5 weeks with BLI by IVIS, and the results demonstrated high-quality follow-up of tumor growth. BxPC3-luc cells induced growth of pancreatic tumors with high take rate in all groups. Gemcitabine and both studied doses of VAL401 decreased tumor volume, and the same trend was seen in tumor weight and the BLI during the study. In conclusion, both gemcitabine and VAL401 decreased tumor volume and the same trend was observed using BLI. Our results demonstrated that S/MAR DNA vectors are able to produce genetically modified cells without the limitations of random genomic integration, whilst providing extra-chromosomal mitotic stability and high levels of sustained transgene expression. When utilized in orthotopic xenograft studies, these luciferase expressing cells formed a reliable and essentially non-invasive imaging platform that substantially improves the efficacy of testing anticancer drug candidates.

#645

The effect of rhBMP-2 on in vitro osteosarcoma tumorigenesis and on pulmonary growth and development.

David S. Geller,1 So Hak Chung,2 Wendong Zhang,3 Sajida Piperdi,3 Carrie Freeman,1 Bang Hoang,1 Rui Yang,1 Michael Roth,1 Jonathan Gill,1 Richard Gorlick1. 1 _Montefiore Medical Center, Bronx, NY;_ 2 _Kosin University College of Medicine, Busan, Republic of Korea;_ 3 _Albert Einstein College of Medicine, Bronx, NY_.

Introduction: Complete surgical resection is a critical component of care for patients with osteosarcoma (OS). Limb-salvage surgery is performed in over 90% of cases. Non-union is a well-recognized complication of allograft reconstructions, frequently performed following tumor extirpation. Bone Morphogenetic Protein-2 (BMP-2) has been shown to be osteoinductive and could theoretically improve allograft-host bone union. The purpose of this investigation is to evaluate the effect of exogenous recombinant human BMP-2 (rhBMP-2) on osteosarcoma.

Methods: Functional assays were performed using five standard osteosarcoma cell lines and three patient derived xenograft cell lines. Random migration motility was measured via wound healing scratch assay. Migration (haptotaxis) was quantitatively measured using Boyden Chamber assay. Invasion was assessed using the Biocoat Matrigel Invasion Kit. Cell proliferation rate was measured by using MTT reagent. Metastatic disease was assessed using a xenograft murine model.

Results: Findings suggest that exogenous rhBMP-2 does not meaningfully affect osteosarcoma migration, invasion or proliferation across all tested lines. Average corrected lung weight in the single-dose rhBMP-2 experiment was 1.56 g in the experimental group versus 1.67 g in the control group (p=0.69). Average corrected lung weight in the triple-dose rhBMP-2 experiment was 1.23 g in the experimental group versus 1.06 g in the control group (p=0.07).

Conclusions and Future Directions: Results support the contention that exposure of osteosarcoma to exogenous rhBMP-2 does not increase either in vitro tumorigenesis or pulmonary disease growth and development within this experimental model. While results are compelling, additional characterization and confirmation of these findings are necessary safety prerequisites prior to proposing clinical application.

#646

Feasibility of assessing circulating tumor cells in patient-derived xenograft tumor models.

Somdutta Roy,1 Kevin Martinez,1 Arturo Ramirez,2 Daniel Campton,2 Joshua Nordberg,2 Eric Kaldjian,2 Scott J. Dylla,1 Holger Karsunky1. 1 _Stemcentrx, South San Francisco, CA;_ 2 _RareCyte Inc., 312 Dexter Ave N, Seattle, WA_.

Background. Patient-derived xenograft (PDX) tumor models more accurately reflect human tumor biology than traditional cancer cell lines or their derived xenografts. Pre-clinical studies in cohorts of PDX lines emulate clinical trials, with each line representing a unique "patient". Stemcentrx has developed more than 600 PDX lines from various cancer types to use in development of novel therapies directed against the cancer stem cells, which can initiate tumors and drive recurrent disease. In order to better understand the biology of metastasis - the primary cause of cancer mortality - a newly developed RareCyte method for detection of human circulating tumor cells (CTCs) in the blood of tumor bearing mice was independently validated.

Methods. For platform validation studies, cells from 5 distinct PDX tumor models were counted and spiked into human blood at approximately 10, 50, 200 or 1000 per 7.5 mL. Blood was processed using AccuCyte cell separation and collection (RareCyte) and the nucleated cells spread onto 8 microscope slides. Slides were stained with anti-human cytokeratin, EpCAM and CD45 antibodies and DAPI (nuclear stain) using an automated instrument. A single slide from each spike-in tube was imaged by the CyteFinder digital fluorescence scanning microscope (RareCyte). Cells with morphology and phenotype (cytokeratin+/EpCAM+, CD45-) consistent with human cancer cells were tallied after review of images and compared to expected counts. For mouse PDX feasibility studies, 0.5-1 mL of blood from PDX tumor-bearing mice (~150-2000 mm3) was collected by cardiac puncture. After red cell lysis and washing, nucleated blood cells were spread onto one slide. Cells were stained, imaged and identified as above substituting anti-mouse CD45.

Results. CTCs were clearly identified by human epithelial cell marker staining in several different PDX lines. Moreover, there was a high correlation between the expected and experimental counts in the validation studies (R = 0.99) that was independent of spike-in number. Regression analysis gave a line of fit with slope 0.95, suggesting 95% recovery efficiency. In the PDX studies, CTCs were identified in 9 of 9 models. The number of CTCs was dependent on PDX line and tumor volume and ranged from 1 to 282 [per 800uL]. Clusters of 4 to 100+ CTCs were seen in blood samples from PDX tumor bearing mice. There was no correlation between number of CTCs and the duration of tumor in mice. However, there was a correlation between total number of CTCs and CTC clusters with the frequency and size of lung metastasis in mice.

Conclusions. Spike-in studies demonstrated linear, high-recovery identification of CTCs from PDX tumor models, using a small volume mouse blood protocol. Captured CTC appear to be present in a high proportion of models across several indications. These results demonstrate the feasibility of evaluating CTCs as a parameter of drug response in pre-clinical studies.

#647

In situ detection of human and mouse species-specific molecules in patient derived xenograft mouse models.

Na Li,1 Xin K. Ye,2 Ming-Xiao He,1 Zhifu Zhang,1 Hongzhe Sun,1 Xin Wang,1 Yuling Luo,1 Xiao-Jun Ma,1 Zhenyu Gu,2 Emily Park1. 1 _Advanced Cell Diagnostics Inc., Hayward, CA;_ 2 _GenenDesign Inc., Shanghai, China_.

Patient derived xenograft (PDX) mouse models are widely used in targeted cancer therapy study and cancer drug development. With human cancer tissues embedded into mouse microenvironment, both human and mouse derived molecules contribute to the proliferation and invasion of the implanted tumor cells. In order to explore the working mechanisms of targeted therapy and evaluate its anti-cancer effects, it is important to uncover the expression and spatial relation of targeted molecules in situ. Although profiling methods such as microarray and RNAseq can address the origin of relevant gene transcripts, they lose tissue spatial and cell morphology information by using tissue lysates. Immunohistochemistry (IHC) can preserve tissue and cell morphology information, however good antibodies are not readily available for every target molecule. In addition, IHC cannot detect long non-coding RNA targets. It is therefore crucial to find a broadly applicable method to separately detect human and mouse derived genes in PDX tissue samples.

Species-specific probes targeting on seven cancer related genes, EGFR, ERBB2, FGFR1, FGFR2, FGFR4, PECAM1 and TGFB1, were designed for RNAscope in situ hybridization assays. The assays were performed in colorectal cancer (CRC) PDX sections and liver cancer PDX TMAs. The RNA expression results were categorized into 5 grades according to manufacturer's scoring guidelines.

Probe species-specificity of human probes and mouse probes was tested in human colon cancers and mouse colons. Both tissues passed quality control by hybridizions with probe-Hs-PPIB/probe-Mm-Ppib and probe-dapB. All human species-specific probes produced negative results in mouse colons, whereas mouse species-specific probes generated signals for Egfr, Erbb2, Fgfr2, Pecam1 and Tgfb1 (score 2 or more than 2) in mouse colons. No mouse species-specific probe signals were observed in human colon cancers. In CRC PDX sections, human species-specific probes detected signals of ERBB2 and FGFR4 (score 3) in colorectal tumor cells whereas mouse species-specific probes detected signals for Fgfr1, Pecam1 and Tgfb1 (score 2 or more than 2) in stroma areas. In liver cancer PDX TMAs, human EGFR, ERBB2, FGFR1, FGFR2, FGFR4 and TGFB1 (score 2 or more than 2) were identified by species-specific probes in liver cancer cells; mouse Egfr, Fgfr1, Pecam1 and Tgfb1 signals (score 2 or more than 2) were observed in the stroma regions. RNAscope duplex assays identified both human FGFR1 and mouse Fgfr1 probe signals in the same sections with different colors.

RNAscope technology using species-specific probes can detect in situ human and mouse genes, respectively, which provides a powerful method to uncover the location of targeted cancer therapy related genes, their expression levels and the spatial correlation of positive cells with surrounding cells. This method will facilitate targeted therapy studies and cancer drug development in PDX mouse models.

#648

Integrated genomic characterization of endometrial cancer tumor grafts: a step toward genomic-guided treatment.

Chieh-Hsiang Yang,1 Elke A. Jarboe,2 Jason Gertz,3 Katherine E. Varley,3 C. Matthew Peterson,1 Margit M. Janát-Amsbury1. 1 _Department of OB/GYN, University of Utah, Salt Lake City, UT;_ 2 _Department of Pathology, University of Utah, Salt Lake City, UT;_ 3 _Huntsman Cancer Institute, University of Utah, Salt Lake City, UT_.

Endometrial cancer (EC) is the fourth most frequently diagnosed gynecological cancer among American Women. Steady increased numbers of newly diagnosed cases and deaths necessitates an added attention. Despite survival rates between 60-80% for localized, early stage disease, only marginal advances have been made in the treatment of distant (16%) and recurrent (15% of early stage) EC over the past two decades. New treatment strategies, guided by more predictive preclinical disease models incorporating molecular characteristics are crucial. Recent findings from The Cancer Genome Atlas (TCGA) Research Network suggested that the current histo-pathologically-guided classification of EC may not suffice to direct treatment recommendations, and introduced the potential adequacy of genomic profiling for reclassifying EC. However, these findings are hampered by the lack of appropriate in vivo models, which adequately facilitate obtaining the necessary preclinical evidence before translating findings to genome-guided clinical trials. Objectives: To establish patient derived orthotopic endometrial tumor grafts recapitulating histology, metastasis, and molecular characteristics of the original tumor specimen, and integrating TCGA genomic characterization to facilitate the development of genomic-guided treatment. Methods: Patient derived EC tissues were orthotopically transplanted to murine uteri and propagated over multiple generations. Generated tumor grafts were characterized for metastases, histopathology, and molecular profiles to ensure grafts resemblance of the original tumor samples. Tumor grafts were then further categorized into the four reported TCGA clusters on the basis of mutation frequency, somatic copy number alterations, microsatellite instability, and POLE mutation. Results: Twenty EC tumor grafts were successfully generated. Histological features, including tumor grade and hormone receptor status, as well as genomic profiles were maintained for up to 7 generations. Xenografts were capable to form metastasis closely parallel to clinical relevant sites. Results from a survival study demonstrated that the established tumor grafts reflected clinical stage-, and grade-based disease progression, and recapitulate TCGA cluster-based survival findings. Conclusions: This study reports the first successful generation of orthotopic EC patient-derived tumor grafts, which retain crucial histo-pathological characteristics, the capacity to form distant metastasis following known clinical patterns, as well as recapitulating molecular features of the original human tumor specimen. Additionally, our model can indeed be used as a tool to investigate the need for reclassifying EC into four clusters rather than two simplified disease subtypes and feasibility in directing genomic-guided treatment based on TCGA recommendations.

#649

Biobank collaboration enables cancer modeling with high quality biospecimens and data.

Lindsay S. Shopland,1 Ivette Emery,2 Todd Hoffert,1 Rise Aalberg,2 Petra Helbig,1 Susan E. LaPierre,2 Vicky Sanders,1 Kevin D. Mills,3 Tara Hill,4 Aysha Sheikh,4 Michael Jones,2 Anne Breggia,2 Jens Rueter1. 1 _Eastern Maine Medical Center, Brewer, ME;_ 2 _Maine Medical Center Research Institute, Scarborough, ME;_ 3 _The Jackson Laboratory, Bar Harbor, ME;_ 4 _Maine Cancer Foundation, Falmouth, ME_.

According to the National Cancer Institute, the lack of standardized, high-quality biospecimens is one of the most significant roadblocks to progress in cancer research. This shortage especially affects the generation of murine xenograft models of human cancers. Maine, with the third largest age-adjusted cancer incidence rate in the nation, has abundant cancer specimens potentially available for research. To meet the need for quality biospecimens, the biobanks at Eastern Maine Medical Center and Maine Medical Center have established a collaboration to jointly procure cancer specimens from Maine patients. Through this collaboration, the cooperating biobanks provide researchers with tissue, blood, bone marrow and other body fluid specimens from a broad spectrum of solid and hematologic cancers. The specimens from hematologic cancer patients have > 90% viability and demonstrate anticipated biomarker composition by flow cytometry. The comparatively high frequency of engraftment of solid tumors in immunodeficient mice is also indicative of the quality of the samples procured by both repositories. Currently, the biobanks are providing fresh ovarian tumors to develop new xenograft models for poorly engrafting tumors in collaboration with the Jackson Laboratory's Patient Derived Xenograft Program. Both biobanks are integrated with clinical and pathology practices and, therefore, the specimens are annotated with in-depth, de-identified patient datasets. With support from the Maine Cancer Foundation, the biobanks have created the Maine Cancer Biospecimen Portal (MBCP), a website that provides one-stop access to annotated cancer biospecimens and to consultation services for translational research study design. The collaboration of the EMMC and MMC biobanks provides a resource of high quality biospecimens for advancement of both in vivo and in vitro translational research such as mouse models of human cancers.

#650

An isogenic experimental model identifies β-catenin as a molecular target in ovarian cancer metastasis.

Sally KY To, Alice ST Wong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Ovarian cancer metastasis is closely associated with unfavorable outcomes, yet the underlying mechanisms remain obscure. Here we establish an isogenic model that could mimic the spontaneous metastasis of human ovarian cancer. Given that tumor cells are heterogeneous in nature, an isogenic pair of highly metastatic (HM) and non-metastatic (NM) cell lines with opposite metastatic capabilities was derived using in vitro and in vivo selection. Incorporation of the luciferase gene into the cell pair allowed non-invasive monitoring of the metastatic events by bioluminescent imaging in vivo. Orthotopic implantation of HM into the ovarian bursa of NOD/SCID mice resulted in metastases within the peritoneum with ascites formation, thus representing the major dissemination pattern of human ovarian cancer cells. However, NM failed to form detectable metastases, although it was tumorigenic at the ovarian bursa. In comparison with NM, HM displayed higher spheroid-forming ability and had higher expression of stemness marker genes, which are characteristics of cancer stem-like population. By proteomic profiling and pathway analysis, HM is found to be enriched in the oncogenic β-catenin signaling, a pathway elevated in ovarian cancer metastases. Tumor initiation and metastasis of HM was dramatically impaired when β-catenin was specifically knocked down. We further demonstrated that β-catenin could down-regulate the expression of Dicer, a major component of the microRNA machinery. These results together suggest that this model could help to delineate the molecular mechanisms for ovarian cancer metastasis, and provide clinically relevant insights to target metastasizing ovarian cancer cells.

#651

Male mouse model of canine and human inflammatory breast cancer (IBC).

Sara Caceres,1 Laura Peña,1 Maria J. Illera,1 Gema Silvan,1 Paloma J. de andres,1 Hui Gao,2 Wendy A. Woodward,2 James M. Reuben,2 Juan C. Illera1. 1 _Universidad Complutense de Madrid, Madrid, Spain;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with poor survival in women. Canine IBC has been proposed as a good surrogate model for the study of the human disease. Male breast cancer is a rare disease, accounting for less than 1% of all malignancies in men. Although rarely, IBC has been also found in men. The aim was to evaluate the capacity of male SCID mice to develop IBC tumors after the injection of canine and human IBC cell lines (IPC-366 and SUM149, respectively). Eighty SCID mice: 40 male and 40 females, 6-8 weeks old were used. Suspensions of 106 IPC-366 and SUM149 cells were implanted subcutaneously into the fourth inguinal mammary gland in 20 male and 20 female mice (of each cell line), respectively. Mice were inspected twice a week for the development of tumors. Tumors were monitored by palpation and measured by calipers. Mice were sacrificed when tumors reached 1.5 cm3 and tumors and organs were removed for immunohistochemical (IHC) characterization. A portion of tumor was used for hormonal analysis (Progesterone, Estradiol, Estrone sulphate, Testosterone, Androstenedione). Results revealed that IPC-366 reproduced tumors in 90% (male) vs 100% (female) after 2 weeks p.i. SUM149 reproduced tumors in 40% (male) vs 80% (female) at 4 weeks p.i. Both cell lines produced metastasis in lungs. Higher metastatic rates found in females than in males (IPC-366: 90% vs 20%; SUM149: 80% vs 50%). EIA hormonal analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors (p< 0.05). IHC analysis revealed that male and female tumors shared immunophenotipic characteristics. All xenografts were highly proliferative (high Ki-67 indexes), triple negative (ER-, PR-, HER-2-), vimentin positive and contained less than 10% of cells positive to AE1-AE3 and CK-14. In conclusion, IPC-366 and SUM149 cell lines can reproduce tumors in male mice capable of metastasize. Male and female xenograft models shared immunophenotypic characteristics. Male mice model would be useful for future IBC research using a different endocrine environment that could help to better understand the androgens function in canine and human IBC. 

### Mechanisms of Tumorigenesis in Animal Models of Cancer 1

#652

Establishment of a novel mouse model of intrahepatic cholangiocarcinoma by liver-specific Kras activation and Pten deletion.

Yumi Terakado, Tsuneo Ikenoue, Kiyoshi Yamaguchi, Yoichi Furukawa. _the institute of medical science the University of Tokyo, Tokyo, Japan_.

The mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K) pathway are intracellular signaling pathways that are involved in carcinogenesis. Various studies suggest that these pathways are important for the development of liver tumors. To investigate the effect of coactivation of MAPK and PI3K pathways in liver tumorigenesis, we generated three types of mouse models by crossbreeding conditional KrasG12D knockin mice and/or conditional Pten knockout mice with Alb-Cre mice (Alb-Cre KrasG12D/+ Pten flox/flox mice, Alb-Cre KrasG12D/+ Pten flox/+ mice, and Alb-Cre KrasG12D/+ mice). Among the three models, mice carrying liver-specific oncogenic Kras knockin and homozygous Pten deletion (Alb-Cre KrasG12D/+ Pten flox/flox) frequently demonstrated weight loss, abdominal distension, and jaundice with median survival of eight weeks. Macroscopic analysis detected diffuse and firm tumorous lesions mainly in the hepatic hilum. Histological examination of the Alb-Cre KrasG12D/+ Pten flox/flox mice disclosed bile duct hyperplasia at four weeks of age, precancerous lesions of intrahepatic cholangiocarcinoma (ICC) at five weeks of age, and advanced ICC lesions that resemble human ICC at seven weeks of age. On the other hand, the Alb-Cre KrasG12D/+ Pten flox/+ mice, the second model, developed ICCs as well as hepatocellular carcinomas (HCCs), and their average survival was eight months. The third mice model, the Alb-Cre KrasG12D/+ mice, developed HCCs but not ICCs, and survived more than one year.

Immunohistochemical analysis of the ICCs in the Alb-Cre KrasG12D/+ Pten flox/flox mice corroborated the activation of the Mapk and the Pi3k pathways in the tumor cells. These data suggest that activity of Pi3k pathway might dictate the fate determination of hepatotumorigenesis into biliary or hepatocyte cell lineage. In addition, oncogenic Kras expression and homozygous Pten deletion cooperate to induce exclusively ICCs. Since a limited number of mouse ICC models have been reported, the Alb-Cre KrasG12D/+ Pten flox/flox mouse model should be a useful tool for the analysis of molecular mechanism(s) underlying ICC.

#653

Identification of metastasis promoting genes using the PiggyBac insertional mutagenesis system.

Qiu N. Tinghu,1 Laura Vera Ramirez,1 Roland Rad,2 Jeffrey E. Green1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Welcome Trust Sanger Institute, Hinton-Cambridge, United Kingdom_.

The Myc oncogene is overexpressed in approximately 30% of human breast cancers. Mice carrying the mammary tumor virus LTR/c-myc (MMTV-c-Myc) develop mammary tumors, but display a relatively low rate of metastases to the lung. However, not all cells that express the transgene develop neoplasms indicating that Myc-induced tumorigenesis requires additional cooperating events for full transformation. To identify genes that cooperate with Myc-induced tumorigenesis leading to the acceleration of tumor progression or an increase in metastases in MMTV-Myc transgenic mice, we have employed the PiggyBac (PB) transposon system.

The PiggyBac system used in our studies was (developed by Rad et al, Science, 2010) is composed of two elements, the ATP-1 PiggyBac transposon and transposase. ATP-1 transposons are mobile DNA elements that can randomly integrate throughout the genome in the presence of transposase resulting in the activation or loss of gene function. The insertion sites can be identified through sequencing. It has been used successfully for genomic modification of cells in vivo leading to cancer gene discovery. In order to better adapt the PB system for studing mammary gland biology, we have designed a system to express a hyperactive transposase and luciferase specifically in the mammary gland using the MMTV LTR (MMTV-hyTPase-luc). The efficiency of MMTV-hyTPase-luc expression was first examined in vitro by transfection with an ATP-1 containing plasmid using the HC11 mouse mammary epithelial cell line. Selection for integration of ATP1 in the cells using puromycin selection and crystal violet staining, RT-QPCR and luciferase assays confirmed that MMTV-hyTPase-luc led to efficient integration of ATP-1 into the HC11 cells. HC11 cell proliferation further increased in the presence of dexamethasone, a hormone that stimulates MMTV promotor function. The MMTV-HyPB-Luc construct was subsequently used to generate transgenic mice and multiple founder lines are being analyzed for mammary specific transposase expression. The best expressing line will be crossed with MMTV-cMyc mice and mice carrying multiple copies of the ATP1 transposon. Transposon integration sites will be determined from tumors or metastases in mice with accelerated tumor development and/or increased metastases compared to controls. Further molecular characterization and validation of candidate genes will identify novel genes that cooperate with Myc to augment tumorigenesis and metastases.

#654

Impact of epithelial cell-specific deletion of mPGES-1 on colon carcinogenesis.

Masako Nakanishi, Daniel W. Rosenberg. _University of Connecticut Health Center, Farmington, CT_.

Activation of the COX-2/mPGES-1/PGE2 signaling axis is a hallmark of many cancers, including colorectal cancer (CRC), prompting the implementation of cancer prevention strategies targeting COX-2 activity. We have shown that targeting the downstream terminal PGE2 synthase, mPGES-1, specifically reduces inducible PGE2 formation without disrupting synthesis of other essential prostanoids. Additionally, global deletion of mPGES-1 (gKO) confers dramatic protection against intestinal cancer development in several mouse models, including the AOM+DSS inflammation-associated colon cancer model. Understanding the role of PGE2 under inflammatory conditions, however, is complicated by the diverse cellular sources and context-dependence of inducible PGE2. To clarify the relative contribution of inducible PGE2 synthesis within distinct cell compartments in colon, we have recently created an mPGES-1 conditional knockout mouse (cKO), where exon 3 of the mPGES-1 gene (mPtges) is floxed for tissue-specific Cre-mediated deletion. To test the role of epithelial-derived PGE2 synthesis on inflammation-associated cancer, we crossed the cKO mice with carbonic anhydrase 1 (Car1)-controlled Cre mice (cKO:Car1), thereby inactivating mPGES-1 function specifically within colonic epithelial cells. Based on our recent observation that global inactivation mPGES-1 sensitizes the colonic mucosa to direct environmental injury, we first examined the sensitivity of the cKO:Car1 mice to DSS-induced acute colitis. Two days after a five-day treatment with 0, 0.5 or 1% DSS, clinical signs of colitis, including the extent of colonic ulceration were determined. Both cKO:Car1 and cKO:WT control mice showed a comparable extent of colonic ulceration, covering ~60% of the epithelial surface. We next examined the impact of epithelial-specific deletion of mPGES-1 on DSS-induced colon carcinogenesis. KO:Car1 and cKO:WT control mice were treated with a single injection of AOM (10 mg/kg bw) followed by five days of 0, 0.5 or 1% DSS, and colons were examined five weeks after the end of DSS treatment. Surprisingly, epithelial deletion of mPGES-1 in the cKO:Car1 mice failed to provide against cancer development in comparison to the cKO:WT mice. This is the first study to show that genetic inactivation of inducible PGE2 formation within a specific cell lineage (e.g. epithelial cells) does not affect both the extent of acute colonic inflammation nor subsequent inflammation-associated carcinogenesis.

#655

Determination of the pro-oncogenic role of PIM1/PIM2 kinases in male reproductive system pre-neoplastic lesions by using conditional transgenic murine models.

Antonio Lucena-Cacace,1 Manuel P Jiménez-García,1 Irene Ferrer,1 Blanca Felipe Abrio,1 Eva M Verdugo-Sivianes,1 Daniel Otero Albiol,1 Marco Perez,1 Sandra Muñoz-Galván,1 José Manuel García Heredía,1 Javier Peinado-Serrano,1 Juan José Marín,1 Maja Narlik-Grassow,2 Carmen Blanco-Aparicio,2 Amancio Carnero1. 1 _INSTITUTO DE BIOMEDICINA DE SEVILLA (IBIS), Sevilla, Spain;_ 2 _CENTRO NACIONAL DE INVESTIGACIONES ONCOLÓGICAS (CNIO), Madrid, Spain_.

Background. PIM proteins belong to a family of ser/thr kinases composed of 3 members, PIM1, 2 and 3, with overlapping functions mainly regulated by pathways controlled by JAK/STAT transcription factors. The PIM family proteins have been implicated in the regulation of apoptosis, metabolism, cell cycle, homing and migration, and they have been found to be overexpressed in many sort of tumors, therefore these proteins are interesting targets for anticancer drug therapy. Although the PIM kinases have been identified as oncogenes in transgenic models, they have weak transforming abilities on their own. However, they have shown to enhance the capacity of other genes or chemical carcinogens to induce tumors. Germinal tumors are among the solid tumors in which both PIM1/2 proteins have been found to be overexpressed.

Objetives. To study the causal role of PIM1/PIM2 proteins and the molecular machinery involved in the generation of male reproductive system tumors.

Methods. We generated two CRE conditional transgenic mice with confined expression of human PIM1 or PIM2 in hormone-dependent tissues, due to MMTV gene promoter which expression is induced by steroid hormones. We fully characterized the hyperproliferative response to these genetic alterations in both TgPIM1 and TgPIM2 models.

Results and conclusions. Conditional transgenic MMTV-Cre/PIM1 and MMTV-Cre/PIM2 models are useful in vivo murine models for studying initiation and progression processes of steroid-dependent tumor development, such as testicle, seminal vesicle and prostate neoplastic alterations. PIM1/2 models showed a reduced survival, a higher percentage of tumors at clinical endpoint and a higher incidence of total tumors percentage, noticing more statistically significant data of tumor incidence, possibly because PIM1/2 expression induces alterations in hormone-dependent tissues that increase proliferation rate and tissue atrophy of certain cyto-architectonic structures embedded in those tissues, leading to physiological mechanisms that shortens life. PIM1/2 conditional models generated hyperplastic changes in testicle and prostate tissues, and epithelial adenomas areas in seminal vesicles, but no carcinomas of male reproductive system, which indicates that PIM1/2 genes are involved in tumor initiation level, but require synergy effects with other oncogenes or carcinogens, which would explain low percentage of male reproductive system tumors registered and no carcinoma formation. Additionally, enhanced inflammation surrounding target tissues were found in PIM1/2 models, which it could be a tumor initiation mechanism led by PIM deregulation of cellular JAK/STAT signaling. Together our data indicate that PIM1/2 over-expression induces a pre-neoplastic phenotype in testicle, seminal vesicles and prostate, pointing a possible role in oncogenic initiation in these tissues.

#656

Histological and 3D morphological evaluation of mammary cancers and primary cells from genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency.

Sahar J. Alothman, Weisheng Wang, Bhaskar V. Kallakury, Priscilla Furth. _Georgetown University, Washington, DC_.

Background: Previously established genetically engineered mouse (GEM) models with spontaneous mammary cancer development have both Brca1 alleles disrupted withTrp53 haploinsufficiency (Brca1f11/f11/MMTV-Cre/Trp53+/-). Cell culture plates with nanoimprinted scaffolds (SCIVAX Life Sciences, Inc) purportedly increase concordance between in vitro/in vivo results but reports are limited to human cancer cell lines. Here we characterized hyperplasia and cancer development in Brca1f11/WT11/MMTV-Cre/Trp53+/- mice and established conditions for 3D culture of primary mammary epithelial cells (MEC) using the imprinted plates. Methods: Time-course of mammary hyperplasia and cancer development was defined in female Brca1f11/WT11/MMTV-Cre/Trp53+/- (n=23) and Brca1f11/f11/MMTV-Cre/Trp53+/- (n=13) mice euthanized at age 6 and 12 months (m) or when largest tumor reached 1 cm3. Mammary tissue was taken at necropsy for histology (age 6/12m) and primary MEC culture (age 6m) using EpiCult-B (StemCell Technologies) on nanoimprinted scaffold plates. Primary normal (wild-type, n=2) and precancerous MEC (Brca1f11/f11/MMTV-Cre/Trp53+/-, n=2; Brca1f11/WT11/MMTV-Cre/Trp53+/-; n= 2) were cultured. Optimal conditions for spheroid growth were established by comparison of spheroid formation from thoracic vs. inguinal mammary glands with different numbers of plated cells and percentage FBS after 7 days culture. Endpoints included sphere number, size and presence/absence of concurrent monolayer growth. Spheres were harvested for histological examination. Hematoxylin and eosin (H&E) stained sections were read for histology and immunohistochemistry (IHC) for Pan-Cytokeratin (Pan-CK) and Ki67 performed for in vivo/in vitro comparisons. Results: Forty-two percent of Brca1f11/WT11/MMTV-Cre/Trp53+/- mice developed mammary cancer by age 12m compared to 100% of Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Cancers appeared by age 6m only in Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Mammary cancers in both models were carcinomas of either adeno, anaplastic or sarcomatoid types. All were Pan-CK positive and proliferation rates were similar between the two genetic models. Spheres developed from all primary MEC attempted. Optimal conditions for sphere formation were plating of 10,000 cells obtained from inguinal mammary gland per well in 5% FBS EpiCult-B. Conclusions: Here we report development of spontaneous mammary cancers in Brca1 insufficiency mice that are more representative of disease development in women who carry only one allele with a BRCA1-mutation and established conditions for use of cell culture plates with nanoimprinted scaffolds for 3D culture of normal and preneoplastic primary murine mammary epithelial cells. Supported by NCI, NIH 1RO1CA112176 (PAF).

#657

N-Myc and STAT Interactor knock out in the mammary epithelium prompts hyper-proliferation.

Hawley C. Pruitt, Brandon J. Metge, Sarah K. Bailey, Lalita A. Shevde, Rajeev S. Samant. _University of Alabama at Birmingham, Birmingham, AL_.

N-Myc and STAT Interactor (NMI) is an evolutionarily conserved protein that is widely expressed in fetal and adult tissues. Early studies implicated its role in regulating the activities of transcription factors (such as MYC, STATs, BRCA1, TIP60 etc.) critical to tumor progression and metastasis. However, the functional relevance of these regulatory activities of NMI remains unknown. Recent findings from our lab have revealed that NMI protein expression is decreased by 70% in primary tumor specimens from patients with metastatic breast cancer. Most recently, we have found that lack of NMI expression in breast cancer cells confers resistance to chemotherapy by blocking autophagy-induced cell death. Thus the status of NMI expression in breast cancer patients may be an important clinical consideration. Additionally, our functional studies have demonstrated that loss of NMI expression allows manifestation of TGFβ and Wnt driven EMT that results in increased invasion and metastatic dissemination. Overall, we have noticed a profound impact of NMI on multiple developmental signaling pathways that are essential for mammary development as well as tumor progression.

To further elucidate the biological role of NMI, we have created a genetically engineered mammary specific Nmi knock out model. We observed that in normal murine mammary tissue Nmi is expressed in the mammary epithelium during all stages of mammary development. However, it's expression is strikingly induced at the onset of pregnancy, implicating an important role of NMI in mammary ductal development and/or lactation. Remarkably, the Nmi knock out mice exhibit distinctly increased number of alveolar structures (30% more than in control mice) during lactation. Moreover, these Nmi-/- mammary glands also show 20% more proliferating mammary epithelial cells (MECs) compared to respective littermate controls. In addition, 3D-alveologenesis of Nmi knock out MECs is highly responsive to induction by growth factors such as TGFα and FGFs when compared to MECs from littermate controls. Hyper-proliferative phenotype has been implicated as one of the key factors for breast cancer initiation as well as progression. Overall, our work describes a direct evidence for the role of NMI as a key regulator of mammary epithelial cell proliferation.

#658

Elucidation of cellular origin of mouse intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion.

Tsuneo Ikenoue, Yumi Terakado, Kiyoshi Yamaguchi, Yoichi Furukawa. _The Institute of Medical Science, The University of Tokyo, Tokyo, Japan_.

Regarding the carcinogenesis of intrahepatic cholangiocarcinoma (ICC), growing interest has been focused on its cellular origin, namely cholangiocytes, progenitor cells, or hepatocytes. We have recently established a novel mouse model of ICC by introducing liver-specific Kras activation and Pten deletion using Alb-Cre mice, in which Cre/loxP-mediated gene recombination was achieved in hepatoblasts and mature hepatocytes. To elucidate the cellular origin of ICC in this mouse model, we utilized hepatocyte or cholangiocyte-lineage specific gene engineering using a tamoxifen (TMX) inducible-Cre/loxP system. The mice carrying Kras activation and Pten deletion by Alb-CreERT2 mice with the TMX treatment at eight weeks of age developed hepatocellular carcinoma and hepatocyte dysplasia within three months. Fluorescence microscopy analyses disclosed that the Cre/loxP-mediated gene recombination was specifically observed in hepatocytes in the mice. In contrast, the mice carrying Kras activation and Pten deletion by Alb-CreERT2 mice at ten days of age induced ICC alone within two months. The gene recombination occurred in cholangiocytes as well as hepatocytes. Expectedly, ICCs were developed in the mice carrying cholangiocyte-specific Kras activation and Pten deletion by K19CreERT mice treated with TMX at 8 weeks after birth. TMX-induced, Cre/loxP-mediated recombination was achieved in epithelia of various organs including intrahepatic bile ducts in these mice. These results suggest that the cellular origin of ICC in mice carrying liver-specific Kras activation and Pten deletion was cholangiocytes but not hepatocytes. This mouse model should be useful for better understanding of human ICC of cholangiocyte-origin and the development of new therapeutic strategies for the disease.

#659

Novel refinements for the autochthonous mouse model of cancer.

Peter K. Cabeceiras,1 Nikhil S. Joshi,2 Tyler Jacks2. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Massachusetts Institute of Technology, Cambridge, MA_.

There are many levels of genetic regulation at play in autochthonous mouse models of cancer. For instance, mouse models can be programed to knockout tumor suppressor genes and enable transcription of oncogenes in order to cause normal cells to transform into cancer cells that can eventually form tumors and metastasize. Concurrently to this, a gene of interest (GOI) under the control of a tetracycline-response element (TRE) promoter can be studied in a doxycycline-dependent fashion. The GOI under the control of the TRE promoter will be transcribed only in the presence of a transcriptional activator fusion protein, rtTA, and a chemical compound, doxycycline. Unfortunately, it is extremely difficult to limit the doxycycline-dependent expression of the GOI only to cancer cells, so normal cells will invariably express the GOI. This inaccuracy can limit the translation of results from mouse model to human patient. In the interest of engineering models that closely recapitulate the disease in question, we propose to limit the expression of rtTA and the GOI to cancer cells by linking rtTA expression to cell cycle regulation. The cell cycle is leveraged because rapid division and proliferation are characteristics intrinsic to proliferative cancer cells, and some cell cycle proteins are abundantly expressed in cancer cells but not expressed in normal cells. By fusing rtTA to cell cycle proteins that are expressed in cycling cancer cells, we hope to improve the accuracy and capabilities of the autochthonous mouse model of cancer.

#660

Modelling conventional and serrated colorectal tumorigenesis by altering the Wnt and MAPK signaling pathways.

Vicki Whitehall,1 Winnie Fernando,1 Diane McKeone,1 Mark Bettington,2 Sally Pearson,1 Catherine Bond,1 Barbara Leggett3. 1 _QIMR Berghofer Medical Research Institute, Herston, Australia;_ 2 _Envoi Specialist Pathologists, Kelvin Grove, Australia;_ 3 _Royal Brisbane and Women's Hospital Department of Gastroenterology and Hepatology, Herston, Australia_.

Background. The majority of conventional colorectal adenomas are initiated by activation of the Wnt signal, whilst activation of the MAPK pathway is associated with progression to cancer. In contrast, sessile serrated adenomas are initiated by activation of the MAPK signaling pathway due to mutation of the BRAF oncogene, whilst the Wnt pathway is activated at the transition to dysplasia. We hypothesised that whilst both the Wnt and MAPK pathways play key roles in colorectal tumorigenesis, the order in which these signaling pathways are activated would determine the morphology of the lesion arising.

Methods. To test this hypothesis, we utilised a conditional BRAF mutant mouse model crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine. The MAPK pathway was activated two weeks after birth, whilst activation of the Wnt signal was achieved using APCMin mice or the intestinal carcinogen azoxymethane. Morphology and number of lesions was confirmed by histologically examination. Nuclear β-catenin immunostaining provided a surrogate for activation of the Wnt signal.

Results. Induction of the BRAF mutation resulted in the rapid and sustained development of widespread intestinal hyperplasia. Murine serrated adenomas (mSA) mimicking the morphology of human traditional serrated adenomas were observed by 5 months and by 10 months 8/18 (44.4%) mice had developed mSA, primarily in the duodenum. Activation of the Wnt pathway using azoxymethane dramatically accelerated the serrated phenotype in both the small intestine (2.9 mSA per mouse, P<0.01) and colon (3.1 mSA per mouse, P<0.05) by 3 months. The MAPK and Wnt pathways also synergized in APCMin mice, increasing the average number of conventional adenomas per mouse from 2.1 to 78.8 (P<0.001).

Conclusion. The rapid induction of hyperplasia and exclusive development of serrated lesions demonstrates that BRAF mutation is a key molecular event underlying initiation of serrated neoplasia. Importantly, we have demonstrated that activation of the MAPK pathway through BRAF mutation is sufficient for commitment to the serrated pathway and that whilst subsequent activation of the Wnt signal can accelerate tumorigenesis, it is not sufficient to override the existing serrated morphology.

#661

Establishment of transgenic mice with liver-specific BRAF V600E mutation.

Hiroki Tanaka,1 Masahiro Yamamoto,2 Kosuke Yamazaki,3 Keiko Shimizu,4 Katsuhiro Ogawa2. 1 _Dpt. of Gatrointestinal Immunology & Regenerative Medicine, Asahikawa Medical University, Asahikawa, Japan; _2 _Dpt. of Pathology, Asahikawa Medical University, Asahikawa, Japan;_ 3 _Dpt of Clinical Medicine, Surgery Area, Japanese Red Cross Hokkaido College of Nursing, Kitami, Japan;_ 4 _Dpt. of Forensic Medicine, Asahikawa Medical University, Asahikawa, Japan_.

Diethylnitrosamine (DEN) is often used to induce hepatocarcinogenesis in rodents to investigate the pathogenesis of hepatocellular carcinoma (HCC). BrafV637E mutation, corresponding to the human BRAFV600E mutation, is highly frequent in DEN-induced hepatic tumors depending on mouse strains.

To investigate the role of BrafV637E mutation in DEN-induced hepatocarcinogenesis, we established the transgenic mice (Alb-Cre/BRAFV600E), in which human BRAFV600E mutation is specifically expressed in the liver, and compared the properties of DEN-induced hepatic tumors and the livers of the transgenic mice. The hepatic tumors were induced by neonatal treatment with DEN, isolated at various time points, and stored as frozen or paraffin-embedded materials. For production of the transgenic mice, we obtained homozygous male BRAFV600E mice, in which a LoxP-flanked cassette containing the human BRAF exons 15-18 cDNA, mouse polyadenilation sequences and the neo gene was inserted into the mouse Braf intron 14 upstream of the human BRAF exon 15 cDNA that encodes the BRAFV600E mutation, and crossed them with female Alb-Cre mice by in vitro fertilization.

RT-PCR and cDNA sequencing revealed that the liver of Alb-Cre/BRAFV600E mice expressed mRNA derived both from normal mouse and mutated human BRAF exon 15. The transgenic mice showed 20% decrease in body weight compared to normal mice, but 5 fold-increase in the liver/body weight. Histological examination revealed that the liver was entirely consisted of basophilic hepatocytes resembling to those in DEN-induced foci of cellular alteration at 8 weeks of birth, and bile duct cells were further increased at 10-12 weeks. The Ki-67 labeling index of hepatocytes in the Alb-Cre/BRAFV600E mice was 3 times higher than normal mice. Immunoblot analysis revealed that the liver of Alb-Cre/BRAFV600E mice expressed the BRAFV600E protein and showed hyperphosphlylation of ERK1 and AktS473 like the DEN-induced hepatic tumors. Furthermore, immunohistochemical staining detected increased C5/C5a expression in the hepatocytes of Alb-Cre/BRAFV600E mice, and by immunoblot analysis C5a, a protease-cleaved form of C5, was specifically detected in Alb-Cre/BRAFV600E liver like DEN-induced tumors. These results indicate that the liver specific expression of mutationally-activated BRAF led to the hepatocytic phenotype mimicking the DEN-induced hepatic tumors.

#663

Factors affecting genome editing using CRISPR/Cas9 in mouse model.

Qi Zheng, Ling-Jie Kong, Huanyu Jin, Jinling Li, Ruby Yanru Chen-Tsai. _Applied StemCell, Inc., Milpitas, CA_.

CRISPR/Cas9 joined genome editing family and became a powerful tool for cancer researches. While CRISPR is effective for generating knock-out, knock-in or point mutation animal models, the protocols and success rates to generate CRISPR mouse model vary significantly among different laboratories. We examined a series of parameters for generating various types of mouse models using CRISPR/Cas9. The efficiency and toxicity of gRNA, Cas9 mRNA and donor concentration, insert size and injection methods were thoroughly analyzed. We also evaluated strategies generating conditional knock-out models using CRISPR. The optimized protocols for successful mouse model generation for knock-in, knock-out, point mutation and conditional knock-out using CRISPR will be proposed.

#664

Insulin receptor signaling contributes to mammary tumorigenesis in mice.

Yekaterina Poloz,1 Samar Mouaaz,1 Sonya Lam,1 Vuk Stambolic2. 1 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre; University of Toronto, Toronto, Ontario, Canada_.

A recent convergence of clinical and epidemiological evidence suggests that hyperinsulinemia associated with obesity and type 2 diabetes leads to higher incidence and poor outcome of breast cancer (BC). To probe the role of insulin signaling in BC, we created the MMTV-driven polyoma middle T BC model mice with an inducible mammary epithelium-specific deletion of the insulin receptor (INSR). Deletion of INSR in the mammary gland considerably reduced the mammary tumour burden in the INSR knockout mice as compared to the INSR wildtype controls, without affecting the tumour onset. The average weight of individual tumours was similar across the genotypes, suggesting that deletion of INSR affected the tumour initiation potential of the epithelial cells rather than tumour growth. Deletion of INSR also affected the metastasis of mammary tumours to the lung, with the INSR knockout mice presenting with half the number of metastases compared to the INSR wildtype controls. Our ongoing efforts are focused on mapping the signaling events downstream of the INSR that govern tumour initiation and metastasis in our mice, as well as modeling the impact of obesity on tumors in this model system, with the ultimate goal of identifying the role of INSR signaling in BC development and progression.

#665

Blocking IL-1-NF-kB axis inhibits Kras-induced pancreatic ductal adenocarcinoma.

Jianhua Ling, Jie Fu, Min Wu, Yu Cao, Paul Chiao. _UT MD Anderson Cancer Center, Houston, TX_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality in the USA with approximately 50 000 new cases and 40 000 deaths in 2015.The 5-year survival rate has remained at 5% for the past three decades and the median survival duration is less than 6 months. The very high rate of mutant Kras occurs in pancreatic cancer (>90%) and pancreatic intraepithelial neoplasia (panIN) (>50%). However, the key signaling pathway of downstream Kras remains unidentified.

We previously uncovered that the NF-κB signaling pathway was constitutively activated in most PDAC specimens and pancreatic cancer cell lines. More recently, our results showed that constitutive NF-κB activity in PDAC was induce by IL-1 autocrine mechanism. IL-1/p62 feedforward loops that initiated by the mutant Kras sustained NF-κB activity and were required for pancreatic cancer development. Therefore, we hypothesize that blocking IL-1 - NF-κB signaling pathway will inhibit Kras-induced pancreatic ductal adenocarcinoma.

To identify the mechanistic role of mutant Kras-induced IL-1 expression in PDAC, we generated genetically engineered mice of Pdx1-cre/Kras LSL-G12D/p53 M/+IL-1α-/-. Our findings revealed that these mutant mice had developed significantly less PDAC number and a significantly delayed PDAC at old ages. Furthermore, we assess the effect of pharmacological IL-1R1 antagonist (anakinra) in human pancreatic cancer cell lines and orthotopic pancreatic cancer mouse. We found that IL-1R1 antagonist significantly decreased NF-κB activity and inhibited cell proliferation, migration and invasion. More interestingly, the combinational treatment IL-1R1 antagonist plus gemcitabine significantly reduced the tumor burden in orthotopic pancreatic cancer mouse.

These results suggested that pharmacologically targeting IL-1α may provide a novel therapeutic approach for pancreatic cacner.

#666

Apc restoration promotes cellular differentiation and reestablishes crypt homeostasis in colorectal cancer.

Kevin P. O'Rourke,1 Lukas E. Dow,2 Scott W. Lowe1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Weill Cornell Medical College, New York, NY_.

Colorectal cancer (CRC) accounts for almost 10% of all cancer mortality in the United States. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. Using a novel mouse model in which Apc can be conditionally suppressed using a doxycycline-regulated shRNA, we previously showed that Apc suppression in adenomas and carcinomas drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. However, this study was unable to explore Apc action in metastasis, which is the most common cause of death in CRC patients, since models do not live long enough for metastatic dissemination to occur. To produce a system that would enable further disease progression, we generated a comprehensive panel of colon organoid cultures that reflect iterative combinations of the 3 most common genetic lesions in human CRC: Apc-mut, Apc-mut;KrasG12D, Apc-mut;p53-mut and Apc-mut;KrasG12D;p53-mut. These cultures can be transplanted orthotopically into the colon of recipient mice or seeded in the circulation to assess primary tumor behavior and metastatic dissemination. We show that Apc silencing in organoid culture results in the dysregulated proliferation of stem and progenitor cells but this is reversible upon Apc restoration or pharmacologic inhibition of the Wnt pathway. When engrafted into the colon of recipient mice, these cells create ~1-3 focal colon tumors that can be tracked using colonoscopy, ultrasound and bioluminescence imaging, and are largely indistinguishable from tumors that develop in transgenic models. Most importantly, triple mutant (Apc/Kras/p53) organoids form invasive adenocarcinoma and colonize the lung following tail vein injection, providing a setting to dissect the importance of Wnt pathway activity in various stages of disease progression. Ultimately, the ability to study cellular and molecular phenotypes in vitro, and assess tumor development in vivo, provides a flexible system to explore the genetic factors underlying CRC progression and metastasis, and overcomes many of the previous technical challenges of modelling advanced CRC.

#667

Tumor cell dormancy is mediated by an increase in RTK signaling in reversible pancreatic cancer models expressing oncogenic K-Ras and c-Myc.

Nirakar Rajbhandari. _University of Nebraska Medical Center, Omaha, NE_.

Recent genetic studies of pancreatic cancer expressing mutant Kras and c-Myc strongly favor targeted therapies against these oncogenic drivers for the successful treatment of advanced staged pancreatic cancer. The potential limitation of these treatments is that they likely leave residual cancer cells that can play a role in tumor recurrence. Our study is focused on understanding the molecular mechanism of the survival of pancreatic cancer cells in the absence of the tumor-driving oncogene in order to successfully eliminate cancer cells and prevent tumor relapse.

Using a reversible c-Myc-induced pancreatic cancer model, we have demonstrated that expression of c-Myc in pancreatic progenitors is sufficient to induce invasive pancreatic cancer, and its expression is required for the maintenance of tumors at primary and metastatic sites. However, despite the complete macroscopic remission of invasive pancreatic tumors at primary and metastatic sites upon downregulation of c-Myc, a few cancer cells survived and remained dormant for a protracted period of time (Lin et al., 2013). In this current study, we have used a reversible Kras-induced pancreatic tumor model and found that cancer cell dormancy is a common attribute in pancreatic cancer following oncogene ablation.

In an effort to identify common, cancer cell-intrinsic molecular pathways that mediate residual disease following the ablation of oncogenic drivers, we performed a genome-wide gene expression analysis (RNA-Seq) of in vivo-derived bulk tumor cells and dormant cancer cells that survived the ablation of c-Myc and oncogenic Kras. Comparison of the gene expression profile of bulk and residual cancer cells revealed that the residual cancer cells have increased receptor tyrosine kinase (RTK)/Akt signaling as a common mechanism for their survival in both c-Myc and Kras-induced tumors. We have experimentally validated the importance of this signaling pathway in cancer cell survival in the absence of oncogenic signaling in both tumor models. In comparison to control mice, we observed a significant slow tumor growth and delayed tumor relapse resulting in longer survival of the tumor bearing mice though a combination of this RTK/Akt pathway inhibition and ablation of Kras or c-Myc. In conclusion, our current study in reversible cancer models highlights the importance for the development of an adjuvant therapeutic strategy targeting this RTK/Akt pathway in addition to oncogenic drivers to effectively eradicate residual cancer cells and to prevent pancreatic cancer recurrence.

#668

Induction of mammary carcinoma in tetracycline-inducible cMET(T1010I ) transgenic mice.

Chuanwei Yang,1 Ana M. Gonzalez,1 Naoto T. Ueno,1 Shuying Liu,1 Jinsong Zhang,2 Gordon B. Mills,1 John Mendelsohn,1 Prahlad Ram1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Saint Louis University School of Medicine, St. Louis, MO_.

Gene amplification and mutations of cMET have been reported in a wide variety of tumors, including colorectal, lung, renal, liver, thyroid, and breast cancer. cMET(T1010I) mutation occurred in 5.2% out of 148 sibling pairs diagnosed with colorectal cancer in one study. This germline mutation occurs in about 1% of breast cancer patients, but its frequency increases to 2% in metastatic breast cancer. Furthermore, 16% of breast cancer cases with PI3KCA mutations have co-mutations of the cMET oncogene. To determine the functional consequences of this germline mutation and elucidate the molecular mechanisms of tumorigenesis induced by its oncogenic mutant protein, we developed a transgenic mouse model expressing human cMET(T1010I) under the control of a Tet-responsive promoter. MMTV promoter - directed expression of reverse tetracycline-controlled transactivator (rtTA) turns on the expression of the cMET(T1010I) transgene in murine mammary glands upon tetracycline administration. Thirty percent of these mice developed early onset mammary carcinoma within 5 months of transgene induction. This contrasts with previous reports of mammary tumor development in MMTV-cMET transgenic mice after more than a year. This strong phenotype is not the result of a random insertional effect of the transgene as no mammary tumors developed in the 29 transgene positive mice without mammary-specific expression of rtTA over a year in our study. Molecular characterization of these tumors is currently underway and the results may reveal mechanisms of tumorigenesis and novel therapeutic targets for patients harboring this mutation. In conclusion, we showed for the first time that the cMET(T1010I) mutation can trigger rapid breast tumorigenicity. By using organ-specific expression of rtTA to direct the expression of cMET(T1010I) in the organ of interest, our transgenic model may be used to understand the impact of cMET(T1010I) on many other cancer types such as colorectal, lung, renal, thyroid, and liver cancer.

#669

Compound heterozygous Sf3b1-K700E mutation and Atm deletion in B cells leads to CLL in mice.

Lili Wang,1 Rutendo Gambe,1 Jean Fan,2 Angela N. Brooks,3 Jing Sun,1 Donna Neuberg,1 Peter Kharchenko,2 Matthew Meyerson,1 Mark D. Fleming,2 Benjamin L. Ebert,2 Ruben Carrasco,1 Catherine J. Wu1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _University of California, Santa Cruz, MA_.

Unbiased next-generation sequencing using primary samples has identified SF3B1 as among the most frequently mutated genes in chronic lymphocytic leukemia (CLL). These mutations localize to a restricted gene region, with more than 50% at the K700E site. The presence of mutation is associated with poor clinical outcomes. These lines of evidence together emphasize the high priority for gaining an understanding of role of SF3B1 in CLL. However, the mechanistic basis for how mutated SF3B1 impacts CLL remains unknown. Prior transcriptomic studies using primary CLL samples have led to the appreciation of altered RNA splicing induced by this mutated gene, but studies of the functional impact of mutated SF3B1 in primary human samples have been complicated by its variable mutant allele frequency across samples as well as its common co-occurrence with other diverse gene mutations. We therefore generated a mouse line that conditionally expressed heterozygous Sf3b1-K700E mutation in B lineage cells.

We sought to characterize the effects of Sf3b1-K700E on RNA splicing and B cell function in this in vivo model. By RNA-sequencing of splenic B cells from wild-type and mutant mice (n=3, each), we detected, classified, and quantified 54 differentially spliced transcripts (p10%) using the tool JuncBASE. Consistent with prior findings in human CLL samples, the splice variants in our mouse model were highly enriched with altered selection of 3' splice sites (49 of 54 events, p<0.001), suggesting that Sf3b1-K700E mutation in the mouse lines cause altered RNA splicing in vivo and functions in a manner faithful to the human leukemia. B cell characterization of the mutant mice revealed that expression of Sf3b1-K700E caused subtle impairments in B cell development and function, and induced a state of cellular senescence based on gene expression and secretome characterization. Mice with heterozygous Sf3b1-K700E did not develop evidence of CLL-associated immunopathologic changes, even with prolonged aging to 24 months. Nevertheless, expression of Sf3b1-K700E with heterozygous deletion of Atm in B cells led to the overcoming of cellular senescence and resulted in the accumulation of clonal B220+CD5+ cells in peripheral blood, marrow, and spleen of elderly mice. We thus dissect the impact of mutated SF3B1 individually and in combination with Atm deletion on paving the path towards B cell leukemogenesis. This novel murine model faithfully recapitulates features of human CLL and has the potential to shed light on mechanisms of cooperation between Atm deletion and SF3B1 mutation in the pathogenesis of CLL.

#670

The Jackson Laboratory Repository: Resource for mouse models of human cancer.

Deborah Boswell, Steve Rockwood, Mike Sasner, The JAX Repository Team. _The Jackson Laboratory, Bar Harbor, ME_.

The mouse is a versatile model organism whose value has been augmented by recent biotechnological innovations in genome editing. While standard inbred and spontaneous mouse strains are still widely utilized, developments in genetic engineering technologies have significantly broadened the strategies available to researchers to generate specific disease models with preclinical applications. The Jackson Laboratory (JAX) has been serving the scientific community as a resource for mouse strains relevant to human disease for more than seven decades. The JAX Repository consists of over 8,000 mouse strains, many with applications for modeling human cancer. A wide selection of models are now available that have applications as hosts for cancer xenograft modeling. Strains have become increasingly specialized for specific purposes: optimized support of human and murine hematopoietic cell engraftment, reduced xenogeneic graft-versus-host disease response, engraftment of human hematopoietic stem cells without irradiation, and immunological tolerance to firefly luciferase and enhanced green fluorescent protein. Mouse strains that recapitulate aspects of specific diseases, such as Chordoma, are also available. In response to the promising potential of CRISPR/cas9 technology, a variety of cas9-expressing lines (constitutive and Cre-inducible expression) are included in the JAX collection for more convenient generation of disease models. Whereas a gene editing approach using viral delivery of cas9 is burdened by packaging size limits, these cas9-expressing mice only require a specific single guide RNA for generating mutations. cas9 expressing mice are available on several congenic strain backgrounds and involve constructs knocked into two different loci, Gt(ROSA)26Sor or Igs2 (also known as Hipp11). The location of the Igs2 locus on chromosome 11 (cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci) occurs within an intergenic region which reduces possible disruption of other endogenous genes.

All Repository mouse strains are cryopreserved and monitored by a robust quality control program to confirm mutation identity and genetic background. A new JAXMice website search interface facilitates queries by disease term as well as by keyword and strain, allele, and gene name or synonym (www.jax.org). Donating a strain to the Repository fulfills the requirements of the NIH's policy for the sharing of research reagents. Researchers wishing to have strains considered for inclusion in the Repository can find the submission form available at: www.jax.org/donate-a-mouse.

The Jackson Laboratory Repository is supported by the NIH, The Howard Hughes Medical Institute, and other private charitable foundations.

#671

Assessing the origins of anaplastic large cell lymphoma in murine models via forced lineage specificity of NPM-ALK expression in T cells.

Tim Malcolm,1 Camilla J. Fairbairn,1 Katherine Hughes,1 Lukas Kenner,2 Amos Burke,1 Suzanne Turner1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _Medical University of Vienna, Vienna, Austria_.

Background:

Anaplastic large cell lymphoma (ALCL) is a T cell lymphoma representing 10-15% of all childhood non Hodgkin lymphomas, whereby those positive for aberrant expression of Anaplastic Lymphoma kinase (ALK) account for 50-80% of cases. The oncogene Nucleophosmin 1 (NPM)-ALK is considered the main driver of pediatric ALCL identified in 83% of ALK+ cases. ALCL cells rarely express a T cell receptor (TCR), CD4 nor CD8 despite displaying an activated cytotoxic T cell phenotype (production of perforin and Granzyme B). Expression of NPM-ALK in mice from the T-cell specific CD4 promoter (which gives rise to expression throughout thymic development) gives rise to thymic lymphomas not mimicking human ALCL suggesting other events are required for peripheral T cell lymphoma development and/or expression of NPM-ALK at different stages of T cell progression: some theories have revolved around the possibility of an infectious aetiology, or subversion of the development of T cells within the thymus in the pathogenesis of ALCL.

Objective:

To assess the potential cell of origin of ALCL through lineage restriction of NPM-ALK to defined T cell lineages (CD4 or CD8) using TCR transgenic mice.

Experimental procedure:

Disease presentation following backcrossing of the CD4/NPM-ALK mouse to a clonal transgenic TCR OT1 (CD4/NPM-ALK/OTI) or OTII (CD4/NPM-ALK/OTII) genetic background in the presence and absence of RAG was monitored. The OTI and OTII transgenic mice express a transgenic TCR which is MHC class I restricted, with a CD8 or MHC class II with a CD4 developmental skew respectively. The TCR in these mice has been engineered to recognise ovalbumin peptides (ova).

Results:

CD4/NPM-ALK/OTI mice develop peripheral disease histopathologically mimicking human ALCL. Conversely, CD4/NPM-ALK/OTII mice develop cortical thymic lymphomas similar to the parental CD4/NPM-ALK strain and mostly of a CD4 single positive T cell phenotype (64%). Intriguingly, the peripheral T cell tumours that develop in the CD4/NPM-ALK/OTI mice do not express the transgenic OTI TCR (or endogenously rearranged TCR). Additionally, haemopoietic tumors only occur on a RAG competent genetic background or in the absence of ova stimulation even though TCR transgene expressing T cells are detected in the periphery of these mice (CD4/NPM-ALK/OTI + MHV-ova administration or CD4/NPM-ALK/OTI/RAG-/-).

Conclusions: These data represent the first murine model resembling human ALCL and suggest that restriction of NPM-ALK to the CD8 T cell lineage results in a disease more closely resembling human ALCL. Furthermore, our data suggest that in the periphery, signaling though the TCR may be pejorative towards peripheral lymphoma development.

#672

Differential effects of p53 mutations on cancer invasion and metastasis in a mouse model of colon cancer.

Ying Feng, Naoya Sakamoto, Maranne Green, Megan Greenn, Kathleen R. Cho, Eric R. Fearon. _University of Michigan, Ann Arbor, MI_.

Inactivation of the APC (adenomatous polyposis coli) tumor suppressor gene plays an important role in initiating most adenomas and colorectal cancers (CRCs). Somatic mutations in the TP53 tumor suppressor gene and KRAS oncogene are found in roughly 60% and 40% of CRCs, respectively, and contribute to tumor progression. In the vast majority of human CRCs, TP53 missense mutations lead to high levels of mutant p53 protein expression and the loss of the other wild type TP53 allele. To evaluate the role of TP53 missense mutations in CRC progression, we generated a genetically engineered mouse model of colorectal carcinoma, via combined targeting of Apc, Kras, and Trp53 alleles in mouse colon epithelium, focusing on comparing phenotypic effects of the murine equivalent of the human R273H mutation (i.e., murine R270H mutation) or a large deletion mutation of mouse Trp53. Inactivation of one allele Apc and activation of an oncogenic Kras allele in colon epithelium generated serrated and hyperplastic morphologic epithelium and adenomas. The addition of either the R270H missense mutation or the Trp53 null mutation to the Apc and Kras mutations led to markedly shortened survival of the mice, due to the development of multiple colon tumors in each mouse ranging from adenomas to late stage adenocarcinomas. Evidence of invasion into the smooth muscle and serosa area was found in both compound mice with missense or deletion mutations in Trp53, with the Trp53R270H mutant mice displaying an increased prevalence of deeply invasive tumors relative to the mice with deletion of Trp53. Furthermore, we found that the missense mutant Trp53R270H allele in combination with Apc and Kras mutations, but not the null-mutant Trp53 allele, was strongly linked to metastases to lymph nodes and lung. We have developed a useful mouse model of metastatic colon cancer that recapitulates the role of TP53 mutations in cooperating with APC and KRAS mutations in human CRC development and progression. In addition, our findings strongly suggest a powerful role for missense mutant p53 proteins compared to TP53 null mutations in promoting invasion and metastasis in CRC progression.

#673

The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma.

Koen Schipper,1 Julian de Ruiter,1 Sjors Kas,1 Eva Schut,1 Marco Koudijs,1 David Addams,2 Jos Jonkers1. 1 _NKI-AVL, Amsterdam, Netherlands;_ 2 _Sanger institute, Cambridge, United Kingdom_.

Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of this type of cancer is the loss of intercellular adhesion which is facilitated by inactivating mutations in E-cadherin. However the loss of E-cadherin alone is not enough to induce ILC. An in vivo Sleeping Beauty insertional mutagenesis screen was performed to identify genes that together with E-cadherin loss contribute to ILC development in mice. In around 85% of the tumors that were analyzed during this screen one of four hits were observed, namely MYPT1, MYPT2, ASPP2 and MYH9. Interestingly these four hits appear to be mutually exclusive indicating a shared mechanism of action.

To investigate how these genes might affect tumorigenesis we first looked at the location of the transposon insertions. This revealed that for MYPT1, MYPT2 and ASPP2 the transposons appeared to localize to a specific region in the gene indicating that these insertions could result in a truncation variant. In the case of MYH9 no clear localization of insertions was found which in combination with the recently published data indicates a loss of function. Northern blot analysis revealed the presence of truncation variants for MYPT1/2 and ASPP2 which for MYPT1 were confirmed to also result in truncated protein variants.

In order to identify the tumorigenic potential of truncated MYPT1 and ASPP2 we overexpressed them in spontaneously immortalized mammary epithelial cell lines HC11 and NMuMG. We are using these cell lines to analyse differences in cell proliferation, anchorage independent growth, sensitivity to apoptosis in vitro and tumor formation in vivo. To further analyse the hits in an E-cadherin negative setting we made use of primary mammary epithelial cells (MECs) isolated from genetically engineered mice with mammary gland specific E-cadherin loss. These MECs normally do not grow past several passages in vitro. However, these MECs when we overexpress truncated MYPT1 or ASPP2, or use shRNA mediated knockdown of MYH9 can be passaged and grown in vitro consistently.

Finally we are generating conditional mouse models with mammary gland specific E-cadherin loss in combination of expression of the truncated variants of MYPT1 or ASPP2, or loss of MYH9. We will to monitor whether these mice are prone to developing ILC.

#674

WHSC1L1 is a driving oncogene in ERα positive breast cancer.

Brittany Turner Ivey. _Medical University of South Carolina, Charleston, SC_.

The 8p11-p12 genomic region is amplified in 15% of breast cancers and is associated with poorer prognosis. This genomic region harbors several oncogenes, including three epigenetic chromatin modifiers: ASH2L, KAT6A, and WHSC1L1. WHSC1L1 is a histone methyltransferase expressed in two isoforms and we have previously demonstrated that this oncogene is involved in overexpression of ERα as well as its activation independent of estrogen signaling in amplicon-bearing SUM44 breast cancer cells. This cell line serves as a model for the subset of breast cancer patients who demonstrate resistance to hormonal therapies in the context of the 8p11 amplicon and WHSC1L1 overexpression. To validate the connection between overexpression of WHSC1L1 and ESR1, we specifically overexpressed the short, non-catalytic isoform of WHSC1L1 in amplicon-bearing cell lines without WHSC1L1 overexpression. Overexpression of WHSC1L1-short resulted in increased expression of ERα in CAMA1 and SUM52 cells, but not in the amplicon null MCF7 cells. To analyze further the oncogenic role of WHSC1L1 in breast cancer, we generated a transgenic mouse model with targeted overexpression of WHSC1L1 in the mammary gland of FVB/N mice. WHSC1L1 transgenic females had normal sized litters, however the pups were profoundly underdeveloped compared to pups born to wild-type females. Pups born to transgenic females displayed delayed hair growth and eye opening and were half the weight of healthy pups nursed by wild-type females. This suggested a lactation deficiency in WHSC1L1-transgenic females. To further investigate this phenotype, mammary glands were harvested for histologic analysis from transgenic and wild-type females at various stages of the reproductive cycle. Mammary glands from virgin WHSC1L1 transgenic females displayed increased branching and terminal bud formation compared to wild-type virgin females. Similarly, alveolar buds of mid-pregnant females were more numerous and densely packed than in wild-type mice. Consistent with a lactation defect, mammary glands from lactating transgenic females showed evidence of decreased alveolar development compared to wild-type females, resulting in mammary glands where half of the gland failed to functionally differentiate. Finally, post-lactation mammary glands from wild-type females underwent involution, whereas glands from transgenic females failed to involute and were hyperplastic. Significantly, we have observed mammary tumor formation in two transgenic female mice thus far. Ongoing studies include further characterization of this novel mouse model, investigation of the effects of WHSC1L1 overexpression on mammary gland development as pertaining to breast cancer tumorigenesis, and the oncogenic role of WHSC1L1 and its effects on ERα. These and other studies are essential for understanding WHSC1L1 and the 8p11 amplicon in ER+ breast cancer, and how this genomic alteration can be exploited to improve patient outcomes.

#675

**Ninjurin1, a target of p53, modulates p53-dependent tumor suppression** in vivo **.**

Hee Jung Yang, Seong Jun Cho, Jin Zhang, Wensheng Yan, Xinbin Chen. _UC Davis, Davis, CA_.

The nerve injury-induced protein 1 (Ninjurin1, Ninj1), a homophilic adhesion molecule, is involved in nerve regeneration, inflammation and vascular regression. Ninj1 is found to be overexpressed in human cancers including hepatocellular carcinoma and acute lymphoblastic leukemia. However, the role of Ninj1 in tumorigenesis has not been elucidated. Recently, we found that Ninj1 is transcriptionally regulated by p53, which in turn regulates p53 expression through mRNA translation. Thus, the mutual regulation between p53 and Ninj1 represents a novel loop in the p53 pathway. To examine the biological significance of the p53-Ninj1 loop, we generated and monitored 4 cohorts of mice: (i) WT, (ii) Ninj1-het, (iii) p53-null, (iv) Ninj1-het; p53-null. We found that Ninj1-het mice were highly prone to skin lesions. We also found that Ninj1 deficiency shortened the life-span but decreased the incidence of sarcoma in mice without p53. Additionally, we found that Ninj1-het; p53-null mice were prone to hydrocephalus and kidney abnormality. To examine the association of Ninj1 with mutant p53, we generated and characterized Ninj1+/-; p53 R270H/- and p53 R270H/- mice. We found that Ninj1 deficiency shortened the life-span and promoted the frequency and aggressiveness of tumors in p53 R270H/- mice. Together, we conclude that Ninj1 differently regulates the tumor susceptibility in a p53-dependent manner in vivo and that the Ninj1-p53 loop plays a pivotal role in tumor suppression and longevity.

#676

Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency enhances tumor burden via increasing HIF-2α expression.

Sarvesh Kumar, Sandra M. Jensen, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. _NIH, Gaithersburg, MD_.

The tissue inhibitor of metalloproteinase family of proteins (TIMPs 1-4) function as natural MMP inhibitors, and have been shown to play a role in maintenance and remodeling of the ECM as well as other cellular processes including proliferation, apoptosis and angiogenesis. A number of studies have shown that the down-regulation or silencing of TIMP2 accelerates tumor development, however, the mechanism is not well understood. High HIF-2α levels in non-small cell lung cancer (NSCLC) correlate with decreased overall survival, while inhibition of HIFs targeted genes VEGF or VEGFR2 are associated with improved clinical outcome. Similarly, TIMP2 mRNA levels were found to be low in NSCLC compared to the corresponding non-neoplastic surrounding lung (p<0.05). The current study was undertaken to examine the molecular mechanisms associated with tumor hypoxia and TIMP2 expression in TIMP2 deficient mice (T2D-/-) and wild type (WT) littermates. Mice were given (1x106)/50μl Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) via intratracheal installation. Tumor progression was monitored by IVIS imaging 2 weeks after administration and continued until week 4 when mice were sacrificed and lungs harvested for further analysis. The IVIS analysis shows higher tumor burden in T2D-/- compared to WT littermates, suggesting that TIMP2 deficiency augments tumor development in these mice (p<0.05). H&E analysis of lung tissue sections revealed a significant increase in the number of tumor nodules in lungs of T2D mice compared to controls (p = 0.003). In an effort to investigate the relationship between TIMP2 and HIF-2α expression; we determined the expression levels of HIF-2α in healthy non-tumor bearing T2D mice compared to WT littermates. Using semi-quantitative real-time PCR, we confirmed a strong basal up regulation of HIF-2α (but not HIF-1α) mRNA in T2D mice lungs compared to WT (p<0.002). In tumor bearing mice, this increase was even higher in the lungs of T2D mice compared to WT (p<0.007). Since VEGF is the direct downstream target of HIF-2α, we determined the expression of VEGF in these mice. As expected, we found a similar trend of VEGF expression in T2D mice. The basal level of VEGF expression was increased in the lungs of healthy T2D mice compared to WT (p<0.006) and even higher in tumors of T2D mice compared WT (p<0.002). Given that the HIF-2α−VEGF signaling pathway plays a major role in neovascularization and cancer progression, microvessel density was assessed in tumor tissue by calculating the mean number of CD31+ vessels. Accordingly, we found a higher number of CD31+ vessels in T2D mice compared to WT (p<0.0001). Collectively, these findings support our previous work on demonstrating TIMP2 as an inhibitor of tumor angiogenesis and also suggest that TIMP2 regulates hypoxic mediators within the tumor microenvironment, therefore offering TIMP2 as a novel bio-therapeutic molecule for lung cancer therapy.

#677

EML4-ALK fusion gene expressing transgenic mouse for lung cancer model.

Kyoung-Ho Pyo,1 Hye Ryun Kim,2 Sun Min Lim,3 Mi Ran Yun,1 Sung-Moo Kim,1 Hwan Kim,1 Han Na Kang,1 Ji Min Lee,1 Sang Gyun Kim,1 Byoung Chul Cho2. 1 _JE-UK laboratory of Molecular Cancer Therapeutics, Yonsei Cancer Research Institute, Seoul, Republic of Korea;_ 2 _Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Division of Medical Oncology, Department of Internal Medicine, CHA Bundang Hospital, Bundang, Seongnam, Republic of Korea_.

Background: EML4-ALK is a fusion-type protein typrosin kinase that is generated in lung cancer. We have established transgenic mouse line. The surfactant protein C gene (SPC) is only expressed in alveolar epithelial cells. We applied SPC promoter on encoded human-EML4-ALK gene. Normally, EML4-ALK gene is silenced when LoxP-PGK-LoxP gene is turned on. When Cre recombinase is present with the specific inducer, tamoxifen, EML4-ALK gene is expressed in alveolar epithelial cell, and converses to lung cancer.

Materials and Methods: For generation of EML4-ALK fusion transgenic mouse, a cDNA fragment encoding FLAG tagged EML4-ALK (variant 1) was ligated to end of the CMV-LoxP-PGK-LoxP. The expression cassette was injected into pronuclear-stage embryos of B6 mouse to generate Tg mouse. Transgenic mouse was prepared for interbreed with KI mouse (SPC-CreER2T-trTA-NeoR). The KI-Tg fusion mouse was confirmed by genotyping, then the mouse was injected with tamoxifen (cre recombinase inducer) intraperitoneally. The lung tumors were observed by MRI or CT. The lung pathology was analyzed by immunohistochemistry and hematoxylin & eosin staining. For confirmation of EML4-ALK protein expression, western blot was performed in different organs. EML4-ALK tumor-bearing mouse was treated with ALK tyrosine kinase inhibitor (TKI) for 2 weeks and tumor shrinkage was measured.

Results: EML4-ALK tumor was observed after one week. Tumor nodules were widely observed in all lung lobes. Pathologically, the histologic type was adenocarcinoma and had homology to patient tissue on H&E stain. We confirmed EML4-ALK expression in different organs including lung, heart, liver, stomach, kidney, colon and brain. EML4-ALK (120kDa) was strongly expressed in lung, but not

in other organs. Indeed, ALK and Flag expression were observed in tumor region by immunohistochemistry. The EML4-ALK tumor bearing mice were treated with ALK TKI for 2 weeks. The ALK TKI-treated mice showed dramatic shrinkage of tumors and showed gain in body weight.

Conclusions: EML4-ALK Tg mouse was successfully established. The benefits of the model compared to a previous conditioned Tg mouse model are (1) no need to treat Cre inducer daily, and (2) short duration (approximately 1 week) of tumorigenesis. We confirmed dramatic tumor shrinkage after treatment with ALK TKI. This model provides a strong preclinical model for testing ALK TKIs in lung cancer.

#678

Lunatic Fringe and p53 cooperatively prevent development of claudin-low breast cancer.

Wen-Cheng Chung,1 Shubing Zhang,2 Keli Xu1. 1 _University of Mississippi Medical Center, Jackson, MS;_ 2 _Central South University, Changsha, China_.

Claudin-low breast cancer (CLBC) is a poor prognosis disease that disproportionately affects younger patients and women with African ancestry, causing significant morbidity and mortality due to chemoresistance and lack of targeted therapy. CLBC shows stemness and mesenchymal features, and is thought to have originated from mammary stem cells. However, little is known on how these tumors initiate and progress. We previously reported that deletion of Lunatic Fringe (Lfng), a modulator of Notch signaling, in the moue mammary epithelial cells induced development of mammary tumors, among them two-thirds are basal-like breast cancer and one-third resemble human CLBC. Deletion of p53 in combination with other mutations (inactivation of Rb or overexpression of oncogenic c-Met) also resulted in mammary tumors with mesenchymal features. We hypothesize that Lfng and p53 may cooperate to suppress CLBC initiation and progression. To this end, we have generated mammary-specific deletion of Lfng and p53 using MMTV-Cre. The vast majority of Lfngflox/flox;p53flox/flox;MMTV-Cre mice succumbed to lymphoma by 3-5 months of age before mammary tumor onset, likely due to the leaky deletion in B- and T cells. Interestingly, deletion of both copies of Lfng and one copy of p53 (Lfngflox/flox;p53flox/+;MMTV-Cre) dramatically accelerated mammary tumor development compared to the Lfngflox/flox;MMTV-Cre model, with the median tumor-free survival shortened from 600 days to 191 days. Of note, all mammary tumors from the Lfngflox/flox;p53flox/+;MMTV-Cre mice were consisted of mesenchymal/spindle-shaped cells and expressed markers of epithelial-mesenchymal transition (EMT), suggesting that deletion of one copy of p53 caused a shift of tumor subtype from basal-like to claudin-low. We found that deletion of Lfng caused up-regulation of E-cadherin in the mammary gland, whereas additional deletion of one copy of p53 decreased E-cadherin but dramatically increased vimentin levels, suggesting that p53 haploinsufficiency promotes EMT in this context. Compared with the wild type gland at 3 months of age, Lfngflox/flox;p53flox/+;MMTV-Cre mutant gland showed down-regulation of Notch downstream targets including Hes5, Hey1, and Hey2, suggesting reduced Notch signaling. Given that Notch signaling is known to restrict mammary stem cell expansion, down-regulation of Notch signaling may lead to inappropriate accumulation of mammary stem cells during CLBC initiation. Taken together, our results reveal genetic interaction between Notch and p53 in the pathogenesis of CLBC. Moreover, we have established a new mouse model for CLBC with complete penetrance and accelerated tumor onset, providing an ideal tool in defining molecular events leading to the initiation and progression of CLBC as well as identifying potential therapeutic targets for this poor prognosis disease.

#679

Dual conditional loss of BLIMP-1 and p53 in B-cells drives B-cell lymphomagenesis.

Aldo M. Roccaro, Yawara Kawano, Antonio Sacco, Jihye Park, Michele Moschetta, Yuji Mishima, Elizabeth Morgan, Ruben Carrasco, Irene Ghobrial. _Dana-Farber Cancer Institute, Boston, MA_.

The transcription factor p53 is a well defined tumor suppressor involved in the modulation of cell proliferation, cell cycle progression and programmed cell death. BLIMP-1 plays a crucial role in modulating B-cell terminal differentiation towards Ig-secreting plasma cells, and it also acts as a tumor suppressor, as documented in both diffuse large B-cell lymphoma and Burkitt lymphoma. Whether B-cell specific loss of both p53 and BLIMP-1 may favor a B-cell lymphoma phenotype remains unanswered. Therefore, we aimed to define the functional relevance of both p53 and BLIMP-1 n B-cell lymphomagenesis in vivo, and generated dual p53/BLIMP-1-floxed conditional inactivation in B-cells, using mice expressing Cre recombinase under the control of CD19 promoter. 100% of the generated CD19Cre/Cre/BLIMPF/F/p53F/F transgenic mice (referred as CD19/Bl-/p53-) presented with diffuse lymphadenomegalies, marked splenomegaly, and hepatomegaly, observed in 90.3% and 77.4% of the cases, respectively. Other clinical manifestations included presence of ascites and hind lymb paralysis that were documented in 12.9% and 19.3% of the cases. The CD19/Bl-/p53- showed worse survival compared to BLIMPF/F/p53F/F mice non-expressing the CD19/Cre recombinase, CD19Cre/Cre/p53F/F, or CD19Cre/Cre/BLIMPF/F (363, 469.5, 460.5, and 770 days, respectively).

H.E. staining of CD19/Bl-/p53--derived lymph nodes, defined a nodal architecture completely effaced by a relatively monomorphic population of large sized atypical lymphoid cells with finely clumped and dispersed chromatin, and multiple basophilic medium sized, paracentrally situated nucleoli. A "starry sky" pattern was also observed. Overall, these features are compatible with a BL-like or high-grade lymphomas. IHC analysis confirmed a marked positivity for B220 staining, whit negativity for TdT, Bcl6, CD138 and CD4, CD8. Tumors were confirmed to be B220+/IgM+, with either Igk- or Ig-lambda-restriction as demonstrated by flow cytometry. Moreover, tumors were shown to be either mono- or bi-clonal, as demonstrated by Southern blotting, thus further confirming the clonal transformation induced by dual BLIMP/p53 deletion in B cells.

Whole exome sequencing was performed from B220-selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice (n: 3) and from matched tail-derived tissues, used as germline. Agilent SureSelectXT was used for library prep. WES was performed on the Illumina HiSeq 2500 platform. Tumor and matched normal DNA sequencing data were processed using the Broad Institute best practices pipeline. We have identified 213 SNVs. Among them, somatic mutations were mapped on genes involved in the regulation of focal adhesion, PDGF singaling, p53-downstream pathway, and lipoprotein metabolism.

These studies demonstrate that the specific dual inactivation of p53 and BLIMP in B-cells promotes oncogenic transformation, resulting in aggressive B-cell lymphoma development.

#680

Diet-induced obesity promotes tumor aggressiveness in genetically engineered mouse models of endometrial hyperplasia/cancer.

Leslie H. Clark, Chunxiao Zhou, Stephanie A. Montgomery, Paola A. Gehrig, Victoria Bae-Jump. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Objective: To determine the effect of diet-induced obesity on the development of endometrial hyperplasia and cancer in genetically engineered mouse models.

Methods: The PTEN heterozygous mouse model for endometrial hyperplasia and the LKB1/p53 endometrioid endometrial cancer mouse model were used. Mice were divided into two dietary groups and fed either a high-fat diet (HFD, 60% calories from fat, obese) or a low-fat diet (LFD, 10% calories from fat, lean) beginning at 3 weeks of age until sacrifice at 32 weeks (PTEN model) and 12 weeks (LKB1/p53 model). At the time of sacrifice, mouse weight and uterine/tumor size were documented. A dedicated mouse pathologist reviewed uterine pathology. For the PTEN heterozygous mouse model, uterine pathology was determined to be simple hyperplasia (SH) if increased number of simple tubular glands were seen without atypia. Complex hyperplasia (CH) was defined as branching, papillary lumenal infoldings, or back-to-back glandular crowding with or without nuclear atypic.

Results: A total of 30 PTEN mice were examined, 14 in the obese group and 16 in the lean group. The HFD-fed mice were larger than the LFD-fed mice with a mean weight of 38.1 gm compared to 29.7 gm (p<0.001). There was no difference in uterine weight between the obese and lean mice, 0.30 versus 0.34 gm, respectively (p=0.76). The lean mice were more likely to develop SH, 44% (n=7) versus 7% (n=1) in the obese group (p=0.04). Of the obese mice, 57% (n=8) developed CH (± atypia) compared to 31% (n=5) of the lean mice (p=0.16). A total of 35 LKB1/p53 mice were examined with 21 in the obese group and 14 in the lean group. The obese LKB1/p53 mice were larger (32.8 gm) than the lean mice (24.0 gm) (p=0.033). The majority of the LKB1/p53 mice developed endometrioid adenocarcinoma with increased tumor size in the obese group (2.1 gm) versus lean group (0.79 gm) (p=0.04).

Conclusions: Diet-induced obesity led to increased mean body weight in both mouse models. In the PTEN heterozygous mouse model, a trend towards more complex endometrial hyperplasia was found in the obese group along with a higher rate of simple hyperplasia in the lean group. Increased endometrial tumor size was seen in the obese versus lean LKB1/p53 mice, suggesting that obesity may lead to increased aggressiveness in endometrial cancer.

#681

Using a genome-editing approach for the stepwise establishment of zebrafish models of pediatric high-grade gliomas and MPNSTs.

Felix Oppel, Shuning He, Ting Tao, Mark W. Zimmerman, Adam D. Durbin, Nina Weichert, Dong H. Ki, A. Thomas Look. _Dana-Farber Cancer Institute, Boston, MA_.

By high-throughput sequencing of cancer patient's genomes, driver mutations have recently been described in high-grade pediatric glioma, an aggressive type of brain tumor in children. The most prominent recurrent mutations were found in four genes, namely NF1, TP53, ATRX and H3F3A. NF1 and TP53 are tumor suppressor genes and their loss of function is characteristic for various human malignancies. However, the mechanisms through which precise missense mutations in H3F3A and loss-of-function of ATRX influence tumor biology remain largely unknown. In our project, we employ the genome-editing technology CRISPR-Cas9 to develop a zebrafish model of pediatric high-grade glioma, in order to examine the impact of these mutations on tumor onset and progression. We previously created a zebrafish line that is deficient for tp53 (tp53-/-) and has a loss-of-function mutation in 3 of 4 alleles of nf1 (nf1a+/-, nf1b-/-), which is duplicated in zebrafish. These fish develop high-grade gliomas with low penetrance and malignant peripheral nerve sheath tumors (MPNSTs) with high penetrance. By CRISPR-Cas9 we further incorporated early frameshift mutations of atrx in the background of tp53-/-, nf1a+/-, nf1b-/-. All analyzed fish injected with Cas9-mRNA and CRISPR guide-RNAs (gRNAs) targeting atrx coding sequences showed germline transmission of mutant alleles, of which one third were frameshift mutations. Primary CRISPR-Cas9 atrx-gRNA injected tp53-/-, nf1a+/-, nf1b-/- fertilized eggs developed as mosaics and exhibited increased tumor penetrance and faster onset of MPNSTs and these tumors harbored atrx mutations in 8/10 analyzed samples. This indicates that atrx loss-of-function mutations cooperate with loss of tp53 and nf1 tumor suppressors. Gliomas were not observed in mosaic atrx mutated fish, and these will be reanalyzed in stable atrx mutant lines. Next, we will add previously described missense mutations of h3f3a. To unravel the synergy between oncogenic mutations in atrx and h3f3a in tumor progression, mutant stable lines for both genes will be investigated independently and in a combined manner. The zebrafish model of pediatric high-grade gliomas and MPNSTs that we are developing will be useful to dissect the mechanisms underlying cooperation among driver mutations and for small molecule screens to identify specific inhibitors of cell growth and survival in these malignancies.

### Molecular Regulation of Tumor Invasion

#682

PKCα mediates FOXC2 transcriptional repression of p120-catenin in breast cancer.

Thao N. Pham. _University of Illinois, Chicago, IL_.

Background: Protein kinase C alpha (PKCα) has been studied as a predictive biomarker for breast cancer aggressiveness and resistance to therapy. In this study, we identified a novel signaling pathway regulated by PKCα in breast cancer cells that involves FOXC2 and p120-catenin (p120), a prominent member of adherens junctions (AJs). We report her that PKCα causes dissolution of AJs, a mechanism which contributes to cancer cells becoming more migratory and invasive, two key features of metastasizing cells. Interestingly, we found that this mechanism is relevant in both estrogen receptor (ER)-positive, endocrine resistant as well as triple negative breast cancer (TNBC) (lack of ER, progesterone receptor, and HER2/neu amplification).

Methods: ER+, endocrine resistant (T47D/PKCα and T47D:A18-TAM1) and TNBC cell lines (HCC1143 and HCC1937) were cultured according to previous publication and ATCC guidelines. Migration and invasion assays were done in Transwell® polycarbonate membranes (Corning). Promoter activity of p120 was measured by luciferase assay using p120 reporter construct obtained from Dr. Fariborz Mortazavi (Division of Hematology/Oncology, UCLA). Chromatin immunoprecipitation (ChIP) to assess binding of FOXC2 on p120 promoter was done using ChIP-grade FOXC2 antibody (Abcam). Lipofectamine®2000 (Invitrogen) was used for transfection of PKCα (Sigma) and FOXC2 (IDT) siRNAs

Results and Conclusions: We found that FOXC2 is a downstream target of PKCα; activation of PKCα by a phorbol ester significantly up-regulated FOXC2 mRNA and knockdown of PKCα by siRNA reduced expression of FOXC2 at both the mRNA and protein level. Immunofluorescence staining showed that depletion of PKCα rescued E-cadherin, secondary to the recovery of p120 expression at the AJ. We demonstrated by ChIP that FOXC2 binds to the promoter region and represses transcription of p120, indicating a novel finding that FOXC2 is a transcriptional repressor of p120 in breast cancer. Therefore, through FOXC2, PKCα can negatively regulate the AJ complex integrity. Correspondingly, knockdown of either PKCα or FOXC2 led to a reduction in the migration and invasion, concomitant with an increase of p120 at the AJ. Data obtained from the Cancer Genome Atlas (TCGA) indicates that high co-expression of PKCα and FOXC2 along with low expression level of p120 are characteristic of TNBC patients, confirming the relevance of this signaling axis in clinical samples. In conclusion, we have identified a new mechanism for migration and invasion in breast cancer cells: in the presence of PKCα, transcriptional repression of p120 by FOXC2 results in down-regulation of E-cadherin and dissociation of the AJs. Notably, we demonstrated that this mechanism is relevant in two distinct molecular subtypes of breast cancer: ER+, endocrine resistant and TNBC. Targeting the PKCα-FOXC2-p120 axis has the potential to reduce the metastatic capacity of these cancer cells.

#683

VEGFA/NRP1 signal contributes to cell adhesion and motility in breast cancer cells.

Marina Kiso, Fumiaki Sato, Sunao Tanaka, Masakazu Toi. _Kyoto Univ. Grad. Sch. of Medicine, Kyoto, Japan_.

Background and objective:

Bevacizumab suppressed tumor angiogenesis by blocking the VEGFA signal into endothelial cells. Although, bevacizumab, in combination with chemotherapy, has exhibited some therapeutic efficacy for metastatic breast cancer, its impact on overall survival has not been proved.

According to recent studies, VEGF is a multi-function molecule targeting not only endothelial cells but also other cell members in tumor microenvironment, including cancer cells, fibroblasts, immune cells, and so on. In this study, we aimed to reveal VEGF-related molecular mechanisms on breast cancer cells itself.

Methods and Results:

A VEGF-expressing basal-like type breast cancer cell line, MDA-MB-231 cells (231 cells) were used. VEGFA of 231 cell was knocked out using Crisper-Cas9 system (VEGFA-KO). Compared to the wild type (WT) 231 cells, VEGFA-KO 231 cells showed rounded and weakened adhesive morphology. Scratch assay and motion picture assay demonstrated the VEGFA-KO 231 cells had impaired cell motility. Bevacizumab treatment to WT 231 cells did not induce this morphologic change.

Next, we generated soluble neuropilin-1 (sNRP1) overexpressed 231 cells. sNRP1 consists of the NRP1 extracellular domain that includes a VEGFA-binding site, traps VEGFA to function as an antagonist. sNRP1-231 cells exhibited rounded and lower adhesive morphology similar to that of VEGFA-KO 231 cells.

Bevacizumab and NRP1 bind VEGFA at amino acid motifs from exon 3-4 and exon 7 of VEGFA, respectively. Thus, our findings suggested that VEGFA could play a role in cell morphology and motility via NRP1 for 231 cells, and that bevacizumab could not block the VEGFA/NRP1 signal.

Discussion

NRP1 is a transmembrane protein that has been identified as a VEGF-A receptor. NRP1 is expressed by endothelial cells and functions as a co-receptor of VEGFRs, enhancing VEGF-A binding and promoting downstream signaling. NRP1 is also expressed in variety of cancer cells including breast cancer. Since NRP1 molecule lacks kinase activity, there have been efforts to find the mechanisms of NRP1 signaling. To date, NRP1 expression of tumor cells has been shown to contribute to proliferative signal transduction from VEGF-A. Previous study showed VEGF-A/NRP1 signals induced the phosphorylation of Akt leading to breast cancer cell survival (Bachelder et al., 2001). And another study showed that the NRP1 cytoplasmic region enhanced the interaction between NRP1 and GIPC1/Syx/RhoA, and promoted tumor cell proliferation. However, the precise mechanisms regarding NRP1 signaling remains unknown.

In conclusion, knocking out of VEGFA and trapping of VEGFA by sNRP1 induced weakened adhesive morphology and impaired cell motility in 231 cells. Because cell adhesion and motility are important factors for tumor metastasis, VEGFA /NRP1 signaling in breast cancer might be related to breast cancer metastasis, and can be a useful therapeutic/preventive target for breast cancer.

#684

Targeting an unconventional kinase-invasion axis in breast cancer metastasis.

Margarite D. Matossian,1 Steven Elliott,1 Van T. Hoang,1 Hope E. Burks,1 Theresa B. Phamudy,1 Doug Chrisey,1 William J. Zuercher,2 David H. Drewry,2 Carrow Wells,2 Bridgette Collins-Burow,1 Matthew E. Burow1. 1 _Tulane University School of Medicine, New Orleans, LA;_ 2 _Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC_.

Triple negative breast cancer (TNBC) is associated with poor survival, metastatic recurrence, increased mortality, and has few viable therapeutic options. Given the high mortality associated with metastasis of this disease, discovery of therapeutics targeting the metastasis axis is imperative. Kinases have been demonstrated to have an essential role in regulation of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Elucidating specific signaling pathways that promote EMT has the potential to provide novel targeting strategies in the treatment of TNBC. Here we demonstrate a partially non-selective polo-like kinase 1 (PLK1) inhibitor, GSK346294, is capable of EMT reversal in phenotypically mesenchymal TNBC cell lines, as evidenced by enhanced E-cadherin expression and loss of cellular migration potential. Structurally analogous but highly specific PLK inhibitors did not exhibit similar EMT changes, which led us to investigate off-target kinases inhibited by GSK346294. Follow-up kinase profiling studies revealed members of the NEver-in-mitosis-related Kinase family (NEK5 and NEK9) as additional kinases inhibited by GSK346294, leading us to investigate their individual roles in the progression of EMT. NEK5 overexpression in a non-invasive breast cancer cell line increased both migration and invasion in trans-well assays. Additionally, enhanced NEK5 expression altered the TNBC gene expression profile, enhancing expression of genes associated with growth factor signaling (MET, EGFR) and the mesenchymal gene signature (SLUG, VIM). To date, no studies exist which delineate the regulatory roles of NEK5 and NEK9 in EMT or cellular invasion/motility. Further investigation into the function of NEK5 and/or NEK9 in the metastatic progression may provide novel therapeutic targets for the treatment of TNBC, the implication of which could impact the treatment and management of neoplastic disease and metastasis beyond breast cancer.

#685

Selective inhibitor of nuclear export (SINE) compounds prevent migration and proliferation of triple negative breast cancer (TNBC) cells by restoring expression of ARRDC3.

Young Hwa Soung,1 Trinayan Kashyap,2 Thalia Nguyen,1 Yosef Landesman,2 Jun Chung1. 1 _Stony Brook Medicine, Stony Brook, NY;_ 2 _Karyopharm Therapeutics, Newton, MA_.

Arrestin-related domain-containing protein-3 (ARRDC3) is one of 6 mammalian arrestins, which has been shown to possess tumor suppressor activity by inducing degradation of phosphorylated β2-adrenergic receptor (β2 AR) and integrin β4 (ITG β4). Our previous studies demonstrated that Sirt2 epigenetically silenced expression of ARRDC3 in TNBC cells and overexpression of ARRDC3 significantly reduced their invasive potential. Therefore, we hypothesized that compounds, which restore ARRDC3 expression levels could serve as novel therapeutic options for TNBC. To test this, we examined the ability of SINE compounds: KPT-185 and KPT-330 (selinexor), to effectively restore ARRDC3 expression. We observed that KPT-185 and selinexor significantly induced ARRDC3 expression and inhibited the proliferation and the invasiveness of the TNBC cell lines MDA-MB-231 and MDA-MB-468 in vitro. In vivo, selinexor inhibited the tumor growth of MDA-MB-231 and MDA-MB-468 xenografts by nearly 100% compared with vehicle treated animals. Furthermore, immunohistochemical analysis of TNBC tumors from selinexor treated mice revealed increased ARRDC3 expression versus vehicle treated animals. Our results suggest that restoration of ARRDC3 expression is an important antineoplastic mechanism of SINE compounds in TNBC and suggest that selinexor could be an effective treatment against tumors with downregulated ARRDC3.

#686

MNK1 promotes the progression of breast ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC).

Qianyu Guo, Sonia V. del Rincon, Christophe Goncalves, Wilson H. Miller. _McGill University, Montreal, Quebec, Canada_.

The mechanism by which breast ductal carcinoma in situ (DCIS) progresses to invasive ductal carcinoma (IDC) is poorly described. Previous studies have revealed that DCIS and IDC share similar genomes and transcriptomes. We hypothesize that one mechanism contributing to the DCIS to IDC conversion involves aberrations in mRNA translation. The Mnk/eIF4E axis has a critical role in promoting the translation of tumor promoting and pro-invasive mRNAs, thus we propose to study whether aberrant activation of this axis promotes the transition of DCIS to IDC. Our model system involved overexpressing constitutively active Mnk1 (caMNK1) in MCF10 variants: (1) MCF10A immortalized mammary epithelial cells and (2) MCFDCIS.com ductal carcinoma in situ cells (termed DCIS cells). Our in vitro results suggest that caMNK1 enables DCIS cells to gain invasive properties. First, overexpression of caMnk1 increased colony formation, larger acinar size in 3D culture, and promoted the migration and invasion of MCF10A and MCFDCIS cells. Second, SEL201, a novel Mnk1/2 inhibitor, can inhibit proliferation, colony formation, 3D acini formation, and the migration and invasion capacity of DCIS-pBABE and -caMNK1 expressing cells. Tumor xenografting in athymic nude mice revealed that caMNK1 facilitates the conversion of DCIS to IDC as the DCIS-caMNK1 expressing tumors progress to IDC, while DCIS-pBABE derived tumors retain DCIS-like structures. Breast tumor samples from patients with DCIS, IDC or mixed DCIS-IDC will also be obtained to examine the levels of phospho-Mnk1 and phospho-eIF4E.

#687

Critical role and mechanism of WASF3 in HER2/HER3 regulation of breast cancer metastasis.

yong teng, Wenhu Pi, John Cowell. _Georgia Regents Univ, augusta, GA_.

WASF3 is overexpressed in high-grade breast cancer and promotes invasion and metastasis but does not affect proliferation. The HER2/ERBB2/NEU gene is also frequently overexpressed in breast cancer and has been shown to promote invasion and metastasis in these tumors. Here we show that WASF3 in present in the HER2 immunocomplex and suppression of WASF3 function leads to suppression of invasion even in the presence of HER2 expression. Overexpression of both HER2 and WASF3 in non-metastatic MCF7 breast cancer cells promotes invasion and metastasis more significantly than either gene alone. HER2 forms homodimers as well as heterodimers with other HER family members and we now show that the ability of WASF3 to promote invasion is highly dependent on the HER2/HER3 heterodimer. The engagement of WASF3 with the HER2/HER3 complex facilitates its phospho-activation and transcriptional upregulation, which is facilitated by HER2/HER3 activation of JAK/STAT signaling. In breast cancer cells overexpressing HER2, therefore, WASF3 is specifically required to facilitate the invasion/metastasis response. Targeting WASF3, therefore, could be a potential therapeutic approach to suppress metastasis of HER2-overexpressing breast tumors.

#688

**Identifying TMEM106B as a novel metastasis driver in non-small cell lung cancers through an** in vivo **gain-of-function screen.**

Samrat Kundu,1 Caitlin Grzeskowiak,2 Chad J. Creighton,2 Kenneth L. Scott,2 Don L. Gibbons1. 1 _UT MD Anderson Cancer Center, Department of Thoracic/Head and Neck Medical Oncology, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Metastatic lung cancer is the leading cause of cancer related death in the world and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Lung cancers commonly demonstrate genetic alteration of potent drivers like Kras, p53 or EGFR accompanied with hundreds of low frequency gene aberrations which are a mix of key driver events and nascent passenger mutations. We established a robust screening platform to selectively identify these functionally critical "driver" genes in lung cancers from a prioritized list of candidates selected by a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models and genomic data of human lung cancers from TCGA, focusing on elevated gene expression and/or gene amplification. We identified 225 putative candidate driver genes which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each gene. These candidate lines were used for a unique in vivo positive selection screen to identify functional metastasis drivers. We transplanted pools of 20 cDNA expressing lines into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences in the genomic DNA from metastatic lesions over primary tumors. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers. We validated our top hits for their individual in vivo metastatic potencies by gain-of-function/loss-of-function studies and identified TMEM106B as one of the primary drivers of metastasis. TMEM106B is a transmembrane protein localizing to the lysosomes and has been shown to drive expression of lysosomal genes. We hypothesize that elevated levels of TMEM106B is able to drive the expression and secretion of lysosomal enzymes thus making cancer cells hyper invasive and metastatic. We found strong correlation for expression of TMEM106B with several lysosomal genes in a panel of 1016 human lung cancers and are currently performing in depth studies to understand these mechanisms of TMEM106B driven metastasis. We are also analyzing the clinical relevance of TMEM106B based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Identification of such novel players will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for effective treatment options for lung cancer patients.

#689

Downregulation of TIMP2 via HIF-1α/miR-210/HIF-3α regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma.

Alan KL Kai, Lo Kong Chan, Regina CL Lo, Joyce Lee, Carmen CL Wong, Jack CM Wong, Irene OL Ng. _The University of Hong Kong, Hong Kong, Hong Kong_.

Cancer metastasis is a multi-step process that involves a series of tumor-stromal interaction, including extracellular matrix (ECM) remodeling which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases 2 (TIMP2) in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly downregulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.1%; 23/56) in human HCCs as compared to the corresponding non-tumorous livers and was significantly associated with direct liver invasion into the adjacent liver parenchyma and poorer survival outcomes of the HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced the cell invasive ability and ECM degradation associated with formation of invadopodia-like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Furthermore, using orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line MHCC-97L not only significantly reduced the incidence of tumor microsatellite formation and venous invasion in the primary hepatic tumor xenografts, but also pulmonary metastasis, suggesting that both extrahepatic and intrahepatic metastasis in HCC was suppressed by TIMP2 expression. Mechanistically, TIMP2 suppression in a hypoxic environment was induced through a regulatory feedback circuit consisting of HIF-1α, miR-210 and HIF-3α. Taken altogether, our findings established that TIMP2 was frequently downregulated in human HCCs and its downregulation was associated with aggressive behavior and poorer patients' outcome. Its suppression was under the regulation of a novel feedback circuit consisting of HIF-1α/ miR-210/ HIF-3α. Overall, our study has provided solid in vitro and in vivo evidence that TIMP2 is an important regulator of ECM degradation and HCC metastasis. These findings demonstrated that perturbation of the dynamic HIF-1a signaling circuit by miR-210 inhibitor abolished TIMP2 downregulation and suppressed HCC cell invasion. They also support the notion that targeting against HIF-1α signaling is a promising direction to tackle the hypoxic responses in HCC elicited by transarterial chemo-embolization (TACE) treatment to HCC patients.

#690

Functional characterization of RPS6KA3 (RSK2) and identification of its naturally occurring mutants in hepatocellular carcinoma.

Lo-Kong Chan, Daniel Wai-Hung Ho, Yung-Tuen Chiu, Alan Ka-Lun Kai, Chun-Ming Wong, Irene Oi-Lin Ng. _Univ. of Hong Kong, Pokfulam, Hong Kong_.

RPS6KA3 encodes the p90 ribosomal S6 kinase 2 (p90RSK2 or RSK2), a protein kinase to relay the upstream transduction signal of the mitogen-activated protein kinase (MAPK) pathway. Currently, no characterization on RSK2 in human HCC has been performed. We determined the RSK2 mRNA expression in paired human HCC samples and their corresponding non-tumorous livers (n=22) by real time PCR. RSK2 over-expression (≥2 folds) was found in 38% of the human HCCs and this over-expression was associated with tumor microsatellite formation, a feature of HCC metastasis. The oncogenic and pro-metastatic roles of RSK2 were further assessed and demonstrated in vivo by an orthotopic liver injection model in nude mice with stable shRSK2 knockdown HCC cells. Stable shRSK2 knockdown cells showed a significant reduction in tumor incidence in liver and lung metastasis. By exome-sequencing and targeted sequencing in a cohort of HBV-associated HCC patient samples (n=111), we found that RSK2 was recurrently mutated in 6.3% of the samples being screened (n=7). All the RSK2 mutants were confirmed by independent Sanger sequencing. Intriguingly, among the identified somatic RSK2 mutants, half of them introduced pre-mature stop gain to the transcript, which leads to the loss-of function of RSK2 through various mechanisms. Taken together, our data suggested that RSK2 may play differential roles in different subsets of HCC patients. Further characterization will definitely derive novel insights on the functional role of mutant RSK2 and its potential use to stratify HCC patients into different molecular subgroups.

(This study was supported by a RGC GRF fund (17116414), SK Yee Medical Research Fund 2011 and University Development Fund of The University of Hong Kong. Ng is Loke Yew Professor in Pathology)

#691

The sweet jaws of malignancy: role of GALNTs glycosylation enzymes in promoting tissue invasion.

Frederic BARD. _IMCB-A*STAR, Singapore, Singapore_.

GalNAc transferases (GALNTs) are enzymes responsible for the initiation of O-linked glycosylation, which occurs on most secreted or cell surface proteins. The GalNAc sugar added on Ser or Thr residues forms the Tn antigen, which is rapidly capped in normal tissues but expressed at high levels in human tumors. The mechanism of high Tn expression was unclear until recently. We found that Src activation induces specifically the relocation of GALNTs from the Golgi apparatus to the endoplasmic reticulum (ER). In hepato-cellular carcinoma (HCC), high Tn expression is also driven by the relocation of GALNTs from Golgi to ER. In a mouse model of HCC, ER targeting of a single enzyme, GALNT1, results in a dramatic stimulation of tumour growth and fast grade progression. Conversely, expression of an ER-targeted inhibitor of GALNTs protects mice from tumor progression. We show that ER relocation of GALNTs induce a profound shift in cellular O-glycosylation pattern, with thousands of sites and hundreds of proteins becoming hyper-glycosylated. Several of these proteins are ER-resident, other are at the cell surface and many are extracellular matrix proteins. We find that MMP14 is one of the key targets of glycosylation and strongly stabilized by glycosylation. We propose that HCC tumor progression is driven by a program of ER-localized protein glycosylation regulating cell surface and ECM proteins.

#692

Identifying genes that regulate bladder cancer progression and invasion.

Amy L. Han, Brendan Veeneman, Scott A. Tomlins, Evan T. Keller. _University of Michigan, Ann Arbor, MI_.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer and can be categorized as either non-muscle invasive (Ta-T1) or muscle invasive (≥T2). Approximately 50% of bladder cancers are T1 at initial diagnosis; however, the recurrence rate for these tumors is high and they may progress into T2. In this study, we aimed to determine if there are specific gene expression differences between T1 vs. T2 bladder cancer that can help identify key regulators in bladder cancer progression and invasion. T1 and T2 bladder cancer tissues were subjected to RNA-Seq to evaluate for differences among these stages. Additionally, the Oncomine database was examined to further narrow down potential candidates that differentiate T1 from T2. These efforts led to the identification of an extracellular matrix glycoprotein, fibulin-3 (FBLN3), as being highly expressed in T2 compared to T1 tissues. To validate these findings, FBLN3 expression was measured using qRT-PCR from formalin fixed and paraffin embedded tissues from patient bladder samples ranging from stages Ta-T4. These studies confirmed that FBLN3 expression was elevated in muscle-invasive compared to non-muscle invasive bladder cancer. Consistent with these findings, FBLN3 expression level correlated with the invasive ability of several bladder cancer cell lines. Specifically, FBLN3 expression was determined using both qRT-PCR and western blotting amongst the T24, UMUC-13, UMUC-3, RT4, and 5637 bladder cancer cell lines. The most invasive cell lines, T24 and UMUC-13, demonstrated the highest FBLN3 expression. In contrast, the least invasive cells, RT4 and 5637, demonstrated the least FBLN3 expression. To determine a functional role for FBLN3 in bladder cancer invasion, we knocked down or increased FBLN3 expression in bladder cancer cell lines using lentiviral transduction. Knockdown of FBLN3 expression in the T24 and UMUC-13 cells inhibited the invasion and migration of these bladder cancer cells; whereas, FBLN3 overexpression in the 5637 cells promoted the invasiveness of the bladder cancer cells. Furthermore, cell viability and growth rates were not affected by manipulation of FBLN3 expression. Our results indicate that FBLN3 serves as a pro-invasive factor in bladder cancer. These findings suggest that FBLN3 could serve as (1) a biomarker to differentiate T1 from T2 bladder cancers and (2) a promising therapeutic target.

#693

Oncogenic and osteolytic function of histone demethylase NO66 in castration resistant prostate cancer.

Rozita Bagheri-Yarmand,1 Nora Navone,1 Xinhai Wan,1 Christopher Logothetis,1 Johnny Huard,2 Krishna Sinha2. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _UT Health Science Center, Houston, TX_.

Androgen ablation by pharmacological use or castration is a standard therapy for treating prostate cancer (PCa), however, at the later stage PCa becomes castration-resistant and is metastasized primarily to bones. Epigenetic changes that cause dysregulated gene expression during progression and metastasis of androgen-independent prostate cancer are not clearly understood. We previously reported that the jumonji C domain-containing NO66 is a histone demethylase specific for H3K4me3 and H3K36me3, which negatively regulates osteoblast differentiation and bone formation. By analyzing a multi-cancer tissue microarray, the serendipitous observation was made that NO66 is overexpressed in a variety of cancers. Our objective was to examine NO66's role in the pathogenesis of prostate cancer and bone metastasis.

Our findings indicate that NO66 knockdown resulted in significant decreases in the proliferation and invasiveness of prostate cancer cells (PC3 and DU145) while overexpressing NO66 promoted their proliferation and invasion. Furthermore, NO66 depletion resulted in a two- to three fold decrease in colony formation and anchorage-independent growth of PCa cells suggesting an oncogenic function of NO66. RNAseq analysis comparing between control and NO66-depleted DU-145 cells revealed a repertoire of differentially expressed genes. NO66 up-regulates genes with proliferative activity such as chemokine CXCL5, growth factor IGFBP5, IL6, WNT and MAPK4 pathway, which are known to drive PCa proliferation. Furthermore, ChIP sequencing for NO66 and H3K9AC in NO66-overexpressing PC3 cells revealed that interaction sites of NO66 overlap with peaks of H3K9AC in majority of the genes. These data clearly indicate that NO66 activates a cohort of genes which are involved in tumor progression and metastasis. Strikingly, implanting NO66-overexpressing prostate cancer cells into the femur of SCID male mice caused massive femoral bone loss after three weeks, which suggests that NO66 causes osteolytic bone lesions similar to the clinico-pathological features of advanced PCa. Finally, analysis of tumor xenografts derived from patients by immunohistochemistry showed high expression of NO66 in high grade advanced PCa compared to low grade and hyperplasia suggesting that NO66 may be used as a biomarker to distinguish aggressive prostate cancer from indolent stage. Our data suggest that NO66 plays an important role in cell proliferation, cell invasion and bone metastasis of prostate cancer.

#694

Axon guidance neuropilin and plexin A2 genes are involved in ERG-associated prostate cancer.

Anthony Turpin,1 Carine Delliaux,2 Tian Tian,3 Nathalie Vanpouille,2 Anne Flourens,2 Rachel Deplus,4 Xavier Leroy,1 Yvan de Launoit,2 Martine Duterque-Coquillaud2. 1 _CHRU de Lille, Lille, France;_ 2 _Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T – Mechanisms of Tumorigenesis and Target Therapies, Lille, France;_ 3 _Center for Genomic Regulation, Barcelona, Spain;_ 4 _Institut Laboratory of Cancer Epigenetics, Faculty of Medicine, ULB, Brussels, Belgium_.

Background: Bone metastases are frequent and severe complications of prostate cancer (PCa), the most common malignancy that affects men in Western countries. Recurrent gene fusions, involving the ERG gene and the androgen-regulated promoter of the TMPRSS2 gene, occur in over 50% of PCa. The abnormal over-expression of the ERG transcription factor disturbs prostate cell transcriptome. We previously identified a series of genes directly and positively regulated by the ERG-fusion in vitro and in vivo. Among them can be found Plexin A2 (PLXNA2), which is a semaphorin receptor family member involved in axon guidance and in many pathophysiological processes, including cancer and bone disorders. We showed that PLXNA2 is involved in TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. These results prompted us to look for genes functionally related to PLXNA2 and involved in ERG-associated PCa.

Methods: Using a PCa cell line (PC3c) stably over-expressing the TMPRSS2:ERG fusion, we first used Ingenuity Pathway Analysis® in silico studies to analyze transcriptomic results and identify axon guidance genes associated with ERG-fusion expression in PCa. Their expression was validated in stable clones by RT-PCR and Immunofluorescence. Secondly, using RNAs of a human PCa sample cohort, we performed qRT-PCR to detect and correlate candidate genes to fusion expression. Finally, to validate the relevance of the identified genes, using immunohistochemistry experiments, we studied the expression of these genes in tissue samples, including primary tumors and lymph nodes or bone metastases.

Results: Using transcriptomic analyzes and expression validation, we identified neuropilin (NRP1 and NRP2) genes, plexin and semaphorin genes deregulated by the ERG fusion expression in stable clones. In human samples, PLXNA2, NRP1 and NRP2 expression were strongly associated to PCa, and particularly to metastastic PCa. We established a significant correlation between both PLXNA2 and NRP1 gene expression and the presence of TMPRSS2-ERG fusion. At protein level, NRP1 and NRP2 were highly detected in human lymph nodes and bone metastasis samples. Since neuropilin proteins are known to interact with plexin to act as coreceptors for semaphorin ligands, protein colocalization in stable clones suggested a functional association of PLXNA2 and NRPs in tumor cells.

Conclusion: Taken together, our results reveal that TMPRSS2-ERG fusion gene expression is able to deregulate axon guidance genes such as NRP1 and PLXNA2 in PCa. Therefore, since the interaction between plexins and their NRP1 and 2 coreceptors often determines the functional specificity of their semaphorin ligand effects, we hypothesize that the ERG fusion expression imbalances the plexin/neuropilin/semaphorin signaling to contribute to PCa progression. These results reinforce the recent interest in targeting neuropilins for cancer therapy.

#695

SHP-2-upregulated ZEB1 is important for PDGFRα-driven glioma epithelial-mesenchymal transition and invasion in mice and humans.

Lei Zhang,1 Weiwei Zhang,1 Yanxin Li,1 Zuoqing Li,1 Yinfang Wang,1 Lina Song,1 Deguan Lv,1 Ichiro Nakano,2 Bo Hu,3 Shi-Yuan Cheng,3 Haizhong Feng1. 1 _Shanghai Jiao Tong University, Shanghai, China;_ 2 _University of Alabama, Birmingham, AL;_ 3 _Northwestern University, Chicago, IL_.

A critical clinical challenge in glioblastoma therapy is robust tumor growth and invasion driven by aberrant activation of oncogenic tyrosine receptor kinases (RTKs) including PDGFRα. Here, we report that a SHP-2-upregulated epithelial-mesenchymal transition (EMT)-inducer, ZEB1 is important for PDGFRα-driven glioma EMT, invasion and glioma stem cell renewal in mice and humans. ZEB1 and activated PDGFRα were co-expressed in invasive regions of mouse glioma xenografts and human clinical glioma specimens. Glioma patients with high levels of both p-PDGFRα and ZEB1 had significantly shorter overall survival compared with those with low expression of p-PDGFRα and ZEB1. Knockdown of ZEB1 inhibited PDGF-A/PDGFRα-stimulated glioma EMT, tumor growth, invasion and glioma stem cell renewal. PDGFRα mutant deficient of SHP-2 binding (PDGFRα-F720) or PI3K binding (PDGFRα-F731/42), knockdown of SHP-2 or treatments of pharmacological inhibitor for PDGFRα-signaling effectors attenuated PDGF-A/PDGFRα-stimulated ZEB1 expression, cell migration and glioma stem cell proliferation. Importantly, SHP-2 acts together with PI3K/Akt to regulate a ZEB1-miR-200 feedback loop in PDGFRα-driven gliomas. Together, our findings uncover a new pathway in which ZEB1 functions as a key regulator for PDGFRα-driven glioma EMT, invasion and glioma stem cell renewal, suggesting that ZEB1 as a potential therapeutic target for human gliomas with high PDGFRα activation.

#696

Targeting the proprotein convertase PCSK6/PAECE4 abrogates human melanoma malignant phenotype.

Geraldine Siegfried,1 Apolline Imbard,1 Serge Evrard,2 Abdel-Majid Khatib1. 1 _INSERM U-1029, PESSAC, France;_ 2 _INSERM U-1029 and Bergonié Cancer institute, Bordeaux, France_.

The proprotein convertases (PCSKs) are involved in the proteolytic maturation/activation of a wide range of protein precursors involved in neoplasia such as adhesion molecules, growth factors, growth factor receptors, and metalloproteinases. Expression analysis of all the PCSKs family members, namely PC1, PC2, furin, PC4, PC5, PACE4, and PC7 in various human and murine melanoma cells revealed increased PCSK6/PAECE4 expression while compared to melanocytes. The use of in vitro digestion assays and cell transfection experiments revealed that targeting the PCSK6/PAECE4 in melanoma cells using small interfering RNA (siRNA) reduced PCSKs activity and repressed the processing the PCSKs substrates proIGF-1R, pro-VEGF-C and proPDGF-A. Theses unprocessed substrates failed to mediate their signaling pathways that associated reduced cell proliferation. Furthermore, PCSK6/PAECE4 gene silencing reduced melanoma cells migration and invasion that paralleled decreased gelatinase MMP-2 and MMP-9 activity and altered expression and secretion of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Taken together, these findings highlight the importance of PCSK6/PAECE4 activity in melanoma cells and suggest PCSK6/PAECE4 inhibition as a potentially promising strategy for the prevention of melanoma invasiveness.

#697

Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines.

Sumit Bhattacharyya, Leo Feferman, Kaoru Terai, Arkadiusz A. Dudek, Joanne K. Tobacman. _University of Illinois at Chicago, Chicago, IL_.

The proteoglycan CSPG4 [chondroitin sulfate proteoglycan 4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP) or neuron-glial antigen 2 (NG2)] has been associated with progression of melanoma, and intervention to reduce CSPG4 has been considered as treatment of melanoma. In recent work, we have reported that decline in the enzyme arylsulfatase B (ARSB, N-acetylgalactosamine 4-sulfatase) is associated with malignancy, including prostate, mammary, and colon. ARSB is the enzyme that removes 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate (DS), and thereby regulates the degradation of the sulfated glycosaminoglycan (GAG) chain. Hence, the absence of ARSB leads to the accumulation of more highly sulfated C4S or DS. In prior published work, decline in ARSB and the associated increase in C4S led to transcriptional events, mediated through a decline in galectin-3 binding to the more highly sulfated C4S present. Transcription factors activator protein 1 (AP-1) and Specificity protein 1 (Sp1) interacted with galectin-3 to mediate the increased transcription of versican and Wnt9A. In this study, the mechanism by which decline in ARSB increased expression of CSPG4 and. increased the invasiveness of melanoma cell lines was addressed. Techniques included: cell culture of normal melanocytes (ATCC PCS 200-013) and melanoma cell lines obtained from radial or vertical growth phase [WM1552C, WM1552C/mock (mock CSPG4 transfection), WM1552C/MCSP (CSPG4 transfected), WM1552C/MCSPΔCD (transfected with CSPG4 without the cytoplasmic domain), WM35] and from metastatic melanoma (1205 Lu); QPCR of CSPG4; ARSB activity assay using the exogenous substrate 4-methylumbelliferylsulfate; silencing of ARSB by siRNA; invasion assay; measurements of total sulfated glycosaminoglycans and C4S using 1,9-dimethylmethylene blue dye; and measurements of matrix metalloproteinase (MMP) activity. The results show that ARSB activity was reduced in the metastatic melanoma cells to 51% of the level in the normal melanocytes. Silencing of ARSB was associated with increase to 2.8 times the baseline level in the mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4) in the normal melanocytes. Invasiveness of the melanoma cells increased by 70% when ARSB was silenced, with corresponding increase in MMP activity. Since CSPG4 has C4S attachments, the effects of ARSB on CSPG4 expression may affect the availability of C4S GAG chains to interact with extracellular matrix components and affect invasiveness. The findings indicate that decline in ARSB and the associated increase in C4S have profound effects on the invasive potential of melanoma and that increased attention to ARSB and C4S may yield new therapeutic interventions.

#698

**Progranulin targeting in urothelial cancer cells inhibits motility, tumor growth** in vitro **and** in vivo **and sensitizes cells to cisplatin.**

Simone Buraschi,1 Shi-Qiong Xu,1 Manuela Stefanello,1 Igor Moskalev,2 Alaide Morcavallo,1 Marco Genua,1 Ryuta Tanimoto,1 Thomas Neill,1 Stephen C. Peiper,1 Leonard G. Gomella,1 Antonino Belfiore,3 Peter C. Black,2 Renato V. Iozzo,1 Andrea Morrione1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Vancouver Prostate Centre, Department of Urologic Sciences University of British Columbia, Vancouver, British Columbia, Canada;_ 3 _Universita' della Magna Graecia di Catanzaro, Catanzaro, Italy_.

Introduction and Objective: Bladder cancer is a major public health problem and affects more than 74,000 Americans with more than 16,000 estimated deaths in 2015. The majority of deaths are due to metastatic spread, commonly to the lungs. Understanding the molecular mechanisms regulating bladder tumor cell invasion and progression toward metastases is essential for developing better therapies to treat bladder cancer patients. The growth factor progranulin has emerged in recent years as an important regulator of transformation in several cancer models. We have previously established a critical role for progranulin in bladder cancer as in fact progranulin acts as an autocrine growth factor and promote motility and invasion of invasive urothelial cancer cells. In addition, progranulin is upregulated in high grade bladder cancer tissues compared to normal tissue controls suggesting that progranulin might work as a novel biomarker with predictive value for bladder cancer progression. However, whether progranulin is important for anchorage-independent growth and in vivo tumor formation of urothelial cancer cells has not been previously established.

Methods: Progranulin depletion was achieved by stably transfecting tumorigenic T24T, UMUC3 urothelial cancer cells with a plasmid expressing an anti-progranulin shRNA. Progranulin-depleted and control UMUC-3 and T24T cells were tested for motility, invasion and anchorage-independent growth by soft-agar assays. Tumor formation in vivo was assessed in various UMUC-3-derived cell lines by xenograft and

orthotopic models. Sensitivity to cisplatin was assessed by cell survival curves. Progranulin expression levels in a bladder tissue microarray were analyzed by HIC.

Results: Progranulin-depleted T24T and UMUC-3 cells were significantly inhibited in their ability to migrate, close a wound and invade through Matrigel compared to control cells in both serum-deprived and 1% serum media. In addition, progranulin targeting strongly reduced the ability of T24T and UMUC-3 cells to grow in anchorage-independency and form colonies in soft-agar. Significantly progranulin-depleted UMUC-3 cells were severely inhibited in tumor formation in vivo as assessed by both xenograft and orthotopic models in immunocompromised mice. Importantly, progranulin depletion sensitized UMUC-3 cells to cisplatin. Finally, progranulin levels correlated with tumor progression in bladder cancer tissues.

Conclusions: Our data are translationally relevant as indicate that progranulin exerts an essential functional role in the regulation of bladder cancer progression. Thus, progranulin may constitute a novel target for therapeutic intervention in bladder tumors. In addition, progranulin may serve as novel biomarker for diagnosis and prognosis of bladder cancer.

#699

Inhibition of cathepsin L1 by U87 KIF9 suppression in glioblastoma multiforme: a potential therapeutic target.

Maria M. Rubinstein,1 Jeffrey Segall2. 1 _Montefiore Medical Center - Albert Einstein College of Medicine, Bronx, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY_.

Glioblastoma multiforme is a fatal primary brain cancer. It has remained one of the most difficult cancers to treat because of glioblastomas' aggressive and local invasive nature. Glioblastoma tumors have many myeloid cells, especially microglia. Increased levels of microglia result in increased glioblastoma invasion and are associated with worse clinical outcomes. Glioblastomas' infiltrative mechanisms are mostly unknown which hinder the efficacy of current treatments. Therefore, further investigations on the mechanisms responsible for local invasion are potential targets for therapeutic interventions.

Recently, there has been interest in evaluating the role of kinesin family members (KIFs) and cathepsins (CTS) as possible targets for therapeutic interventions. KIFs play an essential role in both physiological axonal transport processes and in processes of cell division. Previous studies have investigated the inhibition of KIF11 as a possible therapeutic target in glioblastoma. Preliminary studies in our group have found that the inhibition of KIF9 reduces microglia-stimulated glioma invasion. CTS, which are proteases that function in many pathways and increase neoplastic progression, are other possible therapeutic targets. Specifically in glioblastoma, inhibition of cathepsin L1 (CTSL1) (i) promotes tumor cell death, (ii) decreases apoptosis threshold and (iii) sensitizes glioblastoma cells to radiation. Our study investigated the role of KIF9 suppression in secretion and production of CTSL1.

In order to investigate this relationship, U87 cells were incubated with and without the presence of microglia. Non-targeting and stable KIF9 knockdown cells were incubated with and without the presence of microglia. CTSL1 concentration levels were measured by enzyme immunoassay (ELISA). We investigated CTSL1 concentrations in the cell lysates as well as the cell supernatants. Our results demonstrated an overall decrease in production and secretion of CTSL1 in KIF9 knockdown cells as compared to non-targeting shRNA cells. The effect of KIF9 knockdown was evident in the presence and absence of microglia. Although the exact mechanism needs to be further clarified, inhibition of CTSL1 by KIF9 suppression could be a possible potential target for therapeutic intervention.

#700

Regulation of Fn14 expression by EGFRvIII-STAT signaling enhances glioblastoma cell invasion and survival.

Alison Roos,1 Zachary Mayo,1 Heather Sonnemann,1 Michael Pineda,1 Gil Lambert,1 Harshil D. Dhruv,1 Jeffrey A. Winkles,2 Michael E. Berens,1 Nhan L. Tran1. 1 _Translational Genomics Institute, Phoenix, AZ;_ 2 _University of Maryland School of Medicine, Baltimore, MD_.

Glioblastoma Multiforme (GBM) is the most common malignant brain tumor in adults. Most GBM patients succumb to the disease less than one-year post diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radio-and chemoresistant properties to the tumor cells; therefore, there is a need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the JAK/STAT-signaling pathway. Here we report that GBM cells expressing EGFRvIII show increased expression of a previously established mediator of glioma cell invasion and survival, fibroblast growth factor-inducible 14 (Fn14), at the mRNA and protein level. Treatment with STAT3, STAT5, JAK, or Src inhibitors decreased Fn14 mRNA and protein expression. Finally, knockdown of Fn14 levels in the EGFRvIII-expressing glioma cells decreased both cell survival after temozolomide (TMZ) treatment and cell invasion, which suggests that Fn14, in part, mediates the oncogenic phenotypes conferred by EGFRvIII signaling. Since EGFR inhibitors display limited therapeutic efficacy in GBM patients, we hypothesize that Fn14-targeted therapies could potentially limit invasiveness and chemoresistance in EGFRvIII-dependent GBM tumors.

#701

Loss of Krupple-like factor 10 enhanced epithelial-mesenchymal transition of pancreatic cancer by modulating glucose metabolism via Sirt 6 and PKM2.

Yi-Chih Tsai, Su-Liang Chen, HS Vincent Chang, Hui-Ju Ch'ang. _NHRI, Miaoli County, Taiwan_.

Introduction:

Pancreatic Cancer is well known for its deregulated TGFβ signaling. We have reported that the expression level of Krüpple like factor 10 (Klf10), a TGFβ early response transcription factor, is associated with clinical outcomes of pancreatic cancer patients. In this study, we would like to evaluate the mechanisms of Klf10 in promoting distant metastasis of pancreatic cancer.

Materials and Methods:

Klf10 over-expressing- or depleting-human pancreatic cancer cell lines were established. Western blot was used to evaluate epithelial-mesenchymal transition (EMT) markers and glycolysis related molecules. Cancer cell migration was measured by trans-well assay. Luciferase-labeled Panc-1 cells with or without Klf10 mRNA silencing were injected via tail veins of NOD/SCID mice to evaluate their metastatic ability. The glucose consumption, lactate production and mitochondrial respiration of pancreatic cancer cells were measured by assay kit or XFe Extracellular Flux Analyzers.

Results:

Down-regulating Klf10 in Panc-1 cells was associated with enhanced migration and invasion ability by in vitro and in vivo studies. Immunoblots of cell lysates revealed decreased expression level of E-cadherin and increased Twist, MMP9. Glycolysis activity was elevated while mitochondrial respiration decreased after silencing Klf10 mRNA in Panc-1 cells. The phenomenon was in parallel with decreased Sirt 6 and increased HIF-1α as well as PKM2 expression. We demonstrated that Klf10 bind to the promoter and transcriptionally regulated Sirt 6. Modulating Sirt 6 and PKM2 reversed the EMT and metastatic phenotype of Panc-1 cells with Klf10mRNA silencing.

Discussion:

Loss of Klf10 enhanced the migratory ability of pancreatic cancer cells. Klf10 transcriptionally regulated Sirt 6 which modulated glycolytic enzymes including PKM2 via HIF-1α. Elevated PKM2 promoted EMT phenotype and distant metastasis of pancreatic cancer. Klf10 is a potential prognostic and therapeutic biomarker for pancreatic cancer.

#702

Serglycin promotes cell migration via modulating cytoskeleton reorganization and enhancing focal adhesion turnover.

Jeou-Yuan Chen, Jing-You Guo. _Academia Sinica - Inst. of Biomedical Sci., Taipei, Taiwan_.

Serglycin (SRGN), a hematopoietic cell granule proteoglycan, has recently been shown to be overexpressed in several aggressive cancer types. In nasopharyngeal and hepatocellular carcinomas, elevated expression of SRGN was shown to correlate with poor prognosis. However, the molecular mechanisms underlying SRGN-mediated malignancies remain to be explored. In this study, we showed that SRGN is expressed at elevated levels in several lung cancer-derived cell lines. By gain-of-function and loss-of-function approaches, we showed that SRGN promoted NSCLC cell migration. SRGN modulated actin cytoskeleton reorganization and promoted lamellipodia and filopodia formation at the leading edge, facilitating a directional movement during wound closure in NSCLC cells. In consistence, increased levels of activated Rac1, which is required for lamellipodia formation, and CDC42, which is required for filopodia formation, were detected. Increased focal adhesion (FA) turnover, the process of continuous assembly and disassembly of adhesions, would lead to increased cell migration. We further showed that SRGN promoted Src activation, leading to increased phosphorylation of paxillin (PAX) at Y118 as well as reduced PAX/FAK complex formation, which were well-defined indicators of FA turnover. Taken together, these data suggested that SRGN promotes cell migration through inducing cytoskeleton reorganization and focal adhesion turnover.

#703

Klf4 null MEFs exhibit increased Rho-mediated stress fiber formation associated with migration.

Elizabeth R. Stratton, Kathryn Black, Nathaniel Larson, Engda Hagos. _Colgate University, Hamilton, NY_.

Cell migration is a common process in development, wound healing, normal immune response, and can also occur abnormally in cancerous cells. Migration is coordinated by changes in actin arrangement. The family of proteins called Rho GTPases is known to regulate actin arrangement. How these specific Rho GTPases are activated is cell-context specific and not well known. Krüppel-like factor 4 (KLF4) is a transcription factor that regulates genes involved in proliferation, differentiation, and maintaining cell tissue homeostasis. In our study, we aimed to investigate the role of KLF4 in migration and invasion by evaluating changes in actin filaments and expression of key proteins involved in cell migration.

Klf4 is often mutated in colorectal, gastric and pancreatic cancers types although the underlying mechanisms are not well understood. From our research, we found that Klf4 null MEFs exhibit higher expression of actin structures associated with migration as compared to wildtype. This finding indicates that the increased migration in Klf4 null MEFs could be due to changes in actin arrangement. Specifically, we found that Klf4 null MEFs express higher amounts of Rho, as compared to wildtype MEFs. Our results indicate that KLF4 may act as a regulator of Rho expression, where cells with Klf4 inhibit stress fiber formation.

While movement across 2D surfaces closely resembles migration, it does not encompass the complex process of invasion, a potential precursor for cancerous cells. In order to assess whether Klf4 null MEFs exhibit invasive properties, 3D invasion assays containing a collagen basement membrane were used to mimic the movement of cells from one tissue layer into another. Since Klf4 null MEFs show increased invasion, differences in actin arrangement and expression of Rho in the isolated invasive cells is be crucial in proving the importance of KLF4 in preventing cell migration and invasion.

In order to evaluate the mechanism by which this occurs, we have begun to look at a known pathway in cell migration: the TGF-β/MMP pathway. We found that the SB-505124 inhibitor of TGF-β, showed decreased stress fiber formation and Rho expression in Klf4 null MEFs when compared to wildtype MEFs. In addition, the matrix metalloproteinase MMP3, has also been found to be upregulated in Klf4 null MEFs, demonstrating the role of KLF4 as a tumor suppressor to prevent migration. From these preliminary results, we hope to continue investigating the connection between KLF4 and TGF-β in order to determine a model by which KLF4 mediates cell migration and invasion.

#704

Hyperglycemia stimulates glycolysis and enhances migratory/invasive potential in tongue squamous cell carcinoma.

Anxun Wang,1 Wei Wang,1 Luodan Zhao,1 Qianting He,1 Jingjing Sun,1 Zhonghua Liu,1 Zujian Chen,2 Xiaofeng Zhou2. 1 _First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China;_ 2 _University of Illinois at Chicago, Chicago, IL_.

Objective: The aim of this study was to investigate the role of glycolysis (HK2/PKM2) in the development of tongue squamous cell carcinoma (TSCC) and elucidate how hyperglycaemia stimulate HK2 and PKM2 and enhance migration/invasion potential in TSCC.

Methods: A total of 501 patients which diagnosed as TSCC between 1998 and 2004 were enrolled in the retrospective study. The expression of HK2/PKM2 was examined by IHC and the relationship between TSCC and diabetes mellitus (DM) was investigated. We then investigated the role of HK2/PKM2 in the migration and invasion of TSCC and relevant pathways (the SOD2-H2O2 pathway and miR-138 pathway). Finally, we investigated whether hyperglycaemia enhance migratory/invasive potential of TSCC through stimulating glycolysis.

Results: The prevalent rate of TSCC patients with diabetes was 13.97%, which was significantly higher than the incidence rate of DM (11.6%) in China. Cox analysis revealed that the HR for patients with DM vs without DM was 1.32. IHC results confirmed that the up-regulation of PKM2/HK2 expression was correlated with tumor stage, clinical stages, diabetes, lymph node metastasis and associated with reduced overall survival. PKM2/HK2/AKT1 overexpression increased cell migration and invasion, SOD2 activities and intracellular H2O2. The siRNA-based PKM2/HK2 knockdown inhibited cell migration and invasion, reduced activities of SOD2 and intracellular H2O2, and also inhibited tumor growth and lung metastasis in vivo. Luciferase assays revealed that PKM2/AKT1 was directly targeted by miR-138 and miR-138 can reduce the expression of the PKM2/AKT1 gene. High glucose enhanced the migration/invasion ability, miR-138/PKM2/HK2 expression and increased SOD2 activities and intracellular H2O2.

Conclusions: Our results confirm that DM may be a risk factor in the development of TSCC. PKM2/HK2 plays an important role in the progression of TSCC, and may serve as a biomarker or therapeutic target for patients with TSCC. High glucose stimulates glycolysis (PKM2/HK2) and enhances migration/invasion potential in TSCC.

#705

PRL-3/PTP4A3 phosphatase promotes uveal melanoma cell migration through the regulation of integrin β1 in focal adhesion.

Malika Foy, Oceane Anezo, Nathalie Planque, Simon Saule. _Institut Curie, Orsay, France_.

Using transcriptomic analysis, we found that Phosphatase of Regenerating Liver-3 (PRL-3) overexpression is highly correlated with metastatic tumors and predicts poor prognosis in patients with uveal melanoma. PRL-3 is a dual specific phosphatase with a carboxy-terminal farnesylation motif, allowing anchorage to the plasma membrane. PRL-3 overexpression in ocular melanoma cell line-1 (OCM1) significantly increases cell migration in vitro and invasiveness in vivo, suggesting a direct role of PRL-3 in metastasis (Laurent et al, Cancer Res 2011; 71:666). Despite its role in the metastatic process of various cancers, its mechanism of action and its intracellular substrates remain largely unknown. It has been shown that the anchorage of PRL-3 to the plasma membrane plays an important role in the metastatic process, suggesting that the phosphatase regulates transmembrane substrates. Among potential targets involved in extracellular matrix adhesion, we focused on integrin β1 which we found overexpressed in the transcriptomic analysis of our uveal melanoma cohort.We showed that FTI-277, a farnesyltransferase inhibitor that prevents PRL-3 anchorage to the plasma membrane, abolishes PRL-3-induced OCM1 cell migration on collagen I in vitro, specifically enhances the spreading of OCM1 cells overexpressing PRL-3, and allows the formation of large focal adhesions involving integrin β1. Knockdown of integrin β1 in OCM1-PRL3 cells partially restores the spreading and migration. Furthermore, in focal adhesions, we observed that PRL-3 specifically regulates the aggregation of integrin β1 but does not affect integrin β3. Our results suggest the involvement of integrin β1 in PRL-3-mediated cell migration. To go further, we are currently investigating the phosphorylation status of specific integrin β1 residues, known to be involved in cell adhesion. The present study highlights the importance of PRL-3 membrane localization in uveal melanoma cell migration, by regulating adhesion to the extracellular matrix through integrin β1.

#706

Chemotherapy and inflammation show a p65- and STAT3-dependent pro-tumorigenic potential.

Federica Alessandrini, Laura Pezze, Francesca Precazzini, Silvia D'Ambrosi, Dennis Pedri, Lia Pinto, Yari Ciribilli. _CIBIO, University of Trento, Povo (TN), Italy_.

Tumor microenvironment (TME) plays a critical role in cancer progression and response to therapies. Therapeutic intervention strategies for cancer treatment can induce inflammation, resulting in changes in the TME. To investigate the response of cancer cells to chemotherapy in the context of an inflammatory microenvironment, we studied the effects driven by the chemotherapeutic drug doxorubicin, and the inflammatory cytokine TNFα.

We previously demonstrated using expression microarrays experiments (validated also through qPCR) that the Doxo+TNFα combined treatment determined a strong up-regulation of migration-related genes as well as increased motility in breast cancer cells (MCF7).

We confirmed the synergistic activation of a group of 6 different genes upon Doxo+TNFα combined treatment in different cell line models coming from different cancer types (A549-lung, U2OS-osteosarcoma). Moreover, we demonstrated that this effect was only partially p53-depenedent but strongly Doxo-dependent using p53 mutated MDA-MB-231 breast cancer cells or knocking-out p53 in MCF7 or U2OS cells through the cutting edge technology CRISPR/Cas9.

We also demonstrated that the combined Doxo+TNFα treatment was not only able to disrupt the 3D architecture of mammary acini observed with MCF10A primary cells grown within matrigel, but also to stimulate the tube-forming potential of Human Umbilical Vein Endothelial Cells (HUVEC).

Furthermore, a signature of Doxo+TNFα highly synergistic genes (DT-29) was shown to exhibit prognostic value for luminal breast cancer patients, with adverse outcome correlating with higher relative expression (based on Kaplan-Meier plotter tool). We further used available experimental data sets (RNA-seq measurements from Gene Expression Omnibus) both from breast cancer cell lines (luminal-like vs basal-like breast cancer cells) and breast cancer patients (ER positive vs Triple Negative Breast Cancer, TNBC and vs healthy adjacent tissues). The expression of DT-29 gene signature was analyzed and compared among the different groups of samples. The most statistically relevant genes differentially expressed among the different groups were involved in the STAT3-driven pathways and were prevalently highly expressed in TNBC patients and cell lines.

In order to demonstrate a STAT3-dependent regulation of up-regulated genes upon Doxo+TNFα combined treatment, we pharmacologically inhibited STAT3 using Stattic or we knocked-out STAT3 using CRISPR/Cas9 in MCF7, MDA-MB-231 and U2OS cells. Results demonstrated a STAT3-dependent up-regulation for some of the selected genes and STAT3 inhibition resulted also in a reduced migration potential particularly in U2OS cells.

We propose that the combined treatment with Doxo+TNFα can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy.

#707

High expression of Pol ι promotes invasion and metastases in esophageal squamous cell carcinoma.

Shitao Zou,1 Jinchang Wu,1 Wei-Qun Ding,2 Jundong Zhou1. 1 _Suzhou Municipal Hospital, Suzhou, China;_ 2 _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

.

DNA polymerase iota (Pol iota) is an error-prone DNA polymerase involved in translesion DNA synthesis (TLS) that may play a significant role in the accumulation of DNA mutations. Our previous studies identified the elevated expression of Pol iota in human esophageal squamous cell cancer (ESCC) tissues and revealed that Pol iota contributes to ESCCs' progression. The present study aimed to investigate the molecular mechanism by which Pol iota enhances the invasiveness and metastasis of ESCC cells. We found that the expression of Pol iota was higher in ESCCs with lymph node metastasis compared to those without lymph node metastasis. Kaplan-Meier analysis revealed a negative correlation between the expression of Pol iota and patients' prognosis. Furthermore, the expression of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9), both being essential regulators for cells invasion, were associated with that of Pol iota in ESCCs tissue samples. The wound healing and transwells assay revealed that over-expression of Pol iota enhances motility and invasiveness of ESCC cells. In vivo, up-regulation of Pol iota promoted the colonization of ESCC cells in the liver, lung and kidney. Signaling pathway analysis showed that Pol iota induces the expression of MMP-2/9 and enhances the ESCCs progression via JNK-AP-1 cascade. Altogether, our finding demonstrated the underlying mechanism by which Pol iota promotes ESCCs' development.

#708

**Defensin-alpha 6 (DEFA6) as a protumorigenic function** in vitro **and prognostic marker in colorectal cancer.**

Dongjun Jeong,1 Hyungjoo Kim,1 Seunghyun Oh,1 Seona Ban,1 Sanghee Ji,1 Han Jo Kim,1 Tae Sung Ahn,1 Tae Hyun Kim,2 Hyog Young Kwon,1 Seob Jeon,1 Sang Byung Bae,1 Chang-Jin Kim,1 Moon Soo Lee,1 Moo-Jun Baek1. 1 _College of Medicine, Soonchunhyang Univ., Cheonan-si, Republic of Korea;_ 2 _College of natural science, Soonchunhyang Univ., Cheonan-si, Republic of Korea_.

Objective

DEFA6 also known as human alpha defensing 6 (HD6) is a member of the alpha defensins family that defends the host against bacteria and viruses. Expression of DEFA6 is enhanced in colorectal cancer tissue and cells. The mechanism and function of DEFA6 have not been reported to play an important role in carcinogenesis and cancer progression. The goal of this study was to evaluate the protumorigenic functions of DEFA6 in the colorectal cancer cell line and to evaluate the clinical significance of DEFA6 expression in colorectal cancer patients.

Methods

DEFA6 was highly expressed in colorectal cancer cell lines. Colorectal cancer cell, HCT116, expressing high level of DEFA6 was used for the biological roles of DEFA6 by siRNA transfection. The protumorigenic functions of DEFA6 was evaluated by MTT assay, migration and invasion transwell assay and plate colony forming assay. DEFA6 expression was investigated by immunohistochemistry in 60 cases of colorectal cancer tissue and the association of DEFA6 expression was correlated with patient's survival.

Results

Immunohistochemistry analysis showed that the DEFA6 protein was expressed higher in death group (survival <5 years) than survival group (survival >5 years), (p=0.02). The HCT116 of knockdowned DEFA6 by siRNA showed significant decrease in proliferation by MTT assay (p<0.05) and plate colony forming assay (p=0.00). Also, the ability of migration and invasion was significantly reduced in DEFA6 knockdowned HCT116 compared to control HCT116 cell (both, p<0.05).

Conclusion

DEFA6 plays important roles in invasion and migration of colorectal cancer cell according to the functional study. As patient's survival mostly depends on the migration and invasion of the tumor cells, the high expression of DEFA6 in colorectal cancer is associated with patient's survival and could be a good prognostic marker.

#709

In vitro **functional study of novel oncogene serine protease 33 (PRSS33) and the clinical significance of PRSS33 expression in colorectal cancer patients.**

Dongjun Jeong,1 Seona Ban,1 Hyungjoo Kim,1 Seunghyun Oh,1 Sanghee Ji,1 Han Jo Kim,1 Tae Sung Ahn,1 Tae Hyun Kim,2 Hyog Young Kwon,1 Seob Jeon,1 Sang Byung Bae,1 Chang-Jin Kim,1 Moon Soo Lee,1 Moo-Jun Baek1. 1 _College of Medicine, Soonchunhyang Univ., Cheonan-si, Republic of Korea;_ 2 _College of Natural Science, Soonchunhyang Univ., Cheonan-si, Republic of Korea_.

Background: PRSS33, one of serine protease multigene family, has central roles in the regulation of a wide variety of physiological processes, including inflammation, development and malignancy. However, the function of this gene in colorectal cancer has not been elucidated. The goal of this study was to evaluate the oncogenic functions of PRSS33 in the colorectal cancer cell line and to evaluate the clinical significance of PRSS33 expression in colorectal cancer patients.

Method: PRSS33 was highly expressed in colorectal cancer cell lines, HCT116, SW480 and SW620. The oncogenic functions were evaluated in the cell lines by knocking down PRSS33 with siRNA transfection and compared them with PRSS33 highly expressing control cell lines. The functional studies included cell proliferation assay, invasion assay, migration assay and anchorage-independent semisolid agar colony forming assay. The clinical significance of PRSS33 expression was evaluated in 92 cases of colorectal cancer tissue by immunohistochemistry.

Results: The PRSS33 knockdowned cell lines by siRNA transfection showed significant decreases of proliferation, invasion, migration compared to those of control (p<0.05) respectively. The oncogenic function of PRSS33 was confirmed by anchorage-independent semi-solid agar colony forming assay. The PRSS33 knockdowned cell lines revealed lower colony formation on semisolid agar compared to the PRSS33 highly expressing control cell lines. The disease-free survival rate was decreased in patients of PRSS33 high expression (p=0.001). The overexpression of PRSS33 was associated with survival and death by chi-square test (p=0.002). Multivariate Cox-regression analysis showed an association between PRSS33 expression and prognosis (HR=2.71, 95% CI=1.39-5.27: p=0.003).

Conclusion: This study indicates that PRSS33 is a novel pro-oncogene and the expression is an independent prognostic factor in colorectal cancer patients. In the future, research on the oncogenic signal pathway of PRSS33 in colorectal cancer is necessary.

#710

Epidermal growth factor-induced pyruvate dehydrogenase kinase 1 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation.

Ben-Kuen Chen,1 Jinn-Yuan Hsu,1 Wen-Chang Chang2. 1 _National Cheng Kung Univ., Tainan, Taiwan;_ 2 _Taipei Medical University, Taipei, Taiwan_.

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of pyruvate dehydrogenase kinase 1 (PDK1) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced PDK1 expression in HNSCC. The tumor cell transformation induced by EGF was repressed by the depletion of PDK1. The down-regulation of PDK1 expression or inhibition of its activity significantly blocked EGF-enhanced cell migration and invasion. In addition, depletion of PDK1 impeded EGF-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastastic seeding of the lungs. Knockdown of PDK1 also inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. These results demonstrate that EGF-induced PDK1 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of PDK1 may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.

#711

The essential role of NKX6.3 in gastric cancer cell migration and invasion through targeting of Wnt/β-catenin and Rho-GTPase signaling pathways.

Jung Hwan Yoon, Won Suk Choi, Won Sang Park. _College of Medicine, The Catholic Univ. of Korea, Seoul, Republic of Korea_.

Despite ongoing research and recent progress, the prognosis for patients with advanced gastric cancer remains poor. Wnt/β-catenin and Rho-GTPase signaling pathways are known to play essential roles in malignant transformation and progression of various tumours, including gastric cancer. Here, we identify that NKX6 transcription factor, locus 3 (NKX6.3) binds directly to specific promoter regions of Wnt/β-catenin and Rho-GTPase pathway-related genes, resulting in inhibition of cancer cell migration and invasion. Additionally, we find that the expression level of NKX6.3 is involved in regulation of gastric cancer progression and expression of Wnt/β-catenin and Rho-GTPase pathway-related genes in clinical samples. These results suggest that NKX6.3 prevents EMT and cell migration, implying that NKX6.3 inactivation might be one of the key mechanisms of gastric cancer cell invasion and metastasis and might serve as a potential candidate for targeted gastric cancer therapy. 

### Pro-Tumorigenic Microenvironment

#712

Mechanisms of stromal Lkb1 loss induced tumorigenesis in mouse models of Peutz-Jeghers syndrome.

Saara Ollila, Kari Vaahtomeri, Iris Wong, Kaisa Laajanen, Tomi P. Mäkelä. _University of Helsinki, Helsinki, Finland_.

Germline mutations in tumor suppressor kinase Lkb1 predispose to Peutz-Jeghers syndrome (PJS), with highly penetrant gastrointestinal polyposis and increased cancer risk. PJS polyps display abnormal growth of both stromal and epithelial cells. We have identified clonally expanding fibroblasts as the drivers of tumorigenesis in PJS mouse models by using Fsp1-Cre and Twist2-Cre mice to restrict Lkb1 deletion to stromal cells (unpublished), highlighting the importance of stromal-epithelial signaling in PJS tumorigenesis.

Here, we address the molecular mechanisms of the pathogenesis linked to stromal Lkb1 loss in PJS mouse models. Lkb1 is involved in tissue size control by inhibiting of mTORC1 pathway via AMPK (Shaw et al, Cancer Cell 2004) and by regulating Hippo pathway (Mohseni et al, Nat Cell Biol 2014). First, we investigated the involvement of Lkb1-AMPK-mTORC1 pathway in polyposis. We conditionally deleted both catalytic AMPKα subunits (α1 and α2) in mice using Fsp1-Cre. Surprisingly, Fsp1-Cre-AMPK mice did not develop any tumors by 17 months of age indicating that AMPK is not the critical mediator of Lkb1 tumor suppression in PJS. We next studied the Hippo pathway in polyps of PJS mouse models. Levels of Yap and Taz, the main downstream effectors of Hippo, were elevated in polyps as shown by Western blotting. Immunohistochemical staining revealed profound nuclear Yap/Taz localization indicating transcriptional activity in the stromal compartment throughout the polyps. In contrast, nuclear Yap/Taz staining in epithelial cells was only noted in a restricted polyp base stem cell zone. In addition, we performed RNA-seq analysis of the of Fsp1-Cre;Lkb1flox mouse polyps and noted shared gene expression changes with PJS patient polyps. RNA-seq also revealed significant enrichment of Yap signature, which was validated by qPCR. Finally, we used small intestinal epithelial organoid culture to study the potential of Yap/Taz induced secreted factors to stimulate growth of epithelial cells. We observed that Wnt5a and Epiregulin enhanced epithelial organoid growth, indicating them as candidates mediating the stromal-epithelial signaling and promoting tumorigenesis in PJS polyps.

In conclusion, we show that stromal Lkb1 mutations lead to phenotypes identical to germline Lkb1 heterozygosity in mice and propose increased stromal Yap/Taz activity as a potential mechanism to drive PJS tumorigenes.

#713

Determining the role of tumor-infiltrating B cells in NSCLC.

Sara M. Centuori, Samuel Kim, Cecil Gomes, Charles Putnam, David Mount, Linda Garland, Brandon Larsen, Jesse D. Martinez. _University of Arizona, Tucson, AZ_.

Recent studies in human non-small cell lung cancer (NSCLC) have shown that upregulation of B cell-associated genes strongly correlate with early stage patient survival. After analyzing a gene expression database of lung tumors we showed that CD79A, a pan-B cell marker, had the strongest predictive value, suggesting that B cells are playing a crucial role in NSCLC immunity. In order to evaluate this we first examined where this genetic signature was originating from. We observed that patients with high levels of B cell-related genes also showed high numbers of CD79A+ B cells intimately associated with the tumor, but not being expressed by tumor cells themselves. A closer look at tumor-infiltrating B cells (TIL-B cells) by IHC and flow cytometry of fresh patient tumor samples has confirmed their presence and allowed us to begin elucidating their phenotype. Interestingly, we have found that not all early stage patients have detectable levels of TIL-B cells, but in those that do, TIL-B cells represent a marked proportion of the lymphocytic infiltration. Further evaluation of T cell populations in the same samples indicate that T cell numbers remain relatively consistent but that the numbers of B-cells, especially CD79A+ B-cells, does vary from patient to patient. These data indicate that CD79A+ TIL-B cells are contributing to the generation of an efficient immune response in some but not all patients, and are greatly influencing disease survivability. This information may allow us to stratify patients into low and high risk groups based on the presence or absence of TIL-B cells, respectively.

#714

Macrophage-to-fibroblast transition promotes cancer progression in peritoneal carcinomatosis of gastrointestinal cancer patient.

Mamoru Tanaka,1 Michitaka Nakano,1 Hiroshi Ariyama,1 Kyoko Inadomi,1 Risa Tanaka,2 Shigeo Takaishi,1 Hitoshi Kusaba,1 Eishi Baba,1 Koichi Akashi1. 1 _Kyushu University, Fukuoka, Japan;_ 2 _Hamanomachi Hospital, Fukuoka, Japan_.

[Background] Cancer stromal cell plays an important role in cancer progression. Fibroblasts localized in tumor are especially called cancer-associated fibroblasts (CAFs). CAFs and inflammatory cells form tumor microenvironment and promote cancer growth through the direct or indirect interaction between cancer cells and stromal cells. However, the origin of CAF is not fully understood. Malignant ascites contains not only cancer cells but inflammatory cells including macrophage. Accumulation of macrophages and fibrosis has close relationship. In the research field of fibrotic diseases such as renal fibrosis, some reports indicated macrophages were able to change to fibroblasts phenotypically. Peritoneal carcinomatosis also develops peritoneal fibrosis. We demonstrate that malignant ascites are abundant in macrophages and these macrophages changed to CAFs which promote cancer progression in vivo. [Material and method] Ascitic samples from 44 peritoneal carcinomatosis patients due to gastrointestinal cancer were collected at 5 institutions. This study was approved by each institutional review board. Ascites was separated into cell fraction and supernatant by centrifugation. Supernatant was stored at -20°C. Cells were sorted by FACS using anti-CD45, anti-CD14, anti-CD163 and anti-CD90 antibodies. CD45+CD14+ macrophages were cultured in RPMI medium containing fetal bovine serum (FBS) or supernatant of ascites. Human colorectal cancer cell line DLD-1 cells in combination with the cultured cells from ascites were inoculated to immunodeficient mice subcutaneously. All experiments were conducted following the guidelines of the institutional animal committee of Kyushu University. [Result] CD45+CD14+ macrophage was most frequently observed in CD45+ leukocyte fraction from ascites. Most of macrophages expressed M2 marker (CD163). Some of these macrophages changed to CD45-CD90+ fibroblast-like cells which form spindle shape after 2-3 weeks culture. These fibroblast-like cells expressed fibroblast specific genes such as COL3A1, ACTA2 and FAP. These changes were enhanced by ascites supernatant-containing medium compared with FBS-containing medium. DLD-1 cells with the fibroblast-like cells formed larger tumors in immunodeficient mice, compared with DLD-1 cells alone. [Conclusion] In peritoneal carcinomatosis, macrophage is a potential source of CAF. This macrophage-to-CAF transition is enhanced by malignant ascitic environment. As CAF induced from macrophage enhances tumor progression, inhibition of this transition could be possible therapeutic strategy.

#715

Adipose tissues derived exosomal microRNAs and their variants in ovarian cancer progression.

Chi Lam Au Yeung, Tetsushi Tsuruga, Ngai Na Co, Tsz-Lun Yeung, Cecilia S. Leung, Kwong K. Wong, Samuel C. Mok. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Most ovarian cancers are diagnosed at an advanced stage when the tumor is widely metastatic. The 5-year survival drops to 50% for the cancer cases that spread beyond the pelvis to the omentum. However, the mechanisms underlying the effect of omental adipose tissue on ovarian cancer progression are poorly understood. Recent studies showed that exosomes also contain non-coding RNAs such as microRNAs (miRNAs). Thus, we hypothesize that the transfer of microRNAs and their variants from ovarian cancer-associated omental adipose tissues to ovarian cancer cells via exosomes may contribute to the nearby microenvironment for ovarian cancer metastasis and cancer progression.

Ion Torrent next generation sequencing was performed on miRNAs isolated and enriched from exosomes and cell lysates of ovarian cancer cell lines (OVCA), the epithelial component of microdissected omental ovarian cancer tissues (CT), normal omental adipose tissues (OMN) and ovarian cancer-associated omental adipose tissues (OMT). By integrating the miRNA expression profiles, 65 miRNAs were expressed at significant higher levels in OMT-derived exosomes compared with those in OMN-derived exosomes and OVCA-derived exosomes. A set of miRNAs (miR-32a, miR-221 and miR320a), which had been implicated in controlling cell growth and chemoresistance, was identified. Also, the Ion Torrent results were validated and exosomal transfer of OMT-derived miRNAs was confirmed in vitro. The exosomal communication between adipose tissues and ovarian cancer cells in the omental tumor microenvironment is verified. The transferable miRNAs and their variants may remain functional in the recipient ovarian cancer cells and confer more aggressive phenotypes in these cells.

#716

STAT1 expression in the tumor-stroma microenvironment is influenced by loss of PTEN in prostate cancer.

Thiago Vidotto,1 Clarissa G. Picanço-Albuquerque,1 Fabiano P. Saggioro,1 Rodolfo Borges dos Reis,1 David Robert Siemens,2 Madhuri Koti,3 Jeremy A. Squire3. 1 _São Paulo University, Ribeirão Preto, Brazil;_ 2 _Kingston General Hospital, Kingston, Ontario, Canada;_ 3 _Queen's University, Kingston, Ontario, Canada_.

The prostate cancer (PCa) microenvironment is an admixture of stromal cells, such as lymphocytes, fibroblasts, myofibroblasts and blood vessel endothelium. The reactive stromal microenvironment of PCa facilitates tumor growth through cytokine and chemokine-mediated cell signaling. STAT1, a transcription factor, induces cell cycle arrest, apoptosis and modulates immune and inflammatory response through IFN-α/γ cytokine activation. IFN-α/γ may be secreted in response to PI3K/AKT activation, since this pathway is associated with JAK/STAT signaling. In addition, the PI3K/AKT pathway can become activated when the PTEN prognostic biomarker is deleted in PCa. The rationale for this project is that the immediate stromal microenvironment of PCa may become pro-tumorigenic as a result of induction of inflammatory cytokines caused by PTEN deletions and activation of PI3K/AKT. We performed FISH analysis of the PTEN gene on 172 cores on a PCa tissue microarray (TMA) derived from 43 patients with intermediate risk disease. Analysis of cytoplasmic and nuclear STAT1 in both the tumor and stromal compartments was performed on each TMA core using immunohistochemistry (IHC) to determine whether the tumor PTEN deletion status was associated with STAT1 expression. In each core, tumor and stromal cell histologies were evaluated independently to estimate the percentage area that was stained positive by IHC. The staining was graded as percentage of positive cells with 0 (negative), 1 (less than 10%), 2 (11% to 50%), and 3 (more than 51%). We found that STAT1 expression in stroma was increased when the PTEN gene in the adjacent tumor was homozygously deleted (p=0,04). We also showed that mean STAT1 expression in benign tissue cores was reduced when compared to the average score from tumor tissue with one (p=0.01) or two copies (p=0.03) of the PTEN gene.

STAT1 expression and PTEN gene copy number

---

PTEN copy number | Mean STAT1 in Stroma | Mean STAT1 in Tumor

>2 copies | 0.41 | 0.82

2 copies | 0.51 | 0.65

1 copy | 0.60 | 0.70

0 copies | 0.85 | 0.85

These results suggest that the STAT1 pathway may be influenced by PTEN loss in this tumor, suggesting that the JAK/STAT inflammatory pathway may have future clinical applications in PCa.

#717

Monocytic and granulocytic MDSCs display distinct molecular properties and coordinate the dynamic switches between EMT-MET in breast cancer model.

Eunmi Lee,1 Maria Ouzounova,1 Raziye Piranlioglu,1 Abdeljabar El Andaloussi,1 Mehmet Demirci,1 Ena Novakovic,1 Alicia Hudson,1 Sumeyye Korkaya,2 Gang Zhou,1 Hasan Korkaya1. 1 _Georgia Regents University, Augusta, GA;_ 2 _University of Michigan, Ann Arbor, MI_.

It is widely accepted that the epithelial-mesenchymal plasticity of malignant cells is required during cancer metastatic cascade. The complex phenotypic changes highly depend on the collaboration of various molecular signaling and extracellular cues originating from wide range of stromal cells in the tumor microenvironment. However, the specific mechanisms of how EMT plasticity spatiotemporally regulates metastasis are poorly defined. Myeloid-derived suppressor cells (MDSCs) have been identified in most cancer patients and animal models due to their immune suppressive functions, but recent studies implicate their direct role in promoting metastasis by activating tumor-angiogenesis. To determine the roles of MDSCs in breast cancer metastasis, we utilized murine breast cancer cells, non-metastatic EMT6 and metastatic 4T1 cells. We showed that the metastatic 4T1 murine breast tumors induced early systemic expansion and mobilization of MDSCs in distant sites as well as in the primary tumor. We investigated the direct functions of MDSCs in tumor progression by isolating monocytic and granulocytic MDSCs from primary tumor, lung and bone marrow of tumor-bearing mice and then they were co-cultured with non-metastatic EMT6 cells. We found that tumor infiltrating m-MDSCs from 4T1 tumor-bearing mice increased the expression of Vimentin, Twist1, TGF-β and IL-6 in EMT6 tumor cells. In contrast, flow cytometry sorted lung infiltrating MDSCs from 4T1 tumor-bearing mice enhanced the EpCAM expression and proliferation in EMT6 cells. Cell invasion assay showed that invasive ability of EMT6 cells were significantly increased when they were co-cultured with m-MDSCs while g-MDSCs slightly decreased the number of invaded cells, compared to control group. We utilized immunofluorescence staining and confirmed the increased expression of Vimentin, CK14 (cytokeratin 14) in EMT6 cells co-cultured with m-MDSCs. In contrast, g-MDSCs induced down-regulation of these markers while they increased cell proliferation as assessed by Ki67 staining. Furthermore, flow cytometry analysis showed the increased CD24+CD29+ population, a marker of murine cancer stem cell (CSC) phenotype, in EMT6 cells when co-cultured with m-MDSCs from 4T1 tumor-bearing mice. Tumor sphere assay confirmed that m-MDSCs enhanced sphere forming ability of tumor cells. Taken together, these data suggest that m-MDSCs derived from metastatic 4T1 tumor-bearing mice are able to confer EMT/CSC phenotype on tumor cells while g-MDSCs are more potent in inducing epithelial phenotype and proliferation in tumor cells.

#718

Macrophage PI3Kgamma signaling promotes cancer immune suppression.

Megan M. Kaneda, Judith A. Varner. _UCSD Moores Cancer Center, La Jolla, CA_.

Innate immune cells can switch from a pro-inflammatory state that defends against pathogens and to an anti-inflammatory state that repairs damaged tissue. Pro-inflammatory myeloid cells activate cytotoxic T cells to eliminate pathogens, but anti-inflammatory myeloid cells inhibit T cell mediated immunity, stimulate angiogenesis, and stimulate fibrosis, all of which promote tumor progression. The predominant isoform of PI(3)Kinase in myeloid cells, PI3Kgamma, controls the switch between immune stimulation and immune suppression during inflammation and cancer. Myeloid cell PI3Kgamma and its effectors Akt1, mTor, and S6Kinase stimulate C/EBPbeta-dependent expression of immunosuppressive factors, including Arginase and TGFbeta, but suppress NFkappaB-dependent expression of pro-inflammatory cytokines such as IL12 and IFNgamma, thereby inducing tumor immune suppression and consequent tumor growth and metastasis.

In contrast, inhibition of PI3Kgamma activity by genetic or pharmacological means suppresses C/EPBbeta-mediated transcription of anti-inflammatory factors and stimulates NFkappaB-mediated transcription of the pro-inflammatory cytokines IL12 and IFNgamma, thereby restoring CD8+ T cell-dependent cytotoxicity that inhibits tumor growth and metastasis. Therefore, inhibitory targeting of PI3Kgamma indirectly restores cytotoxic T cell immune responses that elicit tumor suppression without unwanted side effects. Our studies highlight the strong therapeutic potential of targeting this kinase to control inflammation and cancer.

This work was supported by grants to JAV from the NIH (5R01CA126820), the Landon Foundation-AACR (12-60-27-VARN), the Lustgarten Foundation for Pancreatic Research and the Whitworth Foundation for Cancer Research.

#719

ICAM3 and CCL16, inflammation genes screened by high-throughput siRNA library, govern cancer stemness in malignant tumors.

Wenzhi Shen,1 Junling Xie,2 Renle Du,3 Shan Jiang,4 Xiaohe Luo,5 Huiwen He,2 Rong Xiang,3 Yunping Luo6. 1 _The International Collaborative Laboratory for Biological Medicine of the Ministry of Education,School of Medicine, Nankai University, Tianjin, China;_ 2 _Institute of Basic Medical Science, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China;_ 3 _Key Lab of Tumor Microenvironment and Neurovascular Regulation, Nankai University, Tianjin, China;_ 4 _School of Medicine, Nankai University, Tianjin, China;_ 5 _The 2011 Project Collaborative Innovation Center for Biological, School of Medicine, Nankai University, Tianjin, China;_ 6 _The International Collaborative Laboratory for Biological Medicine of the Ministry of Education,School of Medicine, Nankai University, Beijing, China_.

Cancer stem cells (CSCs) have the ability of both self-renewal and differentiation, were thought to be the main root of tumorigenesis, metastasis, recurrence and drug resistance. Current studies have focused on the maintenance of cancer stem cell abilities. Moreover, existing studies have shown that tumor associated inflammation microenvironment (TIM) play a pivotal role in different stages of tumor development. The linkage between inflammation and tumor stemness has been established. However, the evidence of TIM function on maintaining and nourishing CSCs properties remains insufficient. Here, we performed a high throughput siRNA interference platform to systemically screen out the inflammatory genes that regulate tumor stemness via OCT4 promoter linked reporter system in HMLE-Snail cells. 10 candidates were screened out which knockdown could both down-regulate OCT4 expression and decrease ALDH+ subpopulation. Furthermore, we validated the function of genes ICAM3 and CCL16 and found that knockdown ICAM3 or CCL16 inhibit cell migration, sphere formation in 231, A549 and HepG2 cell lines. We also found ICAM3 or CCL16 deficiency decrease side population and reduce chemo-resistance. In addition, mice bearing ICAM3 or CCL16 knockdown breast cancer cells develop smaller tumors and less lung metastases verse controls. Taken together, our study demonstrated that the inflammation related genes ICAM3 and CCL16 govern cancer cell migration and stemness in malignant tumors in vitro and in vivo, and that will provide new possible targets to CSCs for cancer therapy.

#720

Novel prognostic stromal subtypes in triple-negative breast cancer.

Crista Thompson,1 Sadiq M. Saleh,1 Nicholas Bertos,1 Mathieu Gigoux,1 Tina Gruosso,1 Margarita Souleimanova,1 Hong Zhao,1 Michael T. Hallett,2 Morag Park1. 1 _Goodman Cancer Centre, McGill University, Montreal, Quebec, Canada;_ 2 _McGill Centre for Bioinformatics, McGill University, Montreal, Quebec, Canada_.

Breast cancer is a heterogeneous disease in terms of presentation, morphology, molecular profile and response to therapy. Gene expression profiling has identified intrinsic molecular subtypes that are associated with clinical markers (ER, PR, HER2) as well as prognosis and survival. However, it is well established that the intrinsic molecular profiles of breast tumors are not sufficient to perfectly predict disease outcome. Increasing evidence indicates that characteristics of the breast stroma influence tumor progression and response to therapy. Previous work in our lab has demonstrated that gene expression signatures in human stroma can predict outcome of breast cancer patients independently of clinical parameters and molecular subtypes. In this study, we expand our findings by focusing on a previously underrepresented subset of breast tumors that have no detectable ER, PR or HER2 (termed Triple-Negative, TN). TN tumors, which represent approximately 15% of all breast cancers, are typically associated with poor outcome. However, the contribution of the stroma to the underlying heterogeneity of TN breast cancer and its corresponding influence on therapeutic response is not well understood. To address this, we isolated TN tumor epithelial and stromal tissues by laser capture microdissection and subjected them to gene expression profiling. Class discovery revealed distinct gene-clusters (stromal properties) which are associated with prognosis in TNBC whole tumor samples. Analysis of the genes comprising each stromal property suggests that the properties primarily represent the prevalence of distinct cell types, namely T cells, B cells, activated fibroblasts, and myoepithelial cells. Importantly, these properties are not mutually exclusive, i.e. some tumors are associated with multiple stromal properties. While confirming the heterogeneity of TN-associated stroma, this also indicates that a multi-parameter classification better reflects the true nature of the tumor microenvironment. This project provides the first integrated in-depth analysis of the contribution of tumor stromal processes to TN disease heterogeneity, and positions the tumor microenvironment for therapeutic intervention.

#721

Opposite roles of CD11b+ myeloid cells in primary and recurrent brain tumors.

Wu Sheng-Yen. _National Tsing-Hua University, Taoyuan, Taiwan_.

Malignant glioma is one of the toughest tumors to be treated at present due to the complexity of the tumor microenvironment and the intrinsic resistant to irradiation. Previous studies have shown that CD11b+ myleoid cells play essential role in brain tumor recurrence following radiation therapy. Here we used the CD11b-diphtheria toxin receptor (CD11b-DTR) transgenic mouse model to evaluate the role of CD11b+ myleoid cells in TK/GCV suicide gene therapy for brain tumor in a murine astrocytomal tumor model, ALTS1C1-TK. Using this transgenic mouse model, the depletion of peritoneal macrophages (CD11b+F4/80+) and blood monocyte (CD11b+Ly6G-Ly6c-) could be achieved after two injections of DT, but the neutrophil (CD11b+Gr-1+) were increased transiently. Results found that the depletion of CD11b+ myleoid cells enhanced the efficacy of TK/GCV therapy as shown by the increase of median surviving time of GCV-treated ALTS1C1-TK tumor-bearing mice from 30.6 days to 37.5 days. Interestingly, the depletion of CD11b+ myleoid cells enhanced tumor growth as shown that tumor-bearing mice had shorter surviving days and larger tumor size after receiving two doses of DT injections compared to the control PBS group (22.4 days vs 25.0 days, respectively). This indicates that depleted CD11b+ myleoid cells might have anti-tumor function for control tumor, but have pro-tumor activity during TK/GCV therapy. This study also found the effect of CD11b+ myleoid cells was only seen when the depletion was performed during the administration of GCV, but not before or after GCV treatment. Collectively, above findings suggest that CD11b+ myleoid cells might have different roles on primary and recurrent brain tumors.

#722

MYB promotes pancreatic tumor associated-desmoplasia by up-regulating Sonic hedgehog and adrenomedullin.

Arun Bhardwaj,1 Sanjeev K. Srivastava,1 Nikhil Tyagi,1 Sumit Arora,1 Seema Singh,1 James E. Carter,2 Ajay P. Singh1. 1 _Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL;_ 2 _Department of Pathology, College of Medicine, University of South Alabama, Mobile, AL_.

Pancreatic cancer (PC) stands as the fourth leading cause of cancer-related death in the United States. Unfortunately, even with the emergence of tumor-targeted therapies, the 5-year survival rate of PC continues to remain very dismal (̴ 7.0 %). The presence of dense desmoplastic region at the tumor site is a prominent pathological characteristic of PC, and tumor-associated desmoplasia is suggested to limit the therapeutic efficacy by acting as a physical barrier for drug delivery. In addition, it plays pivotal roles in promoting the survival and aggressive phenotypes of tumor cells. Therefore, it is of utmost importance that we identify novel gene targets and associated molecular mechanisms accountable for the development of desmoplasia in pancreatic tumors. Our recent study provided first evidence for a role of MYB, an oncogenic transcription factor, in pancreatic tumor growth and metastasis. Upon histological examination of orthotopic tumor xenografts derived from MYB-overexpressing and -silenced pancreatic tumor cells, we noticed a positive association of MYB expression with the extent of desmoplastic reaction. This finding was further confirmed by staining the tumor xenograft sections with Collagen-I, fibronectin and α-SMA, the well-characterized markers of desmoplasia. In a co-culture experiment, we observed that MYB-overexpressing pancreatic tumor cells promoted greater proliferation of pancreatic stellate cells (PSCs) as compared to the MYB-silenced cells further supporting a role of MYB in desmoplasia. From the mechanistic standpoint, we identified MYB to be novel direct regulator of Sonic hedgehog (SHH) and Adrenomedullin (ADM) expression in PC cells as confirmed by chromatin immunoprecipitation (ChIP) analysis. Treatments of pancreatic tumor (high or low MYB) and stellate cells co-culture with specific inhibitors and/or recombinant SHH and ADM demonstrated that both SHH and ADM cooperatively mediated the effect of MYB on PC and stellate cells via autocrine and/or paracrine mechanisms. Together, our studies suggest that MYB promotes malignant phenotype of PC not only by directly impacting the tumor cells, but also by altering the tumor microenvironment.

#723

Distinguishing macrophages by a unique gene signature to measure response to treatment.

Yasmin A. Lyons, Sunila Pradeep, Jean M. Hansen, Rebecca A. Previs, Hui Yao, Keith A. Baggerly, Anil K. Sood. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Objectives: Tumor-associated macrophage infiltration is associated with poor prognosis, making macrophage depletion an ideal therapy. This study aims to identify a macrophage-specific gene signature to quantify macrophage content and measure response to treatment of high-grade serous cancer (HGSC) with colony stimulating factor-1 receptor (CSF1R) inhibitor.

Methods: 39 murine genes were identified in the literature as being macrophage-specific. Corresponding human orthologous genes were identified using BioMart database. Expression levels (log-transformed normalized RSEM values) of human homologous genes were retrieved from The Cancer Genome Atlas (TCGA) portal. Principal component (PC) and hierarchical clustering (HC) analysis of 305 primary HGSC was performed. Q-PCR was performed on human macrophage cell line, endothelial cell line, and fibroblast cell line. Q-PCR was also performed on tumor tissue obtained from CD57Bl/6 mice injected with murine ovarian cancer and treated with CSF1R inhibitor.

Results: 37 human homologous genes were identified for the macrophage specific murine genes. Of the 37 genes, 12 tracked together tightly and included the top 3 genes with greatest median absolute deviance of RSEM values, thus were chosen for further analysis. Q-PCR using human macrophage cell line revealed high expression in all 12 genes (delta CT <25), with TBXAS, CD14, and FCGR1A being the highest. All 12 genes showed greater expression in macrophages versus fibroblasts and endothelial cells, to varying degrees. Expression of CD14, TLR7, FCGR2C, and FCGR1B was significantly higher (p<0.0001) in macrophages compared to both fibroblasts and endothelial cells. Of these 12 human genes, 4 had homologous murine genes. These genes were used for Q-PCR on murine tumor tissue treated with CSF1R inhibitor. There was a statistically significant decrease in the level of CSF3R (p=0.005), FCGR (p=0.02), and PON3 (p=0.002) between the control group and the group treated with CSF1R inhibitor.

Conclusions: These data suggest that macrophage specific genes could be useful for quantifying macrophage content in tumor samples to monitor therapy-related changes.

#724

MicroRNA-100/mTOR/IL-1ra signaling maintains TAMs phenotype and enhances tumor cell stemness property in mouse breast cancer.

Wei Wang, Yan Liu, Jian Guo, Huiwen He, Junling Xie, Chong Chen, Yunping Luo. _Department of Immunology, Institute of Basic Medical Science, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China_.

Tumor-associated macrophages(TAMs),the main part of immune cells in tumor microenvironment(TME),play a potent role in promoting tumorigenesis. MicroRNAs (miRs) are considered to be crucial regulators in tumor progression. However, the role that miRs play in TAMs phenotype regulation still remains unclear.

In this study, we demonstrated that miR-100 played an important role in maintaining TAMs phenotype and enhancing tumor stemness properties. First, miRNome of TAMs isolated from mouse breast tumor was performed. Compared with macrophages from normal spleen, 40 miRs candidates, in 150 TAMs-high and 169 TAMs-low expressed miRs which were selected from miRNome, were further identified by real-time PCR. Importantly, miR-100 was found with TAMs-high expression pattern(about 70 folds changes). Moreover, we found that miR-100 overexpressed RAW264.7 cells and peritoneal macrophages gained M2 phenotype such as CD206+ cells percentage. Mechanism study demonstrated that miR-100 up-regulated IL-1ra expression detected by cytokine array. Furthermore we found that IL-1ra could enhance 4T1 breast cancer cell stemness properties including increasing tumor cell sphere formation and drug resistance in vitro.

Taken together, our results demonstrate that TAMs-high expressed miR-100 could function to maintain the phenotype of TAMs via targeting mTOR pathway, and promote tumorigenesis by enhancing IL-1ra secretion, which would also serve as a promising therapy target to remodel TME and tumor metastasis.

#725

HMGB1-mediated RAGE activation mechanism in M2 macrophages.

Armando Rojas,1 Paulina Araya,1 Jacqueline Romero,1 Fernando Delgado-López,1 Ramón Pérez-Castro,1 Ileana González,1 Carolina Añazco,1 Erik Morales,1 Jorge Llanos,1 Fernando Vidal-Vanaclocha2. 1 _Catholic University of Maule, Talca, Chile;_ 2 _CEU San Pablo University, Madrid, Spain_.

Tumor-associated macrophages display M2 phenotype and promote immunosuppression, tumor growth, angiogenesis and metastasis. We have previously reported that M2 macrophages express the receptor of advanced glycation end-products (RAGE) and their protumoral activities enhanced by HMGB1, a RAGE ligand highly abundant at the cancer microenvironment. However, RAGE is highly expressed by M1 macrophages and it is unknown if proinflammatory and antitumor cytotoxicity of M1 macrophages become skewed from classical antitumor activities to a tumor permissive profile when M2 macrophages are induced by HMGB1.

In order to clarify whether RAGE contributes to inflammatory response inhibition in M2 macrophages, we look at some microRNAs, a tolerance-like state as well as to some epigenetic changes. Polarized M1 and M2 macrophages were derived from wild-type and RAGE-targeted knock-down THP-1 cells and treated with HMGB1 at different times. NFkB activation was assessed by p65, IKBalpha western blots, and p50 and p65 Transcription Factor Assay. Additionally, we analyzed changes of chromatin activity related to HMGB1 treatment by quantitative chromatin immunoprecipitation (ChIP) with antibodies against acetylated or methylated histone H3 at the IL-10 promoter.

We showed that RAGE activation by HMGB1 did not produce NFkB activation, as assessed by either IKB alpha and phospho p65 western blots or by p65 and p50 trascription factor assays. In addition, HMGB1 did not modify M2 macrophage expression profile of relevant microRNAs for macrophage polarization such as miR-155, miR-21 and miR-125b. However, ChIP analysis showed that HMGB1 induced a transcription-prone histone modification at the IL-10 promoter in M2 macrophages and this epigenetic imprinting correlated with HMGB1-induced IL-10 production, leading to immunosupression. These results demonstrate that RAGE activation on M2 macrophages did not induce NFkB activation and suggest that epigenetic changes are a major mechanism by which activities described for M1 macrophages upon RAGE activation have become skewed during macrophage polarization.

#726

Involvement of proinflammatory chemokines in triple-negative breast cancer (TNBC).

Rosa Mistica C. Ignacio, Carla Gibbs, Eun-Sook Lee, Deok-Soo Son. _Meharry Medical College, Nashville, TN_.

Objective: Triple-negative breast cancer (TNBC) accounts for 12-24% of total breast cancer, and contributes to the more aggressive, therapy-resistant and poorer outcomes. Our analysis based on The Cancer Genome Atlas (TCGA) indicated that basal-like BC subtype representing TNBC has enriched expression profiles for epidermal growth factor receptor 1 (EGFR) and transforming growth factor alpha (TGFα) compared to other subtypes. In addition, TNBC has a higher proinflammatory index compared to non-TNBC. Here we report that the proinflammatory chemokines such as CXCL1-3 and 8 are highly expressed in human TNBC cells and might be potentiated by TGFα-EGFR axis that contributes to cancer progression of TNBC.

Methods: To gauge the role of TGFα-EGFR axis as a proinflammatory driver in TNBC cells, we first checked the expression profiles for EGFR family such as EGFR, HER2, ErbB3, and ErbB4 between TNBC (MCF10A, MB468, MB231, and BT549) and non-TNBC cells (MCF7, T47D). In addition, the expression levels of downstream signaling for EGFR (Akt/Erk) were measured by western blot. To dissect the role of TGFα-EGFR axis in enhancing proinflammatory index in TNBC cells, we utilized a more comprehensive study of the chemokines and chemokine receptors using human chemokine PCR array. Finally, to further confirm the role of TGFα-EGFR axis in inflammatory burden and cancer progression in TNBC, we used EGFR inhibitor and its downstream targets.

Results: TNBC cells expressed more proinflammatory chemokines such as CXCL1-3 and 8 compared to non-TNBC cells. TNBC cells (MB468, MB231, and BT549) showed higher expression of EGFR which led to higher Akt activation as compared to non-TNBC cells. However, Erk is only activated in MB231. To dissect the involvement of Akt, expression of Akt isoforms in TNBC cells was accessed and Akt1 is the predominant isoform expressed in TNBC cells. To further assess the involvement of Akt1 in proinflammatory burden in TNBC cells, we used a commercial siRNA of Akt1 to knockdown Akt1. Knockdown of Akt1 significantly reduced the CXCL2 promoter activity. In addition, afatinib (an EGFR inhibitor) and MK2206 (an Akt inhibitor) significantly reduced the CXCL2 promoter activity.

Conclusion: Thus, higher expression of proinflammatory chemokines CXCL1-3, and 8, via TGFα-EGFR axis-activated Akt, contributes to the inflammatory burden, probably promoting cancer progression, thereby followed by the overall high mortality in TNBC compared to non-TNBC.

#727

TSLP expression and high serum TSLP level indicate a poor prognosis in gastric cancer patients.

Joji Watanabe, Hiroaki Saito, Kozo Miyatani, Masahide Ikeguchi, Yoshihisa Umekita, Shinji Otani, Shunichi Tsujitani. _Tottori university, Yonago, Japan_.

Purpose: Thymic stromal lymphopoietin (TSLP) plays an important role in promoting tumor survival, by manipulating the immune response and angiogenesis. However, the clinical significance of TSLP in gastric cancer is unclear.

Method: Immunohistochemistry was used to investigate TSLP expression in non-cancerous gastric mucosa and gastric cancer tissue from patients with gastric cancer. Serum TSLP levels were measured using an enzyme-linked immunosorbent assay.

Results: Tumors with TSLP expression were significantly larger than those without TSLP expression. TSLP expression was observed more frequently in advanced (T2/T3/T4) than in early (T1) gastric cancer and in stage 3/4 than in stage 1/2. Lymph node metastasis, liver metastasis, positive peritoneal lavage cytology, lymphatic

invasion, and vascular invasion occurred significantly more often in TSLP-expressing than in non-expressing tumors. The prognosis of patients with TSLP-positive tumors was significantly worse than that of patients with TSLP-negative tumors. Patients with high serum TSLP concentrations also had a significantly worse prognosis than those with low concentrations. Multivariate analysis identified serum TSLP level as an independent prognostic indicator.

Conclusion: TSLP is closely related to the progression of gastric cancer and may predict

survival in these patients.

#728

Enhancement of survival and migration of neutrophils by tumor associated a2 isoform vacuolar ATPase.

Safaa A. Ibrahim,1 Arpita Kulshrestha,1 Mukesh K. Jaiswal,1 Gajendra K. Katara,1 Magdy A. Amin,2 Kenneth D. Beaman1. 1 _Rosalind Franklin University of Medicine and Science, North Chicago, IL;_ 2 _Cairo University, Faculty of Pharmacy, Cairo, Egypt_.

Tumors associated neutrophils (TAN) regulate the tumor environment by directly promoting tumor progression and represent a potential therapeutic target for cancer treatment. Identifying tumor associated factors that promote neutrophil recruitment and survival will uncover new targets for cancer therapy. In tumor cells, a secreted peptide from the Nterminal domain of a2 isoform vacuolar ATPase (a2NTD) promotes the protumorigenic properties of neutrophils which in turn, enhance tumor progression. In order to investigate the regulatory role of a2NTD on neutrophils survival, we stimulated freshly isolated neutrophils from healthy volunteers with recombinant a2NTD. a2NTD treatment delayed apoptosis in neutrophils and led to 2.1 fold increase in the percentage survival (P < 0.001). In addition, a2NTD treated neutrophils (a2Neuɸ) showed a significant decreased gene expression of caspase-3, -6, -7 and -8, that was associated with decreased activity of these caspases. Further analysis of the apoptosis related molecules revealed that a2NTD treatment upregulates the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL, c-FLIP, G-CSF and downregulates the gene expression of pro-apoptotic factors; BAX and Apaf-1. Since NF-kB pathway is an important regulator of apoptosis, here we showed by immunofluorescence analysis that a2NTD induces NF-kB p65 activation in neutrophils by nuclear translocation in a reactive oxygen species generation dependent manner. Additionally, treatment of a2Neuɸ with specific NF-kB inhibitor; parthenolide showed a significant abrogation in neutrophil survival as well as downregulation of the gene expression of Bcl2-A1, Bcl-xL, G-CSF and upregulation of BAX. Suggesting that a2NTD enhances neutrophil survival by activating NF-kB pathway in neutrophils. Interestingly, live imaging revealed that a2NTD stimulates neutrophil polarization which was confirmed by the increased assembly of F-actin. Also, flow cytometry analysis revealed that a2NTD treatment stimulates surface expression and activation of Mac-1 integrin. Moreover, important regulators of neutrophil migration; FAK and Src Kinases were activated in a2Neuɸ. Previous studies showed that IL-8; potent neutrophil chemoattractant is secreted by neutrophils. Here, we showed that a2NTD treatment led to upregulation of the gene expression and the secreted protein levels of IL-8 in a time dependent manner. This enhanced secretion of IL-8 was NF-kB dependent. Furthermore, to evaluate the influence of a2NTD on neutrophil migration we did trans-well migration assay. a2NTD stimulation caused 2.8 fold increase in neutrophil migration which decreased to 1.5 fold upon neutralizing IL-8 (P < 0.05). Together, these data demonstrate a novel role of the tumor associated a2-vacuolar ATPase on regulating neutrophil survival and migration, by the action of a2NTD.

#729

Novel role of CB2R in tuning breast tumor microenvironment, EGF/EGFR and IGF-I/IGF-IR pathways.

Mohamad Elbaz,1 Mohd Nasser,2 Janani Ravi,2 Dinesh Ahirwar,2 Ramesh Ganju1. 1 _Ohio State University, Columbus, OH;_ 2 _ohio state university, columbus, OH_.

Cannabinoid receptor-2 (CB2R) is an integral part of the endocannabinoid system. It is upregulated in the primary breast cancer lesions and in different types of immune cells however; its functional role in breast tumorigenesis is not well understood. The present study was aimed at evaluating the mechanistic anti-tumor role of CB2R activation on breast cancer cells and immune cells within the breast tumor microenvironment. First, we analyzed the anti-tumorigenic mechanisms of CB2R activation in ERα- and ERα+ breast cancer cells. Our studies showed that CB2R specific agonist (JWH-015) inhibits EGF and IGF-I-induced migration and invasion of ERα+ and ERα- breast cancer cells. At the molecular level, JWH-015 inhibits EGFR and IGF-IR activation and their downstream targets STAT3, AKT, ERK, NF-kB and MMP-9/MMP-2. Interestingly, We found that JWH-015 significantly reduces breast cancer growth in vivo and the tumors that were derived from CB2R agonist treatment showed reduced activation of EGFR and IGF-IR and their downstream targets compared to control group. Since CB2R is overexpressed in immune cells, we assessed for the role of CB2R activation on modulation of immune cells present in tumor stroma. We observed increased tumor weight, more myeloid derived suppressor cells (MDSCs) (CD11b+/Gr-1+) and less CD3+/CD8+ cells in orthotopically injected CB2R knock out mice compared to wild type mice. Furthermore, we found that JWH-015-treated wild type mice have reduced tumor growth and metastasis, more CD3+/CD8+ cells and less MDSCs within the tumor stroma. For the first time, we show that CB2R activation might suppress breast tumor growth and metastasis through novel mechanisms of inhibiting EGFR and IGF-IR signaling axes on tumor cells and modulating the immune cells' compositions within the breast tumor microenvironment.

#730

The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages.

NOBUHISA TAKASE, Yuichiro Koma, Noriaki Arai, Himiko Kodaira, Masayoshi Hosono, Yumi Ichihara, Mari Nishio, Manabu Shigeoka, Hiroshi Yokozaki. _Division of Pathology, Kobe Univ. Graduate School of Medicine, Kobe, Japan_.

Tumor-associated macrophages (TAMs) have important roles in the angiogenesis, tumor immunosuppression and tumor infiltration of various cancers. Previously, we reported that a large number of infiltrated tumor promoting CD204+ TAMs within esophageal squamous cell carcinomas (ESCCs) contributed to the significant association of clinicopathological malignancy and poor disease free survival. However, the relevant malignant progression mechanisms have not been studied explicitly yet. To elucidate roles of TAMs in ESCCs, we established an in vitro macrophage model using human peripheral blood acquired by healthy volunteer donors. Peripheral blood monocytes were treated with 25 ng/mL recombinant human M-CSF for 6 days to induce macrophage-like differentiation (macrophage-like cells), then exposed to the conditioned media of TE series ESCC cell lines (TECM) for 2 days to induce TAM-like cells. From the cDNA microarray data between macrophages-like cells treated with and without TECM, we interested in upregulated genes. Among highly expressed genes in TAM-like cells, we focused on neural cell adhesion molecule (NCAM) which was a possibility to act on the regulation between tumor and its surrounding stroma. Gene knockdown of NCAM by siRNA in TAM-like cells revealed significant suppression of cell survival and migration via decrease of PI3K/Akt signaling and downregulation of fibroblast growth factor receptor 1 (FGFR1) expression. Interestingly, fibroblast growth factor-2 (FGF-2) derived from ESCCs promotes FGF2/FGFR1 autocrine loop, and activates FGFR1 signaling in TAM-like cells. Furthermore, we analyzed 70 ESCC tissue samples by immunohistochemistry whether the expression levels of FGF-2 were associated with clinicopathological background factors of ESCC patients. Relationships of the expression levels of FGF-2 in human ESCC stromal cells including macrophages had a correlation with the depth of invasion, blood vessel invasion, TNM classification UICC cancer staging and high number of infiltrating CD163 positive and CD204 positive macrophages as a marker for the M2 macrophage phenotype. These findings may indicate novel roles of FGF-2-NCAM/FGFR1 interplay in ESCCs and its microenvironment including TAMs.

#731

Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages: implications for the tumor microenvironment.

Bon Q. Trinh, Nicolas Barengo, Sang Bae Kim, Ju-Seog Lee, Patrick A. Zweidler-McKay, Honami Naora. _UT MD Anderson Cancer Center, Houston, TX_.

Thrombocytosis, characterized by high platelet counts, and anemia, caused by low numbers of red blood cells, are associated with poor outcomes in various solid tumors and also occur in several myeloid disorders. The megakaryocyte and erythroid lineages give rise to platelets and red blood cells, respectively, but the mechanisms that cause increased megakaryopoiesis and concurrently decreased erythropoiesis in malignancies are poorly understood. We and others have previously identified that DLX4, a developmental transcription factor encoded by a homeobox gene, is aberrantly expressed in several types of solid tumors and myeloid malignancies but the mechanisms of DLX4 are poorly understood. In this study, we identified that DLX4 expression increases during megakaryopoiesis and decreases during erythropoiesis. Overexpression and knockdown experiments using normal CD34+ stem/progenitor cells and bipotent K562 cells demonstrated that DLX4 induces lineage markers and morphologic features of megakaryocytes, and concomitantly represses erythroid marker expression and hemoglobin levels. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression induced by DLX4 showed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. Bioinformatic analysis also revealed that DLX4 induces gene signatures that are associated with NF-kappaB signaling. The ability of DLX4 to promote megakaryocyte development and to inhibit erythroid differentiation was diminished by blocking NF-kappaB activity or by repressing IL1B, a transcriptional target of DLX4. These findings indicate that DLX4 promotes megakaryocyte development at the expense of erythroid generation in part by inducing inflammatory signaling. Studies are ongoing to investigate the transcriptional control by homeobox genes of inflammatory signaling in thrombocytosis and anemia that are associated with solid tumors and myeloid disorders.

#732

The impact of aerobic exercise on the tumor microenvironment.

Jennifer M. Wiggins,1 Jennifer Lee,2 Lori Rice,1 Sharon Lepler,1 Christine Pampo,1 Dietmar Siemann1. 1 _University of Florida, Gainesville, FL;_ 2 _National Cancer Institute, Bethesda, MD_.

Intratumoral hypoxia (pO2 < 10mmHg) is associated with aggressive tumor progression, radiation resistance, suppression of the immune anti-tumor response and poor patient outcome. It is often found in solid tumors, and can constitute a major obstacle to anticancer therapy. The primary cause of abnormal oxygen tensions in tumors is the aberrant tumor vasculature, characterized by tortuous, leaky and immature vessels. A number of intervention strategies designed to overcome tumor hypoxia and improve patient outcome have been investigated including high oxygen content breathing and the use of bioreductive prodrugs, but these have had only moderate success in the clinic.

The goal of the present investigations was to determine whether aerobic exercise could be applied to improve tumor perfusion, oxygenation and the immune anti-tumor response in breast cancer and fibrosarcoma models. Such modulation of the tumor physiology and host environment would be expected to lead to enhanced antitumor efficacy when combined with radiotherapy or chemotherapy.

The effects of mild and moderate intensity treadmill running were studied in mice bearing syngeneic murine mammary carcinomas (4T1 and EMT6) or fibrosarcomas (KHT). The exercise intensities were determined by measuring the anaerobic threshold, which was assessed by measuring the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and exercise commenced when tumors reached a size of ~500 mm3 (single) or ~200 mm3 (repeated bouts). Once size was attained, mice were exposed to either mild (12 m/min) or moderate (18 m/min) intensity exercise. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. At the end of the exercise period tumors were analyzed by histology to assess for physiological changes. Specifically, blood was collected and tumors were harvested, sectioned and evaluated by immunofluorescence. The detection of open blood vessels (Hoechst-33342) was used as an indirect indicator of perfusion and while the hypoxia marker (EF5) was used to determine the level of tumor hypoxia. Both markers were quantified using a Chalkley counter and ImageJ NIH software. In addition, the evaluation of tumor infiltrating immune cells after exercise compared to rest is under active investigation by histological analysis. Furthermore, plasma samples from exercised mice are being tested for immune related cytokines, chemokines and growth factors and compared to those from sedentary controls.

Results to date indicate no difference in tumor growth rate between sedentary and exercising mice. Mild daily bouts of aerobic exercise do not affect the total number of tumor blood vessels but do increase the number of blood vessels that are actively perfused. These findings suggest that exercise may have potential utility in overcoming the aberrant microenvironmental conditions associated in solid tumors with therapeutic resistance.

#733

Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5.

Lauren K. Brady,1 Rohil Shekher,2 Vladimir Popov,1 Mircea Ivan,3 Milan Radovich,3 Constantinos Koumenis1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Tulane University School of Medicine, New Orleans, LA;_ 3 _Indiana University School of Medicine, Indianapolis, IN_.

Solid tumors exhibit a range of oxygen concentrations, in part due to abnormal vasculature and limited diffusion of oxygen which lead to hypoxia. Tumor hypoxia is associated with resistance to treatment, metastasis and poor patient outcome for many types of cancer. Assessing the impact of hypoxia in the tumor microenvironment and understanding how it contributes to tumorigenesis and survival is a challenging research goal for several reasons; a chief obstacle has been developing methods to analyze the transcriptome within precise regions of the tumor microenvironment in vivo. To address this task, we used Laser-Capture-Microdissection (LCM) of tumors labeled with the hypoxia probe, EF5, to specifically isolate normoxic and hypoxic regions of tumors from a murine model of head and neck cancer. We then carried out deep RNA-sequencing of the in vivo samples and corresponding in vitro samples grown in 0.5% oxygen conditions.

Here we present the first described study of the hypoxic transcriptome in tumors at base-pair resolution. Our goal is to use these data to evaluate the potential of previously unstudied hypoxia-mediated pathways, such as the regulation of mRNA splicing, to be targeted therapeutically. We identified global changes in several classes of splicing in hypoxic compared to normoxic head and neck cancer cells, including changes in expression of last exons, retained introns and 3' untranslated regions. Many of the alternatively spliced isoforms are in pathways central to hypoxic adaptation, such as glycolysis, cell cycle and translation. Among these genes, we uncovered a previously undescribed splicing event in the master regulator of translation, EIF2B5. The full-length protein encoded by EIF2B5 (eIF2Bε), is a necessary catalytic component of the eIF2B complex, which binds eIF2α and exchanges GDP for GTP to initiate translation. Strikingly, hypoxia induces retention of intron 12 in EIF2B5, which leads to insertion of an early stop codon in frame with the coding sequence; the resulting 65kDa protein isoform lacks a functional guanine nucleotide exchange (GEF) domain compared to the full-length isoform, but retains the region known to bind eIF2B complex subunits. Thus, we predict that the 65kDa isoform of eIF2Bε, may act in opposition to full-length eIF2Bε and inhibit translation initiation in conditions of oxygen deprivation. We are investigating EIF2B5, and other select hypoxia-regulated isoforms, to evaluate how alternative splicing impacts cell growth and survival in hypoxic tumors.

#734

Role of TGF-β in myeloid regulation of tumor-induced bone disease.

Denise Buenrostro, Alyssa Merkel, Julie Sterling. _Vanderbilt University, Nashville, TN_.

Bone is one of the most common metastatic sites for breast cancer, where nearly 70% of patients that die from disease have reported skeletal metastases upon autopsy. Specifically in the bone microenvironment, TGF-β has a complex role in the regulation of tumor growth and is critical for the initiation and continuation of bone destruction, an important parameter of tumor-induced disease. Previous publications have demonstrated that TGF-β can affect myeloid cells by increasing their expansion in the tumor microenvironment and modulating host immune surveillance, thus supporting a tumor promoting environment. Therefore, we hypothesize that inhibition of TGF-β will decrease tumor-induced bone disease by reducing myeloid expansion and tumor growth in bone. To address this we injected mice with the TGF-β inhibitory antibody, 1D11, one week prior to tumor inoculation and continuously treated until sacrifice where myeloid expansion and parameters of tumor-induced bone disease were measured. Two mouse models were used to assess the importance of the immune system, an immunodeficient model inoculated with human breast cancer cells (MDA-MB-231) and a syngeneic model using murine breast cancer cells (4T1). Flow cytometry analysis revealed that TGF-β inhibition decreased a triple positive myeloid population (CD11b+F4/80+Gr-1+) in mice sacrificed at day 28 by five percent. This triple positive myeloid cell population has been associated with supporting tumor progression. Surprisingly our flow cytometry analysis demonstrated that myeloid-derived suppressor cells (MDSCs: CD11b+GR-1+) remained unchanged when TGF-β was inhibited. Micro-CT analysis demonstrated that 1D11 treated mice had a 2-fold increase in bone volume and connective density compared to control mice. X-ray analysis demonstrated that control mice had greater than 2-fold osteolytic bone destruction compared to mice treated with the TGF-β inhibitory antibody. Histomorphometric analyses suggests that TGF-β inhibition reduces tumor burden, increases osteoclast cell numbers and modulates macrophage cell populations. Together these results suggest that inhibiting TGF-β prior to tumor inoculation and subsequently after may prevent expansion of a tumor-promoting myeloid population and promote a bone-phenotype that does not support tumor growth in bone. Future studies will focus on investigating specific myeloid factors and assessing whether TGF-β inhibition decreases expression of these pro-tumorigenic factors.

#736

Hypoxia-induced EGFR tyrosine kinase inhibitor resistance is associated with epithelial-mesenchymal transition in NSCLC.

Yuhong Lu, Peter M. Glazer. _Yale University School of Medicine, New Haven, CT_.

The development of small molecule inhibitors specific for epidermal growth factor receptors (EGFR) with activating mutations has led to a dramatic shift in the treatment of non-small cell lung cancer (NSCLC) patients. Patients with NSCLC carrying such EGFR mutations often show prolonged responses when treated with first or second generation EGFR tyrosine kinase inhibitors (TKIs). However, most patients eventually develop resistance to EGFR TKIs. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis. Studies have linked hypoxia to therapy resistance, including radiation therapy and chemotherapy. In this study, we show that long-term, moderate hypoxia induces gefitinib resistance in the NSCLC cell line, HCC827, that harbors an activating EGFR mutation. The hypoxia-induced gefitinib resistance is associated with reduced BIM induction after gefitinib treatment when compared to normoxic HCC827 cells. In addition, in hypoxic HCC827 cells, gefitinib treatment induces N-cadherin expression, a mesenchymal marker, accompanied by downregulation of E-cadherin, an epithelial maker. These results suggest that epithelial-mesenchymal transition (EMT) may be involved in hypoxia induced gefitinib resistance. Wound healing migration assays further support that hypoxia induces HCC827 cell migration after gefitinib treatment. These results suggest that hypoxia may be a driving force for EGFR TKIs resistance potentially through EMT in NSCLC.

#737

Transcriptome of glioma growing in the brain is simulated by tumor cells cultured under low oxygen pressure.

Ho Jeong Lee, Hyun Kyung Yu, Seung Wook Kim, Sung Il Choi, Junqin He, Isaiah J. Fidler, Sun Jin Kim. _UT MD Anderson Cancer Center, Houston, TX_.

Introduction

Studies using cultured tumor cells are frequently used to generate large scale data. However, the discrepancy between culture media conditions and host microenvironment factors prevent adequate translation to the clinical reality. Since culturing tumor cells in atmospheric oxygen pressure (20% O2) significantly differs from oxygen pressure within different organs (5-7 % O2). We investigated whether the transcriptome of glioma cells cultured under oxygen concentration that is similar to the lesion in the brain (0.5-2% O2) can simulate the transcriptome of tumors growing in vivo.

Materials and Methods

Human glioma, LN229, cells were cultured in MEM supplemented with 10% FBS under 20%, 6% and 1% oxygen pressure. Glioma growing in the brain was established by stereotactic injection of 1x105 LN229 cells into the brain of nude mice, subcutaneous tumors were established by injection of 1x105 cells. Gene microarray was performed using Illumina Human HT-12_v4_BeadChip microarray and data were collected by using an Illumina bead Array Reader

confocal scanner (BeadStation 500GXDW; Illumina). Array data processing and analysis were performed using Illumina BeadStudio software. All statistical analysis was performed using BRB Arraytools Version 4.4.1 under the R language environment. Cluster analysis was performed and Heat Map was generated by Tree view.

Results

Brain and subcutaneous tumors were harvested 3 weeks post injection. RNA was extracted and processed for gene microarray that identified 2187 genes as brain specific transcriptome. Determination of common genes (same direction) between the transcriptome of glioma growing in the brain and cells growing in culture under 20%, 6%, and 1% O2 identified 23, 5, and 174 specific genes, respectively. Ingenuity pathway analysis and gene set enrichment analysis of the 174 common genes (from cells growing in 1% O2) revealed cluster of genes related to cancer and neurologic diseases, cell death/survival, cell-to-cell signaling. Up-regulation of genes associated with survival and resistance such as AKT, MAPK, NFκB1A, VEGF, and GSTA5 were also commonly identified in glioma growing in the brain and LN229 cells cultured under 1% oxygen.

Conclusion

Culturing glioma cells under different oxygen pressures induces different transcriptomes and cells cultured under 1% O2 express transcriptome most similar to cells growing in the brain. These data demonstrate that, to simulate clinical relevance, the oxygen tension of cultures should approximate those found in organs.

#738

Correlation between tumor infiltrating lymphocytes and CCL5 gene expression in chemotherapy-resistant triple negative breast cancer.

Joseph A. Pinto,1 Zaida D. Morante,2 Roberto Salgado,3 Nadezhda Cardenas,4 Jhajaira Araujo,1 Jaime Ponce,1 Franco Doimi,1 Justin Balko,5 Henry Gomez2. 1 _Oncosalud, Lima, Peru;_ 2 _Instituto Nacional de Enfermedades Neoplasicas, Lima, Peru;_ 3 _Institut Jules Bordet, Brussels, Belgium;_ 4 _Universidad Privada San Juan Bautista, Lima, Peru;_ 5 _Vanderbilt University, Nashville, TN_.

Background:

The presence of tumor infiltrating lymphocytes (TILs) have been associated with a better outcome in triple negative breast cancer (TNBC). In the other hand, we have found that CCL5, a chemokine involved in the recruitment of immune cells to the tumor microenvironment is an independent prognostic marker for distant-recurrence free survival (DFRS) in TNBC. The aim of this work was to evaluate the relationship between these two immunological prognostic markers in chemotherapy-resistant TNBC.

Material and Methods:

We evaluated a retrospective cohort (n=74) of TNBC patients with residual disease after neoadjuvante chemotherapy. Residual tumors were profiled with Nanostring for genes previously related to TNBC aggressiveness. Gene expression values for CCL5 were log2 transformed and median centered. TIL count was performed according to the methods published by Salgado et al (2015). Values of TILs count were log2 transformed (values with 0% were removed from the cohort; n=1). Spearman's rank correlation analysis and proportional hazards regression was used to investigate the relationship and impact in the outcome between these two markers.

Results: The median TIL count was 10% (interquartile range: 21.25). A total of 59.5% of cases had TILs <15%. There was a significant correlation between TILs and CCL5 (ρ=0.345, P=0.032). In univariate analysis (as continuous variable), TILs (HR=0.968 per unit of change; 95%IC: 0.946-0.990; P=0.006) and CCL5 (HR=0.95 per unit of change; 95%IC: 0.635-0.996; P=0.046) were both associated with outcome. After adjusting to CCL5 expression, TIL`s count, was the only significant marker with a P-value= 0.007 (HR=0.702 per unit of change; 95%IC: 0.54-0.909), in contrast to CCL5 (HR=0.925, CI95%:0.717-1,194; P-value=0.550).

Conclusions: TILs and CCL5 gene expression are significantly correlated in chemotherapy-resistant TNBC. However, although CCL5 expression is strongly associated with the progression of BC, particularly TNBC, TIL assessment remains the stronger and more significant prognostic immunological marker.

#739

The role of obesity on inflammation markers in the prostate tumor microenvironment.

Charnita M. Zeigler-Johnson,1 Knashawn H. Morales,2 Priti Lal,2 Timothy R. Rebbeck,3 Michael Feldman2. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _Harvard University, Philadelphia, PA_.

Background: Obesity reflects a chronic inflammatory environment that may contribute to prostate cancer progression and poor treatment outcomes. However, it is not clear which mechanisms drive this association within the tumor microenvironment. The aim of this pilot study was to examine prostatic inflammation via tumor infiltrating lymphocytes and macrophages characterized by obesity and cancer severity.

Methods: We studied paraffin-embedded prostatectomy tissue from 99 participants (63 non-obese and 36 obese) from the Study of Clinical Outcomes, Risk and Ethnicity (University of Pennsylvania) Pathologists analyzed the tissue for type and count of lymphocytes and macrophages, including CD3, CD8, FOXP3, and CD68. Pathology data were linked to clinical and demographic variables. Statistical analyses included frequency tables, Kruskal-Wallis tests, Spearman correlations, and multivariable models.

Results: We observed positive univariate associations between the number of CD68 cells and tumor grade (p=0.019), as well as biochemical failure (p=0.033). In multivariable analysis, CD3 and CD8 counts were associated with time to biochemical failure (HR=0.93, 95% CI=0.88-0.99; HR=1.08, 95% CI=1.01-1.17, respectively.) Preliminary results suggested differences in lymphocytes by race. There were no differences in lymphocytes or macrophages by obesity status or BMI.

Conclusions: The number of lymphocytes and macrophages in the tumor microenvironment did not differ by obesity status. However, these inflammation markers were associated with poor prostate cancer outcomes. Further examination of underlying mechanisms that influence obesity-related effects on prostate cancer outcomes is warranted. Such research will guide immunotherapy protocols and weight management as they apply to diverse patient populations and phenotypes.

#740

Silica promotes anchorage-independent growth of human bronchial cells via activation of NF-κB1 p50.

Motoo Katabami, Ichiro Kinoshita, Yasushi Shimizu, Satoshi Takeuchi, Hirotoshi Dosaka-Akita. _Hokkaido University Graduate School of Medicine, Sapporo, Japan_.

Although crystalline silica (hereafter silica) was judged as carcinogen by IARC, the mechanism of silica-induced carcinogenesis remains elusive. As limited studies have been reported to support the direct carcinogenic effect of silica on human lung epithelial cells, silica-induced inflammatory microenvironment may be postulated to facilitate lung neoplastic transformation. To test this hypothesis, we investigate the growth of immortalized human lung bronchial epithelial cell lines NL20, BEAS-2B and 16HBE14o-, following incubation with conditioned media collected 48h after exposed to silica (Silica CM) in each cell line. All cell lines promoted anchorage-independent growth in each Silica CM, compared with those in CM without silica exposure. All the bronchial cell lines in Silica CM increased DNA binding activity of NF-κB1 p50. After incubated with Silica CM, culture supernatants of NL20 and BEAS-2B kept higher concentration of EGF, and those of 16HBE14o- contained higher concentration of TNFα and IGF1, respectively. Recombinant EGF, TNFα and IGF1 are all sufficient to promote anchorage-independent growth of each bronchial cell line, and to increase DNA binding activity of NF-κB1 p50. Neutralizing human EGF and TNFα antibodies dampened the growth of each bronchial cell line. NF-κB inhibitor Bay11-7082 suppressed the growth of all bronchial cell lines incubated with Silica CM. Taken together, silica promoted anchorage-independent growth of human lung epithelial cells via silica-induced secretion of soluble factors, followed by activation of NF-κB1 p50 in an autocrine manner. The present study supports the hypothesis that silica-induced inflammatory microenvironment may be actively involved in the potential carcinogenic effect of silica.

#741

Insulin-like growth factor binding protein-3 (IGFBP-3) enhances obesity-related breast tumorigenesis.

Tiffany Scully,1 Carolyn D. Scott,1 Hasanthi C. de Silva,1 Sue M. Firth,1 Stephen M. Twigg,1 John E. Pintar,2 Robert C. Baxter1. 1 _University of Sydney, Sydney, Australia;_ 2 _Rutgers Robert Wood Johnson Medical School, New Jersey, NJ_.

Introduction and Aim: Epidemiological studies have shown an association between obesity and poor breast cancer prognosis. We have reported that IGFBP-3 influences both breast cancer growth and adipocyte maturation. This study investigated the role of endogenous IGFBP-3 on the development of obesity and subsequently on breast tumor growth. Methods: Wild-type (WT) C57BL/6 or IGFBP-3 knock-out (BP3KO) mice were fed either a high-fat diet (HFD) or control chow diet for 15 weeks, then injected with the syngeneic mouse breast cancer cell line E0771 in the 4th left mammary gland. When the largest tumor reached 1000 mm3, tissues including the mammary fat pad were excised, formalin-fixed and paraffin-embedded for immunohistochemical analysis. Results and Conclusion: Compared to WT mice, BP3KO mice showed both significantly reduced weight gain and mammary fat pad mass (contralateral to tumor) in response to HFD (p < 0.0001, 1-way ANOVA), despite similar food intake between WT and BP3KO mice on HFD. E0771 tumor weight and volume were overall increased by HFD independent of IGFBP-3 status (p = 0.051), and decreased in BP3KO mice independently of diet (p < 0.001, 2-way ANOVA). Despite differences in tumor volumes and weights, tumors from WT and BP3KO mice did not show differences in either the number of mitotically active (Ki67+) or apoptotic (cleaved caspase-3+) cells. Tumors from BP3KO mice however, showed greater infiltration of CD3\+ T-cells compared to WT mice (p = 0.0299, 2-way ANOVA). WT and BP3KO mice showed similar intra-tumoral mean vessel densities (CD31+ area) but differed in terms of the relationship between vessel density and final tumor volume. Increased vessel density was associated with decreased tumor volume in BP3KO mice (r = -0.62, p = 0.03, Spearman's correlation test) in contrast to the increased vessel density associated with increased tumor volume in wild-type mice (r = 0.63, p = 0.02, Spearman's correlation test). These data suggest that endogenous (circulating and/or stromal) IGFBP-3 (i) has a potentially stimulatory role in the expansion of adipose tissue, (ii) can enhance mammary tumor growth in immune-competent mice, potentially by influencing T-cell infiltration into the tumor and (iii) may influence the relationship between tumor vascularity and growth.

### Targeting the Microenvironment

#742

A novel multidrug metronomic chemotherapy significantly delays tumor growth in mice.

Maria Tagliamonte, Annacarmen Petrizzo, Maria Lina Tornesello, Antonio Luciano, Domenica Rea, Antonio Barbieri, Claudio Arra, Maria Napolitano, Gennaro Ciliberto, Franco M. Buonaguro, Luigi Buonaguro. _National Cancer Institute, Naples, Italy_.

The tumor immunosuppressive microenvironment represents a major obstacle to an effective tumor-specific cellular immune response.

We have previously shown that a novel multi-drug chemotherapy administered in a metronomic fashion was able to increase immune response to peptides. The chemotherapy consisted of a cocktail including taxanes (paclitaxel and docetaxel) and alkylating (cyclophosphamide) agents. The newly designed strategy was shown to be safe, well tolerated and significantly efficacious (Tagliamonte et al., CII 2015).

In the present study, the effect on the immunosuppressive tumor microenvironment by the same multi-drug cocktail was evaluated in a mouse model upon sub-cutaneous ectopic implantation of B16 melanoma cells.

Treated animals showed a remarkable delay in tumor growth and prolonged survival as compared to control group. Such an effect was directly correlated with CD4+ T cell reduction and CD8+ T cell increase. A significant reduction in the percentage of both CD25+FoxP3+ and CD25+CD127low regulatory T cell population was found in the spleens as well as in the tumor lesions. An intrinsic CD8+ T cell response specific to B16 naturally expressed Trp2 TAA was observed. The same metronomic chemotherapy combined to a vaccine based on mutated antigens, was subsequently shown to increase immune response to vaccine resulting in a significant delay of B16 tumor growth as well as animal survival.

The novel multi-drug daily metronomic chemotherapy evaluated in the present study was very effective in counterbalancing the immunosuppressive tumor microenvironment and enhancing vaccine efficacy. Consequently, the intrinsic as well as vaccine-induced anti-tumor T cell immunity could exert its function containing tumor growth.

Overall, the described metronomic chemotherapy may represent a promising adjuvant approach to enhance anti-tumor cellular immunity and amplify the biological effects of therapeutic cancer vaccines.

#743

Tyrosine kinase discoidin domain receptor-1 (DDR1) regulates cytotoxicity of recombinant immunotoxin for cancer therapy.

Fatima Ali-Rahmani,1 David Fitzgerald,1 Scott Martin,2 Paresma Patel,2 Marco Prunotto,3 Pinar Ormanoglu,2 Craig Thomas,2 Ira Pastan1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Center for Advancing Translational Science, Bethesda, MD;_ 3 _Roche, Basel, Switzerland_.

Discoidin domain receptor 1 (DDR1) is a collagen-activated tyrosine kinase that facilitates cell adhesion, migration, proliferation and matrix remodeling. The collagen/DDR1 axis is also thought to modulate tumor-stromal interaction and potentially affects tumor response to therapy. Mesothelin is a cell-surface protein over-expressed in several human cancers including mesothelioma, ovarian, lung, breast, and pancreatic cancers with limited expression on normal cells. RG7787 is a clinically optimized recombinant immunotoxin (RIT) consisting of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A in which immunogenic B cell epitopes are silenced. To enhance therapeutic efficacy of RITs we conducted a kinome RNAi sensitization screen which identified DDR1 as a potential target. We hypothesized that DDR1 regulates RIT activity and its inhibition might enhance cell killing by RG7787. Knockdown of DDR1 by siRNA or treatment with inhibitor, '7rh', greatly enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed DDR1 silencing was associated with a decrease in several ribosomal proteins and enhanced inhibition of protein synthesis. Induction of DDR1 expression or stimulation of DDR1 activity by collagen protected cancer cells from RG7787 killing, while DDR1 inhibition enhanced killing by RG7787. Moreover, the combination of immunotoxin and DDR1 inhibitor caused significantly more shrinkage of epithelial A431/H9 and pancreatic KLM1 tumor xenografts than either agent alone. These data demonstrate that DDR1 has a critical role in modulating immunotoxin activity. Inhibition of DDR1 represents a novel strategy to enhance therapeutic efficacy of RITs targeting mesothelin-expressing cancers.

#744

Cancer immunotherapy biomarker profiling assay.

Alex Chenchik, Leonid Iakoubov, Mikhail Makhanov, Costa Frangou. _Cellecta, Inc., Mountain View, CA_.

Selecting patients predisposed to respond to existing and novel immunotherapy treatments requires the development of novel prognostic and predictive biomarkers. Recent reports have identified several gene expression signatures specific for immunity status and immune contexture in solid tumor microenvironments, and these enable predictions of efficacy of a number of chemo- and immunotherapeutics. Robust methods for molecular characterizations of the immune mechanism in the tumor microenvironment are essential to meet the imminent need for diagnostic approaches that identify patient populations responsive to the growing number of immunotherapeutic treatments. Toward this goal, we developed the Cancer ImmuneNet 2500 (CIN2500) assay for profiling cellular composition in the tumor microenvironment and discovery of novel prognostic and predictive immune response biomarkers.

The CIN2500 panel provides signatures for approximately 400 immunity-related genes from 16 predictive and prognostic core genes that have been validated in recent chemo- and immunotherapy clinical trials across several tumor types, including melanoma, colorectal, breast, and lung cancers. In addition, using computational analysis of an immunotherapy network model, we predicted and included in the assay approximately 300 key nodes in pathways specific for antigen presentation and recognition, inhibition, activation and motility of immune cells, and adhesion and apoptosis of cancer cells. The CIN2500 panel also includes a comprehensive set of 1,700 genes specific for detection and quantitative profiling in the tumor microenvironment of different types of activated immune cells of adaptive and innate immunity, and stromal, fibroblast, cancer, endothelial and adipose cell types.

The CIN2500 assay quantitatively profiles the expression levels of approximately 2,500 key cancer genes from 10-100ng of total RNA using multiplex RT-PCR amplification followed by Next-Generation Sequencing (NGS). Built-in standards for each gene target enable sample-to-sample calibration of the NGS data and provide a reference to adjust for background noise that often depends on the quality of samples. Control studies have shown that the CIN2500 assay quantifiably measures 4 orders of magnitude variation in the expression levels of 2,500 immune-related genes from as few as 100 cells in whole lysate, and profiles from frozen biopsies, surgically-removed tumor samples, PBMCs, and FFPE clinical samples show a comparable range and sensitivity. We will present profiling data from infiltrating immune cells and key intact and deficient immune mechanisms in the tumor microenvironment of breast cancer samples to demonstrate the performance and utility of the CIN2500 assay.

#745

Hybrid bioactive nanoparticles for modulating prostate tumor microenvironment and enhancing radiation therapy.

Mohammad Ali Amini, Azhar Z. Abbasi, Claudia R. Gordijo1, Ping Cai, Andrew M. Rauth, Robert G. Bristow, Xiao Yu Wu. _Univeristy of Toronto, Toronto, Ontario, Canada_.

Introduction

Tumor hypoxia is a poor prognostic factor in a number of malignancies such as prostate cancer (PCa). Clinically relevant hypoxic levels are detected in 30-90% PCa with oxygen concentrations below that required for half-maximal radiosensitivity, thus making radiotherapy (RT) ineffective alone [10].

Recently our lab has created pharmaceutically acceptable bioreactive hybrid manganese dioxide nanoparticles (MDNPs) and demonstrated their ability to modulate tumor microenvironment (TME) by reacting with H2O2 and protons and producing O2 in hypoxic tumors, as well as to enhance radiation response of tumor.

Methods

Lipid encapsulated and polymer stabilizedMDNPs (LMD NPs) were prepared by dispersing MnO2 precursor particles in melted lipid and polymer and then characterized for the particle size and zeta potential. In-vitro oxygen generation of LMD NPs was examined by measuring oxygen saturation level (sO2) in the blood using photoacoustic (PA) imaging method with addition of LMD NPs and H2O2.

Biodistribution of LMD NPs in PCa tumor-bearing mice was evaluated by using a Xenogen IVIS Spectrum Imaging System following IV injection of ICG labeled NPs through the tail vein. Mice were monitored non-invasively for up to 24 hours.

To investigate the effect of combination of MDNPs and radiation therapy on tumor growth delay and survival, PCa tumor-bearing mice were treated intravenously with LMD NPs with saline as a control. Four hour post injection, 10 Gy radiation dose was given at the site of the tumor. Mice were monitored every 2 days by measuring tumor size using a caliper. Mice were sacrificed when the tumor size reached 500 mm3.

Results

PA imaging demonstrated a controlled and prolonged generation of O2 in the blood with maximum oxygen saturation (90-95%) being reached within 60 min after incubation with LMD NPs and H2O2. The biodistribution and ex-vivo images of the resected organs showed that LMD NPs accumulated in the prostate tumor sites within 1 h post i.v. injection and remained in the tumor for at least 24 h. The ex vivo optical data of excised tissue showed a significant uptake of LMD nanoparticles by prostate tumor 24 hpost particle administration. The combination of LMD NPs treatment with single dose 10 Gy RT inhibited tumor growth by 24% at 5 days post treatment, whereas the tumor size increased about 47% in mice treated with RT alone. LMD NPs plus RT also improved survival rate of the cancerous mice for up to 53 days, which was about 3.3-fold enhancement in the mean survival rate compared to saline plus RT treatment (30 days).

Conclusion

The new bioreactive MnO2 NPs exhibited desirable oxygen- generating profile and high tumor accumulation and retention after systemic administration. This work has demonstrated, in a preclinical prostate tumor model, that the combination of LMD NPs with radiation therapy is a promising treatment approach for solid tumor. .

#746

Transcriptional reprogramming of pancreatic stroma enhances chemotherapy and halts tumor growth.

Joelle Baddour,1 Sonal Gupta,2 Lifeng Yang,1 Abhinav Achreja,1 Mathew Derichsweiler,1 Thomas Plackemeier,1 Troy Pearson,2 Juan C. Marini,3 Janusz Franco-Barraza,4 Edna Cukierman,4 Anirban Maitra,2 Deepak Nagrath1. 1 _Rice University, Houston, TX;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Baylor College of Medicine, Houston, TX;_ 4 _Fox Chase Cancer Center, Philadelphia, PA_.

Pancreatic cancer remains one of the most lethal of all solid tumors largely due to early dissemination and an aggressive fibrotic stroma. Transcriptional reprogramming of the tumor stroma re-establishes a normal tumor microenvironment with quiescent stellate cells and fibroblasts. A combinatorial transcriptional therapy, developed by our lab, has proven successful at normalizing the tumor microenvironment, both at the phenotypic and genetic levels. Indeed, whole genome methylation profiling in addition to high-throughput transcriptomic analysis of treated/non-treated cells have confirmed stromal reversal from an activated to a quiescent state. This quiescent stromal phenotype induces metabolic changes in tumor cells and hinders their aberrant growth. In addition, in an orthotopic mouse model of pancreatic cancer, stromal reversal enhanced intratumoral drug delivery of gemcitabine and led to reduced tumor growth. These findings suggest that the transcriptional reprogramming of activated stromal cells can lead to a quiescent phenotype that is more suppressive of tumor growth.

#747

Paracrine c-MET/HGF HCC PDX: evaluation of a biologics targeting c-MET.

Dawei Chen, Ziyong Sun, Likun Zhang, Jie Cai, Davy X. Ouyang, Jean-Pierre Wery, Henry Li. _Crown Biosciences, Santa Clara, CA_.

The interaction between the c-MET tyrosine kinase receptor (RTK) on the surface of cancer cells with its ligand, hepatocyte growth factor (HGF) secreted from surrounding tumor stroma, results in paracrine signal transduction in cancer cells to promote growth, invasion, angiogenesis and metastases. c-MET gene amplification/over-expression, or activating mutations, frequently function as oncogenic driver in several types of cancers. c-MET is thus considered a key target(1) for both TKIs or biologics. However, lack of suitable animal models has represented an obstacle for the development of new cMET inhibitors. While patient derived xenografts (PDXs) mirror patients' histopathological/genetic profiles(2-6), they usually lose human stroma, the source of HGF; moreover the HGF originated from murine stroma is incapable of triggering a paracrine signaling because of lack of cross reactivity with the human c-MET. Some PDX grow independent of paracrine mechanisms because of their constitutive c-MET activation by the presence of activating mutations, gene amplification or autocrine signaling, and can be used to evaluate the efficacy of TKIs(5, 7, 8). However they are not suitable to assess biologics, e.g. antibodies disrupting receptor-ligand interaction. Alternatively it is possible to grow tumors in hu-HGF-transgenic immunocompromised mice (humanization) where an artificial paracrine signaling is created. Interestingly, we have identified a unique HCC-PDX, LI0801, which maintains human stroma throughout passages, as demonstrated by immunochemistry staining for human vimentine and human HGF, both markers of stromal components(7, 8). The mechanisms of human stroma maintenance in this PDXremain unclear. We showed that LI0801 responds to different TKIs(7, 8). In addition we evaluated the efficacy of an anti-human c-MET monoclonal antibody in reducing tumor burden in LI0801, and we were able to demonstrate great tumor response to anti-human c-MET antibody, which is significantly more effective than crizotinib. To the best of our knowledge, LI0801 is the first paracrine c-MET PDX reported, and represents an important model to study paracrine cMET/HGF signaling and to evaluate new c-MET inhibitors, both TKIs, and biologics.

References

1. Comoglio PM, et al. Nat Rev Drug Discov. 2008;7:504-16.

2. Ding L, et al. Nature. 2010;464:999-1005.

3.Marangoni E, et al. A new model of patient tumor-derived breast cancer xenografts for preclinical assays. Clin Cancer Res. 2007;13:3989-98.

4.Chen D, Oncotarget. 2015.

5. Yang M, et al. International journal of cancer Journal international du cancer. 2013;132:E74-84.

6. Zhang L, et al. A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy. Sci Rep. 2013;3:2992.

7. Bladt F, et al. Cancers (Basel). 2014;6:1736-52.

8. Chen D. AACR Annual Meeting 2012. 2012;Poster.

#748

Targeting the crosstalk between tumor cells and adipocytes to block breast cancer progression.

Tiziana Triulzi, Valentina Ciravolo, Viola Regondi, Martina Di Modica, Claudia Chiodoni, Elda Tagliabue. _Fondazione IRCCS-Istituto Nazionale dei Tumori, Milano, Italy_.

It is now well-established that the local microenvironment of an emerging tumor plays a vital role in various steps of tumorigenesis. Stromal cells, such as fibroblasts, endothelial and various inflammatory cells, can contribute to tumor progression, and tumor cells can directly model their microenvironment, influencing host cells to secrete factors that favor cancer cell survival and growth. Recent studies have demonstrated the existence of cross-talk between breast cancer (BC) cells and adipocytes, the most abundant stromal cell in the breast, supporting their involvement in BC and consistent with epidemiological studies reporting the association of obesity and insulin resistance/diabetes with a higher risk and poor prognosis of BC.

Based on this evidence, we investigated whether adipocyte de-differentiation is the crucial trigger in inducing pro-tumor microenvironment remodeling and tumor progression.

Co-culture of 4T1 mouse mammary tumor cells using Transwell or their conditioned medium with mature adipocytes derived from the 3T3-L1 mouse cells induced adipocyte de-differentiation, observed as a reduced expression of specific differentiation markers (peripilin, adipocyte protein 2 (aP2), PPARγ and adiponectin) in Western blots and by qRT-PCR, and a reduced triglyceride content as assessed by oil red O staining compared to mature adipocytes. Strong adipocyte de-differentiation, associated with an increase in pro-inflammatory molecules (IL6 and CCL2), was also induced in vitro by tumor interstitial fluids obtained from MMT-PyMT transgenic mice, but not by plasma of these mice. Preliminary studies to identify molecules involved in the cross-talk indicated that the phenomenon is exosome-unrelated.

Based on the relevant role of the PPARγ transcription factor in inducing and maintaining adipocyte differentiation, we co-cultured adipocytes with 4T1 BC cells in the presence of several PPARγ agonists enriched in the diets of many populations displaying a low incidence of cancer (ώ3-fatty acids), and in the presence of glitazones (rosiglitazone and pioglitazone) to force adipocyte stable differentiation. While most of these compounds did not block adipocyte de-differentiation induced by tumor cells, pioglitazone and eicosanoid acid showed some activity in inhibiting this de-differentiation in vitro. Treatment of MMTV-PyMT transgenic mice at tumor onset by oral gavage with pioglitazone did not significantly reduce mammary tumor growth compared to controls, whereas eicosanoid acid treatment significantly reduced tumor multiplicity and tumor burden compared to controls.

Overall, our results suggest that tumor-induced adipocyte de-differentiation participates in tumorigenesis and that the blockade of this program can inhibit tumor progression.

Supported by AIRC and Fondazione Cariplo.

#749

Betaglycan-mediated regulation of mesenchymal stromal cell behavior in the prostate tumor-bone microenvironment.

Leah M. Cook, Jeremy J. McGuire, Conor C. Lynch. _H. Lee Moffitt Cancer Center, Tampa, FL_.

Prostate cancer frequently metastasizes to bone, resulting in increased risk of fractures and severe pain that significantly impacts patient quality of life. Bone metastatic prostate cancer is currently incurable with standard of care therapies being mainly palliative. In bone, prostate cancer generates extensive osteogenic lesions by promoting osteoblast activity. Osteoblasts are specialized bone forming cells derived from mesenchymal stem cells. In a bid to find new molecular targets through which prostate cancer induces osteogenesis, we incubated naïve MSCs with osteogenic bone metastatic prostate cancer cell line C4-2B conditioned media. Gene expression analysis revealed a 3-fold increase in expression of the TGFβ co-receptor, betaglycan. Given the critical role of TGFβ in the context of bone metastatic disease, we examined the role of betaglycan in MSC behavior. To this end, we utilized shRNA to knockdown betaglycan gene expression in mouse bone marrow-derived primary MSCs. Reduced betaglycan expression in knockdown (KD) MSCs had little to no impact on the expression of TGFβ signaling receptors, TGFβRI and TGFβRII, or pan-TGFβ ligand expression. However, using PAI promoter activity, a readout for TGFβ signaling, we observed a 3-fold increase in luminescence in KD-MSCs compared to controls. Likewise, phosphorylated Smad 2 was significantly increased in Betaglycan KD-MSCs in response to TGFβ treatment, collectively demonstrating increased TGFβ signaling in MSCs with reduced betaglycan expression. Using modified Boyden chamber assays, we found that 50% more Betaglycan-KD MSCs migrated towards recombinant TGFβ than controls (100 KD-MSCs cells migrated towards recombinant TGFβ vs. 50 control cells); this change was reversed with the addition of recombinant betaglycan. Using osteogenic differentiation assay, we found that Betaglycan KD-MSC differentiation was reduced by approximately 70% compared to control MSCs as measured by alizarin red staining. Addition of recombinant TGFβ further suppressed differentiation in both KD-MSCs and Control MSCs. This phenomenon was rescued by the addition of recombinant betaglycan, suggesting that MSC-derived betaglycan is critical for driving the osteogenic program. We are currently determining the impact of betaglycan on MSC behavior in the tumor bone microenvironment using in vivo models of bone metastatic prostate cancer. Collectively, these findings suggest that prostate cancer-induced MSC osteogenic programs are regulated in part via the induction of betaglycan.

#750

Nanoliposomal targeting of Ephrin receptor A2 (EphA2): Clinical translation.

Walid S. Kamoun,1 Shinji Oyama,1 Tad Kornaga,1 Theresa Feng,1 Lia Luus,1 Minh T. Pham,1 Dmitri B. Kirpotin,1 James D. Marks,2 Melissa Geddie,1 Lihui Xu,1 Alexey A. Lugovskoy,1 Monica Murphy,3 Carl Morrisson,3 Daryl C. Drummond1. 1 _Merrimack, Cambridge, MA;_ 2 _UCSF, San Fransisco, CA;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Ephrin receptor A2 (EphA2) is part of the Ephrin family of cell-cell junction proteins highly overexpressed in several solid tumors, and is associated with poor prognosis. We developed a novel EphA2-targeted docetaxel nanoliposome, leveraging organ specificity through enhanced permeability effect and cellular specificity through EphA2 targeting. The goal of the study was to develop the diagnostic framework enabling the clinical implementation of EphA2-based exclusion criteria in future MM-310 trials.

We used qFACS and an in vitro assay for liposome (Ls)-cell interaction to identify the minimum number of EphA2 receptors to enable antibody-mediated internalization of Ls. We developed an IHC assay able to differentiate EphA2 - vs + cell lines. We characterized EphA2 staining pattern in tumor samples of various indications and developed a scoring algorithm that allows selection of patients in early clinical trials.

While non targeted Ls do not associate with cells in vitro, there is a strong correlation between EphA2 expression and EphA2-Ls cell association independent of the cell line origin. We used the non-targeted Ls to determine the extent of non-specific binding that can be achieved (~340 Ls/cell) and used partitioning to determine the minimum number of EphA2 receptors necessary to mediate targeting (~3000 receptors/cell). We have developed and validated a qIHC assay for EphA2 (precision ~90%, linearity 0.8 and reproducibility ~5%). We stained a set of ~200 tumor samples from various indications. EphA2 was found to be expressed in tumor cells, tumor-associated myofibroblasts, and tumor-associated blood vessels. Using an inclusive cutoff of 10%, EphA2 prevalence was found to range from 50% to 100% in the tumor types evaluated. No significant difference in staining was seen between metastasis and primary tumors in matched samples.

In summary, we developed a diagnostic framework for prospective selection of EphA2+ patients for MM-310 trials based on a mechanistic single cell cut-off, and a clinical-grade IHC assay.  | |  | |

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

|

Cancer Cells | Tumor associated myofibroblasts | Tumor associated blood vessels | EphA2 Overall Score

Bladder | 19/20 (95%) | 0/20 (0%) | 16/20 (80%) | 19/20 (95%)

Gastric | 18/20 (90%) | 3/20 (15%) | 17/20 (85%) | 20/20 (100%)

Head & Neck | 16/19 (84%) | 0/19 (0%) | 9/19 (47%) | 19/19 (100%)

Lung | 24/41 (58%) | 1/41 (2.4%) | 24/41 (58%) | 28/41 (68%)

Lung-FNA | 7/9 (78%) | \-- | \-- | 7/9 (78%)

Ovarian | 10/18 (55%) | 7/18 (39%) | 17/18 (95%) | 17/18 (95%)

Pancreatic | 15/19 (79%) | 0/19 (0%) | 11/19 (58%) | 17/19 (89%)

Prostate | 7/23 (27%) | 7/23 (27%) | 9/23 (28%) | 12/23 (52%)

TNBC | 6/77 (7%) | 0/77 (0%) | 34/77 (44%) | 37/77 (48%)

#751

Secreted anterior gradient-2 is a potential tumor-microenvironment-related therapeutic target.

Hao Guo,1 Qi Zhu,1 Zhenghua Wu,1 Hitesh B. Mangukiya,1 Xiaoyan Yu,2 Dawei Li1. 1 _Shanghai Jiao Tong University, Shanghai, China;_ 2 _Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China_.

The secreted anterior gradient-2 (AGR2) orthologs in amphibians are critical signaling proteins in anterior specification and limb regeneration. It is also overexpressed in diverse human cancers especially adenocarcinomas. Although tumor-related functions of intracellular AGR2 have been intensively investigated and several AGR2-related signaling pathways have been identified, the full scope of secreted AGR2 functions, especially the exact mechanism of actions, remain insufficiently explored. Here we report that AGR2 induction after SKOV3 xenograft inoculation is tumor microenvironment (TME) dependent. This phenomenon can be mimic in vitro through serum deprivation but not hypoxia treatment. In addition, the secreted AGR2 may in turn influence the development of TME. It scatters the tumor cells and guides the nearby vascular endothelial cells and fibroblasts toward the source of AGR2 through various degrees of crosstalk with VEGF and bFGF pathways. Blocking the AGR2 inhibits the tumor growth and decreases the blood vessel density in SKOV3 xenograft model. Our results show that exogenous AGR2 binds specifically with VEGF and bFGF, and promotes their dimerization. This binding enhances the phosphorylation signals, such as ERK phosphorylation, induced by VEGF and bFGF, and promotes the VEGF- and bFGF- dependent angiogenesis. We also find that heparin may reverse the binding between bFGF and AGR2, indicating that dimerized bFGF that promoted by AGR2 would be released when it is close to the cell surface where heparin sulfate is abundant. Taken together, the present results suggest that tumor-secreted AGR2 may activate the specific growth factors surrounding the tumor cells to promote the angiogenesis and tumor growth, and also act as a short-range chemoattractant to guide surrounding vascular endothelial cells and fibroblasts toward tumor cells in building the TME. This mechanism of action directly links the secreted AGR2 with the cell signaling networks well-known in building TME. This may help to further explain how tumor cells interact with and attract nearby fibroblasts and blood vessel cells to form their microenvironment and support their growth, suggesting a potential anti-TME target.

#752

Fibroblast growth factor receptor 2 - HER2 transactivation: Role of fbroblasts in the acquisition and maintenance of anti-Her2 target therapies resistance in breast cancer.

Patricia Fernandez Nogueira,1 Mario Mancino,2 Gemma Fuster,2 Estel Enreig,1 Elisabeth Ametller,2 Paloma Bragado,2 Vanessa Almendro,3 Pedro Gascón,3 Pedro Gascón3. 1 _Universitat de Barcelona, Barcelona, Spain;_ 2 _IDIBAPS, Barcelona, Spain;_ 3 _Fundació Clinic per la Recerca Biomèdica, Barcelona, Spain_.

INTRODUCTION

Mechanisms underlying tumor progression after chemotherapy are not well understood. Therapeutic treatments can favor the clonal selection of cells with unique properties and different fitness for a given microenvironment. Indeed, tumor cells can induce changes in the structure and composition of the microenvironment to support their growth and spread.

Our aim is to study if clonal selection induced by target therapies like Trastuzumab and Lapatinib favors the outgrowth of cells with different microenvironmental crosstalk capability, and in particular the role of fibroblast in the selection of these particular clones.

MATERIALS AND METHODS

We developed different cell lines resistant to these drugs from the parental SKBR3, BT474 and MDA-MB-453 Her2+ cells lines. We determined their molecular profile and investigated the changes in the expression of selected genes codifying soluble factors known to induce stromal changes, like chemokines, cytokines, matrix remodeling-related enzymes, angiogenic and neurogenic factors.

To study the role of fibroblast in the selection of the resistant clones, and the crosstalk between these two populations, we developed fibroblast immortalized lines derived from breast cancer tumors and normal breast tissue, and study the effect of the fibroblasts in resistant induction, as well as the effect of resistant clones in the fibroblast activation.

RESULTS AND DISCUSION

We found that factors secreted by fibroblast can support cancer growth and progression by influencing and promoting the resistant phenotype. In our proposed model, HER2 positive BC cell lines resistant to Trastuzumab and Lapatinib have an active FGFR2 signalling pathway, able to maintain HER2 signalling by receptor transactivation. This bypasses HER2 inhibition and contributes to the maintenance of the cellular addiction to HER2 pathways even when the protein is being targeted, promoting cell proliferation and survival. Moreover, resistant cell lines secreted factors induced fibroblast activation and also modulated fibroblasts tumour promoting factors secretion pattern. As a result, after exposure to resistant cell lines secreted factors, fibroblasts become more efficient on promoting BC cell lines resistance. Similarly, the supernatant derived from fibroblasts isolated from HER2+ tumours was able to induce the activation of HER2 and FGFR2 pathways. The same effect was found in vivo, were co‐injection with HER2+ tumour associated fibroblasts promoted tumour aggressiveness and resistance through HER2 and FGFR2 activation

CONCLUSION

These results highlight the importance of microenvironment in supporting tumor progression after chemotherapy.

#753

The use of proteomics to analyse whole tumors and identify unique stroma cell targets for antibody-based therapeutics.

James Ackroyd,1 Jason Allen,1 Jo Berry,1 Lisa Roesch,1 Martin Barnes,1 Arnima Bisht,2 Christian Rohlff,1 Keith Wilson,2 Dee Aud,2 Robert Boyd1. 1 _Oxford BioTherapeutics Ltd, Abingdon, United Kingdom;_ 2 _Oxford BioTherapeutics Inc, San Jose, CA_.

Solid tumors comprises of two distinct compartments: cancer cells and the stroma that the cancer cells induce and are dispersed in. This stroma contains stromal (Fibroblasts, endothelial and pericyte cells) and infiltrating immune cells (Lymphocytes, macrophages, granulocytes and myeloid derived suppressor cells). Recent oncology research has implicated these stroma cells as promoters of tumor progression and there is thus a strong need to profile the cell membrane proteins present on these cells. For tumors which do not contain infiltrating T cells (non-immunogenic tumors) it has been recognized that it will be necessary to employ therapeutic agents to disrupt the tumor micro-environment (TME); allowing the release and processing of tumor antigens by antigen presenting cells, entry and migration T cells into intrastromal sites and potent activation of T cell responses to tumor antigens.

We have conducted in-depth proteomic profiling of tumors from 14 different solid cancer indications with varying degrees of stroma involvement to characterize their immune and stromal cell composition. The data from this work was analyzed in OGAP®; a unique proteomic database that integrates information at the tissue, disease and protein isoform level across diseases, indications, and normal tissues to clarify membrane protein expression levels and profiles. It is one of the world's largest proprietary cell-membrane focused proteomic database, with data on over 5000 cancer membrane proteins. Purified stroma cell populations were also profiled to facilitate the interpretation of whole tumor proteomic data.

Cell membrane proteins present in tumors from different cancer indications were analyzed by mass spectrometry. We classified tumors as immunogenic or non-immunogenic using validated T cell markers (CD3, CD4, CD8 and CD69). We then profiled the non-immunogenic tumors for fibroblast, macrophage and B cell type markers to try and identify strong stroma cell expression patterns not detected in normal tissues. Importantly, most current antibody therapeutic stroma cell targets (CSF-1R, FAP and VEGF-A) were re-discovered. Several novel stroma cell therapeutic targets were identified which if targeted by a therapeutic antibody have the potential to enhance the T cell immune response in tumors resistant to current immunotherapy.

#754

Targeting carbonic anhydrase IX in multiple pancreatic ductal adenocarcinoma models results in tumor growth inhibition and increased survival.

Paul C. McDonald,1 Shawn C. Chafe,1 Marylou Vallejo,1 David Schaeffer,2 Don Yapp,1 Claudiu T. Supuran,3 Ben Stanger,4 Mac Parmar,5 Daniel Renouf,6 Shoukat Dedhar1. 1 _BC Cancer Research Ctr., Vancouver, British Columbia, Canada;_ 2 _Vancouver General Hospital, Vancouver, British Columbia, Canada;_ 3 _Universita degli Studi di Firenze, Florence, Italy;_ 4 _University of Pennsylvania, Philadelphia, PA;_ 5 _SignalChem Lifesciences Corp, Richmond, British Columbia, Canada;_ 6 _BC Cancer Agency, Vancouver, British Columbia, Canada_.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive growth, metastatic spread and poor 5 year survival rates of less than 5%. PDAC tumors develop extensive regions of hypoxia that contribute to its aggressive behavior and pose a significant hurdle to therapeutic response. Hypoxia-mediated adaptive responses for tumor cell survival and metastasis include metabolic reprogramming and pH regulation. Carbonic anhydrase IX (CAIX) is a membrane-bound, hypoxia-inducible enzyme that is highly expressed in solid tumors and functions as a critical component of the pH regulatory machinery required by hypoxic cancer cells for survival and metastasis. We have recently developed and validated novel small molecule inhibitors of CAIX and have demonstrated that these inhibitors are effective at reducing breast tumor growth and metastasis. The lead compound, SLC-0111, is currently being assessed in a Phase 1 clinical trial (NCT02215850). In the present study, we have evaluated the efficacy of targeting CAIX in PDAC using genetic and pharmacologic strategies. CAIX expression was found to be induced by hypoxia in a panel of human PDAC cell lines and was also upregulated in cell line-derived xenografts, patient-derived xenografts as well as the KRASG12D/p53-/- KPCY transgenic mouse model of PDAC (Rhim, AD et al. Cell, 2012:148;349-361). shRNA-mediated silencing of CAIX expression in PK-8 PDAC cells significantly reduced hypoxia-mediated cell proliferation (P<0.05) and invasion (P<0.01) in vitro, and dramatically delayed tumor growth (P<0.001) in vivo, resulting in increased median survival of the mice from 36 days to 59 days (P<0.01), similar to that observed following treatment with gemcitabine (36 days to 58 days, P<0.01). Interestingly, gemcitabine treatment in this model increased the fraction of CAIX-positive tumor cells, and silencing of CAIX expression in combination with gemcitabine treatment further delayed tumor growth (P<0.01) and increased median survival to 86 days (P<0.01). Similarly, sequential administration of gemcitabine and the CAIX inhibitor, SLC-0111, significantly delayed tumor growth (P<0.01) and enhanced survival (P<0.01) in PK-8 and PK-1 xenografts, compared to gemcitabine alone. Preliminary data indicate that treatment with SLC-0111, alone and in combination with gemcitabine inhibits the growth of spontaneous PDAC tumors in the KPCY transgenic model of PDAC, as assessed by ultrasound imaging. These results demonstrate that CAIX plays an important role in PDAC progression and is a potential therapeutic target for pancreatic cancer. A focused Phase 1b clinical trial of gemcitabine and SLC-0111 in patients with metastatic PDAC is planned, based on the outcome of the Phase 1 trial.

#755

Antagonistic effect of M-MDSC and N-MDSC during astrocytoma progression.

Chin-NIng Huang. _Department of biomedical and environmental science, Hsinchu, Taiwan_.

The traditional dogma holds that tumor size has a positive correlation with tumor progression. However, on murine orthotopical astrocytoma model ALTS1C1, we found that the tumor growth is not a linear function instead a shrinkage and regrowth wave was noticed. To examine the un-characteristic tumor growth behavior, the relative relationship among tumor cells, brain resident cells, such as astrocytes and microglia, and infiltrating cells, such as monocytic (CD45+CD11b+) or lymphocytic (CD45+CD11b-) lineage cells, was analyzed by flow cytometry and immunohistochemistry (IHC). Not only tumor area, peripheral organs, including spleen, blood, and lymph nodes were also examined. The preliminary data showed that in tumor shrinkage stage, CD3+ lymphocytes increased dramatically, while CD11b+CD45low microglia decreased. Furthermore, we found that the change of two subtypes of myeloid derived suppressor cells (MDSCs), Ly6G+ neutrophilic MDSC (N-MDSC) and Ly6G- monocytic MDSC (M-MDSC), was in opposite direction during tumor progression. The increase of M-MDSCs is associated with the increase of tumor size while the N-MDSC is decreased. More interestingly, the flow cytometry data shows that the total amount of CD11b+ macrophages was constant during tumor growth phase, but IHC shows the different distribution pattern of macrophage subsets within different tumor regions (invasion tumor front vs tumor core). Currently, we are examining the dynamic change of these cells following irradiation to identify the cells that are responsive for tumor shrinkage and re-growth following radiation therapy for brain tumor. These results will be presented in the meeting.

#756

Myo-inositol modulates the tumor microenvironment in a mouse model of lung cancer chemoprevention.

Nese Unver,1 Kirubel T. Zeleke,1 Ximing Tang,2 Samantha Holt,3 Seyed Javad M. Moghaddam,4 Hong Wang,1 Ignacio I. Wistuba,2 Samir M. Hanash,1 Edwin J. Ostrin4. 1 _Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _University of Illinois at Urbana-Champaign, IL;_ 4 _Department of Pulmonary Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Myo-inositol is a nutritional supplement that has been investigated as a chemopreventive agent for lung cancer. We have investigated the effect of myo-inositol in a CC-LR (CCSPCre/LSL-K-rasG12D) mouse lung cancer model. Blood, broncho-alveolar lavage fluid (BALF), and lungs were collected from CC-LR and LR littermate control mice at 14 weeks of age, when mice had a large number of premalignant lesions with only small foci of adenocarcinoma.

Methods: Lesion burden and stage was quantified using macroscopically evident lung surface tumor counts as well as histology from paraffin sections. Serial paraffin sections were also used for immunohistochemistry. Plasma was analyzed using high-sensitivity liquid-chromatography coupled tandem mass spectrometry. Cytokine and chemokine concentrations in BALF were quantitated using multiplex immunobead assays.

Results: Myo-inositol treated mice (n=20) had fewer tumors (p < 0.0001) than mice raised on control diet (n=23). Additionally, lesions were shifted towards lower grade, from adenomas and adenomatous alveolar hyperplasia to bronchial hyperplasia. Several host factors including cytokines and proteins produced by myeloid derived suppressor cells and macrophages were significantly impacted by myo-inositol treatment. In addition to changes in metabolic inflammation derived factors, proteins which are associated with both chronic inflammation and lung tumorigenesis were altered as well.

Conclusions: Myo-inositol treatment both reduced tumor burden and lesion grade in a KRAS model of lung cancer. Our findings of a substantial impact of myo-inositol treatment on the host response suggests novel mechanisms for its chemopreventive activitiy.

#757

p16INK4A negatively regulates Leptin through miR-141 and miR-146b-5p in breast stromal adipocytes.

Huda H. Alkhalaf,1 Abdelilah Aboussekhra2. 1 _King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia;_ 2 _King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia_.

Obesity, a disease linked to increased number of adipocytes in various tissues, is increasingly recognized as a risk factor for the development of breast cancer. Obesity has been reported to be associated with the occurrence of larger, more advanced tumors and aggressive cancer pathological features, including lymph node metastasis, advanced tumor stage, and high grade. Accumulating recent evidence highlights the tumor-surrounding adipose tissue as a key component of breast cancer progression. However, the molecular basis of adipocyte-related breast carcinogenesis remains elusive. To shed light on this important phenomenon, we investigated the autocrine and paracrine roles of the tumor suppressor p16 INK4A protein in adipocytes. We have found that down-regulation of p16INK4A by specific shRNA in breast adipocytes enhanced their migration/invasion abilities and increased the expression/secretion levels of various adipokines including leptin. In addition, we have shown that p16INK4A protein inhibits the pro-carcinogenic effects of breast stromal adipocytes in vitro and in tumor xenografts through repressing the expression/secretion of Leptin. Indeed, p16INK4A suppresses Leptin at the mRNA level. This effect is mediated trough miR-141 and miR-146b-5p, which inhibits Leptin expression through a specific sequence at the Leptin 3'UTR. In addition, we present clear evidence that down-regulation of p16INK4A is sufficient to trans-activate breast stromal adipocytes, which promote epithelial-to-mesenchymal-transition in normal breast luminal cells in a leptin-dependent manner.

These results provide the first indication that p16INK4A and miR-141 and miR-146b-5p represses Leptin and its procarcinogenic effects in breast stromal adipocytes. This indicates that p16 pathway has non-cell-autonomous tumor suppressor function.

#758

Modulating function of tumor associated macrophages by CSF1R inhibitor suppressed tumor growth in hepatocellular carcinoma.

Hui-Chuan Sun, Jian-Yang Ao, Hao Cai, Zong-Tao Chai, Xiao-Dong Zhu. _Zhongshan Hospital, Fudan Univ. Medical Ctr., Shanghai, China_.

Aims: To investigate the effect of colony stimulating factor 1 receptor (CSF1R) inhibitor on macrophages and tumor pathogenesis.

Methods: CSF1 and CSF2 stimulated macrophages were obtained from mouse bone marrow derived monocytes cultured with CSF1 and CSF2. Mouse hepatoma cell line Hepa 1-6, and human hepatoma cell lines, HCCLM3 and HepG2 were used to established a allograft or xenograft tumor models to examine the effect of CSF1R inhibitor on tumor growth and distribution of macrophages. The characteristics of macrophages were determined by flow cytometry, immunohistochemistry, cell motility and Western blotting assays.

Results: CSF1R inhibitor suppressed tumor growth in the tumor models derived from three hepatoma cell lines, and prolonged hosts' survival, but CSF1R inhibitor did not decreased the number of macrophages in Hepa1-6 allograft tumors. in vitro study showed CSF1R inhibitor suppressed proliferation and motility of CSF1 stimulated macrophages but not CSF2 stimulated macrophages. Furthermore, we found CSF1R inhibitor treatment decreased the ability of THP-1 macrophages to promote migration and invasion of tumor cells.

Conclusions: CSF1R inhibitor suppressed tumor growth in hepatoma animal models by modulating function of tumor associating macrophages.

#759

Sunitinib has opposite roles to regulate the myeloid-derived suppressor cells in tumors and peripheral blood.

Fang-Hsin Chen,1 Sheng-Yung Fu,2 Chun-Chieh Wang,1 Chi-Shiun Chiang,2 Ji-Hong Hong3. 1 _Chang Gung University, Taoyuan, Taiwan;_ 2 _National Tsing Hua University, Hsinchu, Taiwan;_ 3 _Chang Gung Memorial Hospital-LinKou, Taoyuan, Taiwan_.

Recent studies indicate that sunitinib, a tyrosine kinase inhibitor, could decease accumulation of myeloid-derived suppressor cells (MDSC) in tumors and also in peripheral blood. This study aims to investigate the behavior and dynamic profile of myeloid-derived suppressor cells in tumor tissues and peripheral blood of sunitinib-treated mice bearing prostate adenocarcinoma TRAMP-C1 tumors. To this end, 3x106 TRAMP-C1 tumor cells were inoculated at shank muscle of C57BL6/J mouse and sunitinib was administered by intraperitoneal injection of a daily dose of 20mg/kg into mice bearing 4 mm diameter tumor. Comparing to tumors in untreated group, sunitinib administration slightly delayed tumor growth delay for 2 days, but significantly decreased micro-vascular density and induced chronic hypoxia in tumors, resulting in the specific accumulation of CD11b+Gr-1+ MDSCs at the tumor necrotic region within CA-IX positive chronic hypoxic area. Flow cytometry was used to analyze the change of CD11b+ myeloid cells within tumor tissues and peripheral blood. Results showed that sunitinib treatment decreased the percentage of CD11b+Ly6G-LyC- tumor-associated macrophages (TAMs), but increased the percentage of CD11b+Ly6G-Ly6C+ monocytic MDSCs (M-MDSCs) while had no effect on CD11b+Ly6G+Ly6C+ neutrophilic MDSCs (N-MDSCs) within tumor tissues. In contrast, both the percentage of N-MDSCs and M-MDSC were expanded in peripheral blood of sunitinib-treated mice. Multiplex immunoassay showed significant increase of CCL2, CCL3, CCL5, CXCL5, IL-17a, GM-CSF, G-CSF and VEGF-A in tumor tissues, but only CXCL5, IL-α, IL-6 and G-CSF in the peripheral blood of sunitinib-treated mice were affected. In conclusion, this study shows that sunitinib has anti-vascular effect and could disrupt the recruitment of CD11b+Ly6G-Ly6C- TAMs into tumors. We also propose that sunitinib might exert different effects on tumor microenvironment and peripheral blood.

#760

Identifying candidate genes that regulate tumor cell dormancy.

Ryan J. Nini,1 Lara H. El Touny,2 Meera Murgai,1 Rosandra N. Kaplan,1 Jeffrey E. Green1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Leidos Biomedical Research, Inc., Frederick, MD_.

Tumor cells disseminate from the primary tumor site early in the disease process. These disseminated tumor cells may remain dormant for years and later become proliferative to cause clinical recurrence and morbidity. The goal of this study is to better understand the biological and genetic mechanisms driving this dormant to proliferative switch. We have developed a 3D culture system using the dormant murine mammary tumor cell line, D2.0R. D2.0R cells remain dormant when cultured on basement membrane extract (BME), and exit dormancy when supplemented with collagen-1 (col-1). We have characterized the gene expression profile of these cells exiting dormancy in 3D through microarray analysis, and developed a fourteen-gene dormancy signature by profile cross-referencing this expression profile to a tumor cell dormancy signature developed by Kim, R.S. et al (PLOS ONE 2012). We hypothesize that knocking down expression of genes upregulated in proliferative cells will hinder the ability of D2.0R cells to break dormancy in 3D culture supplemented with collagen-1. We have validated three genes by qPCR and are screening these upregulated genes for biological effects on proliferation by short-hairpin RNA or molecular inhibition of D2.0R cells in our 3D model. We conclude that this signature will provide useful insight into genetic drivers of the dormant to proliferative switch and potential candidates as therapeutic targets in recurrent disease.

#761

Specific inhibition of prostaglandin E2 production in medulloblastoma.

Filip Bergqvist, Linda Ljungblad, Malin Wickström, Karin Larsson, Anna Kock, Marina Korotkova, John Inge Johnsen, Per Kogner, Per-Johan Jakobsson. _Karolinska Institutet, Solna, Sweden_.

Introduction: Prostaglandin E2 (PGE2) is a key lipid mediator of inflammation and carcinogenesis. Apart from promoting direct growth of cancer cells, PGE2 has been shown to be crucial in maintaining the immunosuppressive tumor microenvironment. Drugs targeting cyclooxygenase (COX)-1 and -2, enzymes upstream in the production of PGE2 and other prostanoids, have successfully reduced tumor growth in many cancer models but these drugs are all associated with adverse long-term effects. The aim of this study is to demonstrate an anti-tumor effect in medulloblastoma by selective inhibition of microsomal prostaglandin E1 (mPGES-1), the enzyme downstream of COX-1/2 specifically required for the synthesis of PGE2.

Experimental procedures: Human medulloblastoma cell lines (DAOY and D283) were treated with a selective small-molecule inhibitor against mPGES-1 or COX-2 in the presence or absence of pro-inflammatory interleukin (IL)-1β. Prostaglandins in supernatants were extracted and quantified with LC-MS/MS. Drug efficacy was determined and cell toxicity was measured with cell viability assay. Mice with human medulloblastoma xenograft will function as proof-of-concept for the anti-tumor activities associated with inhibition of PGE2 production. Key inflammatory enzymes were quantified with Western blot.

Results: Medulloblastoma cells express high levels of COX-2, mPGES-1, and produce PGE2. The increased production of PGE2 by IL-1β was completely blocked when cells were co-treated with inhibitor for mPGES-1 or COX-2. Both inhibitors showed cell toxicity. We did not observe any shunting towards other prostanoids.

Conclusion: We investigate the effect of selective inhibition of mPGES-1 or COX-2 on medulloblastoma growth. We aim to further characterize the mPGES-1 inhibitor and compare to selective COX-2 inhibitor in preclinical medulloblastoma models. Specific inhibition of PGE2 production, rather than general inhibition of prostanoid production, would potentially prove useful as a complement to current medulloblastoma treatments.

#762

The interleukin-1 inflammatory cytokine family modulates p62/SQSTM1 accumulation in breast cancer cell lines.

Afshan Fathima Nawas, Janani Ramachandran, Shayna Thomas, Jananisree Ravichandran, Nikki Delk. _University of Texas at Dallas, Richardson, TX_.

Tumors are likened to wounds that do not heal. Thus, not surprisingly, inflammatory cytokines are present in the primary and metastatic tumor microenvironments due, in part, to infiltrating bone-derived cells (e.g., macrophages). We previously discovered that a bone marrow stromal cell line that secretes the inflammatory cytokine, interleukin-1 beta (IL-1β), upregulates p62 mRNA and protein in prostate cancer cell lines and we showed that p62 can be cytoprotective for prostate cancer cells. p62/Sequestome-1 is a multifunctional scaffold protein that can be seen microscopically as subcellular "speckles" which serve as organizing centers for multiple different signaling cascades, ranging from autophagy-mediated degradation of ubiquitinated proteins to KEAP1-NRF2 antioxidant response. To determine if our previous discoveries might be cancer-type specific and to test our hypothesis that p62 promotes cancer cell survival in response to inflammatory cytokines, we expanded our studies to various breast cancer cell lines of varying receptor status, including ZR57-1, T47D, and MDA-MB-468 and determined cell response two interleukin-1 family members, alpha (α) and beta (β). We treated the breast cancer cells with IL-1α and/or IL-1β and analyzed p62 mRNA and protein levels, subcellular localization, and protein-protein interactions. IL-1α and IL-1β can induce p62 speckle accumulation that can be seen to co-localize with the autophagosome membrane protein, LC3B, suggesting an autophagy-dependent regulation and/or function for p62. In addition, p62 co-immunoprecipitates with KEAP1, suggesting a KEAP1-NRF2-dependent antioxidant function for p62. Taken together, our data implies that cancer cells usurp inflammatory cytokines, such as IL-1, present in the primary or metastatic tumor microenvironment to promote cell survival through proteins such as p62.

#763

Exosomal miR-6126 as a novel tumor suppressor in ovarian cancer.

Pinar Kanlikilicer, Mohammed Mohammed H. S. Rashed, Recep Bayraktar, Rahul Mitra, Cristina Ivan, Burcu Aslan, Xinna Zhang, Rodriguez-Aguayo Cristian, Emine Bayraktar, Martin Picher, Bulent Ozpolat, George A. Calin, Anil K. Sood, Gabriel Lopez-Berestein. _MD Anderson Cancer Center, Houston, TX_.

Tumor microenvironment has been known to play a key role in cancer by the development of drug resistance, epithelial-mesenchymal transition, metastasis and angiogenesis. There is emerging evidence that cancer originated exosomes may contribute to the tumor microenvironment to promote tumorigenesis. Exosomes are nano-sized vesicles that contain non-coding RNAs such as miRNAs or siRNAs. Non-coding RNAs are involved in the pathogenesis of the majority of cancer and reveals a new layer of complexity in the molecular architecture of tumorigenicity. The purpose of this study is to identify miRNA content of both sensitive and resistant ovarian cancer-cell derived exosomes for the identification of exosomal RNA-mediated tumorigenesis in ovarian cancer. Using miRNA microarray profiling, we identified differentially expressed exosomal miRNAs in three sensitive (Heya8, Skov3, A2780) and resistant (Heya8-MDR, Skov3-TR, A2780-CP20) isogenic ovarian cancer cell lines. miR-6126 was found to be only miRNA significantly up-regulated in all cancer cell exosomes released to tumor microenvironment. In vitro functional assays showed that miR-6126 act as a tumor suppressor by inhibiting proliferation, migration, invasion and tube formation. miR-6126 acts as a novel potential tumor suppressor through targeting integrin beta-1, leading to inhibition of PI3K/AKT and Ras/Raf signaling. In vivo experiments also demonstrated that administration of miR-6126 into the nude mice with established tumors inhibited tumor weight. Immunohistochemistry results showed that miR-6126 treatment inhibits many oncogenic functions such as proliferation and angiogenesis of ovarian cancer. We believe that these findings help the identification of exosomal RNA-mediated tumorigenesis in ovarian cancer.

#764

LOX and LOXL2 inhibition as a treatment for ovarian cancer.

Angela Cho,1 Amanda L. Hudson,2 Samuel Yuen,3 Nham Tran,4 Viive M. Howell,2 Emily K. Colvin2. 1 _Bill Walsh Translational Cancer Research Laboratory, Kolling Institute and University of Technology Sydney, St Leonards, Australia;_ 2 _Bill Walsh Translational Cancer Research Laboratory, Kolling Institute, University of Sydney, St Leonards, Australia;_ 3 _Bill Walsh Translational Cancer Research Laboratory, Kolling Institute, St Leonards, Australia;_ 4 _Centre for Health Technologies, University of Technology Sydney, Sydney, Australia_.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women. Despite advances in our understanding of the molecular biology of EOC, patient survival has not significantly improved in decades and new treatments are needed. There is increased interest in therapeutic targeting of the tumor microenvironment (TME). The lysyl oxidase (LOX) family of enzymes are involved in extracellular matrix remodelling through crosslinking of collagen and elastin. They have been shown to play an important role in the TME of other cancers and are regulated by hypoxia, however little is known about their role in EOC. Therefore we aimed to investigate the role of LOX and LOXL2 in EOC using in vitro models.

Tumor epithelial (n=2) and matched cancer-associated fibroblast cell lines (n=2) derived from an SV40-induced mouse model of EOC were generated. MTT assays were performed on all cell lines to assess the effect of LOX and LOXL2 inhibition on cell viability. The effect of LOX and LOXL2 inhibition on cancer cell migration and invasion towards cancer-associated fibroblast conditioned media was also investigated using transwell assays. RNA and protein were isolated and analysed from cells cultured in normoxic and hypoxic conditions to assess whether LOX and LOXL2 gene and protein expression is regulated by hypoxia.

Inhibition of LOX and LOXL2 did not affect cell viability. LOX and LOXL2 inhibition significantly reduced the migration and invasion of EOC cells towards cancer-associated fibroblast conditioned media in vitro (p<0.05). Gene expression analysis comparing Lox and Loxl2 expression in all cell lines demonstrated a significant increase in Lox and Loxl2 expression in cells cultured in hypoxia (p<0.05). Similarly, cellular and secreted LOX and LOXL2 protein expression was increased in cells cultured in hypoxic conditions, supporting hypoxia as a regulator of LOX and LOXL2 expression.

This study demonstrates a role for LOX and LOXL2 in promoting migration and invasion of EOC cells in vitro. The upregulation of LOX and LOXL2 in hypoxia suggests a role in facilitating tumor growth and progression within hypoxic tumors. These results provide support for the utility of LOX and LOXL2 inhibition as promising and novel therapeutic strategies in EOC.

#765

The manipulation of FAP expression and its role in SDF-1 and IL-6 secretion by fibroblasts and melanoma cells.

Zachary B. Fisher, Yuko J. Miyamoto. _Elon University, Elon, NC_.

The cancer microenvironment surrounds tumors and primarily consists of fibroblasts and immune cells that cancer cells utiliaze to support their growth and invasion. Cancer-associated fibroblasts (CAFs) are the most prominent cell type in the microenvironment. They are attracted to tumors and activated by signals from cancer cells, including transforming growth factor-β (TGF-β). Following activation, CAFs display FAP on their surface, which breaks bonds in the extracellular matrix (ECM), creating space for cancer cells to migrate. CAFs also secrete cytokines, including stromal cell-derived factor-1 (SDF-1) and interleukin-6 (IL-6), to initiate this migration. This study seeks to determine if there is a link between the expression of fibroblast activation protein (FAP) and the specific production of SDF-1 and IL-6. Flow cytometry was used to verify the expression of FAP by the Malme-3 fibroblast cell line at an average rate of 50.6% and in the Malme-3M melanoma cell line at 4.8%. TGF-β treatment increased FAP expression in Malme-3M melanoma cells (50% increase), indicating a prominent role in cell transformation. siRNA transfection was also used to reduce FAP expression in both the fibroblasts (48.4% decrease) and the melanoma cells after initial TGF-β activation (11.6% decrease). Preliminary data from intracellular staining and ELISA analysis suggest that SDF-1 and IL-6 production both increased in melanoma cells in direct correlation to FAP expression in, but may be inversely related in fibroblasts. Future studies will further analyze the FAP expression levels and cytokine secretion of both cell types in different microenvironments to elucidate the relationship between FAP expression in cancer and the secretion of SDF-1 and IL-6.

#766

Control of lung cancer with aspirin-triggered stimulation of resolution.

Molly M. Gilligan,1 Megan L. Sulciner,1 Yael Gus-Brautbar,1 Sesquile Ramon,2 Romain A. Colas,2 Sui Huang,3 Charles N. Serhan,2 Dipak Panigrahy1. 1 _Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA;_ 2 _Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 3 _Institute for Systems Biology, Seattle, WA_.

Objective: To establish aspirin-triggered resolvins as a novel treatment to enhance current cancer therapies through the resolution of inflammation and clearance of cancer therapy-induced tumor cell debris. Background: Inflammation in the tumor microenvironment is now recognized as a strong promoter of tumor growth. Substantial epidemiological evidence suggests that aspirin, which suppresses inflammation, also reduces the risk of cancer. However, the mechanism by which aspirin reduces cancer risk and improves survival remains unclear, and toxicity has limited its clinical use. Low-dose aspirin stimulates the endogenous production of aspirin-triggered (AT) resolvins, novel omega-3 fatty acid-derived pro-resolving lipid autacoid mediators. We recently discovered that resolvins have potent anti-tumor activity, making resolvins potential candidates to mediate the anti-cancer activity of aspirin. Thus, we hypothesize that aspirin's anti-cancer activity is mediated in part by AT-resolvins via novel anti-inflammatory and pro-resolution mechanisms. Methods: Flow cytometry confirmed apoptotic and necrotic lung tumor cell debris following chemotherapy (etoposide) or targeted therapy (erlotinib) treatment in vitro. Macrophage phagocytosis was quantified via Relative Fluorescent Units (RFU). Cytokine levels were quantified via ELISA. Results: AT-resolvins counter therapy-induced debris-stimulated tumor growth and spontaneous lung metastases via enhancing endogenous clearance of tumor debris via macrophages and counter-regulating macrophage cytokine production. Pharmacological and genetic antagonism of the AT-resolvin D1 receptor (ALX/FPR2) neutralized aspirin's anti-cancer activity. Conclusions: AT-resolvins may provide a novel pathway and molecular mechanism to explain how aspirin reduces cancer risk so broadly. These anti-inflammatory and pro-resolving lipid autacoid mediators may complement current therapies for lung cancer with minimal toxicity.

#767

TCR sequencing can identify and track tumor-specific T cell populations and is a predictive biomarker of response to DC vaccination in glioblastoma patients.

Shaina Sedighim,1 Melody Hsu,1 Tina Wang,1 Richard G. Everson,1 Alex Tucker,1 Joseph P. Antonios,1 Lin Du,1 Ryan Emerson,2 Erik Yusko,2 Catherine Sanders,2 Harlan Robins,1 William Yong,1 Tom B. Davidson,1 Gang Li,1 Linda M. Liau,1 Robert Prins1. 1 _University of California, Los Angeles, Los Angeles, CA;_ 2 _Adaptive Biotechnologies, Seattle, WA_.

While immunotherapeutic strategies are emerging adjunctive treatments for cancer, sensitive methods of monitoring the immune response after treatment remain to be established. We used a novel next generation sequencing (NGS) approach to determine whether quantitative assessments of tumor infiltrating lymphocyte (TIL) content and the degree of overlap of T cell receptor (TCR) sequences in brain tumors and peripheral blood were predictors of immune response and overall survival in glioblastoma (GBM) patients treated with autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy. A significant correlation was found between a higher estimated TIL content and increased time to progression (TTP) and overall survival (OS). In addition, we were able to assess the proportion of shared TCR sequences between tumor and peripheral blood at time points before and after therapy, and found the level of TCR overlap to correlate with survival outcomes. Higher degrees of overlap, or the development of an increased overlap following immunotherapy, correlated with improved clinical outcome, and may provide insights into the successful, antigen-specific immune response.

#768

The ATP6V1C2 (a vacuolar-ATPase gene) is a novel early prognosticator for colorectal cancer.

Timothy J. Zumwalt, Kunitoshi Shigeyasu, Wenhao Weng, Yoshinaga Okugawa, Jinsei Miyoshi, Ajay Goel. _Baylor University Medical Center, Baylor Research Institute and Charles A. Sammons Cancer Center, Dallas, TX_.

Background: Vacuolar (v) -ATPases are expressed in certain tissues and cell types, and hypothesized to detoxify intracellular environments by expelling protons across plasma membranes, thereby contributing to acidified extracellular environments of solid tumors. Acidic microenvironments suppress anti-cancer cytotoxic T-cells and activate metalloproteinases, promoting tumor cell survival, motility and invasion. Scarce nutrients, hypoxia, and accelerated metabolism that favor pathways that produce excess intracellular protons by promoting glycolysis and lactic acid fermentation, lead to selection of tumor cells that adapted to and countered toxic/acidic cytoplasm. It is unclear whether colorectal cancers (CRCs) upregulate proton-expelling mechanisms, and whether associated genes are prognostic biomarkers.

Aim: We hypothesized that adaptations through upregulation of proton transporting genes is prognostic for CRC. This study aims to identify transcriptional adaptations that CRCs undergo in response to acidic microenvironments and evaluate their prognostic value in multiple cohorts of CRC patients.

Methods: Comprehensive microarray data from 222 CRCs obtained from The Cancer Genome Atlas were used to screen for genes upregulated in CRCs vs. normal mucosae, and stage III and IV vs. early staged tumors. Filtered genes involved in metabolite transport were tested for their ability to predict survival using Kaplan-Meier survival analysis with a stringent median cutoff. A second cohort was used to corroborate initial findings. A third cohort of matching polyps and adenomas was used to determine if candidate gene upregulation can be detected in pre-cancerous lesions. Functional analysis was performed using siRNA knockdown to determine if the candidate gene is critical for CRC proliferation.

Results: Progressive upregulation of ATP6V1C2 from early to late stage CRCs was detected in two separate multinational patient cohorts, where its high expression was correlated with shorter overall survival. Receiver operating characteristic (ROC) curve analysis confirmed that ATP6V1C2 expression successfully discriminated between cancerous and non-cancer tissues. Fisher's exact test confirmed that high ATP6V1C2 expression correlated with advanced depth of invasion (T stage), and distant and liver metastasis. ATP6V1C2 expression was increased in pre-cancerous polyps. SiRNA knockdown of ATP6V1C2 inhibited cell proliferation in high expressing cell lines (HCT116 and HCT15) but not in low expressing RKO and HT29.

#769

Garcinol inhibits cancer stem cell-like phenotype via suppression of the Wnt/β-catenin /STAT3 axis signalling pathway in human non-small cell lung carcinomas.

Wen-Chien Huang. _MacKay Memorial Hospital, Taipei, Taiwan_.

Innate or acquired drug resistance and consequently, tumor relapse in lung cancer patients have been linked to the activities of cancer stem cells (CSCs). Therefore, targeting CSCs is suggested as an effective approach for non-small cell lung cancer (NSCLC) therapy. In this study, we demonstrated that Garcinol, a polyisoprenylated benzophenone isolated from the fruiting bodies of Garcinia indica, possessing anti-inflammatory, antioxidant, acetyltransferase inhibitory and anticancer activities, modulates the activities of lung cancer stem cells (LCSCs) and their associated aggressiveness. Here, we demonstrated the inhibitory effect of Garcinol on LCSC phenotype of human NSCLC cells by using the analytical drug cytotoxicity or cell viability, flow-cytometric and functional assay approach. Garcinol significantly diminished the ability of NSCLC cell lines, H441 and A549 to form spheres. In parallel assays, Garcinol inhibited differentiated lung cancer cell and LCSCs viability in a dose-dependent manner. Consistent with these observations, Flow cytometric data showed that Garcinol reduced putative LCSC pool, evidenced by the dose-dependent decreased proportion of side-population (SP) cells and associated ALDH activity in Garcinol-treated H441 cells, compared to the control group. Additionally, functional assays showed that Garcinol markedly diminished the ability of H441 and A549 cells to form colonies. Mechanistically, Garcinol impaired phosphorylation of LRP6, a co-receptor of Wnt and STAT3. In same assay, Garcinol downregulated β-catenin, Dvl2, Axin2, and Cyclin D1 expressions in NSCLC-generated spheres, suggesting its ability to regulate the Wnt/β-catenin signalling pathway. Put together, we demonstrated here that Garcinol modulates the LCSC phenotype via regulation of the Wnt/β-catenin signalling and inactivation of STAT3, thus, presenting Garcinol as a putative novel anti-LCSC therapeutic agent.

Keywords - Garcinol, non-small cell lung cancer, cancer stem cells, Wnt/β-catenin signalling pathway, anti-cancer therapy, STAT3

#770

Negative regulation of the CCL22/CCR4 axis by TNFR1 improves melanoma outcome.

Caio Abner Leite,1 Paulo Henrique Melo,2 Jose Mauricio Mota,3 Douglas Silva Prado,2 Paula Ramos Viacava,2 Fernando Queiroz Cunha,2 Jose Carlos Alves Filho2. 1 _AC Camargo Cancer Center, Sao Paulo, Brazil;_ 2 _University of Sao Paulo, Ribeirao Preto, Brazil;_ 3 _University of Sao Paulo, Sao Paulo, Brazil_.

Purpose: Immunotherapy for melanoma skin cancer is mainly focused on preventing T cell deactivation through PD1 or CTLA4 inhibition. Another proposed mechanism to increase global T cell response is to prevent Treg immunossupressive function through TNFR1/TNF activation or CCR4/CCL22 inhibition. We aimed to evaluate the role of TNFR1 and CCR4 in an experimental model of melanoma.

Experimental procedures: C57/Bl6 WT, TNFR1 -/- or CCR4 -/- mice were subcutaneously inoculated with 30,000 B16-F10 cells for tumor growth measurement and survival analysis. In a different set of experiments, mice were euthanized fourteen days after inoculation (D14) for spleen, lymph nodes and tumor harvesting for Treg (CD4+CD25+FoxP3+) and TAM (CD45+F4/80+CD206+) quantification by flow cytometry. CCL22 concentrations were quantified in conditioned media from bone marrow-derived macrophages (BMDM) from WT, or TNFR1 -/- mice. Furthermore, TNFR1 and CCR4 gene expression in human melanoma was determined by data mining in deposited datasets (GSE46517).

Results: TNFR1-/- showed increased tumor volumes and reduced survival when compared to their WT counterparts. At D14, we detected increased Treg and TAM accumulation in TNFR1-/- tumor microenvironment. CCL22 concentrations were increased in conditioned media from M2-polarized BMDM from TNFR1-/- mice. CCR4 deficient mice presented reduced tumor volumes and increased survival, accompanied by a reduced Treg accumulation at D14. Data mining in GEO gene expression bank revealed that metastatic melanoma presented reduced TNFR1 and increased CCR4 expression.

Conclusions: CCR4 inhibition is a potential strategy to reduce Treg accumulation and improve outcomes in cancer patients. Further assessments are required to demonstrate the role of TNFR1 pathway in CCR4 activation.

#771

Multi-parametric 3D tumor microtissue-based phenotypic compound classification.

Madhu A. Lal,1 Zoe Weydert,2 Jens Kelm,2 Marc Ferrer1. 1 _NIH-NCATS, Rockville, MD;_ 2 _InSphero, Zurich, Switzerland_.

Here, we report on the development of homo- and heterotypic ovarian and pancreatic microtissue tumor models for a screening of up to 40 compounds selected from the NCATS Oncology focused library of small molecules. The goal of this study was to develop a high throughput compatible drug screening platform that could leverage the best of high throughput and high content screening assays to generate a pharmacological profile of activities that we hope will help better predict activity in vivo .This platform utilizes 3D multicellular spheroids from ovarian (HEY and SKOV) and pancreatic (Panc-1) cancer and enables the discrimination of tumor-specific efficacy and nonspecific cytotoxicity of a drug candidate with subsequent identification and validation of the molecular mechanism of action (MMOA). The biological responses measured over a 10-day drug exposure period included (i) growth kinetic (microtissue size), (ii) potency (IC50ATP_10_days) and efficacy (max. response ATP and size). The biological response of the compounds was compared between the different cell culture formats tested, 2D, 3D homotypic and 3D heterotypic. The comparison of IC50 values among the different ovarian cancer cell cultures showed that 21 out of the 38 compounds tested were more potent in 3D than in 2D. Within the pancreatic models, 13 out of 20 compounds tested were more potent in 3D than in 2D. Interestingly, most of the compounds which showed stronger potency in 3D were targeted small molecule agents. Comparing drug responses of homo vs heterotypic ovarian tumor model systems, 3 compounds were effective only in the heterotypic model including the WNT inhibitor PNU-74654 and GABA uptake inhibitor (Gat-1) SK&F-89976A.

We also evaluated nonspecific cytotoxic effects on the incorporated NIH3T3 fibroblasts by quantifying a secreted reporter over time. The assay system allowed us to discriminate between acute cytotoxic effects (3-day exposure) and sub-chronic toxicity (7-day exposure). Whereas only a very limited number of compounds had acute cytotoxic effects, there were considerable more compounds with sub-chronic cytotoxicity. Based on the single endpoint classifications, we chose an mTOR inhibitor, Torin-2, to further assess molecular mechanism of drug action for all three tumor microtissue models used in this study, applying reverse phase protein array (RPPA) and transcriptome analysis for signaling pathway profiling. Preliminary results of RPPA analysis revealed a clear down-regulation in the cell cycle and PI3K/Akt/mTOR signaling pathway, especially for S6 ribosomal protein phosphorylated at Ser 235, 236, 240 and 244, indicating a strong inhibitory effect on translation and cell growth control.

In summary, we present a 3D tumor microtissue-based phenotypic drug discovery testing scheme which allows a multi-parametric endpoint analysis paired with subsequent molecular pathway identification.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Systems Biology

#772

The molecular landscape of dermal neurofibromatosis.

Sara JC Gosline,1 Pamela Knight,2 Thomas Yu,1 Nripesh Prasad,3 Angela Jones,3 Shristi Shrestha,3 Braden Boone,3 Shawn E. Levy,3 Andrew J. Link,4 Allison C. Galassie,4 Hubert Weinberg,5 Stephen Friend,1 Salvatore La Rosa,2 Justin Guinney,1 Annette Bakker2. 1 _Sage Bionetworks, Seattle, WA;_ 2 _Children's Tumor Foundation, New York, NY;_ 3 _Hudson Alpha, Huntsville, AL;_ 4 _Vanderbilt University, Nashville, TN;_ 5 _Mount Sinai School of Medicine, New York, NY_.

Background: Neurofibromatosis type I (NF1) is a genetic disorder that disrupts neurological tissue growth and can lead to a diverse set of symptoms including systematic growth of benign tumors, learning disorders and bone deformities. It is a rare disease occurring in only 1 in 3,000 people worldwide. While the disease has been linked to loss of function in the NF1 gene - a known tumor suppressor - there is a high degree of phenotypic diversity in the NF1 patient population, making it difficult to identify the underlying cause of the disease and treat it effectively. In this work we seek to improve overall knowledge of dermal NF1 through global molecular characterization of the disease.

Methods: We have collected four dermal neurofibromas and peripheral blood from each of 11 NF1 patients. We analyzed each sample using (1) Whole genome sequencing (WGS) on the Illumina HiSeq X platform, (2) Illumina OMNI2.5 Arrays (3) RNA-Sequencing on an Illumina HiSeq v4 machine and (4) iTRAQ-labeled proteomics. WGS data for both tumor and blood samples from each patient were used to identify patient-specific germ-line mutations as well as tumor-specific somatic mutations in each sample. Single nucleotide polymorphisms identified by the OMNI Arrays were used to identify copy number alterations in both blood and tumor samples. RNA-Seq data and proteomics data were mapped to transcripts and proteins respectively.

Results: Preliminary analysis of this data illustrates a diverse genomic landscape of NF1. Hierarchical clustering of copy number alterations largely show samples clustering by tissue, suggesting that most copy number alterations are somatic and not shared across the germline. However, there are two patients that show germline copy number alterations, including one patient with loss in the NF1 region. WGS analysis suggests similar diversity with each patient possessing a distinct combination of germline and somatic mutations of NF1 and other cancer-related genes. Cluster analysis of the RNA-Seq data shows no patient-specific clusters, suggesting that that each tumor executes a unique transcriptional program.

Conclusion: This work represents a first-ever attempt to profile the diversity of dermal neurofibromatosis at a molecular level. Preliminary analysis of the data underscores the complexity of this disease and explains, in part, previous difficulty in identifying effective treatments. Ongoing work includes expanding the analysis to include more patient samples and other types of NF1-derived tumors. As an orphan disease, NF1 has been poorly characterized compared to more common cancers. To rectify this, the Children's Tumor Foundation and Sage Bionetworks are collaborating to make NF1 data available to the public to accelerate research and the drug discovery pipeline. We expect that this data will be a resource for other NF1 researchers to assist in the study of this disease at the molecular level. All data and preliminary results are publicly available at http://www.synapse.org/dermalNF

#773

Analysis of oncogene affected networks in tumorigenesis of lung cancer.

Adwait Sathe, Yong Chen, Hyuntae Yoo, Michael Q. Zhang. _University of Texas at Dallas, Richardson, TX_.

Genetic abnormality is one of major causes of tumorigenesis by driving the progressive transformation of normal human cells into highly malignant derivatives. Tracing the downstream pathways through which and how the effects of the oncogenic-activating mutations are transmitted is an important and challenging problem in studying disease mechanisms, diagnoses and treatments. In this study, we designed a network-based computational method, called MAXMAP, to not only predict mutation downstream affected pathways, but also quantitatively measure the dynamics of the pathway genes.

We first constructed a heterogeneous network by integrating protein-protein interaction networks and signaling and regulatory networks from available up-to-date curated databases, as well as RNASeq expression data. The RNAseq dataset of these mutated cell lines has 18,096 genes whereas the Protein Protein Interaction and Signalling, regulation dataset has 14,864 genes. We then used the maximal information flow method to globally optimize the effects from the mutations to target downstream genes. The method was applied to analyze the biological network dynamics that is caused by single (TP53), double (TP53+KRAS) and triple (TP53+KRAS+STK11) mutations of HBEC30KT, an immortalized lung cell line, and revealed the core dynamical subnetworks of these mutations. Only the triple mutant is known to have tumor-like properties and as shown in earlier studies these sequential mutation accumulations lead to tumorigenesis in mouse xenograft models. The experimental validation of a small subnetwork was performed using transient knock down of the pathway genes and expression was verified using qPCR.

We showed the dynamical subnetworks through which a gene, SLC7A5 was affected by sequential single, double or triple mutations. We have experimentally validated the predicted small subnetwork for SLC7A5 which is an important transporter of neutral amino acids. It is associated with bladder and bile duct adeno carcinomas and overexpressed in lung cancers. We selected the subnetwork affected due to the single mutation (TP53) for validation, including THBS1, TNFRSF11B and MAPK1 and SLC7A5.

Hence, using the MAXMAP method we have successfully identified a dynamical subnetwork affected by changes in the known network of genes. These results suggest that this computational method may allow us to predict drug targets or biomarkers or to study interesting alternative paths and predict the most likely path leading to certain phenotypic outcomes.

#774

Proteogenomic characterization of high-grade serous ovarian cancer.

Jason E. McDermott, Samuel Payne, Debjit Ray, Vladislav Petyuk, Ronald Moore, Marina Gritsenko, Richard Smith, Karin Rodland. _Pacific Northwest National Laboratory, Richland, WA_.

High grade serous ovarian carcinoma (HGSOC) is a highly lethal gynecological malignancy, in large part because most patients develop resistance to the standard of care chemotherapy with platinum and taxanes. We performed deep proteomic and phosphoproteomic characterization on ovarian high-grade serous carcinomas (HGSC) previously characterized genomically by the The Cancer Genome Atlas (TCGA) as part of the Clinical Proteomic Tumor Analysis Consortium (CPTAC; http://proteomics.cancer.gov/programs/cptacnetwork). We constructed an integrated proteogenomic landscape of HGSC and identified functional modules statistically associated with outcomes including platinum resistance and overall survival.

Integration of genomic data such as copy number alterations (CNA) with global protein abundance data revealed several chromosomal regions that have significant trans effects on protein expression. These genetically affected proteins were used to construct statistical models capable of predicting survival outcomes. Trans-affected proteins were enriched in proliferation, cell motility and invasion, and immune regulation, hallmarks of cancer.

We used statistical methods to identify functional pathways able to discriminate between short surviving and long surviving patients. We found that protein abundance and phosphorylation indicated pathway activity that was able to clearly discriminate between these two groups, but that transcriptional expression and CNAs were not. We integrated proteomic abundance and phosphorylation state to elucidate specific signaling interactions and pathways differentially active in tumors from patients with short overall survival as well as to predict novel kinase substrate relationships important in HGSC. Finally, we identified druggable pathways that are differentially active across proteomic subtypes.

A key point arising from our multimodal analysis is that combining different types of molecular data from these tumors greatly increases the ability to identify biologically relevant differences between groups. Using a Lasso-based regression approach to integrate data we show that the most robustly predictive survival models can be obtained using integrated CNA, transcriptome, proteome, and phosphoproteome data than with any of the individual sources of data.

#775

Combinatorial compound stratification based on integration of LINCS, TCGA and PubChem data.

Vasileios Stathias,1 Bryce Allen,1 Jennifer Clarke,2 Nagi G. Ayad,1 Stephan Schürer1. 1 _University of Miami, Miami, FL;_ 2 _University of Nebraska, Lincoln, NE_.

The purpose of this study is to leverage the large number of perturbation signatures from the NIH Library of Integrative Network-based Cellular Signatures (LINCS) and integrate them with patient data from The Cancer Genome Atlas (TCGA) and PubChem bioactivity data in an effort to prioritize compounds based on their synergistic effect in cancer treatment.

From the large number of LINCS L1000 transcriptional data capturing cellular responses after chemical or genetic perturbations, we extracted gene expression signatures that were indicative of specific LINCS compounds. Moreover, we compared the L1000 transcriptional profiles with ones from TCGA in order to identify characteristic signatures of major cancer types and prioritized small molecule compounds with discordant expression profiles to those cancer types. We then linked the above compounds to protein target annotations and therefore produced a compound-specific protein target profile. For this, we utilized biochemical data produced by the LINCS KinomeScan and KiNative assays and also biochemical data obtained through PubChem. Using the above information, we obtained pairs of compounds that would inhibit unlinked gene sub-networks that were produced through processing of the TCGA transcriptional data.

The above process can be used as a means to suggest compound combinations towards specific cancer types and to prioritize the development of compounds with targeted polypharmacology.

#776

Multiscale treatment response model for triple-negative breast cancer linking drug pharmacokinetics to tumor cell population dynamics.

Matthew T. McKenna, Stephanie L. Barnes, Abigail Searfoss, Darren R. Tyson, Erin Rericha, Vito Quaranta, Thomas E. Yankeelov. _Vanderbilt University, Nashville, TN_.

Introduction

The goal of this study is to establish a predictive model of cytotoxic therapy that incorporates in vitro drug pharmacokinetics and cell-scale therapy response data, on a cell-line specific basis. We report on a series of time-resolved fluorescence microscopy experiments to characterize the uptake of doxorubicin and its effect on the population dynamics of MDA-MB-231 cells, a model of triple negative breast cancer.

Experimental Design

We leveraged the intrinsic fluorescence of doxorubicin to measure its uptake by MDA-MB-231 cells. Cells, labeled with a fluorescent nuclear marker, were seeded in microtiter plates and incubated with doxorubicin concentrations ranging from 10 nM to 10 µM for 6, 12, or 24 hours. These plates were imaged daily via bright field and fluorescent microscopy after addition of doxorubicin. Nuclei were segmented and automatically counted to quantify cell population size. Counts were normalized to population size at time of treatment and converted to population doublings. On a separate channel, extracellular, cytoplasmic, and nuclear doxorubicin fluorescence were quantified. A compartment model describing the movement of doxorubicin from the extracellular space into cells was fit to these data. We then constructed a cell treatment response model and fit it, coupled with the compartment model, to the population data using MATLAB.

Results

MDA-MB-231 cellular response to doxorubicin was tightly linked to both drug concentration and exposure time. Higher doses (> 1 µM) invariably induced rapid cell death. Smaller doses (< 1 µM) induced a concentration-dependent nonlinear response defined by an initial increase in population size that, depending on exposure time, was followed by a protracted decrease in cell number. For example, when treated with 156 nM for 6, 12, and 24 hours, we observed, respectively, an average of 2.6, 2.1, and 0.67 population doublings over the first 150 hours after treatment (p < 0.05 among groups). These populations then either held stable or receded out to 400 hours, when we observed net population doublings of 2.6, 1.7, and 0.037, respectively (p < 0.05). Untreated cells followed a logistic growth pattern, with an average total of 4.4 population doublings.

Conclusion

These time-resolved treatment protocols replicate clinically observed pharmacokinetics of cytotoxic therapies more closely than the constant concentrations in previous dose-response assays. By explicitly considering both drug and population dynamics, our mathematical model enables exploration, in silico, of treatment protocols intractable experimentally. Predictions from model simulations can then be tested experimentally, hopefully allowing for computationally-optimized and experimentally validated treatment regimens that maximize cytotoxic effects of doxorubicin.

#777

The CoGAPS matrix factorization algorithm infers feedback mechanisms from therapeutic inhibition of EGFR that increases expression of growth factor receptors.

Elana J. Fertig,1 Hiroyuki Ozawa,2 Manjusha Thakar,1 Jason Howard,1 Gabriel Krigsfeld,1 Alexander V. Favorov,1 Daria A. Gaykalova,1 Michael F. Ochs,3 Christine H. Chung4. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Keio University, Japan;_ 3 _The College of New Jersey, Ewing Twp, NJ;_ 4 _Moffitt Cancer Center, Tampa, FL_.

Next generation sequencing technologies enable precise personalized medicine. Thus, patients with oncogene driven tumors are currently treated with targeted therapeutics such as EGFR inhibitors. However, drug interactions with other activated signaling pathways in treated tumors often alter predicted therapeutic response. Therefore, bioinformatics algorithms are needed to infer unanticipated molecular interactions from anticipated molecular response to targeted therapeutics in diverse genetic backgrounds. To model heterogeneous genetic backgrounds in HNSCC, we use HaCaT cells with forced overexpression of EGFR, HRAS, and PIK3CA. Previously, the CoGAPS matrix factorization algorithm was shown to infer the specific signaling pathways that were activated in these HaCaT knock-in constructs from gene expression data. In this study, we evaluated whether CoGAPS could also delineate unanticipated signaling changes from anticipated cellular signaling response caused by targeted therapeutic in diverse genetic backgrounds. To delineate these signaling responses, we measured gene expression after treating the modified HaCaT cells with three EGFR targeted agents (gefitinib, cetuximab and afatinib) for 24 hours. The CoGAPS matrix factorization algorithm distinguished a gene expression signature associated with the anticipated silencing of the EGFR network and a signature associated with unanticipated transcriptional feedback in HaCaT constructs that were sensitive to EGFR inhibitors. Notably, the feedback signature showed that EGFR gene expression itself increased in cells that were responsive to EGFR inhibitors. The CoGAPS algorithm further associated such feedback with increased expression of several growth factor receptors by the AP-2 family of transcription factors. Once transcribed, these growth factor receptors may ultimately compensate for EGFR inhibition in these sensitive cells. Our data suggest, that CoGAPS gene expression signatures delineate on target and feedback effects of drugs related to therapeutic sensitivity in diverse genetic backgrounds.

#778

DNA repair capacity, chromosomal damage, methylation and gene expression levels in bladder cancer: An integrated analysis.

Giuseppe Matullo,1 Clara Viberti,2 Barbara Pardini,2 Alessandra Allione,2 Simonetta Guarrera,2 Valentina Turinetto,3 Claudia Giachino,3 Giovanni Fiorito,2 Alessio Naccarati,2 Alessia Russo,2 Rossana Critelli,2 Paolo Destefanis,4 Marco Oderda,4 Paolo Gontero,4 Paolo Vineis,5 Carlotta Sacerdote6. 1 _Turin Univ. and HuGeF, Turin, Italy;_ 2 _Human Genetics Foundation, Turin, Italy;_ 3 _Dept of Clinical and Biological Sciences, University of Turin, Turin, Italy;_ 4 _Dept of Urology, Città della Salute e della Scienza, Turin, Italy, Turin, Italy;_ 5 _MRC-HPA Centre for Environment and Health, School of Public Health, Imperial College London, London, United Kingdom;_ 6 _Center for Cancer Prevention (CPO-Piemonte), Turin, Italy_.

Bladder cancer (BC) is the sixth most commonly diagnosed tumor worldwide. DNA repair capacity (DRC) refers to the ability of a cell to protect the integrity of the genome and DNA repair pathways have been implicated in BC risk. It has been observed that individuals with low DRC tend to accumulate more damage than those with a more efficient DRC. This inter-individual variability is modulated by the genetic background, as well as differential gene expression and epigenetic regulation.

We aimed at studying the relationship between DRC and DNA damage (evaluated by H2AX phosphorylation and micronucleus assays) and BC risk and clinical outcome, integrating with gene expression and epigenetic profile data in 159 BC cases and 159 matched controls, enrolled in the Turin Bladder Cancer Study (TBCS).

We investigated γ-H2AX phosphorylation levels and MN frequencies in cryopreserved peripheral blood mononuclear cells. We found significant differences in micronuclei and nuclear buds frequencies, with higher number of these damages in cases compared to controls (p=0.0002 and p= 0.002 respectively).

On the other hand, we observed a significant association between γ-H2AX basal levels and risk of disease recurrence/progression in both BC patients as a whole and the subgroup of non-muscle invasive BC (NMIBC) (for all BC HR 0.70, 95% CI 0.52-0.94, p=0.02; for NMIBC HR 0.68, 95% CI 0.50-0.92, p=0.01): this suggests a protective effect of DNA double strand breaks signalling in terms of preventing BC recurrence or progression.

In order to evaluate the genetic and epigenetic role in modulation of DRC we performed whole genome methylation and gene expression analyses on the same BC cases and controls. Preliminary analyses on methylation levels did not show any significant difference between cases and controls. Two metalloproteinases (MMP23A and MMP23B) resulted significantly under-expressed in BC compared to healthy controls (logFC=-0.23, p=0.01; logFC=-0.37, p=0.007, respectively). Interestingly, the expression levels of these genes were also significantly correlated with the relative CpGs methylation.

Further analyses focusing on the integration of whole genome data with DRC assays are ongoing to unravel new prognostic biomarkers of disease.

#779

The role of exosome-mediated cell-cell communication in inducing phenotypic changes.

Mingyang Lu,1 Michela Capello,2 Satyendra C. Tripathi,2 Herbert Levine,1 Eshel Ben-Jacob,3 Samir M. Hanash,2 José Onuchic1. 1 _Rice University, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Tel-Aviv University, Tel Aviv, Israel_.

A new class of intercellular communication has recently emerged that involves transfer of exosomes, especially during the interactions between tumor and the immune system. However, the advantage of exosome-mediated cell-cell communication over direct cell to cell communication mechanisms or through soluble factors is unclear. We have performed mass spectrometry based proteomic profiling of exosomes isolated from pancreatic cancer cell lines, for the exosome cell surface and for the protein cargo. The resulting profiles were compared with proteomic profiles of corresponding cell lysates. We uncovered substantial enrichment in protein pairs in exosomes across cell lines but not in cell lysates, suggestive of a mechanism of exosome-based protein sorting and packaging that favor specific protein combinations. Stochastic simulations and analytical analysis provide supportive evidence that exosomes enable recipient cells to rewire gene regulatory networks to induce robust phenotypic transitions among different gene states. Moreover, from the simulation of two-way exosome-mediated communication of two cell types, we show that cells could achieve synchronized decision making through exosomes. Our integrative approach provides new insights into unique features of exosome-mediated cell-cell communication.

#780

Multi-omic profiling of gliomas reveals distinct DNA methylation changes at tumor recurrence.

Lindsay C. Stetson,1 Camila Ferreira de Souza,2 Tathiane Maistro Malta,2 Thais Sarraf Sabedot,2 Quinn Ostrom,1 Peter Liao,1 Daniela Pretti da Cunha Tirapelli,2 Luciano Neder,2 Carlos Gilberto Carlotti,2 Rehan Akbani,3 Sofie Salama,4 Laila Poisson,5 Daniel Brat,6 The Cancer Genome Atlas Network, Houtan Noushmehr,2 Jill Barnholtz-Sloan1. 1 _Case Western Reserve University School of Medicine, Cleveland, OH;_ 2 _Ribeirão Preto Medical School, University of Sao Paulo, Brazil;_ 3 _University of Texas, MD Anderson Cancer Center, TX;_ 4 _University of California, Santa Cruz, CA;_ 5 _Henry Ford Hospital, MI;_ 6 _Winship Cancer Institute of Emory University, GA_.

Varying possibilities of tumor relapse for lower grade glioma (LGG, WHO grade II and III) and glioblastoma (GBM, WHO grade IV) have led to heterogenous clinical outcomes for patients. Our current study aims to establish a molecular profile of recurrence of matched primary and recurrent LGG (n=28) and recurrent GBM (n=24) tumor samples. The Cancer Genome Atlas (TCGA) has comprehensively profiled these matched primary/recurrent tumor sets; whole genomes, coding exomes, methylomes, and transcriptomes have been sequenced, and the samples have undergone targeted profiling of the proteome. While unsupervised analysis techniques have not led to a clear recurrence signature, supervised analysis methods have revealed interesting patterns. Protein profiling has shown that recurrent gliomas retain the overall molecular signature of their primary counterpart, but the DNA damage response, apoptosis and RTK pathways are downregulated in the recurrent gliomas, in contrast to RAS/MAPK, PI3K/AKT, and EMT pathways, which are upregulated. Whole genome sequencing and rearrangement analysis have revealed increased genomic complexity among most recurrent gliomas as well as new fusions of interest in recurrent LGG samples (PTPRZ1-MET and involving ATRX). Using genome-wide Illumina HumanMethylation 450K data we observed that 78.6% of LGGs showed depletion of DNA methylation at recurrence and 50% of GBM tumors showed an enrichment of DNA methylation at recurrence. Patient centric enrichment analysis allowed us to discover a candidate biological subgroup characterized by a subset of LGG recurrences (50%) exhibiting an aberrant CpG methylation loss at inintergenic opensea regions when compared with canonical CpG islands and shores (fold > 1.3 and confidence intervals of 99%). Importantly, inspection of CpG sites significantly hypomethylated at openseas showed that this pronounced epigenetic signature maps to candidate TSS distal and hypomethylated enhancers. The gene-targets of these hypomethylated CpG sites show a corresponding up-regulation of expression. Pathway analysis has demonstrated that these upregulated genes are involved in cellular growth and proliferation, cellular function and maintenance, and cell cycle regulation. Our results provide evidence that DNA methylation may represent a stable signature of glioma recurrence and that the crosstalk between DNA hypomethylation at openseas and chromosomal instability may be involved in glioma recurrence. We plan to further integrate our findings between data types and correlate with treatment and patient clinical outcome.

#781

Modeling the impact of somatic alterations across human cancers.

Hatice U. Osmanbeyoglu, Eneda Toska, José Baselga, Christina Leslie. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Large-scale cancer genomics projects like The Cancer Genome Atlas have generated a comprehensive catalog of somatic mutations and copy number aberrations across many tumor types, but the role of most frequently altered genes remains obscure. To better model the impact of these alterations, we developed a novel computational strategy for exploiting parallel phosphoproteomics and mRNA sequencing data for large tumor sets by linking dysregulation of upstream signaling pathways with altered transcriptional response through the transcriptional circuitry. Our modeling allows us to interpret the impact of somatic alterations in terms of functional outcomes such as altered signaling and transcription factor (TF) activity.

We used this novel machine learning strategy to train phosphoprotein-TF interaction models across 12 human cancers for which large reverse-phase protein array and RNA-seq data sets are available through TCGA. First, we used this approach to identify shared and cancer-specific roles of TF/signaling regulators across cancer types. Then we performed a statistical analysis to associate frequent somatic aberrations with alterations in inferred TF and signaling protein activities. From our analysis, we gained many novel insights into cancer biology. We identified both known (e.g FOXO1 for breast cancer) and novel TF regulators of cancer (e.g. ELK1 for head and neck cancer). Many of these identified TF regulators were significantly associated with survival outcome in bladder urothelial, renal cell clear and endometrial carcinoma.

Next we performed a comprehensive cross-cancer analysis and identified relationships between somatic alterations and downstream transcriptional effects and signaling pathway activation. We observed that specific molecular aberrations have different functional consequences in different cancer types. For example, PIK3CA mutations are associated with altered activities of a diverse set of TFs across cancers that are involved in cell cycle, apoptosis, metabolism and MAPK/ERK signaling. Notably, in cell line models, we validated some of the altered TFs predicted by our model. We showed that PIK3CA mutation leads to ELK1 activation in breast and head and neck cancer models. We further found that different set of TFs are associated with a specific mutation in different cancers due to the different background of genomic aberrations in each cancer. For example, KRAS mutations are associated with a distinct set of TFs depending of cancer-specific co-mutation profiles (e.g. co-mutations of KRAS and TP53 in lung adenocarcinoma and co-mutations of KRAS and APC in colorectal cancer).

Our analysis revealed both known and novel interactions of frequently altered genes with signaling pathways and transcriptional programs in a pan-cancer context. Patterns of co-alterations across cancers may provide new insights relevant to targeted therapy and may be crucial to optimizing combination therapies.

#782

A network approach to study the effect of chemical exposures on gene regulatory system in rats.

Francesca Petralia, Vasily Aushev, Kalpana Gopalakrishnan, Susan Teitelbaum, Jia Chen, Pei Wang. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Exposure to environmental chemicals during early development may increase the risk of developing breast cancer later in life. In this context, we are interested in characterizing which microRNA (miRNA) and mRNA expressions change in a coherent manner across the lifespan, and whether the co-expression pattern is affected by environmental exposures. miRNAs contribute to tumor progression via the regulation of post-transcriptional gene expressions. Thus, information on different interaction patterns among miRNAs and mRNAs measured in mammary tissues from chemical exposed vs. non-exposed rats can cast light on how chemical exposures may alter mammary gland development. Specifically, we consider three common environmental chemicals: diethyl phthalate (DEP), methyl paraben (MPB) and triclosan (TCS). Female Sprague-Dawley rats were treated with these chemicals at four windows of susceptibility (prenatal, neonatal, prepubertal and pubertal) respectively with oral doses shown to produce urinary metabolite levels similar to those measured in US population. We implemented a new algorithm, Joint Random Forest (JRF), for simultaneous estimation of multiple related networks to characterize co-expression patterns among mRNAs and miRNAs. JRF is designed to borrow information across different treatment conditions, so that regulatory relationships shared across conditions can be detected with increased power, while those specific to each condition can be detected with fewer false positives. We focused on 1403 mRNAs and 283 miRNAs with larger variability across rats, and derived four co-expression networks of these mRNAs/miRNAs for each environmental chemical treatment plus a control group. Overall we observed a substantial loss of connectivity in networks of chemical exposed groups (DEP: 1813 edges, MPB: 1539 edges and TCS: 1013 edges) compared to that of the control group (2641 edges). Interestingly, despite the overall loss of connectivity in networks of chemical exposed groups, some microRNAs such as rno-miR-126b (MIMAT0017843) and rno-miR-3596b (MIMAT0017871) showed many connecting edges in networks of chemical exposed groups but zero in that of the control group. In particular, rno-miR-3596b is a member of the Let-7 family which is well known to regulate self-renewal and tumorigenicity in breast cancer cells. Findings like this can lead to better understanding of how chemical exposures could alter gene regulatory activities. Our study also demonstrates the great potential of using JRF to investigate changes in gene regulatory system across different conditions.

#783

Methylation-expression QTLs (meeQTLs) as part of an integrated model of the disruption of gene regulation in cancer.

Jeffrey A. Thompson, Carmen J. Marsit. _Geisel Medical School at Dartmouth College, Hanover, NH_.

This work describes a new approach aimed at untangling the complex relationship that disrupted methylation patterns share with aberrant gene expression in cancer.

Typically, the association between methylation and gene expression is considered through correlation analysis. Anti-correlation of promoter DNA methylation with expression is taken to imply repression of the gene. However, this approach is limited in its ability to consider the effect of methylation on genes other than the one most proximal to a CpG whose methylation value is known. Furthermore, the lack of a statistical model means that covariates may be ignored. Additionally, unnecessary tests may be run (reducing statistical power) when all data points are treated as independent, when in reality CpG loci that are close to each other are often highly correlated. Finally, the effect of copy number variation on gene expression is often not considered.

We propose an approach that addresses these issues and finds significant results with large effects even with relatively small sample sizes. The core of our method involves eQTL analysis, but not based on the typical SNP-gene association. Instead, matched tumor/normal data are used to model differentially methylated regions (DMRs) throughout the genome. Highly correlated values are aggregated, resulting in a more stable measure of methylation and fewer tests. The extant of differential methylation for an individual is then assigned as that individual's 'genotype' for a DMR. These genotypes are then used for an eQTL analysis, modeling the association of methylation and expression both in cis and in trans. We call this meeQTL analysis, for methylation-expression QTLs. Covariates can also be included in the model. Furthermore, the effect sizes from meeQTL analysis are adjusted, to compensate for the effect of copy number variation on the results.

As a proof of concept, we ran our meeQTL analysis on breast cancer data from TCGA, and found thousands of significant meeQTLs, using just 78 matched samples. The top 10 cis-meeQTLs (by effect size) had absolute log-odds between 6 and 9 (i.e. a 1 unit increase in methylation is associated with a 6 to 9 unit change in expression), and include genes such as HIF3A, a negative regulator of hypoxia response genes, or KLF15, which regulates TP53 and NF-kappa B. The top 10 trans-meeQTLs had absolute log-odds between 16 and 19 and include multiple genes associated with a DMR in the first intron of CRADD, a death-domain containing protein. Our model also found that DMRs experience significantly fewer somatic mutations than other loci and identified regions of copy number variation associated with aberrant gene expression in trans. These results suggest our approach can provide robust delineation of methylation and expression relationships arising from the complex structure of the genome and the epigenetic mechanisms regulating its activity.

#784

Overshoot during phenotypic switching of cancer cell populations.

Alessandro Luigi Sellerio,1 Emilio Ciusani,2 Noa Bossel Ben-Moshe4,3 Stefania Coco,4 Andrea Piccinini,4 Christopher R. Myers,5 James Sethna,5 Costanza Giampietro,1 stefano zapperi,1 Caterina Anna Maria Laporta1. 1 _Center for Complexity & Biosystems, University of Milan, Milan, Italy; _2 _Istituto Neurologico Carlo Besta, Milan, Italy;_ 3 _Weizmann Institute, Rehovot, Israel;_ 4 _University of Milan, Milan, Italy;_ 5 _Cornell University, Ithaca, NY_.

Whether tumor cell are organized hierarchically or stochastically is still debated. Here we address this question by sorting human melanoma cells using three distinct cancer stem cell (CSC) markers —CXCR6, CD271 and ABCG2— and observing that negative cells re-express their marker by first overshooting and then returning to the level of unsorted cells. Combining experimental measurements with theoretical modeling and numerical simulations, we show that the population dynamics of cancer cells is associated to a complex miRNA network regulating the Wnt and PI3K pathways. Hence phenotypic switching is not stochastic but tightly regulated by the balance between positive and negative cells in the population. Reducing the number of CSCs below a threshold triggers massive phenotypic switching suggesting that a therapeutic strategy based on CSC eradication is unlikely to succeed.

#785

The prepubertal mammary gland transcriptome suggests a role for the immune system in hormone-independent breast cancer.

Hillary Stires, Catina Crismale-Gann, William J. Belden, Wendie S. Cohick. _Rutgers University, New Brunswick, NJ_.

Identifying signaling pathways involved in mammary gland development has contributed to our understanding of breast cancer. While growth of the gland is isometric between birth and puberty, ductal expansion is significant and morphological changes such as terminal end bud formation occur during this developmental window. While estrogen receptor alpha (ERα) and progesterone receptor (PR) expression are evident during this time, pre-pubertal levels of free estradiol are low and growth is independent of ovarian hormones. Basal-like breast cancers represent a subset of breast cancers (10-15% of all women with breast cancer) that often lack ERα, PR and HER2 (i.e. triple negative), are ovarian hormone independent, and have a worse prognosis than other tumor subtypes. Animal models for basal-like breast cancer are limited. Therefore identifying gene changes during prepubertal mammary gland development may give insight into the mechanisms that drive these breast cancers. Offspring from individual Sprague Dawley dams were sacrificed on postnatal days (PND) 2, 10 and 20 (n=5 to 6 for each PND) and the fourth inguinal glands were removed. RNA was isolated and sequenced by RNASeq using 25 million paired end reads. Bioinformatics analysis utilizing TopHat, Bowtie2, and CummeRbund revealed >7000 genes that changed during this time (alpha=0.05). Analysis by DAVID/KEGG and Ingenuity Pathway Analysis demonstrated differences in immune pathways over time whereby immune genes increased from PND 2 to 10 to 20. Specifically, T Cell Receptor Signaling and B Cell Development were two of the most affected pathways. These results are interesting as recent studies have indicated that the immune system may influence basal-like breast cancers. Molecular signatures defining subtypes of basal-like breast cancer have been described and include basal-like immunosuppressed (BLIS) and basal-like immune activated (BLIA). A better understanding of the role of the immune system in prepubertal mammary gland development may help guide treatment strategies for hormone independent breast cancers.

#786

Project Survival: Interrogative Biology® platform mediated discovery of molecular markers for detection, stratification and outcomes in pancreatic cancer.

Niven R. Narain,1 A James Moser,2 Ramesh Ramanathan,3 John Crowley,4 Amy Stoll-D'Astice,4 Yezhou Sun,1 Leonardo O. Rodrigues,1 Eric M. Grund,1 Emily Chen,1 Vivek K. Vishnudas,1 Michael A. Kiebish,1 Viatcheslav R. Akmaev,1 Rangaprasad Sarangarajan1. 1 _Berg, LLC, Framingham, MA;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA;_ 3 _Mayo Clinic, Scottsdale, AZ;_ 4 _Cancer Research and Biostatistics, Seattle, WA_.

Pancreatic adenocarcinoma generally presents with a poor prognosis and an extremely low response rate to first line therapies. There is a critical unmet need to discover and implement effective diagnostic panels to stratify outcomes and tailor treatment strategies to improve survival. The BERG Interrogative Biology® platform utilizes Artificial Intelligence to analyze and integrate multi-omic profiles with clinical annotation to define novel biomarkers and improve treatment interventions. Herein, we analyzed the serum proteome, signaling lipidome, structural lipidome, and metabolome of 163patients at multiple timepoints (pancreatic cancer: 115; pancreatitis: 15; and age-matched healthy controls: 33). Utilizing the power of the Bayesian Network learner, bAIcis™ (BERG Artificial Intelligence Clinical Information System), multi-omic profiles were aligned to the longitudinal clinical information and subjected to AI-algorithms to infer probabilistic cause-and-effect relationships among molecular and clinical variables explicitly explaining pancreatic cancer status, cancer progression, and survival, and defining the interconnectivity of molecular features with clinical phenotype. Network features linking into clinical endpoints and key network pressure points were identified as molecular drivers. The drivers of clinical endpoints were analyzed to rank potential biomarkers. Novel biomarkers were discovered that demonstrate diagnostic potential for pancreatic cancer detection, disease prognosis including metastatic disease progression, patient stratification associated with survival, and response to standard chemotherapy agents like Gemcitibine. A prospective study is underway to validate the biomarkers and discover additional diagnostic and therapeutic molecules to improve the outcomes for patients affected by adenocarcinoma of the pancreas.

#788

Transcriptional landscape of drug response guides the design of potent and synergistic drug combinations.

Marc Hafner,1 Mario Niepel,1 Qiaonan Duan,2 Evan Paull,3 Josh Stuart,3 Aravind Subramanian,4 Avi Ma'ayan,2 Peter K. Sorger1. 1 _Harvard Medical School, Boston, MA;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _University of California, Santa Cruz, Santa Cruz, CA;_ 4 _Broad Institute of MIT and Harvard, Cambridge, MA_.

Transcriptional profiling of drug-treated cells yields high dimensional response signatures that allow drugs to be compared with each other. For example, the Connectivity Map collects signatures that are aggregated across multiple cell types. However, most therapeutic drugs are effective only against a subset of disease genotypes, particularly in the case of anti-cancer drugs. Here we ask how transcriptional signatures vary across cell lines and dose and correlate these signatures to the phenotypic response (growth inhibition). Using these cell line specific signatures, we inferred which signaling pathways are perturbed by specific kinase inhibitors and identified synergistic drug combinations.

We treated 6 breast cancer cell lines with more than 100 targeted inhibitors at 6 doses and measured their transcriptional response at 2 time points. We focused on inhibitors targeting key the PI3K and MAPK signaling pathways, as well as receptor tyrosine kinases (RTKs) and cyclin-dependent kinases (CDKs); many of them are currently studied in clinical trials. We identified that ~40% of the perturbations induce a significant difference in their gene expression profile. Clustering revealed the signatures are time point specific. Some clusters contain perturbations from multiple cell lines, like CDK inhibitors that down regulate genes related to the cell cycle in all six lines. In contrast, clusters comprising inhibitors of the PI3K and MAPK pathways are specific to each cell line and pathway. The perturbations induced by RTK and non-RTK inhibitors cluster with either the PI3K or the MAPK inhibitors depending on the cell line. Thus, the transcriptional response allows us to identify differences in pathway usage between cell lines, in particular to which pathway RTKs signal predominantly.

We found that the significance of the transcriptional signature is not necessarily related to growth inhibition. In particular, some inhibitors have little effect on growth, yet induce a significant transcriptional signature. The most striking case is the inhibition of MEK and EGFR in BT20 that induces strong transcriptional and biochemical responses but inhibits growth by only ~20%. Based on the transcriptional signature we inferred and validated experimentally that FoxO, which is generally regulated by the PI3K pathway, is partially activated by MEK or EGFR inhibition. This suggests that EGFR and PI3K inhibitors act synergistically in BT20, which we validated experimentally both at the level of FoxO activation and growth inhibition. We validated the most promising drug pair by treating xenografts.

We have shown how we can use measurements of expression signatures and cellular phenotypes following single drug perturbations to identify drug combinations that are synergistic in individual cell lines. This approach is a step toward the rational design of co-drugging strategies with differential effect and larger therapeutic windows.

#789

Leveraging transcriptomic and genomic data to better select models for preclinical oncology therapeutic development to identify cell lines most similar to patient tumors.

Yoonjeong Cha,1 Adam Labradorf,2 Joseph Perez-Rogers,2 Brian Haas,3 Andrew Lysaght,1 Brian Weiner,1 Fadi Towfic,1 Kevin Fowler,1 Benjamin Zeskind,1 Sarah Kolitz,1 Badri Vardarajan,4 Maxim Artyomov,5 Rebecca L. Kusko1. 1 _Immuneering, Cambridge, MA;_ 2 _Boston University, Boston, MA;_ 3 _Broad Institute, Cambridge, MA;_ 4 _Columbia, New York City, NY;_ 5 _Washington University St Louis, St Louis, MO_.

Cancer cell lines represent the front line of new compound testing, and results from these experiments often decide which compounds go on for further testing. Genomic context plays a critical role in drug response and now genomic data for tumors and cell lines are widely available. However, cell lines are often chosen based on ease of access, literature prevalence, and ease of culture. We combined gene expression and CNV/mutation profiling from four pancreatic cancer tumor datasets (GSE21501, GSE28735, ICGC, TCGA,) and three pancreatic cancer cell line datasets (Klijn et al, Collisson et al, and CCLE) to identify which cell lines best match patient tumors.

CNV comparison revealed that popular cell lines do not always have the best CNV correlation with tumors: when comparing pancreatic cancer tumors to cell lines, the citations of the top five cell lines by CNV correlation were less than 10% of the pancreatic cancer cell line total. Next we filtered for driver mutations including SMAD4 and CDKN2A using mutation scoring algorithms and clustered tumors and cell lines. We found that many cell lines with few citation counts clustered readily amongst tumors (such as L33). Leveraging the hypothesis that different hits in the same pathway can have a similar downstream effect, we combined CNV, expression and mutation data and clustered cell lines together with tumors based on overall aberrations in MSigDB cancer pathways. L33 and YAPC clustered near tumors while the majority of other cell lines clustered together.

To identify coexpressed gene clusters, we ran WGCNA individually in all seven datasets and discovered modules consistent in cell line and tumor datasets using iGraph. One of the most interesting modules (interferon regulated genes) is expressed highly in the majority of tumors profiled. About half of cell lines also express this module highly, suggesting that they may be more ideal models for high interferon expression tumors than other cell lines.

Here we present evidence demonstrating that certain cell lines mimic pancreatic tumor genomes more closely while others represent patterns of genomic features not commonly observed in vivo. We also show that certain biologically relevant tumor subtypes may be better represented by some cell lines than others. Our analysis highlights the emerging role of genomics in advancing the clinical success of therapeutic trials.

#790

Coupled dynamics of drug synergy, gene expression, and alternative splicing in combination therapies in breast cancer.

Xintong Chen,1 Gustavo Stolovitzky,2 Bojan Losic1. 1 _Icahn School of Medicine at Mount Sinai, NEW YORK, NY;_ 2 _IBM research, NEW YORK, NY_.

Drug combination therapies in the cancer setting often succeed where mono-therapies fail, facilitating durable and robust responses that may curtail metastases and even be accompanied by milder side-effects. Predicting synergistic and antagonistic combinations based on the gene expression data of mono-therapy drug-tumor response is an important open problem wherein the role of transcriptional splicing dynamics is often ignored or too poorly correlated with phenotypes to be useful.

In this work we leverage the inherent transcript/exon level resolution of RNA-seq data to infer combination-specific splicing signatures associated with additive and synergistic (subadditive) drug combinations as defined by canonical viability measurements in a time-course experiment. We integrate splicing information into a gene regulatory network (GRN) to compute leading order effects of splicing on the GRN by adaptive network refinement. Briefly, we use RNA-seq to study the transcriptional response over time (0, 3, 6, 9, 12, and 24 h) for three drugs (A, B and C) and their combinations (AB, AC and BC) in MCF-7 (ER+) breast cancer cells lines. Cell viability measurements show that one of the combinations (AB) is strongly synergistic, whereas the other two (AC and BC) are merely additive. We show via rigorous linear modeling of RNA-seq count data at the exon level that in addition to a novel transcriptional signature driven by differential expression, the combination AB transcriptional landscape is characterized by persistent alternative splicing signatures mostly comprised of genes which are not differentially expressed with respect to A or B but whose functional role has been dramatically changed by the addition (deletion) of a key regulatory protein domain encoded by the extra(missing) exon. We construct an isoform-level co-expression network (IRN) to probe the regulatory changes this dynamical splicing induces and show that it crucially contributes to the emergence of extensive transcriptional cascades by creating and removing key gene-gene correlations and altering the modular structure of the network. Using this approach we show that a key isoform of Protein beta-arrestin-1 (ARBB1), which participates in desensitization of G-protein coupled receptors through intracellular response to Estrogen, is enriched in the calcium ion-dependent exocytosis module of IRN, while ARRB1 remains functionally uninformative in the regular GRN. Our results suggest that any gene-signature based drug synergy prediction algorithm must take into account alternative splicing in order to effectively characterize the novel pathways being activated in the synergistic drug-tumor interaction. 

## EPIDEMIOLOGY:

### Genes and Function and Risk

#791

Examining the roles of deleterious and potentially damaging variant alleles in African and Caucasian ancestral populations.

Myron K. Gibert, Luisel Ricks-Santi, John T. McDonald. _Hampton University, Newport News, VA_.

The purpose of this study was to analyze the overlap between potentially damaging Single Nucleotide Polymorphisms (SNPs) for coding gene regions across African American and Caucasian populations. Allele frequency and genotype data from the International HapMap Project was acquired for ancestral groups from Utah Residents with Northern and Western European Ancestry (CEU), Yoruba in Ibadan, Nigeria (YRI) and African Americans from the Southwest (ASW) resulting in 34,844 unique variants. Using the Variant Effect Predictor, non-synonymous SNPs in overlapping transcripts were filtered and found in 12,367 unique genes. SIFT or Polyphen-2 scores were used to determine which variants were likely to prove damaging to protein function, resulting in 10,917 variants. Functional annotation of biological pathways in genes affected by these variants was analyzed using DAVID. There was enrichment in olfactory pathways/g-protein coupled receptor function, cell membrane function and epidermal growth factor activity. Comparisons of genes affected by these deleterious variants were overlapped with the catalog of published genome-wide association studies (GWAS) with SNP-traits associated with cancer. There were 90 of 463 genes in common with this data that were examined for linkage disequilibrium with the damaging variants. Finally, SNPs with a variant allele frequency differences >±5% between CEU versus YRI or ASW resulted in with 2,381 SNPs with 55.7% overlap between groups. Analysis of patient genotypes found those in the CEU population were more likely to possess a homozygous allele. Using Chi-Squared tests, populations were examined for risk allele frequencies by genotype. This study demonstrated variants with potentially damaging consequences in cancer pathways are disproportionally represented in different ancestral populations.

#792

Super-transactivation TP53 variant in the germline of a family with Li-Fraumeni variant.

Badr Id Said,1 Han Kim,2 James Tran,2 Ana Novokmet,1 David Malkin1. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _The University of Toronto, Toronto, Ontario, Canada_.

Li-Fraumeni Syndrome (LFS) is a rare autosomal dominant familial cancer syndrome, characterized by multiple malignancies and frequent germline alterations in TP53. In this study, we highlight four unclassified exonic p53 variants detected in patients with a suspected diagnosis of LFS. We report for the first time the discovery of two novel functional variants in codons 191(c.572C>G; p.P191R) and 360 (c.1079G>T; p.G360V), located, respectively, in the DNA binding domain and in a linker region near the tetramerization domain of TP53. Our data revealed that while the P191R variant decreased the transactivation levels of several p53 targets, it failed to segregate with the disease state. The G360V variant, on the other hand, behaved in a paradoxical fashion by causing a stark upregulation in the activity of several p53 response elements. This tumor suppressive effect was also observed at the level of colony formation and c-Caspase 3 activation. While unlikely to be disease-causing, we propose that these variants may represent novel p53 polymorphisms and potential phenotypic modifiers in LFS. In the future, the enhanced transactivation effects of G360V-p53 may also prove useful in designing more efficacious p53-based gene therapies.

#794

A genetic polymorphism of ALDH2 and breast cancer risk: an analysis of 6,624 cases and 5,750 controls from the Breast Cancer Association Consortium.

Tomotaka Ugai,1 Roger L. Milne,2 Hidemi Ito,1 Keitaro Matsuo1. 1 _Aichi Cancer Center Research Institute, Nagoya, Japan;_ 2 _School of Population and Global Health, The University of Melbourne, Melbourne, Australia_.

Background.

Numerous epidemiological studies consistently indicate that consumption of alcoholic beverages is an independent risk factor for female breast cancer. Although the mechanism underlying this effect remains unknown, it has been hypothesized that ethanol is mutagenic via its first metabolite, acetaldehyde. An impact of acetaldehyde on the carcinogenesis of several types of cancer has been shown in experimental models and in epidemiological studies in East Asian countries where a functional aldehyde dehydrogenase 2 (ALDH2) polymorphism Glu504Lys (rs671) is prevalent. However, its association with breast cancer risk has not been fully elucidated. We conducted an association analysis of original data from Asian studies participating in the Breast Cancer Association Consortium (BCAC).

Patients and Methods.

Nine studies were used for this analysis, including 6,624 cases and 5,750 controls. The pooled odds ratio (OR) with 95% confidence interval (CI) for breast cancer risk associated with the ALDH2 polymorphism was estimated using logistic regression models. We estimated unadjusted ORs and adjusted ORs for potential confounders.

Results.

In the unadjusted (crude) analysis, no significant association between ALDH2 polymorphism and breast cancer was observed, with ORs relative to Glu/Glu, of 1.02 (95% CI=0.95-1.11; P=0.51) for Glu/Lys, 1.13 (0.96-1.32; 0.14) for Lys/Lys and 1.04 (0.97-1.12; 0.29) for carriers of any Lys allele. In addition, after adjustment for all potential confounding factors, this lack of significant association was consistently observed, with ORs relative to Glu/Glu, of 0.95 (95% CI=0.86-1.04; P=0.25) for Glu/Lys, 0.99 (0.82-1.20; 0.93) for Lys/Lys and 0.95 (0.87-1.04; 0.29) for carriers of any Lys allele. These findings were consistent across ER, PR and HER2 status.

Conclusions.

No significant association between ALDH2 polymorphism and breast cancer was observed in this study. Considering the established impact of ALDH2 504Lys on the carcinogenesis of several types of alcohol-induced cancer, our findings suggest that acetaldehyde is less influential to the breast carcinogenesis induced by alcohol, as is the case for upper-aerodigestive tract cancers.

#796

ERCC3 R109X is a moderate risk breast cancer risk variant in Ashkenazi Jews.

Joseph Vijai,1 Sabine Topka,1 Kara Maxwell,2 Vignesh Ravichandran,1 Tinu Thomas,1 Danylo Villano,1 Ann Maria,1 Pragna Gaddam,1 Anne Lincoln,1 Steven Hart,3 Susan Neuhausen,4 Mark Robson,1 Jeffrey Weitzel,4 Mark Daly,5 Katherine Nathanson,2 Fergus Couch,3 Gadi Rennert,6 Kenneth Offit1. 1 _Mem. Sloan-Kettering Cancer Ctr., New York, NY;_ 2 _Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _Beckman Research Institute of the City of Hope, Duarte, CA;_ 5 _Broad Institute of MIT and Harvard, Boston, MA;_ 6 _CHS National Israeli Cancer Control Center, Haifa, Israel_.

Background:

Known gene mutations account for approximately 50% of the risk for breast cancer. However, a considerable fraction of heritable risk remains unexplained. Fifteen percent of the risk is accounted for by BRCA1/2 and another 3% by TP53, PTEN, LKB1 and CDH1. CHEK2, ATM, PALB2, BRIP1, RAD51C, RAD51D and BARD1 account for 4%, while SNPs discovered from large multicenter genome-wide association studies explain another 14% of the heritable risk. Founder mutations in the DNA repair pathway genes such as BRCA1 and BRCA2 account for the majority of AJ breast cancer mutations.

Methods:

We performed exome sequencing of 49 early onset (age <35) breast cancer cases, and 85 BRCA wild type familial breast cancer cases, all of Ashkenazi Jewish (AJ) from the New York City area. A recurrent truncating mutation was then analyzed in 3131 breast cancer cases and 2716 unaffected women of Ashkenazi ancestry from New York and Israel. Using CRISPR and overexpression systems on the human mammary epithelial cell line HMLE, transcript and protein levels were assayed for the mutant and compared to wild-type. Treatment with IlludinS and UVC were performed to assess DNA damage response. Finally, clonogenic survival assay was also performed.

Results: Amongst the DNA repair pathway genes, exome sequencing revealed a heterozygous recurrent truncating mutation in ERCC3 (R109X) in 2 of 49 early onset breast cancer cases of AJ ancestry and 4 familial AJ probands. Taqman genotyping in a case control setting from New York and Israel revealed 54 mutation carriers in 3131 cases and 32 in the 2716 controls. In total, there were 60 heterozygotes detected in 3209 cases and 32 in 2716 controls [OR 1.59 (95% CI 1.01-2.50)]; p=0.02 Fisher one- tailed). Functional studies using CRISPR and overexpression systems on human mammary epithelial cells, show that the mutation results in lower transcript levels and this reduction is effected by nonsense mediated decay of the mutant transcript. Western blotting showed that the mutation resulted in a smaller protein (~12kDa). Clonogenic assays showed similar survival rate of mutant and wildtype under UVC exposure, however the mutant cell line showed significantly smaller colony size demonstrating a growth disadvantage that was further increased upon DNA damage. Treatment with fungal sesquiterpene IlludinS, a known sensitizer to mutant ERCC3 cell lines, showed drastically reduced survival when compared to the wild type human mammary epithelial cells.

Conclusions:

We demonstrate that ERCC3 is a moderate risk gene for breast cancer in individuals of Ashkenazi ancestry. ERCC3 is somatically mutated in multiple cancers including breast, ovarian and pancreatic cancers, however its role as a cancer susceptibility gene requires further elucidation. Additional functional and population genetic studies to further characterize this novel ERCC3 variant are underway.

#797

A splicing variant of TERT identified by GWAS interacts with menopausal estrogen therapy in risk of ovarian cancer.

Alice W. Lee,1 Ashley Bomkamp,2 Elisa V. Bandera,3 Allan Jensen,4 Susan J. Ramus,1 Marc T. Goodman,5 Mary Anne Rossing,6 Francesmary Modugno,7 Kirsten B. Moysich,8 Jenny Chang-Claude,9 Anja Rudolph,9 Aleksandra Gentry-Maharaj,10 Kathryn L. Terry,11 Simon A. Gayther,5 Daniel W. Cramer,11 Jennfier A. Doherty,12 Joellen M. Schildkraut,13 Susanne K. Kjaer,4 Roberta B. Ness,14 Usha Menon,10 Andrew Berchuck,15 Bhramar Mukherjee,2 Lynda Roman,1 Paul D. Pharoah,16 Georgia Chenevix-Trench,17 Anna H. Wu,1 Malcolm C. Pike,18 Celeste L. Pearce2. 1 _University of Southern California, Los Angeles, CA;_ 2 _University of Michigan, Ann Arbor, Ann Arbor, MI;_ 3 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 4 _Danish Cancer Society Research Center, Copenhagen, Denmark;_ 5 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 6 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 7 _University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 8 _Roswell Park Cancer Institute, Buffalo, NY;_ 9 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 10 _University College London, London, United Kingdom;_ 11 _Brigham and Women's Hospital, Boston, MA;_ 12 _The Geisel School of Medicine at Dartmouth, Hanover, NH;_ 13 _The University of Virginia, Charlottesville, VA;_ 14 _The University of Texas School of Public Health, Houston, TX;_ 15 _Duke University Medical Center, Durham, NC;_ 16 _University of Cambridge, Cambridge, United Kingdom;_ 17 _QIMR Berghofer Medical Research Institute, Herston, Australia;_ 18 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Menopausal estrogen-alone therapy (ET) is a well-established risk factor for serous and endometrioid ovarian carcinoma. Genetics also plays an important role in ovarian carcinoma etiology, which is partly attributable to 18 confirmed susceptibility loci identified by genome-wide association studies (GWASs). Each of these common variants is associated with a modest relative risk estimate, but it is possible that interactions between non-genetic and genetic risk factors exist, thereby putting some women at higher risk. This interplay among the 18 loci, ET use, and ovarian carcinoma risk has yet to be evaluated.

In our analysis, we used individual questionnaire data from 1,414 serous cases, 337 endometrioid cases, and 4,051 controls across 10 case-control studies participating in the Ovarian Cancer Association Consortium (OCAC). The genotype data was based on information from three GWASs, their replication efforts, and two large-scale arrays. Conditional logistic regression was used to determine the association between the 18 confirmed variants and risk of serous and endometrioid ovarian carcinoma among ET users and non-users separately. A likelihood ratio test was used to test for statistical interaction (i.e., departure from a multiplicative model).

After controlling for well-established ovarian carcinoma risk factors as well as genetic ancestry, a splicing variant in TERT, rs10069690, showed a significant interaction with ET use for risk of serous ovarian carcinoma (pint=0.014). ET users carrying the T allele had a 50% increased risk of disease (OR=1.50, 95% CI 1.16-1.93); the impact of this allele was even stronger for long-term ET users of 10+ years (OR=2.13, 95% CI 1.39-2.37, pint=0.036). Non-ET users showed essentially no association with the disease (OR=1.09, 95% CI 0.97-1.22). In addition, two SNPS, rs7207826 (C allele) and rs56318008 (T allele), in other genomic regions had significant interactions with ET use for the endometrioid histotype (pint=0.030 and pint=0.042, respectively).

Overall, we have shown evidence of statistical interactions between postmenopausal ET use and three confirmed ovarian cancer susceptibility alleles with risk of serous and endometrioid ovarian cancer. We observed four statistically significant interactions, which is twice as many as would be expected by chance at the p≤0.05 level, although none survived correction for multiple comparisons. This is the first study, to our knowledge, to suggest potential gene-environment interactions in ovarian carcinoma in the context of hormone therapy use with confirmed susceptibility alleles. It is also intriguing that the identified interactions include confirmed variants that are located in or adjacent to genes in which estrogen is biologically involved. These findings, if replicated, may be critical for future risk prediction modeling.

#798

Effects of low-dose environmental chemicals on the mammary transcriptome at critical windows of development in a rodent model.

Kalpana Gopalakrishnan,1 Francesca Petralia,1 Pei Wang,1 Fabiana Manservisi,2 Laura Falcioni,2 Luciano Bua,2 Fiorella Belpoggi,2 Luca Lambertini,1 James Wetmur,1 Susan Teitelbaum,1 Jia Chen,1 Vasily Aushev1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Ramazzini Institute, Italy_.

Exposure to environmental chemicals, including those commonly found in personal care products has been linked to mammary cancer at high doses using animal models. Their effects at low doses comparable to human exposure, especially during critical windows of development remain poorly understood. We investigated the effects of three prevalent environmental chemicals - diethyl phthalate (DEP), methyl paraben (MPB), triclosan (TCS) - and their mixture (MIX) on the transcriptome of normally developing mammary at low doses mimicking human exposure. Using a female Sprague-Dawley rat model, we targeted four early developmental exposure windows - prenatal, neonatal, prepubertal and pubertal, as well as continuous exposure from birth to adulthood (both parous and nulliparous). Control rats were exposed to vehicle only. All exposures were by oral gavage. Whole-transcriptomes of mammary glands were profiled by Affymetrix rat gene arrays. Differentially expressed genes were identified by linear models. Despite dynamic transcriptome changes in the normal developing mammary, exposure to environmental chemicals induced detectable gene expression changes in a window-specific fashion. We discovered that puberty represented a window of heightened sensitivity to MPB and DEP exposure with 341 and 175 altered genes relative to controls, respectively (false discovery rate (FDR) < 0.25, fold change (FC) ≥ 1.5). Chronic DEP exposure from birth to adulthood resulted in changes in 1151 and 427 genes in parous and nulliparous rats, respectively, compared to corresponding controls (FDR < 0.25, FC ≥ 1.5). Importantly, the number of differentially expressed genes across development in exposed rats was significantly reduced compared to control rats, suggesting possible alteration in developmental pace by environmental chemicals. We used a joint random forest algorithm to construct co-expression networks and identified gene modules with distinct connectivity patterns between treatments and controls. Results indicated a loss of correlation structure in exposed rats compared to controls. For example, genes such as Ntn4, Tmcc3, Lalba and Lcn2 showed fewer connected edges in the co-expression network of the DEP, MPB and TCS groups compared to controls. These results highlight critical windows of exposure and implicate the potential health effects of these ubiquitous environmental chemicals in human populations.

#799

Interaction between meat and fish intake and genes involved in hormones, inflammation and energetic factors, and risk of breast cancer among Hispanic and Non-Hispanic White women: The Breast Cancer Health Disparities Study.

Andre E. Kim,1 Juan Pablo Lewinger,1 Shirong Zhang,1 Roger K. Wolff,2 Laura Fejerman,3 Esther M. John,4 Gabriela Torres-Mejia,5 Jennifer S. Herrick,2 Stephanie D. Boone,6 Avonne E. Connor,6 Lisa M. Hines,7 Kathy B. Baumgartner,6 Anna Giuliano,8 Martha L. Slattery,2 Mariana C. Stern1. 1 _University of Southern California, Los Angeles, CA;_ 2 _University of Utah, Salt Lake City, UT;_ 3 _University of California San Francisco, San Francisco, CA;_ 4 _Cancer Prevention Institute of California, Fremont, CA;_ 5 _Instituto Nacional de Salud Publica, Cuernavaca, Mexico;_ 6 _University of Louisville, Louisville, KY;_ 7 _University of Colorado at Colorado Springs, Colorado Springs, CO;_ 8 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Previously, we reported associations between breast cancer (BC) risk and tuna intake in non-Hispanic White (NHW) and Hispanic women, and processed meat intake among Hispanic women only. We also reported associations with SNPs in genes involved in the Convergence of Hormones, Inflammation, and Energy-related Factors (CHIEF) network. Given the multifactorial nature of BC etiology, we tested for interactions (GxE) between meat/fish intake and SNPs in selected CHIEF pathways using a set-based method that aggregates SNPs into groups for GxE association testing.The study population included Hispanic (1046 cases, 1331 controls) and NHW (1234 cases, 1468 controls) women from two US population-based case-control studies that are part of the Breast Cancer Health Disparities Study. Harmonized meat intake variables were dichotomized, and include red meat, processed meat, poultry, fish, and tuna. 734 SNPs were grouped into 107 genes based on loci. Genes were further grouped into 7 pathways based on biological function: CHIEF core, ALOX/COX, Interleukins, JAK/STAT/SOC, TLR/TNF, MAPK, and TGF-β. We performed the analysis in a top-down fashion, sequentially testing for interactions between meat/fish intake and pathways, genes within pathways, and SNPs within genes. To test pathways and genes, we used a set-based GxE approach that extends the adaptive rank truncated product (ARTP), and to test individual SNPs we used logistic regression. At gene and SNP level analyses, we applied Bonferroni corrections for the number of genes or SNPs tested. Analyses were stratified by ethnicity. Models were adjusted for study, age, education, menopausal status, family history of breast cancer, parity, body mass index, physical activity, education, intake of alcohol, fiber, calories, fat, carbohydrates, proteins, and genetic ancestry. We report results that showed statistically significant interactions at both pathway and gene levels. Analyses were conducted using the R programming language. Among NHW women, pathway-level analyses using ARTP identified an interaction between poultry intake and the interleukin/cytokine pathway, which was driven by the IL17A gene (2 SNPs); poultry intake was inversely associated with BC risk among carriers of the minor alleles in this gene. Among Hispanics, ARTP analyses identified an interaction between tuna intake and total fish intake and the Jak/Stat/SOC pathway, which was driven by STAT5A (tuna: 2 SNPs) and STAT5B (total fish and tuna: 3 SNPs). The positive associations between fish or tuna and BC risk were restricted to carriers of minor alleles in these two genes. Overall, our findings suggest a role for poultry and fish intake in chronic inflammation and tumor growth promotion in breast carcinogenesis and highlight specific genes that may play roles in these carcinogenic pathways.

#800

ALDH2 **rs671 polymorphism and alcohol consumption in the risk of gastric cancer: A Mendelian randomization study.**

Jeongseon Kim,1 Sarah Yang,1 Jeonghee Lee,1 Il Ju Choi,1 Young Woo Kim,1 Keun Won Ryu,1 Joohon Sung2. 1 _National Cancer Ctr. Korea, Goyang-si, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea_.

Background: The effect of increased alcohol consumption on the risk for gastric cancer (GC) has not been fully elucidated, due to the presence of confounders and gender differences. In this study, aldehyde dehydrogenase 2 (ALDH2) rs671, the genetic polymorphism related to the elimination of acetaldehyde, was used as an instrumental variable (IV) for Mendelian randomization analysis to dissect the role of alcohol in GC risk.

Methods: The study included Korean 450 GC cases and 1,050 controls recruited at the National Cancer Center. Data of 795 GC patients and 4,893 controls from the Korean Centers for Disease Control were used for further validation. The odds ratios (OR) for the ALDH2 genotype and its interaction with alcohol consumption were estimated using multivariate logistic regression. The two-stage control function method was performed using rs671 as the IV for alcohol consumption.

Results: All AG carriers had an OR of 1.26 (95% confidence interval, 1.11-1.42) compared with GG carriers. In a quantitative analysis of alcohol intake, the conventional OR was 1.21(1.09-1.36) and the IV-OR was 0.90(0.63-1.28). Females had low alcohol consumption and did not show a significant risk for GC while males had a high risk for GC.

Conclusions: Alcohol consumption is not associated with a risk for GC by itself, and ALDH2 polymorphism should be considered simultaneously when assessing the risk. Females did not display an evident risk for GC regarding alcohol, partly due to their lower level of alcohol consumption. However, further dissection of gender difference is necessary.

#801

Chromosomal damage as markers of genotoxicity and carcinogenesis.

Pavel E. Vodicka,1 Ludmila Vodickova,1 Zdena Polivkova,2 Ludovit Musak,3 Maria Dusinska,4 Sona Vodenkova,1 Veronika Vymetalkova,1 Michal Kroupa,1 Alessio Naccarati,5 Rajiv Kumar,6 Kari J. Hemminki6. 1 _Institute of Experimental Medicine ASCR, Prague, Czech Republic;_ 2 _Third Medical Faculty, Charles University, Prague, Czech Republic;_ 3 _Clinic of Occup. Med. Toxic., Univ. Hospital Martin, Martin, Slovakia;_ 4 _NILU, Lillestroem, Norway;_ 5 _HUGEF, Torino, Italy;_ 6 _German Cancer Research Center, Heidelberg, Germany_.

Human cancers arise from cells unable to maintain genomic and chromosomal stability, mainly as a sequential consequence of altered DNA repair mechanisms (base and nucleotide excision, mismatch and double-strand breaks). Chromosomal aberrations (CAs) are a marker of cancer risk and many specific CAs represent causative events in malignant transformation. Non-specific CAs arise as a result of direct DNA damage by ionizing radiation (chromosome-breaks; CSA) or replication on a damaged DNA template (CSA and chromatide-breaks; CTA). Frequencies of CAs in blood lymphocytes (PBL) are predictive for cancer risk in prospective epidemiological studies and patients with many types of cancer show elevated CAs at diagnosis.

We have recently disclosed associations of CAs with variants in genes encoding DNA repair and xenobiotic metabolizing enzymes on 1800 healthy subjects exposed and unexposed to potentially carcinogenic compounds. Notably, lower frequencies of CAs and CTA are associated with high activity EPHX1 and XPD Lys751Gln homozygous variant genotypes and several pair-wise interactions significantly modulated CA, CTA and CSA frequencies. On the contrary, increased CAs and CSA frequencies were observed in subjects bearing splicing A variant in CCND1 G870A. Current investigations aim at understanding the genetics underlying CAs as intermediary cancer biomarkers, such as variants in genes encoding kinetochores, mitotic apparatus regulating enzymes, variants in genes encoding polymerases in DNA repair synthesis and the CAs dynamics in subjects repeatedly analysed over time. We have also explored miRNA binding sites in 3´UTR of DNA repair genes (BER, NER and DSB) both in colorectal cancer patients and in healthy subjects with CAs.

Since shortening of telomeres in each cell division may lead to telomere crisis and complex CAs, relative telomere length (RTL) was determined in 187 individuals and compared to their CA count in PBL. Further analyses investigated RTL in incident cancer patients (breast, colorectum and lungs) and in patients (breast and colorectum) with the estimated double-strand breaks repair capacity. CAs detected in patients with above solid cancers were associated with clinicopathological characteristics and analysed as a potential prognostic factors.

Our studies suggest that CAs in PBL may represent perspective transient marker in carcinogenesis and we hereby provide a biological basis for the link between CAs and cancer risk along with the genetic control of the overall CA frequency.

Grant support: GA CR 15-14789S, AZV 15-27580A and COST LD14050.

#802

**Genetic polymorphism in** SLC7A2 **interacts with calcium:magnesium intake ratio in risk of colorectal neoplasia in a two-phase study.**

Xiangzhu Zhu,1 Pin Sun,2 Martha J. Shrubsole,1 Reid M. Ness,1 Elizabeth A. Hibler,1 Qiuyin Cai,1 Jirong Long,1 Zhi Chen,1 Guoliang Li,1 Lifang Hou,3 Walter E. Smalley,1 Todd L. Edwards,1 Edward Giovannucci,4 Wei Zheng,1 Qi Dai1. 1 _Department of Medicine, Vanderbilt University, Nashville, TN;_ 2 _Department of Occupational Health and Toxicology, School of Public Health, Fudan University, Shanghai, China;_ 3 _Institute for Public Health and Medicine, Northwestern University, Chicago, IL;_ 4 _Departments of Nutrition and Epidemiology, Harvard University, Boston, MA_.

Colorectal cancer (CRC) remains the third most common cancer in men and the second in women worldwide; thus, preventive strategies for CRC are critically needed. Solute carrier family 7, member 2 (SLC7A2) gene encodes a protein called cationic amino acid transporter 2, which mediates the transport of arginine, lysine and ornithine. L-arginine is necessary for cancer development and progression, including an important role in CRC pathogenesis. Furthermore, previous studies found both calcium (Ca) and magnesium (Mg) inhibit the transport of arginine. Thus, Ca, Mg or Ca:Mg intake ratio may interact with polymorphisms in the SLC7A2 gene in risk of CRC. To test this hypothesis, we conducted a two-phase case-control study within the Tennessee Colorectal Polyps Study (TCPS) among participants who completed a semi-quantitative 108-item food frequency questionnaire. In the first phase, 23 tagging single-nucleotide polymorphisms (SNPs) in the SLC7A2 gene were analyzed for 725 colorectal adenoma cases and 755 controls. In the second phase conducted in an independent set of 607 cases and 2113 controls, we evaluated for replicationthe significant findings from the first phase. We observed that no SNPs in SLC7A2 were significantly associated with the risk of colorectal adenoma at P < 0.05. However, rs2720574 significantly interacted with Ca:Mg intake ratio in association with adenoma risk, particularly multiple/advanced adenoma in both the first and second phases. In the combined analysis, among those with a Ca:Mg intake ratio below 2.78, individuals who carried GC/CC genotypes were at a higher risk of adenoma [odds ratio (OR, 95% CI)=1.36(1.11-1.68)] and multiple/advanced adenoma [OR (95% CI) =1.68(1.28, 2.20)] than those who carried the GG genotype. Among those with the GG genotype, a high Ca:Mg ratio was associated with increased risks of colorectal adenoma (OR (95%CI): 1.73(1.27-2.36)) and advanced/multiple adenomas (1.62(1.05-2.50)) whereas, among those with the GC/CC genotypes, high Ca:Mg ratio was related to reduced risks of colorectal adenoma (0.64(0.42-0.99))and advanced/multiple adenomas (0.55(0.31-1.00)); p(interactions) = 0.002 and 0.0001 for total and advanced/multiple adenomas, respectively. These findings indicate the Ca:Mg ratio, instead of Mg or Ca alone, interacted with SLC7A2 polymorphism in risk of colorectal neoplasia.

#803

Obesity and associated lifestyles modify the effect of glucose metabolism-related genetic variants on impaired glucose homeostasis among postmenopausal women.

Su Yon Jung, Eric M. Sobel, Jeanette C. Papp, Carolyn J. Crandall, Alan N. Fu, Zuo-Feng Zhang. _University of California Los Angeles, Los Angeles, CA_.

Background: Impaired glucose metabolism-related genetic variants likely interact with obesity-modifiable factors in response to glucose intolerance, yet their interconnected pathways have not been fully characterized. We assessed in postmenopausal women whether obesity, physical activity, and high dietary fat intake interact with the single-nucleotide polymorphisms (SNPs)-glucose variations.

Methods: We used data from 1,027 postmenopausal participants of the Genomics and Randomized Trials Network study and 15 SNPs associated with glucose homeostasis. We used regression analysis plus stratification and graphic approaches.

Results: Fasting levels of glucose, insulin, and homeostatic model assessment-insulin resistance (HOMA-IR) were higher in obese, inactive, and high fat-diet women than in their respective counterparts. Carriers within subgroups differently demonstrated the direction and/or magnitude of the variants' effect on glucose-relevant traits. Variants in GCKR, GCK, DGKB/TMEM195 (P for interactions=0.02, 0.02, and 0.01), especially, showed interactions with obesity: obese, inactive, and high fat-diet women had greater increases in fasting glucose, insulin, and HOMA-IR levels. Obese carriers at TCF7L2 variant had greater increases in fasting glucose levels than non-obese carriers (P for interaction=0.04), whereas active women had greater decreases in insulin and HOMA-IR levels than inactive women (P for interaction = 0.02 in both levels).

Conclusions: Our data support the important role of obesity in modifying glucose homeostasis in response to glucose metabolism-relevant variants. These findings may inform research on the role of glucose homeostasis in the etiology of disease, particularly obesity-related cancer, and the development of intervention strategies to reduce risk in postmenopausal women.

#804

Gene-environment interactions and the risk of Barrett's esophagus in three US cohorts.

Marta Crous-Bou,1 Diane Feskanich,1 Meir J. Stampfer,1 Charles S. Fuchs,2 Immaculata De Vivo,1 Brian C. Jacobson3. 1 _Brigham and Women's Hospital - Harvard Medical School, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Boston University Medical Center, Boston, MA_.

Background: Several single nucleotide polymorphisms (SNPs) have been associated with Barrett's esophagus (BE) risk. Additionally, environmental factors including smoking, alcohol consumption and heartburn have been shown to increase BE risk. However, no studies have evaluated whether interactions between these genetic and environmental factors influence BE risk. Understanding how genes and environmental risk factors interact may provide key insight into the pathophysiology of BE.

Objectives: To examine the main effects and the potential effect-modification between known genetic loci (SNPS) and established environmental risk factors for BE.

Patients and Methods: We performed a nested case-control study using data on 401 incident BE cases and 436 age-matched controls from the Nurses' Health Study, Nurses' Health Study II, and Health Professionals Follow-up Study cohorts, who gave blood and completed biennial questionnaires. We genotyped SNPs identified in previous BE GWAS as well as SNPs in candidate genes related to BE susceptibility (i.e., related to excess body fat, fat distribution, factors associated with insulin resistance, and inflammatory mediators). A genetic risk score (GRS) was constructed to evaluate the combined effect of the selected SNPs on BE risk. Interactions between SNPs and BE risk factors were also assessed.

Results and Conclusions: We found a borderline association between our GRS and BE risk: a one-allele increase in the unweighted GRS increased the risk of Barrett's esophagus by a factor of 1.20 (95%CI=1.00-1.44; p=0.057). We observed a significant interaction between smoking and BE genotypes (p-interaction=0.016). However, while alcohol consumption and heartburn duration were strongly associated with BE, we did not observe any significant gene-environment interactions.

#805

The molecular mechanisms of obesity driving breast cancer etiology and prognosis in post-menopausal women.

Yu Jing Jan Heng,1 Jun Wang,2 Aditi Hazra,3 David J. Hunter,3 A. Heather Eliassen,3 Rulla M. Tamimi,3 Susan E. Hankinson,2 Andrew H. Beck1. 1 _Beth Israel Deaconess Medical Center Harvard Medical School, Boston, MA;_ 2 _University of Massachusetts Amherst, Amherst, MA;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA_.

This study aims to gain insights into the molecular mechanisms of obesity driving breast cancer etiology and prognosis as post-menopausal women with high body mass index (BMI) have increased breast cancer risk and poorer prognosis.

We examined the differential gene expression of breast tumors from 441 post-menopausal women part of the Nurses' Health Studies. Primary analysis was performed between women with BMI of <25, ≥25 to <30, ≥30 to 35 and ≥35 while secondary analysis was performed using BMI as a continuous variable. Analyses were adjusted for age of diagnosis, year of diagnosis, post-menopausal hormone therapy, alcohol consumption, microarray batch and ER status.

In the primary analysis, differentially expressed genes associated with BMI were identified in pairwise analyses comparing women with BMI ≥35 with women in lower BMI categories (<25 vs. ≥35, n=58 significant genes; ≥25 to <30 vs. ≥35, n=29; ≥30 to 35 vs. ≥35, n=12; FDR<0.05); no significant genes were identified in the other pairwise comparisons. In the secondary analysis which considered BMI as a continuous value, two genes, NCOA3 and PTPN1, were significantly up-regulated in breast tumors with increasing BMI (FDR<0.05). Gene set enrichment analyses suggested that tumors from post-menopausal women with increasing BMI have increased inflammation and decreased carcinogen/anti-cancer drug metabolism.

These molecular insights have further elucidated breast cancer etiology in post-menopausal women with high BMI.

#806

SNPs in vitamin D-related genes are associated with prostate cancer aggressiveness in the North Carolina-Louisiana Prostate Cancer Project (PCaP).

Susan E. Steck,1 Anna Woloszynska-Read,2 Samuel Antwi,1 Hongmei Zhang,3 Lenore Arab,4 Elizabeth T.H. Fontham,5 James Mohler,2 L. Joseph Su,6 Feifei Xiao,1 Gary Smith,2 Donald Trump,2 Candace Johnson,2 Jeannette Bensen7. 1 _University of South Carolina, Columbia, SC;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _University of Memphis, Memphis, TN;_ 4 _UCLA, Los Angeles, CA;_ 5 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 6 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 7 _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Introduction: African Americans have higher incidence of, and mortality from, prostate cancer (PCa) compared to other racial/ethnic groups. Frequency of polymorphisms in genes involved in vitamin D metabolism, transport, and activity differ by race/ethnicity. Examining the association between polymorphisms in vitamin D-related genes and PCa aggressiveness may explain differing susceptibility to aggressive PCa among individuals and across different racial/ethnic groups.

Methods: TagSNPs (n=315) in 13 genes (VDR, GC, CYP24A1, CYP27A1, CYP27B1, CYP2R1, CYP3A4, DHCR7, CASR, NADSYN1, RXRA, RXRB, RXRG) were genotyped using Illumina GoldenGate or Sequenom assays in 524 African-American and 657 European-American men with newly-diagnosed PCa from PCaP. DNA extracted from blood samples collected at enrollment was stored at -80C prior to analyses. Research subjects were classified as high aggressive if Gleason sum ≥8, or Gleason score (4+3), or PSA >20 ng/ml, or Gleason score (3+4) AND clinical stage = T3-T4. The comparison group (low aggressive) included research subjects with Gleason sum <7 AND Stage T1-T2 AND PSA < 9 ng/ml. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated for high aggressive PCa for each SNP using logistic regression with adjustment for age and proportion of African ancestry. Associations were considered statistically significant at p<0.05. A polygenic score based on a previous study of SNP predictors of serum 25-hydroxy-vitamin D levels was calculated utilizing SNPs in the GC, CYP24A1, CYP2R1, and NADSYN1 genes.

Results: Among African Americans, 21 SNPs were associated with PCa aggressiveness. The variant allele was associated negatively or positively with high aggressive PCa in eleven and ten SNPs, respectively. For example, two SNPs in the vitamin D binding protein gene known as GC, were inversely associated (rs222054: OR, 0.55, 95%CI, 0.38-0.80; and rs16847028: OR, 0.61, 95%CI, 0.39-0.94). Among European Americans, the variant allele was inversely associated with high aggressive PCa for four SNPs in three genes (CASRrs3863977; CYP24A1rs4809960; RXRArs1007971; and RXRArs3118526), and positively associated with high aggressive PCa for three SNPs in the CYP27B1 gene (rs703842, rs4646536, and rs10877013). There was no association between higher number of 'low vitamin D' alleles in the four SNPs that comprised the polygenic score and PCa aggressiveness for either race.

Conclusions: Polymorphisms in genes involved in vitamin D etabolism and activity, the vitamin D binding protein and calcium sensing receptor were associated with PCa aggressiveness, and there was no overlap in SNPs between race groups. Our ongoing work will examine interaction between polymorphisms of vitamin D-related genes and vitamin D metabolite levels on PCa aggressiveness.

#807

Polymorphisms in DNA-repair genes and its association with pain among breast cancer patients receiving adjuvant radiation therapy.

Eunkyung Lee,1 Jean L. Wright,2 Cristiane Takita,1 Eden Martin,1 Susan Slifer,3 Jennifer J. Hu1. 1 _University of Miami, Miami, FL;_ 2 _Johns Hopkins University, Baltimore, MA;_ 3 _University of Miami, MIAMI, FL_.

Pain related to cancer or treatments is a critical quality of life (QOL) issue for breast cancer survivors. This study aimed to evaluate the association between the polymorphisms in DNA-repair genes and radiation therapy (RT)-associated pain among breast cancer patients receiving adjuvant RT. Pain score was assessed at pre- and post-RT as the mean of four pain severity items (i.e., pain at its worst, least, average, and now) from the Brief Pain Inventory with 11-point numeric rating scale (0-10) and RT-associated clinically relevant pain was considered present when pain score increased from <4 to ≥ 4 during RT. We evaluated 3,747 single-nucleotide polymorphisms (SNPs) in 119 DNA repair-related genes; 15 genes in base excision repair (BER), 11 in mismatch repair (MMR), 26 in nucleotide excision repair (NER), 15 in homologous recombination, 5 in non-homologous end-joining (NHEJ), 15 in DNA polymerase, and 47 in other DNA repair-related pathways. Multiple logistic regression analyses were conducted to assess the associations between SNPs and RT-associated pain. In a prospective study of 359 breast cancer patients, 81 experienced RT-associated pain. After controlling for significant covariates, 232 SNPs were found significant at p <0.05 and 48 at p < 0.01. Significant associations (p<0.01) were found in 11 SNPs in XRCC4 and 3 in XRCC5 in NHEJ, 7 in ATR in DNA damage response pathway, 6 in MGMT in direct reversal of damage pathway, 6 in MSH3 and 1 in PMS1 in MMR, 3 in XRCC1 genes in BER, 2 in POLB and POLB, and 1 in REV1 in DNA polymerase pathway, 1 in XPC and RPA3 in NER, and 1 in UBE2V2 in Rad6 pathway. Among these, most significant association was found in SNP in ATR serine/threonine kinase (rs13099184) which showed that carrying one or two doses of minor T allele was associated with increased risk of experiencing RT-associated pain (odds ratio [OR]=2.33; 95% confidence interval (CI)=1.53-3.55, p=8.52x10-5). For XRCC4 (rs2089565), carriers with one or two minor C allele were more likely to experience RT-associated pain (OR=1.93, 95% CI=1.31-2.84, p=8.1x10-4). This study demonstrated that genetic variations in multiple genes in DNA-repair pathways may contribute to inter-individual variation in pain experience among cancer patients during RT. After validation with a larger sample, these findings may be used as predictive biomarkers of radiation sensitivity which allows to identify patients at high risk of pain. Also the results can provide biological targets for early intervention for pain management to improve QOL in breast cancer survivors.

#808

Microsatellite instability in metastatic colorectal cancer (mCRC).

Jee Hung KIM, Chang-gon Kim, Joong Bae Ahn, MinKyu Jung, Seung Hoon Beom, Joo Hoon Kim, Soo Jin Heo, Sang Joon Shin. _Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Seoul, Republic of Korea_.

Background: Although early stage colorectal cancer (CRC) with microsatellite instability (MSI) phenotype has a more favorable prognosis, impact on outcome of MSI status in metastatic CRC (mCRC) is rarely known. We aim to explore patterns of metastatic spread and prognosis of CRC with MSI-H (high-level MSI) in comparison to MSI-L (low-level of MSI)/MSS.

Methods: Patients with mCRC who underwent testing for MSI were identified using retrospective cohort of Yonsei Cancer Center. Patients were categorized to MSI status (MSI-H vs. MSS-L/MSS). Patterns of metastatic spread, surgical resection after recurrence and outcome between two groups were compared.

Results: A total of 944 patients were evaluated. Of all, 34 patients (3.6%) had MSI-H tumors. A distinct pattenrs of metastatic spread was observed in MSI-H tumors, namely higher rates of peritoneal metastasis (41% vs 10%, P < 0.001), intraabdominal lymph node metastasis (14.7% vs 4.8%, P = 0.011), and lower rates of lung metastasis (8.8% vs 24.6%, P = 0.034), liver metastasis (20.6% vs 53.6%, P < 0.001). Patients with MSI-H tumors were less likely to receive metastectomy (24.8% vs 55.9%, P < 0.001). Median overall survival was inferior in MSI-H tumor compared with MSI-L/MSS tumor (16.3 months vs 38.1 months, P = 0.005).

Conclusions: Different patterns of metastatic spread and receipt of surgical resection in MSI-H mCRC is demonstrated in this study. In addition, MSI-H phenotype is associated with poorer survival in mCRC on the contrary to early stage disease. Future studies focusing on deeper understanding of tumor biology and therapeutic response in this rare disease entity are warranted.

#809

Dietary iron intake, genetic variants in microRNA related iron regulatory pathway genes, and the risk of non-small cell lung cancer.

Liren Zhang,1 Yuanquing Ye,1 Huakang Tu,1 Michelle A.t. Hildebrandt,1 John V. Heymach,2 Jack A. Roth,3 Jian Gu,1 Xifeng Wu1. 1 _Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Thoracic Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Department of Thoracic & Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX_.

As an essential nutrient that facilitates DNA synthesis, cell proliferation and metabolism, iron is involved in carcinogenesis. While epidemiological studies suggest a J-shaped model for the relationship of serum iron level and cancer risk with both too low and too high levels associated with increased cancer risks, the effect of genetic variants related to intracellular iron regulatory pathway and dietary iron intake on non-small cell lung cancer (NSCLC) risk is little known. In this study, we evaluated a panel of miRNA related genetic variants in iron regulatory pathway genes on the risk of NSCLC. In discovery phrase, 157 miRNA related single nucleotide polymorphisms (SNPs) from 81 genes of iron regulatory pathway were genotyped in 1656 NSCLC cases and 1486 healthy controls. Eight SNPs in 6 genes were significantly associated with risk of NSCLC. A SNP in 3' UTR of Iron-Responsive Element Binding Protein 2 (IREB2) gene at 15q25 region was subsequently replicated in dbGaP lung cancer GWAS dataset. Combining all subjects (7,352 cases, 7,296 controls), the overall P-value was 4.9 x 10-9 (Odds ratio (OR) = 0.86). eQTL analysis showed that the SNP on IREB2 alters IREB2 gene expression (P = 3.0 x 10-11). The SNP is predicted to alter a miR-29 binding site on IREB2 gene and indeed the expression of miR-29 is inversely correlated with IREB2 expression in tumor tissues. We then determined the expression of serum miR-29a in 150 early-stage NSCLC patients and 172 healthy controls and found that miR-29a was significantly associated with higher risk of cancer (OR = 1.78, P = 0.03). By analyzing dietary intake information of the discovery population, iron intake was significantly associated with risk of NSCLC in logistic regression analysis and there was also evidence of a synergistic interaction with between the SNP and iron intake in modulating NSCLC risk (P for interaction = 0.01). Taken together, SNPs at miRNA binding sites of iron regulatory pathway genes and dietary iron intake may modify risk of NSCLC individually and jointly.

#810

The CARRIERS consortium: Establishing refined breast cancer risk estimates in known predisposition genes.

Jenna Lilyquist,1 Peter Kraft,2 Steven N. Hart,1 Emily J. Hallberg,1 Chunling Hu,1 Raymond Moore,1 Rohan Gnanaolivu,1 Susan M. Domchek,3 Jeffrey N. Weitzel,4 Katherine L. Nathanson,3 David E. Goldgar,5 Fergus J. Couch1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Harvard University, Boston, MA;_ 3 _University of Pennsylvania, Philadelphia, PA;_ 4 _City of Hope, Duarte, CA;_ 5 _University of Utah, Salt Lake City, UT_.

Breast cancer is the most common type of cancer, and has a strong heritable component, with approximately 15 percent of new cases having family history of breast cancer. Genetic panel testing for patients with a family history of breast cancer has become a popular clinical resource. However, the risk of breast cancer associated with inactivating mutations in the predisposition genes is largely undefined, creating critical limitations in the interpretations of the results from panel testing and subsequent medical management. The Cancer Risk Estimates Related to Susceptibility Genes (CARRIERS) consortium was established to determine the risk of cancers associated with truncating mutations in the known predisposition genes and to extend these studies to the clinical classification of variants of uncertain significance (VUS) in the relevant genes.

CARRIERS involves screening of approximately 30,000 population-based breast cancer cases and 30,000 study matched controls for mutations in 28 established or proposed breast cancer predisposition genes. Population-based risks for breast cancer associated with mutations in each gene will be estimated. In parallel, 10,000 breast cancer cases from moderate and high-risk breast cancer families will be similarly screened for mutations and the penetrance of the mutations in high-risk families will be estimated. Furthermore, VUS identified by mutation screening will be characterized by functional and family-based studies and models for classification of the clinical relevance of VUS in each gene will be developed. These data are expected to lead to improvements in cancer risk assessment and improved management of patients found to carry mutations or VUS in breast cancer predisposition.

#811

Mosaic chromosome Y loss is not associated with testicular germ cell tumor risk.

Mitchell J. Machiela, Casey L. Dagnall, Anand Pathak, Jennifer T. Loud, Stephen J. Chanock, Mark H. Greene, Katherine A. McGlynn, Douglas R. Stewart. _Division of Cancer Epidemiology and Genetics, Bethesda, MD_.

Background

Testicular germ cell tumors (TGCTs) affect approximately 6 in 100,000 men with peak incidence in men 15 to 40 years of age. Established risk factors include family history of disease, undescended testis, contralateral TGCT, impaired fertility, and 21 GWAS susceptibility loci. Recent evidence suggests a 1.6 Mb deletion in the AZFc region ("gr/gr" deletion) of chromosome Y previously associated with male infertility is also associated with two- and three-fold increased risks of sporadic and familial TGCT, respectively. Furthermore, population-based studies have suggested mosaic chromosome Y loss in blood-derived DNA may be associated with increased risk of various solid tumors. Our aim was to investigate if mosaic loss across the entire Y chromosome is a TGCT risk factor.

Methods

We obtained blood and buccal-derived DNA from two studies: the NCI Familial Testicular Cancer Study (FTC, Cases=172, Controls=163) and NCI's US Servicemen's Testicular Tumor Environmental and Endocrine Determinants Study (STEED, Cases=506, Controls=611). We designed a quantitative polymerase chain reaction (qPCR) with 15 probes spanning the Y chromosome to assess Y loss. Samples were run in triplicate across three batches and normalized to a standard curve. We quantified probe signals as a target to reference (T/R) ratio, and averaged ratios across the 15 probes to yield an overall Y chromosome signal.

Results

On average, men with TGCT had slightly lower mean T/R ratios compared with control men lacking TGCT (1.02 vs. 1.03). Multivariate logistic regression models adjusted for study batch effects detected no significant relationship between mean Y chromosome T/R ratio and TGCT risk (OR=0.34, 95% CI=0.10-1.17, P=0.09). A similar null relationship was observed when using a threshold of 1.5 standard deviations below the mean T/R ratio to dichotomize between mosaic Y loss and no mosaic Y loss (OR=0.59, 95% CI=0.21-1.63, P=0.31).

Conclusion

Our study suggests that mosaic chromosome Y loss, as measured by 15 probes spanning the Y chromosome, is not associated with either sporadic or familial TGCT risk. Further studies aimed at investigating the AZFc region of the Y chromosome are needed to better assess whether this locus is directly associated with TGCT risk or is indirectly associated by means of a shared relationship with impaired fertility.

#812

Association between variants in genes involved in the immune response and prostate cancer risk in men randomized to finasteride in the Prostate Cancer Prevention Trial.

Danyelle A. Winchester,1 Cathee Till,2 Jianfeng Xu,3 Ian M. Thompson,4 Scott M. Lippmann,5 Howard L. Parnes,6 Angelo M. De Marzo,7 Charles G. Drake,7 Elizabeth A. Platz,8 The PCPT P01 Project 4 Research Team. 1 _Johns Hopkins Bloomberg School of Public Health, Hyattsville, MD;_ 2 _2SSWOG Statistical Center, Fred Hutchinson Cancer Research Center,, WA;_ 3 _Cancer Genomic Research, NorthShore University HealthSystem, IL;_ 4 _University of Texas Health Sciences Center San Antonio, TX;_ 5 _Moores Cancer Center, University of California San Diego, CA;_ 6 _Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Department of Health and Human, MD;_ 7 _James Buchanan Brady Urological Institute and Department of Urology, Johns Hopkins University School of Medicine, MD;_ 8 _Johns Hopkins Bloomberg School of Public Health, MD_.

Background: We reported that some, but not all single nucleotide polymorphisms (SNPs) in select immune response genes are associated with prostate cancer in the Prostate Cancer Prevention Trial* (PCPT) placebo arm. Here, we investigated whether these same SNPs are associated with risk in men randomized to finasteride, which is known to increase intraprostatic inflammation. Methods: 16 candidate SNPs in IL1, IL2, IL4, IL6, IL8, IL10, IL12(p40), IFNG, MSR1, RNASEL, TLR4, and TNFA and 7 tagSNPs in IL10 were genotyped in 625 prostate cancer cases, and 532 controls negative for cancer on an end-of-study biopsy nested in the PCPT finasteride arm. Cases and controls were non-Hispanic white men. We used logistic regression to estimate log-additive odds ratios (OR) and 95% confidence intervals (CI) adjusting for age and family history (matching factors). Results: Minor alleles of rs2243250 (T) in IL4 (OR=1.46, 95% CI 1.03-2.08, P-trend=0.03), rs1800896 (G) in IL10 (OR=0.77, 95% CI 0.61-0.96, P-trend=0.02), rs2430561 (A) in IFNG (OR=1.33, 95% CI 1.02-1.74; P-trend=0.04), rs3747531 (C) in MSR1 (OR=0.55, 95% CI 0.32-0.95; P-trend=0.03), and possibly rs4073 (A) in IL8 (OR=0.81, 95% CI 0.64-1.01, P-trend=0.06) were associated with higher- (Gleason 7-10; N=222), but not lower- (Gleason 2-6; N=380) grade prostate cancer. In men with low PSA (<2 ng/mL), these associations were attenuated and/or no longer significant, whereas inverse associations with higher-grade disease were apparent for minor alleles of rs1800795 (C: OR=0.70, 95% CI 0.51-0.94, P-trend=0.02) and rs1800797 (A: OR=0.72, 95% CI 0.53-0.98, P-trend=0.04) in IL6. While some IL10 tagSNPs were associated with lower- and higher-grade prostate cancer, distributions of IL10 haplotypes did not differ from controls, except possibly among those with low PSA (P=0.07). Conclusion: In the PCPT finasteride arm, variation in genes involved in the immune response, including possibly IL8 and IL10 as in the placebo arm, may be associated with prostate cancer, especially higher-grade disease, but we cannot rule out PSA-associated detection bias or chance due to multiple testing.Funding: P01 CA108964, U10 CA37429, UM1 CA182883, T32 CA009314. *A SWOG-Coordinated Study S9217

#813

The association of androgen metabolizing enzymes and prostate cancer in a multiethnic study.

Delores J. Grant,1 Lauren Howard,2 Emily Wiggins,3 Amanda De Hoedt,3 Adriana Vidal,4 Skyla T. Carney,1 Jill Squires,5 Clara E. Magyar,5 Jiaoti Huang,5 Stephen J. Freedland4. 1 _North Carolina Central University, Durham, NC;_ 2 _Duke University, Durham, NC;_ 3 _Durham Veterans Administration Medical Center, Durham, NC;_ 4 _Cedars-Sinai Health System, Los Angeles, CA;_ 5 _University of California at Los Angeles, Los Angeles, CA_.

Introduction: Uridine 5'-diphosphate-glucuronosyltransferase 2B (UGT2B) genes code for enzymes that catalyze the clearance of testosterone, dihydrotestosterone (DHT), and DHT metabolites in the prostate basal and luminal tissue. The expression of the UGT2B15, UGT2B17, and UGT2B28 enzymes has not been evaluated in prostate tissue samples from hormone therapy-naïve patients.

Methods: We determined the expression of the three enzymes in 190 prostate tissue samples from surgical specimens of a multiethnic cohort of patients undergoing radical prostatectomy at the Durham Veterans Affairs Medical Center. The association between each biomarker's percent positive and H-score and the risk of biochemical recurrence (BCR) was tested using separate Cox proportional hazards models. In an exploratory analysis, UGT2B17 total positive and H-score were split at the median and we tested the association between UGT2B17 group and risk of BCR.

Results: The median follow-up was 118 months (IQR: 85-144). We found no association between UGT2B15 or UGT2B28 and risk of BCR. However, there was a trend between UGT2B17 and BCR (HR=1.01, 95% CI 1.00-1.02, p=0.11), though not statistically significant. Upon further investigation, we found patients with UGT2B17 total positive above the median had a significant increased risk of BCR on univariable analysis (HR=1.57, 95% CI 1.02-2.43, p=0.041) but this association was attenuated in the adjusted model (HR=1.50, 95% CI 0.94-2.40, p=0.088).

Conclusions: Our findings suggest that UGT2B17 overexpression was associated with a significant increased risk of BCR. These results are consistent with previous reports that show UGT2B17 significantly expressed in prostate cancer metastases.

#814

Gene-based analysis of the IGF signaling pathway and risk of breast cancer in African American women: The AMBER consortium.

Edward A. Ruiz-Narvaez,1 Kathryn L. Lunetta,1 Chi-Chen Hong,2 Stephen A. Haddad,1 Song Yao,2 Ting-Yuan D. Cheng,2 Jeannette T. Bensen,3 Elisa V. Bandera,4 Christopher A. Haiman,5 Andrew F. Olshan,3 Christine B. Ambrosone,2 Lynn Rosenberg,1 Julie R. Palmer1. 1 _Boston University, Boston, MA;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 4 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 5 _University of Southern California Keck School of Medicine, Los Angeles, CA_.

Dysregulation of the IGF (insulin-like growth factor) signaling pathway plays a key role in cancer development. It is still unclear whether germline variation in genes in the IGF pathway may affect risk of breast cancer. We conducted a gene-based analysis of 184 genes in the IGF signaling pathway to identify genes carrying genetic variation affecting risk of breast cancer and the specific estrogen receptor (ER) subtypes. Tagging single nucleotide polymorphisms (SNPs) for each gene were selected and genotyped on a customized Illumina Exome Array. Imputation was carried out using 1000 Genomes haplotypes. The analysis included 91,627 SNPs in 3,663 breast cancer cases (including 1,983 ER positive,1,098 ER-negative) and 4,687 controls from the African American Breast Cancer Epidemiology and Risk (AMBER) consortium, a collaborative project of four large studies of breast cancer in African American women (Carolina Breast Cancer Study, Black Women's Health Study, Women's Circle of Health Study, and Multiethnic Cohort). We used a multi-locus adaptive joint (AdaJoint) test to determine the association of each gene in the IGF signaling pathway with overall breast cancer and ER subtypes. None of the 184 tested genes, including IGF, IGF binding protein (IGFBP), and IGF receptor (IGFR) genes were associated with disease after adjustment for multiple testing. At the nominal level of P≤0.01, BAIAP2 (P=0.003), and CALM2 (P=0.009) genes were associated with overall breast cancer; BAIAP2 (P=0.001), and CSNK2A1 (P=0.01) with ER+ breast cancer; and BRAF (P=0.003), BAD (P=0.005), and MAPK3 (P=0.009) with ER- breast cancer. The intronic rs9913477 SNP in the BAIPA2 gene was significantly associated with overall (P=3.2x10-7) and ER+ (P=4.4x10-7) breast cancer at the pathway-wide level after adjusting for the total number of tested SNPs. Odds ratios and 95% confidence intervals of the risk G-allele were 1.44 (1.25, 1.66), and 1.53 (1.30, 1.81) for overall and ER+ breast cancer, respectively. BAIAP2 codes an adaptor protein, IRSp53, which functions as a substrate of the insulin receptor and IGF-1 receptor tyrosine kinases and links Rho-family small GTPases such as Rac. In vitro studies have shown that activation of Rac promotes metastatic behavior of breast cancer cells. In conclusion, we identified a SNP in BAIAP2 associated with overall and ER+ breast cancer.

These results highlight the importance of the IGF signaling pathway in the pathogenesis of breast cancer.

#815

Patterns of metagene activation in ovarian cancer subtypes.

James Rudd,1 Emily K. Shea,2 Gregory P. Way,3 Casey S. Greene,3 Jennifer A. Doherty1. 1 _Dartmouth College, Lebanon, NH;_ 2 _Williams College, Williamstown, MA;_ 3 _University of Pennsylvania, Philadelphia, PA_.

High grade serous ovarian cancer (HGSC) is a complex and aggressive disease. Recently, three or four gene expression-based subtypes, which may be differentially associated with survival, have been reported in several populations. To identify the biological functions that define the subtypes, we determined the extent to which metagenes—linear combinations of gene expression vectors—were differentially activated across subtypes, could be reliably identified across populations, and showed consistent associations with survival.

We previously clustered HGSC samples using gene expression data from TCGA, Tothill (GSE9891), Yoshihara (GSE32062), and Mayo (GSE74357) to identify subtypes across populations. We found subtype-specific genes within each population through differential expression analysis (p < 4.6x10-6). Using the intersection of differentially expressed genes for parallel subtypes across these four populations, we applied non-negative matrix factorization to identify metagenes. To determine whether the metagenes were consistently observed across populations, we performed leave-one-dataset-out cross validation. For each metagene, we performed gene set enrichment analysis against the National Cancer Institute pathway interaction database to annotate metagene pathways. We examined whether increasing tertiles of metagene activity, which we termed low, medium, and high activity, were associated with survival using a random effects meta-analysis of Cox regression estimates adjusting for age at diagnosis, tumor stage, tumor grade, and debulking status.

Five metagenes were consistently identified and significantly associated with HGSC subtypes (p < 0.0001). Of these, a metagene weakly enriched for the CMYB pathway was associated with subtype 1; three metagenes (one significantly enriched with the IL12 pathway and the others weakly enriched with the FCER1 and CXCR4 pathways) were associated with subtype 2; and a metagene weakly enriched with the AVB3 Integrin pathway distinguished between all 3 subtypes. Neither the CYMB metagene nor the IL12 metagene was significantly associated with survival. High activity of the CXCR4 and AVB3 metagenes was associated with poorer survival (hazard ratios (HR) and 95% confidence intervals (CI) are, respectively: 1.21, 0.99-1.48 and 1.34, 1.09-1.64). In contrast, high activity of the FCER1 metagene was associated with improved survival (HR 0.76, 95% CI 0.62-0.93).

Metagenes that are consistently and statistically significantly associated with subtype may be indicative of functional differences between HGSC subtypes. The contrast in hazard estimates for metagenes associated with subtype 2 may indicate that the metagenes capture survival signal distinct from the subtype association. Future work associating metagene activity with subtype uncertainty may better enable the refinement of subtype definitions and the development of subtype specific treatment strategies.

#816

Genetic variants in the dopaminergic system and bladder cancer clinical outcomes.

Jeanne A. Pierzynski,1 Yuanquing Ye,1 Alma Rodriguez,2 Michelle A.T. Hildebrandt,1 Ashish M. Kamat,3 Colin P.N. Dinney,3 Xifeng Wu1. 1 _Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _2. Department of Urology, Division of Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Bladder cancer is estimated to be the fourth most common cancer diagnosed and the eighth most common cancer related death in men in 2015. A hallmark of bladder cancer outcomes is the high rates of recurrence and progression in patients diagnosed with non-muscle invasive bladder cancer (NMIBC) and poor survival rates in those that have progressed to or are diagnosed with muscle invasive bladder cancer (MIBC). Because of the poor outcomes, research for predictors of clinical outcomes is necessary. Recent studies show that current depressive symptoms at time of bladder cancer diagnosis are associated with bladder cancer mortality. We are interested in investigating the genetic mechanism underlying this relationship. The dopaminergic pathway is of interest because of its associations with depression and smoking addiction (with smoking being the main risk factor for bladder cancer). Therefore we aimed to do a candidate pathway analysis examining 256 single nucleotide polymorphisms (SNPs) in 15 genes from the dopaminergic system. We analyzed the association of genetic variants with recurrence and progression in NMIBC patients (N=497) and the association with survival in MIBC patients (N=399). Overall, there were four SNPs with a p-value of less than 0.01associated with recurrence in NMIBC. The most significant variant being rs2797853 located in DBH under the recessive model (HR=1.77, 95% CI: 1.28- 2.46. P=0.0012). Eight polymorphisms had a statistically significant association with progression in NMIBC patients, with COMT: rs5993891 being the most significant variant showing an increased odds of bladder cancer progression (HR= 2.54, 95% CI: 1.50- 4.28, P=0.0013). Interestingly, the variant rs174675 located in COMT (an enzyme responsible for degrading dopamine) was associated with an increased risk of recurrence (HR= 1.23, 95% CI: 1.01-1.49, P=0.046) and progression (HR= 1.55, 95% CI: 1.13-2.12, P= 0.007). Finally, five SNPs were statistically significant for bladder cancer survival in MIBC patients. BDNF: rs2203877 was associated with a 71% increased risk of death (HR= 1.71, 95% CI: 1.25-2.34, P= 0.001). There was a significant difference in median survival time (MST). Those with the homozygous dominant or heterozygous genotype had a MST of 66.3 months and those with the homozygous recessive genotype had a MST of 31.8 months (Plog-rank=0.02). The variant rs2239395 in COMT was associated with an increased risk of progression (HR= 2.54, 95% CI: 1.49-4.34, P=0.002) and death (HR= 1.63, 95% CI: 1.05-2.53, P=0.041). In addition to validation studies that are underway, the next steps include analyzing the association between these factors and BMI (a factor associated with depression, the dopamine system, and bladder cancer). The results of this study have the potential to contribute to identifying bladder cancer patients who may have poor clinical outcomes as well as the development of interventions that may improve outcomes.

#817

Mendelian randomization and mediation analysis of 5p15.33, telomere length and lung cancer risk.

Linda Kachuri,1 George Davey Smith,2 Geoffrey Liu,3 Maria Teresa Landi,4 David C. Christiani,5 Neil E. Caporaso,4 James D. McKay,6 Xifeng Wu,7 Melinda C. Aldrich,8 Gad Rennert,9 Dawn Teare,10 Chu Chen,11 Gary E. Goodman,11 Jennifer A. Doherty,12 John K. Field,13 Lambertus A. Kiemeney,14 Adonina Tardón,15 Aage Haugen,16 Stephen Lam,17 Loic Le Marchand,18 Matthew B. Schabath,19 Angeline S. Andrew,12 Mattias Johansson,6 Jonas Manjer,20 Philip Lazarus,21 Susanne Arnold,22 Gordon Fehringer,1 Xuchen Zong,1 Paul Brennan,6 Stig E. Bojesen,23 Christopher I. Amos,24 Rayjean J. Hung1. 1 _Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada;_ 2 _University of Bristol, School of Social and Community Medicine, Bristol, United Kingdom;_ 3 _Ontario Cancer Institute, Princess Margaret Cancer Center, Toronto, Ontario, Canada;_ 4 _Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD;_ 5 _Departments of Environmental Health and Epidemiology, Harvard School of Public Health, Boston, MA;_ 6 _International Agency for Research on Cancer, Lyon, France;_ 7 _Department of Epidemiology, Division of Cancer Prevention and Population Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 8 _Department of Thoracic Surgery and Division of Epidemiology, Vanderbilt University Medical Center, Nashville, TN;_ 9 _Technion, Israel Institute of Technology, Haifa, Israel;_ 10 _School of Health and Related Research, University of Sheffield, Sheffield, United Kingdom;_ 11 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 12 _Department of Community and Family Medicine, Geisel School of Medicine, Dartmouth College, Lebanon, NH;_ 13 _Roy Castle Lung Cancer Research Programme, University of Liverpool Cancer Research Centre Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom;_ 14 _Radboud University Medical Centre, Nijmegen, Netherlands;_ 15 _University Institute of Oncology of Asturias, Oviedo, Spain;_ 16 _STAMI – The National Institute of Occupational Health, Oslo, Norway;_ 17 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 18 _University of Hawaii Cancer Center, Honolulu, HI;_ 19 _Moffitt Cancer Center, Tampa, FL;_ 20 _Skåne University Hospital, Lund University, Lund, Sweden;_ 21 _College of Pharmacy, Washington State University, Spokane, WA;_ 22 _Markey Cancer Center, University of Kentucky, Lexington, KY;_ 23 _Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, Herlev, Denmark;_ 24 _Center for Genomic Medicine, Department of Community and Family Medicine, Geisel School of Medicine, Dartmouth College, Lebanon, NH_.

Background: Telomere length (TL) is a predictor of lung cancer risk, but the direction of this association differs between and prospective and case-control studies. This discrepancy may be attributed to reverse causation in the latter, due to disease-related changes in TL that is measured after diagnosis or treatment. To overcome these limitations and characterize the relationship between TL and lung cancer risk we carried out observational and mediation analyses, and a 2-stage Mendelian Randomization (MR) analysis, where we developed novel genetic instruments for TL and tested the association with lung cancer using 20 OncoArray studies in the Transdisciplinary Research in Cancer of the Lung group of the International Lung Cancer Consortium.

Methods: The observational analysis examined TL measured using qPCR in 1128 cases and 928 controls. Odds ratios (OR) for TL were adjusted for age, sex and cigarette pack-years. Mediation analysis was used to estimate the % of the lung cancer association in 5p15 that operates through TL. To develop novel TL instruments, variants identified through deep sequencing of the 5p15 locus were genotyped in 900 controls. Variants that met MR criteria were combined into a single instrumental variable (IV), and its association with TL was estimated. We also used 6 previously identified TL predictors (p<5×10-8) as genetic instruments: rs10165485 (ACYP2), rs10936599 (TERC), rs11100479 (NAF1), rs9420907 (OBFC1), rs6028466 (DHX35), rs755017 (RTEL1). For these SNPs association estimates for TL were obtained from the literature. To estimate the association with lung cancer risk, we tested all 7 IVs using data from 14324 lung cases and 10783 controls in OncoArray. Lastly, to obtain a summary estimate for the causal effect of TL on lung cancer risk, ORs from OncoArray were combined with β-TL estimates using a likelihood-based MR model.

Results: The observational analysis suggested that longer TL is associated with decreased lung cancer risk (OR=0.94, p=0.04). This was more pronounced for squamous carcinoma (OR=0.77, p=1.1×10-4). We also showed that TL mediates up to 8% (p<0.05) of the lung cancer signal in 5p15. In the first stage of the MR analysis, we identified 8 5p15 SNPs that were associated with TL (p<5×10-3), including 6 novel rare variants, not previously associated with TL. Together these variants were reliably associated with TL (β=0.15, p=1.8×10-7) and explained 2.3% of variance in TL. Using this new instrument and 6 other SNPs as IVs, our MR analysis showed that longer TL is a risk factor for lung cancer (OR=1.77, 95% CI: 1.32-1.54), especially adenocarcinoma (OR=2.02, 95% CI: 1.29-3.71).

Conclusions: We developed novel genetic instruments for TL, and confirmed that genetically predicted longer TL is associated with increased lung cancer risk. These findings suggest that previously reported associations of long TL with decreased risk were likely due to residual confounding by smoking, age and/or reverse causation.

#818

Shortened telomere length in hepatocellular carcinoma in the United States.

Baiyu Yang,1 Fatma M. Shebl,2 Lawrence R. Sternberg,1 Andrew C. Warner,1 David E. Kleiner,1 Daniel C. Edelman,1 Allison Gomez,1 Casey L. Dagnall,3 Belynda D. Hicks,3 Sean F. Altekruse,1 Brenda Y. Hernandez,4 Charles F. Lynch,5 Paul S. Meltzer,1 Katherine A. McGlynn1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Yale University, New Haven, CT;_ 3 _National Cancer Institute/Frederick National Laboratory for Cancer Research, Bethesda/Frederick, MD;_ 4 _University of Hawaii, Honolulu, HI;_ 5 _University of Iowa, Iowa City, IA_.

Background: Telomeres play an important role in the maintenance of chromosomal stability. It has been previously reported that telomere length is shortened in hepatocellular carcinoma (HCC) compared to paired non-tumor tissue. However, most studies have been conducted in high-risk regions such as Asia. To date, no prior study has examined telomere length and HCC in the United States, a low risk region where HCC etiology may differ from that of high risk regions.

Methods: We measured telomere length in 127 paired tumor and non-tumor tissue samples from persons with HCC in Iowa, Hawaii and Connecticut. Formalin-fixed, paraffin-embedded tissues were collected at the time of diagnosis. Relative telomere length was measured by a monochrome multiplex quantitative PCR assay. The Wilcoxon signed-rank test was used to compare telomere length between tumor and paired non-tumor tissues. Cox proportional hazards modeling was used to estimate the association between telomere length and mortality, adjusting for age at diagnosis, sex, and tumor stage and size at diagnosis.

Results: Of the 127 pairs of samples, telomere length was shorter in the tumor in 88 pairs (69%), longer in the tumor in 33 pairs (26%) and the same length in the tumor and non-tumor tissue in 6 pairs (5%). Overall, the HCC tissues had statistically significantly shorter telomere length than their matched non-tumor tissues (p < 0.01). The proportion of pairs in which the tumor telomere was shorter than non-tumor was higher among those with localized stage tumors (81%) compared to those with regional or distant stage tumors (50%) (p < 0.01 after adjusting for age at diagnosis, sex, and source of the case). In addition, we observed a 119% higher risk of mortality (adjusted relative risk 2.19) among persons whose tumors had shorter telomeres than their non-tumor tissue, compared to persons whose tumors had longer telomeres; however, the confidence interval was relatively wide (0.94-5.12).

Conclusion: Using tissue samples from persons with HCC in the United States, we found that telomere length was shorter in tumor tissue compared to paired non-tumor tissue, especially among localized stage tumors. In addition, there was a suggestion that tumor telomere shortening might be associated with higher risk of mortality. Together, these results may provide insight into the role of biological mechanisms associated with telomere shortening and into the prognosis of HCC in lower risk populations.

#820

Molecular epidemiological studies of germinal center-associated nuclear protein (ganp) in the development of human sporadic breast cancers.

Kazuhiko Kuwahara,1 Hidemi Ito,1 Kiyotaka Kuzushima,1 Hiroji Iwata,2 Hideo Tanaka,1 Keitaro Matsuo1. 1 _Aichi Cancer Center Research Institute, Nagoya, Japan;_ 2 _Aichi Cancer Center Hospital, Nagoya, Japan_.

[Introduction]

The altered expression of germinal center-associated nuclear protein (GANP), a member of mammalian TREX2 complex, has been observed in various tumors. Previously, we demonstrated that deficiency of GANP developed mammary gland tumors resembling human breast cancers in two mouse models. In addition, we showed that decreased expression of GANP was an independent prognostic factor of human breast cancer in a retrospective cohort study. These results suggested that GANP might be associated with breast cancer development. Although a little is know about functional significance of ganp polymorphisms, we explored possible association.

[Methods]

We conducted a case-control study to examine association between ganp polymorphisms and risk of breast cancer among Japanese population within the framework of the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC). Cases are 694 incident cases who had been diagnosed as breast cancer at Aichi Cancer Center Hospital (ACCH) during 2001-2005. A total of 1,376 non-cancer controls were patients who visited ACCH at same period as cases and were confirmed to have no evidence of cancer. Controls were individually matched for age and menopausal status with cases. ganp polymorphisms were assessed by iCOGS chip (Reference: Michailidou K et al. Nat. Genet. 2013;45:353-361). Multivariable conditional logistic regression models were applied to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for each locus.

[Results]

Ten out of 13 polymorphisms, in high linkage disequilibrium, showed statistically significant associations with breast cancer risk. For instance, in rs2839186, the OR for those with AA alleles compared with those with GG alleles is 0.41 (95% CI, 0.23-0.72, p=0.002) in genotype model. As compared with those with G allele, the OR for those without was 0.40 (95% CI, 0.23-0.71, p=0.002) in recessive model. On the other hand, in dominant model, the OR for those with A allele was 0.95 (95% CI, 0.78-1.16, p=0.635) in comparison to those without that.

[Conclusion]

In this study, we clearly show that mutant SNPs in rs2839186 decrease breast cancer risk. We are currently analyzing if the ganp SNPs affect the prognosis of breast cancer patients.

## PREVENTION RESEARCH:

### Models and Mechanisms in Cancer Prevention

#821

Discovery of catechol moiety-containing natural compounds as direct ERK2 inhibitors by in vitro kinase assay and co-crystallography.

Seung Ho Shin,1 Margarita Malakhova,1 Igor Kurinov,2 Do Young Lim,1 Ann M. Bode,1 Zigang Dong1. 1 _University of Minnesota, Austin, MN;_ 2 _Cornell University, NE-CAT, APS, Argonne, IL_.

Catechol (pyrocathechol, 1,2-dihydroxybenzene) is a chemical that is used by industry in the manufacture of certain products. Many natural compounds contain catechol as a functional group found in various fruits and vegetables such as apple, apricot, grape, bananas, soybean, peanut, pear, plum, mango, avocado, and mushroom. Extracellular signal-regulated kinase 2 (ERK2), a protein kinase that belongs to MAPK family, modulates many important functions such as cell growth, apoptosis and transcriptional regulation in cancer. The expression levels of ERK2 mRNA and protein are reportedly very high in many human cancers, including leukemia, colon, breast, lung and skin cancer. Previously, we reported that norathyriol and caffeic acid, which possess the catechol moiety, and catechol itself directly bind and inhibit ERK2 kinase activity. Those findings led us to test catechol moiety-containing natural compounds, luteolin, quercetin, fisetin, 7,3',4'-trihydroxyisoflavone, and cyanidin, against ERK2 kinase activity. We found that all of these compounds directly bind to ERK2 and inhibit its activity. We further confirmed the results by resolving a co-crystal structure of ERK2 bound with luteolin in the ATP binding site. To apply this finding to cancer, the effect of the compounds was tested on the K562 human myelogenous leukemia cell line where ERK2 is highly expressed and knock-down of the protein reduced anchorage-independent growth. Five newly-found ERK2 inhibitors (luteolin, quercetin, fisetin, 7,3',4'-trihydroxyisoflavone and cyanidin) and three known ERK2 inhibitors (norathyriol, caffeic acid and catechol) decreased cell viability of K562 at 40 μM. When the eight compounds were used at the same dose in combination (5 μM each and 40 μM total), the mixture inhibited ERK2 and reduced cell viability of K562 cells. In summary, using in vitro kinase assays and co-crystallography, we identified new ERK2 inhibitors that contain the catechol functional group, and showed anti-cancer effects of the compounds by cell viability assay and soft agar assay. This work impacts the cancer prevention community by showing that many natural compounds can work together targeting ERK2, an important target in cancer.

#822

Role of c-Myc in prostate cancer stem-like cell inhibition by sulforaphane.

Su-Hyeong Kim, Avani R. Vyas, Shivendra V. Singh. _University of Pittsburg Cancer Institute, Pittsburgh, PA_.

Chemopreventive efficacy of D,L-sulforaphane (SFN) against prostate cancer has been shown in a transgenic mouse model but the underlying mechanism is not fully understood. The present study demonstrates that c-Myc, which is an oncogenic transcription factor frequently upregulated in human prostate cancer, is a molecular target of SFN. Exposure of human prostate cancer cells (LNCaP, C4-2, and PC-3) and a cell line derived from prostate adenocarcinoma of a transgenic mouse (Myc-CaP) to SFN resulted in a marked decrease in c-Myc protein level. Naturally-occurring thio, sulfinyl, and sulfonyl analogues of SFN were also effective in causing suppression of c-Myc protein level. The c-Myc is a known regulator of cellular metabolism but basal glycolysis was not altered by SFN treatment in empty vector transfected or in c-Myc overexpressing PC-3 cells. The effect of SFN treatment on prostate cancer stem-like cells (pCSC) was also determined as c-Myc is implicated in their maintenance. Analysis of aldehyde dehydrogenase 1 activity, CD49f+ fraction, and sphere forming efficiency revealed dose-dependent inhibition of pCSC in SFN-treated cells. The pCSC inhibition by SFN was partially but significantly attenuated by overexpression of c-Myc. Expression profiling revealed upregulation of a set of stemness genes by c-Myc overexpression but their downregulation by SFN treatment. Finally, oral administration of SFN to Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice resulted in a marked decrease in c-Myc protein expression in the adenocarcinoma. In conclusion, this study indicates that c-Myc downregulation contributes to pCSC inhibition by SFN. This investigation was supported by grant CA115498 awarded by the National Cancer Institute.

#823

Prevention of pancreatic cancer by cAMP control.

Jheelam Banerjee, Arokya M. Papu John, Mohammed H. Al-Wadei, Hildegard M. Schuller. _University of Tennessee, Knoxville, TN_.

Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). Using a hamster model of pancreatitis-associated PDAC induced by treatment with ethanol in the drinking water and the nicotine-derived nitrosamine 4-methylnitrosamino-(3-pyridyl)-1-butanone (NNK) by subcutaneous injection as well as immunohistochemistry of human PDAC tissue microarrays, we have previously shown that these cancers overexpressed stress neurotransmitters and cAMP while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) was suppressed. Using our hamster model, the current study has tested the hypothesis that cAMP control by GABA supplementation in the drinking water prevents the development of pancreatitis-associated PDAC. Our data reveal strong preventive effects of GABA supplementation on the development of PDAC and pancreatic intraductal neoplasia (PanIN). ELISA assays and immunohistochemistry revealed significant decreases in the levels of cyclic adenosine monophosphate (cAMP) and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers, phosphorylated signaling proteins associated with cell proliferation and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP control by GABA supplementation inhibits multiple cancer supporting pathways at the level of cancer stem cells, differentiated cancer cells and the immune system, identifying this approach as promising novel tool for the prevention of PDAC in individuals with a history of smoking and alcoholism.

#824

Effects of metfornin on mammary carcinogenesis and metabolic profiles in methylnitrosourea-treated rats on standard or Western diets.

Mark S. Miller,1 Matthew D. Thompson,1 Ronald A. Lubet,1 Vernon E. Steele,1 Harold E. Seifried,1 Clinton J. Grubbs2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

The methylnitrosourea-induced model of ER+ mammary cancer in female Sprague-Dawley rats has been routinely used to screen for chemopreventive agents. We recently reported that metformin was ineffective in reducing cancer formation in rats on a standard (4% fat) diet (Thompson, et al., Cancer Prev Res. 8, 231, 2015). In the present studies, rats were placed on a standard (Teklad) diet or on a Western diet (20% fat, low calcium) at 43 days of age (DOA), and administered MNU at 50 DOA. Rats were administered metformin or vehicle daily by gavage beginning at 57 days of age. Serum from the rats of the various groups was obtained both at 21 days following initial administration of metformin (or vehicle) and at the end of the study (when tumors had developed in most rats). We found an increase in the final body weights, number of mammary cancers, and the final weights of tumors in rats on western diet compared with standard diet. Furthermore, metformin failed to decrease tumor multiplicity in either diet, in fact, it increased final cancer weights (P<.05). Serum from rats in the various groups were examined for approximately 500 metabolites (Metabolon; Research Triangle Park, NC). The Metabolites which were differentially expressed when comparing Western vs standard diets at either time point included myoinositol, scyllo-inositol, 2-hydroxydecanoate, carnitine, tocopherol, and nicotinamide. Certain of these were fatty acids and lipid soluble vitamins that one would expect to be altered by diet. We also compared serum from animals on standard or Western diets at the early or late time points to determine metabolites associated with cancer development. Metabolites whose levels changed in tumor bearing rats included 4-OH-butyrate, acetyl-carnitine, uranate, and threonate. Finally, we compared the effects of metformin treatment in rats on Western or standard diet and found that the metabolic changes associated with metformin treatment were dependent on the diet employed. Thus, metformin altered levels of arachidate and serotonin only in rats on standard diet, while carnnitine and N-delta-acetylornithine were only modulated in rats on Western diet. Again, the most striking result may be that we still failed to observe any efficacy of metformin in animals on standard or Western diet.

#825

Myc-nick promotes efferocytosis through M2 macrophage polarization.

Xiancai Zhong, Ha-Na Lee, Young-Joon Surh. _Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, Republic of Korea_.

Resolution of inflammation is an active process which is important for maintaining or restoring homeostasis in response to inflammatory insult. Failure in resolution results in persistent and chronic inflammation which increases the risk of cancer development. Efferocytosis, an essential part of resolution of inflammation, is defined as phagocytic removal of apoptotic cells, especially neutrophils, at the inflamed site, thereby preventing exposure of tissue to toxic intracellular components. It is carried out by a distinct group of phagocytes including anti-inflammatory macrophages (M2 type). Therefore, M2 macrophages represent a key component of the cellular proresolving mechanism and a potential factor for prevention of inflammation-associated cancer. Dysregulation, often overexpression, of c-Myc drives dedifferentiation of cancer cells to acquire capacity for self-renewal and metastasis, which accounts for poor prognosis in cancer patients. A cytoplasmic N-terminal part of full length c-Myc, termed Myc-nick, has been reported to promote differentiation of myoblasts. In the present study, we investigated whether Myc-nick is involved in M2 macrophage polarization and promotion of efferocytosis. We found that ectopic expression of Myc-nick in mouse bone marrow derived macrophages (BMDM) promoted the M2 macrophage polarization through upregulation of interleukin 10 (IL-10) and peroxisome proliferator activated receptor gamma (PPARɣ). Furthermore, overexpression of exogenous Myc-nick in BMDM increased capture of apoptotic cells by BMDM through upregulation of a class of phosphatidyl serine (PS) receptors including T-cell immunoglobulin and mucin domain containing 4 (Timd4). PS constitutes one of 'eat-me' signals carried by apoptotic cells. PS receptors present on the surface of macrophages are important to recognize, engulf and finally clear apoptotic cells via phagocytosis. In addition, Myc-nick-induced efferocytosis was found to be tightly associated with K (lysine) acetyltransferase 2A (Kat2a/Gcn5). This may lead to the acetylation of transcription factors that regulate expression of PS receptor proteins. In conclusion, activation of M2 macrophages by Myc-nick facilitates efferocytosis and hence resolution of inflammation, and this may suppress inflammation-associated cancer progression.

#826

Benzyl isothiocyanate inhibits breast cancer-induced osteoclastogenesis.

Subrata K. Pore, Anuradha Sehrawat, Shivendra V. Singh. _UNIVERSITY OF PITTSBURGH, Pittsburgh, PA_.

Breast cancer is a major public health issue for women worldwide and 70% of the disease preferentially metastasizes to bone resulting in its destruction. Advanced breast cancer releases osteoclastogenic factors to promote bone resorption. As bone metastasis with breast cancer is associated with high mortality, osteoclastogenesis is a potential therapeutic target. We have shown previously that benzyl isothiocyanate (BITC), which is present in edible cruciferous vegetables, inhibits breast cancer development in a transgenic mouse model. The present study was designed to determine effect of BITC on breast cancer-induced osteoclastogenesis. We employed well-established in vitro osteoclast co-culture system of Raw 264.7 murine macrophage cells with human breast cancer cells for this assessment. BITC treatment significantly inhibited the number as well as size of osteoclasts in a dose dependent manner in every co-culture experiment. In addition, BITC downregulated both mRNA and protein levels of macrophage colony stimulating factor (MCSF) and receptor activator of NF-κB ligand (RANKL) which are key factors for formation of osteoclasts. Moreover, receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation as well as bone resorption was suppressed in the presence of BITC. On the other hand, BITC induced osteoprotegerin (OPG) acting as a decoy receptor of RANKL and negatively regulated osteoclast formation. Quantitative PCR analysis revealed that mRNA levels of osteoclastogenesis-related cytokines and proteins (IL-1β, IL-6, TNF-α, GM-CSF, OPG, RUNX2) were repressed in BITC-treated breast cancer cells. Taken together, BITC showed inhibitory activity on breast cancer-mediated osteoclastogenesis. This study was supported by the grant CA129347 awarded by the National Cancer Institute.

#827

Polyphenon E treatment affects p73 mRNA levels.

L. Michael Carastro,1 Ricardo A. Cordova,1 Christopher D. Cole,1 Jong Y. Park2. 1 _University of Tampa, Tampa, FL;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Prostate cancer (PCa) is the most common malignancy among men in the USA. Polyphenon E (PolyE) is a standardized blend of polyphenols found in green tea extract, which has been shown to have chemoprevention value in PCa models, but the molecular mechanism(s) have not been elucidated. The p73 gene is a member of the p53 tumor suppressor family. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and deltaNp73 (DNp73) isoforms, respectively. These p73 mRNA isoforms, TAp73 and αNp73, encode protein isoforms which differ in their N-termini. TAp73 isoforms include the full-length N-terminal sequence and are transcriptionally active. αNp73 isoforms lack the N-terminal trans-activation domain and are dominate negative. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive PCa and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. We hypothesize that exposure to PolyE induces changes in TAp73/DNp73 mRNA isoform ratios. Our goal for this study is to assess the effect of PolyE on total p73 mRNA levels and TAp73/DNp73 mRNA isoform ratios using two human PCa cell lines with different p73 DNP genotypes: DU145 (wild-type) and PC-3 (heterozygous). Cultured cells were treated with PolyE (100 or 200 mg/L). Cellular RNA was isolated, converted to cDNA and used in TaqMan RT-PCR assays to detect total p73, TAp73 or DNp73 isoform mRNAs. Our data from PolyE treatments of both DU145 and PC-3 cells are consistent with increased p73 mRNA levels (2.05 and 1.60 fold, respectively) and higher TAp73/DNp73 mRNA ratios (1.13 and 1.29 fold, respectively) at the low Poly E level (100 mg/L). At the high PolyE level (200 mg/L), the TAp73/DNp73 mRNA ratios significantly decreased for both DU145 and PC-3 (0.54 and 0.49 respectively). However, the overall p73 mRNA levels was not changed in DU145 (0.91 fold), but increased in PC-3 (1.60 fold) as compared with level in negative control at the high Poly E concentration. Therefore, in this exploratory study, PolyE treatment of the two PCa cell lines tested using a more moderate concentration of 100 mg/L results in higher overall p73 levels and increased TAp73/DNp73 mRNA ratios. This data could provide a potential molecular rationale for the observation that Poly E treatments provide chemopreventative impact against PCa.

#828

ɑ-Lipoic acid inhibits human hepatoma cell proliferation by Grb2-mediated downregulation of EGFR activity.

Lan Yang,1 Yuntao Lin,1 Ya Wen,2 XiaoJuan Sun1. 1 _Biobank of Shenzhen Second People's Hospital, ShenZhen, China;_ 2 _Guangzhou Medical University, Guangzhou, China_.

Background: It has been reported that ɑ-Lipoic acid (ɑ-LA) as a naturally occurring anti-oxidant, suppresses the growth of hepatoma cells, but the mechanisms remains unclear.

Aims: To investigated whether ɑ-LA inhibited hepatoma cells proliferation through EGFR-Grb2.

Methods and Results: The results showed that Hu7, Bel7402 and HepG2 proliferation were dramatically decreased after ɑ-LA (2 mM) treatment for 24 and 48 hours, which were measured by CCK8 assay. Moreover, phosphorylation levels of EGFR was decreased by ɑ-LA treatment, and its associated with an inhibition in cell-cycle transition. Next, we found that growth factor receptor bound protein 2 (Grb2), which is adaptor protein, was also significantly reduced by RT-PCR and western blot after ɑ-LA treatment at 24 and 48 hours. Consistent with previous report, silenced of Grb2 dramatically blocked cell proliferation. To test whether the downregulation of Grb2 mediates ɑ-LA induced proliferation inhibition, Grb2 was transfected into hepatoma cells to recover the reduction of Grb2 induced by ɑ-LA. It was showed that overexpression of Grb2 also successfully inhibited ɑ-LA induced decrease of cell proliferation. In addition, the data showed that phosphorylation levels of ERK1/2 was decreased by cell treated with ɑ-LA. These results suggested that the Grb2 through binds to phosphorylated site in EGFR medicated ɑ-LA suppress cell proliferation.

Conclusion: Our study showed ɑ-LA inhibited cell proliferation through Grb2 suppressed phosphorylation levels of EGFR, ERK1/2 was involved in this pathway.

#829

Chemoprevention of oxidative stress-associated oral carcinogenesis by sulforaphane depends on NRF2 and the isothiocyanate moiety.

Aixian Lan,1 Wenjun Li,1 Yao Liu,1 Xinyan Zhang,1 Shanshan Zhou,2 Olesya Palko,3 Hao Chen,3 Mayanga Kapita,3 Justin Prigge,4 Edward Schmidt,4 Xin Chen,2 Zheng Sun,1 Xiaoxin Luke Chen3. 1 _Capital Medical University, Beijing, China;_ 2 _Changzhou University, Changzhou, China;_ 3 _North Carolina Central University, Durham, NC;_ 4 _Montana State University, Bozeman, MT_.

Oxidative stress is known to play an important role in oral cancer development. In this study we aimed to examine whether a chemical activator of NRF2, sulforaphane (SFN), may have chemopreventive effects on oxidative stress-associated oral carcinogenesis. We first showed that Nrf2 activation and oxidative damage were commonly seen in human samples of oral leukoplakia. With gene microarray and immunostaining, we found 4-nitroquinoline 1-oxide (4NQO) in drink activated the Nrf2 pathway and produced oxidative damage in mouse tongue. Meanwhile whole exome sequencing of mouse tongue identified mutations consistent with 4NQO's mutagenic profile. Using cultured human oral keratinocytes and 4NQO-treated mouse tongue, we found that SFN pre-treatment activated the NRF2 pathway and inhibited oxidative damage both in vitro and in vivo. On the contrary, a structural analogue of SFN without the isothiocyanate moiety did not have such effects. In a long-term chemoprevention study using wild-type and Nrf2-/- mice, we showed that topical application of SFN activated the NRF2 pathway, inhibited oxidative damage, and prevented 4NQO-induced oral carcinogenesis in an Nrf2-dependent manner. Our data clearly demonstrate that SFN has chemopreventive effects on oxidative stress-associated oral carcinogenesis, and such effects depend on Nrf2 and the isothiocyanate moiety.

#830

**Pin1 is a novel target of withaferin A, a mammary cancer chemopreventative steroidal lactone from** Withania somnifera **.**

Suman K. Samanta, Shivendra V. Singh. _University Of Pittsburgh, Pittsburgh, PA_.

We have shown previously that naturally occurring steroidal lactone withaferin A (WA) inhibits breast cancer development in a transgenic mouse model. Mammary cancer chemoprevention by WA in vivo was associated with suppression of peptidyl-prolyl isomerase Pin1 protein. Pin1 is known to regulate diverse cellular processes including cell proliferation, cell cycle progression, apoptosis, and migration. Moreover, Pin1 is overexpressed in breast tumor and its expression is positively correlated with tumor grade. Thus, we studied functional significance of Pin1 downregulation by WA. Consistent with in vivo observations, Pin1 protein and mRNA expression was decreased upon WA treatment in human breast cancer cells irrespective of ER, HER-2 or p53 status. Overexpression of Pin1 partly reversed the growth inhibitory effect of WA in both MCF-7 (wild-type p53) and SUM159 (mutant p53) cells. However, WA-induced mitotic arrest and apoptosis were partly abrogated only in MCF-7 cells upon ectopic expression of Pin1 perhaps due to p53 stabilization. Molecular docking suggested covalent binding of WA with Cys113 of Pin1, which was confirmed by mass spectrometry. Overexpression of mutant (C113A) Pin1 augmented WA-mediated inhibition of cell survival as well as mitotic arrest and apoptosis in MCF-7 cells indicating that decreased catalytic activity of Pin1 is essential for growth inhibition by WA in breast cancer cells. Taken together, the present study suggests a critical role for Pin1 in WA-mediated growth inhibition of breast cancer. This study was supported by the grant CA142604 awarded by the National Cancer Institute.

#831

c-Myc is a novel target of prostate cancer cell growth inhibition by honokiol.

Krishna B. Singh, Eun-Ryeong Hahm, Shivendra V. Singh. _University of Pittsburgh Cancer Institute, PITTSBURGH, PA_.

Honokiol (HNK) has been shown to inhibit human prostate cancer cell growth in vitro and in vivo, but the underlying mechanism is not fully understood. The present study demonstrates that c-Myc, a key mediator of prostate cancer growth, is a novel target of HNK. Exposure of prostate cancer cells to plasma achievable concentrations of HNK resulted in a marked decrease in levels of total and/or phosphorylated c-Myc. HNK-mediated suppression of c-Myc protein level was due in part to repression of its transcription or protein degradation. We also observed down-regulation of c-Myc in tumors from PC-3 xenografted mice upon oral administration of HNK when compared with control; although the difference was not significant. Stable overexpression of c-Myc conferred protection against HNK-mediated inhibition of colony formation and G0-G1 cell cycle arrest in PC-3 cells. Moreover, HNK suppressed the expression of c-Myc downstream target genes (Cyclin D1 and EZH2) and these effects were restored in Myc-overexpressing PC-3 cells. Consistent with these results, cells with stable knockdown of EZH2 were more sensitive to growth inhibition by HNK compared with control cells. Because androgen receptor (AR) is implicated in regulation of c-Myc expression, we explored the possibility whether AR could affect the expression of c-Myc and its target genes. Indeed, AR overexpression in PC-3 cells enhanced the expression of both c-Myc and its target genes. In conclusion, it is reasonable to postulate that HNK may be effective in retarding growth of c-Myc-driven prostate cancer. This study was supported by the grants CA101753 and CA115498 awarded by the National Cancer Institute.

#832

Preventing the progression of estrogen receptor-negative/HER2-overexpressing early stage breast disease by lapatinib.

Sunil Acharya,1 Jia Xu,1 Xiao Wang,1 Shalini Jain,1 Hai Wang,1 Qingling Zhang,1 Joseph Bower,1 Rebecca Richards-Kortum,2 Dihua Yu1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Rice University, Houston, TX_.

Tamoxifen and aromatase inhibitors (AIs) have shown efficacy to prevent estrogen receptor positive (ER+) breast cancer; however, there is no effective prevention strategy for ER- breast cancer. HER2 overexpression (HER2+) can be detected in up to 40% of ER- breast cancers and it has been reported to promote the progression of early stage breast cancer. Here, we tested the feasibility of lapatinib to prevent ER-/HER2+ early stage breast disease. Our data showed that lapatinib reduced the proliferation and increased the apoptosis in an in vitro ER-/HER2+ early breast disease model. Furthermore, lapatinib treatment in the HER2-driven MMTV-neu mouse model from early stage of disease development (atypia, at 10 weeks of age) significantly delayed tumor initiation and progression as detected by mammary gland biopsy and fiber optic micro-endoscope (FOME) imaging. Mechanistically, we found that lapatinib inhibited GLUT4 upregulation and increased glucose up-taking in HER2-overexpressing cells. These results suggested that lapatinib is a promising agent for the prevention/intervention of ER- and HER2+ breast cancers. Our study brings better understanding of the mechanism underlying ER-/HER2+ early stage breast disease progression to cancer and our strategy of cancer prevention with low-dose kinase inhibitor could be rapidly translated into the clinic for other cancer types in the near future.

#833

Benzyl isothiocyanate mediates glucose uptake through AKT activation in breast cancer cells.

Ruchi Roy, Shivendra V. Singh. _University of Pittsburgh, Pittsburgh, PA_.

We have shown previously that benzyl isothiocyanate (BITC) administration retards the development of mouse mammary tumors driven by Her-2 oncogene proving in vivo evidence for its chemopreventive efficacy. However, we also observed an increase in glucose uptake by tumor in BITC treatment group compared with control mice. Proteomics using tumors from control and BITC-treated mice revealed changes in proteins related to metabolism. Thus, we studied the mechanism by which BITC increases glucose uptake using four cell lines with different genetic backgrounds (e.g., ER+, ER-, HER-2+, triple negative). Consistent with in vivo findings, BITC treatment enhanced the protein levels of glucose transporters (GLUT1 and GLUT4) regardless of ER, HER-2 or p53 status. On the other hand, lactate dehydrogenase (LDH) was downregulated in response to BITC treatment. Pyruvate kinase (PKM2) as well as tricarboxylic cycle (TCA cycle) related enzymes (IDH1, PDH, MDH) were upregulated in BITC treated cells compared with control. In agreement with these results, BITC treatment increased glucose, pyruvate, and acetyl-CoA levels but decreases lactate level. These results suggested that elevated glucose after BITC treatment was largely utilized through TCA cycle. Because AKT is known to regulate tumor metabolism including glycolysis and oxidative phosphorylation, we explored its involvement in BITC-mediated metabolic alterations. To our surprise, BITC treatment resulted in a marked increase in activation of AKT. Pharmacological inhibition of AKT antagonized BITC-mediated increase in GLUT1 expression as well as increase in glucose and pyruvate levels. In addition, AKT inhibition augmented BITC-mediated inhibition of cell survival (colony formation), induction of apoptosis, and suppression of migration in human breast cancer cells. Based on these findings, we propose that mammary cancer chemopreventive efficacy of BITC may be augmented by a combination regimen involving an AKT inhibitor. This study was supported by the grantCA129347 awarded by the National Cancer Institute.

#834

Constitutive omega-3 fatty acid production in fat-1 transgenic mice protects against UVB-induced skin inflammation and carcinogenesis.

Hye-Won Yum,1 Joydeb Kumar Kundu,2 Yong-Yeon Cho,3 Young-Joon Surh1. 1 _Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, Republic of Korea;_ 2 _College of Pharmacy, Keimyung University, Daegu, Republic of Korea;_ 3 _Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, Catholic University of Korea, Bucheon, Gyeonggi-do, Republic of Korea_.

Exposure to solar radiation, especially ultraviolet B (UVB), is the most prevalent cause of skin photocarcinogenesis. Omega-6 (ω-6) and omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are known to have opposite effects on the inflammatory and carcinogenesis processes. Generally, ω-6 PUFAs promote inflammation and tumor development, whereas ω-3 PUFAs possess anti-inflammatory as well as antioxidative activity and enhance host immune response. In the present study, we investigated the protective effects of ω-3 PUFAs on UVB-induced skin inflammation and carcinogenesis using fat-1 transgenic mice harboring ω-3 desaturase gene capable of converting ω-6 to ω-3 PUFAs. The ω-3/ω-6 PUFA ratio was significantly higher in the dorsal skin of fat-1 mice than that in the wild-type (WT) C57BL/6 mice. In comparison to WT animals, fat-1 mice expressed heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1 and their mRNA transcripts to a greater extent, which was attributable to the elevated activation of nuclear factor erythroid-related factor-2 (Nrf2) responsible for upregulation of cytoprotective and stress-responsive proteins. Upon single exposure to UVB (500 mJ/cm2) irradiation, fat1 mice exhibited a significantly lower degree of skin inflammation and phototoxicity and the expression of the pro-inflammatory enzyme, cyclooxygenases-2 (COX-2) as compared to those observed in WT mouse skin. The protection of fat-1 mice from UVB-induced skin inflammation was associated with decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3), one of the major redox-sensitive transcription factors, whose overactivation is implicated in the pathogenesis of inflammation and subsequent development of skin cancer. To determine whether the elevated ω-3 PUFA level also protects against skin papillomagenesis, hairless fat-1−/− albino and fat-1+/− albino mice were generated by cross-breeding of male fat-1+/− transgenic mice with female SKH-1 hairless albino mice. After repeated UVB irradiation (180 mJ/cm2) thrice a week for 23 weeks, the incidence and the multiplicity of papillomas were markedly reduced in the fat-1+/− albino group as compared to the fat-1−/− albino group. Moreover, the expression of COX-2 and activation of STAT3 in papillomas from fat-1+/− albino mice were significantly lower than those found in UVB-irradiated fat-1−/− albino mice. Taken together, our results indicate that functional fat-1 potentiates cellular defense against UVB-induced skin inflammation and tumor development through elevation of Nrf2-mediated upregulation of cytoprotective gene expression.

#835

Comparison of direct transplantation of colon cancer cells vs. tumor xenografts using orthotopic mouse model for colon cancer progression and metastasis.

Anathbandhu Chaudhuri,1 Liying Geng,2 Jing Wang2. 1 _Stillman College, Tuscaloosa, AL;_ 2 _Eppley Cancer research, Nebraska Medical center, Omaha, NE_.

Orthotopic transplantation is the process of implantation of cancer cells or tumor xenograft tissues into the organ of origin of the cancer. This procedure enables cells to grow and proliferate in their natural microenvironment. The orthotopic transplantation model has been observed to be highly amenable to cancer cells and recapitulates the human disease with high fidelity. Orthotopic transplantation is very common in clinical cancer research using athymic nude mouse as a model system. A major advantage of the orthotopic transplantation model is its ability to study the development and progression of cancer metastasis. The identification and therapeutic validation of anti-metastatic agents is of urgent need since till date there are no effective anti-metastatic therapies available for colon cancer.

The procedure of xenograft transplant is time consuming and costly because of the involvement of more number of mice. Popular method for orthotopic transplant is to grow a subcutaneous tumor injecting human cancer cells and then transplant a small piece of xenograft to the immune deficient mouse for clinical study. In this study, we directly injected the tumor cells below the sub-serosal layer of the caecum to develop colorectal cancer bypassing the formation of xenograft and its subsequent transplantation. We compared the effectiveness in tumor progression and metastasis between the cell transplant and xenograft implant to the caecum.

GFP labelled HCT116 cells were used for this study to develop colorectal cancer. First, we injected 8 million cells subcutaneously to the athymic nude mice to generate xenograft for further transplant to the caecum. On another set of experimental animals, a total of 2 million cells were directly injected below the sub-serosal membrane to develop primary tumor on the colon. Whole body GFP imaging revealed that within a week of transplantation of cancer cells or xenografts, primary tumor starts to develop to the colon and metastasized within 4-5 weeks after implantation. In both the cases, we record a 100% of primary tumor formation with 50-60% liver and 75-80% lung metastasis. To confirm the metastasis in liver and lung we studied GAPDH expression on the tissues and conduct H&E /immunofluorescence staining. Whole body or organ GFP imaging also reveals that pancreas, kidney and associated intestinal lymph glands are severely affected by both the orthotopic transplantation methods.

In conclusion, direct sub-serosal injection of cells to develop colorectal cancer primary and metastatic deposits is cost effective and time saving and thus seems to potentially have an advantage over xenograft transplant method.

#836

Effect of voluntary running on metastasis in a mouse model of breast cancer.

Sara Mijwel,1 Helene Rundqvist,1 Carolin Lindholm,1 Carina Strell,1 Pernilla Roswall,1 Kristian Pietras,2 Randall S. Johnson,1 Arne Östman1. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _Lund University, Lund, Sweden_.

Introduction: Emerging evidence states that regular physical exercise provides a risk reduction for breast cancer by approximately 25 %. In addition, recent studies suggest that physical activity may also have an effect on recurrence and mortality. Mouse models are suitable tools to study anti-neoplastic mechanisms.

Aim: to evaluate the effect of voluntary running on tumor initiation, growth and metastasis in the Polyoma Middle T (PyMT) model of breast cancer.

Methods: PyMT mice were housed with access either to wirelessly recording running wheels or locked control wheels. Running distances and tumor volumes were recorded. At 12 weeks, mice were sacrificed. Mammary glands were histologically staged and pulmonary metastases were quantified. In a follow up study, pre-trained mice were injected intravenously with tumor cells derived from the PyMT model and after an additional 10 weeks of voluntary running, pulmonary metastases were quantified.

Results: PyMT mice ran significantly more than wildtype mice (6.4 vs 3.4 km/day). No significant effects of voluntary running on tumor- initiation, volume or stage were found. However, a trend for reduced metastasis was observed. After intravenous injections of tumor cells, runners had a significantly lower pulmonary metastasis frequency than non-runners.

Conclusion: In this aggressive breast cancer model, an average of 6 km/day of voluntary running did not induce any effect on tumor formation or growth. However, the findings suggest that physical activity has impact on the metastatic process.

#837

Reproducibility of efficacy results for chemopreventive agents in the methylnitrosourea-induced rat mammary cancer model.

Vernon E. Steele,1 Ronald A. Lubet,1 Clinton J. Grubbs2. 1 _National Cancer Institution, Bethesda, MD;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Perhaps the greatest question in biological science is: Can results be reproduced? This problem is of particular concern for a group whose primary mission is to screen for potentially effective agents and rank results by activity. The reproducibility of testing specific agents for chemopreventive activity, testing the same agent in various protocols, and testing multiple agents of a given mechanistic class in the MNU-induced model of ER+ breast cancer in Sprague-Dawley rats is presented. The model employs female virgin Sprague-Dawley rats on a standard 4% Teklad diet which develop ER+ mammary cancers (adenocarcinomas) following MNU injection at 50 days of age (DOA). Preventive agents were given by gavage or diet starting at 55 DOA, and animals monitored for tumor development until approximately 170 DOA. Seven agents which we had previously studied and which were strongly positive [tamoxifen, vorozole (an aromatase inhibitor), Targretin, gefitinib] or strongly negative (naproxen, Lipitor, metformin) were examined in the assays presented. These study results confirmed our previous data. We next tested these same agents in animals given a Western diet (high fat, low calcium) and again obtained the same results. We had thought that the altered physiology and potentially altered pharmacokinetics of rats on a Western diet might alter the observed chemopreventive efficacy. These studies show that the direct repetition of clearly positive and negative agents can be achieved and, in fact, repeated with a significant variation in diet. Importantly, in terms of reproducibility, we find that multiple agents of the same mechanistic class give similar results. Thus, various SERMs (tamoxifen, toremifene, Arzoxifene), RXR agonists (targretin, UAB-30, 4Me-UAB-30), EGFR inhibitors (gefitinib, Erlotinib, lapatinib) are all highly positive (70-95% reduction of cancer multiplicity). In contrast, the negative classes COX inhibitors (naproxen, sulindac, aspirin) or statins (atorvastatin or simvastatin) have been consistently negative (<20% reduction). Therefore, reproducibility of efficacy results appears to be quite solid in our chemoprevention agent development program for rat mammary cancer testing, and we can, with significant certainty, trust our results.

#838

Natural plant extracts induced DNA damage and apoptosis through oxidative stress in transgenic mouse model.

Clement G. Yedjou,1 Paul B. Tchounwou,1 Lucio Miele,2 Marinelle Payton1. 1 _Jackson State University, Jackson, MS;_ 2 _LSU School of Medicine, Department of Genetics, New Orleans, LA_.

One of the most frightening events that women will ever face is being diagnosed with breast cancer. One in eight women in the United States will be diagnosed with this life-threatening disease during her lifetime. Even with all the advances in pharmaceutical technology, mortality rates for breast cancer have remained stagnant for the past few decades. The development of new drugs from natural products is considered important. The specific aim of the present study was to use transgenic mouse mammary tumors as a test model to explore the therapeutic mechanisms of a novel natural product as anti-cancer agent in the treatment of breast cancer. To achieve our specific aim, we performed both in vitro and in vivo studies. Acridine orange and propidium iodide (AO/PI) staining were used to visualize live and dead cells with the means of Cellometer Vision. Tumor volume and weight were measured. Tumor histology was assessed by immunohistochemistry, and enzymatic activities were determined by spectrophotometry. The extent of DNA damage was evaluated by the Comet assay. Cell/tissue apoptosis was measured by the flow cytometry. Data obtained from the AO/PI dye assessment indicated that the tested natural product significantly reduced the number of live cells in a dose-dependent manner, showing a gradual increase in the loss of viability in treated cells. We observed tumor growth inhibition after 4 weeks of daily intraperitoneal administration of plant extracts to trangenic mice. There was a significant increase in DNA damage in treated mice compared to the control mice. Flow cytometry data showed a strong dose-response relationship between plant extracts treatment and annexin V/PI positive cells. Similarly, a statistically significant and dose-dependent increase (p< 0.05) was recorded with regard to caspase 3 activity. Data from enzyme analysis (alaninine transaminase, aspartate transaminase, and creatinine) revealed that plant extract administration is not toxic to treated mice. These results suggest that induction of cell death, DNA damage, and cell/tissue apoptosis may be involved in the therapeutic mechanisms of plant extracts in breast cancer. Collectively, the findings from this study provide convincing evidence that the tested plant extracts may represent a potential anticancer candidate against breast cancer. Research supported by NIH Grant No. NIMHD-G12MD007581) and Grant No. P20GM103476.

#839

Novel polypectomy system in a preclinical model of colon cancer.

Furkan U. Ertem,1 Wan-Mohaiza Dashwood,1 Praveen Rajendran,1 Gottumukkala Raju,2 Asif Rashid,3 Roderick Dashwood1. 1 _Texas A &M Health Science Center Houston, Houston, TX; _2 _Department of Gastroenterology, Hepatology & Nutrition, MD Anderson Cancer Center, Houston, TX; _3 _Department of Pathology, MD Anderson Cancer Center, Houston, TX_.

Colonoscopy has been used for over a decade to monitor tumor progression in live animals. However, murine models typically have not involved routine resection of entire lesions, to parallel the clinical scenario in which polyps are observed and removed during a colonoscopy examination. A polyp resection (polypectomy) protocol was developed in the polyposis in rat colon (Pirc) model. Because a major cause of morbidity in such rodent models involves blockage of the colon, polypectomy can be used as a preventive strategy, helping to prolong the lifespan of the animal. Polypectomy in murine models opens the door to longitudinal prevention studies, and determining the molecular changes in resected tumors. A means of specifying the location of each lesion within the colon also was developed in order to track individual polyps in a temporal manner. The "PLC" classification system has three components: P, polyp number from the tail-end; L, location of the polyp from the tail-end; and C, clock face orientation of the polyp within the gut lumen. In summary, we developed a novel murine polypectomy protocol that closely parallels the human clinical scenario during a screening colonoscopy. The procedure employed the Pirc model, but it is directly applicable to longitudinal studies in other murine models of colon cancer. This work was supported by P01 grant CA090890 from the National Cancer Institute, by P30 grant ES023512 from the National Institute of Environmental Health Sciences, by the John S. Dunn Foundation, and by a Chancellor's Research Initiative from Texas A&M University.

#840

The modifying effects of the extract from Okinawan sweet potato leaves in mouse colon carcinogenesis.

Saori Nakachi,1 Ai Tokeshi,1 Reika Takamatsu,1 Kazunari Arakaki,1 Masatsugu Uehara,2 Akira Iguchi,2 Junsei Taira,2 Naoki Yoshimi1. 1 _University of the Ryukyus, Okinawa, Japan;_ 2 _Okinawa National College Of Technology, Okinawa, Japan_.

A) Introduction

Okinawa is the southernmost islands in Japan. The climate is subtropical and largely different from mainland of Japan. There are various kinds of Okinawan specific plants and they have been eaten as food. Okinawa is also known as the region where has large number of centenarians. Okinawan specific plants could be the reason of longevity. Okinawan sweet potato is one of these specific plants. Since old times, Okinawan people have eaten the sweet potato leaves but this custom has not be seen in mainland of Japan. In fact, Okinawan sweet potato contains much higher polyphenols such as caffeoylquinic acid (CQA) derivatives than other vegetables. It is reported that CQA derivatives have anti-oxidative activity and anti-mutagenic activity. We examined the modifying effect of Okinawan sweet potato leave extract in two-step mouse colon carcinogenesis.

B) Experimental procedures

71 male ICR mice, 5 week-old, were divided into 5 groups. Mice in groups 1-3 were given azoxymethane (AOM ; 10 mg/kg body weight) by intraperitoneal injection once at experimental first week, and one week after AOM injection they were administrated 1.5% dextran sulfate sodium in drinking water for 7 days. Mice in group 1 were fed control diet (CE-2, CLEA Japan, Inc),and mice in groups 2 and 3 were fed diet contained Okinawan sweet potato leaves extract (200 ppm and 1000 ppm respectively). Mice in groups 4 and 5, which were not injected AOM, were fed diet contained Okinawan sweet potato leaves extract (1000 ppm), and control diet respectively. At 12 experimental week, all mice were sacrificed and the colon tissues were removed to count the number of preneoplastic lesion, aberrant crypt foci (ACF), and were histologically analyzed the nodules larger than 3mm on colonic mucosa.

C) Results

The mean number of ACF was 1.64±0.95 (/colon length cm) in group 1, 1.18±0.79 in group 2, 1.14±0.60 in group 3, respectively. In high dose treated rats (group 3), the appearance of ACF was slightly lower than that in group 1, although not significant (P=0.09). Histopathologically, the most nodules were lymphoid follicles but some showed neoplastic lesions. The neoplastic lesions were revealed as adenoma or adenocarcinoma. The number of the neoplasm was 9, 8 and 2 in groups 1, 2 and 3, respectively. The tumor incidence in group 3 was inhibited those in groups 1 and 2 (P<0.05 by Fisher exact test). No ACF and neoplastic lesions were observed in groups 4 and 5.

D) Conclusion

It was shown that Okinawan sweet potato leave extract has a possibility to prevent the development and progression of neoplasms in mouse colon carcinogenesis model.

#841

Effect of equol on hormone-dependent rat mammary carcinoma induced by ethyl methanesulphonate (EMS).

MISAKI ONO, Takako Higuchi, Mikako Takeshima, Shuji Nakano. _Nakamura Gakuen University, Fukuoka, Japan_.

Soy isoflavones such as daidzein and its bioactive metabolite equol converted from daidzein by human intestinal bacterial flora, have been proposed to reduce the risk of breast cancer incidence in Asian women probably due to higher number of the inhabitants capable of producing equol in these countries. Although several in vitro studies shows the anti-cancer activity of equol against human breast cancer cells, there have been few reports that have directly showed the preventive role of equol in the experimental animal model of mammary carcinoma. We investigated here the effects of equol on the development of breast cancer using the EMS chemically induced rat model of hormone-dependent mammary carcinoma. Female WKA rats (n=30) were equally divided into 2 groups, subsequently given EMS orally for 12 weeks and fed the CE2 diets with or without 50 mg equol/g diet throughout the experiments. All EMS-treated rats fed either diet developed ER- and/or PgR- positive hormone-dependent mammary carcinoma by 24 weeks. Compared with the equol-free diet, the diet supplemented with equol could not significantly delay the development of mammary carcinoma, showing any preventive activity on the development of EMS-induced hormone-dependent mammary carcinoma. The body weight, total EMS uptakes, and urine estradiol concentrations were not significantly different between these 2 groups. These data indicate that high dose administration of equol alone does not exert clear preventive effects on hormone-dependent EMS-induced mammary carcinogenesis, and may require other soy isoflavone components such as genistein that has previously been shown to exhibit a great synergy when combined with equol. The in vivo combination studies using genistein and equol are underway.

#842

The NALP3 inflammasome and IL-1β signaling link asbestos-induced inflammation with the development of malignant mesothelioma.

Yuwaraj Kadariya,1 Craig W. Menges,1 Jacqueline Talarchek,1 Qi Cai,1 Andres J. Klein-Szanto,1 Ralph A. Pietrofesa,2 Melpo Christofidou-Solomidou,2 Brooke T. Mossman,3 Arti Shukla,3 Joseph R. Testa1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _University of Pennsylvania Perelman School of Medicine, Philadelphia, PA;_ 3 _University of Vermont College of Medicine, Burlington, VT_.

Exposure to asbestos is causally associated with the development of malignant mesothelioma (MM), a cancer of cells lining the internal body cavity. MM is an aggressive cancer that is resistant to all current therapies. Once inhaled or ingested, asbestos causes inflammation in and around tissues that come in contact with these carcinogenic fibers. Recent studies suggest that inflammation is a major contributing factor in the development of many types of cancer, including MM. The NALP3 inflammasome, including the component ASC, is an important mediator of inflammation that senses extracellular insults, such as infection or asbestos, and activates a signaling cascade resulting in release of mature IL-1β and recruitment of inflammatory cells. To determine if inflammasome-mediated inflammation contributes to asbestos-induced MM, we chronically exposed Asc-deficient mice to asbestos and evaluated tumor incidence, latency and overall survival. Asc-deficient mice showed a significant delay in tumor onset compared to wild-type animals, suggesting that the NALP3 inflammasome plays a role in MM carcinogenesis. We also tested whether inflammation-related release of IL-1β promotes tumor development in an accelerated mouse model of asbestos-induced MM. Nf2+/-;Cdkn2a+/- mice exposed to asbestos in the presence of the IL-1β antagonist, anakinra, showed a 50% longer disease-free survival than in similarly exposed mice given vehicle control, consistent with IL-1β signaling contributing significantly to MM development. Collectively, these studies provide evidence for a link between asbestos-associated inflammation/IL-1β signaling and the development of MM; furthermore, these findings provide rationale for chemoprevention strategies targeting inflammation-related IL-1β signaling in high risk, asbestos-exposed populations.

#843

Spontaneous immortalization of human mammary epithelial cells from a woman with a germline STK11 mutation.

David Euhus,1 Dawei Bu,2 Michael Considine,1 Leslie Cope1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _University of Texas Southwest Medical Center, Dallas, TX_.

Spontaneous immortalization of benign mammary epithelial cells (MEC) in culture is very rare. Models of MEC immortalization suggest that cells escape M0 growth arrest (selection) by silencing p16 expression after

which they encounter a second growth arrest, M1 (agonescence), characterized by telomere shortening and profound genomic instability. Pre-selection, post-selection, and spontaneously immortalized MEC from a woman with a germline STK11 mutation were subjected to a comprehensive "OMICS" evaluation including miRNA, transcriptome, methylome, exome, and comparative genomic hybridization (CGH) in order to understand the molecular events associated with MEC immortalization. Principle component analysis of gene expression identified 4 significant components, the first of which showed the greatest separation between the different time points. The most notable expression changes were upregulation of FOXQ1, IL1-related genes, Ras-related genes, and fibronectin and downregulation of COL1, COL6, CK19, and CDH1. MEC gradually acquired global hypomethylation of non-CpG DNA and regional hypermethylation of CpG islands (including p16). A classifier based on methylation pattern showed that immortalized cells moved away from identity with The Cancer Genome Atlas (TCGA) benign samples towards TCGA breast cancer samples. Immortalization was associated with a modest accumulation of mutations and a modestly greater number of DNA copy number alterations than was observed in earlier passage cells but no evidence of general genomic instability. Immortal cells showed a large deletion at 10q13-15 and deletions at 10p11-15 and 17p11-13 that were associated with reduced expression of genes in those regions. Post-selection bulk cell populations are quite distinct from early passage cells, but are not direct precursors of the immortal cells as some DNA copy number alterations acquired by post-selection cells were not observed in the immortalized cells. Rather, in this instance, it is likely that post-selection cells established conditions conducive to the immortalization of a rare cell in the mix. This has implications for the development of tissue-based approaches for breast cancer risk stratification.

#844

High-resolution microbiome profiling and meta-analysis yields insight into microbial consortia associated with colorectal cancer.

James R. White,1 Julia Drewes,2 Cynthia L. Sears2. 1 _Resphera Biosciences, Baltimore, MD;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Colorectal cancer (CRC) continues to be the second leading cause of cancer-related deaths globally. While multiple environmental factors are associated with incidence of CRC, multiple studies have reported that CRC is frequently linked with an altered colonic microbiota both in terms of taxonomic composition as well as structural organization. However, differences in study design and analysis methodologies among CRC microbiome profiling studies have generated inconsistent findings. To identify bacterial taxa consistently associated with CRC, we applied a uniform microbiome analysis methodology to sequence datasets followed by meta-analysis.

Raw 16S rRNA amplicon sequence datasets from 12 studies including two new Malaysian cohorts were collected and analyzed using the Resphera Insight high-resolution taxonomic assignment protocol. Taxonomic abundances within each dataset were then assessed for significant enrichment or depletion in colorectal carcinoma samples versus controls. Alpha diversity and differences in predicted functional capacity were assessed utilizing QIIME and PICRUSt, respectively.

Several bacterial species were consistently enriched in colorectal carcinoma samples relative to healthy controls including Fusobacterium nucleatum, Fusobacterium necrophorum, Leptotrichia trevisanii, Bacteroides fragilis, Parvimonas micra, Peptostreptococcus stomatis, and Gemella morbillorum. In contrast to other studies that have generally described Fusobacteria as enriched in CRC relative to controls, we identified multiple fusobacterial members that were often not strongly linked with CRC including Fusobacterium varium and Cetobacterium somerae. Evaluating functional predictions inferred from taxonomic composition, we observed consistent enrichment of specific KEGG categories in CRC including glycosyltransferases and carbohydrate digestion and absorption.

Applying a uniform high-resolution analysis protocol for microbiome profiling enabled detection of specific species that were strongly associated with CRC across multiple studies. These findings demonstrate that higher level analyses (e.g. those at the genus or family level) may be insufficient for detection of indicator taxa in some populations.

#845

Diet stamps on bugs: early life dietary energy intake impacts gut microbiota.

Jinyu Xu,1 Jeffrey Galley,1 Michael Bailey,2 Jennifer Thomas-Ahner,1 Steven Clinton,1 Susan Olivo-Marston1. 1 _Ohio State University, Columbus, OH;_ 2 _The Research Institute at Nationwide Children's Hospital, Columbus, OH_.

The complex and dynamic interactions between diet, gut microbiota (GM) structure and function, and colon carcinogenesis are only beginning to be elucidated. We examined the colonic microbiota and aberrant crypt foci (ACF) in C57BL/6N female mice fed various dietary interventions (control, energy restricted and high-fat) provided during two phases (initiation and progression) of azoxymethane (AOM)-induced early colon carcinogenesis. During progression (wks. 22- 60), a high-fat diet enhanced ACF formation compared to a control or energy restricted diet. In contrast, energy restriction during initiation phase (wks. 3-21) enhanced ACF burden at 60 weeks, regardless of the diet in progression phase. Alterations in GM structure during the initiation phase diet were partially maintained after changing diets during the progression phase. However, diet during the progression phase had major effects on the mucosal GM that energy restriction in progression phase increased Firmicutes and reduced Bacteroidetes compared to a high-fat diet, regardless of initiation phase diet, suggesting that diet may have both transient effects as well as a lasting impact on GM composition. Integration of early life and adult dietary impacts on the colonic microbial structure and function with host molecular processes involved in colon carcinogenesis will be key to defining preventive strategies.

#845A

Novel twin method estimates genetic prevalence of breast cancer.

Stuart G. Baker. _National Cancer Institute, Bethesda, MD_.

The standard twin method of the last one hundred years uses data from fraternal and identical twins to estimate heritability, which is the fraction of the variance attributed to genetic effects under an additive genetic model. Using the same data, the latent class twin method estimates a more easily interpretable quantity, the genetic prevalence, which is the fraction persons with an outcome who are in a genetic susceptibility latent class. A key assumption is that the genetic susceptibility latent class has relatively large independent probabilities of outcome as compared with an environmental susceptibility latent class with smaller and possibly dependent probabilities of outcome. Unlike the standard twin method which assumes additive genetic inheritance, the latent class twin method also allows for autosomal recessive, autosomal dominant inheritance. The method handles survival data by treating loss-to-follow up and death from competing risk as non-informative censoring. Applying this methodology to registry data from 45,000 pairs of Nordic twins, we estimated a genetic prevalence for breast cancer of only 1%. Because BRCA1 and BRAC2 have a combined prevalence of around 1%, there are likely no other high-penetrance genes contributing to breast cancer

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Novel and Integrative Analyses of Cancer Genome Data

#846

Improving T-cell receptor clonotyping of T-cell lymphomas using hybrid-Capture and next-generation sequencing.

Etienne Mahe,1 David Mulder,2 Mark Dowar,2 Mahadeo Sukhai,3 Linh Nguyen,2 Pamela Ohashi,2 Jan Delabie,4 Tracy L. Stockley,3 Trevor Pugh,3 Suzanne Kamel-Reid3. 1 _Calgary Lab Services, Calgary, Alberta, Canada;_ 2 _University Health Network, Toronto, Ontario, Canada;_ 3 _Princess Margaret Hospital, Toronto, Ontario, Canada;_ 4 _Toronto General Hospital, Toronto, Ontario, Canada_.

Background

T-cell clonality assays (TCAs) support a variety of clinical and research interests including T-cell malignancy clone identification, minimal-residual disease (MRD) testing and T-cell receptor (TCR) repertoire characterization for the purposes of immunotherapy. Unfortunately, many TCAs are unable to interrogate alpha, beta, gamma, and delta TCR loci simultaneously. Aiming to overcome these limitations, we designed a T-cell clonality assay (the "NTRA"), capable of identifying T-cell gene rearrangements from all four TCR loci, using hybrid-capture followed by deep next-generation sequencing (NGS). A novel informatics package exploiting the Burrows Wheeler Alignment algorithm and finding of CDR3 seed sequences was also deployed. We then validated the assay using orthogonal methods and in a series of clinical T-cell malignancy specimens.

Methods

We used DNA probes for hybrid-capture of all V and J segments in the TCR loci, followed by NGS on the Illumina NextSeq 500 platform. Analytical validation used a series of 10 specimens, including 6 flow-cytometry characterized T-cell specimens of variable degrees of immunophenotypic uniformity and 4 cell-lines with known TCR rearrangements. PCR/gel electrophoresis and Sanger sequencing were used for orthogonal validation, using primer sets designed to test each specimen for all 90th-centile V-J configurations. Subsequently, 61 clinical T-cell lymphoma specimens were tested, each previously assessed for T-cell clonality using the clinical gold standard (i.e. PCR/ electrophoresis using the BIOMED-2 consensus primers for the TCR-beta (TRB) and TCR-gamma (TRG) loci).

Results

PCR confirmed the NTRA-identified V-J configurations with an area-under-the-curve (AUC) by receiver-operator characteristic (ROC) analysis of 0.91. Relative to single-strand Sanger sequencing, the NTRA CDR3 sequence results showed a ROC AUC of 0.83. In a DNA dilution series employing the results of a "clonal" specimen (a Jurkat cell line) spiked into a "polyclonal" specimen (a mononuclear peripheral blood specimen), we could identify "clonal" specimen-specific V-J combinations and error-corrected CDR3 sequences down to less than 10^-5. In the final clinical validation, the NTRA performed with a ROC AUC of 0.82 relative to the current TRB & TRG BIOMED-2 clinical assay.

Conclusions

Our novel assay can overcome the current TCR locus data-yield limitations resulting from primer-based TCAs in a single-tube. The NTRA shows comparable sensitivity and specificity relative to current standard assays and excellent performance relative to current MRD techniques when applied to clinical T-cell lymphoma specimens.

#847

Molecular characterization of breast tumor T-cell infiltration in exome datasets.

Olivier Harismendy,1 Eric D. Levy,1 Valentine Garate-Calderon,2 Brian Woo,1 Ricardo Armisen3. 1 _UCSD, La Jolla, CA;_ 2 _Universidad de Chile, Santiago, Chile;_ 3 _Pfizer, Santiago, Chile_.

Tumors are heterogeneous, containing tumor-infiltrating lymphocytes (TILs) which presence has been associated with favorable prognosis in multiple tumor types. Importantly, most of the T-cell specificity for tumor neo-antigen lies in a unique sequence of DNA (CDR3) resulting from the recombination of the V, D, and J segments of the T-cell receptor beta (TCRB clonotype). The Cancer Genome Atlas (TCGA) is the most comprehensive molecular dataset of tumors but does not include detailed information about TILs or tumor immunity in general. In order to characterize the T-cell infiltration in public cancer genomic datasets, we hypothesize that such a unique CDR3 DNA fragment can be recovered from bulk tumor genome-wide DNA or RNA sequencing and that the information can be used for clinical association studies.

We first establish the feasibility of the approach by comparing the deep CDR3 sequencing (ImmunoSeq) and CDR3 reads recovered in tumor exome data. The clonotypes were detected using three CDR3 read detection tools and results were compared to the ImmunoSeq results. Each tool identifies between 10 and 38 reads. As expected, most of the CDR3 reads are misaligned to the naïve reference genome (60% clipped). Across all three tools, we identify 26 unique clonotypes, 15 of which are detected in the ImmunoSeq experiment. IMSEQ shows the strongest overlap with the other tools (13/14 clonotypes) and with ImmunoSeq (9/14 clonotypes) showing that our strategy identifies bona fide CDR3 reads from exome datasets. We applied the approach to a cohort of 1,078 TCGA samples. We identified CDR3 reads in 473 samples which are more likely to have a higher fraction of TILs as detected by histological review (p=1.14x10-8). Using the RNA-seq datasets, 906 samples had CDR3 reads and also associated with higher TIL content (p=4.41x10-6). Tumors with CDR3 reads in both DNA and RNA datasets tend have a higher TIL fraction. A set of 66 tumors shares a unique TCRβ clonotype with however no association with tumor subtypes or HLA haplotype. When restricted to Her2+ tumors, high CDR3 read counts, but not high fraction of TILs, was associated with better overall survival (p=0.016), suggesting that CDR3 reads are predictive of anti-Her2 response. In contrast, the presence of CDR3 reads in Her2- tumors is not prognostic. A detailed analysis of 22 immune meta-gene expression signatures in these samples complements CDR3 reads information and suggests mechanisms of immune-tolerance.

Our results demonstrate the feasibility of identifying genetic evidence of T-cells in bulk tumor samples using deep sequencing, providing information about the T-cell abundance and clonal identity. This novel type of tumor immune biomarker enriches the comprehensive dataset collected through TCGA, and reveals correlations with other molecular features and clinical outcomes.

#848

Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer.

Malgorzata A. Komor,1 Annemieke C. Hiemstra,1 Thang V. Pham,2 Sander R. Piersma,2 Robert P. Sebra,3 Bo W. Han,4 Meredith Ashby,4 Beatriz Carvalho,1 Gerrit A. Meijer,1 Connie R. Jimenez,2 Remond JA Fijneman1. 1 _Netherlands Cancer Insitute, Amsterdam, Netherlands;_ 2 _VU medical center, Amsterdam, Netherlands;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 4 _Pacific Biosciences, Menlo Park, CA_.

Introduction

Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test detecting blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection.

Materials and methods

RNA and proteins were isolated from CRC cell line SW480 before and after siRNA-mediated down-modulation of the splicing machinery: SF3B1, U2AF1, and SRSF1. To identify splice variants, mRNA was sequenced (Illumina HiSeq) and analyzed. RNA-seq analysis included quality checks (FASTQ, RSeQC), reads mapping (STAR), differential gene expression (DESeq2) and differential expression of splicing isoforms between conditions (MATS). Results from the in silico analysis were validated by qRT-PCR. Proteins were analyzed by in-depth tandem mass spectrometry (QExactive). A proteogenomic data analysis pipeline was established to enrich the sequence database, against which the mass spectra are searched, with the predicted protein splice variants and identify protein isoforms. To further extend the splice-variant database, PacBio sequencing of full-length transcripts is being performed for the SW480 siSF3B1- and control-samples.

Results

Differential expression analysis on RNA and protein level proved that the knock-down experiments were performed successfully. The RNA-seq analysis revealed hundreds of mRNA splice variants, including positive controls described in literature. For example, down-modulation of SF3B1 resulted in quantitative mRNA changes of spliced isoforms of ADD3 both in RNA-seq and RT-PCR data. The proteomics experiment yielded over 6000 proteins per sample, among which a number of protein isoforms resulting from alternative splicing.

Conclusions and discussion

We established a proteogenomic pipeline for the analysis of alternative splicing and provided experimental proof of concept.

We expect, however, that the true complexity of RNA variant information remains highly underestimated. We therefore are performing PacBio long-read RNA-seq to validate our approach and to identify additional (novel) splicing events. In future studies we will apply the mRNA-seq based proteogenomic pipeline for detection of protein alterations to a series of adenomas at low- and high-risk of progression, and CRCs to validate the isoforms in a clinically relevant setting. Novel findings will be evaluated for their performance as screening markers for CRC.

This work was financially supported by KWF project nr: VU 2013-6025.

#849

Improved geometric deconvolution of bulk tumor genomic data.

Theodore Roman, Lu Xie, Russell Schwartz. _Carnegie Mellon University, Pittsburgh, PA_.

We develop new methods for improved computational deconvolution of clonal populations from heterogeneous bulk genomic data. Deconvolution methods have become an important tool in resolving intratumor heterogeneity from bulk tumor genomic data, but prevailing methods are still able only to resolve only coarse-grained approximations of true clonal substructure. We seek to improve a genomic deconvolution strategy called "simplicial complex inference" that is designed to take advantage of similarity between the clonal subpopulations of single tumors or tumor regions to better infer portraits of those clonal subpopulations and their distribution across tumor samples.

We evaluate the method through application to DNA copy-number (CN) data from a 472-tumor ovarian cancer (OV) panel and a 408-tumor lung squamous small cell carcinoma (LUSC) panel from The Cancer Genome Atlas (TCGA). We deconvolved the OV data into a mixture of six clonal models, two per data sample, each interpreted as a consensus copy number profile of one recurring progression stage found in a subset of OV samples. We analyzed the resulting models for enrichment of labels derived from RNASeq-based subtyping (Verhaak et al., 2013), revealing significant correspondence by chi-squared test between tumor subclusters identified by the deconvolution and OV subtypes (p = 0.0168). We further analyzed the clonal models to identify regions of significant amplification or loss, which we tested for enrichment of gene ontology (GO) terms and pathway terms through the DAVID toolkit. Ontological analysis showed significant enrichment for GO terms connected to genetic regulation and proliferation including "DNA binding," "DNA packing," and "protein-DNA complex" (p < 0.001). The LUSC data was partitioned into nine clonal models, three per sample. A log-rank test showed significant correspondence between mixture subclustering and survival for LUSC patients (p < 0.001). Regions of significant CN gain or loss identified in the clonal models were found by DAVID to be significantly enriched for pathway terms including "Cytokine network" (p=0.0025) and "Cytokines and Inflammatory Response" (p=0.0043). These findings suggest that more sophisticated models of clonal population structures can enhance our ability to deconvolve heterogeneous tumor genomic data and lead to better inference of clonal substructure.

#850

Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using single molecule sequencing.

Maria Nattestad,1 Karen Ng,2 Sara Goodwin,1 Timour Baslan,1 Fritz Sedlazeck,1 James Gurtowski,1 Elizabeth Hutton,1 Yogi Sundaravadanam,2 Tyler Garvin,1 Marley Alford,3 Elizabeth Tseng,4 Philipp Rescheneder,5 Jason Chin,4 Timothy Beck,6 Melissa Kramer,1 John McPherson,6 James Hicks,1 Michael C. Schatz,1 William R. McCombie1. 1 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _Bard College, Annandale-On-Hudson, NY;_ 4 _Pacific Biosciences, CA;_ 5 _University of Vienna, Vienna, Austria;_ 6 _University of California Davis, CA_.

Genomic instability is one of the hallmarks of cancer, leading to widespread copy number variations, chromosomal fusions, and other structural variations in many cancers. The breast cancer cell line SK-BR-3 is an important model for HER2+ breast cancers, which are among the most aggressive forms of the disease and affect one in five cases. Through short read sequencing, copy number arrays, and other technologies, the genome of SK-BR-3 is known to be highly rearranged with many copy number variations, including an approximately twenty-fold amplification of the HER2 oncogene, along with numerous other amplifications and deletions. However, these technologies cannot precisely characterize the nature and context of the identified genomic events and other important mutations may be missed altogether because of repeats, multi-mapping reads, and the failure to reliably anchor alignments to both sides of a variation.

To address these challenges, we have sequenced SK-BR-3 using PacBio long read technology. Using the new P6-C4 chemistry, we generated more than 70X coverage of the genome with average read lengths of 9-13kb (max: 71kb). Using Lumpy for split-read alignment analysis, as well as our novel assembly-based algorithms for finding complex variants, we have developed a detailed map of structural variations in this cell line. Taking advantage of the newly identified breakpoints and combining these with copy number assignments, we have developed an algorithm to reconstruct the mutational history of this cancer genome. From this we have characterized the amplifications of the HER2 region, discovering a complex series of nested duplications and translocations between chr17 and chr8, two of the most frequent translocation partners in primary breast cancers. We have also carried out full-length transcriptome sequencing using PacBio's Iso-Seq technology, which has revealed a number of previously unrecognized gene fusions and isoforms. Combining long-read genome and transcriptome sequencing technologies enables an in-depth analysis of how changes in the genome affect the transcriptome, including how gene fusions are created across multiple chromosomes. This analysis has established the most complete cancer reference genome available to date, and is already opening the door to applying long-read sequencing to patient samples with complex genome structures.

#851

Analysis of cancer-initiating mosaic mutation in germline samples of pediatric cancer patients by next generation sequencing.

Xiaotu Ma, Donald Yergeau, Bhavin Vadodaria, Joy Nakitandwe, John Easton, Sheila Shurtleff, Jinghui Zhang. _St Jude Children's Research Hospital, Memphis, TN_.

A cancer predisposition mutation can occur during post-zygotic stages resulting in a mosaic mutation pattern across tissues. Mosaic germline mutations such as those affecting RB1, TP53 and AKT1 are important for clinical testing. However, it is challenging to detect mosaic mutation by genome-wide sequencing approaches as a mutant allele present at a low fraction in a germline sample can be caused by either tumor-in-normal contamination or mosaicism. Here we propose a two-step analytical approach to distinguish between these two possibilities. The first involves assessing the level of tumor-in-normal contamination using all clonal somatic mutations identified through the analysis of tumor purity and chromosome ploidy. The second step determines the probability of mosaicism for a candidate variant by calculating the binomial probability of its mutant allele fraction (MAF) under the contamination hypothesis. We applied this process to 1,172 paired tumor-normal pediatric cancer samples analyzed by whole-genome or whole-exome sequencing. Six mosaic variants were identified, including three pathogenic TP53 mutations with a mutant allele fractions (MAF) ranging from 0.2 to 0.3, one RB1 mutation in a retinoblastoma case with a MAF of 0.08, one IDH1 p.R132H mutation in an acute myeloid leukemia‎ case with a MAF of 0.21, and one mosaic RYR2 p.E1690D mutation in an osteosarcoma case with a MAF of 0.11. At the same time, we were able to reject three candidate TP53 variants caused by contamination with MAFs ranging from 0.06 to 0.33. All predicted mosaic mutations were verified by ultra deep sequencing at 10,000X coverage. This experience demonstrates the feasibility of detecting germline mosaicism from whole-genome or whole-exome sequencing of paired tumor-normal samples and would caution accurate analysis of tumor-in-normal contamination when evaluating candidate germline mosaicism.

#852

Modeling the emergence of resistance to chemotherapeutics with virtual tumor.

Frances A. Brightman, Eric Fernandez, David Orrell, Christophe Chassagnole. _Physiomics Plc, Oxford, United Kingdom_.

Drug resistance is a major cause of treatment failure in cancer, and understanding and overcoming mechanisms of resistance is a key challenge in advancing cancer therapy. Although the progression from cytotoxic chemotherapy to drugs aimed at specific molecular targets has improved response rates and reduced adverse effects, in the majority of cases there is still no effective treatment for metastatic disease; resistance constrains the effectiveness of both conventional chemotherapy and targeted agents. Resistance arises from mutations in the genome of cancer cells and/or epigenetic changes. The problem is compounded by considerable intra- and inter-tumour genetic heterogeneity, dictated by the genetic background and history of each cancer cell. It is therefore not surprising that patients with apparently the same cancer can respond differently to the same treatment, and it is becoming increasingly clear that cancer should be managed through personalized medicine.

Some steps have already been taken in this direction, such as the development of CancerDR (Cancer Drug Resistance Database), but the approach requires large-scale genomic profiling, which is unlikely to be widespread in clinical practice in the immediate future. In the interim, recent studies have shown that the emergence of drug-resistant disease can at least be delayed through treatment with novel dosing regimens.

Physiomics has developed a 'Virtual Tumour' (VT) technology that can predict how a tumor will respond to drug exposure. This integrated PK/PD simulation platform can be used to optimize drug dosing and scheduling, and to design new combination therapies. The VT technology integrates pharmacokinetic and pharmacodynamic effects, and models the way individual cells behave within a tumor population. These agent-based methods are particularly suitable for modeling multiple cell populations, and representing the heterogeneity of a clinical tumor. Given the significance of cancer drug resistance, and the form that future cancer therapy is likely to take, Physiomics is actively engaged in developing personalized medicine solutions. As a first step, we have incorporated chemotherapeutic resistance into our VT platform.

The VT has been extended by the addition of a resistance module, which has been developed and calibrated using data taken from the literature. This module captures the fundamental mechanism by which resistance arises. Through a case study also derived from the literature, we demonstrate that the extended VT can be applied to model the emergence of resistance in patient-derived xenografts. Furthermore, we show that the VT can be used to identify and optimize therapeutic strategies for delaying the emergence of drug resistance.

Our enhanced VT capability represents the first step towards a ground-breaking tool for developing personalized treatment, which is set to revolutionize cancer therapy in the near future, especially for patients with resistant disease.

## CLINICAL RESEARCH:

### Biomarkers to Direct Cancer Therapy

#853

Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients.

Jennifer Bordeaux,1 Douglas Johnson,2 Jeffrey Sosman,2 Ju Young Kim,1 Christine Vaupel,1 Bashar Dabbas,1 Justin Cates,2 Jeff Hall,1 Jelveh Lameh,1 Shabnam Tangri,1 Naveen Dakappagari1. 1 _Genoptix, Inc., a Novartis Company, Carlsbad, CA;_ 2 _Vanderbilt University, Nashville, TN_.

While PD-1/L1 axis-targeted therapies have shown promising clinical responses, their use in combination therapies is associated with both benefits and safety concerns. Response rates for single-agent anti-PD-1 therapies are significantly higher in biomarker positive patients, therefore there is a need to utilize predictive diagnostics to enhance benefit-risk profiles and guide treatment decisions. To address this, we developed a novel quantitative multiplexed immunohistochemistry assay that provides objective quantitation of PD-L1 positive cells, but more importantly assesses interactions with immune cells (PD-1+ or CD8+) in formalin-fixed paraffin embedded (FFPE) clinical specimens. The clinical validity of this assay was verified in a small series of melanoma patients treated with anti-PD1 targeted therapies/agents.

FFPE melanoma tissues from patients who received anti-PD-1 therapy were fluorescently stained with a combination of anti-PD-1, PD-L1, and S100 antibodies plus DAPI or a combination of anti-CD8, PD-L1, and S100 antibodies plus DAPI. Each slide was then imaged on Vectra platform and the frequencies of biomarker positive cells (PD-L1, PD-1, and CD8) and their interaction scores were objectively evaluated using proprietary Automated Quantitative Analysis (AQUA®) algorithms. Analytical sensitivity, precision, and accuracy were established using standardized PD-L1 and PD-1 tissue control arrays composed of cell lines and lymphoid organs, while range of biomarker expression was verified on archived melanoma clinical specimens (n=30), including samples taken from melanoma patients prior to anti-PD-1 therapies (n=21).

Frequencies of PD-L1 positive cells could be accurately quantified within 1% to 100% range in predefined control cell line mixtures. PD-L1 and PD-1 measurements were highly reproducible (R2 = 0.98 and 0.97, respectively). A broad range of PD-L1 and PD-1 expression and interaction scores were observed in archival clinical specimens (n=53). In a cohort of 21 advanced melanoma patients treated with nivolumab (n=5) or pembrolizumab (n=16), the PD-1/L1 interaction score was found to reliably distinguish responders from non-responders (p = 0.03) while PD-L1 alone (p = 0.15) or CD-8 alone (p = 0.23) did not. Additionally, patients exhibiting higher PD-1/L1 interaction scores had superior response rates (78% vs. 17%, p = 0.03). Responders experienced significantly longer median progression-free survival (177 vs. 85 days, p = 0.014), and fewer deaths (22% vs 58%) compared with patients having lower PD-1/L1 interaction scores.

In terms of diagnostic utility, the PD-1/L1 multiplex test showed superior predictive power (78% Positive Predictive Value, 83% Negative Predictive Value) compared with PD-L1 expression alone. Additional studies are underway to fully establish diagnostic utility and aid in treatment guidance.

#854

Mutation load measured using a 315 gene panel predicts genome-wide mutation load.

Artur Veloso, Z. Alexander Cao, Derek Y. Chiang, Hans Bitter, Kenzie D. MacIsaac. _Novartis Institutes for Biomedical Research, Cambridge, MA_.

High tumor mutation load has been associated with better response to immunotherapy. Measuring mutation load via whole-genome DNA sequencing can be cost prohibitive and requires extensive analysis and data management. Targeted genomic, on the other hand, are an appealing alternative. However it is unclear how mutation load assessed using a sample of hundreds of genes relates to genome-wide mutation burden. Here we use mutation data from The Cancer Genome Atlas (TCGA) to investigate if the exonic mutation load in a small set of genes can be used to predict the genome-wide exonic mutation load.

Using a gene panel composed of 315 genes, we observed a strong (R=0.72) positive correlation between the total mutation burden and the gene panel mutation burden. To determine whether these genes allow high and low mutation burden samples to be accurately identified, we derived various classifiers on a training set of TCGA samples and evaluated their performance on held-out test data. High mutation load was defined as greater than 181 non-synonymous mutations. This threshold best distinguished microsatellite instable (MSI) high training set samples from MSI low and microsatellite stable (MSS) samples (95% true positive rate and 15% false positive rate).

Receiver operating characteristic (ROC) curve analysis revealed that the 315-gene panel had excellent power to discriminate high and low mutation samples, with the area under the ROC curve evaluated on held-out test data ranging from 0.85 to 0.97 across indications. Our analysis suggests that the optimal threshold for identifying high mutation load samples may vary by indication.

Our models were trained and tested using data generated by the TCGA consortium on pre-treatment patient biopsies. Direct application of our classifiers to clinical data would assume similar mutation calling sensitivity between platforms and similar underlying patient populations. To test these assumptions, we compared the mutational loads between TCGA and clinical samples within the same indication. We further performed a sensitivity analysis using a simulation-based approach which showed how deviations from these underlying assumptions would be expected to affect classification performance.

These results demonstrate the feasibility of using mutation burden in a cancer related gene panel as a biomarker for genome-wide mutation load. This approach may be useful in identifying patients more likely to respond to cancer immunotherapies, but may require development be tailored to the specific sequencing platform and patient-population of interest.

#855

p53 isoform Δ133p53β triple negative breast cancer and increased relapse with neoadjuvant taxanes.

Jean-Christophe Bourdon,1 Alexandra Diot,1 Valerie Meuray,1 Leen Slaets,2 Richard Iggo,3 Herve Bonnefoi,3 David Cameron,4 Alastair M. Thompson5. 1 _Dundee Cancer Centre, Dundee, United Kingdom;_ 2 _EORTC, Bruselles, Belgium;_ 3 _Institute Bergonie, Bordeaux, France;_ 4 _Edinburgh Cancer Centre, Edinburgh, United Kingdom;_ 5 _MD Anderson Cancer Center, Houston, TX_.

Background:

Studies of TP53 mutation, protein expression and modifications of the p53 network in breast cancer yield a complexity which has so far hindered substantive clinical progress. The effects of p53 mutation and p53 protein expression in breast cancer is dependent on the breast cancer subtype and stressors involved. Since identifying differential expression of p53 isoform mRNAs in breast cancer a decade ago, we have demonstrated associations between individual p53 isoforms (p53β, p53γ, Δ133p53α, Δ133p53β), breast cancer cell behaviour and clinical outcomes. Co-expressed combinations of isoforms may influence canonical p53 (p53α), whether mutant or wild type. Patterns of p53 isoform co-expression may hold the key to understanding p53 functionality, responses to therapy and disease behaviour in breast cancer.

Methods:

Expression of p53α, p53β, p53γ, Δ133p53α, Δ133p53β, Δ133p53γ by reverse-transcription PCR was analysed for the EORTC 10994 neoadjuvant breast cancer trial. In 469 available primary breast tumors, 223 patients had been randomised to a preoperative anthracycline regimen (fluorouracil, epirubicin, cyclophosphamide [FEC]) and 246 patients to receive a taxane based regimen (three cycles of docetaxel followed by three cycles of docetaxel + epirubicin [T-ET]). TP53 status was assessed by use of the yeast functional assay (FASAY) on biopsy samples that contained at least 20% tumour cells. Breast tumor subtypes: HER2 positive, triple-negative (TNBC), luminal-A, luminal-B (HER2 negative) and luminal-B (HER2 positive) were determined using the pathological markers recommended by the St Gallen breast cancer consensus guidelines. The primary endpoint was progression-free survival (PFS) with 9 years median follow up at the time of analysis.

Results:

All cancers expressed multiple p53 isoform mRNAs; none expressed canonical p53 mRNA (p53α) alone. There was a strong association between the p53 isoforms with 4 combinations of co-expressed p53 mRNAs predominated. Expression patterns included: (a) 285 tumours co-expressed p53α, p53β, p53γ, Δ133p53α, and Δ133p53γ; (b) 122 tumours co-expressed p53α, p53β, p53γ, Δ133p53α, Δ133p53β and Δ133p53γ (c) in 25 Δ133p53 negative tumors, p53α, p53β and p53γ were co-expressed (d) 22 p53β negative tumors co-expressed p53α, p53γ, Δ133p53α and Δ133p53γ. While no individual p53 isoform expression was associated with PFS, among the 104 TNBC expressing all p53 isoforms (pattern (b), Δ133p53β positive tumors), patients had 11.31 times greater risk of relapse when treated with T-ET than when treated with FEC (95%CI 1.91-66.99, p<0.0075).

Conclusions:

Corroborating the experimental and animal model data that p53 isoform expression, p53 mutation and p53 protein expression influence the pluripotent nature of p53, patients bearing Δ133p53β positive TNBC may benefit more from treatment with FEC than T-ET in the neoadjuvant setting.

#856

Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib.

Emma Pailler,1 Marianne Oulhen,1 Kirsty Ross,1 Fanny Billiot,1 Nathalie Auger,2 Isabelle Borger,2 Maud NgoCamus,2 Jordi Remon-Masip,2 David Planchard,2 Jean-Charles Soria,2 Benjamin Besse,2 Françoise Farace1. 1 _Gustave Roussy / INSERM U981, Villejuif, France;_ 2 _Gustave Roussy, Villejuif, France_.

The duration of clinical response is unpredictable in ALK-rearranged NSCLC patients treated by crizotinib but all patients invariably develop resistance. Using filter-adapted fluorescence in situ hybridization (FA-FISH) we previously reported ALK-rearrangement detection in circulating tumor cells (CTCs) from ALK-rearranged patients. Here we report the monitoring of ALK-rearranged (≥1x3'/5', ≥1x(3' and 5')) and ALK-amplified (>2x3'/5') CTCs at baseline and at an early time point under crizotinib therapy in an extended cohort of ALK-rearranged patients. The correlation between CTC subsets harboring distinct FISH patterns and clinical parameters including progression-free survival (PFS) and overall survival (OS) is presented.

Forty ALK-rearranged patients were recruited. Blood samples were collected at baseline to crizotinib and at 1-3 months. Abnormal ALK-FISH patterns were examined in CTCs using immunofluorescence staining (DAPI/CD45) combined with FA-FISH after isolation by size of epithelial tumor cells (ISET) filtration. CTCs were defined into five distinct subsets according to the number of FISH 'break-apart' signals (3' and 5') or isolated red signals (3') and/or native copies (3'/5'). All ALK-abnormal cells were validated by a cytogenetician. Clinical data was collected retrospectively.

Confirming our previous data, ALK rearrangement was detected in CTCs: 32/39 patients (82%) had ≥4 ALK-rearranged CTCs per 1 ml of blood. Age >60 years and smoking status >15 pack years were associated with a higher mean number of ALK-amplified CTCs (p=0.0423 and p=0.0407 respectively). No statistically significant correlation was observed between the different CTC subsets with abnormal FISH patterns at baseline and PFS or OS. However we observed a statistically significant correlation (p=0.0196) between the evolution under crizotinib of the numbers of ALK-amplified CTCs and PFS. An increase in the numbers of ALK-amplified CTCs during treatment is associated with a PFS of 6.5 months, while a decrease in this subset is associated with a PFS of 25 months. There was no corresponding correlation with OS.

The evolution under crizotinib therapy of numbers of ALK-amplified CTCs is significantly correlated with PFS. Although not dominant, amplification of the ALK gene has been reported to be one mechanism of acquired resistance to crizotinib therapy in tumor biopsies. CTCs with an amplification of ALK do not express the oncogenic ALK fusion protein and are therefore not targeted by crizotinib. FISH patterns suggested these CTCs may have a high degree of ploidy and chromosomal instability which may contribute to promote the emergence of CTC subclones with a high metastatic potential. Our data suggest that number of ALK-amplified CTCs may be a predictive biomarker allowing prediction of ALK-rearranged NSCLC patients who are at risk of early resistance to crizotinib.

#857

Diagnostic test system for sensitive, specific and reproducible detection of EML4-ALK RNA fusion transcripts in the blood of patients with NSCLC.

Hestia Mellert,1 Leisa Jackson,1 Dianna Maar,2 Dawne Shelton,2 Gary Pestano1. 1 _Biodesix, Inc., Boulder, CO;_ 2 _BioRad Digital Biology Center, Pleasanton, CA_.

Clinical testing for the detection of RNA fusions in tissue currently include FISH, IHC, PCR and NGS. However, approximately 30% of patients with advanced non-small cell lung cancer (NSCLC) are not candidates for tissue biopsies and in some cases where tissue is obtained, it is not always of sufficient quantity for molecular testing. For these reasons, the detection of nucleic acids in circulation has become increasingly relevant to clinical testing. In this study, we have focused on the development of an EML4-ALK diagnostic test system that includes the prospective collection of whole blood and the reproducible detection of mRNA fusion transcripts by a PCR-based technology. The focus was on the detection of EML4-ALK transcripts from donors with and without previously diagnosed NSCLC. Pre-analytic complexity was reduced by restricting the handling of samples to 72 hours from time of sample receipt to test result. Recovery methods for RNA extraction from donor plasma were then optimized to enrich for RNA recovered from both circulating-free, and RNA within blood vesicles, including platelets and exosomes. A two-step reverse transcription and Droplet Digital™ PCR (RT-ddPCR) method was evaluated in detection testing using a multiplexed EML4-ALK assay that includes variants 1, 2 and 3. Analytic assay specificity and sensitivity was examined for RT-ddPCR efficiency using a cell-line positive for EML4-ALK variant 1 and in vitro RNA designed to mimic variants 1, 2, and 3. Analytic sensitivity was determined to be 0.2% of fusion RNA spiked into a background of normal plasma. Precision studies were conducted with two different amounts of input RNA (high and low), over three consecutive days, three runs in one day and with two operators. The SD of detected fusion transcripts in this study did not exceed 25%. Finally, normal (n = 10) and FISH positive NSCLC donor (n = 9) plasma samples were processed for the recovery of RNA and tested for EML4-ALK fusions. All FISH positive cases were accurately detected by the RT-ddPCR test with EML4-ALK variant copies ranging from 13 to 150 copies. EML4-ALK fusion transcripts were not identified in normal donor plasma. We conclude that the developed test system is highly suited for a 72 hour test to result turnaround, reproducible and sensitive detection of diagnostic fusion RNA variants, including EML4-ALK in blood in the clinical laboratory.

#858

Combining sensitivity markers to identify triple-negative breast cancer patients most responsive to veliparib/carboplatin: results from the I-SPY 2 TRIAL.

Denise M. Wolf,1 Christina Yau,1 Ashish Sanil,2 Lamorna Brown-Swigart,1 Gillian Hirst,1 Meredith Buxton,1 Melissa Paoloni,3 I-SPY 2 TRIAL Investigators, Olufunmilayo Olopade,4 Hope Rugo,1 Angela De Michele,5 Fraser Symmans,6 Don Berry,2 Laura Esserman,1 Laura van 't Veer1. 1 _University of California, San Francisco, CA;_ 2 _Berry Consultants, LLC, Austin, TX;_ 3 _QuantumLeap Healthcare Collaborative, San Francisco, CA;_ 4 _University of Chicago, Chicago, IL;_ 5 _University of Pennsylvania, Philadelphia, PA;_ 6 _University of Texas, MD Anderson, Houston, TX_.

BACKGROUND: In the I-SPY 2 TRIAL, HER2-negative patients received standard chemotherapy alone or with the PARP inhibitor veliparib and carboplatin (VC). VC graduated in the triple-negative (TN) subtype, and we've previously shown that MammaPrint High1/High2 (MP1/2) risk class and the PARPi-7 signature may predict VC response. Here we evaluate whether combining these signatures identifies a subset of TN patients especially likely to respond to VC.

METHODS: This analysis includes 60 TN patients (VC: 39 and controls: 21). PARPi-7 and MP1/2 signature scores are computed from Agilent 44K arrays. We further stratify TN patients by VC-sensitivity biomarkers (MP2, PARPi7-high). We use Bayesian modeling to estimate pCR rates in each arm and the predictive probability of VC demonstrating superiority to control in a 1:1 randomized phase 3 trial of 300 'biomarker-positive' patients. Our study is exploratory and does not adjust for multiplicities of biomarkers outside this study.

RESULTS: Though 90% of TNs are PARPi7-high or MP2, concordance between these biomarkers is 50%. The estimated pCR rates to VC are 69% in PARPi7-high and 64% in MP2 TN patients, compared to 53% in the entire TN subgroup. TN patients positive for both sensitivity markers (assessed as PARPi7-high and MP2) achieved an estimated pCR rate of 79% in the VC arm vs. 23% in the control arm, with a predictive probability of success in phase 3 of 99.6%. In contrast, TN patients negative for at least one VC sensitivity marker (PARPi7-low and/or MP1) only had an estimated response rate to VC of 35%.

Estimated pCR rates in biomarker subsets

---

Biomarker subsets within TN | Estimated pCR rate in V/C [95% CI] | Estimated pCR rate in controls [95% CI] | Predictive probability of phase 3 success (300 pt)

Unselected TN (n=60) | 53% [39 - 67] | 27% [13 - 43] | 0.904

PARPi7-high and MP2 (n=24; 40%) | 79% [60 - 93] | 23% [5.8 - 49] | 0.996

PARPi7-low and/or MP1 (n=36; 60%) | 35% [17 - 55] | 29% [13 - 49] | 0.386

CONCLUSION: Our analysis suggests TN patients who are also MP2 and PARPi7-high may be more sensitive to V/C than patients with fewer markers in the 'sensitive' state.

#859

Gene and pathway differences between MammaPrint High1/High2 risk classes: results from the I-SPY 2 TRIAL in breast cancer.

Denise M. Wolf,1 Christina Yau,1 Lamorna Brown-Swigart,1 Gillian Hirst,1 Meredith Buxton,1 Melissa Paoloni,2 I-SPY 2 TRIAL Investigators, Olufunmilayo Olopade,3 Angela De Michele,4 Fraser Symmans,5 Hope Rugo,1 Don Berry,6 Laura Esserman,1 Laura van 't Veer1. 1 _University of California, San Francisco, CA;_ 2 _QuantumLeap Healthcare Collaborative, San Francisco, CA;_ 3 _University of Chicago, Chicago, IL;_ 4 _University of Pennsylvania, Philadelphia, PA;_ 5 _University of Texas, MD Anderson, Houston, TX;_ 6 _Berry Consultants, LLC, Austin, TX_.

BACKGROUND: Further stratification of the 70-gene MammaPrint(TM) prognostic signature into 'high' and 'ultra-high' risk groups may help predict chemo-sensitivity. In I-SPY 2, patients were classified as MammaPrint High1 (MP1) or MammaPrint (ultra) High2 (MP2), using a threshold predefined by the median cut-point of I-SPY 1 participants who fit the eligibility criteria for I-SPY 2. MP1/2 classification was added to HR and HER2 to define the subtypes used in the I-SPY 2 adaptive randomization engine. The first two experimental agents/combinations to graduate from I-SPY 2 were veliparib/carboplatin (V/C) in the TN subset, and neratinib (N) in the HR-HER2+ subset. MP2 was found to be a sensitivity marker for V/C but not N, whereas MP1 class appears associated with resistance to N within the HER2- subset. Despite these associations with response, it remains unclear whether MP1/MP2 classification represents differences in tumor biology. Here, we present exploratory analysis to elucidate the pathway differences between the MP classes.

METHODS: 263 patients (V/C: 71, N: 115, and controls: 77) with pre-treatment Agilent 44K microarrays and MP1/2 class assessments were considered in this analysis. To identify signature genes associated with MP1 vs. MP2 class, we (1) apply a Wilcoxon rank sum test and (2) fit a logistic model. P-values are corrected for multiple comparisons using the Benjamini-Hochberg (BH) method, with a significance threshold of BH p<0.05 from both tests. We then perform pathway enrichment analysis using DAVID. In addition, we perform multivariate analysis adjusting for receptor subtype. Our study is exploratory and does not adjust for multiplicities of other biomarkers in the trial but outside this study.

RESULTS: 63% (165/263) of patients are MP1 class and 37% (98/263) MP2. MP1/2 class is associated with receptor subtype (Fisher's exact test: p<2E-16), where 71% of TN patients are MP2 and 96% of HR+HER2+ patients are MP1. Of the 70 signature genes, 86% (60/70) differ in expression between MP1 and MP2, with 70% (42/60) expressed at a higher level in MP2, including CDCA7, MELK and CENPA. In a whole transcriptome analysis, 10,500 genes (of ~30,000) appear differentially expressed. Following adjustment for HR and HER2 status, 4368 genes are significantly differentially expressed between MP1 and MP2. By DAVID enrichment analysis, the biggest pathway-level differences are found in cell cycle, proliferation, and DNA repair, with the MP2 set showing higher expression.

CONCLUSION: MP2 class appears associated with higher expression of cell cycle & DNA repair genes. Association between MP2 class and response to V/C suggests that higher cell cycle activity may contribute to V/C sensitivity.

## ENDOCRINOLOGY:

### Molecular Pharmacology of Hormone-dependent Malignancies

#860

ESR1 coregulator binding inhibitor (ECBI): a novel agent for treating hormone therapy-resistant breast cancer.

Ratna K. Vadlamudi,1 Gangadhara Reddy Sareddy,1 Suryavathi Viswanadhapalli,1 Tae-Kyung Lee,2 Shi-Hong Ma,3 Wan Ru Lee,3 Monica Mann,1 Samaya Rajeshwari Krishnan,1 Vijay Gonugunta,3 Yang Liu,2 Douglas W. Strand,3 Rajeshwar Rao Tekmal,1 Jung-Mo Ahn,2 Ganesh V. Raj3. 1 _UT Health Science Center at San Antonio, San Antonio, TX;_ 2 _UT Dallas, Dallas, TX;_ 3 _UTSW, Dallas, TX_.

Background: Estrogen contribute to the progression of breast cancer via estrogen receptor 1 (ESR1) and current therapies involve either antiestrogens (AE) or aromatase inhibitors (AI). However, most patients develop resistance to these drugs. In resistant tumors, activation of ESR1 in the absence of ligand or mutations in ESR1 allow interaction between the ESR1 and coregulators leading to sustained ESR1 signaling and proliferation. Here we, developed a novel ESR1 coregulator binding inhibitor (ECBI) that targets persistent ESR1 signaling that commonly occur in therapy resistant breast tumors.

Methods: Using rational design, we synthesized and evaluated a small organic molecule (ECBI) that mimics the ESR1 coregulator nuclear receptor box motif. Mechanistic studies were conducted using reporter gene assays, RT-qPCR., ChIP, and RNA-Seq analysis. Xenografts and patient derived tumors were used for preclinical evaluation and toxicity.

Results: In estrogen induced proliferation assays using several ESR1+ve model cells, ECBI significantly inhibited growth and promoted apoptosis. Importantly, ECBI showed little or no activity on ESR1 negative cells. Further, ECBI also reduced the proliferation of several ESR1 positive hormonal therapy resistant cells. Mechanistic studies showed that ECBI interacts with ESR1, efficiently blocks ESR1 interactions with coregulators and reduces the ESR1 driven ERE reporter gene activity. Further, ECBI directly interacted with mutant-ESR1 with high affinity and significantly inhibited mutant-ESR1 driven oncogenic activity. RNA sequencing analysis revealed that ECBI blocks multiple ESR1 driven pathways, likely representing the ability of a single ECBI compound to block multiple ESR1-coregulator interactions. Treatment of ESR1-positive and therapy resistant as well as syngeneic xenograft tumors with ECBI (10 mg/kg/day/oral) significantly reduced the tumor volume compared to control. Using human primary breast tissue ex vivo cultures, we have provided evidence that ECBI has potential to dramatically reduce proliferation of human breast tumors.

Conclusions: The ECBI is a novel agent that targets ESR1 with a unique mechanism of action. ECBI has distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Remarkably, ECBI block both native and mutant forms of ESR1 and have activity against therapy resistant breast cancer cell proliferation both in vitro and in vivo and against primary human tumor tissues ex vivo. This first-in-class agent with its novel mechanism of action overcomes the limitations of current therapies.

#861

Global transcription factor repression by the coactivator SRC-1 mediates disease progression in endocrine-resistant breast cancer.

Damir Vareslija,1 Elspeth Ward,1 Ailis Fagan,1 William Gallagher,2 Arnold DK Hill,1 Leonie S. Young1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _UCD, Dublin, Ireland_.

Despite the clinical utility of endocrine therapies to treat ER positive breast cancer, 40% of patients eventually develop resistance, leading to disease progression. Steroid receptor co-activators have been described as "the powerhouses of transcription" with a role in mediating hormone receptor responsiveness. There is now substantial evidence that steroid receptor coactivator-1 (SRC-1) is central to the ability of endocrine tumours to adapt and overcome targeted therapy thereby facilitating metastatic disease progression. Recent work highlighted the potential ability of SRC-1 to also act as transcriptional repressor that can bi-directionally regulate genes that help it promote the resistant phenotype.

This study employed a genome-wide multi-omics sequencing approach to determine the SRC-1 repression signature in endocrine resistance and elucidate the mechanism by which SRC-1 mediates this repression.

RNA-sequencing was used to investigate the transcriptional network affected by SRC-1 in an endocrine resistant cell line model. This approach identified 1,495 genes significantly downregulated by SRC-1, with common functional pathways such as differentiation, cell morphogenesis and extracellular matrix enriched in the gene set. Parallel global methylation sequencing analysis revealed methylation as a possible mechanism by which SRC-1 was repressing target genes, which was confirmed with mechanistic studies utilising 5-AZA-2'-deoxycitidine. Through combined analysis of our global sequencing data we identified a SRC-1 regulated transcriptional hub of differentiation genes, that have protective roles in normal and treatment- sensitive breast cancer cells and which associate with poor disease-free survival in breast cancer patients. Furthermore, our work demonstrated that SRC-1 can re-programme cells to become poorly differentiated by supressing this normally protective transcriptional hub, and thereby promote aggressive endocrine resistant phenotype.

Here we report a novel mechanism by which SRC-1 can drive endocrine resistant tumorigenesis. Our genome-wide discovery approach revealed an epigenetic re-programming pathway, whereby concerted differential DNA methylation is potentiated by the activation of SRC-1 in the endocrine resistant setting. This study suggests that therapeutic strategies of combined targeted epigenetic therapy with estrogen deprivation could be successful strategy to prevent acquired resistance to endocrine therapy.

#862

WNT4 mediates endocrine response and resistance in invasive lobular carcinoma cell lines and patient tumor explants.

Matthew J. Sikora,1 Courtney L. Andersen,1 Caroline M. Alexander,2 Priscilla M. McAuliffe,1 Steffi Oesterreich1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _University of Wisconsin, Madison, WI_.

Invasive lobular carcinoma (ILC) is a breast cancer subtype affecting ~30,000 U.S. women annually. Over 90% of ILC are estrogen receptor (ER)-positive; however, endocrine therapy may have poorer efficacy in a subset of ILC patients versus invasive ductal carcinoma (IDC) patients. This prompted us to assess global ER activity in ILC cell lines MDA MB 134VI (MM134) and SUM44PE (44PE) to identify novel mediators of ER signaling. These analyses identified the Wnt ligand WNT4 as an ILC-specific ER target gene, with an ILC-specific ER binding site (ERBS) at the WNT4 locus. Considering the critical role of WNT4 in normal mammary gland expansion, we hypothesize that ILC cells utilize WNT4 signaling to drive endocrine response and resistance.

We assessed whether WNT4 is necessary for ILC cell growth using siRNA. WNT4 knockdown completely blocked estrogen-induced growth in ILC cells but not IDC cells. In parallel, the WNT4 ERBS was only occupied in ILC cells in response to estrogen, but progesterone-induced WNT4 in IDC was not associated with this ERBS. This suggests that, via the ILC-specific WNT4 ERBS, ILC cells drive estrogen-regulated proliferation by hijacking a developmental Wnt pathway. Wnt pathways typically activate β-catenin; however, we observed β-catenin dysfunction in ILC cells and that WNT4 cannot activate β-catenin. Thus, WNT4 signals in ILC cells via a novel non-canonical pathway.

Using long-term estrogen-deprived (LTED) variants of MM134 and 44PE (4 and 2 lines, respectively), we assessed WNT4 in ILC endocrine resistance. WNT4 is over-expressed, but uncoupled from ER, in all MM134-LTED. Conversely, WNT4 is reduced in 44PE-LTED but remains ER-regulated; ER occupies the WNT4 ERBS only in 44PE-LTED cells and not MM134-LTED. Using siRNA, MM134-LTED (high WNT4) are growth-inhibited by WNT4 knockdown, while 44PE-LTED (low WNT4) are insensitive. However, WNT4 knockdown sensitizes 44PE-LTED to endocrine therapy. Taken together, uncoupling and upregulating WNT4 or WNT4/ER cross-talk may represent convergent endocrine resistance mechanisms in ILC.

To assess the role of WNT4 in patient ILC, we examined WNT4 protein in archival breast tumors and observed that WNT4 is frequently expressed in ILC and IDC tumors. We also examined WNT4 regulation and endocrine response in patient tumor explants. We observed ER regulation of WNT4 directly in ILC tissues that correlated with sensitivity to fulvestrant but resistance to tamoxifen; this may mimic clinical endocrine resistance. Ongoing studies are assessing WNT4 as a biomarker and mediator of endocrine resistance in ILC.

Clinical observations suggest that ER regulates unique pathways in ILC. We identified WNT4 as a downstream effector of endocrine signaling in ILC, with critical roles in both estrogen-induced growth and endocrine resistance. WNT4 signaling may represent a novel target to modulate endocrine response specifically for ILC patients.

#863

Differential activity and SERD sensitivity of clinical ESR1 mutations.

Weiyi Toy,1 Hazel Weir,2 Pedram Razavi,1 Michael Berger,1 Wai Lin Wong,1 Elisa De Stanchina,1 Jośe Baselga,1 Sarat Chandarlapaty1. 1 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 2 _AstraZeneca UK Limited, London, United Kingdom_.

Background: Mutations in the ligand binding domain of ESR1 have been identified as recurrent alterations in ER+ metastatic breast cancer patients previously treated with aromatase inhibitors. Selective estrogen receptor degraders (SERDs) such as fulvestrant have been speculated to be a rational therapeutic approach to inhibiting these mutants by promoting receptor inhibition and degradation. Whether such drugs can potently and durably inhibit the distinct ESR1 LBD mutants both in vitro and in vivo has not been well characterized. In this study, we investigated the activities of different SERDs against a set of ESR1 LBD mutants identified in the clinic.

Methods: The diversity of ESR1 LBD mutations was analyzed by next generation sequencing of metastatic breast tumors from over 900 patients treated at MSKCC. Laboratory models of these different mutants were generated to interrogate their activities and drug sensitivity. We utilized in vitro reporters of ER driven transcription, proliferation assays, and both cell line derived and patient derived models to characterize the different mutants.

Results: In addition to previously characterized mutations, D538G, Y537S/N/C, L536R, and E380Q, we identified a number of novel ESR1 LBD mutations in this series such as S432L, L469V, and Y537D among others. We found that while most ESR1 LBD mutations promoted estrogen independent ER function, some did not. We also found that different activating ER mutants had significant differences in the degree to which they induced estrogen independent signaling. Consistent with these findings, we found that while most SERDs could antagonize all of the mutant receptors, there were significant differences in potency. Whereas certain ESR1 mutants such as E380Q were inhibited at similar concentrations as wild type receptors, other mutants such as Y537S required higher concentrations. These differences appeared to impact the in vivo efficacy of the FDA approved SERD, fulvestrant, which is known to have poor pharmacokinetic properties. However, a SERD with high bioavailability and potency, AZD9496, was found to be sufficient to fully inhibit mutant driven tumor growth from WT, D538G and Y537S expressing tumors in vivo.

Conclusions: Altogether, the data suggest that clinical ESR1 LBD mutations have distinct effects in activating the receptor and differentially impact the efficacy of ER antagonists. In order to be broadly effective against different ESR1 LBD mutants, SERDs may require highly optimized pharmacokinetic properties.

#864

Androgen receptor stability in prostate cancer is regulated by the cochaperone Bag-1L.

Laura Cato,1 Antje Neeb,2 Adam Sharp,3 Scott Ficarro,4 Victor Buzon,5 Xavier Salvatella,5 Jarrod A. Marto,4 Johann S. de Bono,3 Andrew C. Cato,2 Myles Brown1. 1 _Dana-Farber Cancer Institute and the Center for Functional Cancer Epigenetics, Boston, MA;_ 2 _Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany;_ 3 _The Institute of Cancer Research and the Royal Marsden Hospital, Sutton, United Kingdom;_ 4 _Dana-Farber Cancer Institute and the Blais Proteomics Center, Boston, MA;_ 5 _Institute for Research in Biomedicine and the Barcelona Institute of Science and Technology, Barcelona, Spain_.

The androgen receptor (AR) is an important determinant of normal and malignant prostate growth and its function is usually regulated by circulating levels of androgens, which act by binding to the ligand-binding domain (LBD) of the receptor. Currently available therapeutic intervention in prostate cancer concentrates on reducing the androgen-mediated activation of the receptor by either blocking the production of androgens or by competing with endogenous androgens for the ligand-binding pocket. However, these treatments are often palliative as almost all patients eventually develop castration-resistant prostate cancer (CRPC). Therefore a great need exists to discover alternative modes of AR inhibition, outside of targeting the LBD. Factors that control AR stability and receptor turnover may constitute new regulatory targets for inhibiting AR action.

Bag-1L is a nuclear-resident cochaperone with the ability to control AR transactivation function by directly interacting with the receptor. Here we show that a conserved domain (BAG) within the C-terminus of Bag-1L induces significant structural changes within the intrinsically disordered N-terminal domain of the AR upon binding the receptor. A consequence of this may be increased accessibility of either domain for additional protein interactions. Using a mass spectrometry approach involving the combination of tandem affinity purification (TAP) and rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) we could show that Bag-1L, besides its ability to interact with AR, forms a complex involving the E3 ubiquitin-protein ligase CHIP and its known interaction partner Hsp70/Hsc70. Both proteins are involved in the folding and proteolytic turnover of AR, implying a role of Bag-1L in both processes. To further test this, we investigated the function of Bag-1L in human patient samples and in prostate cancer cell lines. We observed that nuclear Bag-1 protein levels are increased from hormone naïve to CRPC status in prostate cancer patients and this correlates with an increase in AR protein level. TALEN-mediated loss of Bag-1L in a CRPC cell line model leads to a stabilization of the AR protein, yet a drastic reduction in genome-wide AR binding and about a 50% decrease in AR-regulated target genes critical for cell proliferation and migration. Correspondingly, we observed a significant reduction in prostate cancer growth, which could be rescued when Bag-1L was re-expressed. Combined these results demonstrate the importance of Bag-1L for AR stability and function, and the potential of this protein for the therapeutic intervention in prostate cancer.

#865

Isolation of WDR77-mediated interaction between androgen receptor and p53 uncovers novel treatment strategy for prostate cancer.

Sangeeta Kumari,1 Simon Schlanger,1 Dan Wang,2 Song Liu,2 Hannelore Heemers1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

The ~30,000 prostate cancer (CaP) deaths in the United States annually are due to failure of androgen deprivation therapy (ADT). ADT prevents ligand-activation of androgen receptor (AR), which is the main target for treatment of non-organ-confined CaP. ADT fails while CaP remains dependent on AR. Recent NextGen sequencing approaches on ADT-recurrent CaP specimens have confirmed AR amplification, and emergence of gain-of-function AR mutations and forms of AR that do not require ligand-activation. The same sequencing studies also identified a striking enrichment in gain-of-function p53 mutations during ADT. AR and p53 have been recognized as the 2 major genomic drivers of lethal CaP progression, do not interact directly and are not targeted efficiently for therapy in ADT-recurrent CaP. Recently, using a customized gene expression oligoarray, we defined the contribution of 18 clinically relevant coregulators to androgen regulation of 452 AR target genes. Coregulator contribution to AR action was highly gene-specific and context-dependent. Selectivity in dependence of AR target genes on individual coregulators was reflected in the composition of associated AR binding sites, as well as in CaP cell biology and clinical CaP progression that was controlled by these AR target genes. The existence of diverse AR-coregulator-transcription factor (TF) transcriptional codes was evidenced by WDR77-dependent interaction between AR and p53. In this novel AR-, p53- and WDR77-dependent transcriptional mechanism, the coregulator WDR77 physically and functionally bridged the action of AR and p53. WDR77 controlled selectively androgen regulation of 96 AR target genes that are associated with aggressive CaP and that mediate cell death, DNA replication, recombination and repair. Using Cistrome project tools, binding motifs for p53 were found close to AR binding sites in WDR77-dependent AR target genes, and ChIP validated androgen-dependent recruitment of p53 and WDR77 to those genomic regions. Genome-wide gene expression profiling confirmed significant overlap (n=262) in p53- and WDR77-dependent androgen-responsive genes in CaP cells. Co-immunoprecipitation and mammalian-2-hybrid assays demonstrated p53 and WDR77 interaction, and delineated the p53-WDR77 interacting domains. In conclusion, WDR77-dependent interaction between AR and p53 provides the rationale for a novel CaP treatment strategy, namely disruption of functional interaction(s) between AR and TFs, such as p53, that control the CaP biology responsible for lethal disease progression.

#866

Delineation of novel CYP24A1 transcriptional regulators.

Wei Luo, Yingyu Ma, Mikhail Chernov, Donald L. Trump, Candace S. Johnson. _Roswell Park Cancer Institute, Buffalo, NY_.

The anti-proliferative and pro-apoptotic effects of 1α,25 dihydroxycholecalciferol (1,25D3) are well-documented in various tumor model systems in vitro and in vivo. However, limited anti-tumor effects of 1,25D3 have been observed in clinical trials. One potential reason may be the over-expression of CYP24A1, the primary 1,25D3 degrading enzyme, in tumors, and the subsequent rapid local inactivation of 1,25D3. A CYP24A1 inhibitor might be added to improve the anti-tumor activity of 1,25D3. However, most CYP24A1 inhibitors are non-specific, and their use is accompanied by a striking increase in the CYP24A1 expression level. In this study, we screened a small molecule library to identify novel CYP24A1 transcriptional inhibitors using a luciferase reporter assay in a human prostate cancer PC3 cell line stably expressing CYP24A1 promoter-driving luciferase reporter. Screening of DIVERSet™, a diverse library of 55,230 compounds, resulted in the identification of 150 hits, each of which had over 50% inhibition of 1,25D3-induced CYP24A1 promoter activity. After confirming initial observations, four hits (CPI3, CPI8, CPI11 and CPI17) displaying the strongest inhibitory effect were chosen to further examine their effects on endogenous and 1,25D3-regulated CYP24A1 expression in cancer cells. Prostate cancer (PC3) cells, bladder cancer (RT112, RT112/D21) cells, kidney cancer (A498, ACHN and 786-O) cells were treated with CPI3, CPI8, CPI11 and CPI17 alone or followed by 100 nM of 1,25D3. qRT-PCR analysis showed that 1,25D3 induced CYP24A1 mRNA expression in these cancer cells and the induction of CYP24A1 was significantly inhibited by the treatment with CPI8 and CPI17 (P < 0.01). Western blot showed the reduction of 1,25D3-induced CYP24A1 protein expression in CPI8 and CPI17 treated cancer cells. These results indicate that CPI8 and CPI17 inhibit CYP24A1 expression at the transcriptional level. To investigate whether these compounds affect the transcriptional expression of vitamin D receptor (VDR) and other vitamin D target genes, the expression of VDR and p21Waf1 were measured by qRT-PCR in kidney cancer A498, ACHN and 786-O cells treated with CPI8, which displayed the strongest inhibitory effect on CYP24A1 expression. Treatment with CPI8 did not affect VDR expression in kidney cancer cells. In contrast with the reduction of CYP24A1 expression by CPI8, treatment with CPI8 markedly increased p21Waf1 mRNA and protein expression compared to 1,25D3 alone. These results indicate that CPI8 differentially affects the expression of vitamin D target genes. In summary, we identified novel CYP24A1 inhibitors, which differentially regulate 1,25D3 target genes. Inhibiting CYP24A1 expression, while increasing the expression of other anti-proliferative 1,25D3-target genes, may be a useful approach to enhance 1,25D3 antitumor activity.

Supported by NIH/NCI grant CA67267.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Antibody-targeted Therapy

#867

**ABT-165 is a first-in-class therapeutic Dual Variable Domain Immunoglobulin (DVD-Ig** TM **) that targets DLL4 and VEGF for the treatment of cancer.**

Yingchun Li, Jonathan Hickson, Dominic Ambrosi, Deanna Haasch, Kelly Foster-Duke, Lucia Eaton, Fang Jiang, Surekha Akella, Wenqing Gao, Sherry Ralston, Jijie Gu, Susan Morgan-Lappe. _AbbVie, North Chicago, IL_.

The first generation anti-angiogenic drugs designed to block the VEGF/VEGFR pathway lend modest clinical benefit for cancer patients. Other than VEGF, DLL4 is the only known angiogenic factor with a haploinsufficiency phenotype, underscoring its essential role in vascular function. Indeed, both the VEGF/VEGFR and the DLL4/Notch signaling axes are known to cooperate during pathological angiogenesis. DLL4 is also implicated in VEGF-independent pathways, cancer stem cell survival, and immune suppression that could collectively contribute to tumor cell resistance. Given both intrinsic and acquired patient resistance mechanisms exist, targeting the DLL4/Notch pathway represents a unique opportunity for a combination strategy to improve upon current VEGF/VEGFR pathway inhibitor therapies. To this end, ABT-165 was developed as a first-in-class dual specific biologic using AbbVie's proprietary dual-variable domain immunoglobulin (DVD-IgTM) technology. ABT-165 is capable of simultaneously binding to DLL4 and VEGF with nanomolar affinities and blocking the cognate ligand-receptor interactions that result in the potent inhibition of DLL4-mediated Notch1 activation and VEGF-stimulated endothelial cell proliferation. ABT-165 is functionally superior in vitro compared to the combination of parental anti-VEGF and anti-DLL4 antibodies. In human tumor xenograft models, ABT-165 induced significant inhibition of tumor growth and survival benefit compared to single anti-DLL4 or anti-VEGF antibody treatments at equivalent doses. Mechanistically, this enhancement of anti-tumor efficacy is due in part to the disruption of new tumor vasculature coupled with blockade of vessel perfusion. Furthermore, ABT-165 in combination with cytotoxic chemotherapy agents induced tumor regression, which outperformed bevacizumab plus chemotherapy in both human breast and colon xenograft models. ABT-165 displays non-linear, dose-dependent pharmacokinetic profiles in mice and cynomolgus monkeys, with an apparent terminal half-life > 5 days in both species at a target saturation dose. In a GLP monkey toxicity study, ABT-165 at doses up to 200 mg/kg was well-tolerated with non-adverse treatment-related histopathology findings limited to the liver and thymus. In contrast, adverse and non-adverse findings were observed in the hearts of rats and monkeys, respectively, with an in-house proprietary anti-DLL4 antibody. Given that coupling of anti-DLL4 with anti-VEGF activities into a DVD-Ig may lend improved safety and/or efficacy profiles compared to antibodies, ABT-165 was advanced into a Phase 1 clinical trial.

Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie.

AbbVie participated in the interpretation of data, review, and approval of the publication.

#868

Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors.

Dangshe Ma,1 Bitha Narayanan,1 Kim Marquette,2 Edmund Graziani,3 Frank Loganzo,1 Manoj Charati,1 Nadira Prashad,1 Nathan Tumey,3 Jon Golas,1 Christine Hosselet,1 George Hu,1 Frank Barletta,1 Alison Betts,3 Judy Lucas,1 Chris O'Donnell,3 Lioudmila Tchistiakova,2 Hans-Peter Gerber,1 Puja Sapra1. 1 _Pfizer, Pearl River, NY;_ 2 _Pfizer, Cambridge, MA;_ 3 _Pfizer, Groton, CT_.

Antibody-drug conjugates (ADCs) have emerged as an important class of cancer therapeutics. The FDA approval of Kadcyla (T-DM1), a single agent for treatment of HER2-positive advanced metastatic breast cancer, was a significant milestone in the field of targeted therapy, as the first and only ADC for treatment of solid tumors. Despite the 3-month improvement over standard of care in the median survival, almost all the patients eventually became refractory to T-DM1. We have identified several possible areas for improvement: 1) The potency of T-DM1 as confirmed by the Phase III clinical data is restricted to high HER2 tumors which leaves moderate or low HER2 expressing patients without access to T-DM1 treatment; 2) The 48% overall response rate is indicative of intrinsic resistance to T-DM1 and all T-DM1 treated patients eventually relapse. 3) The randomized lysine conjugation in T-DM1 generates heterogeneity of the product. We have developed a novel, site-specific anti-HER2 ADC (NG-HER2 ADC) and evaluated it in comparative preclinical studies with T-DM1. The results show that the NG-HER2 ADC is ~ 10 fold more potent than T-DM1 in HER2 3+ xenograft models of breast and gastric cancers. Our proprietary cleavable and permeable linker-payload can mediate bystander effect and this enables potent anti-tumor activity in non-HER2 amplified breast cancer and heterogeneous low HER2 NSCLC PDX models, where T-DM1 is ineffective. Our ADC can overcome T-DM1 resistance in in vitro and in vivo models.. Our site-specific ADC at HNSTD of 9 mg/kg in cynomolgus monkeys showed high AUC, long half-life and had normal clinical observations with no marked neutropenia. On the contrary, conventional conjugates with cleavable linker payloads typically have severe bone marrow toxicity as DLT above 5 mg/kg. The therapeutic index for NG-HER2 ADC is significantly greater than T-DM1 in all models tested. NG-HER2 ADC has a projected clinical efficacious dose of ~1 mg/kg, compared to 3-5 mg/kg for T-DM1, based on PK/PD modeling. In addition, the activity of the NG-HER2 ADC shows increased infiltration of CD8 positive effector cells, an essential component for immuno-oncology (IO) efficacy, in a syngeneic HER2 overexpressing model. This property potentially allows the combination of the ADC with IO drugs to improve the long-term, overall survival. Our data provides preclinical proof of concept for NG-HER2 ADC with best-in-class potential and is currently being tested in preparation for clinical trials for treatment of HER2 solid tumors.

#869

Meditope enablement and structural analysis of anti-CD33 antibodies.

Calin D. Dumitru,1 Michael Matho,2 Elisabeth Gardiner,2 John C. Williams,3 Krzysztof Bzymek3. 1 _Meditope Biosciences Inc., San Diego, CA;_ 2 _Meditope Biosciences, San Diego, CA;_ 3 _City of Hope, Duarte, CA_.

Meditope Biosciences' SnAP (Site-specific novel Antibody Platform) technology provides a novel and specific way to create antibody drug conjugates (ADCs). Cyclic peptides, termed 'meditopes', have been designed to selectively bind to a 'meditope binding site' engineered into the Fab of an antibody. The process of conferring meditope binding is referred to as "meditope enablement". Enabling antibodies for meditope binding does not interfere with antigen recognition or antibody integrity due to the unique location of the binding site. Meditope peptide binding can be utilized to attach cytotoxic payloads to an enabled antibody in a specific and consistent manner, and has the potential to dramatically improve the consistency of ADC production. For advanced hematological cancers such as CD33 positive acute myeloid leukemia (AML), using an anti-CD33 monoclonal antibody conjugated to a potent cytotoxic agent may be a useful tumor reduction strategy, especially when patients have failed other treatments. To this end, meditope enablement of lintuzumab and gentuzumab permits a consistent, site-specific attachment of cytotoxins with a highly controllable drug antibody ratio (DAR). Structural and biophysical characterization of the meditope enabled anti CD33 antibodies has provided key insight to the meditope enablement process. Experiments confirm that payload conjugation and meditope peptide binding does not interfere with antibody integrity or antigen recognition. The meditope peptide drug complexes were tested in cell-based assays for CD33-targeted cell killing. Using X-ray crystallographic analysis and guided docking, we propose modifications to further enhance affinity of the meditope peptide conjugate : CD33 antibody interaction.

#870

Derivation and characterization of antibodies from immune checkpoint blockade treated cancer patients.

Mark Branum, Laura Smith, Heather Brett, Paul Algate, Brad Greenfield, William Brown, Kristine M. Swiderek. _Theraclone Sciences, Seattle, WA_.

The IgG+ memory B cell repertoire of cancer patients demonstrating durable responses to immune checkpoint blockade therapy has been interrogated to identify tumor specific antibodies. Matched serum and PBMC samples have been collected from 10 melanoma patients with durable responses to treatment with ipilimumab. A panel of 7 well characterized melanoma cell lines with a diverse set of oncogenic driver mutations has been employed to assess durable responder patient serum reactivity to cell surface determinants in a live-cell flow cytometry assay. IgG+ memory B cells obtained from several prioritized durable responder melanoma patients have been plated out on 384 well plates at monoclonal density. The memory B cells have been activated in a short term culture system optimized to expand the clonal B cells and induce secretion of IgG. Antibodies secreted from clonally expanded B cells have been screened using live cell high throughput multiplex flow cytometry assays against a panel of melanoma cell lines. A prioritized subset of antibodies, selected on the basis of tumor cell line binding profiles and antibody v-region sequences, has been cloned and expressed as recombinant IgG1. A cell surface target for a prioritized antibody has been identified and the in vitro analysis and characterization of the antibody including functional activity and immunohistochemistry staining on tumor and normal tissues has been carried out in preparation for in vivo testing of efficacy.

#871

Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale.

Walid S. Kamoun,1 Lia Luus,1 Christine Pien,1 Tad Kornaga,1 Shinji Oyama,1 Zhaohua Richard Huang,1 Suresh Tipparaju,1 Dmitri B. Kirpotin,1 James D. Marks,2 Alexander Koshkaryev,1 Melissa Geddie,1 Lihui Xu,1 Alexey Lugovosky,1 Daryl C. Drummond1. 1 _Merrimack, Cambridge, MA;_ 2 _UNCSF, San Fransisco, CA_.

Ephrins receptors are cell to cell adhesion molecules that mediate signaling and are implicated in neuronal repulsion, cell migration and angiogenesis. Ephrin receptor A2 (EphA2) is part of the ephrin family and was shown to be overexpressed in several solid tumors including ovarian, gastric and lung cancer and is associated with poor prognosis. MM-310 is a novel EphA2 targeted docetaxel nanoliposome. Similar to other nanoparticles, MM-310 leverages organ specificity through enhanced permeability and retention, but can also leverage cellular specificity through its EphA2 targeting arm.

Binding of targeted liposomes to cells in vitro was assessed using flow cytometry in a large panel cell lines. 3D spheroids were used to assess targeting as well as liposome penetration. In order to test the effect of targeting on liposome microdistribution in vivo, primary and metastatic tumor bearing animals were injected with a mixture of EphA2-targeted liposome (EphA2-Ls) and non-targeted liposome (NT-Ls) labeled with two different lipophilic fluorescent dyes. Tissues were assessed using fluorescent microscopy. In order to evaluate the contribution of EphA2-targeting to efficacy, four gastric xenograft models were treated with either MM-310 or a non-targeted version of the drug NT-310, and compared to free docetaxel.

In cell suspension models, we observed a high level of specificity for EphA2-Ls, with more than one hundred-fold increase in liposome cell association. 3D spheroid assays showed that EphA2-Ls binds and penetrates EphA2+ spheroids, while non-targeted liposomes show minimal penetration. Tissue microdistribution analysis in triple negative breast and esophageal tumor models following injection of the EphA2-Ls/NT-Ls mixtures showed a target mediated shift in the microdistribution of liposomes. EphA2-Ls penetrated deeper within the lesions while the NT-Ls deposited at high levels in areas close to the microvasculature. The target mediated shift in microdistribution was also observed in lung metastasis model, with a pattern of distribution that potentially matches disseminated tumor cells. In the same animals, targeting did not affect microdistribution in normal organs such as liver, spleen and skin. Four models of gastric and esophageal cancers were used to test the potential link between cell targeting and tumor growth control. While NT-310 and MM-310 were both able to control tumor growth leading to regression in most models, in three out of four models there was a statistically significant difference between MM-310 and NT-310 at 25 mpk. Biomarker analysis is underway to evaluate the key parameters necessary to mediate targeting.

In conclusion, the data suggests clear evidence of targeting in 2D cell suspension, 3D spheroids, and in primary as well as metastatic tumor models in vivo. In vivo efficacy data showed evidence of the contribution of EphA2 targeting to tumor growth control and regression in several gastric cancer models.

#872

In vivo activity of a novel CDH6 targeting antibody-drug conjugate, including population-scale ovarian PDX clinical trial.

Carl U. Bialucha, Scott D. Collins, Yeonju Shim, Xiamei Zhang, Roberto Velazquez, Colleen Kowal, Caroline Bullock, Hongbo Cai, Stacy M. Rivera, Julie M. Goldovitz, Esther Kurth, Alice T. Loo, Guizhi Yang, John Green, Lance Ostrom, Matthew J. Meyer, Rebecca Mosher, Hui Gao, Juliet Williams, Emma Lees. _Novartis Institutes for Biomedical Research, Cambridge, MA_.

The Cadherin-6 (CDH6) gene was found to be frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CHD6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the tubulin-binding maytansinoid payload DM4. CDH6-ADC was evaluated across multiple linker-payload combinations with the sulfo-SPDB-DM4 format being selected based on a superior combined profile pertaining to activity, selectivity and tolerability.

To gain a broader understanding of CDH6-ADC activity in vivo we profiled the lead candidate against a panel of 31 unselected patient derived ovarian xenograft (PDX) models in a 1x1x1 PDX clinical trial, similar to that described in Gao et al., 2015. In this unbiased high throughput in vivo screen, CDH6-ADC demonstrated robust antitumor activity, with an overall response rate of 39%. Responses were generally durable beyond 150 days and were achieved at doses yielding exposures anticipated to be achievable in humans and observed in PDX models featuring a range of CDH6 expression level and degree of tumor heterogeneity. Retrospective analysis of individual PDX responses and molecular profiling data demonstrate that sensitivity to CDH6-ADC is highly correlated to CDH6 transcript and protein levels. These findings suggest an ability to prospectively identify patients most likely to benefit from this novel targeted therapy. Furthermore, CDH6-ADC demonstrated robust tumor regressions in a representative PDX xenograft model that was refractory to carboplatin/paclitaxel standard of care therapy. These data suggest that CDH6-ADC may benefit both treatment naïve patients and patients that have progressed on prior therapy containing tubulin-targeting anti-mitotics. Extending beyond ovarian cancer, we found CDH6 to be frequently overexpressed in renal cancer. CDH6-ADC was active against RCC PDX models featuring patient relevant levels of CDH6 expression. Data described herein suggest that this novel ADC may be an effective treatment for patients with CDH6 expressing tumors, including ovarian and renal cancer - both indications with a high unmet medical need.

#873

A novel MET-EGFR bispecific antibody LY3164530 shows advantage over combining MET and EGFR antibodies in tumor inhibition and overcome resistance.

Ling Liu,1 Wei Zeng,1 Marcio Chedid,1 Yi Zeng,1 Sheng-hung Tschang,1 Yu Tian,1 Ying Tang,2 Jirong Lu1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Eli Lilly and Company, San Diego, CA_.

The epidermal growth factor receptor (EGFR) and the mesenchymal-epithelial transition factor (MET) are receptor tyrosine kinases that each plays a key role in cancer signaling.

Co-expression and activation of MET and EGFR are found in a number of tumor types, including non-small cell lung, colorectal, gastric, and head and neck cancers (Nanjo et al. 2013). Blocking one receptor tends to up-regulate the other, leading to resistance to single-agent treatment (Engelman et al. 2007). Amplification of MET and/or high levels of HGF expression has been observed in non-small cell lung cancer (NSCLC) patients with intrinsic or acquired resistance to tyrosine kinase inhibitors of EGFR (Engelman et al. 2007; Yano et al. 2011). Conversely, MET-amplified lung cancer cells exposed to MET-inhibiting agents for a prolonged period develop resistance via the EGFR pathway (McDermott et al. 2010). The crosstalk between the MET and EGFR pathways suggests that dual inhibition of these targets may lead to improved outcomes for patients with MET- and EGFR-positive cancers, and that simultaneous inhibition may overcome or delay resistance compared to the blockade of just a single pathway. LY3164530 is an engineered mAb-scFv bispecific antibody that consists of an immunoglobulin G4 (IgG4) antibody to MET and a single-chain variable fragment (scFv) to EGFR fused to the N-terminus of each heavy chain (HC). LY3164530 binds to extracellular domains of MET and EGFR with high affinity and inhibits signaling via both MET and EGFR receptors by blocking ligand binding and internalizing and degrading both receptors. In tumor cells, it binds and co-immunoprecipitates both receptors. LY3164530 has increased avidity binding to MET in cells expressing higher level of EGFR. This increased avidity binding leads to better neutralization of HGF compared to parental MET antibody in these cells. Surprisingly, LY3164530 has superior activity in internalizing/degrading EGFR (wild type and mutant forms) in vitro and in vivo relative to the combination of LY2875358 (i.e., emibetuzumab) and cetuximab in cells expressing high MET and EGFR. In addition, LY3164530 has superior activity in overcoming HGF-mediated resistance to erlotinib, gefitinib, lapatinib, or vemurafenib as compared to the combination of individual monoclonal antibodies targeting these receptors in cell-based assays. In vivo, administration of LY3164530 results in dose-dependent antitumor activity in multiple cell line-derived NSCLC and gastric xenografts. The antitumor activity of LY3164530 is equivalent, and in some cases superior to the combination of emibetuzumab and cetuximab in NSCLC and gastric tumor models. The Phase 1 study with LY3164530 is on-going (NCT02221882).

### Approaches to Elucidating and Overcoming Drug Resistance

#874

Circadian/melatonin disruption by dim light at night drives paclitaxel resistance in breast cancer via activation of stat3.

Steven M. Hill, Shulin Xiang, Robert T. Dauchy, Lulu mao, Lin Yuan, Adam Hauch, Victoria P. Belancio, Melissa A. Wren-Dail, David Pointer, Peter W. Lundberg, Whitney M. Summers, David E. Blask. _Tulane University School of Medicine, New Orleans, LA_.

Resistance to chemotherapy is a significant impediment to the treatment of breast cancer. More than 30% of breast cancer patients present with intrinsic resistance to chemotherapy; almost all who initially respond will develop acquired resistance. Resistant tumors frequently exhibit constitutive activation of numerous survival signaling pathways, including ERK, AKT, NF-kB, and STAT3. We have reported that the circadian hormone melatonin inhibits the growth of both ERα+/ERα- breast cancers and , as well as the daytime induced phospho-activation of ERK1/2, AKT and NF-kB in breast tumor xenografts. We also demonstrated that dim light at night (dLAN), by decreasing nocturnal melatonin, resulted in constitutive phospho-activation of ERK1/2, CREB, NF-kB, and STAT3, promoting resistance to tamoxifen and doxorubicin therapy. Here we tested the hypothesis that dLAN, via phospho-activation of ERK1/2 and STAT3, promotes resistance to paclitaxel (Pax). Female nude rats with "tissue-isolated" MCF-7 breast cancer xenografts were housed in photoperiodic conditions of either LD 12:12, 12:12dLAN (0.2 lux), or 12:12dLAN supplemented with nighttime melatonin (0.05 υg/ml) in the drinking water, with lights on at 0600 hrs and off at 1800 hrs. When estimated tumor weights reached 2.5 g, animals were treated daily with either diluent or Pax i.p. (4υγ/kg) 2 h prior to onset of dLAN or dLAN with nighttime melatonin supplementation. Blood samples collected during the mid-dark phase (2400 hrs) showed elevated nocturnal melatonin in the LD 12:12 group, but significantly suppressed melatonin in the dLAN group. Tumor xenografts from rats housed in dLAN showed a 3-fold decrease in latency-to-onset and a 2.8-fold increased growth rates vs. those from rats receiving melatonin supplementation. Tumor cAMP levels, linoleic acid, and tumor metabolism (Warburg effect) were significantly elevated in dLAN tumors. Numerous signaling pathways including ERK1/2, RSK2, and STAT3, were phospho-activated and others including AKT and HER2/3 were elevated at 2400 hrs by dLAN but repressed in dLAN melatonin supplemented tumors. Tumors from dLAN rats showed intrinsic resistance to Pax, whereas those in LD 12:12 or dLAN and supplemented with nighttime melatonin rapidly regressed. These findings show that temporally coordinated and integrated metabolic and signal transduction mechanisms, particularly the STAT3 pathway, underlying human breast cancer growth, can be activated by the host's exposure to LAN with profound effects culminating in rapid tumor progression and the development of resistance to chemotherapy.

#875

Acquisition of chemoresistance in tumor cells requires crosstalk between dying and remnant live tumor cells via HMGB1.

Junmin Zhou, Xianghong Chen, Danielle L. Gilvary, Melba Marie Tejera, Erika A. Eksioglu, Thu Le Trinh, Sheng Wei, Julie Djeu. _H. Lee Moffitt Cancer Center, Tampa, FL_.

Chemoresistance often develops during drug treatment in cancer patients but its mechanism remains unclear. To understand acquisition of drug resistance, we investigated whether tumor cells dying during drug treatment might signal to neighboring live cells to program themselves for survival in the drug-containing environment. The experimental design was to identify a factor in the supernatants of docetaxel (DTX)-treated human DU145 and PC3 prostate tumor cells that could cause live tumor cells to resist apoptosis and cell death under docetaxel. We discovered that dying cells release HMGB1 which can bind TLR4 or RAGE on live tumor cells causing the induction of cytoplasmic Clusterin (CLU). We demonstrated that CLU is a potent prosurvival protein that can trap Bax from translocating to the mitochondria to cause caspade 3 activation and subsequent apoptosis. Proof of this pathway came from the ability of anti-HMGB1to prevent supernatants from dying cells to induce CLU in newly-plated tumor cells. The receptors for HMGB1 were identified by use of anti-TLR4 and anti-RAGE, which prevented recombinant HMGB1 or dying cell supernatants from inducing CLU in newly-plated tumor cells, causing them to subsequently succumb to DTX toxicity. Moreover, overexpression of anti-sense CLU in tumor cells abrogated their ability to respond to recombinant HMGB1 or dying cell supernatants to induce drug resistance. Most importantly, standard chemotherapeutic agents including gemcitabine, taxol, Ara-C, doxorubicin, cisplatin, etoposide and carboplatin, can cause the release of HMGB1 from dying tumor cells, implicating this crosstalk between dying and live tumor cells as a common pathway towards multi-drug resistance.

#876

Genome-scale genetic knockout screen identifies modifiers of EGFR dependence in non-small cell lung cancer cells.

Jon DiMaina, Chris Duckworth, Hiu Wing Cheung. _Medical University of South Carolina, Charleston, SC_.

Non-small cell lung cancers with epidermal growth factor receptor (EGFR) gene mutations can exhibit a strong dependence on its signaling for growth and survival. They are also sensitive to EGFR tyrosine kinase inhibitors (TKIs), which provide superior clinical benefits to conventional chemotherapy. However, despite initial response, most patients experience relapse with resistant tumors within a year. This study aims to identify modifiers of dependence on mutant EGFR signaling and the mechanisms by which they do so in order to improve therapeutic strategies and outcomes.

We first performed a genome-scale CRISPR-Cas9 genetic knockout screen. In this system, gene silencing occurs by site-specific double-strand-breaks induced by the Cas9 nuclease that is localized to its target by a single-guide RNA (sgRNA). The error-prone repair pathway introduces insertion/deletion mutations leading to frameshifts or premature stop codons. We constructed a pooled sgRNA library targeting more than 18,000 protein-coding human genes with multiple sgRNA vectors. The cell line HCC827 was used in our screen as it is EGFR-mutant and sensitive to EGFR TKIs. We transduced HCC827 lung cancer cells with the pooled sgRNA library and cultured them in the presence of Erlotinib, an EGFR TKI, or DMSO control for 17 days. At the end of the treatment period we identified sgRNAs that were enriched in Erlotinib-treated groups over control groups, indicating genes whose loss-of-function confer TKI resistance. We applied the RNAi gene enrichment ranking (RIGER) algorithm to identify gene hits with enrichment of multiple sgRNAs.

Top-ranked candidates include previously confirmed genes PTEN, NF1, NF2, TSC1, and TSC2, validating this system as a means to identify modifiers of EGFR dependence in HCC827 cells. A novel candidate gene is the E3 ubiquitin ligase HUWE1. We showed that suppression of HUWE1 by sgRNAs or inducible short hairpin RNA (shRNA) in HCC827 cells re-activated AKT and ERK1/2 signaling pathways and increased cell proliferation and survival in response to EGFR inhibition. These findings were confirmed in vivo by implanting mouse xenografts of HCC827 cells expressing shHUWE1 and monitoring tumor development in response to EGFR-TKI therapy. Tumors expressing HUWE1 led to durable tumor regression throughout the treatment period but tumors with suppressed HUWE1 continued to grow into large tumors. Immunohistochemistry analysis of excised tumors also revealed increased AKT and ERK1/2 signaling as well as increased proliferation and decreased apoptosis.

We have shown that dependence on EGFR signaling can be decreased in EGFR-mutant lung cancer cells through mechanisms that involve the activation of AKT and ERK1/2 signaling pathways. Ongoing studies involve identifying HUWE1 substrates/interactions that participate in tumor cell response to EGFR inhibitors, revealing a novel mechanism of resistance to EGFR-targeted therapy.

#877

Evolution of resistance to EGFR inhibition from drug tolerant cancer cells.

Aaron N. Hata, Yotam Drier, Maria Gomez-Caraballo, Faria Siddiqui, Lecia Sequist, Bradley Bernstein, Jeffrey Engelman. _Massachusetts General Hospital, Boston, MA_.

Acquired drug resistance to targeted cancer therapies remains a significant clinical problem. Although mechanisms of acquired resistance of EGFR mutant non-small cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. We demonstrate that acquired resistance caused by the T790M gatekeeper mutation can occur either by selection of pre-existing T790M clones or via evolution of T790M-negative drug tolerant cells that develop the mutation during drug treatment. Additionally, the path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells have a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR. To characterize drug tolerant cells in patients undergoing EGFR inhibitor therapy, we performed single-cell RNA-Seq on EGFR mutant NSCLC samples prior to and after initiation of EGFR inhibitor. These results offer novel insights into the evolution of acquired resistance to EGFR inhibitors and point to new therapeutic opportunities to prevent or overcome resistance in the clinic.

#878

Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer.

Mariangela Russo,1 Giulia Siravegna,1 Lawrence S. Blaszkowsky,2 Giorgio Corti,1 Giovanni Crisafulli,1 Leanne G. Ahronian,2 Benedetta Mussolin,1 Eunice L. Kwak,2 Michela Buscarino,1 Luca Lazzari,1 Emanuele Valtorta,3 Mauro Truini,3 Nicholas A. Jessop,4 Hayley E. Robinson,4 Theodore S. Hong,2 Mari Mino-Kenudson,4 Federica Di Nicolantonio,1 Ashraf Thabet,5 Andrea Sartore-Bianchi,3 Salvatore Siena,3 A. John Iafrate,4 Ryan B. Corcoran,2 Alberto Bardelli1. 1 _University of Turin, Depart. of Oncology, Candiolo (TO), Italy;_ 2 _General Hospital Cancer Center, Boston, MA;_ 3 _Niguarda Cancer Center, Ospedale Niguarda Ca' Granda, Milan, Italy;_ 4 _Department of Pathology, Massachusetts General Hospital, Boston, MA;_ 5 _Department of Radiology, Massachusetts General Hospital, Boston, MA_.

How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent lines of therapy is unknown. Exposure to therapy may result in selection of sub-clonal cell populations, capable of growing under drug pressures. Therefore, a single-lesion biopsy at disease progression may vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient and may fail to detect the existence of distinct but important resistance mechanisms that could impact clinical response.

We identified a colorectal cancer (CRC) patient in whom multiple tumor biopsies were obtained at resistance following prolonged response to with the anti-EGFR antibody cetuximab. Full-exome sequencing of 1000 cancer genes of both primary tumor and progression biopsy revealed a TP53 mutation in all samples and a novel MEK1 p.K57T mutation in one of the progressing liver biopsy. A mutation at the same MEK1 codon was identified in the cetuximab-resistant HCA46 CRC cell line. Biochemical analysis showed constitutive activation of MEK and ERK despite cetuximab treatment. However, the combination of the MEK inhibitor trametinib with either cetuximab or panitumumab restored sensitivity, suggesting a potential therapeutic strategy to overcome resistance to EGFR blockade caused by this mutation.

Accordingly, the patient was treated with the combination of panitumumab and trametinib. After 3 months, imaging demonstrated a reduction in size of the biopsied liver metastasis harboring the MEK1 mutation, but revealed that some other lesions had progressed. Plasma collected prior to therapy was analyzed by next-generation sequencing confirming the presence of both TP53 and MEK1 variants, but surprisingly unveiling a previously unrecognized KRAS mutation. ddPCR analysis of longitudinal timepoints of ctDNA unveiled that TP53 mutant levels dropped after initiation of therapy, but rose later during treatment in parallel with CEA tumor marker levels. However, MEK1 mutant levels declined sharply, indicating effective suppression of MEK1 mutant clones by panitumumab and trametinib; while KRAS mutant levels rose, indicating outgrowth of a resistant KRAS-mutant clone. Biopsy of a different liver metastasis that progressed despite panitumumab and trametinib revealed the same KRAS mutation identified in ctDNA.

In summary these findings illustrate how distinct acquired resistance mechanisms can arise concomitantly in separate metastases within the same patient, leading to mixed lesion-specific responses to subsequent targeted therapies. Liquid biopsy approaches, in association with single-tumor biopsies, have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent targeted therapies.

#879

Single cell analysis resolves combinatory targeted therapy for arresting the BRAFi-induced cellular dedifferentiation of metastatic melanomas.

Yapeng Su,1 Wei Wei,2 Min Xue,1 Lidia Robert,2 Jennifer Tsoi,2 Thomas Graeber,2 Antoni Ribas,2 James Heath1. 1 _Caltech, PASADENA, CA;_ 2 _UCLA, Los Angeles, CA_.

Targeted therapy that inhibits the BRAF (V600E) protein has shown impressive initial responses in metastatic melanoma patients. However, acquired drug resistance, derived from tumor heterogeneity, always limits its efficacy and compromises its treatment outcomes. Melanocyte to neural crest transition (MNT) has been observed to play crucial role in early stage adaptive drug tolerance, and the mathematic modeling indicates the transition is contributed from both stochastic phenotypic transition and non-genetic selection. Whole transcriptomic analysis enriched important signaling proteins that are involved in the resistance development process. Single cell proteomic analysis of those proteins was performed on patient-derived melanoma models to further study the early stage drug tolerance. This resolves how protein signaling network coordination rewires in the process, which cannot be resolved from proteomic analysis of bulk tumor tissue. Signaling network coordination analysis indicates that co-inhibiting the hubs of the network (MEK, NFKB-p65) could keep cells in the drug-susceptible stages. The combination therapy that co-inhibits mutant BRAF, MEK and NFKB-p65 together successfully arrested the MNT transition and induced a sustained tumor growth suppression in multiple patient-derived melanoma cell lines. This study provides us a brand-new way of using single cell proteomic analysis to infer effective combinations by resolving protein signaling network coordination that is inaccessible through traditional bulk-level proteomic measurement.

#880

Mapping the metastatic colorectal cancer phospho-proteome for predicting response to cetuximab.

Robin Beekhof,1 Mariette Labots,2 Jaco C. Knol,1 Tim R.A. Schelfhorst,1 Nicole van Grieken,3 Sander R. Piersma,1 Thang V. Pham,1 Andrea Bertotti,4 Livio Trusolino,4 Henk M.W. Verheul,2 Connie R. Jiménez1. 1 _VUmc - OncoProteomics Laboratory, Department of Medical Oncology, Amsterdam, Netherlands;_ 2 _VUmc - Department of Medical Oncology, Amsterdam, Netherlands;_ 3 _VUmc - Department of Pathology, Amsterdam, Netherlands;_ 4 _University of Torino Medical School, Candiolo, Torino, Italy_.

Introduction: The discovery of the key role of the epidermal growth factor receptor (EGFR) and its downstream signaling effectors in the pathophysiology of colorectal cancer (CRC) has resulted in the clinical use of targeted therapies in the treatment of metastatic CRC (mCRC). However, clinical benefit to EGFR blockade is observed in only a subgroup of CRC patients wild type for KRAS andNRAS. Genomic characterization of patient derived xenograft (PDX) models of mCRC have revealed additional resistance mechanisms to the EGFR MAb cetuximab including amplification of MET, HER2 and mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 (Bertotti et al. 2011, 2015). However, not all resistance is understood and the biochemical signaling correlates of sensitivity and resistance remain incompletely understood. Phosphoproteomics allows for comprehensive protein phosphorylation profiling, and thereby may shed new insights into the biology of CRC and the molecular basis of cetuximab response.

Aim: To identify aberrant signaling pathways, predictive biomarkers, and alternative drug targets in genomically-annotated CRC PDX models by mass spectrometry-based phosphoproteomics.

Methods: We performed LC-MS/MS based phosphoproteome profiling (van der Mijn et al. 2015; Piersma et al. 2015) on untreated CRC PDX models lacking KRAS, NRAS or BRAF mutation (n=20). For comparison, KRAS, BRAF or NRAS-mutant and MET or HER2 amplified models (n=21) were analyzed. Phosphopeptides were captured by phosphotyrosine antibodies and titanium dioxide. MS/MS data was searched using MaxQuant. Phosphoproteome data (ion intensities and spectral counts) were correlated with drug sensitivity. Dedicated bioinformatics was used to identify hyperactive kinases and signaling pathways in individual tumors.

Results: The dataset contained 13.418 tyrosine-, serine- and threonine-phosphorylated peptides derived from 4588 proteins, including 237 phosphorylated kinases. Unsupervised hierarchical clustering of phosphopeptide intensities revealed subclusters enriched for sensitive and resistant models. Among cetuximab-resistant models, HER2 and MET were identified as hyperphosphorylated and hyperactive driver kinases, confirming the genomic annotation. Sensitivity to combination treatment of both EGFR and driver kinases was observed previously, providing proof-of-principle of the use of phosphoproteomics for the identification of driver kinases.

Conclusion/Future: This project is uncovering the phosphoproteomic landscape of colorectal tumors and will yield a better understanding of signaling pathways involved, with an emphasis on identifying activated drug targets and predictive biomarkers in this disease, in relation to KRAS/NRAS/BRAF status and response to anti-EGFR therapy.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Disordered Gene Regulation and Chromatin State in Malignant Transformation

#881

Dissecting chromatin dynamics in malignant progression.

Hanseul Yang,1 Daniel Schramek,2 Rene Adam,1 John Levorse,1 Brice Keyes,1 Ping Wang,3 Deyou Zheng,3 Elaine Fuchs1. 1 _The Rockefeller University, New York, NY;_ 2 _The Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada;_ 3 _Albert Einstein College of Medicine, New York, NY_.

Tumor-initiating cells within a cancer exhibit distinct patterns of transcription factors and gene expression compared to stem cells within their healthy tissue. Little is known about the key transcription factors that dictate chromatin remodeling and the accompanying transcriptional changes that ultimately hardwire the malignant behavior of tumor-initiating stem cells. Here, by in vivo chromatin and transcription profiling, we show that dramatic shifts in large open-chromatin (super-enhancer) landscapes underlie these differences. Focusing on one of the most common and life-threatening cancers world-wide, squamous cell carcinoma (SCC), we show that super-enhancers of SCC-stem cells contain the binding sites for a distinct set of putative master transcription factors. Many of their genes, including Ets2 and Elk3, are themselves regulated by SCC super-enhancers, suggesting a cooperative feed-forward loop. Malignant progression requires these genes, whose knockdown severely impairs tumor growth and prohibits progression from benign papillomas to SCCs. Interestingly, ETS2 is known to be phosphorylated and activated by RAS/MAPK signaling, and knockdown of ETS2 results in loss of expression of SCC super-enhancer-associated genes. Conversely, in vivo forced expression of an active ETS2 version harboring a phosphomimetic substitution at the MAPK-activation residue is sufficient to trigger cellular transformation without oncogenic RAS. Moreover, ETS2-overactivation in epidermal progenitors rewires the super-enhancer landscape and induces SCC super-enhancer associated genes including Fos, Junb, Klf5 and Elk3. Finally, we identify dramatic remodeling of the Cxcl1/2 locus from an H3K27me3-repressed to an ETS-regulated, super-enhancer-activated state, and provide evidence for their autocrine oncogenic role in SCCs through a cognate receptor Cxcr2. Together, our findings unearth an essential regulatory network required for the SCC chromatin landscape and unveil its importance in malignant progression.

This work was funded by grants from the National Institutes of Health (R01-AR31737) and NYSTEM #CO29559.

#882

TMPRSS2-ERG drives global mistargeting of mammalian SWI/SNF (BAF) complexes in prostate cancer.

Gabriel J. Sandoval,1 John L. Pulice,1 David Y. Takeda,1 Monica A. Schenone,2 Marius Pop,2 Gaylor Boulay,3 Miguel N. Rivera,3 Lucienne Ronco,2 William C. Hahn,1 Cigall Kadoch1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _The Broad Institute of Harvard and MIT, Boston, MA;_ 3 _Massachusetts General Hospital, Boston, MA_.

Prostate cancer remains one of the leading causes of cancer-related death in men. Chromosomal rearrangements resulting in the fusion of the androgen regulated gene TMPRSS2 and the ETS-family transcription factor ERG occur in over 50% of all prostate cancer cases. Recent studies enabled by genome-wide methodologies have implicated altered epigenomic landscapes and changes in DNA accessibility as major contributors to ERG-driven oncogenesis, however the precise mechanism underlying the ERG transcriptional signature has to date remained unclear. Here we performed the first endogenous purification and SILAC-mass spectrometric analysis of ERG in TMPRSS2-ERG prostate cancer cells. Remarkably, we demonstrate that ERG directly interacts with the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which was recently shown to be mutated in >20% of human malignancies. ERG co-localizes with BAF complexes genome-wide, resulting in specific ERG-dependent BAF complex targeting to sites enriched in known ERG, FOXA1, and HOXB13 motifs; additionally, loss of ERG in TMPRSS2-ERG driven cell lines results in dramatic retargeting of BAF complexes away from ERG-dependent sites, to sites enriched in known AR and CTCF motifs. Importantly, ERG-driven BAF complex retargeting contributes to activation of TMPRSS2-ERG prostate cancer gene expression signatures. We map the ERG-BAF interaction to a specific region within the ERG amino acid sequence and find that this region is required to bind BAF complexes. These studies reveal a novel, unexpected mechanism of action of ERG-driven oncogenesis and offers new strategies for therapeutic intervention.

#883

NUP98-fusion proteins interact with the NSL/MLL1 complexes to drive leukemogenesis.

Haiming Xu, Daria G. Valerio, Meghan E. Eisold, Amit Sinha, Richard . P. Koche, Wenhuo Hu, Chun-Wei Chen, Scott H. Chu, Gerard L. Brien, Scott A. Armstrong. _Memorial Sloan Kettering Cancer Center, New York, NY_.

The Nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in an array of hematopoietic malignancies via chromosomal translocations involving 11p15. NUP98-rearranged leukemias show elevated HOXA and HOXB cluster genes and mouse model systems have recapitulated this high-level expression independent of whether the leukemias are derived from mouse or human bone marrow. However, the molecular mechanisms of NUP98-fusion mediated leukemogenesis and elevated HOX gene expression in this leukemia are unclear. Recent studies in Drosophila show that nucleoplasmic Nup98 functions as a potential transcriptional activator, and physically interacts with the non-specific lethal (NSL) and trithorax (Trx)/mixed lineage leukemia (MLL) complex (Kalverda et al., 2010; Pascual-Garcia et al., 2014). To test whether the intranuclear localized NUP98-fusion proteins interact with NSL/MLL1 complexes, coimmunoprecipitation (co-IP) experiments using 293T cells with overexpression of NUP98 fusions and wild type (WT) NUP98 were performed. NUP98-fusion proteins NUP98-HOXA9, NUP98-HOXD13, NUP98-NSD1 and NUP98-PHF23, but not the full-length WT NUP98, show physical interaction with MLL1 and the NSL histone acetyltransferase (HAT) complexes. Genome-wide chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) illustrated that NUP98-HOXA9 and MLL1 co-localize at Hoxa and Hoxb gene promoters, which correlates with the presence of activating chromatin modifications such as H4K16ac and H3K4me3. In vitro and in vivo functional assays further showed that Mll1 is crucial for the growth of NUP98-HOXA9-transformed cells, and for the initiation and maintenance of NUP98-HOXA9 driven AML. These findings were further supported by transcriptome analyses performed in mouse NUP98-HOXA9 transformed cells lacking Mll1. We found a significant enrichment of co-bound targets of MLL1 and NUP98-HOXA9 and genes downregulated in the absence of Mll1. Gene set enrichment analysis (GSEA) demonstrated strong similarity between Mll1-dependent gene expression signature in our murine NUP98-HOXA9 AML model and the expression profile of human NUP98-fusion AML, indicating that our findings in murine AML models can be extended to human AML. Finally, the overexpression of Hoxa9 and Meis1, direct binding targets of NUP98-HOXA9 and MLL1 that are downregulated upon Mll1 loss, rescues the in vitro transformation defects in NUP98-HOXA9 Mll1-/- cells. In conclusion, our findings support a model where NUP98-fusion proteins recruit the NSL/MLL1 complex as an important step during leukemogenesis. The deregulated HOX gene expression in NUP98 fusion transformed cells remains dependent on MLL1. The discovery of this common leukemogenic pathway for NUP98-fusion proteins opens up new avenues for potential therapeutic opportunities to treat patients with leukemia that harbor various NUP98 rearrangements.

#884

NSD3-short is an adaptor protein that couples BRD4 to the CHD8 chromatin remodeler.

Chen Shen, Jonathan J. Ipsaro, Junwei Shi, Christopher R. Vakoc. _Cold Spring Harbor Lab, Cold Spring Harbor, NY_.

The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain. We show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by utilizing a PWWP chromatin reader module, and by employing an acidic transactivation domain. Genetic targeting of NSD3 or CHD8 mimics the phenotypic and transcriptional effects of BRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome and are each released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4-NSD3-CHD8 complexes from chromatin to suppress transcription.

#885

Epigenetic regulation of estrogen receptor transcription by the PI3K pathway in breast cancer.

Toska Eneda,1 Hatice Osmanbeyoglu,1 Moshe Elkabets,2 Carmen Chan,1 Pau Castel,1 Maura Dickler,1 Scott Armstrong,1 Christina Leslie,1 Maurizio Scaltriti,1 Baselga José1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Ben-Gurion University of the Negev, Israel_.

Mutations in the PIK3CA gene are the most frequent genomic alterations in estrogen receptor (ER)-positive breast cancers. Direct pharmacological inhibition of PI3K signaling is therefore an attractive clinical strategy and a number of PI3K pathway inhibitors are currently under clinical development. Unfortunately, although the majority of ER-positive PIK3CA-mutant patients respond, mechanisms of resistance to these inhibitors inevitably emerge. By studying both mouse models and human samples, our laboratory has previously uncovered that inhibition of PI3K pathway increases ER transcriptional activity, which in turn renders cells more susceptible to endocrine therapy. The mechanisms by which ER and PI3K signaling pathway regulate each other in breast cancer cells, however, remain elusive.

To better understand the cross-talk between the PI3K pathway and the ER transcriptional program, we developed an unbiased transposon activation mutagenesis screen with the goal of identifying modulators of resistance to PI3K inhibitors in ER-positive tumors. Among the genes identified, we found a number of key regulators of ER function including the pioneer transcription factors FOXA1 and PBX1. We further confirmed that FOXA1 and PBX1 expression and transcriptional activity was enhanced upon PI3K inhibition and validated these observations in both xenograft models and samples from patients undergoing treatment with the PI3Kα inhibitor BYL719. Moreover, chromatin imunoprecipitation (ChIP)-sequencing against ER and FOXA1 demonstrated that these factors occupy the same genomic regions, and their binding is increased upon PI3K inhibition. Silencing FOXA1 or PBX1 impaired the activation of the ER-dependent transcriptional program following PI3K blockade and sensitized cells to PI3K inhibition.

To better understand the role of FOXA1 and PBX1 in the ER-PI3K crosstalk, we have then studied in detail the chromatin changes upon BYL719 treatment using transposase-accessible chromatin using high-throughput sequencing (ATAC-seq) in breast cancer cells. Epigenomic profiling using ATAC-seq is also being done on patient samples collected before BYL719 administration (pre-treatment) and during therapy (on-treatment). ATAC-seq and RNA-seq data from the same patients will be integrated using novel computational approaches.

These analyses will help to dissect the ER-dependent epigenetic changes occurring upon PI3K inhibition and how the cells use these chromatin modifications to adapt to the pharmacological stress. Elucidating the interconnection between the PI3K pathway and ER activity may uncover novel mechanisms of resistance to either PI3K inhibitors or endocrine therapy in ER-positive breast cancer patients.

#886

Notch signaling activates B-cell specific enhancers to drive oncogene targets in B-cell lymphoma.

Russell J. Ryan,1 Jelena Petrovic,2 Dylan M. Rausch,1 Winston Lee,3 Michael Kluk,4 Laura Donohue,1 Shawn Gillespie,1 Jon C. Aster,3 Warren S. Pear,2 Bradley E. Bernstein1. 1 _Massachusetts General Hopsital, Boston, MA;_ 2 _University of Pennsylvania School of Medicine, Philadelphia, PA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Weil Cornell Medical College, New York, NY_.

Recurrent gain-of-function mutations in genes encoding Notch receptors are associated with poor clinical outcome in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but genome-wide functional targets of Notch signaling in B cells have not been characterized.

We identified genome-wide binding sites for Notch transcription factor (TF) complexes, using antibodies specific for intracellular NOTCH1 and its binding partner RBPJ, in CLL and MCL lymph node biopsies, and lymphoma cell lines. We employed a gamma-secretase inhibitor washout strategy to rapidly modulate mutant NOTCH1 protein activation in complementary lymphoma cell line models of ligand-dependent and ligand-independent Notch signaling, and performed RNA-sequencing and genome-wide enhancer acetylation profiling in both the "notch on" and "notch off" states. These data identify a common set of functional Notch target genes in B-cell lymphoma that includes both canonical Notch targets as well as B-cell-specific target genes. The latter are frequently associated with adjacent B-cell-specific enhancers that show direct Notch TF binding and Notch-signaling-dependent histone acetylation. Notch-activated target genes include MYC and other TFs implicated in B-cell lymphoma, and are significantly enriched for mediators of targetable oncogenic lymphoma signaling pathways, (B-cell receptor, toll-like receptor, JAK-STAT, MAP kinase, and G-protein signaling pathways, all FDR q-value < 1e-3 in KEGG and / or Reactome analyses). Many B-cell Notch target genes show significantly increased expression in lymph nodes from patients with NOTCH1 mutant vs. wild-type CLL. Notch target genes are also up-regulated in lymph node- versus peripheral blood-derived CLL cells, consistent with immunohistochemical evidence for microenvironment-dependent Notch signaling activation in the CLL lymph node. Combined small-molecule inhibition of Notch and B-cell receptor signaling showed a synergistic anti-proliferative effect in Notch-dependent lymphoma cells.

Our data reveal the functional regulome of Notch signaling in small B-cell lymphoma, and may have implications for the role of Notch signaling in normal B-cell development. These findings suggest novel strategies for rational combination therapy in CLL and MCL.

#887

N-Myc drives neuroendocrine prostate cancer.

Etienne Dardenne,1 Himisha Beltran,1 Kaitlyn Gayvert,1 Matteo Benelli,2 Adeline Berger,1 Loredana Puca,1 Joanna Cyrta,1 Andrea Sboner,1 Zohal Noorzad,1 Theresa MacDonald,1 Cynthia Cheung,1 Dong Gao,3 Yu Chen,3 Martin Eilers,4 Juan Miguel Mosquera,1 Brian D. Robinson,1 Mark A. Rubin,1 Olivier Elemento,1 Francesca Demichelis,1 David S. Rickman1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Center for Integrative Biology, Trento, Italy;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _Theodor Boveri Institute, Würzburg, Germany_.

Emerging observations from clinical trials suggest that a subset of castration resistant prostate adenocarcinomas (CRPC) eventually evolve or progress to a predominantly neuroendocrine phenotype (NEPC). This transition is emerging as an important mechanism of treatment resistance. This cell plasticity is characterized by loss of androgen receptor (AR) and prostate specific antigen (PSA), and significant over-expression and gene amplification of MYCN (encoding N-Myc). While N-Myc is a bona fide driver oncogene in several rare tumor types, the molecular mechanisms that underlie N-Myc driven NEPC have yet to be characterized.

Integrating a novel genetically engineered mouse (GEM) model of prostate specific N-Myc overexpression, human prostate cancer cell line modeling, and human prostate cancer transcriptome data, we found that N-Myc over-expression leads to the development of poorly differentiated, invasive prostate cancer that is molecularly similar to human NEPC tumors. To determine if N-Myc plays a causal role in driving the NEPC phenotype, we generated GEM lines that carry a CAG-driven lox-stop-lox human MYCN gene integrated into the ROSA26 (LSL-MYCN) locus and either a Tmprss2 driven tamoxifen-activated Cre recombinase (T2-Cre) or probasin (Pb)-Cre. Since PTEN deletion is a frequent alteration in CRPC and PI3K/AKT signaling can enhance N-Myc protein stability we also engineered the mice with a floxed Pten locus. N-Myc over-expression in the context of Ptenf/+ at 3 months post-induction leads to focal mouse high-grade prostatic intraepithelial neoplasia (mHGPIN). T2-Cre; Ptenf/f; LSL-MYCN+/+ mice develop highly proliferative, diffuse mHGPIN which consists of proliferations of cells with nuclear atypia that expand the glands, imparting irregular borders and inducing a mild stromal response, mitotic figures, and incipient necrosis. RNAseq data from N-Myc these mHGPIN lesions show they are molecularly similar to NEPC based on RNAseq data from 203 human CRPC and NEPC samples. At 6 months, Pb-Cre; Ptenf/f; LSL-MYCN+/+ mice develop poorly differentiated, highly proliferative, invasive prostate cancer.

Based on the RNAseq data from the N-Myc GEM line, GEM-derived mouse prostate cancer organoid cultures and isogenic cell lines, we found that N-Myc regulates a specific NEPC-associated molecular program that includes a repression of AR signaling, enhanced AKT signaling and repression of Polycomb Repressive Complex 2 target genes. We further showed that N-Myc interacts with AR and this interaction depends on Enhancer of Zeste Homolog 2 (EZH2). Finally, N-Myc expressing cell lines and organoids displayed an enhanced sensitivity to inhibitors targeting the AKT pathway, EZH2 and Aurora-A.

Altogether, our data shows that N-Myc drives the neuroendocrine phenotype in prostate cancer and provides rationale for the development of new therapeutic strategies for treating this aggressive subtype of prostate cancer.

### Oncogene and Tumor Suppressor Function and Targeting

#888

Inactivation of the DLC1 RhoGAP tumor suppressor by point mutation occurs commonly in human cancer and can result from Rho-dependent or Rho-independent mechanisms.

Xiaolan Qian, Dunrui Wang, Beatriz Sanchez-Solana, Brajendra K. Tripathi, Marian E. Durkin, Alex Papageorge, Douglas R. Lowy. _National Cancer Institute, Bethesda, MD_.

The Rho GTPases, which are frequently activated in advanced cancer, stimulate their downstream effectors to regulate a variety of fundamental cellular process, e.g., cell cycle, cell migration, cell-cell adhesion, and cytoskeleton organization. The down-regulation of RhoGAPs is one mechanism of increased Rho activity, and members of the tumor suppressor DLC Rho-GAP family (DLC1-3) are frequently down-regulated by gene deletion or DNA methylation in human cancers. Here we report that, in addition, point mutations in the DLC1-3 coding regions occur frequently in certain cancers, most of the mutants we have analyzed have reduced tumor suppressor activity, at least two distinct mechanisms can account for this reduction, and one mechanism appears to be Rho-independent. In the TCGA database, DLC1 was mutated in 11% of gastric cancer (GAC), 10% colorectal cancer (CRC), 7% of melanomas (MEL), and 6% of lung adenocarcinoma (LAD). Most alterations were point mutations encoding a single amino acid change. Mutation of DLC2 and DLC3 also occurred in these tumor types, but were less frequent. The mutation frequency of p190-A, which is a Rho-GAP from a different gene family, was even less frequent, ranging from 4% in LAD to zero in GAC. Taken together, mutation of at least one DLC gene or p190-A was found in about 20% of LAD, CRC, and MEL, and 15% of GAC. In LAD, the DLC1 expression levels from the patients with DLC1 mutations were lower than the patients with DLC1 wild type, suggesting selection for decreased DLC1 expression in tumors harboring mutant DLC1. We constructed expression vectors for many of the DLC1 mutants and analyzed their phenotypes in human tumor cell lines. Most were found to be loss of tumor suppressor function mutants, as they were deficient for reducing growth in soft agar, cell migration, and focal adhesion turnover. One class of deficient mutants had lesions in the RhoGAP domain, which directly inactivated its RhoGAP activity and increased the activity of Rho downstream effectors. A second class had lesions in the DLC1 START (StAR-related lipid-transfer) domain, which is known to bind caveolin. although the RhoGAP activity was normal, complex formation with caveolin was much lower than with wild type DLC1. We conclude that, in addition to the frequent down-regulation of DLC1 expression via gene deletion or DNA methylation in tumors, point mutation of its coding sequence commonly inactivates the tumor suppressor activity of DLC1 at the protein level by at least two alternate mechanisms: attenuation of it binding to caveolin or of its RhoGAP activity, which up-regulates RhoA and its downstream effectors. (# Contributed equally)

#889

Dissecting the role of MYC in BRCA1-associated breast cancer.

Chiara Svetlana Brambillasca, Linda Henneman, Julian de Ruiter, Anne Paulien Drenth, Jos Jonkers. _NKI-AVL, Amsterdam, Netherlands_.

Breast cancer is the most common cancer among women in the Western world. Approximately 8% of all breast cancer cases may be caused by inheritance of mutations in breast cancer susceptibility genes. Of these, BRCA1 is the most important one, contributing to approximately half of all familial breast cancer cases. BRCA1-related breast tumors show a triple-negative (TBNC) basal like phenotype, that correlates with aggressive characteristics and bad prognosis. A number of BRCA1 associated genes have been identified; among those MYC overexpression seems to have an important role in aggressive TBNC. In line with this, we found MYC to be frequently amplified in both mouse and human BRCA1-deficient breast cancers. Thus, evaluating the contribution of MYC to BRCA1-associated breast cancer might unravel new mechanistic insights and potentially open the way to new therapeutic approaches.

To pursue this, we generated a WAPCre;Brca1F/F;Trp53F/F ;Myc (WB1P-MYC) mouse model to monitor tumor development. Whereas the WAPCre;Brca1F/F;Trp53F/F (WB1P) mouse model develops tumors after 300 days, Cre-conditional overexpression of the MYC transgene leads to a dramatic acceleration in tumor development with a median survival of 100 days. Tumors of both models were histopathologically classified as solid carcinomas. Further characterization revealed that WB1P-MYC tumors show higher levels of apoptosis and DNA damage. Moreover, we also observed a strong difference in immune infiltrate between WB1P and WB1P-MYC tumors. To better characterize these differences between WB1P and WB1P-MYC tumors we obtained tumor organoids. Importantly, these tumor organoids maintained characteristics of the original tumor like Brca1, P53 deletion status and MYC overexpression. Moreover, they can be orthotopically transplanted in vivo, leading to formation of solid carcinomas. Therefore, they represent a versatile platform to test new drug combinations both in vitro and in vivo.

To test if MYC overexpression is also required for maintenance of established tumors, we are developing a "double-layered" system for Cre-conditional and doxycycline-regulatable overexpression of the MYC gene in the WAPCre;Brca1F/F;Trp53F/F model. By administering doxycycline we will induce MYC only in Cre-expressing mammary epithelial cells. After tumor formation MYC will be switched-off to assess its role in tumor maintenance and progression. Furthermore, we would like to test new therapeutic approaches, like combining Myc down-regulation with administration of PARP inhibitors. In addition, tumor organoids of this inducible WB1P-MYC model will be a powerful system to study the MYC-associated phenotype, drugs combinations and forward genetic screens.

#890

TGFβ upregulation mediates growth retardation in EGFR T790M mutant non-small cell lung cancer.

Claire E. Repellin,1 Pinar O. Eser,2 Marzia Capelletti,3 Takeshi Shimamura,4 Dalia Ercan,5 Chunxiao Xu,6 Nathanael S. Gray,2 Kwok-Kin Wong,2 Pasi A. Jänne2. 1 _SRI Biosciences, Menlo Park, CA;_ 2 _Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Loyola University Chicago Stritch School of Medicine, Maywood, IL;_ 5 _Dana-Farber Cancer Institute, Northeastern University, Boston, MA;_ 6 _EMD Serono, Inc., Boston, MA_.

Introduction:

A subset of non-small cell lung cancer is driven by activating mutations in the epidermal growth factor receptor (EGFR). The majority of EGFR-driven lung cancers respond to EGFR kinase inhibitors gefitinib and erlotinib, although the clinical efficacy these inhibitors is limited by the development of drug resistance. A secondary missense mutation encoding EGFR T790M is the most prevalent resistance mechanism observed in patients, accounting for nearly 60% of relapse following gefitinib or erlotinib treatment. Although the T790M mutation enhances EGFR kinase activity, T790M harboring tumors exhibit indolent growth and are associated with favorable prognosis compared to tumors that acquire alternative mechanisms of drug resistance. The mechanism(s) underlying the indolent growth mediated by EGFR T790M are not well characterized.

Methods:

To identify the factor(s) mediating T790M associated growth retardation, we characterized T790M amplified drug resistant cells. EGFR mutant parental lung cancer cell lines PC9 and H3255 were selected through sequential treatment with gefitinib and the irreversible EGFR inhibitor dacomitinib. The resulting drug resistant cells, PC9DR and H3255DR, harbored amplified EGFR T790M and exhibited marked growth retardation. These T790M amplified models were characterized to identify factors conferring indolent growth. T790M expressing patient tumor samples were also analyzed to validate T790M associated changes.

Results:

Ectopic overexpression of EGFR T790M induced growth retardation in EGFR inhibitor-naïve cells. Incubation of parental PC9 cells in media conditioned on PC9DR cells was sufficient to slow PC9 growth rates, suggesting that a secreted factor was responsible for the growth retardation observed in the T790M amplified cells. A Luminex assay of PC9DR cells revealed upregulation of transforming growth factor beta 2 (TGFβ2), which was confirmed at the transcript level by qPCR. Treatment of parental PC9 cells with recombinant TGFβ was sufficient to induce slowed growth, while inhibition of TGFβ signaling using a TGFβ receptor inhibitor rescued growth rates in T790M amplified cell lines. Finally, T790M harboring patient tumor samples showed a trend of TGFβ2 transcript upregulation.

Conclusions:

We have demonstrated that TGFβ2 is upregulated in EGFR T790M amplified lung cancer cells, and is sufficient to elicit T790M associated growth retardation. The correlation between TGFβ expression and indolent growth of T790M expressing tumors identifies TGFβ as a potential biomarker to predict patient prognosis and outcome.

#891

Dual defects of MDM2/MDMX-p53 pathways cause global metabolic disruption and enhance tumorigenesis.

Hua Lu, Yi-wei zhang, Peng Liao, Wenjuan Liao, Jiaxiang Chen, Shelya X. Zeng. _Tulane University School of Medicine, New Orleans., LA_.

Although recent discoveries have linked the tumor suppressing functions of p53 to its regulation on cellular metabolism, the exact signaling pathways underlying how p53 is activated in response to various metabolism stresses, and how p53 coordinates the systemic metabolism homeostasis and further blocks tumorigenesis remain largely unclear. Previous studies have shown that the mice containing a single knock-in at cysteine residue 305 of MDM2 (MDM2C305F) is defective in ribosomal stress (RS) induced p53 activation and display severe nutrient shortage-induced hepatosteatosis. And very recently we found that in response to metabolic stress (MeS), AMPK directly phosphorylates serine 342 of MDMX, leading to p53 activation, which is defective in MDMX3SA mice harboring a mutant MDMX with S341A, S367A and S402A mutations. In the current study, we want to determine if the dual defects in the MDM2/MDMX-p53 pathways might thoroughly impair p53's ability to regulate metabolic homeostasis and thus favor metabolism-related tumorigenesis, by generating a double knock-in (DKI) MDMX3SA/MDM2C305F mouse line. Our results indicate that DKI mice on normal chow diet exhibit mild, but significant, metabolism abnormalities, including increased body weight, higher fasting-glucose level and impaired glucose tolerance, which is exacerbated by high-fat-diet feeding. Consistently, MEFs isolated from DKI mice possess higher capacity of differentiation into adipocytes, and less beige fat is induced in the subcutaneous white adipose tissue of DKI mice after chronic cold exposure. Furthermore, we observe that the survival rate of DKI mice is significantly lower compared to either control or single knock-in groups, which may be due to the higher morbidity of cancers in DKI mice, including hepatoma, lymphoma and lung carcinoma. Because of these observations, we further specifically investigate if dual defects of these p53 activation pathways will accelerate hepatocellular carcinogenesis or not. We indeed find that treatment of carcinogen DEN causes more and bigger tumor nodules in the liver of DKI mice. These results suggest that both RS-MDM2-p53 and MeS-MDMX-p53 pathways play important roles in regulation of global metabolism, and dual defects of these pathways benefit metabolism-related tumor formation and growth. Further study is required to establish the direct effect of the p53 pathway defects-mediated metabolic disruption on tumorigenesis, and determine the underlying mechanisms.

#892

Functional characterization of EIF1AX mutations in thyroid cancer predicts for gain of function by increasing translational rate with concomitant derepression of upstream inputs from mTOR.

Gnana P. Krishnamoorthy,1 Inigo Landa,1 Jeffrey A. Knauf,1 James Nagarajah,1 Gunnar Rätsch,2 Hans-Guido Wendel,3 James A. Fagin1. 1 _Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY;_ 2 _Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY;_ 3 _Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY_.

EIF1AX (eukaryotic translation initiation factor 1A) is a component of the translation pre-initiation complex (PIC). Recurrent EIF1AX mutations, first reported in uveal melanomas, are found in ~1% of papillary thyroid cancers in a mutually exclusive manner with other oncogenic driver events (BRAF, RAS, and oncogenic fusions). By contrast, they are enriched in advanced thyroid cancers (9% of anaplastic and 11% poorly-differentiated thyroid cancers), and are strongly associated with RAS mutations (p<0.0001). EIF1AX mutations cluster in the N-terminal (NTT) or C-terminal tails (CTT). EIF1AX NTT missense mutations in thyroid cancer occur within the first 15 amino acids, whereas the CTT mutation disrupts a splice acceptor site at exon 6 (A113splice). A113splice is the most prevalent defect and is private to this disease, and results in two differentially spliced mRNAs: (1) Cryptic splice variant: by use of a cryptic acceptor site in exon 6 that leads to a 132 AA protein that excludes 12 AA; (2) Truncated splice variant: by retaining intron 5, leading to a 115 AA truncated protein. EIF1AX mutants retain the ability to recruit the ternary complex, as shown by co-IP with EIF2 after ectopic expression of NTT or A113splice EIF1AX mutants in HEK293T cells, or after CRISPR-mediated knock-in of A113splice into Cal62 RAS-mutant thyroid cancer cells. The EIF1AX mutants had greater affinity to EIF5 compared to WT, consistent with a more stable PIC. As translation initiation is a rate-limiting step, the altered affinity of EIF1AX mutants to PIC components could impact the rate of protein synthesis. We tested this by L-azidohomoalanine labeling, which showed contrasting roles of the two A113-generated splice variants expressed at endogenous levels: i.e. the cryptic splice variant increased protein synthetic rate, whereas the truncated splice variant strongly inhibited protein translation. Despite inhibiting translation, the truncated splice variant showed a paradoxical increase in 4EBP1 phosphorylation. Upon A113splice knock-in, where both variants are expressed, translation is increased, which we hypothesize results from the combined effects of 4EBP1 phosphorylation, caused by relief of negative feedback events upstream in the pathway, with increased PIC assembly caused by the cryptic splice variant. We are currently determining whether the altered rate of protein synthesis is global or selective. Of note, cells expressing the cryptic, but not the truncated splice form, showed a transforming phenotype as assessed by soft agar colony formation. As EIF1AX mutations co-occur with RAS mutations in advanced thyroid cancers, it is likely that RAS-induced PI3K-AKT/mTOR signaling may provide a further cooperative benefit and play a key role in disease progression, and in generating specific tumor cell dependencies.

#893

**Diffuse gastric adenocarcinoma often harbors** KMT2C **mutations resulting in malignant phenotypes and worse overall survival.**

Soo-Jeong Cho,1 Changhwan Yoon,2 Jun Ho Lee,2 Kevin K. Chang,2 Bulent A. Aksoy,2 Do Joong Park,3 Sam S. Yoon2. 1 _National Cancer Center, Goyang, Republic of Korea;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Bundang Hospital, Seongnam, Republic of Korea_.

Purpose: The Lauren diffuse type of gastric adenocarcinoma (DGA) is genomically stable compared to the intestinal type, but the primary driver mutations in DGA are still being defined. Lysine (K) -specific methyltransferase 2C (KMT2C), also known as mixed lineage leukemia 3 (MLL3), is a histone methyltransferase involved in transcriptional coactivation and can be mutated in gastric cancer. The purpose of this study was to determine the frequency of KMT2C mutation in DGA and to elucidate the role of KMT2C in DGA tumorigenesis and progression.

Methods: Whole exome sequencing was performed on matched tumor samples of 27 patients with DGA who underwent potentially curative surgical resection. Immunohistochemistry (IHC) was performed for KMT2C protein expression on these same tumors and on an additional 289 tumors from a separate cohort of patients with DGA. KMT2C overexpression in DGA cell lines (MKN-45 and SNU-668) using lentiviral transduction and KMT2C shRNA knockdown in normal gastric epithelial cells were examined in several in vitro assays.

Results: Thirty-eight percent of the 27 sequenced DGA specimens harbored somatic mutations in KMT2C. The next most frequent mutation, CDH1, which encodes E-cadherin, was found in 28% of specimens. Patients with KMT2C mutation tended to have worse overall survival (OS) compared to patients without the mutation (p = 0.194). Additional tumor tissue for IHC analysis was only available from 11 of 27 patients. Among this group, patients with tumors expressing KMT2C protein had better OS compared to patients whose tumors did not express KMT2C (p = 0.034). In an external validation set of 289 DGA samples, KMT2C expression again correlated with better OS (p = 0.009). In the DGA cell lines, KMT2C overexpression reduced proliferation by 32-39%, soft agar colony formation by 75-77%, spheroid cell formation by 77-78%, cell migration by 52-60%, and cell invasion by 50-74%. KMT2C overexpression also increased resistance to 5-FU and cisplatin chemotherapy. KMT2C shRNA knockdown in gastric epithelial cells increased proliferation, soft agar colony formation, migration, and invasion by 1.7- to 25-fold.

Conclusions: KMT2C is frequently mutated in DGA, and loss of KMT2C protein expression correlates with worse OS. Restoration of KMT2C expression in DGA cell lines reduces cancer stem-like cell and malignant phenotypes. Thus, KMT2C may be a novel therapeutic target for patients with DGA.

#894

Discovery of YAP-TEAD protein-protein interaction (PPI) inhibitors for cancer therapy.

Anne Soude, Martine Barth, Stephanie Bocart, Frederic Thoreau, Philippe Masson, Isabelle Braccini, Christian Montalbetti, Pierre Broqua, Claudia Fromond. _Inventiva, Daix, France_.

The Hippo pathway is emerging as a critical player in several processes involved in cancer progression, including cell proliferation and EMT. YAP mediates the downstream effects of Hippo signaling, and high nuclear expression of YAP has been documented in many tumors types. YAP translocates to the nucleus, binds to TEAD transcription factor and drives the expression of growth factors, including CTGF, Cyr61 and surviving, suggesting that therapies targeting YAP-TEAD interaction are likely to have clinical impact for the treatment of cancer patients. However, due to the challenging nature of protein-protein interactions, a potent inhibitor that surpasses the affinity of the YAP-TEAD interactions has not been developed. All the critical residues for the YAP-TEAD interaction belong to interface S3. In order to identify interface S3 binders able to disrupt the YAP-TEAD interaction, we started a drug discovery program based on combined FBS/HTS strategy. TEAD protein drugabbility was assessed by fragment screen using NMR and SPR technologies. This approach, supported by assigned HSQC protein NMR information, provided fragment hits which were used as starting points for the identification of more potent binders. We also designed an AlphaLisa assay to validate the ability of our compounds to disrupt the YAP-TEAD interaction. 240,000 compounds of our proprietary library were screened using AlphaLisa assay. Several hits in the µM range were identified and further confirmed using SPR. Successful chemistry optimization generated compounds showing inhibitory activity in the AlphaLisa assay at 50-100 nM for the best compounds. To confirm the validity of our inhibitors, we constructed plasmids encoding TEAD protein fused to Gal4 DNA-binding domain. We transfected HEK293 cells with these plasmids together with Gal4-driven luciferase reporter plasmid in the presence of YAP S127A/S397A. Our compounds showed marked inhibition of cell-based transactivation assay at 3-10 µM. In order to establish a panel of cancer cell lines whose proliferation and target gene expression were either dependent or independent of YAP/TEAD, we selected several cell lines based on their mutational status, YAP nuclear localization, and their proliferative response to a siRNA targeting either YAP or TEAD. Our data show that YAP-TEAD inhibitors block both cell proliferation and target gene expression only in YAP/TEAD-dependent cell lines. In conclusion, we have successfully demonstrated that YAP-TEAD PPI can be inhibited by small molecules, and our compounds show activity in cell-based assays. Medicinal chemistry optimization on various series is ongoing to improve potency and DMPK parameters for further in vivo evaluation in cancer xenograft models.

## PREVENTION RESEARCH:

### Highlights in Cancer Prevention Advances

#895

Genomic characterization of premalignant lung squamous cell carcinoma lesions.

Joshua D. Campbell,1 Catalina Perdomo,2 Sarah Mazzilli,2 Yaron Geshalter,2 Samjot S. Dhillon,3 Gang Liu,2 Sherry Zhang,2 Hangqio Lin,2 Jessica Vick,2 Christopher Moy,4 Evan Johnson,2 Matthew Meyerson,1 Suso Platero,4 Marc Lenburg,2 Mary Reid,5 Avrum Spira,2 Jennifer Beane2. 1 _Broad Institute, Cambridge, MA;_ 2 _Boston University Medical Center, Boston, MA;_ 3 _Roswell Park Cancer Institute, Buffalo, NY;_ 4 _Jannsen Pharmaceuticals, Horsham, PA;_ 5 _Roswell Park Cancer Institute, Buffalo, MA_.

Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions. However, not all premalignant lesions will progress to lung SqCC and many of these lesions will regress without therapeutic intervention. Understanding the molecular events that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention.

Methods: Bronchial brushings and biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject (n=30), both premalignant lesions (PMLs) and the cytologically normal mainstem bronchus were sampled repeatedly over time (n=288 samples). DNA and RNA were isolated from a total of 197 bronchial biopsies of PML (average of 5 per subject) and 91 bronchial brushings. DNA was also isolated from the blood to serve as a matched normal. Exome capture was performed using the Agilent SureSelect Human All Exon+UTR 70MB kit and sequenced to a mean depth of coverage of 75x (n=85 samples from 22 subjects). RNA libraries were prepared with Illumina TruSeq (mRNA-Seq: n=288 samples from 30 subjects and miRNA-Seq: n=183 samples from 26 subjects).

Results: We identified gene and miRNA expression changes associated with histological grade as well as progressive/stable disease. The Hippo pathway, Wnt signaling, p53 signaling, and immune-related pathways are modulated with histological grade and disease progression. Genes associated with histological grade in the cytologically normal airway and in the biopsies were significantly concordantly enriched (FDR<0.05) demonstrating a strong relationship between the PMLs and the field of injury. The somatic mutation rate of PMLs displayed no significant association with histological grade (p=0.65). Mutations in previously characterized lung cancer genes included TP53 (3%), CREBBP (3%), FAT1 (3%), and NOTCH1 (9%). Examining copy number alterations revealed a single metaplastic lesion with an arm-level amplification on chr5p containing TERT. The two lesions with the highest mutation rates (>3/Mb) were taken from adjacent sites over two time points in the same individual with a history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations (p=2.2 x 10-17) indicating a common evolutionary ancestor, and contained mutations in CREBBP and FAT1, suggesting they are at increased risk for progressing to frank malignancy.

Conclusions: We performed genomic profiling of PMLs in the airways of high-risk smokers. The gene expression and somatic alterations that were observed in known cancer genes may be among the earliest events in cancer development.

#896

The airway field of injury reflects gene expression changes associated with the presence of lung squamous premalignant lesions.

Sarah A. Mazzilli,1 Ania Tassinari,1 Yaron Gethalter,1 Gang Lui,1 Mary Pine,2 Stephen Lam,3 Mary Reid,2 Suso Platero,4 Marc Lenburg,1 Avrum Spira,1 Jennifer Beane1. 1 _Boston University, Boston, MA;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _University of British Columbia, British Columbia- British Columbia Cancer Agency, British Columbia, Canada;_ 4 _Janssen Research and Development, PA_.

Lung squamous cell carcinoma (SCC) arises in the epithelial layer of the bronchial airways and is preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs that develop go on to form carcinoma. In addition, the majority of lung cancer chemoprevention agents tested to date are ineffective. Molecular characterization of the airway field of injury in individuals with PMLs could provide novel insights into the earliest molecular events associated with carcinogenesis and identify biomarkers to guide lung cancer detection and chemoprevention.

RNA-sequencing was conducted on cytologically normal airway brushings from current and former smokers with (n=50) and without (n=25) PMLs as part of the British Columbia Lung Health Study. Linear modeling strategies were used to identify 280 differentialy expressed genes at FDR<0.002 between subjects with and without PMLs. Pathway analysis using GSEA and ROAST revealed enrichment of genes involved in the electron transport chain and oxidative phosphorylation pathways in subjects with PMLs. These findings were validated by measuring the cellular bioenergetics of cultured epithelial cells from biopsies of PMLs and non-lesion areas. Baseline oxygen consumption rates were 2.5 fold higher (p<0.001) and the spare respiratory capacity was 1.5 fold higher (p<0.001) in PML cultures. These data suggest that metabolism-associated gene expression observed in the field of injury of PMLs is correlated with PMLs. In addition, there is a significant concordant enrichment (FDR<0.05) between the signature and gene expression in PMLs adjacent to SCC tumors, in SCC tumors, and in the field of individuals with lung cancer. This concordance led to the development of a 200-gene biomarker that accurately predicts the presence of PMLs (AUC=0.90 n=17 independent samples). Importantly, this biomarker was also predictive (AUC -.72) of progression/stability vs. regression of these premalignant lesions in an independent cohort of cytologically normal airway brushings collected as part of the RPCI screening clinic (n=18).

This is the first study to comprehensively profile gene expression changes in airway epithelial cells in the presence of PMLs. A subset of these changes reflects the earliest changes in the process of lung squamous cell carcinogenesis including the genes involved in cellular metabolism. However, the molecular alterations in the field of injury are dynamic as bronchial lesions either progress or regress these changes may be leveraged to monitor efficacy in chemoprevention trials. In addition monitoring molecular changes in high-risk smokers may identify smokers with PMLs that should receive lung cancer screening as well as lay the foundation for personalized lung cancer chemoprevention.

#897

Methionine restriction alters functional polarization of macrophages in a murine model of prostate cancer.

Ashley Orillion,1 Remi Adelaiye-Ogala,1 Li Shen,2 Eric Ciamporcero,3 May Elbanna,1 Sreenivasulu Chintala,1 Sreevani Arisa,1 Ben Elzey,4 Chinghai Kao,1 Luigi Fontana,5 Roberto Pili1. 1 _Indiana University - Purdue University Indianapolis, Indianapolis, IN;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _University of Turin, Turin, Italy;_ 4 _Purdue University, Lafayette, IN;_ 5 _Washington University, St. Louis, MO_.

Background: Epidemiological studies link prostate cancer (CaP) to dietary intake. Recent research has shown that individuals consuming a Western diet, high in protein, have higher circulating IGF-1 (insulin-like growth factor 1) which feeds into the nutrient sensing mTOR pathway. This evolutionarily conserved pathway is central to the development of advanced stage and castration resistant CaP. Our previous published studies have shown that dietary protein restriction inhibits tumor growth via mTOR pathway alteration and reduces circulating IGF-1 levels in vivo. The current study hypothesizes that dietary methionine restriction will alter the functionality of immune cells enhancing the ability of the immune system to respond to cancerous insults.

Methods: Mice were fed low (7% protein) or control protein (21% protein) diets. In a prevention study mice consumed modified diets for four weeks prior to being inoculated with CaP tumors. In a simultaneous intervention study mice were inoculated with CaP tumors first then four weeks later were placed on the modified diets.

Results: In the low protein diet group, we observed that pro-tumor M2 polarized macrophages were significantly reduced in the tumor microenvironment (TME) for both prevention (p=0.012) and intervention diet (p=0.0003) studies as compared to the control diet, in addition to observation of mTOR inhibition and IGF-1 reduction. Besides protein content in diet, we also analyzed the amino acid composition. Multiple groups have shown that methionine restriction (MR) inhibits mTOR activation, alters the immune system, and prolongs the survival of rodents. The mechanism by which MR impacts the innate immune system is still poorly understood. In our preliminary in vitro studies, we observed that modification of a single amino acid (AA), such as methionine, is sufficient to inhibit activation of both the mTOR pathway and M2 polarization (Arginase1 qRT-PCR of two samples in quadruplicate (p=0.0732)), while increasing the presence of M1 macrophage marker iNOS (p=0.2689) in bone marrow derived macrophages (BMDMs). Thus, we hypothesize that modulating specific amino acids in the diet is sufficient to alter macrophage expression of M1/M2 characteristics, and their functions in the TME.

Conclusions: Preliminarily data shows that altering one specific AA intricately linked to the mTOR pathway is sufficient to change macrophage polarization status both at the protein and gene expression levels, suggesting that dietary alteration of a single AA is capable of altering macrophage function. The results of the study provide the basis for translational use of dietary means to alter the immune system and improve the therapeutic effects of immunotherapies.

#898

Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy.

Joao Incio,1 Priya Suboj,1 Shan M. Chin,1 Chen Ivy,1 Mei Ng,1 Hadi Nia,1 Jelena Grahovac,1 Hao Liu,1 Shannon Kao,1 Suboj Babykutty,1 Yuhui Huang,1 Keehoon Jung,1 Nuh Rahbari,1 Xiaoxing Han,1 Vikash Chauhan,1 John Martin,1 Julia Kahn,1 Peigen Huang,1 Raquel Soares,2 Yves Boucher,1 Dai Fukumura,1 Rakesh Jain1. 1 _Harvard Medical School/MGH, Boston, MA;_ 2 _FMUP, Portugal_.

INTRODUCTION: With the current epidemic of obesity, the majority of pancreatic cancer patients are overweight or obese at diagnosis. Importantly, obesity worsens treatment outcomes in pancreatic cancer patients. Therefore, understanding the mechanisms that underlie the poorer prognosis of obese cancer patients is of paramount importance. Obesity causes inflammation and fibrosis in the normal pancreas due to the accumulation of dysfunctional hypertrophic adipocytes. Importantly, desmoplasia - a fibroinflammatory microenvironment - is a hallmark of pancreatic ductal adenocarcinoma (PDAC), and we have shown that activation of pancreatic stellate cells (PSCs) via angiotensin-II type 1 receptor (AT1) pathway is a major contribution to tumor desmoplasia. Whether obesity affects desmoplasia in PDACs, and interferes with delivery and response of chemotherapeutics is currently unknown.

EXPERIMENTAL DESIGN: Using both human samples and mouse models of PDAC - multiple syngeneic models of PDAC: PAN02, AK4.4, KPC, iKRAS in diet-induced and genetic obese mouse models -, we determined the effects of obesity on desmoplasia and inflammation/immune cell infiltration, tumor growth and delivery and response to chemotherapy.

RESULTS: We found that obesity aggravates desmoplasia in PDACs in both patient samples and multiple mouse models. In addition, tumors in obese mice presented with elevated levels of activated PSCs and fibrosis, as well as inflammatory cytokines and TANs,. These alterations in the tumor microenvironment in obesity associated with accelerated tumor growth, reduced tumor blood perfusion and increased hypoxia, and impaired delivery and efficacy of chemotherapeutics. Genetic ablation and pharmacological inhibition (losartan) of AT1 signaling reversed obesity-augmented desmoplasia and tumor growth, and improved the response to chemotherapy to the level observed in lean mice. We further discovered the underlying mechanisms: 1) obesity increases intra-tumor adipocytes and IL-1ß secretion by these cells; 2) increased IL-1ß induces TAN recruitment; 3) recruited TANs activate PSCs; and 4) activated PSCs enhance desmoplasia. Conversely, activated PSCs also secrete IL-1ß that recruits further TANs. Of clinical relevance, we found that metformin not only normalizes the abnormal systemic metabolism, but also reprogramms PSCs and immune cells and alleviates the fibroinflammatory microenvironment in pancreatic cancer in obesity/diabetes.. Importantly, the strategies described above were not effective in the normal weight setting.

CONCLUSION: Here we successfully demonstrated that targeting desmoplasia, including immunomodulation with anti-IL-1ß, or treatment with generic drugs such as losartan and metformin are potential strategies to potentiate treatments in PDAC patients with excess weight.

#899

Cancer protection associated with dietary methyl donor deficiency is characterized by persistent changes to epithelial proliferation and metabolism.

Matthew P. Hanley, Daniel W. Rosenberg. _UConn Health, Farmington, CT_.

The role of folate one-carbon metabolism in colorectal cancer development is incompletely understood, and nutritional intervention studies have produced conflicting results. We previously demonstrated that a diet deficient in the methyl donors folic acid, methionine, choline and vitamin B12, and supplemented with homocysteine, reduces intestinal tumor incidence by greater than 95% in ApcMin/+ mice. Here we extend these findings to a second mouse tumor model, ApcΔ14, and further show that the cancer protection afforded by even short-term dietary methyl donor deficiency (MDD) is long-lasting. Eleven weeks of MDD followed by methyl donor repletion was sufficient to maintain tumor suppression for at least 7 additional weeks (22.2 ± 3.5 vs 70.2 ± 4.6 intestinal tumors; p < 0.001). Sustained tumor protection was associated with altered intestinal crypt homeostasis. MDD increased the proportion of intestinal epithelial cells undergoing apoptosis in normal crypts and in tumors (4.9- and 3.2-fold, respectively), while reducing cell proliferation (Ki-67) and mitosis (PHH3). In addition, metabolomic profiling and metabolite set enrichment analysis (MSEA) revealed that tumor protection is associated with persistent alterations to metabolic pathways related to cellular proliferation, including glutamine metabolism, biogenic amine synthesis, nucleotide salvage and glutathione synthesis. Taken together, these results indicate that even a temporary dietary methyl donor restriction in cancer-prone mice can induce persistent changes to the intestinal epithelium that provide long-lasting protection in adult mice.

#900

In vivo **modeling of** NRAS **-mutant melanoma reveals differential preventative efficacy amongst SPF30 sunscreens.**

Andrea M. Holderbaum, Rebecca C. Hennessey, James E. Gillahan, Anamaria Bonilla, Conor Delaney, Raleigh D. Kladney, Kathleen L. Tober, Tatiana M. Oberyszyn, Christin E. Burd. _The Ohio State University, COLUMBUS, OH_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2016 Official Press Program. It will be posted online following its presentation.

#901

Transgenerational inheritance of increased mammary cancer risk in the offspring of high fat diet fed dams: Changes in oxidative stress pathways.

Nguyen M. Nguyen, Fabia O. Andrade, Sonia DeAssis, Idalia Cruz, Carlos Benitez, Roger Godschalk, Leena Hilakivi-Clarke. _Georgetown University, Washington, DC_.

Maternal high fat diet has been shown to increase mammary cancer risk among female offspring. We investigated here if the increase is seen across multiple generations. For that purpose, pregnant C57BL/6NTac dams (F0) were fed either a control (CON, 6% corn oil) or an isocaloric high n-6 polyunsaturated fat (HF) diet (18% corn oil) between gestation days 8 and 21. Offspring in subsequent generations (F1-F3) were not exposed to any further dietary modifications, and kept on CON diet. Mammary tumors were initiated by priming the mammary glands with 15 mg medroxyprogesterone (MPA) at week 6, followed by a 1 mg dose of 7,12-dimethylbetz(a)anthracene (DMBA) given on weeks 7-9 for a total of 3 doses. Animals were monitored for 20 weeks after the final dose of DMBA for tumors. Both F1 and F3 generation female HF offspring exhibited a significantly higher mammary tumor incidence and tumor burden than the control offspring. In F3 generation, the increase was seen from week 12 of tumor monitoring until the end of the study. The increase in risk was preceded by a higher number of terminal end buds in the F1 and F3 generation mammary glands. Malondialdehyde (MDA) adduct levels were found to be significantly elevated in the HF group of F3 offspring, suggesting that the increased mammary cancer risk in this generation was attributed to oxidative stress. Western blot analysis of mammary glands identified significantly elevated Keap1 (a repressor of the antioxidant response) and PERK levels in HF F3 offspring; and elevated levels of Beclin-1 and p62 in both F1 and F3 generations compared with CON offspring, corroborating with the idea that oxidative stress was the culprit. miRNA analysis of F3 mammary glands showed decreased expression of mir-324-5p, mir-136 and mir-378 that target Keap1 and p62, potentially explaining the increase in Keap1 and p62 levels. Our results suggest that oxidative stress and antioxidant response plays a role in explaining transgenerational increase in mammary cancer risk among offspring exposed to high fat diet through F0 dams.

## TUMOR BIOLOGY:

### Immunomodulation in Cancer

#902

A small molecule glycomimetic antagonist of E-selectin and CXCR4 (GMI-1359) delays pancreatic tumor metastasis and significantly alters the pancreatic tumor microenvironment.

Maria M. Steele,1 William E. Fogler,2 John L. Magnani,2 Michael A. Hollingsworth1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _GlycoMimetics, Inc., Rockville, MD_.

The cellular and molecular composition of the pancreatic tumor microenvironment includes a complex matrix that encases tumor cells and presents a conundrum in respect to therapy. The dense desmoplasia that accompanies tumor growth reduces blood flow to tumors and creates an environment that compromises delivery of therapeutics. As well, elimination of certain stromal elements enhances the aggressiveness and metastatic potential of pancreatic cancer cells. Recent studies attempt to target stroma in an effort to enhance delivery and efficacy of therapeutic agents and to block metastasis. In this context we have investigated the activity of GMI-1359, a potent dual antagonist that targets both E-selectin and CXCR4. Adhesion protein E-selectin is crucial for regulating vascular and lymphatic endothelial cell (EC) interactions with tumor cells during transmigration. The CXCL12-CXCR4 chemokine axis contributes to the formation of the tumor microenvironment including fibroblast and immune cell recruitment, lymph- and angiogenesis, tumor cell proliferation/survival, and tumor stem cell mobilization. Our in vitro results demonstrate that pancreatic fibroblasts secrete significant amounts of CXCL12 which promotes tumor cell and lymphatic and vascular EC directional migration; this migration toward CXCL12-secreting fibroblasts was completely blocked by GMI-1359. Additionally, CXCL12-stimulated ECs facilitated increased transendothelial migration (TEM) by pancreatic tumor cells. Dual antagonist GMI-1359 inhibited this CXCL12-dependent increase in pancreatic tumor cell TEM and was more effective than an independent small molecule E-selectin only inhibitor. Using an in vivo orthotopic model of pancreatic cancer in athymic mice, we evaluated the ability of GMI-1359 (with and without co-administration Gemcitabine (Gem)) to suppress tumor progression, modulate tumor microenvironment composition, and prolong survival. Our work demonstrated GMI-1359 slightly inhibited tumor growth when used alone or in combination with Gem but did not prolong survival in this immune compromised model. However, GMI-1359 inhibited tumor metastasis to the spleen, liver, and lungs. Interestingly, GMI-1359 significantly modulated the cellular composition of the tumor microenvironment. Immunohistochemical analysis revealed mice treated with GMI-1359 (with or without Gem administration) had drastically reduced desmoplasia and reduced lymphatic and blood vessel densities compared to mice treated with vehicle control or Gem alone. The E-selectin only inhibitor had no effect on tumor microenvironment composition. Further studies of GMI-1359, particularly in immune competent models, are warranted to understand its potential for disrupting the pancreatic tumor microenvironment, inhibiting dissemination, and enhancing anti-tumor immune responses.

#903

IFN-γ induced PD-L1 on tumor and host cells co-operatively prevents tumor immune elimination after cancer immunoediting.

Takuro Noguchi, Jeffrey P. Ward, Matthew M. Gubin, Cora Arthur, Robert D. Schreiber. _Washington University in St. Louis, St. Louis, MO_.

Tumor cells can escape immune destruction via engaging the PD-1/PD-L1 inhibitory pathway. However since PD-L1 is induced on many different cell types by IFN-γ, the importance of PD-L1 on tumor versus host cells in mediating immune escape remains unclear. In this study, we assessed the role of PD-L1 expression on two closely-related but distinct clones derived from the same d42m1 methylcholanthrene (MCA)-induced sarcoma line. Edited d42m1-T3 sarcoma cells grow progressively in wild type (WT) mice but are rejected in mice treated with anti-PD-L1. Upregulation of PD-L1 was observed on both immune and tumor cells in vivo. To assess the role of PD-L1 specifically on tumor cells, we generated d42m1-T3 cells lacking PD-L1 (T3ΔPDL1) using CRISPR-Cas9. T3ΔPDL1 cells were spontaneously rejected in naïve syngeneic WT mice but formed progressively growing tumors in Rag2-/- mice. To explore the role of host-cell expressed PD-L1, we challenged WT mice with 3-fold higher numbers of T3ΔPDL1 cells and found that the tumor cells formed progressively growing tumors in WT mice. However, when tumor bearing mice injected with the high dose of T3ΔPDL1 cells were treated with blocking PD-L1 mAb, they all rejected their tumors. Thus, PD-L1 expression on both tumor and host cells plays a critical role in preventing immune elimination of edited MCA sarcoma cells. Alternatively highly immunogenic, unedited d42m1-T9 sarcoma cells were spontaneously rejected in WT mice due to the presence of the strong rejection antigen, mutant Spectrin-β2. Enforced expression of PD-L1 on unedited d42m1-T9 cells to levels similar to those induced under physiologic in vivo conditions did not alter the regressor phenotype of these cells. In contrast, enforced overexpression of PD-L1 on d42m-T9 cells to levels that were 20 fold higher than physiologic led to formation of progressively growing tumors in WT mice. Thus, physiologic levels of PD-L1 expression on highly immunogenic unedited tumor cells are not sufficient to counteract immune effector mechanisms driven by "strong" tumor antigens. This result shows that adaptive immune resistance occurs only on tumor cells that have undergone immunoediting.

#904

Heterozygous ATG7 inhibition enhances endocrine therapy responsiveness through regulation of damage associated molecular patterns and priming the immune system in ER+ breast tumors.

Robert Clarke,1 Pamela A.G. Clarke,1 Anni Wärri,1 Katherine L. Cook2. 1 _Georgetown University, Washington, DC;_ 2 _Wake Forest Univ. School of Medicine, Winston-Salem, NC_.

Breast cancer is the most prevalent cancer in women, with over 230,000 new cases diagnosed annually. The most common type of breast cancer, comprising over 70% of all cases, express the estrogen receptor (ER); these tumors are usually treated with an endocrine therapy such as an antiestrogen or aromatase inhibitor. Unfortunately, many of these tumors develop resistance, or in some instances express de novo resistance, which limits the curative potential of these therapies. Autophagy, a cellular process of "self-eating", is implicated as possible contributor to endocrine therapy resistance in breast cancer. We previously showed that inhibiting autophagy through low-dose chloroquine treatment reversed antiestrogen therapy resistance in ER+ orthotopic breast tumors. We now show that heterozygous deletion of ATG7 decreased breast tumor multiplicity and increased endocrine targeting therapy sensitivity in a DMBA-model of ER+ mammary carcinogenesis. Knockdown of ATG7 through RNAi increased cytoplasmic to nuclear high-mobility group binding protein B1 (HMGB1) protein ratio, suggesting targeting ATG7 promotes damage associated molecular patterns (DAMP) signaling. Heterozygous deletion of ATG7 also increased cytoplasmic HMGB1 protein expression in tumor epithelial cells indicating targeting ATG7 promotes DAMP signaling in vivo. Furthermore, tumors from ATG7 heterozygous mice displayed increased CD68 staining indicating elevated macrophage infiltration. Corroborating previous literature showing the critical role of autophagy in T-cell maturation, splenic CD8+ T-cells were significantly reduced in tumor bearing ATG7 heterozygous mice. However, ATG7 heterozygous mice displayed increased tumor CD8+ T-cell population. These data suggest that while targeting ATG7 reduces overall circulating T-cell populations, inhibiting ATG7 enhances tumoral cytotoxic T-cell recruitment. Tumors from ATG7 heterozygous mice also showed a significant decrease in foxp3+ cells, indicating targeting ATG7 reduces immuno-suppressive T-reg population. Serum from ATG7 heterozygous mice showed increased circulating MDC, MCP-1, IL-6, and eotaxin cytokines. Taken together, these data suggests that ATG7 inhibition promotes an anti-tumor pro-inflammatory cytokine profile to enhance tumoral immune cell recruitment and promote endocrine therapy responsiveness.

#905

Pterostilbene (PTER) suppresses breast cancer brain metastasis by targeting a c-Met mediated inflammation network.

Fei Xing, Yin Liu, Sambad Sharma, Kerui Wu, Kounosuke Watabe. _wake forest baptist health, Winston-Salem, NC_.

At the late stage of breast cancer, most patients develop metastatic lesions and the brain is one of the major metastatic sites. Brain metastasis profoundly affects the cognitive and sensory function as well as morbidity of patients, and the one year survival rate among these patients remains less than 20%. The result of our newly developed gene set enrichment analysis (GSEA)-based pathway screening indicates that c-Met pathway is highly activated among the patients who developed early brain metastasis. To identify a novel target of c-Met pathway in brain metastasis, we performed the microarray analysis in two brain metastatic cell lines with the expression of doxycycline-inducible sh-cMet. Our results indicate that a group of inflammatory cytokines including IL1b, IL8 and CXCL1 were significantly induced by c-Met activation. Furthermore, we found that IL1b was able to induce the secretion of HGF (hepatocyte growth factor) from tumor associated astrocytes (TAA) which in turn activates c-Met pathway in cancer cells. Our results of endothelial cell tube formation assays also strongly suggest that cMet-induced IL8 and the activation of its receptor, CXCL1, promote tumor angiogenesis which is essential for the metastatic growth of cancer cells. Natural compounds (NC) have been extensively studied for their anti-tumor effects. However, much less studies have been done on NC for the treatment/prevention of brain tumors mainly due to the obstacle of Blood-brain-barrier (BBB) permeability. To identify the NCs that can be used for treating brain metastasis, we performed pathway analysis which only focused on BBB-permeable NC. The results of GSEA indicate that Resveratrol-targeting genes were significantly enriched among those patients who developed brain metastasis compared to metastasis-free patients. Furthermore, we found that the one of derivatives of Resveratrol, Pterostilbene (PTER), showed significantly more potent activity than Resveratrol and suppressed brain metastasis in vitro and in vivo by targeting the c-Met oncogene. These findings suggest that TAA-mediated c-Met activation plays a key role in brain metastasis and that PTER is a potential therapeutic agent to treat brain metastasis by suppressing the c-Met expression.

#906

Exosomes from mutant TP53 cancer cells polarize tumor associated macrophages.

Tomer Cooks,1 Ioannis S. Pateras,2 Keval M. Patel,3 Tim Forshew,4 Vassilis G. Gorgoulis,2 Curtis C. Harris1. 1 _NCI, Bethesda, MD;_ 2 _University of Athens, Athens, Greece;_ 3 _4- Addenbrooke's Hospital, Cambridge, United Kingdom;_ 4 _UCL cancer Institute, London, United Kingdom_.

Introduction: TP53 mutants are involved in the pathogenesis of most solid tumors and are known to gain oncogenic functions distinct from their original wild-type form. The existence of such gain-of-function (GOF) activities is supported by ample evidence, however only in a cell-autonomous fashion. Since tumor-associated-macrophages (TAM) are also a hallmark of solid tumors typically correlated with poor prognosis, we investigated the link between mutations in the TP53 gene (mutp53) occurring in epithelial tumor cells and the formation of a surrounding TAM population in-situ.

Methods: By designing a co-culture system we incubated human primary monocytes together with colorectal cancer (CRC) cells differing in their p53 status. Relevant macrophages markers were evaluated on RNA level and protein level. In addition, co-cultured macrophages were subjected to various functional assays (phagocytosis, migration, and invasion). In attempt to confirm clinical relevance, samples from a cohort of human CRC patients were analyzed using genomic and immunohistochemical methods. To identify the interaction between the tumor cells and the macrophages, we isolated exosomes from the CRC cells and subjected them to a Nanostring analysis to learn about their microRNAs composition.

Results: In this study, we discovered that mutp53 exerts a non-cell-autonomous effect over neighboring macrophages by using specific microRNAs (miRs) which are shuttled through an exosomal transfer resulting with a phenotype change of the affected macrophages. Mutp53-specific exosomes containing cargoes such as miR-1246 were shown to be used by macrophages at the receiving end, thus promoting the formation of TAM subset also observed in surgical specimens resected from cancer patients.

Conclusions: Altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" p53 mutants tightening the interaction between the tumor cell and the immune compartment. Additionally, this study is the first to show a non-cell-autonomous role played by mutant p53 - the most common form of mutation found in human cancers. Deciphering the intricate regulation shared by the tumor cell and its surrounding macrophages may give rise to novel prognostic and diagnostic tools as well as to therapeutic approaches targeting TAM, specific tumor-promoting miRs and mutp53-specific subsets of exosomes.

#907

Brain microenvironment induced PTEN loss by microRNAs promotes brain metastasis.

Lin Zhang,1 Siyaun Zhang,2 Jun Yao,1 Frank J. Lowery Lowery,1 Qingling Zhang,1 Wen-Chien Huang,1 Ping Li,1 Min Li,1 Xiao Wang,1 Chenyu Zhang,1 Hai Wang,1 Kenneth Ellis,1 Mujeeburahiman Cheerathodi,1 Joseph McCarty,1 Diane Palmieri,3 Patricia Steeg,3 Jodi S Saunus,4 Sunil Lakhani,4 Suyun Huang,1 Aysegul Sahin,1 Kenneth Aldape,1 Dihua Yu1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Notre Dame, Notre Dame, IN;_ 3 _Woman's Malignancies Branch, National Cancer Institute, Bethesda, MD;_ 4 _University of Queensland Centre for Clinical Research, Herston, Australia_.

Metastasis is the number one cause of cancer-related mortality. Major neoplastic diseases such as melanoma, lung, breast, and colon cancers have high incidences of brain metastases. One-year survival after diagnosis of brain metastasis is less than 20%. Cancer cells dynamically interacts with specific organ microenvironments to establish metastasis as depicted by the "seed and soil" hypothesis. Yet it is unclear when and how disseminated tumor cells acquire the essential traits from the brain microenvironment that primes their subsequent metastatic outgrowth.

Here we found that primary tumor cells with normal PTEN expression lose PTEN after dissemination to the brain, but not to other organs. Metastatic brain tumor cells that have experienced PTEN loss have PTEN levels restored once they leave the brain. This brain microenvironment-dependent, reversible PTEN mRNA and protein down-regulation is epigenetically regulated by microRNAs (miRNAs) from astrocyte-derived exosomes. Furthermore, this adaptive PTEN loss in brain metastatic tumor cells leads to an increased secretion of cytokine chemokine (C-C motif) ligand 2 (CCL2), which recruits Iba1+ myeloid cells that reciprocally enhance outgrowth of brain metastatic tumor cells via enhanced proliferation and reduced apoptosis.

Our findings signify the dynamic and reciprocal cross-talk between tumor cells and other brain stromal cells. Disseminated tumor cells acquire the essential traits from the microenvironment of brain that prime their outgrowth. Importantly, our finding provides new opportunities for effective anti-metastasis therapies: inhibiting CCL2 might be an effective therapeutic intervention of life-threatening brain metastases.

#908

Immune regulation of tumor dormancy in syngeneic mouse model.

Raziye Piranlioglu,1 Maria Ouzounova,1 Eunmi Lee,1 Alicia Hudson,2 Sumeyye Korkaya,3 Ali Arbab,1 Hasan Korkaya1. 1 _Cancer Research Center, Georgia Regents University, Augusta, GA;_ 2 _Georgia Regents University, Augusta, GA;_ 3 _Michigan University, Ann Harbor, MI_.

Metastatic disease -end stage of tumor progression- is the major cause of cancer-related death.It is widely accepted that malignant cell plasticity between epithelial-mesenchymal-transition (EMT) and mesenchymal-epithelial-transition (MET) is required for metastasis to occur. The classical model of metastasis suggests tumor cell dissemination occur late in tumor development, however emerging studies strongly indicates that dissemination is an early process and provide a striking evidence that tumor cells start to disseminate during the initial steps of tumor development. Late appearing metastases arise from these early-disseminated tumor cells. The mechanism by which some early-disseminated tumor cells colonize and generate metastatic growth while some remain dormant is not well known. In order to understand the underlying factors, we utilized murine mammary tumors (4T1 as metastatic and EMT6 as less metastatic) in a syngeneic mouse model. We performed time course experiments to determine the early factors that may contribute to the metastatic growth. 4T1 or EMT6 tumor cells were implanted orthotopically into the fat pads and tumor cell dissemination was analyzed over 3 weeks time points. We determined that both 4T1 and EMT tumor cells disseminated as early as one week post-implantation, however only 4T1 tumor cells develop metastasis in distant organs. Furthermore, we also resected primary tumors 1,2 and 3 week post implantation of EMT6-Luci or 4T1-Luci tumors and observed distant metastasis via optical imaging of luciferase expression in live animals. Although the majority of 4T1 tumor bearing mice (>80%) develop pulmonary metastasis when 4T1 tumors resected 2 and 3 weeks post-implantation, only 10% of mice develop metastasis when primary tumor resected one week post implantation. In contrast, EMT6 tumors following resection only relapsed in the primary tumor site but failed to develop metastasis. We investigated the possible mechanism of efficient pulmonary metastatic growth following the resection of tumors 2-3 week post implantation. Interestingly we found a significant infiltration of granulocytic subset of myeloid derived suppressor cells (g-MDSC) in 4T1 tumor bearing mice by week 2 and 3. Furthermore, we found that lung infiltrated g-MDSCs promote tumor cell growth via paracrine factors. In co-culture studies we found that there is a reciprocal secretion of panel of inflammatory cytokines, growth factors and matrix metalloproteases between tumor cell and g-MDSCs suggesting that these cells in the lung microenvironment support the metastatic growth. Our studies provide a new paradigm in the understanding of metastatic growth and the role of microenvironment in distant organs.

### The Relevance of Stemness Properties in Cancer

#909

How glioma stem cells maintain stemness outside their niche.

Jian Hu, Takashi Shingu, Liang Yuan. _UT MD Anderson Cancer Center, Houston, TX_.

Glioma stem cells (GSCs) have higher self-renewal capacity than neural stem cells (NSCs). However it is still not clear how GSCs maintain their self-renewal outside their niches and whether or not enhanced self-renewal alone without affecting cell proliferation can promote gliomagenesis. Here we report the generation of a novel conditional KO allele of QKI, a tumor suppressor in GBM with RNA binding motif. When QKI was deleted in NSCs isolated from subventricular zone of Nestin-CreERT2 QKIL/L mice, multiple NSC markers were dramatically increased, including Notch1, Sox2,beta-catenin, ID1 and BLBP. When QKI was deleted in adult Nestin-CreERT2 QKIL/L mice, the NSC population, which is characterized by long-term BrdU retention and GFAP+/Nestin+ double positive staining, was greatly increased, indicating that deletion of QKI enhances NSC self-renewal. Surprisingly, we found that deletion of QKI also decreases cell proliferation rate of NSCs/GSCs, suggesting that QKI deletion makes NSCs/GSCs more quiescent. When QKI-/- NSCs were cultured in differentiation medium, they were not able to differentiate like QKI+/+ NSCs, indicating that QKI is required for NSC lineage determination. To determine whether QKI KO promotes gliomagenesis, we generated a Nestin-CreERT2 QKIL/L PtenL/L P53L/L cohort, in which 92% of the mice developed glioblastoma starting at 2 months. However, the Nestin-CreERT2 PtenL/L P53L/L cohort did not develop any glioma up to a year; therefore QKI deletion greatly promotes gliomagenesis. Transcriptomic and proteomic profiling coupled with PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) analyses revealed that genes involved in receptor trafficking were greatly enriched by QKI deletion. Specifically, 34% of the genes up-regulated by QKI deletion are involved in receptor delivery, and 39% of the genes down-regulated by QKI deletion are subunits of receptor degradation machineries such as endosomes and lysosomes. High level of receptor delivery and low level of receptor degradation concomitantly enrich receptors on the membrane and enhance the activity of the receptors that are involved in maintaining stemness, including RTK, Notch1 and Frizzled. Inhibition of the activity of receptors such as EGFR can diminish the enhanced self-renewal and decreased differentiation caused by QKI deletion. Taken together, our data suggest that QKI deletion increases receptor delivery and decreases endocytosis-mediated degradation, consequently enhancing the receptor activity and self-renewal capacity. Therapeutically, because QKI deletion greatly reduces lysosome level and protein degradation capacity, QKI-low cells are under much higher ER-stress compared with QKI-high cells; hence high level of ER-stress may provide an Achilles' heel for QKI-low glioblastoma, and pharmacologically inducing more ER-stress could render cytotoxicity to QKI-low glioma cells while keep normal cells intact.

#910

Genetic subclone heterogeneity of the human colon cancer initiating cell compartment.

Klara M. Giessler,1 Kortine Kleinheinz,2 Gnana Prakash Balasubramanian,3 Daniel Hübschmann,4 Taronish D. Dubash Rai,1 Sebastian D. Dieter,1 Christine Siegl,1 Christopher M. Hoffmann,1 Sarah Weber,1 Raffaele Fronza,1 Saira Afzal,1 Manfred Schmidt,1 Martin Schneider,5 Alexis Ulrich,5 Juergen Weitz,6 Wilko Weichert,7 Matthias Schlesner,2 Benedikt Brors,8 Claudia R. Ball,9 Hanno Glimm9. 1 _Dep. of Translational Oncology, National Center for Tumor Diseases (NCT) and the German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _Div. of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _Div. of Applied Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg and the National Center for Tumor Diseases (NCT), Heidelberg and the German Cancer Consortium (DKTK), Germany;_ 4 _Dep. for Bioinformatics & Functional Genomics, Div. of Theoretical Bioinformatics, DKFZ, IPMB and BioQuant, Heidelberg University, HBIGS Graduate School, Heidelberg University, Dep. of Pediatric Immunology, Hematology and Oncology, University Hospital, Heidelberg, Germany; _5 _Dep. of Surgery, University Hospital Heidelberg and the German Cancer Consortium (DKTK), Germany;_ 6 _Dep. of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus of the Technical University Dresden and the German Cancer Consortium (DKTK), Germany;_ 7 _Institute of Pathology, Ludwig Maximilians University Munich and the German Cancer Consortium (DKTK), Germany;_ 8 _Div. of Applied Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany and the National Center for Tumor Diseases (NCT), Heidelberg and the German Cancer Consortium (DKTK), Germany;_ 9 _Dep. of Translational Oncology, National Center for Tumor Diseases (NCT) and the German Cancer Research Center (DKFZ), Heidelberg and the German Cancer Consortium (DKTK), Germany_.

A subpopulation of tumor-initiating cells (TIC) drives colorectal cancer (CRC) progression in serial xenotransplantation. Strikingly, the CRC TIC compartment itself is heterogeneous and comprised of a hierarchy of long-term (LT-) TIC, tumor transient amplifying cells (T-TAC) and delayed contributing (DC-) TIC. Whether this functionally heterogeneous TIC compartment is genetically homogenous or whether distinct genetic subclones drive the functional heterogeneity of the TIC compartment is yet unknown. To address this question, we performed high coverage (91-126 fold) whole genome sequencing on primary CRC patient tumors (n=3), corresponding serially passaged TIC enriched spheroids as well as spheroid derived serial xenografts. Sequenced samples harbored between 22.800 and 232.000 synonymous or non-synonymous single nucleotide variants (SNVs). In addition, all samples contained multiple focal or large-scale somatic copy number alterations (CNAs). Clustering of SNVs as well as subclonal copy numbers from serial xenografts and spheroids were used to define SNV- and CNA-based subpopulations. Next, cellular fractions of identified subpopulations were determined and combined applying maximal parsimony to create models of the minimal number of subclones present in each sample. Using this strategy, we found that multiple subclones were present in each sample analyzed. Subclone heterogeneity was maintained during serial in vitro passaging and serial xenografting. Importantly, tumor initiation in xenografts was driven by at least 3 distinct genetic subclones whose relative contribution dynamically changed over time. Strikingly, in serial samples from the same patients, different genetic subclones grew out in vivo and in vitro.

To test whether functional heterogeneity of the TIC compartment is related to the presence of genetic subclones, we assessed the contribution of different TIC subtypes - LT-TIC, T-TAC and DC-TIC - at early and late time points of xenografting using secondary genetic marking. Therefore, 1x10^5 cells derived from early and late generation xenografts were transduced with an integrating lentiviral vector, thereby generating a stable barcode-like genetic mark which differs in each transduced cell. Following serial transplantation of transduced cells for 3 mouse generations, tumors were harvested and lentiviral integration sites were determined using highly sensitive LAM-PCR and high-throughput sequencing. Assessing the relative contribution of LT-TIC, T-TAC and DC-TIC revealed that the functional heterogeneity of the TIC compartment was preserved despite profound genetic subclone dynamics in serial xenotransplantation. These results strongly indicate that multiple genetic subclones drive long-term tumor formation and that functional heterogeneity of the CRC TIC compartment is not based on specific genetic subclones.

#911

BRM loss promotes tumor progression through extracellular matrix remodeling and elevated mammary epithelial stem/progenitor activity.

Jason J. Northey, Laura Damiano, Valerie M. Weaver. _University of California, San Francisco, San Francisco, CA_.

BRM expression is progressively lost in breast cancer and low levels are predictive of poor patient prognosis. We have previously shown that oncogenic downregulation of BRM promotes malignancy of mammary epithelial cells (MECs) through the CEBP/β-mediated induction of α5-integrin. BRM and BRG1 are necessary but mutually exclusive subunits of the SWI/SNF ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions to alter accessibility of specific genomic regions. To explore the physiological role of BRM in the normal mammary gland and in breast cancer, we studied BRM germline knockout mice (BRM KO). We first backcrossed BRM KO mice onto a clean genetic background and observed a 40% increase in animal size compared to wild type littermates. Histological examination of developing BRM KO mammary glands revealed enhanced ductal branching and a 60% increase in numbers of terminal end buds. Concordantly, immunohistochemistry for phospho-histone H3 exhibited significantly elevated proliferation of BRM KO MECs. Picrosirius red staining of mammary glands further demonstrated an accumulation of collagen around epithelial ducts in BRM KO mice that correlated with an upregulation of genes encoding the extracellular matrix (ECM) proteins Collagen-I and Fibronectin as well as the collagen remodeling enzymes Loxl1 and Loxl2 as assessed by quantitative PCR (qPCR). Subsequent flow cytometry analysis of mammary glands to sort luminal versus basal MEC populations demonstrated a preference for expansion of the basal lineage in BRM KO mice compared to controls. Basal MECs are in direct contact with the ECM and mammary stem cells are proposed to exist within this population. To investigate the possibility that BRM KO augments mammary stemness, we performed colony formation and limiting dilution transplantation assays, which served to confirm that the expanded basal population was indeed associated with elevated MEC stem/progenitor activity and also to verify the MEC dependence of these effects. To gain more mechanistic insight, we sorted luminal/basal MEC populations for qPCR analysis and found that MECs from BRM KO mice produce more abundant transcripts of genes associated with stemness. Our preliminary data now suggests a compensatory upregulation of BRG1 in BRM KO MECs and elevated expression of several known YAP/TAZ target genes involved in the control of cell proliferation. Finally, to describe a role for BRM in tumor progression, we examined the outcome of BRM KO in MMTV-NEU mice and observed more rapid tumor growth with a trend to higher tumor incidence and metastasis in mice lacking BRM. Taken together, our data indicate that SWI/SNF chromatin remodeling regulates YAP/TAZ control over MEC growth and a loss of BRM expression leads to BRG1-directed ECM remodeling, MEC proliferation and elevated mammary stemness, resulting in heightened mammary tumor aggression.

#912

Mist1+ secretory progenitor cells can give rise to cancer in the intestine and colon.

Hayakawa Yoku,1 Kosuke Sakitani,1 Woosook Kim,1 Yagnesh Tailor,1 Karan Nagar,1 Kazuhiko Koike,2 Samuel Asfaha,1 Timothy C. Wang1. 1 _Columbia University, New York, NY;_ 2 _The University of Tokyo, Tokyo, Japan_.

Intestinal crypts are maintained by long-lived intestinal stem cells (ISCs) which reside near the base of glands. Above the ISC pool, there are short-lived progenitors that can supply lineage-specific differentiated cell types into the villus. Notch and Wnt pathways play a key role for determining the stem/progenitor cell function and their cell fate, and regulating cancer development in the gastrointestine. Although it has been well established that ISCs are a major origin of intestinal cancer, it remains unknown whether intestinal progenitor population can give rise to cancer. We use Mist1-CreERT mice and induce Notch and Wnt activation by mating with LSL-Notch1-IC mice and/or Apc flox mice. We found that a bHLH transcriptional factor Mist1 is expressed in Paneth cells (Lysozyme+CD24high) as well as short-lived secretory progenitors (Lysozyme-CD24low) in the intestine. Mist1+ secretory progenitors are radio- and chemo-resistant, but do not show stem cell interconversion even after intestinal injury or Lgr5 ablation. However, aberrant Notch activation changes Mist1+ cells to Lgr5+ long-lived enterocyte progenitors, with loss of secretory lineage differentiation. Mist1+CD24low progenitor population can form intestinal organoids with Notch activation in vitro. Mist1+ secretory progenitors can give rise to intestinal cancer by simultaneous Notch and Wnt activation by loss of Apc gene, while loss of Apc in Mist1 lineage alone does not develop cancer. In the colon, Mist1 marks colonic secterory progenitors that become to lineage trace and can be a cancer-initiating cell after Notch activation or DSS-induced colonic injury. These results provide the clear evidence of cellular plasticity dependent on Notch signaling in the gut, and suggest short-lived progenitors as another cellular origin of cancer besides ISC pool.

#913

Novel leukemia stem cell-targeted therapy for acute myeloid leukemia based on dual inhibition of EZH1/EZH2.

Shuhei Fujita,1 Daisuke Honma,2 Kazushi Araki,2 Kazumasa Aoyama,3 Atsushi Iwama,3 Issay Kitabayashi1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Oncology Research Laboratories, Daiichi Sankyo Co., Ltd, Tokyo, Japan;_ 3 _Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Tokyo, Japan_.

Acute myeloid leukemia (AML) is a clonal malignant disorder originating from a small number of leukemic stem cells (LSCs). Selective targeting of LSCs is a promising strategy for the prevention and treatment of AML relapse. Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the stemeness of ES cells and hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) is catalytic components of PRC2 that trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Mutations and overexpression of EZH1/2 are associated with cancers including hematopoietic malignancies. Here, we used genetic deletion of EZH1/2 or a novel dual inhibitor of EZH1/2 activity to show that loss or inhibition of EZH1/2 eradicates dormant AML stem cells.

To examine the effects of genetic deletion of EZH1/2 on AML cells, Ezh1-null, Ezh2-conditional and double knock-out mice were generated. Hematopoietic stem/progenitor cells prepared from single knock-out mice or double knock-out mice were transduced with various types of AML fusion-genes, such as MOZ-TIF2, MLL fusions, AML1-ETO, and others by retroviral infection, and cultured in vitro or transplanted into irradiated recipient mice to induce AML in vivo. When the cells were cultured in vitro, double deletion of Ezh1/2 induced cell differentiation and apoptosis more severely than single deletions in all subtypes of AML tested, resulting in complete loss of cells. In AML mice, deletion of Ezh1/2 induced AML cell differentiation and complete remission of AML, which was not achieved by single deletion of Ezh1 or Ezh2. Genetic deletion of both Ezh1 and Ezh2 on the LSC fraction dramatically reduced the number of LSCs (L-GMP), especially quiescent LSCs, whereas deletion of either Ezh1 or Ezh2 did not have such a strong effect. The transcriptional profiles of LSCs deficient in both Ezh1 and Ezh2 were characterized by the upregulation of cell cycle-related genes such as Cyclin D1/D2, which is the main regulator of G0/G1 transition, along with differentiation-related genes. These results suggested that deletion of both Ezh1 and Ezh2 is required for eradication of LSCs.

To investigate whether pharmacologic inhibition of EZH1/2 could serve as a therapeutic strategy in AML, we developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1 and EZH2. The drug induced cell differentiation and apoptosis in most subtypes of AML tested in vitro and its effects were similar to those of genetic depletion of EZH1/2. Oral administration of the EZH1/2 dual inhibitor reduced the number of LSCs effectively in AML mice in a manner similar to the effect of genetic deletion of EZH1/2.

Taken together, these results strongly suggest that dual inhibition of EZH1 and EZH2 is a promising therapeutic strategy to eradicate LSCs in a wide range of AMLs, which could lead to important advances in the treatment of AML.

#914

Sox10 regulates stem- and mesenchymal-like features in mammary cells.

Christopher Dravis, Geoffrey M. Wahl. _Salk Institute for Biological Sciences, La Jolla, CA_.

To discover mechanisms that mediate the initiation and progression of aggressive and stem-like breast cancers, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor, Sox10. Here we report that Sox10 is specifically expressed in mammary cells that exhibit the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence, while its overexpression increases stem/progenitor activity. Intriguingly, we also discover that Sox10 overexpression elicits epithelial-to-mesenchymal transition (EMT) in mammary organoids in an FGF signaling-dependent manner. Further, modulation of Sox10 levels can induce sequential EMT, migratory, and clonogenic behaviors that strikingly resemble proposed mechanisms of metastasis. Consistent with these findings, we report that Sox10 is preferentially expressed in the most stem- and EMT-like triple negative breast cancer subtypes, which suggest that Sox10 may reprise these same functions during tumorigenesis. Indeed, we find that in an autochthonous mouse model of basal-like breast cancer, Sox10 is expressed at high levels and that these Sox10+ tumor cells exhibit characteristics that are similar to mammary cells in which Sox10 is ectopically expressed. Collectively, these results demonstrate a signaling mechanism through which stem and mesenchymal-like states are acquired in mammary cells, and indicate possible therapeutic targets to counter these functions in breast cancers for which targeted therapies are currently unavailable.

#915

RNA processing signatures of normal versus malignant progenitor cell aging predict leukemia stem cell sensitivity to RNA splicing modulation.

Leslie A. Crews,1 Larisa Balaian,1 Heather S. Leu,1 Nathaniel P. Delos Santos,1 Angela C. Court,1 Anil Sadarangani,1 Maria A. Zipeto,1 James J. La Clair,2 Reymundo Villa,2 Sheldon R. Morris,3 Rainer Storb,4 Anna Kulidjian,5 Edward D. Ball,6 Michael D. Burkart,2 Catriona H.M. Jamieson1. 1 _UCSD Moores Cancer Center, La Jolla, CA;_ 2 _UC San Diego Department of Chemistry and Biochemistry, La Jolla, CA;_ 3 _UC San Diego Department of Medicine, La Jolla, CA;_ 4 _Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA;_ 5 _UC San Diego Department of Orthopedic Surgery, La Jolla, CA;_ 6 _UC San Diego Division of Bone Marrow Transplantation, La Jolla, CA_.

Introduction

Human bone marrow aging is typified by decreased cellularity, stem cell exhaustion and myeloid lineage bias that may set the stage for development of myeloid malignancies. Secondary AML (sAML) is a malignancy that has been associated with alterations in RNA processing genes and currently has few effective treatment options available. A central goal of future therapeutic strategies is to prevent disease relapse and therapeutic resistance by selectively targeting unique gene products that are essential to LSC but not normal HSC function. Therefore, we established whole gene, long non-coding RNA (lncRNA), splice isoform, and RNA editing signatures of benign versus malignant bone marrow progenitor cell aging, and evaluated the therapeutic efficacy of splicing-targeted agents in pre-clinical humanized in vitro and in vivo model systems.

Methods

Whole transcriptome sequencing (RNA-Seq) was performed on FACS-purified hematopoietic stem (CD34+CD38-Lin-) and progenitor cells (CD34+CD38+Lin-) from aged (average age = 65.9 ± 6.8 years old) versus young (average age = 25.8 ± 3.0 years old) adult healthy bone marrow samples, and in leukemia stem cells (LSC) from patients with sAML (average age = 71.4 ± 7.9 years old). Comparative gene set enrichment analyses (GSEA), splice isoform, lncRNA, and RNA editing profiles were identified for normal and malignant progenitor cell aging. Then, we evaluated the spliceosome modulatory agent 17S-FD-895 in splicing reporter activity, PCR, and functional in vitro hematopoietic progenitor and in vivo LSC primagraft assays.

Results

Disruption of pre-mRNA splicing activity has recently been implicated as a therapeutic vulnerability in some types of cancer. Comparative whole transcriptome RNA sequencing (RNA-seq) analyses revealed pre-mRNA splicing factor gene expression was significantly disrupted in human AML LSC compared with age-matched normal progenitors. Comparative splice isoform RNA-seq and qRT-PCR validation revealed recurrent intron retention and exon skipping in expressed transcripts, such as PTK2B and several protein phosphatase gene products. Notably, transcription factor profiling of AML LSC demonstrated downregulation of key tumor suppressor genes, such as IRF8 and TP53. We then investigated the LSC inhibitory efficacy of a stable and potent splicing modulatory agent, 17S-FD-895, in humanized stromal co-culture and AML LSC primagraft assays. Pharmacological spliceosome modulation disrupted AML LSC maintenance in vivo by altering splicing of stem cell survival and AML-associated transcripts at doses that spared normal hematopoietic progenitors.

Conclusions

Detection and targeted modulation of aberrant RNA processing provides an innovative strategy for AML LSC eradication with implications for treatment of a variety of human malignancies and other age-related disorders. 

# Monday, April 18, 2016

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Cell-Cell Interaction and Tumor Microenvironment

#916

Slit2 inhibits breast cancer growth and metastasis by modulating tumor microenvironment.

Dinesh K. Ahirwar, Mohd W. Nasser, Mohamad Elbaz, Kontestine Shilo, Ramesh Ganju. _The Ohio State University, Columbus, OH_.

Breast cancer (BC) continues to be a major health issue particularly in developed countries. The Slit2 gene is hyper-methylated in BC and acts through Roundabout Homolog1 (Robo1) receptor. Slit2/Robo1 signaling has been shown to inhibit the migration of a variety of cells including BC cells. Recently, it has also been reported to improve chemotherapy outcome in intestinal cancer. However, Slit2/Robo1-mediated mechanisms that regulate tumor growth and metastasis are not well known. Using syngeneic mouse model, we report that the over-expression of Slit2 using Adeno-Slit2 transduction successfully restrains tumor growth and also restrict the metastasis to lung, compared to Adeno-null control. To explore the Slit2 mediated cellular events, we analyzed the stromal cells recruitment to the tumor and observed reduced number of tumor associated macrophages (TAMs) in the Adeno-Slit2 group compared to Adeno-null group. We further analyzed the underlying molecular mechanisms and found that the soluble Slit2 inhibits TGF-β1 mediated alternative activation of macrophages to TAMs. Treatment with TGF-β1 significantly enhances the markers of TAMs by upregulating β-catenin levels. Interestingly, we observed that Soluble Slit2 inhibits TGF-β1 mediated activation of TAMs by inhibiting downstream signaling molecules Smad2 and β-catenin. We also analyzed Slit2 expression in human breast cancer TMA and an inverse correlation of Slit2 expression was observed with the incidence of breast cancer, metastasis and macrophage recruitment. This study highlights the ability of Slit2 to prevent macrophage alternative activation to TAMs, thereby restrict tumor growth and lung metastasis. Anti-cancer activity of Adeno-Slit2 suggests therapeutic potential of Slit2 to treat advanced breast cancer.

#917

The E-cadherin/catenin complex and the intercellular adhesion of breast cancer cells are positively regulated via phosphorylation by the Syk tyrosine kinase in breast cancer cells.

Toufic Kassouf,1 Philippe Montcourrier,1 Romain Larive,1 Anne Morel,2 Serge Urbach,3 Peter J. Coopman1. 1 _INSERM U1194, Montpellier, France;_ 2 _CNRS UMR5237, Montpellier, France;_ 3 _Functional Proteomics Platform, CNRS, Montpellier, France_.

The spleen tyrosine kinase Syk was mainly studied in immunoreceptor-activated signaling in hematopoietic cells. We first demonstrated that Syk is also present in mammary epithelial cells and that its expression is lost in malignant breast cancer cells. Using mouse xenograft models injected with Syk-transfected cells we and others established that Syk acts as a tumor and metastasis suppressor. Moreover, clinical studies reveal a correlation between reduced Syk expression and a decreased survival and increased metastasis risk in breast cancer and other carcinomas. The main objective of our investigations is to unravel the mechanisms of the anti-oncogenic activity of Syk. For this, identification of its substrates and signaling pathways is crucial. A quantitative phospho-proteomic approach allowed to identify novel potential Syk substrates involved in intercellular adhesion and epithelial polarity, both characteristics of cell differentiation that are lost during tumor invasion and metastasis.

Phosphorylation assays with recombinant proteins demonstrated that E-Cadherin (E-Cdh), α, β and p120-catenin (Ctn) are direct Syk substrates. Using mass spectrometry we identified the tyrosine residues phosphorylated by Syk and generated phospho-Tyr-peptide-specific (pY) antibodies. Using immuno-fluorescence Syk was shown to colocalize with E-Cdh at adherens junctions (AJ). Syk transfection increased E-Cdh, α- and β-Ctn phosphorylation at AJ in a kinase-dependent manner, whereas non-phosphorylatable mutant Syk exhibited a decreased AJ localization. This was biochemically confirmed by immunoprecipitation and Western blot experiments. Phosphorylated Ctns are still associated with E-Cdh and vice versa. In 2D culture, Syk transfection stimulated intercellular contact formation. Correspondingly, in Syk-knockdown cells the 2D and 3D cell-cell re-aggregation was decreased. Conversely, cell migration and invasion were inhibited by Syk transfection and stimulated by Syk knockdown. At the molecular level, Syk transfection increases the interaction between the E-Cdh/Ctn complex with zonula occludens proteins and the actin cytoskeleton.

In conclusion, Syk seems to play a positive role in the formation and maintenance of AJ via the phosphorylation of the E-Cdh/Ctn complex. Loss of Syk expression or function might lead to the disruption of these complexes and consequently promote invasion and metastasis. (Supported by the Fondation ARC SL220110603480 and Plan Cancer/INCa ASC14021FSA)

#918

TAK1: a potential target in the treatment of breast cancer metastasis.

Oihana Iriondo, Yarong Liu, Mostafa Elhodaky, Lin Li, Julie E. Lang, Pin Wang, Min Yu. _University of Southern California, Los Angeles, CA_.

TGFβ-activated kinase 1 (TAK1) is a serine/threonine kinase from the MAPKKK family. Its activity is regulated by different cytokines, including interleukin 1, TGFβ, TNFα and BMPs, and it is therefore a key player in the cellular responses induced by changes in the microenvironment. Once activated, TAK1 activates other intracellular kinases, including the p38 MAPK, JNK and IKK, regulating processes such as cell survival, differentiation or inflammatory responses. TAK1 activation has been shown to influence the progression of several types of cancer, including lymphomas and pancreatic, colon, liver and breast cancer. The goal of this study is to elucidate the mechanisms by which TAK1 regulates breast cancer progression. We performed xenotransplantation experiments with MDA-MB-231 cells using two different approaches to study the effect of TAK1 inactivation in breast cancer metastasis. First, we used (5Z)-7-Oxozeanol, a compound that can irreversibly inhibit the kinase activity of TAK1. Due to poor solubility, (5Z)-7-Oxozeanol is not easily deliverable in vivo and previous animal studies showed toxicity in the animal gut using traditional oil based intraperitoneal injections. Therefore, we synthesized the nanoparticle lipOxo, which is a (5Z)-7-Oxozeanol-encapsulating crosslinked multilamellar liposome. Characterization of these nanoparticles showed enhanced loading efficiency, vesicle stability and sustainable release kinetics of the encapsulated drug. Moreover, nanoparticle packaged drugs reduced viability of cancer cells in vitro. Treatment of mice injected with MDA-MB-231 cells with (5Z)-7-Oxozeanol containing nanoparticles reduced metastasis formation, while growth of the primary tumor was not affected. In order to corroborate the involvement of TAK1 in breast cancer metastasis, we generated MDA-MB-231 cells overexpressing either the wild type TAK1 or a dominant negative form of TAK1, and confirmed that expression of wild type TAK1 promotes metastatic growth, while dominant negative TAK1 reduces it. Using these stable cell lines, we confirmed that TAK1 is required for activation of p38 by several cytokines, which could be responsible for increased metastatic growth in vivo. Importantly, we found that these cytokines can be expressed in the lung microenvironment, suggesting that a crosstalk between cancer cells and cells from the lung microenvironment could play an important role in the influence of TAK1 on breast cancer metastasis.

#919

A genome-wide screen in C. elegans identifies cell non-autonomous suppressors of let-60/RAS mediated oncogenic over-proliferation.

Komal Rambani,1 Raleigh Kladney,1 Serena Chang,1 Markus Harrigan,1 James Dowdle,1 Xing Tang,1 Huayang Liu,1 Elizabeth Brunner,2 Helen Chamberlin,1 Gustavo Leone1. 1 _The Ohio State University, Columbus, OH;_ 2 _Davidson College, Columbus, NC_.

Coordinated proliferative signals from the mesenchymal cells play a crucial role in the regulation of proliferation of epithelial cells during normal development, wound healing and several other normal physiological conditions. However, when epithelial cells acquire a set of malignant mutations, they respond differently to these extrinsic proliferative signals leading to enhanced abnormal proliferation of mutant epithelial cells and hence tumor growth. Despite mounting evidence that stromal cells influence the growth of tumors and cancer progression, it is unclear which specific genes in these mesenchymal cells regulate the molecular signals that promote the over-proliferation of the adjacent mutant cells. The complexity of several cell types and their interactions in vivo in the cancer mouse models and human tumor samples limits our ability to identify mesenchymal genes important in this process. Thus, we took a cross species approach to use C. elegans vulval development as a model to better understand the impact of mesenchymal (mesodermal) cells on the proliferation of the epithelial (epidermal) cells. This model includes well-described signaling interactions between mesodermal (anchor cell, gonad, and muscle) cells and epidermal (vulva precursor cells, VPCs) cells. In this system, three of the six equipotent VPCs normally acquire vulva fates (termed 1° and 2°) upon receiving coordinated EGF and Wnt signals from the mesodermal cells, and lateral Notch signaling between the VPCs. Then, they divide 3 times to produce a symmetric vulva comprised of 22 daughter cells. Abnormal activation of the EGF pathway, such as by introducing gain-of-function (gf) mutations in let-60/Ras, leads to the promotion of vulval fates in the remaining three VPCs, which then divide inappropriately resulting in development of multiple vulval protrusions (Muv phenotype).To identify genes that function in mesodermal cells to inhibit the proliferation of Ras-activated epidermal cells, we genetically engineered C. elegans to develop a strain that possesses let-60(gf)/Ras mediated over-proliferation (Muv phenotype) and RNA interference (RNAi) competence specific to the mesodermal tissues. This strain was subjected to a genome-wide RNAi screen. Our screen identified 47 genes that, upon RNAi knock-down of a gene in mesodermal cells, suppress over-proliferation of let-60(gf)/Ras epithelial cells in a genotype-specific manner. Notably, these genes encode chromatin remodeling proteins, ribosomal proteins, proteins involved in vesicle transport, and metabolic factors, rather than secreted molecules. Importantly, candidate genes emerged from this screen are significantly enriched for the conserved genes from C. elegans to humans. These results form an initial understanding of molecular factors in mesenchymal cells that impact oncogenic cell proliferation.

#920

Migratory cancer side population cells induces stem cell altruism in bone marrow mesenchymal stem cells to resist therapy, and enhance tumorigenic potential of non-tumorigenic cells.

Joyeeta Talukdar,1 Rashmi Bhuyan,2 Jaishree Garhyan,2 Bidisha Pal,2 Sora Sandhya,1 Sukanya Gayan,1 Anupam Sarma,3 Reza Bayat-Mokhtari,2 Hong Li,2 Jyotirmoy Phukan,4 Wael Tasabehji,2 Seema Bhuyan,1 Amal Ch Kataki,3 Rika Tsuchida,2 Herman Yeger,5 Debabrata Baishya,6 Bikul Das2. 1 _KaviKrishna Laboratory, Guwahati Biotech Park, Guwahati, India;_ 2 _Forsyth Institute, Cambridge, MA;_ 3 _B Borooah Cancer Institute, Guwahati, India;_ 4 _Guwahati Medical College, Guwahati, India;_ 5 _Hospital for Sick Children, Toronto, Ontario, Canada;_ 6 _Gauhati University, Guwahati, India_.

Stem cell may exhibit altruistic behavior that may benefit cancer cells. We recently demonstrated altruistic phenotype in human embryonic stem cells (Das B et al. Stem Cells 2012). The phenotype exhibited reversible induction of high HIF-2alpha and low p53 expression, associated with high glutathione secretion. We speculated that cancer stem cell might induce a similar altruistic phenotype in human bone marrow (BM) derived stem cells (hematopoietic, mesenchymal and endothelial cells). The altruistic reprogrammed BM cells may then facilitate tumor growth, as well as resist the toxicity of oxidative-stress inducing anti-cancer agents. To investigate this possibility, we have obtained conditioned media (CM) from migratory side-population (SPm) and non-SP cells of a diverse panel of tumors including epithelial tumors. The SPm cells exhibit very high tumorigenic capacity (Das B et al, Stem Cell, 2008). The CM was added to in vitro bone marrow (BM) derived CD133+ cells that contain hematopoietic, endothelial, and mesenchymal stem cells. We found that SPm derived CM (henceforth known as SPm-CM) treatment increased the self-renewal capacity of CD271+/CD45- BM-MSCs. Importantly, the reprogrammed CD271+ BM-MSCs (henceforth known as R-MSCs) phenotype exhibited enhanced stemness reprogramming, a cytoprotective mechanism associated with stem cell altruism (Das B et al. Stem Cells, 2012). In contrast, the treatment with CM obtained from non-SP cells did not exhibit R-MSCs. We found that VEGF/VEGR1 autocrine signaling may be involved in R-MSCs mechanism. Importantly, the R-MSCs derived CM reprogrammed non-CSCs to CSCs, and reduced the toxicity of chemotherapy on non-SP cells. In vivo, R-MSCs derived CM, when injected to mice, exhibited mobilization of CD271+ BM-MSCs to circulation. The circulatory CD271+ BM-MSCs exhibited distinct phenotype of R-MSCs including high expression of HIF-2alpha, and VEGFR1. Finally, in human cancer patients, we identified R-MSC phenotype in the peripheral circulation. These studies suggest that cancer stem cells may exploit stem cell altruism to reprogram BM-MSCs for their own benefit. The reprogrammed BM-MSCs gene expression may have the potential as a diagnostic marker for CSC-induced stem cell altruism.

#921

Pericellular-interactions promote malignant progression of glioblastoma via novel NOTCH-1/HIF-1α pathway.

Eunsoo Kim,1 Jungwhoi Lee,2 Seung-Wook Ryu,1 Chulhee Choi,1 Kyungsun Choi1. 1 _Korea Advanced Inst. of Science & Tech., Daejeon, Republic of Korea; _2 _Jeju National University, Jeju-do, Republic of Korea_.

Malignant tumors generally develop hypoxic condition in the core region of tumor mass, which induces stabilization of hypoxia inducible factor-1 alpha (HIF-1α) by preventing proteasome-dependent degradation. HIF-1α has been shown to regulate various oncogenes for malignant progression of many types of tumors including glioblastoma multiforme (GBM). In the present study, we showed that cell-to-cell interactions at high density can induce malignant transformation of tumor cells by activation of HIF-1α even under normoxic condition. To delineate the biological effect of cell culture density, we maintained cancer cells in different density. Four types of GBM cell lines (U251-MG, LN215-MG, U373-MG, and CRT-MG) were used to examine the oncogenic phenotypes of cancer cells cultured in different densities. Cells cultured in a high-density condition showed higher rate of proliferation and migration activity and higher resistance to temozolomide, an anti-cancer drug commonly used for GBM treatment. We found that the pericellular interactions stimulate the cleavage of notch-1 intracellular domain (NICD), and the cleaved NICD protects the HIF-1α from hydroxylation and subsequent proteasomal degradation, resulting in nuclear translocation of HIF-1α. Collectively, we suggest that the cell-cell interactions in high-density culture conditions induce NICD-mediated HIF-1α activation, which leads to malignant phenotypic changes. Therefore, treatment of gamma-secretase inhibitor (GSI), which can block NOTCH-1 signaling pathway, in combination with TMZ could be a promising therapeutic strategy in TMZ-resistant tumor cells.

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI14C0042)

#922

PGE2/HuR/CEBPD axis in macrophages contributes to immunosuppression and inhibits phagocytosisof nasopharyngeal carcinoma.

Yu-Wei Hsiao, Ju-Ming Wang. _Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Tainan, Taiwan_.

The association of inflammation and cancer progression via modulation of phagocytosis and suppression of adaptive immunity by tumor-associated macrophages (TAM) to promote tumorigenesis has been implicated in a number of recent studies. To date, the underlying mechanisms involving the interactions of these two cellular processes remain elusive, particularly in the involvement of posttranscriptional regulation. In this study, we demonstrate that prostaglandin E2 (PGE2) activates transcription factor CCAAT/enhancer binding protein delta (CEBPD) in macrophages by inducing nucleocytoplasmic shuttling of RNA binding protein Hu antigen R (HuR), which plays a direct role in stabilizing CEBPD mRNA in macrophages. Increase of CEBPD resulted in elevated expression of IL-10, a well-studied immunosuppressor, and PTX3, which contribute to suppression of phagocytosis of cancer cells by macrophages. In addition, the conditioned medium of CEBPD-transfected macrophages showed an immunosuppressive effect to attenuate phagocytosis of cancer cells by macrophages, suggesting an autocrine mode of regulation. Immunohistochemistry studies demonstrated that the cytosolic level of HuR protein correlates with increased CEBPD in TAM and malignant nasopharyngeal carcinoma (NPC). Collectively, these results provide new evidences that the inflammatory PGE2/HuR/CEBPD axis in macrophages plays protumor roles in controlling immunosuppression and attenuating phagocytosis of cancer cells by macrophages.

#923

Exploring the role of the ovarian stromal extracellular matrix in early tumor development.

Jamie T. Goodner-Bingham, Yang Yang-Hartwich. _Yale University, New Haven, CT_.

Background: Advancements in ovarian cancer research have shown evidence for extra-ovarian origins of malignant cells. The fallopian tubes (FT) are thought to be a source of the most common and aggressive type of ovarian tumor, high grade serous which are more common in women with increased ovulation. We have previously demonstrated that the ovulation wound site provides an ideal environment for malignant FT cells to implant into the ovary (1). However, while malignant cells implant during the reproductive stage, the highest instance of ovarian cancer occurs in peri- and postmenopausal women. We hypothesize that the ovarian environment provides the necessary factors to keep implanted malignant cells quiescent until peri- and post-menopause when the changes in the ovarian environment trigger proliferation and growth. The extracellular matrix (ECM), secreted by the ovarian stroma, is a major part of this environment which regulates cell-to-cell communication and maintains structural integrity. The intent of this study is to determine the critical role of the pre- and postmenopausal ECM and elucidate the effects of matrix remodelling on malignant cells.

Methods: We generated malignant cells by introducing p53 mutations to healthy FT and ovarian surface epithelial (OSE) cell lines. In vitro, we isolated ECM from pre-menopausal ovarian stroma. Additionally, we overexpressed matrix metalloproteinase 2/9 (MMP2/9) in stromal cells to induce matrix remodelling, resulting in an ECM resembling postmenopause. These matrices were used to develop a new 3D culture method which allowed us to evaluate the effect menopausal ECM remodelling has on malignant cell pathways.

Results: The 3D ovarian stromal ECM maintained and delayed growth of the co-cultured malignant cells compared to their monolayer counterparts. Postmenopausal remodelling of the ECM, via upregulation of MMPs, triggered proliferation and expansion of malignant cells. We discovered, through pathway screening, that AKT, ERK 1/2, PTEN, WNT and PYK2 were associated with these ECM-related changes in the malignant cells.

Conclusion: These results support the use of a 3D model in culturing pre-tumor cells to create an in vitro model that closely resembles an ovarian environment. We have also shown that the ECM plays a role in maintaining malignant cells by supressing proliferation and growth. Additionally, we have demonstrated that the remodelling of the ECM that occurs with menopause-associated increase in MMP2/9 expression triggers expansion of malignant cells. It is clear that the ECM plays an important role in the early development of ovarian cancer and that MMP could be a contributing factor to the postmenopausal presentation of these malignancies. Going forward, we plan to further elucidate the specific molecular mechanisms involved in postmenopausal tumor development.

#924

Upregulation of the MET transcript is consistently associated with invasion and tumor budding in colorectal cancer.

Conor Bradley, Philip Dunne, Stephen McQuaid, Victoria Bingham, Mark Lawler, Manuel Salto-Tellez, Patrick Johnston, Sandra Van Schaeybroeck. _Queens University Belfast, Belfast, United Kingdom_.

Background:

The c-MET proto-oncogene is frequently overexpressed (50-60%), amplified (1-3%), and mutated (1-3%) in colorectal cancer (CRC). Hepatocyte growth factor (HGF)-dependent and independent activation of c-MET has been associated with increased survival and resistance to targeted therapies. This study aimed to investigate the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples.

Methods:

In order to model CRC tumour cell invasion, we have generated invasive CRC subpopulations using Boyden Invasion chambers. To model the CRC microenvironment, we have used a range of co-culture techniques with CRC cells and colon fibroblasts. Migration/invasion was determined using xCELLigence System (Roche). c-MET expression in parental and invasive cell lines was measured using Western blotting and qRT-PCR. c-MET expression in CRC FFPE tissues was measured using IHC and RNAScope®.

Results:

We identified marked upregulated expression of c-MET at both the protein and transcript levels in our invasive CRC cell line models. Importantly, both parental and invasive subpopulations were found to be inherently dependent on c-MET for migration, as RNAi against c-MET abrogated migration/invasion in both parental and invasive models. We also demonstrated that stimulation of CRC cells with rh-HGF resulted in increased CRC cell migration/invasion. In addition, co-culture of CRC cells with colonic myofibroblasts, resulted in marked increases in migratory and invasive capacity, and this was dependent on HGF/c-MET signaling. Interestingly, stimulation with myofibroblast conditioned medium or HGF promotes rapid degradation of c-MET at the protein level, followed by recycling, while MET transcript remains unaltered, illustrating a dynamic expression of c-MET protein in response to activation. We further showed that MET is transcriptionally upregulated in tumour budding foci at the invasive front of a cohort of stage III CRC tumors. Intriguingly, c-MET protein levels do not correlate with the transcript, most likely due to a similar protein degradation process observed in our aforementioned in vitro models.

Conclusions:

We show for the first time a key role for transcriptional upregulation of MET as a molecular driver of tumour invasion, both in vitro and in stage III CRC tumours.

#925

IL-4 / IL-13 mediated cell-to-cell fusion in prostate cancer progress.

Berna Uygur, Evgenia Leikina, Leonid Chernomordik. _NIH, Bethesda, MD_.

Poorly understood interactions between prostate cancer (PCa) cells and their microenvironment direct the epithelial to mesenchymal transition (EMT) during disease progression. Cell-to-cell fusion, such as cell fusion in muscles and placenta, is one of the ways in which cells can drastically change their properties. Cell fusion has been proposed to promote tumor cell diversity and the ability to metastasize. Metastatic disease has been also suggested to depend on the interleukin 4 (IL-4) signaling. Patients with hormone-refractory PCa have elevated levels of IL-4 and it has been proposed that targeting IL4 -receptor interactions limits metastatic disease. Here, to mimic conditions at the interphase between rhabdosphincter myofibers and apical anterior regions of prostatic gland we explored interactions of PC3 cells (human PCa cell line) with skeletal muscle cells. Co-culturing of these cells resulted in an elevated concentration of related cytokines IL-4 and/or IL-13 in the tissue culture medium from the co-cultured cells but not from muscle cells cultured on their own. The co-culturing (i); the application of the conditioned medium from the co-cultured cells (ii); and treating PC3 cells with recombinant IL-4 (iii) resulted in a robust fusion between PC3 cells, in a significant increase in the percentage of PC3 cells expressing stem cell marker CD133, in the drug resistance, in an increase of the expression of mesenchymal-characteristic markers pAKT, MMP9, desmin, and in the loss of E-cadherin that is another marker for EMT. The described effects required co-culturing of the PC3 cells and muscle cells. Moreover, the co-culturing and recombinant IL-4 application also upregulated the expression of the two proteins - annexin A5 and syncytin that play important role in several cell-cell fusion processes. Significantly, application of either (i) IL-4-neutralizing antibodies or (ii) peptide inhibitors of syncytin; and (iii) lowering annexin A5 expression suppressed cell proliferation and fusion. Our findings substantiate a hypothesis that IL-4 and IL-13 in the microenvironment of PCa cells promote the EMT and raise metastatic potential of PCa cells by their fusion mediated by upregulated syncytin- and annexin A5.

#926

Pentraxin 3 is responsive to the activation of CCAAT/enhancer binding delta and promotes cancer progression in the tumor microenvironment.

Jhih-Ying Chi,1 Ju-Ming Wang2. 1 _Institute of Basic Medical Science, National Cheng Kung University, Tainan, Taiwan;_ 2 _Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Tainan, Taiwan_.

This lack of understanding especially impacts the design of anticancer treatments. It is a common observation in the clinic that recurrent cancers, and particularly anticancer drug-resistant cancers, are more difficult to treat because they spread faster than the original tumor. CCAAT/enhancer binding protein delta (CEBPD) was activated in M2 macrophages and myofibroblasts/cancer-associated fibroblasts (CAFs) and contributed to metastasis, invasion and recurrence of cancer cells in response to PGE2 and the anticancer drugs. We further found that pentraxin 3 (PTX3) was activated in response to CEBPD induction and involved in migration and invasion of breast cancer cells, and the same phenomenon was also observed in drug-resistant cancers. Interestingly, we found that a recombinant PTX3 protein containing the C-terminal region (pPTX3) showed an opposite tumorigenic effect to the mammalian PTX3. The results suggest that PTX3 could be a promising target for preventing the metastasis/invasion of cancers and treating drug-resistant cancers.

#927

Fibroblast-induced switching to the invasive phenotype and PI3K-mTOR signaling protects melanoma cells from BRAF inhibitors.

Kotryna Seip, Karianne Giller Fleten, Anna Barkovskaya, Vigdis Nygaard, Mads Haugen Haugen, Birgit Øvstebø Engesæter, Gunhild Mari Mælandsmo, Lina Prasmickaite. _Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway_.

Phenotypic heterogeneity of cancer cells can reason diversity in therapy responses within the same tumor, which might influence the overall efficacy of the treatment. Tumor stroma is an important contributor to intratumoral heterogeneity and can play a significant modulatory role in therapy response/resistance. Through studies on biological mechanisms of stroma-promoted resistance, novel targets could be identified for combination therapies aimed to eradicate both stroma-dependent and independent counterparts of the tumor.

In this study we explore how the efficacy of the BRAF inhibitor (BRAFi) vemurafenib, a targeted agent commonly used against BRAF-mutant malignant melanoma, is modulated by stromal cells. By using multiple co-culture systems and experimental metastasis models, we showed that in the presence of lung fibroblasts, adjacent melanoma cells respond poorly to BRAFi. The protective influence of stroma was associated with stroma-induced changes in the melanoma cell phenotype, which was mapped by global gene expression and proteome analysis. We revealed that under the influence of fibroblasts, melanoma cells underwent a phenotype transition to the invasive, mesenchymal-like state characterized by down-regulation of melanocytic markers (MITF and its targets), up-regulation of receptor tyrosine kinases (RTKs)/RTK-linked signaling (like AXL or PDGFR activating down-stream PI3K) and elevation of extracellular-matrix fibronectin. We propose that these alterations allow melanoma cells to utilize alternative signaling pathways, like RTK-PI3K-mTOR instead of BRAF-driven MAPK, which reduces sensitivity to BRAFi. This scenario is further supported by the observations that: i) upon BRAFi treatment, stroma-protected melanoma maintained high levels of phospho-ribosomal protein S6 (pS6), a mTOR effector protein; ii) inhibition of mTOR or the upstream pathway PI3K together with BRAF eradicated pS6high subpopulations and enhanced the anti-proliferative effect in stroma-interacting melanoma; iii) the benefit of mTOR and BRAF co-inhibition was also seen in early-stage lung metastases in vivo.

In conclusion, our findings signify the importance of stromal cells, specifically lung fibroblasts, in regulating melanoma cell phenotype and signaling, which impairs response to BRAFi. The stroma-induced invasive phenotype determinants that facilitate RTK-PI3K-mTOR signaling (e.g. AXL, PDGFR or fibronectin-binding integrins) could represent potential targets for overcoming stroma-mediated resistance to BRAFi. Currently, we are testing BRAFi in combination with several novel inhibitors of the identified RTKs and performing a cancer drug sensitivity screen to reveal the most effective drug combinations against stroma-interacting melanoma cells.

#928

Myofibroblasts induce poorly differentiated tumor-like spheroids in small intestinal organoids by Wnt-independent mechanism.

Agnieszka Pastula,1 Klaus-Peter Janssen,2 Stefanie Hauck,3 Roland M. Schmid,1 Michael Quante1. 1 _Gastroenterologie II, Klinikum rechts der Isar TUM, Munich, Germany;_ 2 _Chirurgische Klinik, Klinikum rechts der Isar TUM, Munich, Germany;_ 3 _Helmholtz Zentrum München, Munich, Germany_.

Recently, three dimensional (3D) organoid cultures were established from different organs; however those organoid cultures are exclusively composed of epithelial cells, thus missing the stromal niche. The later one is believed to play a crucial role during all stages of tumor development and resistance to therapy. One of the most abundant and the most important stromal cell in the tumor microenvironment is a myofibroblast. The contribution of myofibroblasts to tumor progression is well established, however little is known about their role during epithelial cell homeostasis and tumor initiation.

The niche was reconstructed by combining small intestinal (SI) crypts together with different types of mesenchymal cells in 3D culture systems. As mesenchymal cells the following cells were used: murine small intestinal fibroblasts, murine gastric carcinoma associated fibroblasts (CAFs), human fetal esophageal fibroblasts and human cardia myofibroblasts. While SI organoid culture is almost exclusively composed of budding structures, in the co-culture with intestinal myofibroblasts ~50% of crypts were growing as non-budding spheroids. Those spheroids were positive for Ki-67, and independent of external R-Spondin, EGF and Noggin. Surprisingly, no differences between CAFs and other mesenchymal cells could be observed. Gene expression analysis revealed that myofibroblasts significantly upregulated Sox-9 (~3 fold) and CD44 (~4-fold) mRNA levels, and promoted proliferation rather than differentiation in the SI organoids. Clonogenicity assay showed that in the co-culture crypt cells exhibited about 3 times greater self-renewal capacity. Interestingly, by morphology and decreased number of PAS positive cells, the spheroids resembled intestinal organoids from Apc +/1638N mouse tumors. However, in the spheroids induced by co-culture the expression of Axin-2 was not increased, and Wnt inhibitor studies (IWP-2, C59) provided additional evidence that spheroid induction can be mediated by other mechanism than canonical Wnt. Indirect co-culture and conditioned media experiments revealed that spheroid formation was mediated by soluble factors. Mass spectrometric analysis of the secretome from the co-culture suggested the involvement of extracellular matrix-receptor pathway and focal adhesion pathway.

Taken together, our studies demonstrate that the microenvironment shapes intestinal crypts in a similar way as cell-autonomous genetic alterations. The data point out to the contribution of myofibroblasts to tumor initiation phenotype in the intestinal crypts by a mechanism independent of canonical Wnt. The precise signaling pathway remains to be elucidated in the future. Increased knowledge on the cellular communication between stroma and epithelium can contribute to the development of anti-cancer therapeutics that target not only cancer cells, but also the stromal niche.

#929

Tunneling nanotube conduits facilitate the bystander effect after oncolytic viral infection.

Emil Lou,1 Justin Ady,2 Venugopal Thayanithy,1 Kelly Mojica,2 Joshua Carson,2 Prassanna Rao,1 Yuman Fong3. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 3 _City of Hope Medical Center, Duarte, CA_.

Background: Oncolytic viruses have come to the forefront of cancer therapeutics following FDA approval for treatment of metastatic melanoma in 2015. Efficacy of viral therapy is enhanced by the bystander effect, a cellular phenomenon that amplifies the effects of the virus following activation of the prodrug ganciclovir (GCV) by virally-expressed thymidine kinase (TK) and intercellular spread of GCV via gap junctions. In the complex and stroma-rich tumor microenvironment, gap junctions may not completely account for cell-to-cell communication. Tunneling nanotubes (TNTs) are a novel and recently characterized alternative form of direct cell-to-cell communication in the tumor matrix. TNTs are fine, long, F actin-based cell extensions that serve as short and long-range conduits for efficient transfer of cellular cargo. Here, we investigate the ability of TNTs to mediate a TK-based bystander effect after oncolytic viral infection and administration of GCV.

Methods: We infected 3 mesothelioma cell lines with NV1066, a mutant replication-competent strain of herpes simplex virus-1 (HSV-1) that encodes viral eGFP and viral TK. Confocal microscopy and time-lapse imaging were performed 12-36 hours later. A modified Transwell assay was used to separate infected cells from uninfected cells to assess TNT propagation of eGFP-tagged NV1066. GCV was added to infected cells in the top chamber to assess TNT propagation of TK-activated GCV. Apoptosis was measured using TUNEL assay in the bottom chamber to quantify the extent of the bystander effect.

Results: Confocal microscopy demonstrated effective intercellular transfer of GFP-tagged virus between cells via TNTs prior to oncolysis. Quantification of TUNEL-positive cells at 48 hours indicated that addition of NV1066 to the top chamber resulted in 33% of the initially uninfected cells in the bottom chamber dying by 48 hours. The addition of GCV to virally infected cells in the top chamber significantly increased apoptosis in recipient cells in the bottom chamber, from 33% to 71%, producing a 2.3-fold increase in cell killing attributed to TNT transfer of viral TK-activated GCV (p=0.007). Thus, TNTs were shown to transfer viral TK-activated GCV to non-infected cells, leading to cell death via a long-range form of the bystander effect.

Conclusions: Here we demonstrate that TNTs provide a previously unknown and alternative mechanism for the bystander effect in which viral TK-activated GCV is transferred via TNTs and induces apoptosis in non-infected recipient cells.

#930

Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth.

Wenjuan Wu, Julie Stewart, Constance King, Bonita Jones, Robert Flack, Susan Pratt, Randi Berryman, Michelle Swearingen, Diane Bodenmiller, Xi Lin, Mark Uhlik, Beverly Falcon, Anthony Fischl, Jason Manro, Ramon Tiu, Sudhakar Chintharlapalli, Bronislaw Pytowski, Shripad V. Bhagwat, Sean Buchanan, Sheng-Bin Peng. _Eli Lilly and Company, Indianapolis, IN_.

Lung cancer is the leading cause of cancer death worldwide. MAPK activation via KRAS mutation is present in up to 30% of lung cancer patients. NSCLC patients with KRAS mutation is associated with poor prognosis and represents an unmet medical need. LY3009120, a pan-RAF and RAF dimer inhibitor which is in phase I clinical trial, was previously demonstrated to have anti-tumor activities in BRAF or RAS mutant tumor cells in vitro and in vivo. Ramucirumab, a fully-human antagonist monoclonal antibody to human VEGFR-2 was recently approved as an anti-angiogenic treatment for several cancer indications including second-line NSCLC. Combination strategies in cancer including targeting both tumor cells and the surrounding stroma cells have been shown to be effective in various disease subtypes. In this study, the combination effect of LSN3074753 (a surrogate and an analogue of LY3009120) with VEGFR-2 inhibitor DC101 (a monoclonal antibody specific for murine VEGFR-2 and a surrogate for ramucirumab) were evaluated in KRAS mutant NSCLC models, including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). LSN3074753 treatment alone resulted in 66.9% and 82.4% tumor growth inhibition in H2122 and A549 xenograft tumors, respectively; and 41.4% tumor regression in H441 xenograft tumors. DC101 treatment alone resulted in 64.5%, 75% and 102.2% tumor growth inhibition in H2122, A549 and H441, respectively. The combination of LSN3074753 and DC101 led to more significant tumor growth inhibition (87.2% of tumor growth inhibition for H2122, p<0.001) or tumor regression (16.3% and 51.5% tumor regression for A549 and H441, respectively, p<0.001) when compared to single agent treatment. The molecular mechanism was further investigated in H2122 and H441 tumor xenografts in terms of MAPK signaling and its gene signatures, tumor vascularization, cell proliferation and apoptosis. Treatment with LSN3074753 or DC101 alone reduced the tumor vessels and pericytes, but the combination treatment showed better effects. Similarly, the analysis of multiple cell cycle markers indicated that the combination treatment inhibited cell proliferation more than the single agent alone. Moreover, the combination effect of LSN3074753 with ramucirumab was also investigated in vitro, including endothelial cell sprouting, cord formation and tumor cell driven cord formation assays to further understand the molecular mechanisms underlying the combination benefit on different tumor compartments including tumor and stroma cells. Overall, combined inhibition of pan-RAF and VEGFR-2 enhanced both antiangiogenesis and antitumor effects, and the data support that combining a panRAF inhibitor with ramucirumab represents a promising strategy for the treatment of KRAS mutant NSCLC.

#932

AR signaling differentially regulates the growth of prostate epithelial cells.

Xiuping Yu, Shu Yang. _LSU Health Sciences Center, Shreveport, LA_.

The prostate is an androgen-dependent organ. Androgen stimulates the growth of normal prostatic cells, as well as prostate cancer (PCa). However, AR signaling does not always promote cell proliferation, in some cases, it may even inhibit the growth of prostatic cells. In this study, we used NHPrE, a cell line derived from normal human prostate epithelia, to study how AR signaling modulates the growth of prostatic cells and to explore the mechanisms involved in AR's differential function in vitro and in vivo. Our results showed that inducing AR signaling in NHPrE cells suppressed their growth in vitro, concomitant with a reduction of Myc. In contrast, ectopic expression of AR in NHPrE cells induced the expression of Myc and pSTAT3 and drove the development of invasive PCa in vivo, suggesting a distinct in vivo function of AR signaling in prostatic cells. Furthermore, our data showed that the presence of prostate stromal cells over-rode androgen-mediated growth inhibition on NHPrE/AR cells in vitro; and IL-6/pSTAT3 was implicated in this stromal/epithelial communication. In conclusion, our study showed that AR signaling differentially regulated the growth of prostatic cells in vitro and in vivo through a mechanism that involved stromal/epithelial interactions.

#933

Protein kinase a regulation of malignant brain tumor migration.

Jennifer N. Ausland, Robert H. Newman, Patrick M. Martin. _North Carolina Agricultural & Technical State University, Greensboro, NC_.

Glioblastoma multiforme (GBM) is the most common and deadly type of primary brain tumor, accounting for over 13,000 new cases in the U.S. annually and a two-year survival rate of < 5%. GBMs are highly invasive and classified by the World Health Organization as a grade IV astrocytoma. Recent studies have shown that protein kinase A (PKA), a key regulator of signal transduction and various biological functions, is essential for maintaining polarity during cell migration and that localized activation of PKA at the leading edge is an early event in directional cell migration of other cell types. Though PKA has previously been shown to promote proliferation and migration in lung, colorectal, adrenocortical, and prostate cancer cell lines, the molecular mechanisms that link PKA activity with GBM physiology remain unclear. Wound healing assays in human GBM cell lines U-251 MG, U-87 MG and A172, demonstrated that GBM cell migration increased following the addition of 20 μM epidermal growth factor (EGF). Similar results were found with 5 μM treatment of the adenylate cyclase activator, forskolin, and inhibited by pre-treatment with 10 μM of the PKA inhibitor, H89. In order to assay the amount of PKA activity, we measured the phosphorylation of cAMP response element binding protein (CREB) in these cell lines. Western blot studies revealed an increase in CREB phosphorylation in cells treated with EGF and forskolin. Phosphorylation of CREB was decreased with the addition of H89 treatment. Our western blot analysis supports our wound healing data demonstrating a correlation between increased PKA-mediated CREB phosphorylation and migration in the absence of serum. Taken together, these data suggest that PKA activation by EGF is involved in GBM cell migration. Moreover, our findings describe a mechanism by which PKA regulates the migration of human brain tumors.

#934

Tributyltin-induced dysregulation of inflammatory cytokine levels in human and mouse immune cells.

Shanieek Lawrence,1 Samuel T. Pellom,2 Anil Shanker,2 Margaret Whalen1. 1 _Tennessee State University, Nashville, TN;_ 2 _Meharry Medical College, Nashville, TN_.

The effects of Tributyltin (TBT), a widespread environmental contaminant, on the induction of in vivo and ex vivo cytokine production and secretion were investigated. Human monocyte-depleted peripheral blood mononuclear cells and different groups of Balb/C mice were stimulated with desired concentrations of TBT (200-2.5 nM for human studies and 200-25 nM for mouse studies). The quantitative determination of IFNγ, TNFα and IL-1β was performed in mouse sera by MagPix analysis and western blot. The levels of these cytokines were measured at different time points: 6, 12, 24 and 48 h after injection. Analysis of IFNγ and TNFα in human cells were conducted by ELISA and western blot. MAPK and NF-κB pathways were inhibited to determine TBT-induced dysregulation of IFNγ and TNFα. Inhibition of the p38 and p44/42 pathways blocked the TBT-induced elevation of IFNγ, while the inhibition of the p38 pathway blocked the TBT-induced elevation of TNFα. Results showed that TBT increased the levels of IFNγ, TNFα and IL-1β in the sera of mice. IFNγ and TNFα secretion from human monocyte-depleted peripheral blood mononuclear cells also increased, showing striking agreement in the response to TBT between the mouse and human systems. These data suggest that TBT exposure can result in an inflammatory environment, which could predispose the host to various immunopathologies.

Grant U54CA163066 from the National Institute of Health

#935

Oral cancer cells may hijack stem cell altruism to survive during extreme hypoxia, and exposure to chemotherapeutic drugs.

Rashmi Bhuyan,1 Hong Li,1 Sukanya Gayan,1 Bidisha Pal,1 Reza Bayat-Mokhtari,1 Jyotirmoy Phukan,2 Debabrata Baishya,3 Anupam Sarma,4 Joyeeta Talukdar,5 Manaf Muhammad Alkurdi,1 Wael Tasabehji,1 Seema Bhuyan,5 Gayatri Gogoi,5 Ista Pulu,5 Herman Yeger,6 Bikul Das1. 1 _Forsyth Institute, Cambridge, MA;_ 2 _Guwahati Medical College, Guwahati, India;_ 3 _Gauhati University, Guwahati, India;_ 4 _B Borooah Cancer Institute, Guwahati, India;_ 5 _KaviKrishna Laboratory, Guwahati Biotech Park, Guwahati, India;_ 6 _Hospital for Sick Children, Toronto, Ontario, Canada_.

The mechanism of tumor hypoxia induced reprogramming of cancer cells are not understood well. Hypoxia might activate evolutionary preserved cellular defense mechanism that could contribute to tumor progression and metastasis. We recently described that during hypoxia/oxidative stress, human embryonic stem cells (hES) exhibit an altruistic defense mechanism, where a few cells reprogram to a highly undifferentiated state, and secrete glutathione to protect rest of the community of cells from oxidative stress induced DNA damage (Das B et al, Stem Cells, 2012). The altruistic stem cells phenotype exhibited high HIF-2alpha and low p53 activity. After a few weeks, the reprogrammed cells, although highly fit to survives, underwent spontaneous apoptosis/differentiation by re-activating the p53/MDM2 oscillation. Thus, the reprogrammed cells sacrificed its own fitness to enhance the fitness of the rest of the community, an altruistic behavior (Das B et al. Stem Cells, 2012). Here, we investigated the potential hijacking of the altruistic defense mechanism by oral cancer cells during exposure to hypoxia. We exposed the four oral cancer cell lines SCC-25, SCC-15, SCC-9 and SCC-5 to extreme hypoxia followed by re-oxygenation for 72 hours. We found that while majority of cancer cells underwent apoptosis, a few cancer cell lines survived, exhibited side-population (SP) phenotype, high level of HIF-2alpha, Nanog, Sox-2 and MYC. The SP cells exhibited migratory activity, as well as high tumorigenic and metastatic activity in NOD/SCID mice. ChIP assay indicated that HIF-2alhpa interact with MYC and NOTCH1. The conditioned media of SP cells exhibited high level of glutathione, and ability to protect non-SP cells from cisplatin-induced toxicity. These results indicate that HIF-2alpha and MYC may cooperate to reprogram a few oral cancer cells to altruistic stemness phenotype. The altruistic phenotype that exhibited cytoprotective activity against cisplatin mediated toxicity. Thus, similar to bacteria, where altruistic biofilm exhibit novel drug resistance mechanism, stem cell altruism may serve as a novel drug resistance mechanism in oral cancer.

#936

Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome.

Cristina Cristofoletti, Mario Picozza, Antonella Bresin, Maria Cristina Picchio, Enrico Scala, Giuseppe A. Lombardo, Francesca Passarelli, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. _Ist. Dermopatico dell'Immacolata-IRCCS, Rome, Italy_.

Introduction

Sézary Syndrome (SS) is a rare and aggressive variant of Cutaneous-T-Cell Lymphoma (CTCL) characterized by the presence of malignant lymphocytes named Sezary (SS) cells in the skin, lymph nodes, and peripheral blood. With a poor prognosis, SS has not a specific therapy still available. As a role of skin in SS pathogenesis is not elucidated yet, here we study the contribution of this microenvironment by comparing matched skin and blood derived SS cells for tumor cell proliferation and activation levels of PI3k/AKT pathway. Using our previous SNP array data we also verified the genomic status of members belonging to this pathway in a large cohort of SS patients.

Methods

Expression of Ki67 proliferation marker was evaluated in perfect-paired blood and skin-paraffin biopsies

obtained from eleven SS patients by flow cytometry analysis and immunohistochemistry respectively. KI67+ neoplastic cells were calculated as percentage within neoplastic CD4+ cells recognized by a co-staining with the specific TCRVb rearrangement.

Phosphorylation levels of members of PI3k/AKT pathway were compared between matched circulating and skin resident SS cells proteins derived from 2 SS patients using an AKT kinase array (Cell Signaling). Affymetrix SNP6.0 arrays was used to investigate the copy number (CN) status of members of mTORC 1 pathway in a cohort in 37 SS samples derived from 23 SS patients and 3 cell lines.

Results

Skin derived SS cells showed a significant higher proliferation index (PI) respect to SS cells obtained from blood (12%±11 vs 1,24%+1,18; P=0,00025). In order to identify the signals responsible for SS proliferation, we used a PI3K/AKT kinase array that revealed an enhanced phosphorylation levels of many components of this cascade, particularly of PRAS40 with a Fold change (Fc) of 6,15; GSK3a (Fc=4.83), mTOR (Fc=4.61) mP70S6K (Fc=4.64) BAD (Fc= 7.09) and 4EBP1 (Fc=5.40)in skin-SS cells respect to circulating ones.

We next deepen our observations in mTORC1 signaling because of this and earlier observations made in CTCL cell lines. Using our SNP6 array data we have verified the genomic status of members of this pathway. Results obtained showed that P70S6K, the kinase downstream TORC1 that plays a crucial role in protein synthesis, showed a mono-allelic gain in 9 out 23 patients (39%) whereas PDCD4, a protein that inhibits protein translation displayed a mono-allelic loss in 10 of 23 individuals (43%). 4 of these patients showed a concomitant P70S6K gain and PDCD4 loss. Overall these genetic defects suggest that an abnormal protein synthesis can occur in these patients..

Conclusion:

Our data demonstrate that skin microenvironment enhances SS cell proliferation index and activates mTORC1 signaling, an unbalanced pathway that reveals novel potential therapeutic targets for Sezary Syndrome.

#937

Nef-M1 peptide inhibits oncogenic signaling and tumor angiogenesis in patient-derived xenografts of colorectal cancer.

Venkat R. Katkoori, Harvey L. Bumpers. _Michigan State University, CHM, E. Lansing, MI_.

Introduction: The Nef-M1peptide competes effectively with the natural ligand of CXCR4, SDF-1α, to inhibit tumor angiogenesis in colorectal and breast cancer cell models. However, these established cell models may not signify an adequate means for evaluating signaling mechanisms and therapeutic sensitivities. In this study, we evaluated the antiangiogenic effect of Nef-M1 and its inhibitory role in oncogenic signaling in patient-derived xenograft (PDX) of colorectal cancer (CRC).

Experimental Design: Surgical specimens of human CRC were cut into 2-to 3-mm3 pieces in antibiotic-containing RPMI 1640 medium. Non-necrotic tumor pieces were selected and implanted subcutaneously in severe combined immunodeficient (SCID) mice. Tumors were harvested when they reached a size of 1500-mm3 (PDX1). The xenografts were repassaged multiple times with retention of genetically human characteristics. One week after implantation the mice started receiving intraperitoneal Nef-M1 injections at 2μg biweekly. Sections from PDX were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial marker (CD31). MVD was determined by light microscopy in areas of invasive tumor containing the highest numbers of microvessels per high power field. Western blot analyses were performed on lysates of both PDX and CRC cell lines to assess the effect of Nef-M1 on the oncogenic signaling pathways.

Results: Histological analysis revealed that the surgical specimens and the corresponding PDXs were morphologically similar. However, differences were seen in the untreated and treated PDXs. In mice bearing subcutaneous PDX tumors, Nef-M1 treatment inhibited tumor growth. Immunostaining analyses indicated that control PDXs had well established vascularity, but Nef-M1 treated PDXs had poor vascularization. The average MVD was reduced in Nef-M1 treated PDXs (n=530) compared to control PDXs (n=1132) (p<0.017).Western blot analyses of lysates of PDXs indicated that Nef-M1 effectively suppressed the expression of VEGF-A and the activation of p38 and extracellular signal-regulated kinase (ERK) MAP kinases. Suppression of Akt and Wnt/beta-catenin activation was significantly higher in Nef-M1 treated PDXs. Furthermore, Nef-M1 treated CRC cells displayed inhibition of Wnt/beta-catenin signaling as demonstrated by an increased expression of p21 and decreased expression of beta-catenin, cyclin D1, Axin 2 and survivin. The signaling appears linked to the CXCR4 receptor as cells expressing CXCR4 became susceptible to Nef-M1 induced inhibition of MAP kinase, Akt or Wnt/beta-catenin signaling pathways.

Conclusions: Treatment with Nef-M1, a peptide antagonist of CXCR4, is a highly promising novel experimental therapeutic strategy against CRC. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251.

#938

Activation of ATR/CHK1 checkpoint is critical for mediating p53-dependent autophagy and pro-inflammatory VEGF production in human bronchial epithelial cells under fine particulate matters (PM2.5) exposure.

Lun Song. _Beijing Inst. of Basic Medical Sciences, Beijing, China_.

The epidemiological and clinical studies have increasingly shown that fine particulate matters (PM2.5) exposure is associated with exacerbation of airway inflammation, which might results in the occurrence of lung cancer. Thus, understanding how PM2.5 triggers inflammatory responses in the respiratory system is a crucial issue for the study of PM2.5 toxicity. In the current study, we found that exposure of human bronchial epithelial cells (Beas-2B) to the winter PM2.5 collected in Wuhan, the south city of China, induced an significant up-regulation of VEGF production, a typical signaling event functionally in controlling chronic airway inflammation and vascular remodeling. Further investigations showed that autophagy was induced upon PM2.5 exposure and then mediated VEGF up-regulation by activating Src/STAT3 pathway in the Beas-2B cells. When exploring the upstream signaling events responsible for autophagy induction, we disclosed the requirement of p53 activation and its downstream target DRAM expression for the induction of autophagy, which results thus have extended the role of p53/DRAM-dependent autophagy beyond cell fate determination under genotoxic stresses and to the control of pro-inflammatory cytokine production. Moreover, PM2.5 exposure strongly induced activation of ATR/CHK1 axis, which subsequently triggered p53-dependent autophagy and VEGF production in Beas-2B cells. Therefore, these findings suggest a novel link between processes regulating genome integrity and the airway inflammation via autophagy induction in bronchial epithelial cells under PM2.5 exposure.

#939

Cell-free DNA associated with extracellular vesicle: Biomarker or bioactivity.

Ganesh V. Shelke,1 Su Chul Jang,1 Yanan Yin,2 Cecilia Lässer,1 Jan Lötvall1. 1 _Krefting Research Centre, Gothenburg, Sweden;_ 2 _Shanghai First People's Hospital, Jiao Tong University,, Shanghai, China_.

Extracellular vesicles (EVs) including exosomes transfer bioactive cargo, including proteins and nucleic acids. Recently, EVs associated DNA were shown to be surrogate biomarker present in body fluids harboring various oncogenic mutations. Cell-free, extracellular DNA is reported in cell cultures and many body fluids, but its relative location in relation to EVs and function is not known. Our study demonstrates that DNase sensitive nucleic acids were present on the surface of EV isolates from mast cell lines. Association of EV-DNA was revealed by increase of zeta potential and particle number upon DNase treatment. Additionally, cytoplasmic DNA traces were found in recipient mesenchymal stem cell after EVs treatment. In conclusion, we define that surface association of DNA with EVs that could regulate recipient cellular function.

#940

The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines.

Raia Doumit, Roula Tahtouh, Rita Ammoury, Riad Sarkis, Nada Alaaddine, George Hilal. _Saint-Joseph University, Beirut, Lebanon_.

Background: Hepatocellular carcinoma (HCC) is the third largest contributor to cancer mortality in the world. Alphafetoprotein (AFP) is synthetized and secreted by the majority of HCCs. Despite its controversial value as classical HCC diagnostic and follow-up marker, the AFP was lately shown to correlate with the volume of liver cancer. It is also useful as a predictive marker for radiotherapy treatment response and patients prognosis. On the molecular level, AFP has a proliferative role in HCC since it acts on its own receptor on the surface of hepatocytes; however, its mechanism of action remains unclear. Our laboratory have previously demonstrated that telomerase and protein kinase C (PKC) modulate AFP secretion in HCC cell lines and this effect is additive. The aim of this study is to elucidate the PKC isoforms involved in AFP secretion modulation and telomerase expression. Methods: Two AFP secretory cell lines, HepG2/C3a and PLC/PRF/5, and one non-secretory AFP cell line, SNU-387, were cultured in DMEM (RPMI-1640 for SNU-387) media with 10% FBS and 1% Penicillin/Streptomycin and incubated in humid 5% CO2 incubator. The RNA was extracted; the RT-PCR was performed to characterize the PKC isoforms and the modulation of hTERT by these isoforms. The role of each isoform on AFP secretion was elucidated using different PKC isoform modulators; the AFP concentration was measured using ELISA technique. The viability and toxicity of cells were assessed using WST-1. Results: The three cell lines express the PKC alpha, delta and epsilon isoforms. Whereas the other isoforms Beta 1 and 2 are slightly expressed in C3a, the gamma and beta isoforms are slightly expressed in PLC and SNU respectively. Dose-dependent inhibition of PKC alpha and beta by GO6976 decreased AFP secretion by 40 % at 10 uM by the HepG2/C3a cell line. The inhibition of PKC delta by Rottlerin dose-dependently decreased AFP secretion by 30% at 5 uM. The PKC epsilon modulator FR236924 dose-dependently inhibited AFP secretion by 50% at 1 uM. Pan PKC inhibitor, GO6983, decreased AFP secretion by 45% at 5 uM. The same profile was obtained for PLC/PRF/5 with a maximum 30% of AFP down-regulation except for Rottlerin which showed a 75% of AFP inhibition. The catalytic subunit expression of telomerase, hTERT, was only inhibited by Rottlerin at 5 uM. Our experiments did not show any cell death nor cell proliferation modulation when incubating the three cell lines with PKC inhibitors. Conclusion: Combining the above results with our previous ones, we suggest for the first time, that PKC delta modulates AFP secretion through telomerase however the mechanism by which PKC alpha and epsilon modulate AFP secretion is to be elucidated. However, further experiments are needed to support this statement.

#941

Differential roles for membrane-bound and soluble CD109 in breast cancer progression.

Priyanka Sehgal, Anie Philip. _McGill University, Montreal, Quebec, Canada_.

Introduction: Our group has recently reported the identification of CD109 (a GPI anchored cell surface glycoprotein) as a TGF-β co-receptor and potent antagonist of TGF-β signaling and responses in human epithelial cells in vitro and in vivo. Proteinase mediated shedding converts CD109 from a membrane-bound coreceptor into a soluble effector capable of binding TGF-β. Our results indicate that CD109 overexpression inhibits TGF-β -induced Epithelial Mesenchymal Transition (EMT) and migration in breast cancer cells in vitro. However, in breast carcinomas, CD109 overexpression correlates with poor prognosis and an aggressive phenotype. Our findings together with the recent report of over expression of CD109 in breast cancer provide compelling reasons to examine whether membrane anchored or soluble CD109 plays an essential role in breast cancer progression and thus represent a molecular target for the treatment of breast cancer.

Methods: To investigate the role of CD109 in breast cancer cells that overexpress membrane-anchored (m)CD109 or soluble (s)CD109, or in which endogenous CD109 expression is blocked, or breast cancer cells treated with recombinant CD109 protein, were analysed for SMAD signaling and EMT markers (Fibronectin, PAI, Vimentin, N-cadherin, α-sma, Snail) using immunoblotting. Cell migration and invasion potential in response to TGF-β were assessed using scratch assay and boyden chamber assay, respectively.

Results: To determine the role of CD109 in breast cancer progression, human MDA-MB-231 breast cancer cells were transfected with plasmid overexpressing wild-type (WT) which has the potential to shed CD109. Overexpression of WT CD109 decreased TGF-β induced phospho-SMAD signaling, EMT, migration and invasion of breast cancer cells. Conversely, treatment of Hs578T breast cancer cells with CD109 siRNA increased TGF-β induced phospho SMAD signaling, EMT, migration and invasion of cancer cells. In an alternate approach breast cancer cells treated with recombinant CD109 (resembling the shed form of CD109) also showed decreased TGF-β induced EMT, migration and invasion of cancer cells. However overexpression of membrane bound (furinase-mediated shedding resistant) form of CD109 in MDA-MB-231 breast cancer cells showed increased EMT, migration and invasion of cancer cells. Analysis of downstream signaling showed that although both membrane bound and soluble form of CD109 attenuates TGF-β signaling, the membrane bound CD109 promotes tumor aggressiveness.

Conclusion: Taken together, these results suggest that membrane anchored and soluble form of CD109 have different role in breast cancer. Both membrane bound and soluble CD109 inhibit TGF-β signaling in breast cancer however the membrane bound form of CD109 can promote cancer progression. Mechanisms underlying the differential effect of these two forms of CD109 in breast cancer need to be deciphered.

#942

Role of IL-8/CXCR2 network in the tumor cell proliferation of esophageal squamous cell carcinoma.

Masazumi Inoue, Hiroya Takeuchi, Sachiko Matsuda, Tomohiko Nishi, Kazumasa Fukuda, Rieko Nakamura, Tsunehiro Takahashi, Norihito Wada, Hirofumi Kawakubo, Yuko Kitagawa. _Keio University, Tokyo, Japan_.

Background: IL-8, a pro-inflammatory cytokine, and its receptor, CXCR2, are expressed invarious cancer cells. The IL-8/CXCR2 network is believed to be involved in angiogenesis, tumor cell proliferation and invasion. However, its role inesophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we investigated the role of the IL-8/CXCR2 network in the tumor microenvironment of ESCC.

Methods: IL-8 and CXCR2 expression was investigated immunohistochemically in 63 primary ESCCs with R0 resection but without any preoperative treatment. We evaluated the intensity of the staining in four grades; grades 3 (strong) and 2 (medium) were deemed "positive", and 1 (weak) and 0 (negative) as "negative". The concentration of IL-8 and

CXCR2 were investigated using the human ESCC cell lines TE1, 4, 5, 6, 8, 9, 10, 11, 14 and 15. IL-8/CXCR2 signaling was stimulated by IL-8 addition, and suppressed by SB225002 (Cayman Chemical) treatment, a selective antagonist of CXCR2, and IL-8 specific siRNA. Stable IL-8 over-expressing TE4 cells were established, and cell proliferation assays were performed. To evaluate tumor growth in vivo, we subcutaneously inoculated 1.0×106 TE4 cells or transfectant into nude mice, and half of the

transfectant-inoculated mice received 1mg/kg of SB225002 by intraperitoneal injection thrice weekly. The size and incidence of subcutaneous tumors were recorded.

Result: Eight (12.7%) patients were positive for IL-8/CXCR2 network immunostaining. IL-8/CXCR2 network expression was significantly

related to poor survival in ESCC patients (P<0.05). IL-8 concentration was high in TE4, 5, 10 and 14 supernatant, and low in that of TE1, 6, 8, 9, 11 and 15. CXCR2 expression was high in TE1, 4, 5, 6, 8, and 9 cells, and low in TE10, 11, 14, and 15. We chose TE1 and TE4 cells for further investigation. Stimulation or inhibition of the IL-8/CXCR2 network resulted in significant enhancement or suppression of cell proliferation in both cell lines (P<0.05) in a dose-dependent manner. IL-8 over-expressing TE4 cells showed significantly higher proliferative activity than the parental line (P<0.05), which was markedly suppressed by inhibition of the IL-8/CXCR2

network using SB225002 (P<0.05). In vivo, tumor sizes were smaller in the SB225002-treated group than in the untreated group.

Conclusion: Our results demonstrated that stimulation of the IL-8/CXCR2 network clearly enhanced ESCC cell proliferation, while its inhibition obviously suppressed ESCC cell proliferation in vitro. These results indicate that control of IL-8/CXCR2 network signaling may be a new therapeutic strategy for ESCC.

#943

Retinoid sensitizes Tumor Necrosis Factor (TNF) -related apoptosis inducing ligand -induced cytotoxic effect in breast cancer by upregulating cell surface receptor Death Receptor-4 expression.

Annekatrine Kratz, Chuanbing Zang, Jan Eucker, Hongyu Liu. _Charite Campus Benjamin Franklin, Berlin, Germany_.

Tumor Necrosis Factor (TNF) -related apoptosis inducing ligand (TRAIL, also called APO-2L) is a member of the TNF super family which is known to induce apoptosis selectively in tumor cells, whereas its cytotoxicity to non-transformed cells turned out as low to moderate. The potential of TRAIL as a pharmacotherapeutic agent against cancer is therefore widely pre-clinically and clinically investigated. However, the test results from a wide range of in vitro models revealed that a great portion of tumor cell cultures displayed inherent or acquired resistances towards TRAIL-mediated cell death. Mechanisms underlying these resistances comprise, among others, downregulation of cell surface receptors of TRAIL, namely Death Receptors (e.g. DR4, DR5). Hence, enhancing the DRs expression by application of TRAIL in combination with other therapeutics, is one of the diverse approaches to augment the TRAIL-sensitivity and overcome the resistance.

The term Retinoids comprises different polyisoprenoids of the Vitamin-A-group. The derivatives all-trans- and 9-cis retinoic acid (ATRA and 9-cis-RA) function as activators of transcription initiating nuclear hormone receptors and hereby regulate key components of carcinogenesis like proliferation, differentiation and apoptosis. Furthermore, Retinoids have proven potential in the secondary prevention of breast cancer. In this study, we investigated whether the combination of TRAIL with Retinoids could be a new therapeutic approach for breast cancer treatment. Our results indicate that simultaneous incubation with TRAIL and ATRA resulted in a synergistic inhibition of proliferation in the Retinoids-sensitive breast cancer cells. This synergistic combination effect on cell proliferation correlated with augmented cell apoptosis as detected with different methods. Immunoblotting assays revealed that the apoptosis-regulaing factors for both intrinsic and extrinsic apoptotic pathways including Bcl-2 family members, Bid, survivin, and diverse caspases were involved in the combination treatment-triggered apoptosis folowing the Apo2L/TRAIL activation. Analysis of cell surface DRs expression by flow cytometry demonstrated that ATRA contributed to the enhancement of TRAIL-sensitivity most possibly by increasing the TRAIL receptor DR4 but not DR5 expression and thus leading to more strong apoptotic effect when cells were upon TRAIL treatment.

Taken together, we demonstrated for first time that Retinoid derivatives can be used to enhance the cytotoxic effect of TRAIL in some sub-entities of breast cancer by upregulating the DR4 cell surface expression. Our study provides a rationale for future clinical trials.

#944

The effects of GnRH-(1-5) on endometrial cancer cell lines.

Darwin O. Larco, Madelaine J. Cho-Clark, Maya Sorini, Cameron Lee, T. John Wu. _USUHS, Bethesda, MD_.

The Gonadotropin-Releasing Hormone (GnRH) is a central regulator of reproductive function and behavior. In the periphery, GnRH is secreted by gynecologic tissues to exert local effects. Furthermore, GnRH is processed in the extracellular environment by the metalloendopeptidase EP24.15 to generate the bioactive metabolite, GnRH-(1-5). We have previously demonstrated that GnRH-(1-5) transactivates the epidermal growth factor receptor (EGFR) pathway to promote cell migration by binding the orphan receptor GPR101 in the Ishikawa and ECC-1 endometrial cancer cell lines. In this study, we sought to determine whether the effects of GnRH-(1-5) were dependent on the stage of the endometrial cancer. Cell lines ACI-181, ACI-52, and ACI-80 derived from endometrial cancers representative of stages 1, 2, and 3 respectively were tested in their response to GnRH-(1-5) treatment. Furthermore, the expression of GPR101 was confirmed by western blot analysis. Treatment with 100nM GnRH-(1-5) did not have significant changes in phospho-EGFR (pEGFR) levels in all ACI cell lines. However, subsequent measurement of ERK phosphorylation demonstrated that ACI-181 cells treated with GnRH-(1-5) had significantly (p < 0.05) increased levels in the pERK2 subunit but not pERK1, which parallels our initial findings in the Ishikawa cell line. No changes in pERK1/2 levels were determined in the other cell lines. To measure the effect of GnRH-(1-5) on cell migration, we implemented a wound closure assay. Interestingly, ACI-181 and ACI-52 cells had increased migration in response to GnRH-(1-5) treatment. To determine whether another pro-migratory pathway is regulated by GnRH-(1-5) in these cells, we measured TGF-β bioactivity using a cell-based assay from conditioned media of cells treated with GnRH-(1-5). We found that ACI-181 and ACI-52 cells had significantly (p<0.05) increased TGF-β bioactivity in response to GnRH-(1-5), indicating that these cells produced higher levels of bioactive TGF-β. It is likely that TGF-β and/or other growth factors may promote pro-survival and/or pro-migratory signaling cascades via an autocrine mechanism. These results further suggest that GnRH-(1-5) may exert differential effects on endometrial cancer cells that are not dependent on cancer progression.

Funding Sources: This research was supported by grants from the John P. Murtha Cancer Center at Walter Reed National Military Medical Center through the Uniformed Services University and the National Science Foundation (IOS-1052288).

#945

Tissue tranglutaminase-2 promotes gastric cancer progression via ERK1/2 pathway.

Liping Su, Xiaofeng Wang, Quan Zhou, Xiongyan Wu, Bingya Liu. _Shanghai Jiao Tong University, Shanghai, China_.

Gastric cancer is one of the most common tumors worldwide with extensive local tumor invasion, metastasis and poor prognosis. Thus, understanding the mechanisms regulating progression of gastric cancer is the key for developing effective therapeutic strategies. Tissue tranglutaminase-2 (TG2), a multifunctional member of the transglutaminase (TGase) family, has already been proved to play an important role in tumor initiation and progression. However, the specific role and underlying molecular mechanisms of TG2 in the progression of gastric cancer (GC) remain unknown. Here we showed that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain and lose-of function approaches, we found that TG2 promotes GC cell proliferation, migration and invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with activating of ERK1/2 pathway, and ERK1/2 inhibitor U0126 inhibited cell proliferation, migration and invasion induced by overexpression of TG2. Taken together, these findings suggest that TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer. 

### Circulating RNAs, Epigenetics, and Novel Non-coding RNAs in Cancer

#946

Epigenetic silencing of tumor suppressor long non-coding RNA BM742401 in chronic lymphocytic leukemia.

Lu Qian Wang, Kwan Yeung Wong, Zhen Hai Li, Chor Sang Chim. _The University of Hong Kong, Hong Kong, China_.

Background: B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. Long non-coding RNAs (lncRNAs) emerge as a new class of ncRNAs measuring longer than 200 nucleotides in length, and implicated in a wide range of biological processes including normal development, differentiation or carcinogenesis [1]. Recently, deregulated expression of lncRNAs has been found in many human cancers, indicating its potential oncogenic or tumor suppressor role in carcinogenesis [2]. However, data on the role of lncRNA in CLL is scanty. BM742401 is a tumor suppressor lncRNA downregulated in gastric cancer [3]. As the promoter region and the entire transcript are embedded in a CpG island, we postulated that BM742401 is a tumor suppressor lncRNA inactivated by DNA methylation in CLL.

Methods: Methylation of BM742401 was studied by methylation-specific polymerase chain reaction (MSP) in nine healthy donor normal controls including three bone marrow, three peripheral blood buffy coats, and three CD19-sorted peripheral B-cells, in addition to seven CLL cell lines, and ninety-eight diagnostic CLL marrow samples.

Results: By MSP, promoter of BM742401 was unmethylated in normal controls, but methylated in four out of seven (57%) CLL cell lines. MSP statuses, including complete methylation (MM), partial methylation (MU), and complete unmethylation (UU), were verified by quantitative bisulfite pyrosequencing. Methylation of BM742401 was inversely correlated with expression. In the completely methylated WAC3CD5+ CLL cells, treatment with 5-Aza-2'-deoxycytidine led to promoter demethylation and re-expression of BM742401 transcript. Functionally, stable overexpression of BM742401 by lentiviral transduction resulted in inhibition of cellular proliferation and enhanced apoptosis through caspase-9-dependent intrinsic apoptosis pathway, suggesting a tumor suppressor role of BM742401 in CLL. In primary CLL samples, methylation of BM742401 was detected in 43/98 (44%) of patients. Moreover, among CLL patients with standard-risk cytogenetic aberrations, methylation of BM742401 correlated with advanced Rai stage (≥ stage 2) (P = 0.002). Furthermore, methylation of BM742401 was significantly associated with methylation of miR-129-2 (P = 0.034).

Conclusions: BM742401 is a tumor suppressor lncRNA frequently methylated in CLL. The functional mechanism of BM742401 in triggering tumor suppressive phenotype and regulation of other protein-coding genes warrant further studies.

References:

1. Esteller, M., Non-coding RNAs in human disease. Nature Reviews Genetics, 2011. 12(12): p. 861-874.

2. Prensner, J.R. and A.M. Chinnaiyan, The emergence of lncRNAs in cancer biology. Cancer discovery, 2011. 1(5): p. 391-407.

3. Park, S.-M., et al., A known expressed sequence tag, BM742401, is a potent lincRNA inhibiting cancer metastasis. Experimental & Molecular Medicine, 2013. 45(7): p. e31.

#947

Polycomb protein SUZ12 is regulated by a novel miRNA like RNA (miR-G655A) present in extracellular vesicles secreted by mesenchymal stem/tromal cells.

Patrice Penfornis, Joseph D. Fernandes, Radhika R. Pochampally. _University of Mississippi Medical Center, Jackson, MS_.

Introduction: Role of human mesenchymal stem/stromal cells (hMSCs) in cancer progression is well studied. Previously, we showed that hMSCs use autophagy to survive in adverse environment (e.g. serum deprivation). Using this model to mimic the solid tumor core, we¹ve shown that the secretome of these stressed hMSCs is also tumor supportive. Furthermore, we showed that extracellular vesicles (EVs) secreted by cells contain small RNAs, proteins and lipids that are tumor supportive. In this study, we focused on a new and unknown small RNA contained in EVs, its potential mRNA targets in recipient cancer cells and its regulation in parent hMSCs and other cancer cells.

Methods: EVs were isolated from serum deprived human mesenchymal stem/stromal cells (hMSCs) using a concentrator followed by serial ultracentrifugation. RNA molecules less that 200 bases were isolated and a purified cDNA library was used for cluster generation then sequenced on Illumina GAIIx. Human cancer cells (osteosarcoma, fibrosarcoma and breast cancer) miRNA knockdown models were obtained using agomirs and shRNA strategies against miRNA-G655A. Cell proliferation assays were performed using trypan blue and Cyquant DNA detection. In vivo tumor progression in nude mice was tracked by caliper measurement and relative GFP signal detected with in vivo imaging station. Small RNA targets were revealed by Affimetrix Human gene 2.0 ST array analysis and confirmed by 3¹UTR luciferase assays. Cross linked-ChIP assays for EZH2 and SUZ12 were used to identify interactions with the new small RNA promoter region.

Results: Next generation sequencing of EVs from hMSCs identified an unknown small RNA, temporarily named miR-G665A. We report here that miR-G665A is conserved in mammals and its expression is confirmed, but varies between tissues or cell line¹s. EVs secreted by hMSCs are notably enriched with miR-G665A. Both in vitro and in vivo knockdown studies using agomirs and shRNA strategies indicate that miR-G665A participate in cell proliferation and tumor progression. Its expression is also regulated through cell cycle. Microarray data from these knockdown studies revealed potential end targets of miR-G665A (e.g. interleukins,MMPs). Some direct targets are validated using 3¹UTR luciferase assays. One of the direct target is SUZ12, a component of the polycomb group complex PRC2, a master epigenetic regulator which contributes to the neoplastic phenotype. We also show that PRC2 complex (SUZ12 and EZH2) targets the regulation of the expression of this novel small RNA in a feedback loop mechanism.

Conclusion: The role of this novel small RNA on the regulation of an essential transcription factor could lead to a better understanding of the crosstalk mechanisms between mesenchymal stem/stromal and cancer cells.

This work is supported by NIH grant CA1515851 and the UMMC Cancer Institute startup funds.

#948

Microenvironmental origin of circulating miRNAs in lung cancer.

Orazio Fortunato, Cristina Borzi, Davide Conte, Giovanni Centonze, Mattia Boeri, Carla Verri, Linda Calzolari, Ugo Pastorino, Gabriella Sozzi. _Fondazione IRCCS Istituto Tumori Milano, Milan, Italy_.

INTRODUCTION We reported the identification of diagnostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Circulating miRNAs are released into the bloodstream by different mechanisms such as passive leakage from broken cells or active secretion through microvesicles or protein complexes.

MATERIAL and METHODS To evaluate the origin and the release of the 24 miRNAs we analyzed their expression by real-time PCR in different lung cell types including fibroblasts, hematopoietic, endothelial, bronchial epithelial (HBECs) and tumor cells and in the respective culture medium (CM) as well as in plasma samples from heavy smokers and patients.

RESULTS and DISCUSSION The analysis of lung cancer plasma samples in patients showed that miR-106a, miR-140, miR-142-3p, miR-17, miR-660, miR-320, miR-197 and mir-28-3p were those significantly returning to a physiological level after surgery (p<0.05). On the other hand, mir-126 and mir-451 remained deregulated after tumor resection suggesting an "host-related" origin and likely indicating the persistence of a risk profile. To evaluate which lung cell type express these miRNAs we performed in situ hybridization and observed that mir-451 is specifically expressed in lung interstitial alveolar walls by cells of the fibro-monocytic infiltrate whereas mir-126 seems to be expressed by endothelial cells.

The analysis of the 24-miRNAs composing the signature in the various lung cell types showed an increased expression in specific cellular components such as that of mir-16-17-19b-106-145 in fibroblasts, mir-126 in endothelial cells, mir-451 and mir-142-3p in hematopoietic cells. The analysis of miRNAs released in CM showed that miRNAs were secreted differently and independently from their cellular levels and that tumor cells were the less involved in this release. Interestingly, we identified miRNAs that were expressed and released from blood cells only such as mir-451, mir-133 and mir-142-3p. To better characterize the origin of plasma miRNAs, we isolated exosomes from CM of the different cell types. We detected the presence of the 24 miRNAs in exosomes and identified exosomal miRNAs that were secreted by specific cellular compartments (mir-101 from blood cells, mir-145 from fibroblasts and mir-126 from endothelial cells). Furthermore, by analyzing the exosome fraction of 29 plasma samples of 21 smokers and 8 patients we confirmed the presence of all the 24 miRNAs inside exosomes and comparing miRNAs content in plasma and in their respective exosome fraction we observed a good degree of correlation (Pearson's R=0.89, p for Pearson's R <0.001) indicating that miRNAs expression in exosomes may reflect miRNA levels in total plasma.

CONCLUSION Our findings on the origin of circulating miRNAs support the conclusion that plasma miRNAs are heterogeneous and are actively secreted inside exosomes by different cellular components and not specifically by tumor cells.

#949

Epigenetic silencing of microRNA-34a promotes cholangiocarcinoma growth by regulating Notch pathway.

Hyunjoo Kwon, Kyoungsub Song, Chang Han, Jinqing Zhang, Nathan Ungerleider, Lu Yao, Tong Wu. _Tulane University, New Orleans, LA_.

Aberrant expression and regulation of microRNAs (miRNAs) have been found in multiple cancers and their dysregulation importantly contribute to the tumorigenic processes. Among these miRNAs, miRNA-34a (miR-34a) functions as a tumor suppressor and its dysregulation has been reported in several cancers. In this study, we investigated epigenetic regulation and biological function of miR-34a in human cholangiocarcinoma cells. Our data indicate that miR-34a expression is epigenetically silenced in human cholangiocarcinoma cells (CCLP1, SG231, Hucct1 and TFK1) compared to the nonmalignant biliary epithelial cells (H69). Treatment of human cholangiocarcinoma cells with the DNA methylation inhibitor, 5'aza-2'deoxycytidine (5-Aza-CdR), or the EZH2 (enhancer of zeste 2 polycomb repressive complex 2) inhibitor, GSK126, enhanced the expression of miR-34a. In parallel, knockdown of EZH2 by shRNA also significantly decreased miR-34a expression in human cholangiocarcinoma cells. We further showed that DNA methylation and EZH2-mediated histone H3 lysine 27 trimethylation (H3K27Me3) repressed miR-34a gene expression, as determined by methylation-specific PCR and chromatin immunoprecipitation. The DNA methylation and EZH2-mediated H3K27Me3 independently repress miR-34a expression in cholangiocarcinoma cells since treatment of 5-Aza-CdR and GSK126 had no effect on H3K27me3 and DNMT1 levels, respectively. Functional analyses revealed that miR-34a mimic decreased cholangiocarcinoma cell proliferation, colony formation and migration, in vitro. Moreover, we identified Notch1, Notch2 and Jagged 1, which are the major receptors and ligands of the Notch pathway, as miR-34a target genes in cholangiocarcinoma cells. Accordingly, forced overexpression of miR-34a significantly decreased the expression of Notch1, Notch2 and Jagged1, as determined by Western blotting and qRT-PCR analyses. Our results provide novel evidence that miR-34a expression is epigenetically silenced by DNA methylation and EZH2 in human cholangiocarcinoma cells. Thus, epigenetic induction of miR-34a or miR-34a replacement therapy may represent a novel therapeutic approach for the treatment of cholangiocarcinoma.

#950

Bistable switching of c-KIT by estrogen-mediated ceRNA and epigenetic silencing of miR-193a predicts survival in ovarian cancer.

Frank Hsueh-Che Cheng,1 Baltazar D. Aguda,2 Hon-Yi Lin,3 Je-Chiang Tsai,4 Marek Kochanczyk,5 Ru-Inn Lin,3 Jora M. J. Lin,1 Gary C. W. Chen,6 Cheng-Chang Chang,7 Hung-Cheng Lai,8 Kenneth P. Nephew,9 Tzy-Wei Hwang,4 Michael W. Y. Chan1. 1 _Department of Life Science and Advanced Institute of Manufacturing with High-Tech Innovations, National Chung Cheng University, Chia Yi, Taiwan;_ 2 _Disease Pathways LLC, Bethesda, MD;_ 3 _Departments of Radiation Oncology Buddhist Dalin Tzu Chi Hospital, Chia Yi, Taiwan;_ 4 _Department of Mathematics, National Chung Cheng University, Chia Yi, Taiwan;_ 5 _Institute of Fundamental Technological Research, Polish Academy of Sciences, Warsaw, Poland;_ 6 _Institute of Molecular Biology, National Chung Cheng University, Chia Yi, Taiwan;_ 7 _Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical, Taipei, Taiwan;_ 8 _Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan;_ 9 _Medical Sciences, Department of Cellular and Integrative Physiology, Bloomington, IN_.

Ovarian cancer is one of the most lethal cancers in the female reproductive system. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by the surface antigen CD44 and c-KIT (CD117). Previous study also suggested that long term treatment of estrogen such as hormonal replacement therapy (HRT) may increase the risk of ovarian cancer, however the role of estrogen in ovarian carcinogenesis is still controversial. To unravel this complexity, we propose a mathematical model to explore how the ER signaling pathway contribute to c-KIT expression during ovarian carcinogenesis: one through a ceRNA competition of an ER target,E2F6 and c-KIT for their targeted miRNA, miR-193a; second by binding of E2F6 protein, in association with the polycomb complex, to the promoter of miR-193a to downregulate miR-193a transcription by epigenetic modifications. Our model found that epigenetic silencing of miR-193a generates a bistable switching of c-KIT during ovarian carcinogenesis based on the level of EZH2. To confirm our results, we performed ectopic expression of miR-193a and 3'UTR luciferase in ovarian cancer cell lines and confirmed that E2F6 and c-KIT are the targets of miR-193a. Importantly, treatment of E2 or bisphenol A (BPA) resulted in the up-regulation of E2F6 and c-kit mRNA in IOSE cells in which no or low methylation at the promoter CpG island of miR-193a was found. On the contrary, promoter hypermethylation of miR193a could be observed in miR-193a-underexpressed CP70 ovarian cancer cells but not in HeyC2 cells which showed similar expression level of miR-193a as in IOSE cells. Treatment of demethylating agent (5azaDC) or EZH2 inhibitor (GSK343) resulted in a reexpression of miR-193a in CP70 ovarian cancer cells. Overexpression of miR-193a inhibited tumor growth in vitro and in an animal model. Further ChIP-PCR assay also found that open chromatin mark H3K4me3 was enriched in the promoter region of miR-193a in HeyC2 but not in CP70 cells. On the contrary, repressive chromatin marks H3K9me3 and H3K27me3 were only enriched in CP70 cells. Clinically, ovarian cancer patients (n=109) with higher promoter methylation of miR-193a were associated with poor survival (p>0.05). Additional analysis of the TCGA ovarian cancer dataset demonstrated that ovarian cancer patients with low expression of EZH2, a polycomb-group protein, showed positive correlation (p<0.05) between E2F6 and c-KIT which resembles the ceRNA phenomenon between these two mRNAs. Importantly, ovarian cancer patients with low expression of EZH2 tended to have lower expression of c-KIT. In conclusion, our mathematical model and experimental data suggests that miR-193a can be epigenetically regulated by both ceRNA and promoter methylation. Simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.

#951

Role of the MSC-derived exosomal and endogenous JAK2-SET/PP2A-β-catenin-modulator miR-300 in leukemic stem/progenitor proliferation and survival in CML.

Rossana Trotta,1 Giovannino Silvestri,1 Lorenzo Stramucci,1 Justin J. Ellis,2 Justine Yu,1 Jason J. Harb,3 Paolo Neviani,4 Guido Marcucci,5 Klara Srutova,6 Polakova K. Machova,6 Denis-Claude Roy,7 Peter Hokland,8 Michael Deininger,9 Ravi Bhatia,10 Carlo Gambacorti-Passerini,11 Dragana Milojkovic,12 Alistair Reid,12 Jane Apperley,12 Ferenc Livak,1 Jianfei Qi,1 Maria R. Baer,1 Danilo Perrotti1. 1 _University of Maryland Baltimore School of Medicine, Baltimore, MD;_ 2 _The Ohio State University, Columbus, OH;_ 3 _Blood Center of Wisconsin, Milwaukee, WI;_ 4 _Norris Comprehensive Cancer Center, Los Angeles, CA;_ 5 _City of Hope Comprehensive Cancer Center, Duarte, CA;_ 6 _University of Prague, Prague, Czech Republic;_ 7 _University of Montreal, Montreal, Quebec, Canada;_ 8 _Aarhus University Hospital, Aarhus, Denmark;_ 9 _University of Utah, Salt Lake City, UT;_ 10 _University of Alabama Birmingham, Birmingham, AL;_ 11 _University of Milano-Bicocca, Monza, Italy;_ 12 _Imperial College, London, United Kingdom_.

MiR-300 is a microRNA predicted to target multiple components of the BCR-ABL1/JAK2/hnRNPA1/SET/PP2A/β-catenin pathway, which is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs). Nanostring arrays analysis of bone marrow (BM) cells from healthy individuals (n=5) and CML patients (n=10) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300).

MiR-300 transduction in CMLCD34+ cells and BCR-ABL1+ cell lines decreased JAK2, β-catenin, hnRNPA1 and SET expression and increased PP2A activity. Targets were confirmed by miR-300 expression in BCR-ABL1+ cells expressing Flag-tagged miR-300-targets lacking or carrying a wild-type or mutated 3'UTR. Restored miR-300 expression in CMLCD34+ cells and/or BCR-ABL1+ cell lines impaired proliferation and clonogenic potential, markedly reduced LTC-ICs, and increased TKI sensitivity. Notably, miR-300 expression was inhibited by BCR-ABL1 in proliferating cells. Accordingly, imatinib restored miR-300 expression in CD34+ dividing progenitors and BCR-ABL1+ cell lines without altering miR-300 levels in quiescent (CFSEMAX) CMLCD34+ cells (n=3), consistent with the BCR-ABL1 kinase-dependent activation of the Jak2/SET/PP2A/β-catenin pathway in CML progenitors but not quiescent Ph+ HSCs. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4).

To determine whether enhanced miR-300 expression in quiescent cells depends on cell autonomous events or is induced by the BM microenvironment, we exposed BCR-ABL+ cells to conditioned medium (CM) of HS-5 or hTERT mesenchymal stem cells (MSC). CM strongly decreased proliferation, induced imatinib but not FTY720 (PP2A activator) resistance, increased miR-300 levels, decreased BCR-ABL1 activity and Jak2 expression but not its activity, and did not alter β-catenin levels or PP2A activity. Interestingly, miR-300 was found in MSC-derived exosomes, and its expression increased in BCR-ABL1+ cells exposed to exosomes. Accordingly, proliferation of CML-BCCD34+and LAMA-84 cells was strongly reduced upon exposure to MSC-derived exosomes. These effects were abolished when we used CM from MSCs transduced with a miR-300 antagomir.

Altogether our results indicate that downregulation of miR-300 appears necessary for the activation of JAK2/SET/PP2A/β-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence.

#952

Genome-wide enhancer analysis identifies PVT1 as an oncogenic enhancer of MYC.

Kunitoshi Shigeyasu,1 Shusuke Toden,1 Takeshi Nagasaka,2 Toshiyoshi Fujiwara,2 C. Richard Boland,1 Ajay Goel1. 1 _Center for Gastrointestinal Research,Baylor University Medical Center, Dallas, TX;_ 2 _Gastroenterological Surgery, Okayama University, Okayama, Japan_.

Introduction: An enhancer is a short stretch of DNA that can be targeted by specific proteins to activate gene transcription, and recent evidence suggests that enhancers can act as key regulators of oncogenes in cancer. Recently, the FANTOM5 project comprehensively characterized enhancer activities in various cell types. Interestingly, several enhancers coding for lncRNAs were commonly up-regulated in multiple cancer types, suggesting that these enhancers may play an important oncogenic role through enhancer-promoter-lncRNA interaction, and may help mediate chemoresistance. However, specific lncRNAs with oncogenic enhancer function and their clinical significance in colorectal cancer (CRC) remains unexplored.

Aims: We analyzed genome-wide cancer-specific enhancer activation in multiple cancers to identify potential oncogenic lncRNAs, and assessed their clinical significance in two independent patient cohorts with CRC. We thereafter evaluated the functional role of candidate enhancer lncRNAs in CRC.

Methods: Using the FANTOM5 enhancer database, cancer-specific enhancer activity was analyzed in cancers of the colon, stomach, lung, prostate and melanoma. LncRNA candidates that encode these cancer-specific enhancers were identified. We then screened these lncRNAs in two independent CRC cohorts, as well as the APC(Min/+) mouse model. The functional role of target lncRNAs was assessed in parental and chemoresistant CRC cell lines. The oncogenic enhancer activity was confirmed using the chromatin conformation capture (3C) assay.

Results: We identified ten lncRNA candidates with consistent enhancer activities in all five cancer types. In particular, PVT1 appeared to have one of the highest enhancer activities. Using two independent CRC cohorts, we confirmed upregulation of PVT1 in cancer tissues. Patients with high-PVT1 expression had shorter overall survival in Stage II and III CRCs, suggesting that the PVT1 expression could discriminate high-risk patients, and identify those who may benefit from adjuvant chemotherapy. Intriguingly, we discovered that PVT1 was also upregulated in human colorectal adenomas as well as adenomas isolated from APC(Min/+) mice, indicating that PVT1 may be involved in the early stages of the adenoma-carcinoma sequence. The expression level of PVT1 significantly correlated with c-MYC expression in CRC specimens and the 3C assay showed that the enhancer region in PVT1 can target c-MYC. Finally, we demonstrated that PVT1 and c-MYC were co-overexpressed in several chemoresistant CRC cell lines, and that suppression of PVT1 led to a significant reduction in proliferation of chemoresistant cells, further suggesting that PVT1 may play a key role in chemoresistance.

Conclusion: PVT1 acts as an oncogenic enhancer of c-MYC in CRC. High expression of PVT1 served as a predictive marker for identifying high-risk Stage II and III CRC patients. PVT1 could serve as a potential therapeutic target in chemoresistant CRCs.

#953

miR-31 targets ARID1A to enhance the progression of oral carcinoma.

Wen-Cheng Lu,1 Shu-Chun Lin,1 Kuo-Wei Chang2. 1 _National Yang-Ming Univ. Institute of Oral Biology, Taipei, Taiwan;_ 2 _National Yang-Ming Univ. School of Dentistry, Taipei, Taiwan_.

miR-31 is an oncogenic microRNA in several malignancies including oral squamous cell carcinoma (OSCC). However, the function of miR-31 in modulating the stem cell properties in OSCC has not yet been explored. AT-rich interacting domain (ARID) containing protein members play pluripotent roles in modulating chromatin accessibility of the transcription machineries for gene expression. ARID1A has been shown suppressive to several malignancies and the driver mutations in ARID1A gene are noted in tumors. In this study, we identified that miR-31 targets ARID1A in OSCC, and a reverse expression between miR-31 and ARID1A was noted in OSCC tumors. Furthermore, the miR-31 induced migration, invasion, CSC cell population and stemness gene expression were rescued by ARID1A expression in OSCC cells. ARID1A suppressed the transcription activity of a panel of stemness markers through its affinity to A-T rich region in the promoters. A reverse correlation between ARID1A and these stem cell markers was also seen in OSCC tumors. miR-31+/ARID1A- OSCC cell subclone exhibited higher potency in sphere formation, as well as tumorigenesis and neck metastasis in nude mice relative to other cell subclones. In addition, tumors carrying high miR-31 expression and low ARID1A expression had the worst survival. In summary, this study provides novel mechanistic clues demonstrating that miR-31 targets ARID1A to enrich the stemness properties for OSCC progression.

#954

Analysis of noncoding RNA expression in a mouse model of PTEN-deficient prostate cancer.

Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Yuji Hatanaka, Yoshihiko Fujita, Yosuke Togashi, Masato Terashima, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. _Kinki University Faculty of Medicine, Osaka-Sayama, Japan_.

Prostate cancer is a heterogeneous disease that is driven by combined genetic and epigenetic alterations. A significant portion of the mammalian genome consists of non-coding regions of RNA. Increasing evidence has shown that these noncoding RNAs (ncRNAs) have significant roles in the epigenetic regulation of several cellular processes, and their dysregulation can contribute to tumorigenesis and promote disease progression in many cancer types. In order to gain better insights into the potential roles of ncRNAs in prostate cancer, we used a genetically engineered mouse model of prostate cancer to perform a comparative analysis of the cancer transcriptome and the landscape of ncRNAs in castration-naïve prostate tumors and the progression to castration-resistant disease. Whole transcription arrays were used to explore both messenger (mRNA) and long intergenic non-coding RNA (LincRNA) transcript expression in normal prostate tissue from wildtype mice and prostate tumors from conditional PTEN-knockout mice. Comparative analyses were performed between prostate tumor samples from castration-naïve mice and, at 4 weeks and 10 weeks post-surgical castration. Altered expression of ncRNAs was most commonly observed and accounted for 56.2% (2370/4216), 47.6% (4460/9366) and 41.57% (1545/3717) of the total genes differentially expressed between normal mouse prostate and prostate tumors from castration-naïve, 4 weeks post-castration and 10 weeks post-castration, respectively. We also profile the expression of relevant cancer-related lincRNAs present in these models. Overall, our analyses provide data to support a role of LincRNA dysregulation in the pathogenesis of PTEN-deficient prostate cancer, and suggest that these mouse models might provide a potential preclinical tool to test candidate prospective therapies targeting ncRNAs.

#955

Inhibition of SEC24D decreases exosome release of the tumor suppressor miR-605 in renal cell carcinoma.

Sreenivasulu Chintala, Remi Adelaiye-Ogala, Ashley Orillion, Sreevani Arisa, May Elbanna, Roberto Pili. _Genitourinary Program, Division of Hematology & Oncology, IU Melvin and Simon Bren Cancer Center, Indiana University School of Medicine, Indianapolis, IN_.

Background: MicroRNA 605 (miR-605) has been recently reported as a putative tumor suppressor and its overexpression decreases tumor cell proliferation, migration and clonogenicity. To date, the role of miR-605 in renal cell carcinoma (RCC) has not been investigated. We recently showed the decrease of circulating miR-605 in serum of clear cell renal cell carcinoma (ccRCC) patients who responded to the treatment with the histone deacetylase (HDAC) inhibitor vorinostat, and the VEGF blocker bevacizumab. The current study was designed to investigate the expression of miR-605 and understand the mechanism(s) responsible the extracellular release of miR-605 using RCC cells. Methods: 786-0 cells were treated with and without vorinostat for 24h and condition media were collected, briefly centrifuged to settle the cells and debris, and processed to isolate exosomes using the ExoQuick exosome isolation kit (Systems Biology, CA). Purified exosomes and 786-0 cells were used to isolate RNA and further prepared cDNA to utilize for quantitative RT-PCR analysis. Expression of miR-605 in exosomes and cells was determined by Quantitative RT-PCR using TaqMan MicroRNA Assays with miR-605 primers obtained from Applied Biosystems, NY. To determine the role of secretory protein 24 family member D (SEC24D), a catalytic component of coat protein complex (COPII) involved in the secretory pathway, 786-0 cells treated with vorinostat were used to determine SEC24D expression by QRT-PCR and Western blot analysis. TCGA data was used to determine correlation of SEC24D expression clear cell renal cell carcinoma patients' survival. Results: Vorinostat treatment resulted in a significant decrease of miR-605 expression in exosomes and increase (100 fold) of intracellular expression. Furthermore, the increased intracellular miR-605 was associated with the inhibition of SEC24D mRNA and protein expression in 786-0 cells treated with vorinostat. Cancer Genomic data analysis of c-Bioportal from MSKCC of TCGA revealed the overall poor survival of ccRCC patients with alteration of SEC24D. Conclusion: Taken together, our preliminary data suggest that the HDAC inhibitor vorinostat inhibits SEC24D and exosome mediated extracellular secretion of miR-605 in RCC cells. These results suggest that vorinostat treatment retained intracellular miR-605 that target genes involved in cell survival and proliferation in RCC. Further studies will evaluate circulating miR-605 as a predictive biomarker to determine the efficacy of vorinostat in ongoing trials with RCC patients.

#956

Anticancer effects of silibinin-induced small nucleolar RNA 11B on bladder cancer cells.

Soichiro Yamamura, Yozo Mitsui, Shahana Majid, Hannah Nip, Nathan Bucay, Sharanjot Saini, Guoren Deng, Varahram Shahryary, Rajvir Dahiya, Yuichiro Tanaka. _San Francisco Veterans Affairs Medical Center, San Francisco, CA_.

Silibinin is the major active constituent of silymarin, an extract of milk thistle seeds. Silibinin has been shown to have significant anti-cancer effects in a variety of malignancies. However, the molecular mechanisms of silibinin action in bladder cancer have not been reported. In the present study, we found that silibinin (10 µM) significantly suppressed proliferation and invasion of T24 and UM-UC-3 bladder cancer cells. We analyzed various pathways as possible mechanisms of silibinin action using RNA-Seq technology in bladder cancer cell lines. Analysis of RNA-seq data identified silibinin targets that down-regulate the actin cytoskeleton pathway. Analysis of RNA-seq data also identified the mitogen-activated protein (MAP) kinase signaling pathway as a silibinin-down-regulated pathway. These pathways were also found to crosstalk through Ras and phosphatidylinositide 3-kinase (PI3K). These data show that silibinin anti-cancer effects are through down-regulation of actin cytoskeleton and MAP kinase pathways in bladder cancer. We also found that silibinin induced small nucleolar RNA 11B (SNORD11B) and that SNORD11B suppressed proliferation and invasion of bladder cancer cells. We will discuss the pathway of the anti-cancer effects of SNORD11B.

#957

Regulation of PCGEM1 in castration resistant prostate cancer.

Tsui-Ting Ho, Yin-Yuan Mo. _University of Mississippi Medical Center Cancer Institute, Jackson, MS_.

PCGEM1 has been shown to be prostate cancer specific long non-coding RNA (lncRNA). Our previous study indicates that PCGEM1 plays a role in castration resistance through regulation of androgen receptor (AR) splice variants. Although it is known that PCGEM1 is often upregulated in prostate cancer, little is known how PCGEM1 is regulated. In the present study, we show that p54/nrb is a positive regulator for PCGEM1. PCGEM1 is higher in castration resistant CWR22Rv1 than in androgen sensitive LNCaP cells. Moreover, androgen deprivation induces PCGEM1 in LNCaP cells. Histone ChIP assays suggest that the active binding site is within 500 bp upstream of putative transcriptional start site. By using biotin-labelled PCR product and pulldown assays combined with mass spectrometry analysis, we show that p54/nrb interacts with the promoter of PCGEM1. While ectopic expression increases, suppression of p54/nrb by RNAi or knockout (KO) suppresses PCGEM1, supporting a role for p54/nrb in PCGEM1 regulation. Moreover, rescue experiments indicate that re-expression of p54/nrb in KO cells restores the ability to induce PCGEM1. Finally, 3, 3'-Diindolylmethane (DIM), a known chemoprevention agent derived from plants such as broccoli, is capable of suppressing PCGEM1 expression by preventing the interaction of p54/nrb with the PCGEM1 promoter. In particular, DIM reduces tumor growth by suppression of PCGEM1 and promoting apoptosis in the castrated xenograft mouse model. Together, these results demonstrate a novel mechanism of how PCGEM1 and AR splicing are regulated. As a result, PCGEM1 may ultimately contribute to castration resistance.

#958

MiR-196b is an epigenetically silenced tumor suppressor for lung cancer.

Carmen S. Tellez,1 Daniel E. Juri,1 Kieu Do,1 Maria A. Picchi,1 Teresa Wang,2 Gang Liu,2 Avrum Spira,2 Steven A. Belinsky1. 1 _Lovelace Respiratory Research Institute, Albuquerque, NM;_ 2 _Boston University, Boston, MA_.

Silencing of microRNAs by promoter hypermethylation could play a significant role in lung cancer initiation and progression. A pre-malignancy model using carcinogen transformed human bronchial epithelial cells (HBECs) treated with the demethylating agent 5-aza-2'deoxycytidine followed by next-generation sequencing identified miR-196b and miR-34c-5p as being epigenetically regulated. Bisulfite sequencing confirmed silencing via dense promoter hypermethylation that was commonly replicated in malignant cell lines and primary tumors. Functional studies of miR-196b revealed that it was epigenetically silenced by chromatin modification and DNA methylation during transformation of HBECs and re-expression modulated genes involved in cell cycle regulation. Stable re-expression led to a cell cycle arrest mediated in part through transcriptional regulation of the FOS oncogene that was replicated in vivo by a profound reduction in growth of tumor xenografts. Luciferase assays demonstrated that forced expression of miR-196b inhibits FOS promoter and AP-1 reporter activity. A case-control study showed that methylation of miR-196b in sputum was strongly associated with lung cancer (OR = 4.7, p<0.001). These studies identify miR-196 as a tumor suppressor whose silencing early in lung carcinogenesis may provide a selective growth advantage to pre-malignant cells. Targeted delivery of this microRNA could benefit prevention and therapy for the management of lung cancer.

#960

A translational investigation of piRNAs in glioblastoma multiforme.

Daniel I. Jacobs,1 Qin Qin,1 Alan Fu,1 Zeming Chen,2 Jiangbing Zhou,2 Yong Zhu1. 1 _Yale School of Public Health, New Haven, CT;_ 2 _Yale University Department of Neurosurgery, New Haven, CT_.

Background: PIWI-interacting RNAs (piRNAs) are highly abundant small noncoding RNAs that partner with PIWI proteins to protect germline tissues from destabilizing transposon activity. While aberrant expression of PIWI proteins has been linked with poor outcomes for many cancer types, far less is known about the expression or function of piRNAs in a cancer context. Here we provide evidence supporting a novel role of piRNAs in gliomagenesis that may be leveraged for therapeutic benefit.

Methods: In order to characterize piRNA expression patterns in glioblastoma multiforme (GBM), we performed array-based piRNA expression profiling in seven pairs of human normal and tumor tissue specimens with validation by qPCR. In vitro (cellular proliferation, colony formation, cell cycle, apoptosis, transcriptional profiling) and in vivo (orthotopic xenograft mouse model) assays were performed to characterize the phenotypic impact of differentially expressed piRNAs using cell line models of GBM. For all functional experiments, synthetic piRNA mimics or negative control oligos were transiently delivered into cells via Lipofectamine RNAiMAX transfection reagent.

Results: piRNA profiling revealed expression of over 350 piRNAs in both normal and tumor brain tissues including several with dysregulated expression in GBM tissue. Individual transfection of four down-regulated piRNA mimics revealed antiproliferative effects on glioma cells, consistent with tumor-suppressive roles. Notably, two other piRNAs that were expressed at equivalent levels in tumor and normal tissues did not affect cell proliferation when delivered into glioma cells, indicating specificity of piRNA functionality. Upregulation of the most underexpressed piRNA (~10-fold lower expression in GBM tissue) was found to induce cell cycle arrest and apoptosis and to alter transcriptional levels of several genes involved in cell stress and survival pathways in glioma cell lines, yet it did not affect normal astrocyte proliferation. Finally, the volume of intracranial mouse xenograft tumors was significantly reduced for approximately two weeks after pre-implantation cell transfection with the piRNA as compared to negative control RNA, and median survival was extended by two days for piRNA-treated mice (n=9/treatment group).

Conclusions: Although piRNAs have not traditionally been thought to be expressed in healthy somatic tissues, our results are consistent with recent studies that have detected expression of a similar number of piRNAs in other normal tissues. Our results show that a subset of these piRNAs exhibit dysregulated expression in GBM and have cancer-relevant phenotypic properties in glioma cell line and mouse models. Taken together, our study reveals a previously unidentified functional role for piRNAs as tumor suppressors in gliomagenesis, and suggests that restoration of normal piRNA levels may be a viable GBM therapy.

#961

Comprehensive catalog of culture-derived exosomal microRNA cargo from 4 discrete head and neck carcinoma cell lines.

Scott M. Langevin,1 Damaris Kuhnell,1 Tess L. Parry,2 Jacek Biesiada,1 Xiang Zhang,1 Mario Medvedovic,1 Susan Kasper1. 1 _University of Cincinnati College of Medicine, Cincinnati, OH;_ 2 _Bethany College, Bethany, WV_.

Background: Exosomes are nano-sized (40-100 nm), membrane encapsulated vesicles that are released by cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including non-coding RNA. MicroRNA (miRNA) are small (18-25 nucleotides in length), evolutionarily conserved, non-coding RNA molecules that are involved in regulation of gene expression in essentially all eukaryotic organisms through post-transcriptional degradation and translational inhibition of messenger RNA (mRNA). The purpose of this study was to catalog exosomal miRNA cargo that are differentially secreted from head and neck squamous cell carcinoma (HNSCC) cells in vitro using Next Generation miRNA-sequencing.

Methods: We obtained 4 commercially available HNSCC cell lines: (1) H413 (female, buccal); (2) Detroit 562 (female, pharynx metastatic to pleura); (3) FaDu (male, hypopharynx) and (4) Cal 27 (male, tongue). We additionally obtained HGEPp primary human gingival epithelial cells (pooled from 3 healthy female donors) for comparison. Cells were cultivated in supplier-recommended media with 10% fetal bovine serum (FBS) that was super-depleted of exosomes via 18 hour ultracentrifugation at 100,000g and 1% penicillin/streptomycin at 37°C with 5% CO2 in 150 cm2 flasks with 25mL media. To achieve adequate volume for exosome isolation (50mL), cells were cultured in 2-pair sets of flasks in triplicate (6 flasks total per cell line). Media was collected when cells reached 80-90% confluence. Exosomes were isolated and purified from exosome-depleted cell culture media by differential ultracentrifugation and the presence of purified exosomes was verified via nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument. Total RNA was extracted from each exosome pellet using the miRNeasy Micro kit, and Next Generation miRNA-sequencing was subsequently performed by the University of Cincinnati Genomics, Epigenomics and Sequencing Core (Cincinnati, OH) using the Illumina HiSeq platform. Differential exosomal miRNA secretion was assessed between each HNSCC cell line and the non-pathologic gingival epithelial control cells, adjusting for false-discovery rate using the Benjamini and Hochberg method.

Results: We observed 93 miRNA transcripts that were differentially secreted (Q < 0.05) in exosomes derived from at least one of the HNSCC cell lines relative to the non-pathologic epithelial control cells; 23 miRNA transcripts were differentially secreted by all 4 cell-lines.

Conclusions: This provides evidence for differential secretion of miRNA in HNSCC-derived exosomes. These exosomal miRNA warrant further evaluation as potential biomarkers for HNSCC.

#962

Deregulated transcription of mouse satellite sequences accelerates oncogenic processes via functional inhibition of YBX1 protein.

Takahiro Kishikawa, Motoyuki Otsuka, Kazuhiko Koike. _University of Tokyo, Tokyo, Japan_.

Highly repetitive tandem arrays located at centromere and pericentromere regions of the chromosomes had been considered epigenetically silent by heterochromatin modification. However, the deregulated transcription of the non-coding satellite sequences exists in human and mouse pancreatic cancer tissues. This aberrant expression can be detected even in Kras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are pre-cancerous lesions to invasive pancreatic cancer. To examine the biological roles of these aberrantly expressed satellite RNAs during the carcinogenesis steps, we established genetically-modified mouse PanIN-derived cells, in which mouse major satellite (MajSAT) RNAs were ectopically expressed. MajSAT RNA expressing PanIN-derived cells showed the increased chromosomal instability and the rate of spontaneous point mutations in the genomic as well as in the mitochondrial DNAs. We identified YBX1 protein as a binding protein specifically with MajSAT RNA by RNA immunoprecipitation. MajSAT RNA inhibited nuclear translocation of YBX1 under oxidative damage, which reduced the DNA damage repair activity of YBX1. The mutation rate of genomic and mitochondrial DNAs, and transformation rate were rescued by YBX1 overexpression in these cells. These findings indicate that satellite transcripts at the early stage of cancer development may act as "mutagens" and accelerate oncogenic processes.

#963

Epigenetic silencing of miR-199a-3p is associated with increased cell survival, enhanced cell motility, and sensitivity to tivantinib through regulation of c-Met expression in hepatocellular carcinoma.

Kohichiroh Yasui, Akira Tomie, Naoto Iwai, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Itoh. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and can act as tumor suppressors or oncogenes. To identify miRNA involved in the development of HCC, we performed a genome-wide miRNA gene expression analysis using the human miRNA microarray (Agilent) in paired tumor and non-tumor tissues from patients with primary HCC. The array-based miRNA expression profiles were validated by quantitative PCR. We also screened for genes with promoter DNA hypermethylation using a genome-wide DNA methylation microarray analysis (the Illumina HumanMethylation27 BeadChip) in primary HCCs. We found that miR-214-3p, miR-199a-5p, and miR-199a-3p were significantly down-regulated by aberrant promoter hypermethylation in HCC tumors. The c-Met gene was identified as the direct target of miR-199a-3p. Our study showed that reduced expression of miR-199a-3p was associated with increased cell survival through the c-Met/Akt pathway and enhanced cell motility through phosphorylation of FAK. The transfection of miR-199a-3p mimic reduced the expression of c-Met and enhanced the sensitivity to the c-Met inhibitor tivantinib in HCC cells. In conclusion, our results suggest that epigenetic silencing of tumor suppressor miR-199a-3p may be involved in hepatocarcinogenesis through regulation of c-Met expressionand and be associated with the sensitivity to tivantinib.

#964

Circulating biomarker miRNA-150 promotes tumor cell proliferation and progression by targeting SRC kinase signaling inhibitor 1 (SRCIN1) in non-small cell lung cancer.

Jing Lin, Liren Zhang, Yuanqing Ye, Taro Oba, Emanuela Gentile, Emanuela Gentile, Jing Wang, Yang Zhao, Jian Gu, Ignacio Wistuba, Jack A. Roth, Xifeng Wu, Lin Ji. _The University of Texas M.D. Anderson Cancer Center, Houston, TX_.

Circulating microRNAs (c-miRNAs) expression ratio (ER) signatures were evaluated as prognostic biomarkers and therapeutic targets in early stage non-small cell lung cancer (NSCLC) patients. In a population study with 171 stage I and II NSCLC patients, the miR-150 and associated ER signature miR27A/miR-150 (ER ≥0.86, HR =3.92, p =1.46E-4) were significantly associated with 5-year survival. The upregulated expression of miR-150 was detected in primary tissue samples (n=245) by miRNA microarray profiling analysis and significantly associated with cancer recurrence (p =0.027) and overall survival (HR =0.88, p =0.04). Ectopic expression of miR-150 in NSCLC H1299, A549, and H2023 cells transfected by miR-150 expression plasmids promoted tumor cell proliferation, tumor cell-induced colony formation and migration/invasion, while blocking the action of miR-150 with a synthetic miR-150 inhibitor or transduction with a miRZip-150 inhibitor expressed by a lentiviral vector significantly inhibited these biological activities. The SRC kinase signaling inhibitor 1 (SRCIN1) was predicted to be a potential target of miR-150 by bioinformatics tools.miR-150-targeted binding and cleavage in the 3'-UTR of SRCIN1 mRNA were confirmed by a novel stem-loop array-reverse transcription-PCR assay. Both SRCIN1 gene and protein expression were significantly downregulated by ectopic expression of miR-150 and upregulated by miR-150 inhibitors in H1299 and H2023 NSCLC cells, as demonstrated by qRT-PCR and Western-blotting assays, respectively. Modulation of key elements in the SRCIN1 signaling pathway by ectopic expression or inhibition of miR-150 were further determined by qRT-PCR and predicted by ingenuity pathway analysis. Our findings suggest that circulating miR-150 and its associated ER signatures could serve as novel prognostic biomarkers for early stage NSCLC and function as an oncogenic miRNA that promotes lung cancer cell proliferation, invasion, and metastasis by direct targeting SRCIN1signaling pathway thus identifying SRCIN1 as a potential therapeutic target.

#965

Exosome-derived microRNAs contributes to prostate cancer chemoresistance.

JIng Li,1 Xiaozhen Xu,1 Hao Guan,1 Atsushi Mizokami,2 Evan T. Keller,3 Xin Yang,1 Xia Liu,1 Jiyong Tan,1 Longyuan Hu,1 Yi Lu,1 Jian Zhang4. 1 _Key Laboratory of Longevity and Ageing-related Diseases, Ministry of Education, China & Center for Translational Medicine, Guangxi Medical University, Nanning, China; _2 _Department of Urology, Kanazawa University, Kazanawa, Japan;_ 3 _Department of Pathology and Urology, University of Michigan, Ann Arbor, MI;_ 4 _Key Laboratory of Longevity and Ageing-related Diseases, Ministry of Education, China & Center for Translational Medicine, Guangxi Medical University, Guangxi, China & Department of Pathology and Urology, University of Michigan, Ann Arbor, MI_.

Accumulating evidence has demonstrated that certain miRNAs play important roles in cancer cell chemoresistance. However, the pleotropic functions of exosome-derived miRNAs on developing chemoresistance remain unknown. In this study, we aimed to build potential networks of miRNAs, which derived from the exosome of chemoresistant prostate cancer (PCa) cells, with their known target genes using miRNA expression profiling and bioinformatic tools. Global miRNA expression profiles were measured by microarray. Twelve miRNAs were initially selected and validated by qRT-PCR. Known targets of deregulated miRNAs were utilized using DIANA-TarBase database v6.0. The incorporation of deregulated miRNAs and target genes into KEGG pathways were utilized using DIANA-mirPath software. To construct potential miRNA regulatory networks, the overlapping parts of two selected KEGG pathways were visualized by Cytoscape software. We identified 29 deregulated miRNAs, including 19 up and 10 down, in exosome samples derived from two kinds of paclitaxel resistance PCa cells (PC3-TXR and DU145-TXR) compared with their parental cells (PC3 and DU145). The enrichment results of deregulated miRNAs and known target genes showed that a few pathways were correlated with several critical cell signaling pathways. We found that hub hsa-16-5p, hsa-miR-3915, hsa-miR-488-3p, hsa-miR-5004-5p, hsa-miR-23c, hsa-miR-3673, hsa-miR-3654, and hsa-miR-32-5p were potential targets to hub androgen receptor (AR) and phosphatase and tensin homolog (PTEN). Hub TCF4 target genes were mainly regulated by hub hsa-miR3176, hsa-miR-141-3p, hsa-miR-606, hsa-miR381, and hsa-miR-429 miRNAs. These results may provide a linkage between PCa chemoresistance and exosome regulatory networks and thus lead us to propose that PTEN and TCF4 genes may be the important genes which regulated by exosome miRNAs in chemoresistance cancer cells. Supported by NSFC Key Project 81130046; NSFC Project 81171993 and NSFC81272415; Guangxi Key Projects 2013GXNSFEA053004 and 2012GXNSFCB053004; Guangxi Ministry of Education grants 201202ZD022 and 201201ZD004.

#966

Circulating exosomal miRNAs as potential biomarker in liposarcoma.

Lucia Casadei,1 Chad Creighton,2 Kara Batte,1 Dina Chelouche-Lev,3 Carlo M. Croce,4 Raphael E. Pollock1. 1 _The Department of Surgical Oncology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Sheba Medical Center, Surgery B, Tel Aviv, Israel;_ 4 _The Department of Molecular Virology Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH_.

Liposarcoma (LPS) is the most common soft tissue sarcoma histological subtype. Despite the development of combined modality treatments in recent years, a significant proportion of patients respond poorly to chemotherapy, leading to local recurrence or distant metastasis. Thus, early detection of recurrent or metastatic disease or early decision making could improve patient prognosis. However, there are no useful biomarkers for these purposes. Thus, the discovery of novel biomarkers to detect tumors, predict their drug sensitivity, and monitor their progression is one of the most important challenges that must be overcome.

miRNAs are short (approximately 22 nucleotides in length) non-coding RNAs (ncRNAs) that regulate gene expression by binding to specific mRNA targets and promoting their degradation and/or translational inhibition; since their discovery in body fluids (secreted in cell-borne membrane vesicles or associated with macromolecular complexes), circulating blood-borne microRNAs are being investigated as potent minimally invasive biomarkers of several diseases including tumors.

In this study, our goal is to identify potential biomarkers in the peripheral blood of LPS patients that could be useful for LPS diagnostic, prognostic and therapeutic purposes.

To determine the miRNA expression profiles of LPS in peripheral blood (PB) and in peripheral blood exosomes (PBX), a series of 16 human LPS patient specimens and 8 healthy controls was analyzed on a miRNA microarray platform capable of detecting 800 human miRNAs (nCounter Human microRNA expression Assay Nanostring v3). Validation of the array was performed by qRT-PCR on a new set of samples (10 human LPS and 9 healthy controls).

To identify differentially expressed miRNAs, we compared miRNAs expression profiles from PBX of 15 patients to 8 healthy samples. A total of 26 miRNAs were significantly upregulated and 3 miRNAs were significantly down regulated (p<0.004). When we compared miRNA expression profiles from PB to healthy controls, fewer miRNAs were consistently deregulated (18 miRNAs were upregulated and 3 miRNAs were downregulated, p<0.02). Moreover the miRNAs found to be consistently deregulated in PB were all also significantly deregulated in the PBX. Validation of the miRNA panel in an independent cohort of LPS patients confirmed this signature.

Our study has identified a specific miRNA signature in circulating exosomes that may have a novel role in the diagnosis, prognosis and or treatment of LPS patients in a clinical setting.

#967

Vemurafenib regulates miRNA-211 expression in melanoma - effects on extracellular vesicle RNA cargo and function.

Taral R. Lunavat,1 Lesley Cheng,2 Berglind Einarsdottir,1 Cecilia Lässer,1 Jonas A. Nilsson,1 Andrew F. Hill,2 Jan Lötvall1. 1 _University of Gothenburg, Gothenburg, Sweden;_ 2 _La Trobe University, Melbourne, Australia_.

In melanoma, more than 50% of the patients harbor BRAF somatic missense mutations, most affecting the V600 region. Vemurafenib, a potent BRAF inhibitor used for the treatment of late stage melanoma, has shown promising results by causing programmed cell death in melanoma cells. Recently, extracellular vesicles including apoptotic bodies, microvesicles and exosomes has been shown to contain genetic information especially RNA with distinct repertoire of RNA, which could be transferred from one cell to another. Here we investigate the effects of Vemurafenib on melanoma cells and the RNA content in their subsets of extracellular vesicles. Upon vemurafenib treatment, we found that the melanoma cells harboring the BRAF mutation significantly increases the RNA and protein cargo in the different subsets of extracellular vesicles. The RNA profile also changes substantially in apoptotic bodies and microvesicles with significant changes in the ribosomal RNA ratio. Further, using small RNA sequencing, we found that extracellular vesicles from treated cells harbor unique miRNAs. The significantly different miRNA detected with sequencing analysis was validated in vitro with PCR and confirmed that miR-211 is upregulated in cells as well as subsets of extracellular vesicles after treatment. Furthermore, miR-211 was validated to be significantly upregulated in tumors tissues harvested from patient derived xenograft (PDX) as well as cell line derived xenograft and its vesicular subsets. This shows that vemurafenib induces miR-211 upregulation in BRAF mutant cells and PDX especially in the subsets of extracellular vesicles. miR-211 induction upon oncogene treatment could be potentially used as a biomarker in melanoma patients with BRAF mutation.

#968

Circulating microRNA-1290 as a novel diagnostic and prognostic biomarker in human colorectal cancer.

Yuji Toiyama,1 Hiroki Imaoka,1 Hiroyuki Fujikawa,1 Junichiro Hiro,1 Susumu Saigusa,1 Koji Tanaka,1 Yasuhiro Inoue,1 Yasuhiko Mohri,1 Takao Mori,2 Toshio Kato,3 Ajay Goel,4 Masato Kusunoki1. 1 _Institute of Life Sciences, Mie University Graduate School of Medicine, Tsu, Japan;_ 2 _Moriei Hospital, Kuwana, Japan;_ 3 _Tohyama Hospital, Tsu, Japan;_ 4 _Center for Gastrointestinal Research & Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX_.

Background & Aims:

MicroRNAs (miRNAs) play a crucial role in diverse cellular biological processes such as differentiation, proliferation, migration, invasion and survival. MiRNA expression patterns are found to be frequently dysregulated in human tumors, which is currently being exploited both from a basic science perspective and for its clinical usefulness as disease biomarkers. Circulating miRNAs are attracting major interest as potential noninvasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early detection and/or evaluating prognosis of CRC patients.

Methods:

Comprehensive miRNA array analysis was performed using serum samples from patients with colorectal neoplasia (CRC: n=3, adenoma: n=3) and healthy controls (n=3). Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects.

Results:

Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly upregulated in patients with colorectal adenomas (P < 0.0001) and cancers (P < 0.0001). Serum miR-1290 levels could robustly distinguish adenoma (area under the curve [AUC] = 0.718) and CRC patients (AUC = 0.830) from normal subjects. Serum miR-1290 levels significantly diminished after surgical resection of the primary tumor in the same patients' serum specimens (P < 0.0001). In addition, we observed a statistically significant positive correlation of miR-1290 expression levels between primary CRC tissues and matched serum samples (ρ = 0.482; P < 0.0001). High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor in CRC patients (hazard ratio = 4.51; 95% confidence interval = 1.23-23.69; P = 0.0096).

Conclusions:

Serum miR-1290, which might be secreted from primary CRC, is a novel diagnostic and prognostic biomarker in CRC.

#969

Circulating cell-free microRNA as a novel biomarker for synovial sarcoma patients.

Koji Uotani,1 Tomohiro Fujiwara,1 Aki Yoshida,1 Takuya Morita,1 Yutaka Nezu,2 Tadashi Komatsubara,1 Kazuhisa Sugiu,1 Takenori Uehara,1 Toshinori Omari,1 Ken Takeda,1 Toshiyuki Kunisada,1 Junya Toguchida,3 Akira Kawai,4 Takahiro Ochiya,2 Toshifumi Ozaki1. 1 _Okayama University, Okayama city, Japan;_ 2 _Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan;_ 3 _Kyoto University, Kyoto, Japan;_ 4 _Division of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan, Japan_.

INTRODUCTION

Synovial sarcoma (SS) is characterized by the SS18-SSX fusion gene, and the presence of this chromosomal translocation is clinically useful as a diagnostic marker. However, SS18-SSX does not reflect disease progression and can be detected only with tumor specimens surgically resected. Therefore, novel non-invasive biomarkers would help clinicians to diagnose or detect tumor progression, which could improve patients' prognosis. We investigated the expression of the serum microRNA (miRNA) of SS patients, and evaluated its significance using in vitro and in vivo experimental models.

METHODS

In the screening phase, global analysis by miRNA microarray was performed using extracted RNA derived from patients' serum and culture medium. The serum samples were collected from SS patients, age-matched non-SS patients, and healthy volunteers. The culture mediums were obtained from human SS cell lines and human mesenchymal stem cells (hMSCs) as controls.

In the validation phase, the expression levels of miRNA derived from culture medium were evaluated by quantitative real-time PCR (qRT-PCR) to determine the secretory potential of miRNA candidates. SS cell lines and hMSCs were cultured and the culture medium was collected at 0, 24, and 48 hours. Furthermore, serum miRNA levels of SS-bearing mice were analyzed by qRT-PCR according to the tumor growth and tumor resection.

RESULTS

A miRNA microarray profiling revealed that four miRNAs were significantly highly expressed (p < 0.05) in SS patients' serum and cell culture medium, compared to control samples, which were selected as candidates for further analysis. We then identified that one miRNA expression among four candidates in the culture medium from all SS cell lines increased with time and with increasing numbers of tumor cells. These data suggested that the detected miRNA is a secretory miRNA derived from SS cells.

Next, we investigated whether the serum expression of the detected miRNA correlates with tumor progression using SS-bearing mice, which demonstrated that the detected miRNA expression in mice serum increased size-dependently (R = 0.78, p < 0.05). Moreover, it significantly reduced after tumor resection (p < 0.05), indicating that the detected serum miRNA level reflects tumor burden in vivo. Furthermore, the expression of the detected miRNA in serum collected from SS patients was significantly higher compared with that in serum collected from other soft tissue sarcoma patients.

CONCLUSIONS

Our results indicate that the serum expression of the detected miRNA could be useful for tumor monitoring as a non-invasive biomarker for SS. Moreover, the detected miRNA may contribute to SS progression.

#969A

The histone methyltransferase EZH2 is a novel target of anticancer therapy in endometrial cancer.

SHINYA OKI, KENBUN SONE, KATSUTOSHI ODA, AKIRA NISHIJIMA, MAKOTO TAKEUCHI, CHUWA AGAPITI, KAYO ASADA, CHINAMI MAKII, KEI KAWANA, YUTAKA OSUGA, TOMOYUKI FUJII. _Department of Obstetrics and Gynecology, Graduate School of Medicine, the University of Tokyo, BUNNKYO-KU TOKYO, Japan_.

Besides genetic disorders, dysregulation of epigenetic machinery has been proved to play a crucial role in human carcinogenesis, and the histone methylation/demethylation process has been considered as one of key regulators for this event. However, the roles of these alterations in cancer progression have not been well characterized in endometrial cancer. The aim of this study is to elucidate that one of methyltransferases , enhancer of zeste homolog 2 (EZH2) is a novel target of anti-cancer therapy in endometrial cancer.

We demonstrate that EZH2 plays pivotal roles in human endometrial cancer. To confirm these roles, we performed the expression profile of EZH2 in 11 endometrial cancer cell lines and 50 clinical endometrial cancer specimens, under informed consent and approval of our ethics committee. In addition, we performed the functional analysis of

EZH2 using endometrial cancer cell lines. The expression Quantitative real-time PCR revealed that EZH2 is aberrantly overexpressed in endometrial cancer cell lines and clinical samples compared with immortalized endometrial cell line and normal endometrial tissues (P<0.05). The reduction of EZH2 expression by small interfering RNAS resulted in suppression of growth of cancer cells and caused apoptotic cell death in endometrial cancer cell lines.

Our results suggest that dysregulation of EZH2 plays an important role in the growth regulation of endometrial cancer cells, and further functional studies may affirm the importance of EZH2 as a promising therapeutic target for endometrial cancer.

#970

Circulating microRNA signature in group 4 medulloblastoma patients.

Evelina Miele,1 Vincenzo Alfano,1 Zein Mersini Besharat,2 Giuseppina Catanzaro,2 Angela Mastronuzzi,3 Andrea Carai,3 Agnese Po,2 Luana Abballe,2 Antonella Cacchione,3 Franco Locatelli,3 Elisabetta Ferretti2. 1 _Istituto Italiano di Tecnologia, Rome, Italy;_ 2 _University of Rome Sapienza, Rome, Italy;_ 3 _Ospedale Pediatrico Bambino Gesù, Rome, Italy_.

Medulloblastoma is the most common malignant brain tumor in childhood. Four main clinically and molecularly distinct MB subtypes have been identified: WNT, SHH, Group 3, and Group 4. Among them, Group 4 MB (G4MB) appears to have the most skewed age ratio, is predominant in males and shows an intermediate prognosis. In spite of being the most frequent (35%), it is also the least understood of all the subgroups. Diagnosis is based on patients' clinical symptoms and neuro-imaging techniques with high risk of false positive and/or late tumor discovery. The early detection of the disease is essential to reduce both cancer mortality and long time therapy related toxicity. To date, there is no reliable marker for G4MB that could facilitate the early diagnosis and allow the evaluation of the response to radio and chemotherapy treatment. Therefore, the discovery of new biomarkers as a specific signature of G4MB patients, potentially useful as diagnostic factor, would be extraordinarily worthwhile.

MicroRNAs are single-stranded non coding RNA molecules of 19-24 nucleotides in length involved in the post-transcriptional regulation of gene expression, whose expression is strongly deregulated in several types of tumors. Recent studies have shown that microRNAs are released into the bloodstream and are stably detectable thanks to their association with extracellular vescicle, or complex with RNA-binding protein that protect them from RNAse degradation. The minimally invasive liquid biopsy flanked to the reliability and specificity of microRNAs in cancer detection indicates circulating microRNAs not only as promising biomarkers to get early diagnosis, but also to follow treatment response and detect recurrence.

Here we report a high-throughput screening of microRNAs in G4MB plasma samples before surgery and during follow-up. Two different approaches (DeepSeq and Low Denstity Array) were used to detect the differentially expressed circulating microRNAs in 4 patients and in 4 age- and sex- matched healthy donors. Baseline microRNA expression profiling allowed us to disclose a signature of 34 miRNAs able to identify patients versus healthy children. Moreover, selected microRNAs were validated by RT-qPCR technology on a wider cohort of G4MB patients (n=8) and healthy donors (n=8). Specifically, we identified two important oncogenic microRNA clusters (miR-17/92 and miR-106b/25) significantly upregulated in G4MB plasma patients. Therapy reduced the expression of these microRNAs up to levels of healthy donors.

In conclusion we identified circulating microRNA signature in G4MB.

#970A

Using a radiogenomic approach to classify pancreatic cancer precursors.

Jennifer B. Permuth,1 Jung Choi,1 Yoganand Balarunathan,1 Jongphil Kim,1 Dung-Tsa Chen,1 Kun Jiang,1 Sonia Orcutt,1 Lu Chen,1 Kimberly Quinn,1 Rodrigo Carvajal,1 Guillermo Gonzalez-Calderon,1 Michelle Fournier,1 Mahmoud Abdalla,1 Alberto Garcia,1 Amber Bouton,2 Danny Yakoub,3 Suzanne Lechner,3 Jose Trevino,2 Nipun Merchant,3 Robert Gillies,1 Mokenge Malafa1. 1 _Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _University of Florida Health Sciences Center, Gainesville, FL;_ 3 _Sylvester Comprehensive Cancer Center at the Universtiy of Miami, Miami, FL_.

Background: Intraductal papillary mucinous neoplasms (IPMNs) are cystic pancreatic cancer precursors incidentally detected by imaging in more than 75,000 Americans each year. There is an unmet need to discover noninvasive approaches to differentiate 'benign' IPMNs that can be monitored from 'malignant' IPMNs that warrant surgery. We previously developed a blood-based 'miRNA genomic classifier (MGC)' that helps predict malignant IPMN pathology. The goal of this study was to evaluate whether novel radiomic features from preoperative computed tomography (CT) scans may improve prediction of IPMN pathology beyond that provided by standard radiologic features, either alone or in combination with the MGC.

Methods: Preoperative CT images were obtained for 37 surgically-resected, pathologically-confirmed IPMN cases with matched preoperative miRNA expression data. Images were reviewed for standard radiologic features characterized to be 'high-risk' or 'worrisome' for malignancy according to consensus guidelines. The region of interest within the pancreas was identified and segmented using a semi-automated algorithm. A total of 112 two-dimensional (2D) quantitative texture features (which measure tumor size, shape, and location) and non-texture features (which measure smoothness, coarseness, and regularity) were extracted. Logistic regression models were used to explore associations between non-redundant radiomic features and IPMN pathology. Principal component analysis was also performed to generate an index score (defined by the first principal component of the most promising radiomic features) that was evaluated for its association with malignant pathology. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the new radiomic features individually and in combination with the MGC were estimated and compared to values obtained for standard radiologic features.

Results: The MGC and standard 'high-risk' and 'worrisome' radiologic features had area under the receiver operating characteristic curve (AUC) values of 0.83, 0.84, and 0.54, respectively. Analysis of 112 extracted preoperative radiomic CT features revealed 14 textural and non-textural features that differentiated malignant from benign IPMNs (P<0.05). Collectively, the 14 radiomic features had an AUC=0.77. A model that combined radiomic features and the MGC had an AUC= 0.92 and estimates of sensitivity (83%), specificity (89%), PPV (88%), and NPV (85%) that were superior to models not based on these novel data types.

Conclusions: Our preliminary findings suggest that clinical decision-making models that integrate novel quantitative radiomic features with a blood-based miRNA classifier may more accurately predict IPMN pathology than models that rely on standard clinical data or worrisome radiologic features alone. Larger, prospective, multi-center studies are planned to explore this topic further.

### LncRNAs and Mechanisms in Cancer

#971

A novel long noncoding RNA, onco-lncRNA 230, induces apoptosis and invasion in lung squamous cell carcinoma.

Cynthia Y. Tang, Jessica M. Silva-Fisher, Ha X. Dang, Nicole M. White, Christopher A. Maher. _Washington University in St. Louis, St. Louis, MO_.

Long non-coding RNAs (lncRNAs) are RNAs that are longer than 200 base pairs and do not translate into proteins but have been shown to play roles in all aspects of biological regulation. LncRNAs have also been shown to regulate gene expression including chromatin organization, transcriptional regulation, and post-transcriptional control. In addition, they are now emerging to be important players in our understanding of cancer and have been shown to promote tumorigenesis. However, the mechanisms driving the advancement of tumor progression to metastasis is poorly understood. To identify lncRNAs critical to tumorigenesis, our lab conducted a pan-cancer analysis of normal and primary tumor tissues from The Cancer Genome Atlas to discover lncRNAs that are commonly altered across cancer types, which we termed 'onco-lncRNAs'. During our analysis, we discovered a novel unannotated onco-lncRNA, onco-lncRNA-230, that was significantly up regulated in head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) tumors relative to matched adjacent normal tissues, when available. We chose to focus on onco-lncRNA-230 as it displayed more than seven fold increase in LUAD and more than thirty seven fold change in LUSC tumors relative to adjacent normal. First, we validated this finding by quantitative PCR which revealed greater than a two fold enrichment of onco-lncRNA-230 expression in two out of seven LUAD and all six LUSC cells lines compared to a normal lung cell line. Because we observed the greatest increase in expression in HCC95 LUSC cells, we next sought to determine if modulating the expression of onco-lncRNA-230 promotes oncogenic phenotypes. Silencing of onco-lncRNA-230 in HCC95 cells did not change cellular proliferation but significantly increased apoptosis. Furthermore, silencing onco-lncRNA-230 resulted in a decrease in cellular invasion by transwell assay. Taken together, this represents the first study reporting that onco-lncRNA-230 is dysregulated in multiple cancer types, significantly up-regulated in lung cancer patient samples and lung cancer cell lines, and confers oncogenic phenotypes. Moving forward, we intend to explore how onco-lncRNA-230 mechanistically promotes lung cancer with the objective of utilizing onco-lncRNA-230 as a novel cancer diagnostic and therapy.

#973

A long noncoding RNA-regulating enhancer links Ewing sarcoma susceptibility to stress response.

Dave NT Aryee,1 Eleni Tomazou,2 Maximilian Kauer,2 Thomas Grünewald,3 Franck Tirode,4 Olivier Delattre,4 Heinrich Kovar1. 1 _St. Anna Children's Cancer Research Inst. & Department of Pediatrics, Medical University, Vienna, Austria; _2 _St. Anna Children's Cancer Research Inst., Vienna, Austria;_ 3 _Institute of Pathology, Ludwig-Maximilian University, Munich, Germany;_ 4 _INSERM, U830 Génétique et Biologie des Cancers, Institut Curie, Paris, France_.

Ewing sarcoma is the second most frequent bone cancer in Caucasian children and young adults, but hardly occurs in Africans. It is characterized by a relatively quiet genome with only one highly recurrent chromosomal aberration, a EWS-ETS (predominantly EWS-FLI1) gene rearrangement. The resulting fusion gene encodes for an aberrant ETS transcription factor which drives sarcomagenesis. In a large GWAS study, three Ewing sarcoma susceptibility loci were discovered on chromosomes 1, 10 and 15, but the molecular mechanisms, how these loci affect tumorigenesis, remained largely unknown.

By epigenomic mapping, EWS-FLI1 ChIP-seq, and reporter gene assays we found one of the susceptibility loci to localize to an EWS-FLI1 bound enhancer in the intron of a long non-coding antisense RNA, which we termed lncESST1 (Ewing Sarcoma Susceptibility Transcript 1). Deletion of the ETS binding region in the enhancer by CRISPR/Cas9 mediated gene editing lead to a drastic decrease in lncESST1 expression. Modulation of lncESST expression by siRNA or enhancer deletion significantly reduced soft agar colony formation of Ewing sarcoma cells without affecting growth under adherent culture conditions. Detachment of Ewing sarcoma cells from the plastic surface and growth under spheroid culture conditions induced stress granule formation as an immediate response, and a steady increase in lncESST1 expression over a period of at least 72 hours. We report that lncESST1 binds to a stress granule component with which it shares the promoter, and that loss of lncESST1 expression due to enhancer deletion perturbs stress granule formation. Our study therefore unveils a novel mechanism of lncRNA mediated cancer susceptibility.

Supported by FWF grant I1225-B19

#974

Characterization of the novel lncRNA, PCAT14, clinically associated with metastatic prostate cancer.

Nicole M. White,1 George Zhao,2 Jin Zhang,1 Elias Davicioni,3 Christopher A. Maher1. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _GenomeDx Biosciences Inc., Vancouver, British Columbia, Canada_.

Each year, over 180,000 men are diagnosed with prostate cancer in the United States. Advances in research have established a molecular stratification of prostate cancer disease improving screening and treatment options. However, some patients lack these genetic aberrations, indicating that prostate tumors may harbor disease-associated noncoding RNAs that further characterize molecular subtypes. Long non-coding RNAs (lncRNAs) are largely unexplored and are emerging as a new aspect of cancer biology through advances in sequencing technologies. To discover novel transcripts, and overcome the shortcomings of relying on incomplete or inaccurate annotations, we focused on an approach using a genome-wide annotation-independent method to identify regions of differential expression named SWORD (Sliding WindOw Region Discovery tool). We applied SWORD to recently generated data from aggressive prostate tumors and adjacent normal tissue. We discovered ten novel lncRNAs including the previously annotated Prostate Cancer Associated Transcript-14 (PCAT14). PCAT14 was consistently altered in an integrative analysis performed across three patient cohorts consisting of primary tumors with matched control transcriptome sequencing and Affymetrix gene expression including metastatic tumors. Utilizing the clinical data associated with the Affymetrix cohort, PCAT14 significantly associated with both high (9) and low (6) Gleason scores. Interestingly, PCAT14 is highly upregulated in primary tumors relative to control tissue and its expression is downregulated in metastatic tumors relative to primary tumors. These data suggest that PCAT14 expression promotes a metastatic phenotype. Therefore, we assessed PCAT14 expression within a cohort of 1008 radical prostatectomy specimens from three independent patient cohorts across institutes. We found that patients with high versus low PCAT14 expression showed significantly different rates of distant metastasis free survival, biochemical recurrence free survival, prostate cancer specific survival, and overall survival. Moreover, PCAT14 was implicated with protein-coding genes involved in biological processes promoting aggressive disease. In vitro experiments in prostate cancer cell lines further supported the clinical data associating PCAT14 with aggressive disease. Overall, we discovered that PCAT14 is broadly deregulated, promotes aggressive oncogenic phenotypes, and is significantly prognostic for multiple clinical endpoints supporting its significance for predicting metastatic disease. Due to its tissue-specific expression PCAT14 may serve as a valuable biomarker to define a subgroup of high-grade prostate carcinomas and improve disease management and patient prognosis.

#975

Hypoxic long noncoding RNA NDRG1-OT1 inhibit NDRG1 in MCF-7 breast cancer cell line.

Hsin-Chen Lin, Liang-Chuan Lai, Hsing-Guang Chen, Hung-Hsin Chen, Mei-Hung Chang, Mong-Hsun Tsai, Eric Y. Chung. _National Taiwan University, Taipei, Taiwan_.

Hypoxia is a crucial factor that associated with solid tumor progression by driving multiple signaling pathway. Our lab previously reported that NDRG1 was oxygen-responsive genes and could affect cellular function in a breast cancer cell line MCF-7. Long-non coding RNAs (lncRNAs) have recently emerged as important regulators of tumor progression by participating in several process of gene expression. However, it is unclear whether lncRNAs are responsed to oxygen concentrations in breast cancer. Therefore, the aim of this study is to identify lncRNAs which are responsive upon changes in oxygen concentrations, and its regulatory mechanism in the breast cancer MCF-7 cells. We investigated the differentially expressed lncRNAs in different oxygen concentrations using next-generation sequencing (NGS) and validated these results through quantitative reverse transcription polymerase chain reaction (qRT-PCR). We found an lncRNA, NDRG1-OT1, that was up-regulated during hypoxia and down-regulated during reoxygenation. Next, we identified the differentially expressed genes regulated by NDRG1-OT1 using microarrays and confirmed these results through qRT-PCR and Western blotting. We found an oxygen responsive gene, NDRG1, was inhibited by NDRG1-OT1. Furthermore, Ingenuity Pathway Analysis(IPA) indicated that NDRG1-OT1 could involve in the Ubiquitin-proteasome pathway. We considered NDRG1-OT1 might regulate NDRG1 stability and ubiquitination, and we are proving these possibility by immunoblotting and immunoprecipitation. These findings may provide new insights into epigenetic regulation of breast cancer during hypoxia.

#976

Long non-coding RNA Fendrr as biomarker for lung cancer.

Antonio Herrera,1 Marta Cuadros,1 Sandra Rodriguez,2 Raul Torres,3 Esther Farez,1 Pedro Medina1. 1 _Universidad de Granada, Granada, Spain;_ 2 _Centro Nacional de Investigaciones Oncologicas, Madrid, Spain;_ 3 _Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain_.

Background. Carcinogenesis has been extensively studied at a molecular level however, only since some years ago this research field has been fed by the discovery of a new type of long non-coding RNAs that are able to regulate gene expression and control various cellular mechanisms: Long non-coding RNA (LncRNA). In this study we have focused on the LncRNA Fendrr, which is located on chromosome 16 (16q24.1). This gene produces a long non-coding RNA transcribed bidirectionally with the Forkhead box-F1 (Foxf1) transcription factor on the opposite strand. The distance between Fendrr and FoxF1 is around 1700 bp and it seems to contain the promoter of both genes. In addition, the function of LncRNA Fendrr is currently unknown.

There is controversial data about the possible correlation between Fendrr and FoxF1. Szafranski et al (Genome Res. 2013) showed that Fendrr downregulates FoxF1 (by using siRNA against Fendrr). On the other hand, Cabili et al (Genome Biol. 2015) demonstrated that the mRNA of Fendrr and Foxf1 are in the same proportion and localization (nucleus and cytoplasm) and Grote et al (RNA Biol. 2013) indicated that Fendrr interacts with PRC2 complex, which is able to inhibit FoxF1 expression. However, Xu et al (J Hematol Oncol. 2014) showed that, regardless of an overexpression or knockdown of Fenderr, any change in FoxF1 expression was found.

Objective. One of the principal aims of this study is to investigate how Fendrr and FoxF1 are silenced in lung cancer and which are their functions.

Results. Our preliminary results show that 17 out of 20 (85%) human lung cancer cell lines tested, didn´t express neither Fendrr nor Foxf1. However, to date, it has not been identified any mutation in Fendrr or FoxF1 in these cell lines.

In addition, it has been seen that methylation of the promoter and the response of 5-aza is dependent of the p53 status. Moreover, we have observed that ectopic overexpression of LncRNA Fendrr increases the expression of FoxF1 in some lung cancer cell lines and, interestingly, inhibition of FoxF1 gene increases expression of Fendrr. In contrast, inhibition of Fendrr does not seem to produce any change in FoxF1 expression. This could mean that Fendrr regulates FoxF1 expression but requires the help of other components.

Our main goal is to investigate whether Fendrr and FoxF1 are silenced in lung cancer cell lines and how demethylating agents can re-express Foxf1 and Fendrr genes. In addition, we will explore the epigenetic patterns of lncRNAs in lung cancer cell lines using sequencing-based methylome data.

Conclusion. We demonstrate that Fendrr and FoxF1 are silenced by methylation in p53 mutated human lung cancer cells lines and we also have observed that Fendrr regulates the expression of FoxF1. All there results could support the hypothesis the role of Fendrr in regulating FoxF1 expression, a gene that could be a putative tumor suppressor.

#977

RNA-sequencing analysis implicates novel non-coding RNAs in human papillomavirus-associated head and neck squamous cell carcinoma.

Angela E. Zou,1 Aswini R. Krishnan,1 Yinan Xuan,1 Maarouf A. Saad,1 Avinaash Korrapati,1 Sunil J. Advani,2 Jessica Wang-Rodriguez,3 Weg Ongkeko1. 1 _University of California, San Diego, San Diego, CA;_ 2 _Moores Cancer Center, San Diego, CA;_ 3 _VA Medical Center, San Diego, CA_.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. Given the recent emergence of human papillomavirus (HPV) as a major etiological factor in head and neck cancers, there exists a pressing need to better resolve the molecular heterogeneity of HNSCCs, define more precise diagnostic and prognostic tools, and develop a wider repertoire of targeted therapies. Non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), have been increasingly viewed as promising sources of insight into cancer onset and progression, with their diverse regulatory roles, tissue specificity, and aberrant expression in various human diseases now established by a multitude of studies. Using RNA-sequencing data from 466 HNSCC patients in The Cancer Genome Atlas (TCGA), we sought to investigate the transcriptome-wide dysregulation of both classes of ncRNAs in HPV-associated HNSCCs. Differential expression analyses controlled for patient smoking status revealed 5 lncRNAs and 9 miRNAs exhibiting significantly altered regulation between HPV-negative and HPV-positive HNSCC tumors (FDR < 1 x 10-10). Among these, 8 ncRNAs were additionally correlated with HNSCC tumor stage, invasion, and anatomic site, among other clinical characteristics (Kruskal-Wallis, p < 0.05). We subsequently validated the dysregulation of several candidate HPV-associated ncRNAs in vitro after ectopically expressing HPV16 E6/E7 in a panel of normal oral epithelial and HPV-negative HNSCC cell lines. To explore the functional consequences of our identified HPV-associated ncRNAs, we further investigated lnc-SP9-1:1, an lncRNA upregulated by 73.89 fold in HPV-positive compared to HPV-negative HNSCCs (FDR < 1 x 10-10). Cell proliferation, migration, and matrigel invasion assays were performed to evaluate the effects of siRNA-mediated lnc-SP9-1:1 knockdown in E6/E7 overexpressing cell lines. While the specific roles of the vast majority of non-coding transcripts remain unknown, their consistent dysregulation in HPV-positive HNSCCs, coupled with their ongoing functional characterization, promises to unravel new layers of understanding of the molecular underpinnings of HPV-associated HNSCCs and nominate new biomarkers and therapeutic candidates for this specific disease.

#978

Integrated analysis of long noncoding RNA and mRNA expression profile in laryngeal squamous cell carcinoma.

Ru Wang, Jugao Fang, Ling Feng. _Beijing Tongren Hospital, Beijing, China_.

Objective: The purpose of this study is to investigate the aberrant expressed lncRNA and mRNA in laryngeal squamous cell carcinoma (LSCC), and identify the potential lncRNA correlated with functional mRNA in LSCC.

Methods: A total number of 9 pairs of primary Stage IV LSCC and adjacent non-neoplastic tissues were surgically removed for integrated microarray analysis of lncRNA and mRNA using the Aglient GeneChip with 46506 lncRNA probes and 30656 mRNA probes. Random Variance Model(RVM) t-test was applied to filter the differentially expressed genes. Hierarchical clustering analysis was used to reveal the expression patterns of lncRNA and mRNA. GO analysis was applied to analyze the main function of the differentially expressed genes according to the Gene Ontology. Pathway analysis was used to find out the significant pathway of the differentially expressed genes according to KEGG, BioCarta and Reactome. Gene-gene interaction network was constructed based on the data of differentially expressed genes to build and analyze molecular networks. LncRNA-mRNA-network was built according to the normalized signal intensity of specific expression in mRNA and lncRNA to identify the interactions between mRNA and lncRNA. Then the significantly altered lncRNA and its correlated mRNA were validated by the qRT-PCR in an independent cohort of 30 pairs of LSCC tumor tissue and adjacent normal tissue samples.

Results: The integrated microarray analysis of lncRNA and mRNA identified 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs(fold change≥2, p<0.05). To identify typical mRNA with core functions from the identified mRNAs, the GO, KEGG pathway analysis and Gene-gene interaction network selected three mRNAs HIF1A, ITGB1, and DDIT4, which mainly involved in biological processes, such as pathways in cancer, HIF-1 signaling pathway, Focal adhesion, ECM-receptor interaction, and PI3K-Akt signaling pathway. QRT-PCR validated that the three mRNAs exhibited higher expression in cancer tissues. According to lncRNA-mRNA-network,we found that lncRNA MIR31HG was positive correlated with HIF1A, lncRNA NR-027340 was positive correlated with ITGB1, lncRNA SOX2-OT-015 was positive correlated with DDIT4. QRT-PCR further validated that the 3 lncRNAs were overexpressed in cancer tissues compared to adjacent non-neoplastic tissues of the 30 samples.

Conclusions: lncRNA MIR31HG, NR-027340, SOX2-OT-015 and their correlated mRNAs were identified in LSCC tissues by microarrays, and further validated by qRT-PCR. These up-regulated lncRNAs may act as novel biomarkers in LSCC and may be potential therapeutic targets for LSCC patients.

#979

Identification of a novel long noncoding RNA within the LCK gene locus that regulates prostate cancer cell growth.

Huy Q. Ta, Samuel R. Jackson, Hilary Whitworth, Shriti Bhadel, Daniel Gioeli. _University of Virginia, Charlottesville, VA_.

Prostate cancer remains the second most common type of cancer and frequent cause of cancer-related mortality in American men. Even though many patients with metastatic prostate cancer will initially respond to androgen deprivation therapy, virtually all patients will relapse and develop lethal castration-resistant prostate cancer. Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and there is increasing evidence demonstrating that dysregulation of lncRNAs is associated with many human cancers, including cancers of the prostate, breast, and lung. We have discovered in a high-throughput RNAi screen identifying regulators of prostate cancer cell growth that knockdown of lymphocyte-specific protein tyrosine kinase (LCK) significantly decreases growth of prostate cancer cells in the presence and absence of androgen. Surprisingly, immunoprecipitation and western blot analyses show that LCK is not expressed at the protein level in prostate cancer cells. Rapid amplification of cDNA ends (RACE) and sequencing have revealed that a previously unannotated lncRNA lies within exon six and the 3'UTR of the LCK gene. While short hairpin RNAs (shRNAs) targeting the carboxy-terminus of the LCK gene decreases cell growth, expression of shRNAs specific for the amino-terminus has no effect on growth. Furthermore, only quantitative polymerase chain reaction (qPCR) primers directed toward the 3' section of the LCK gene yield detectable levels of transcript. These data provide further validation for the existence of a lncRNA within the LCK gene locus. Remarkably, the lncRNA situated within the LCK gene is dramatically upregulated in response to androgen. Therefore, we have labeled this lncRNA "HULLK" for Hormone-upregulated lncRNA within LCK. Cellular fractionation and qPCR show that HULLK predominantly localizes to the cytoplasm. In addition to the effects on prostate cancer cell growth, we have data that alludes to the increase in transcript levels of several Src family members following depletion of HULLK. Thus, these studies indicate that the LCK gene contains a lncRNA involved in the regulation of prostate cancer cell growth and perhaps transcription. While additional analyses will be required to fully characterize HULLK, our data suggest that it may serve as a novel regulator of prostate cancer proliferation.

#980

The long non-coding RNA CAI2 is a tumor suppressor gene.

Olga Cohen,1 Ruey-Jen Lin,2 Alice L. Yu,2 Mitchell B. Diccianni1. 1 _UCSD, San Diego, CA;_ 2 _Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital, Taoyuan, Taiwan_.

We have recently reported on the discovery of a long non-coding RNA imbedded in intron 2 of the p16/ARF (CDKN2A) locus at 9p21. CAI2 is a non-conserved, RNA pol II regulated gene with whose expression is highly correlated with parent genes p16 and ARF. However, CAI2 is expressed in cell lines deleted for p16, ARF and even p15, and is expressed cell lines epigenetically silenced for p16 and/or ARF. Furthermore, treatment of p16 and/or ARF epigenetically silenced cell lines with 5-aza-2'-deoxycytidin allows p16 and/or ARF re-expression while having no influence on CAI2, confirming independent regulation. Speculating, therefore, that the regulatory region of CAI2 lies imbedded in the non-transcribed portion of intron 2, we created expression constructs of the immediate 5' region of CAI2. However, consistent with a bioinformatical analysis, no regulatory activity was detected. This suggests that this non-coding RNA is non-traditionally controlled by remote but yet to be identified regulatory element(s) and/or mechanisms. By colony formation assay, CAI2 overexpression can inhibit cell growth in the p16 intact HEK293T and SK-BR-3 breast cancer cell lines. RNA Immunoprecipitation has revealed that CAI2 interacts with H3K27me3, a recruiter of Polycomb Repressive Complex 1 (PRC1) that modulates chromatin structure and gene expression, and BMI1, a PRC1 complex protein that is a negative regulator of p16. Consistent with this effect, CAI2 can induce p16 expression in HEK293T cells, and this was hypothesized to be the mechanism by which CAI2 inhibits cell growth. However, we now show that CAI2 can also inhibit colony formation in the p16/ARF deleted MCF7 and MDA-MB-231 breast cancer cell lines. Interestingly however, CAI2 overexpression had little to no effect on 2D cultures of HEK293T or MCF7 cells including DNA synthesis, XTT metabolic activity or cell count.

As CAI2 is imbedded in the p16/ARF gene, standard siRNAs may inadvertently influence p16 and/or ARF expression due to actions on their pre-mRNAs. Therefore, we created a series of partial CAI2 knockouts using CRISPR technology and compared their growth by colony formation assay. Notably, clones containing a partial knockout of the 5' region of CAI2 proliferated faster than mock transfected clones, consistent with the growth suppressive properties observed with CAI2 overexpression. In 2D cultures, a small increase in DNA synthesis was also observed in the partial deletions. The HEK293T clones containing partial deletions of CAI2 also exhibited small increases in expression of the remaining intact portions of the gene with a concomitant decrease in p16 expression, all consistent with an influence of CAI2 on p16 expression and cell growth; no influence on ARF expression was observed.

Taken together, the data demonstrate that CAI2 is capable of influencing p16 expression, but this influence is insufficient to fully account for the ability of CAI2 to regulate growth, and we conclude that CAI2 is a bona fide tumor suppressor gene in its own right.

#981

Loss of TGFβ signaling leads to increased susceptibility to HCC in mice: Evidences of lncRNA H19's effects in HCC promotion.

jinqiang Zhang, Chang Han, Weina Chen, Kyoungsub Song, Ying Wang, Lu Yao, Nathan Ungerleider, Hyunjoo Kwon, Tong Wu. _Tulane University, New Orleans, LA_.

The output of a TGF-β response is highly contextual depending on specific tissue types and diseases. This is especially true in hepatocellular carcinoma (HCC) initiation and progression, where the tumor-initialing and malignant cells are highly influenced by the background liver environments. To delineate the role of TGF-β in the process of hepatocarcinogenesis, we employed a novel tumor-initiating cell transplantation system that avoids the possible effects incurred from genetic manipulation of the background liver. Specifically, TGF-β receptor II (Tgfbr2) flox/flox mice at 14 days of age were given one dose (25µg/g bodyweight) of the hepatic carcinogen diethylnitrosamine (DEN) via intraperitoneal injection. Three months later, the initiated hepatocytes from Tgfbr2 flox/flox mice were isolated and transplanted to the same strain (C57Bl/6) wild type recipient mice. Four weeks after transplantation, the recipient mice received one does of Cre recombinase adenovirus (Ad-Cre) through tail vein injection to delete Tgfbr2 in the transplanted tumor-initiating hepatocytes; separate group of the recipient mice were injected with the control adenovirus (Ad-GFP). The recipient mice were closely monitored for liver tumor burden. We observed that deletion of Tgfbr2 in tumor-initiating hepatocytes by tail vein injection of Ad-Cre led to formation of more and bigger liver tumors (compared to Ad-GFP group). This finding suggests that TGF-β inhibits the malignant potential of tumor-initiating hepatocytes. In parallel, we further analyzed the effect of TGF-β in tumor initiating hepatocytes in vitro. Specifically, tumor-initiating hepatocytes isolated from DEN-treated Tgfbr2 flox/flox mice were infected with Ad-Cre or Ad-GFP prior to TGF-β1 treatment in vitro; RNAs were then isolated for transcriptome sequencing analysis. We identified a group of lncRNAs that were noticeably regulated by TGF-β, including the lncRNA H19. We found that deletion of Tgfbr2 by Ad-Cre in tumor initiating hepatocytes led to a 5-fold increase of H19 expression. We observed that deletion of H19 by siRNA decreased the tumor-initiating cell property, whereas forced overexpression of H19 enhanced it. Chromatin immunoprecipitation assay showed that SOX2 bound to the promoter region of H19 gene. While SOX2 overexpression in tumor-initiating hepatocytes enhanced H19 transcription, SOX2 knockdown reduced it. Furthermore, TGF-β treatment reduced the protein level of SOX2 in tumor-initiating hepatocytes. Taken together, our findings disclose a novel mechanism that importantly regulates hepatocarcinogenesis --- TGF-β inhibits H19 expression via SOX2 in tumor-initiating hepatocytes and this effect leads to inhibition of HCC development.

#982

Long non-coding RNA PICSAR promotes growth of cutaneous squamous cell carcinoma by regulating ERK1/2 activity.

Minna Piipponen, Liisa Nissinen, Mehdi Farshchian, Pilvi Riihilä, Atte Kivisaari, Markku Kallajoki, Juha Peltonen, Sirkku Peltonen, Veli-Matti Kähäri. _University of Turku, Turku, Finland_.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and its incidence is increasing globally. Long non-coding RNAs (lncRNAs) are involved in various biological processes, and their role in cancer progression is emerging. The aim of this study was to find and characterize new biomarkers for evaluating the risk of progression and metastasis of cSCC and identify novel therapeutic targets for recurrent and metastatic cSCC. RNA-sequencing of cSCC cells (n=8) and normal human epidermal keratinocytes (NHEKs, n=4) revealed overexpression of a previously uncharacterized long intergenic non-coding RNA (lincRNA), LINC00162 in cSCC cells. Analysis of tissue sections by RNA in situ hybridization showed that LINC00162 is specifically expressed by tumor cells in cSCCs but not by keratinocytes in normal skin in vivo. The expression of LINC00162 in cSCC cells was upregulated by inhibition of the p38α and p38δ MAPKs using BIRB796 or p38α and p38δ specific siRNAs. Knockdown of LINC00162 inhibited proliferation and migration of cSCC cells, and suppressed the growth of human cSCC xenografts in vivo. Furthermore, knockdown of LINC00162 inhibited ERK1/2 activity and upregulated expression of dual specificity phosphatase DUSP6 in cSCC cells. Based on these observations, LINC00162 was named PICSAR (P38 Inhibited Cutaneous Squamous cell carcinoma Associated lincRNA). Our results provide mechanistic evidence for the role of PICSAR in promoting cSCC progression via activation of ERK1/2 signaling pathway by downregulating DUSP6 expression. These results also identify PICSAR as a novel biomarker and putative therapeutic target in cSCC.

#983

Integrative analysis of androgen receptor regulated long non-coding RNA in prostate cancer.

Rohit Malik, Yajia Zhang, Marcin Cieslik, Yashar S. Niknafs, Sethuramasundaram Pitchiaya, Yasuyuki Hosono, Shruthi Subramaniam, Sahr Yazdani, Xuhong Cao, Dan Robinson, Arul Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer. AR regulates a large repertoire of genes; however, the effect of androgen signaling on the regulation of long non-coding RNAs (lncRNA) remains incompletely understood. Using transcriptome sequencing (RNA-seq) of AR-positive cell lines VCaP and LNCaP treated with dihydroxytestosterone (DHT), we identified genes, including lncRNAs, which were strongly regulated by AR. To confirm direct regulation by AR, we interrogated AR-ChIP-seq data from VCaP and LNCaP cells, identifying the lncRNAs with direct AR binding. Existence of these lncRNAs in prostate cancer tissue samples was confirmed by analysis of RNA-seq data from prostate tumors, and the degree of differential expression in prostate tumors (localized and castration resistant metastases) versus benign was determined. The most highly overexpressed lncRNA in this analysis was a 2.7kb multi-exonic transcript present on chromosome 16 called ARlnc1. RACE was utilized to determine the exact exon structure of this gene, and its expression levels in various prostate cancer cell lines as well as independent prostate cancer tissue cohorts was assessed. Further, knockdown of ARlnc1 in AR dependent cell lines inhibited cell proliferation and induced apoptosis. Knockdown of ARlnc1 affected molecular signatures related to cell cycle, mitosis and DNA damage. Interestingly, ARlnc1 knockdown also suppressed global androgen signaling as determined by Gene set enrichment analysis using AR gene signature. Upon investigation of the mechanism through which PRCAT47 regulate AR signaling, we discovered that ARlnc1 regulates AR at the level of translation. Taken together, our data suggests that many lncRNAs are regulated by androgen signaling, and we identify one such lncRNA that is involved in a protein-lncRNA positive feedback loop.

#984

LncRNA LINC00668 predicts a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically silencing of CKIs.

Jinfei Chen. _Nanjing First Hospital, Nanjing Medical University, Nanjing, China_.

Recently, long noncoding RNAs (lncRNAs) have been shown to have important regulatory roles in human cancer biology. By utilizing publicly available lncRNAs expression profiling data of gastric cancer and integrating analyses, we screened out LINC00668, whose expression is significantly increased and is correlated with outcomes in gastric cancer (GC). Further experiments revealed that LINC00668 knockdown significantly repressed the proliferation both in vitro and in vivo. Mechanistic investigations showed that LINC00668 was a direct transcriptional target of E2F transcription factor 1 (E2F1). Moreover, we also found that LINC00668 plays a key role in G0/G1 arrest, and further demonstrated that LINC00668 was associated with PRC2 and that this association was required for the epigenetic repression of cyclin-dependent protein kinase inhibitors (CKIs), including p15, p16, p21, p27 and p57, thus contributing to the regulation of gastric cancer cell cycle. To our knowledge, this is the first report showed that the role and molecular mechanism of LINC00668 in cancer. Together, our results suggest that E2F1-regulated LINC00668, as a cell cycle regulator, enriches a mechanistic link between lncRNA and E2F1-mediated cell cycle regulation pathway and may serve as a candidate prognostic biomarker and target for new therapies in human gastric cancer.

Key words: E2F1; LINC00668; cell cycle; proliferation; gastric cancer

Acknowledgements: The work was partly supported by the National 973 Basic Research Program of China (Grant No. 2013CB911300) grants from the National Natural Science Foundation of China (Grant No. 81272469, 81502071 and 81572928) and the clinical special project for Natural Science Foundation of Jiangsu Province (Grant No. BL2012016), and the grant from Nanjing 12th Five-Year key Scientific Project of medicine to Dr. Jinfei Chen.

#985

Downregulation of long noncoding RNA TINCR promotes invasion and metastasis and predicts poor prognosis in tongue squamous cell carcinoma.

Zehang Zhuang, Cheng Wang, Nan Xie, Yue Wu, Jinsong Hou, Xiqiang Liu, Hongzhang Huang. _Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China_.

Background and aim: Recently long non-coding RNAs (lncRNAs) have emerged as new gene regulators involving in a number of developmental and tumorigenic processes. However, few lncRNAs have been well characterized in tongue squamous cell carcinoma (TSCC). The aim of this study is to determine whether lncRNAs play a role in TSCC progression.

Methods: The expression profiles of lncRNAs and protein-coding genes in TSCC tissues and paired adjacent non-tumor tissues from 10 patients were compared by using lncRNA microarrays. TSCC-specific gene co-expression networks were constructed by differential expression analysis and weighted gene co-expression network analysis (WGCNA). Levels of these differentially expressed lncRNAs were verified in TSCC tissues (n=45) using Quantitative real-time PCR (qRT-PCR) and further confirmed in another patient cohort (n=103) by in situ hybridization (ISH). The effects of lncRNA on tumor cell invasion and migration were assessed by silencing or overexpressing the lncRNA in vitro.

Results: A total of 815 lncRNAs and 1783 protein-coding genes were differentially expressed between TSCC tissues and paired adjacent non-tumor tissues. WGCNA showed that one of the co-expression networks was significantly enriched for biological process that was relevant to epithelial cell differentiation, among which, TINCR was significantly down-regulated. Both PCR and ISH analyses validated that the down-regulation of TINCR in tumor tissues compared with controls. The expression level of TINCR was associated with tumor size, differentiation, cervical lymph node metastasis status, clinical stage and outcomes in patients with TSCC.Patients with TINCR low expression had a reduced overall survival. COX regression analysis showed that TINCR can be served as an independent prognostic factor for patients with TSCC. More, ectopic transfection of TINCR dramatically suppressed cell invasion and migration in vitro. On the contrary, knockdown of TINCR led to p63 upregulation, and enhanced cell invasion and migration.

Conclusion: Our study suggests that downregulation of TINCR plays a role in TSCC progression, at least partially, by regulating epithelial differentiation-associated genes.

#986

Long noncoding RNA CCAT2 cooperates with miR-145 and miR-21 to regulate colon cancer stem cells.

Yingjie Yu,1 Pratima Nangia-Makker,2 Lulu Farhana,1 Adhip P. N. Majumdar2. 1 _Wayne State University and Department of Veterans Affairs Medical Center, Detroit, MI;_ 2 _Wayne State University, Karmanos Cancer Center and Department of Veterans Affairs Medical Center, Detroit, MI_.

Colon cancer-associated transcript-2 (CCAT2), a long noncoding RNA (lncRNA), has been reported to be over-expressed in colorectal cancer to promote tumor growth and metastasis and also reduces sensitivity to chemotherapy, a property related to cancer stem cells (CSCs). MicroRNAs, small endogenous noncoding RNAs, negatively control the expression of target genes by cleaving mRNA or through translation repression. We have recently reported cooperation of miR-21 and miR-145 to regulate colon CSCs proliferation and differentiation. Down-regulation of miR-21 enhances CSC enriched chemo-resistant colon cancer cells' susceptibility to chemo- therapeutic regimens. Although both CCAT2 and miR-21 increase WNT signaling activity and reduce chemosensitivity to 5-FU, a critical evidence on how CCAT2 regulates stemness leading to chemo-resistance is lacking. The primary goal of this investigation was to examine whether CCAT2 associates with miR-145 and miR-21 and whether this association plays a role in regulating stemness of colon cancer cells. We have observed that CCAT2 transcript level is dependent on p53 expression and activity, as evidenced by a marked increase in CCAT2 expression in p53 wild type harboring HCT-116 colon cancer cells compared with p53 null HCT-116 and p53 mutant HT-29 cells. We further revealed that knockdown of CCAT2 increased the expression of miR-145 and negatively regulates miR-21 in HCT-116 cells and impairs proliferation and differentiation of these cells. On the other hand, upregulation of CCAT2 induces proliferation of HCT-116 cells and also increases the expression of CSC markers CD44 and CD166. We have also observed that miR-145 and miR-21 cooperation plays a role in regulating colon cancer stem cells by down-regulating Sox2 and increased activity of Wnt/β-catenin pathway. Our current results suggest that lncRNA CCAT2 cooperates with miR-145 and miR-21 to regulate colon cancer stem cells, which provides a potential therapeutic target for chemoresistance in colorectal cancer.

#987

A novel cell cycle-associated lncRNA, HOXA11-AS, is transcribed from 5-prime end of HOXA transcript and a biomarker of progression in glioma.

Qixue Wang,1 Junxia Zhang,2 Yanwei Liu,3 Junhju Zhou,1 Chunsheng Kang,1 Lei Han1. 1 _Tianjin Medical Univ. General Hospital, Tianjin, China;_ 2 _The First Affiliated Hospital of Nanjing Medical University, Nanjing, China;_ 3 _Beijing Tiantan Hospital, Capital Medical University, Beijing, China_.

The comprehensive lncRNA expression signature in glioma remains fully unknown. We performed a high throughput microarray to detect ncRNA expression profile in 225 human glioma tissues. One novel lncRNA, HOXA11-AS with unknown function, is the antisense transcript of HOX11 gene. The results showed that HOXA11-AS was closely associated with glioma grade and poor prognosis. Multivariate cox regression analysis revealed that HOXA11-AS was an independent prognostic factor in glioblastoma multiforme patients and its expression was correlated with glioma molecular subtype of the Cancer Genome Atlas. Gene set enrichment analysis indicated that gene sets most correlated with HOXA11-AS expression involved in cell cycle progression. Overexpression of HOXA11-AS transcript promoted cell proliferation in vitro while knockdown of HOXA11-AS expression manifested a repressive function during these cellular processes via regulating cell cycle progress. The growth promoting and inhibiting effect of HOXA11-AS was also demonstrated in a xenograft mouse model. For the first time, our data confirms that HOXA11-AS is an important long non-coding RNA that primarily serves as a prognostic factor for glioma patient survival, a biomarker for identifying glioma molecular subtypes, and therapeutic target for glioma patient.

#988

A novel long non-coding RNA, TSA-LINC2, regulates cellular growth and is associated with poor prognosis in breast cancer.

Martin Pichler,1 Stefanie Cerk,1 Verena Stiegelbauer,1 Daniela Schwarzenbacher,1 Hui Ling2. 1 _Medical University of Graz, Graz, Austria;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Long non-coding RNAs (LINCs) are an emerging class of molecules in cancer diagnosis and prognosis. The number of LINCs exceeds the number of protein-coding genes and their role in breast cancer is largely unknown.

Methods: In this study we used non-adherent growing tumor spheres ("mammospheres") as a model system to identify tumor-sphere associated (TSA) gene expression patterns. We used microarrays to profile different breast cancer cell lines and selected the most up/down-regulated differentially expressed genes by RT-PCR. Clinical correlations including survival analysis of almost 900 breast cancer patients in two independent cohorts and experimental evaluation of the biological function were done.

Results: Among several TSA-genes, one novel not previously reported LINC, (that we termed TSA-LINC2) was significantly up-regulated in mammospheres (up to 50 fold, p<0.05). In patient samples, TSA-LINC2 was significantly up-regulated in cancer tissue compared to normal breast tissue, and high expression was associated with poor survival in different molecular breast cancer subtypes (p<0.05). Knock-down experiments of TSA-LINC2 in a panel of breast cancer cell lines led to significantly altered cellular growth, anchorage-independent growth and mammosphere formation in triple negative (p<0.05). Molecular profiling with gene expression arrays shows that TSA-LINC2 regulates cell cycle-associated genes.

Conclusion: This novel long non-coding RNAis involved in breast cancer progression and might be useful as a prognostic marker in breast cancer patients.

#989

A novel function of LncRNA AK023948—regulation of PI3K signaling.

Pratirodh Koirala, Yin Yuan Mo. _University of Mississippi Medical center, Jackson, MS_.

The human genome expresses a large number of long non-coding RNAs (lncRNAs) and increasing evidences suggest their physiological functions.However, the roles vast majority of lncRNAs are elusive in breast cancer. In the present study, we identified a set of lncRNAs that were differentially expressed in breast cancer tissues as compared to normal tissue. AK023948 is one of such lncRNAs which is up-regulated in breast cancer tissue and in breast cancer cell lines. Ectopic expression of AK023948 in breast cancer MCF-7 cells promotes proliferation and anchorage independent cell growth along with activation of AKT phosphorylation. On the other hand, knockdown or knockout of AK023948 in MCF-7 cells reduces the proliferation of cells and phosphorylation of AKT. Furthermore, there is a positive correlation between AK023948 and pAKT expression in breast cancer tissue microarray. To determine the underlying mechanism of AK023948-mediated activation of AKT, we performed RNA precipitation assays using biotin-labeled AK023948 RNA probe combined with mass spectrometry analysis and identified that ATP dependent RNA helicase A (RHA/DHX9) is AK023948. The interaction of AK023948 with DHX9 was confirmed by Western blot and RNA immunoprecipitation (IP), respectively. Co-IP indicated that DHX9 interacts with PI3K regulatory subunit p85. Further characterization revealed that AK023948 works as scaffold for p85-DHX9 interaction, which is essential for stability of p85 and AKT activity. Finally, we showed that AK023948 is essential to AKT activation induced by growth factors and acidosis, but it has no effect on activation of ERK phosphorylation, suggesting that AK023948 specifically regulate AKT signaling via PI3K. Together, these results suggest that AK023948 contributes to breast cancer pathogenesis by activating AKT and AK023948 may serve as a novel target for breast cancer therapy.

#990

Key tumor growth controlling long non-coding RNA (lncRNA) expression alterations in the colorectal adenoma-carcinoma sequence.

Alexandra Kalmar,1 Zsofia B. Nagy,1 Orsolya Galamb,2 Barnabas Wichmann,2 Barbara K. Bartak,1 Zsolt Tulassay,2 Bela Molnar2. 1 _2nd Dept of Internal Medicine, Semmelweis University, Budapest, Hungary;_ 2 _Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary_.

Background: Long non-coding RNAs may play role in colorectal cancer (CRC) development, however, lncRNA expression profile in the colorectal adenoma-carcinoma sequence (C-ACS) and its relation to the complex epigenetic regulation system still remain incomplete.

Aims: We aimed the whole genomic lncRNA expression profiling with up- and downstream epigenetic analyses of the C-ACS in order to explore the underlying mechanisms and consequences of aberrantly expressed lncRNAs.

Materials&methods: lncRNA expression levels were analyzed on 60 colonic biopsy samples (20 CRCs, 20 adenomas, 20 normals) by Human Transcriptome Array (HTA) 2.0 (Affymetrix). Data analysis was perfomed using Expression Console and Transcriptome Analysis Console. Expression of certain candidates was verified in silico on HGU133 Plus 2.0 array data and also by qRT-PCR. DNA methylation status of lncRNA promoter regions was studied by methyl-capture sequencing. miRNA targets of lncRNAs were predicted with miRCODE algorithm and miRNA expression was analysed using GeneChip miRNA 3.0 Array. In the respective regulatory networks mRNA expression changes were also evaluated on the basis of the above-mentioned whole genome expression arrays.

Results: According to HTA results analyzing 40.914 non-coding transcripts on whole genome level, in adenomas 12 lncRNAs (e.g. CCAT1, LINC00278) were significantly upregulated and 6 lncRNAs (e.g.FLJ22763, RP11.747D18.1) were downregulated compared to the healthy controls, while in CRC samples 1 lncRNA (UCA1) was overexpressed and 8 lncRNAs (e.g. LINC00350, LINC00261) were underexpressed compared to adenomas (p<0.05; -2≥Fold change≥2). In CRC samples 8 lncRNAs (e.g. MACC1, AC123023.1) were upregulated and 11 lncRNAs (e.g. RP13-497K6.1) were downregulated compared to normal controls. Furthermore, 42% of lncRNAs upregulated in CRC samples showed significantly elevated expression (p<0.05) already in adenoma samples (e.g. LINC350, CCAT1 were upregulated and LINC01133, FLJ22763 were downregulated compared to healthy controls). Promoter DNA methylation showed inverse relation with lncRNA expression along the C-ACS (e.g. CCAT1). In line with aberrant lncRNA expression in tumors, miRNA and mRNA targets' expression showed systematic alterations, e.g. UCA1 upregulation in CRC samples in parallel with miR-1 downregulation accompanied by MET proto-oncogene target mRNA overexpression (p<0.05).

Conclusion: The defined lncRNA sets (including MACC1, CCAT1, UCA1) have a central regulatory role in colorectal adenoma development and in tumor cell growth pathways. The underlying DNA methylation changes and miRNA and mRNA target expression alterations were proven using whole genomic array technologies. The identified lncRNA candidate sets can be further investigated as early diagnostic biomarkers and as potential therapeutic molecular targets for CRC.

#991

Long non-coding RNA LINC01133 inhibits epithelial-mesenchymal transition and metastasis in colorectal cancer by interacting with SRSF6.

Jianlu Kong, Honghe Zhang, Enping Xu, Qiong Huang, Jian Chen, Maode Lai. _Zhejiang University, Hangzhou, China_.

Objective Long non-coding RNAs (lncRNAs) play crucial roles in many biological and pathological processes, including tumor metastasis. The aim of this study was to investigate the expression and molecular function of LINC01133 in colorectal cancer (CRC).

Design LINC01133 expression was assessed in CRC cell lines and 219 clinical tissue samples by quantitative RT-PCR. Small interfering RNA-mediated knockdown was used to probe the biological functions of LINC01133, and its binding protein was isolated and identified using ChIRP-MS combined with western blot, and further validated by RNA immunoprecipitation assays.

Results We found that LINC01133 inhibited CRC cell migration and invasion by regulating the epithelial-mesenchymal transition (EMT), and was downregulated by TGF-β. We then showed that SRSF6 directly bound to LINC01133. Furthermore, we confirmed that that the EMT process regulated by LINC01133 in CRC cells depended on the presence of SRSF6. The expression of LINC01133 was markedly downregulated in CRC tissues relative to their corresponding normal mucosal tissues, and patients with tumors displaying higher LINC01133 expression had a better clinical outcome.

Conclusion These data suggest that LINC01133 inhibits the EMT and metastasis by directly binding to SRSF6 as a target mimic, and may serve as a prognostic biomarker and/or an effective target for anti-metastasis therapies for CRC.

#992

Long noncoding RNA UPAT promotes colon tumorigenesis by inhibiting degradation of UHRF1.

Kenzui Taniue, Yasuko Takeda, Tetsu Akiyama. _Institute of Molecular and Cellular Biosciences, Tokyo, Japan_.

Increasing evidence suggests that lncRNAs play critical roles in a diverse set of biological processes, including proliferation, differentiation, embryogenesis, neurogenesis and stem cell pluripotency. It has been reported that many lncRNAs regulate gene expression and various post-transcriptional processes, including splicing, transport, translation and degradation of mRNA. Furthermore, recent studies have shown that Many lncRNAs play critical roles in tumor development and progression.

We attempted to identify lncRNAs critical for the tumorigenicity of colon tumor cells. We have found that a lncRNA termed UPAT is required for the survival and tumorigenicity of colorectal cancer cells. We also show that UPAT interacts with and stabilizes the epigenetic factor UHRF1 by interfering with its β-TrCP-mediated ubiquitination. UHRF1 plays key roles in multiple biological processes, including proliferation and development. Moreover, UHRF1 is overexpressed in various tumors, including colon cancer, and plays a critical role in the proliferation and survival of tumor cells. Furthermore, we demonstrate that UHRF1-UPAT complex upregulate SCD1 and SPRY4, which play critical roles in the survival and proliferation of colon cancer cells.

Our findings suggest that UPAT-mediated stabilization of UHRF1 is critical for the proliferation and tumorigenicity of colon tumor cells. Our results suggest that UHRF1-UPAT axis may be a promising molecular target for colon cancer therapies.

#993

Identification of estrogen receptor alpha 1 bound lncRNAs in aggressive breast cancer.

Jessica M. Silva-Fisher,1 Abdallah M. Eteleeb,1 Torsten O. Nielsen,2 Charles M. Perou,3 Jorge S. Reis-Filho,4 Mathew J. Ellis,5 Elaine R. Mardis,1 Christopher A. Maher1. 1 _Washington University in St.Louis, St.Louis, MO;_ 2 _Vancouver Hospital & Health Sciences Centre, Vancouver, British Columbia, Canada; _3 _University of North Carolina Chapel Hill, Chapel Hill, NC;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _Baylor College of Medicine, Houston, TX_.

Breast cancer (BC) is the second most common newly diagnosed cancer and the second leading cause of cancer death among women in the United States. Around 70% of diagnosed BCs are estrogen receptor positive (ER+). Despite the proven benefits of adjuvant endocrine therapy in women with hormone receptor positive breast cancer, relapses still occur even after initial treatment with endocrine therapy for 5 years, referred to as late stage relapse. While existing studies have focused on the role of protein-coding genes, long non-coding RNAs (lncRNAs) are an emerging and under-characterized class of transcripts that have been shown to be dysregulated in breast cancer. Recently, lncRNAs have been shown to function by interfacing with corresponding RNA binding proteins to play critical regulatory roles in chromatin remodeling and diverse cellular processes by acting as decoys, guides, and scaffolds. As estrogen receptor expression is controlled mostly by epigenetic and post-transcriptional mechanisms, and very rarely at the genomic level, we hypothesize that lncRNAs may interact with ER to promote aggressive disease. To address this, we aimed to identify lncRNAs bound to the estrogen receptor alpha 1 (ESR1) that promote late stage breast cancer. To accomplish this, we first used transcriptome sequencing to identify altered expression levels of lncRNAs between primary tumors and late-stage relapse breast cancer patients. We detected 2086 altered lncRNAs when comparing the metastatic to the primary samples with an FDR <0.05, of which 202 were novel. As expected, Gene Set Enrichment Analysis of differentially expressed protein-coding genes revealed an enrichment of biological concepts associated with breast cancer and metastasis. Next, in order to identify ESR1 bound lncRNAs associated with aggressive disease, we conducted RNA Immunoprecipitation Sequencing (RIP-Seq) of all transcripts bound to ESR1 as compared to an IgG control in the ER+ T47D cell line in triplicate. We identified 210 transcripts bound to ESR1, which we term ESR1 bound lncRNAs (ESRlncs). The identified ESRlncs consisted of both novel (unannotated) and known (annotated) lncRNAs. Interestingly we found ~50% of ESRlncs had significantly altered expression in the metastatic samples, with 96% (105) having more than a two fold increase in expression. Further characterization of these ESRlncs is ongoing to decipher how they interact with ESR1 to promote aggressive and metastatic disease. Overall, this is the first study to discover ESR1 bound lncRNAs that may be contributing to late stage relapse in breast cancer patients.

#994

Long noncoding RNAs generated from the BRCA1 pseudogene regulate genomic instability and homologous recombination repair.

Y. Jane Han,1 Jing Zhang,1 Jennifer M. Mason,1 Aleix Prat,2 Toshio Yoshimatsu,1 Charles M. Perou,3 John Kwon,1 Prasanth Kannanganattu,4 Olufunmilayo I. Olopade1. 1 _University of Chicago, Chicago, IL;_ 2 _Valld´Hebron Institute of Oncology, Barcelona, Spain;_ 3 _University of North Carolina, Chapel Hill, NC;_ 4 _University of Illinois at Urbana-Champaign, Urbana, IL_.

Background: Genomic instability is frequently associated with cancer and can be indicative of a poor prognosis for breast cancer. While the significant role of BRCA1 in regulating DNA damage repair and checkpoint activation is well defined, little is known about the biological properties of the BRCA1 pseudogene (BRCA1P1) in neoplastic processes. The BRCA1P1 pseudogene was created by a recent evolutionary event, in which a partial duplication of the BRCA1 gene was followed by an insertion of a processed pseudogene of RPLP1P4.

Methods: To functionally annotate BRCA1P1, we conducted DNA-FISH, RNA-FISH, a luciferase reporter gene assay, bisulfite sequencing, microarray analysis, radiation-induced foci by immunofluorescence, I-SceI-induced double strand break repair assay, Caspase 3/7 assay, flow cytometry, qRT-PCR, and Western blot analysis. Depletion of BRCA1P1-lncRNA was achieved using LNA-antisense oligonucleotides (ASO), while overexpression of BRCA1P1 was attained using the CRISPR-On activation system.

Results: BRCA1P1 pseudogene is transcribed as a ~1.6 kb unspliced and nucleus-retained long non-coding RNA (lncRNA) using a bidirectional promoter between BRCA1P1 and the neighbor of BRCA1 gene 1 (NBR1). In contrast to the BRCA1 promoter, which is sensitive to hypoxia and subject to CpG island methylation, the pseudogene promoter did not respond to hypoxia nor did it undergo epigenetic modification. Expression of the BRCA1P1 pseudogene varies among 16 breast cell lines with higher expression in some lines, due in part to chromosome 17 polysomy. Interference of BRCA1P1-lncRNA expression with ASO increased genomic instability and spontaneous DNA damage as indicated by an increased spontaneous 53BP1 and RAD51 foci. Breast cancer cells with BRCA1P1 depletion exhibited a defect in DNA damage repair and increased apoptosis in response to irradiation and DNA damaging drugs, respectively. I-SceI-based assays confirmed a decrease in homologous recombination (HR) repair in cells with BRCA1P1 silencing. Interestingly, inhibition of BRCA1P1-lncRNA increased mRNA and protein expression of BRCA1, perhaps to compensate for the HR defect in the cells. The effects of BRCA1P1 overexpression on regulating DNA repair processes are currently under investigation in cells with BRCA1 mutations.

Conclusion: These findings suggest a novel role for the BRCA1P1 pseudogene in regulation of DNA damage repair in human breast cancer cells. It's potential role as a therapeutic target remains to be explored.

#995

The functional involvement of H19, a Long noncoding RNA, in human colorectal cancer.

HUI LING,1 Masahisa Ohtsuka,1 Martin Pichler,2 Cristina Ivan,1 Daisuke Matsushita,1 George Calin1. 1 _MD ANDERSON CANCER CENTER, HOUSTON, TX;_ 2 _Medical University of Graz, Graz, Austria_.

The clinical significance of long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 bp that do not code for proteins, in colorectal cancer (CRC) remains largely uncharacterized. We selected 10 lncRNAs associated with CRC, analyzed their expression association with patient survival using The Cancer Genome Atlas (TCGA) CRC data, and identified H19 as the one significantly associated with CRC patient survival. Experimental data using CRC cell line models showed that downregulation of H19 significantly reduced cancer cell proliferation, anchorage-independent growth, and cell migration capacity. Flow cytometric analysis suggested a clear G1 arrest of CRC cells following depletion of H19 expression. Our mechanism study suggests that H19 is pivotal in maintaining Wnt signaling and notch signaling. These finding represent a novel mechanism of H19 function in colorectal cancer, and strongly support the translational importance of this lncRNA as cancer marker and therapeutic target.

#996

STAT3 modulates HOTAIR transcription to influence HNSCC growth in vitro and in vivo.

Xuan Zhou. _Tianjin Medical University Cancer Institute & Hospital, Tianjin, China_.

Background

Squamous cell carcinoma is the most common cancer type of head and neck (HNSCC) with poor prognosis. Signal transducer and activator of transcription 3 (STAT3) activation is involved in HNSCC carcinogenesis as a transcript factor. A new class of transcripts, long noncoding RNAs (lncRNAs), has been recently found to be pervasively transcribed in the genome. HOTAIR, lncRNA Hox transcript antisense intergenic RNA, has been characterized as a novel hall marker to predict poor prognosis in HNSCC. HOTAIR regulates downstream target gene transcription by recruiting polycomb repressive complex 2 (PRC2) to the cell nucleus and triggers triple methylation of H3K27.

Methods

RNA sequencing, qPCR and CHIP assay were used to determine HOTAIR is a down-stream gene of STAT3. MTT and western blot were employed to examine the regulatory mechanism between STAT3 and HOTAIR in HNSCC cancers in vitro. Additionally, a cell derived xenograft tumor model was used to further validate the anti tumor effect of targeting STAT3/HOTAIR axis in vivo.

Results

STAT3 depleted HNSCC cell showed decreased HOTAIR expression by qPCR assay. CHIP assay indicated STAT3 bind to the promoter region of HOTAIR encoding gene. Combined inhibition of STAT3 and HOTAIR significantly inhibited cell proliferation, sensitive to cisplatin in HNSCC. Up-regulating HOTAIR partially compensated anti-tumor effect of targeting STAT3 in HNSCC.

Conclusion

Our data suggested that HOTAIR overexpression is depended on constitutive STAT3 activation in HNSCC and targeting STAT3/HOTAIR axis showed significant therapeutic potential in vitro and in vivo.

#997

Hotair promotes glioma cell cycle through a b-catenin mediated mRNA network.

Qixue Wang,1 Kai Huang,1 Yu Ren,2 Yunfei Wang,1 Bingcong Zhou,1 Yanli Tan,3 Chuan Fang,4 Jie Li,5 Chunsheng Kang1. 1 _Tianjin Medical University General Hospital, Tianjin, China;_ 2 _Tianjin Medical Universiy, Tianjin, China;_ 3 _Hebei University, Hebei, China;_ 4 _The Affiliated Hospital of Hebei Universiy, Hebei, China;_ 5 _Moores Cancer Center, University of California, San Diego, CA_.

Background:

LncRNA hotair is an oncogene that involves in the progression of several cancers. Previously we and others have reported that hotair promotes glioblastoma progression by regulating cell cycle. However, further mechanism still needs to be explored. The wnt/b-catenin signaling pathway is a crucial factor in the development of many cancers. B-catenin is a nuclear transcription factor that regulates multiple genes involved in cell proliferation, survival and EMT that contribute to glioma development.

Methods:

Positive correlation genes of hotair were picked up from CGGA (Chinese Glioma Genome Atlas) database by bioinformatics analysis. Real Time PCR is used to test the mRNA expression levels in U87 cells and astrocytes. Western blot is emplored to determine PKM2 expression after knocking down hotair. Further more, and nude mouse glioma intracranial model is employed to examine in vivo impact of hotair on GBM.

Results:

Hotair is a cell cycle related lncRNA as previously reported. Bioinformatics analysis indicated that various genes play important roles on cell cycle are positively correlated with hotair. Theses genes might be the executors of hotair on cell cycle regulating. We constructed a top 18 hotair-related genes network by connectivity. To test the hypothesis, we knock down hotair expression in U87 cells, and found out that FOXM1, CCNA2, CEP55, CENEP, CCNB2, HMMR, NCAPG, NUSAP were down-regulated significantly. In addition, overexpression of hotair in astrocytes could upregulate the mRNA of those genes. To further explore the regulatory mechanism, we analyed the promoter of these hotair-regulated genes and found out b-catenin/TCF4 binding site on all of them. This indicated that hotair might upregulate cell cycle associated genes through b-catenin/TCF4 pathway. FH535 is an inhibitor of wnt/b-catenin. The mRNA expression of these genes could be suppressed by FH535 treatment, which confirms the promoter analysis. It is reported that PKM2 could promote b-catenin nuclear translocation. Interestingly, we found out that binding site of miR-330 exists on both of hotair and PKM2 mRNA. Knocking-down of hotair inhibited PKM2 mRNA and protein expression in U87, U87vIII, U251 and LN229 cells. Further more, hotair siRNA could inhibit glioma growth in vivo.

Conclusion:

Our data indicated that hotair promotes glioma cell cycle through a b-catenin mediated mRNA network, and hotair could be a a biomarker and therapeutic target in glioblastoma. Meanwhile, there are still a lot of work to be done.

#998

LncRNA WDFY3-AS2 contributes to the EMT and metastasis in breast cancer.

Edward Richards,1 Sridevi Challa,2 Yajuan Li,2 Jennifer Permuth-Wey,2 Marilyn Bui,2 Domenico Coppola,2 Thomas Sellers,2 Jin Cheng2. 1 _Koch Institute, Cambridge, MA;_ 2 _Moffitt Cancer Center, Tampa, FL_.

The role of TGFβ in promoting mammary cell EMT and breast cancer metastasis was well established. However, the involvement of long non-coding RNA (lncRNA) in this process remains elusive. Here we demonstrated that mouse lncRNA BB179049 and its human ortholog WDFY3-AS2 were significantly increased upon TGFβ-induced EMT. WDFY3-AS2 was frequently elevated in invasive, metastatic and triple-negative breast cancer. Depletion of WDFY3-AS2 abrogated TGFβ-induced EMT and breast cancer metastasis. WDFY3-AS2 positively regulates STAT3 and WDFY3 through interaction with hnRNP-R. These data indicate that WDFY3-AS2 could play a pivotal role in TGFβ-induced EMT and metastasis and serves as a critical therapeutic target in aggressive breast cancer.

#999

P16 DNA methylation inactivates transcription of IncRNA ANRIL.

Ying Gan, Baozhen Zhang, Chenghua Cui, Dajun Deng. _Peking Univ Cancer Hospital & Inst, Beijing, China_.

The exonic ANRIL (P15AS) is a 3.8-kb lncRNA transcribed from the antisense strand of the P14 promoter and flanking regions. Recently, we have constructed a P16 promoter DNA methyltransferase (P16-Dnmt) using the pTRIPZ vector that can specifically methylate human P16 CpG islands and found that P16 methylation directly leads to gene transcription silence [Cui et al. Genome Biology 2015, 16:252]. However, whether ANRIL transcription is affected by P16 DNA methylation has not previously been reported. In the present study, correlation between endogenous P16, P15, P14 mRNA and ANRIL levels in a panel of cell lines were determined using quantitative RT-PCR. Unexpectedly, the results showed that ANRIL transcriptional level was positively and significantly correlated with the P16 mRNA level (r=0.774, P=0.003), but not correlated with the P15 or P14 mRNA levels (r=0.09 or 0.30, P=0.78 or 0.35). These phenomena were confirmed using the public available transcriptome data in the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). Furthermore, transcription of both ANRIL and P16 mRNA were observed only in cancer cell lines in which the P16 alleles are unmethylated (Caski, SGC7901, BGC823, GES1, Siha and HeLa), but not in these cell lines in which the P16 alleles are fully methylated (Colo205, PC3, MHCC97H, RKO, SW480 and AGS). These suggest the possibility that methylation of the P16 promoter may inactivate transcription of ANRIL as well as P16. To study the possible causality between P16 DNA methylation and transcriptional silence of ANRIL, we employed P16-Dnmt to induce P16-specific DNA methylation in gastric cancer BGC823 cells, and found that transcription levels of both P16 and ANRIL were significantly decreased by the induced P16 DNA methylation (P<0.001 and 0.024, respectively). Besides, transcription level of P14 was also reduced (P=0.043) while the P14 promoter CpG islands remained to be unmethylated. In contrast, P16 DNA demethylation induced by an artificial P16-specific transcription factor [Zhang et al. Human Gene Therapy 2012, 23:1071-81] re-activated transcription of both ANRIL and P16 in H1299 cells (P<0.001). The mechanism of inactivation of ANRIL expression by P16 DNA methylation is under investigation. In conclusion, transcription of ANRIL is regulated by P16 DNA methylation.

#1000

The long non-coding RNA HIF1A-AS2 regulates mesenchymal glioma stem cell tumorigenicity.

Marco Mineo, Franz Ricklefs, Arun Rooj, Shawn M. Lyons, Pavel Ivanov, Ennio A. Chiocca, Jakub Godlewski, Agnieszka Bronisz. _Brigham and Women's Hospital/Harvard Medical School, Boston, MA_.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults, which initiation and progression is driven by a subset of self-renewing GBM stem-like cells (GSCs). Long-non coding RNAs (lncRNAs) have been recently shown to play important roles in regulating numerous biological processes both in physiologic and pathologic condition. Identification of functional lncRNAs important for GBM initiation and progression may shed new light on understanding pathophysiology of the disease. We used a custom made lncRNA Nanostring platform to profile the expression of lncRNAs in subtype-characterized collection of patient-derived GSCs. We demonstrated that lncRNA signature may distinguish between GSC subtypes. Out of 73 lncRNAs we found 7 that were overexpressed specifically in the most aggressive mesenchymal (M) GSC subtype. Among them, HIF1A-AS2 was the most differentially expressed lncRNA. HIF1A-AS2 was reported to be overexpressed in many types of cancers; however its biological function and its role in GBM progression are unknown. Knockdown of HIF1A-AS2 in M GSCs resulted in reduced growth, increased cytotoxicity, and it strongly inhibited their neurosphere formation capability. Using more global approach we found out that knockdown of HIF1A-AS2 in M GSCs caused deregulation of several out of 730 cancer-related genes. Functional bioinformatic analysis revealed that these differentially expressed mRNAs are closely related to proliferation, transcriptional regulation and cell division. RNA pull-down assay showed that HIF1A-AS2 may exert its effects through specific binding of RNA helicase DHX9, a multifunctional protein with important roles in transcription, pre-mRNA processing and translation. We also demonstrated that HMGA1, a gene known to be regulated by DHX9, was specifically down-regulated in HIF1A-AS2 knockdown cells both at mRNA and protein level. Finally, we showed that silencing of HIF1A-AS2 blocked M GSC tumor growth in vivo resulting in significant survival benefits. Taken together, our results suggest HIF1A-AS2 as an important lncRNA in pathophysiology of GBM.

### Metabolic Reprogramming and Autophagy

#1001

Expression of cytochrome C oxidase 4 (COX4) in thyroid cancer cells.

Vasyl V. Vasko,1 Athanasios Bikas,2 Aneeta Patel,1 John Costello,1 Rok Tkavc,1 Kenneth D. Burman,2 Kirk Jensen1. 1 _Uniformed Services Univ. of the Health Sci., Bethesda, MD;_ 2 _Washington Hospital Center, Washington, DC, 20010, DC_.

Background:

Targeting cell metabolism has emerged as a therapeutic strategy for the treatment of cancer. Metabolically active thyroid cancers are resistant to treatment with radioactive iodine. Aberrant expression of genes controlling glycolysis was demonstrated in thyroid cancer cells, but little is known regarding the role of mitochondrial proteins in thyroid carcinogenesis. Cytochrome c oxidase 4 (COX4) plays pivotal roles in oxidative phosphorylation and the cellular response to oxidative stress. COX4 may thus represent a promising therapeutic target.

Objectives:

We examined expression of COX4 in human thyroid tumors and performed functional studies using thyroid cancer cell lines.

Material and Methods

Expression of COX4 was examined by immunostaining in 25 follicular adenomas (FAs), 22 follicular cancers (FTCs), 90 papillary cancers (PTCs) and in 48 samples from normal thyroid tissue. FTC-derived (FTC133) and PTC-derived (BCPAP) cell lines were used to create COX4-deficient cells by lentiviral transfection. The efficiency of COX4 inhibition was examined. Mitochondrial membrane potential was examined by JC-1 staining. DNA-damage signaling was examined after cell exposure to γ radiation (6-18 Gy). We also determined the effects of 2-deoxyglucose (2DG) on viability of thyroid cancer cells with compromised COX4. Caspase-3 and PARP cleavage assays were performed to measure apoptosis.

Results

Positive immunostaining with anti-COX4 was detected in 6/48 (12.5%) normal tissue, 5/20 (25%) FAs, 7/22 (31%) FTCs, and 51/90 (56%) PTCs. The intensity of COX4 immunostaining was significantly higher in thyroid cancers than in either normal thyroid (p=0.0001) or benign FAs (0.001). COX4 expression was more frequently detected in PTCs than in FTCs (p=0.03). The mRNA level of COX4 was higher in BCPAP and FTC133 cells compared to normal thyroid. In both cell lines, silencing of COX4 altered intra-cellular distribution of JC-1 staining. In control cells, JC-1 staining was perinuclear, but in COX4-deficient cells it became diffusely cytoplasmic. COX4 silencing affected cell growth and response to γradiation in a cell type specific manner. In BCPAP cells, downregulation of COX4 was associated with inhibition of cell growth, block in G1 phase and inhibition of Cyclin D1. In BCPAP cells, COX4 silencing activated DNA-damage signaling and increased sensitivity to γ radiation. Inhibitor of glycolysis (2DG) was more efficient against COX4-deficient than COX4-expressing BCPAP cells. In FTC133 cells, silencing of COX4 increased the rate of growth and induced expression of Cyclin D1. COX4 silencing did not increase FTC133 cell sensitivity to γradiation nor to treatment with 2DG.

Conclusion

COX4 is implicated in regulation of thyroid cancer cell growth and response to DNA damaging or metabolic treatments. These data suggest that evaluation of COX4 in thyroid cancer could serve as a biomarker of response to treatment with metabolic agents.

#1002

IDH1 promotes tumor growth and resistance to targeted therapies in the absence of mutation.

Alexander H. Stegh. _Northwestern University Lurie Comp. Cancer Center, Chicago, IL_.

Metabolic abnormalities of cancers provide opportunities for novel tumor-specific therapies. Isocitrate dehydrogenases (IDHs) are enzymes of the tricarboxylic acid (TCA) cycle that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and the reduction of NADP+ to NADPH. Oncogenic mutations in two IDH-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico analysis of The Cancer Genome Atlas (TCGA) data combined with wet-bench analysis of tumor extracts indicate that non-mutated IDH1 mRNA and protein is commonly overexpressed in primary GBM. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs survival of animal subjects bearing patient-derived xenografts (PDXs). On molecular levels, diminished IDH1 activity results in reduced α-KG and NADPH production, which is paralleled by deficient metabolic flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common mechanism of metabolic adaptation of GBM to support macromolecular synthesis, aggressive growth, and therapy resistance.

#1003

Targeting liver-tumor initiating cells via hampering the lipogenesis pathways through stearoyl - CoA desaturase.

Kin Fai Ma,1 Jessica Lo,1 Eunice Yuen-Ting Lau,1 John A. Copeland,2 Irene Oi-Lin Ng,1 Terence Kin-Wah Lee1. 1 _University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Mayo Clinic Jacksonville, Jacksonville, FL_.

Hepatocellular carcinoma is one of the most lethal cancers in the world. Most of the patients are diagnosed at advanced stage, and they receive sorafenib as their treatment modality. However, sorafenib can only extend patients' survival to a median of 3-4 months. Hence, there is an urgent need to identify a new therapeutic target for treatment of this disease. Increasing evidence showed that tumor-initiating cells (T-ICs) are intrinsically resistant to conventional treatments. Identification of the signalling pathways that maintain the functions of T-ICs provides potential targets for treatment of HCC. For this purpose, we cultured serial passages of hepatospheres combined with chemotherapeutic regimens as a strategy to enrich liver T-IC population. Using cDNA microarray, we found upregulation of several key enzymes in lipogenesis in enriched T-IC population, among which Stearoyl CoA desaturase-1 (SCD1), the enzyme involved in the conversion of saturated into monounsaturated fatty acids, was the most significant. In HCC clinical samples, 62% (36/58) was found to be overexpressed (> 2-fold) when compared with non-tumor counterparts and patients with SCD1 overexpression had shorter disease free survival. Using overexpression and knockdown approaches, SCD1 was found to regulate the traits of T-ICs, including tumorigenicity, self-renewal, drug resistance and expression of liver T-IC markers. SCD1 was found critical in survival of T-ICs, as enriched T-IC population are more sensitive to the inhibition of SCD1 compared to adherent lineages. Interestingly, SCD1 was found to be upregulated in sorafenib-resistant HCC cells. Pharmacological inhibition of SCD1 by A939572 not only suppressed self-renewal ability but also consistently enhanced the sensitivity towards sorafenib. Using a patient-derived xenograft model, we found that a new inhibitor against SCD1 demonstrated significant growth suppressive effect. By comparing the genetic profiles between SCD1 knockdown cells and control cells by RNA sequencing analysis, we found that upregulation of ER stress signature genes may be the potential downstream targets of SCD1 to maintain the properties of T-ICs. In conclusion, we demonstrated the crucial role of SCD1 in maintenance of liver T-ICs, and targeting SCD1 in combination with sorafenib might be a novel therapeutic regimen against HCC.

#1004

GLS inhibitor CB-839 modulates cellular metabolism in AML and potently suppresses AML cell growth when combined with 5-azacitidine.

Tianyu Cai,1 Philip L. Lorenzi,1 Dinesh Rakheja,2 Michael Pontikos,1 Lina Han,1 Qi Zhang,1 Helen Ma,1 Thomas D. Horvath,1 Amit K. Verma,3 Marina Konopleva1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _UT Southwestern Medical Center, Dallas, TX;_ 3 _Albert Einstein College of Medicine, Bronx, NY_.

Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions.

First, we performed metabolic analysis of HL-60 cells co-cultured with bone marrow-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids, possibly reflecting inhibition of global protein synthesis. CB-839 suppressed Cys consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis. CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, demonstrating activity under conditions mimicking BM microenvironment.

We have previously shown that the leukemic bone marrow microenvironment is highly hypoxic (Benito PLoS One 2011), and hypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG (measured by LC-MS/MS) in HL-60 (6.2+/- fold) and OCI-AML3 cells (2.9+/- fold) with wt-IDH. That increase in L-2HG was potently inhibited by CB-839. AML cells produced very little D-2HG, and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies.

Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1µM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and primary AML cells (n=6).

In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth.

#1005

Mitochondrial inhibition decreases the malignant phenotype of esophageal adenocarcinoma cells through the induction of autophagy.

Deborah R. Depew, Paul L. Feingold, Kate Brown, Yuan Xu, Mahadev Rao, Michael Moses, Leonard M. Neckers, David S. Schrump, R Taylor Ripley. _NIH/NCI, Bethesda, MD_.

OBJECTIVE

Esophageal adenocarcinoma (EAC) is a lethal disease for which novel therapies are needed. Malignant cell metabolism often shifts from mitochondrial respiration to aerobic glycolysis (Warburg effect). However, glucose alone does not meet the metabolic requirements of cancer cells; therefore, glutamine becomes an essential nutrient. A critical step in glutamine metabolism is the conversion of glutamine to glutamate by the enzyme glutaminase-1 (Gls-1), a potential therapeutic target. The present study was undertaken to identify the effect of targeting glutamine utilization by the mitochondrion in EAC.

METHODS

Assays were performed in 3 EAC and 1 Barrett's cell lines: Flo-1, NCI-SB-ESC2 (Esc2), OE33, and CP-C. qRT-PCR and immunoblot were used for Gls-1 expression. Cyquant® and Millipore Invasion Kit measured proliferation and invasion. Flow cytometry, SA-β-galactosidase assay, and immunoblot assessed cell cycle, apoptosis, senescence, and autophagy. Seahorse Extracellular Flux Analyzer was used to quantitate mitochondrial respiration and glycolytic capacity. Cells were treated with bis-2 [5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethylsulfide (BPTES), an inhibitor of Gls-1, and/or metformin, a mitochondrial electron transport chain inhibitor. Glycolysis was inhibited with 2-deoxyglucose (2-DG). Flo-1 and Esc2 were transduced with shRNA targeting Gls-1.

RESULTS

Glutamine withdrawal decreased proliferation of Flo-1, OE33, and CP-C more than Esc2. Similarly, BPTES caused a more significant decrease in proliferation in Flo-1, OE33, and CP-C than in Esc2. BPTES growth inhibition was reversed by α-ketoglutarate, a metabolite of glutamate. Unlike BPTES, metformin decreased proliferation in all cell lines which was augmented with 2-DG inhibition of glycolysis. Metabolic and knockdown experiments were performed in Flo-1 and Esc2. Glucose partially blocked mitochondrial spare capacity in Flo-1, but completely abrogated the spare capacity in Esc2 suggesting a higher glycolytic dependency in Esc2. Also, knockdown of Gls-1 blocked mitochondrial spare capacity and increased glycolysis in both cell lines. Knockdown of gls-1 decreased proliferation and invasion. Glutamine withdrawal induced autophagy as evidenced by increased LC3 and pAMPK and decreased p70 S6 Kinase in both cell lines. Apoptosis and senescence were not observed. To test glutamine's effect on glycolysis, glutamine repressed the negative regulator of glycolysis, thioredoxin interacting protein (TXNIP), suggesting that glutamine can increase glycolysis while simultaneously fueling the mitochrondria.

CONCLUSIONS

Mitochondrial respiration is mediated by both glucose and glutamine in EAC cells. Glutamine exerts an effect on glycolysis through repression of TXNIP. Whereas EAC cells have unique metabolic profiles, targeting common metabolic steps such as Gls-1 may be novel strategies to treat EAC.

#1006

Novel adjuvants in triple negative breast cancer chemotherapy.

Antonio Rampoldi, Adriana Harbuzariu, Tia L. Harmon, Ruben R. Gonzalez-Perez. _Morehouse School of Medicine, Atlanta, GA_.

Background: Obese patients develop more frequently triple negative breast cancer (TNBC) that has not target therapy and are commonly treated with chemotherapeutics. Leptin, whose levels are elevated in obesity, can induce TNBC proliferation, angiogenesis and resistance to chemotherapeutics. An antagonist for leptin receptor OB-R, leptin peptide receptor antagonist 2 (LPrA2), has been shown to block leptin signaling and decrease tumor progression. LPrA2 also countervails leptin induced resistance to chemotherapeutic agents. We used pegylation or iron oxide nanoparticle (IONPs) as adjuvants to increase LPrA2 solubility, stability and effectiveness.

Hypothesis: Conjugated LPrA2 will decrease TNBC cells proliferation, expression of leptin target molecules and tumorigenesis.

Methods: Human TNBC and murine breast adenocarcinoma derived cell line E0771 (progesterone and HER2 receptor negative) were used. The E0771 cell line was generated from an estrogen receptor positive (ER+) mammary adenocarcinoma isolated from a C57BL/6J mouse. E0771 was made insensitive to estrogen by treatment with drug Tamoxifen (TAM) mimicking TNBC. To specifically assess the role of RBP-Jk (CBS/CSL, an essential transcription factor for Notch signaling) the CRISPR/Cas9 system was used to disrupt RBP-JK gene expression in both wild-type and TAM treated E0771 cells. Cells were treated with chemotherapeutics (Paclitaxel, Doxorubicine, Ciclophosphamide) in presence of leptin and LPrA2-conjugates. The expression of leptin-targeted molecules, Breast Cancer Stem Cells (BCSC) and EMT markers were analyzed after cell treatment.

Results: Chemotherapeutics effects on proliferation, survival and molecular markers were modified by the addition of LPrA2-conjugates. Notch molecules (receptors and ligands) were downregulated by LPrA2 treatment.

Conclusion: The current data indicate that Leptin inhibition could be used as an adjuvant for chemotherapeutic treatments in TNBC patients. LPrA2-conjugates could increase the efficacy of chemotherapy.

Acknowledgements: This work was partially supported by the National Institute of Health and National Cancer Institute Grant U54 CA118638, NIH/SBIR 1R41CA183399-01A1, and DOD Idea Award BC W81XWH-13-1-0382 to RRGP; and facilities, and support services at Morehouse School of Medicine (NIH RR03034 and 1C06 RR18386) and NIH/NCRR grant 1G12RR026250-03.

#1007

The amino acid transporter SNAT4: Potential role as a tumor suppressor in melanoma.

Nicholas J. Otte,1 Angelika Broer,2 Patrick O'Young,1 Michelle van Geldermalsen,1 Qian Wang,1 Charles G. Bailey,1 Stefan Broer,2 Jeff Holst1. 1 _Centenary Institute, Campderdown, Australia;_ 2 _Research School of Biology, Canberra, Australia_.

Cancer cells utilize amino acids to satisfy their need for nutrients and fuel their accelerated growth. The amino acid glutamine has been identified as one of the key building blocks in cancer cells, utilized for macromolecular synthesis and energy production. Due to the increased requirement for glutamine and other amino acids, cancer cells commonly increase expression of amino acid transporters, such as SLC1A5 (ASCT2). Amino acid transporters are membrane transport proteins that are used by cells to move important amino acids in and out of the cell. Interestingly, most amino acid transporters are upregulated in cancer to ensure continued access to nutrients.

We have recently shown that ASCT2 plays a critical role in regulating glutamine uptake in melanoma, prostate and breast cancer. Many other glutamine transporters are upregulated in different cancers, including SLC38 family members SNAT1, SNAT2 and SNAT3. The role of SLC38A4 (SNAT4) in amino acid uptake and cancer growth, however, has not been examined. Like the other SNATs, SNAT4 is a sodium-dependent amino acid transporter that transports neutral amino acids, such as alanine, across the plasma membrane, although it has low affinity for glutamine. Interestingly, unlike the other SNAT family members, our analysis showed that SNAT4 expression is downregulated in 879/917 cancer cell lines in Oncomine. Analysis of SNAT4 mutations in patient samples using cBioPortal showed infrequent mutations in the majority of cancers. However, the TCGA melanoma data (cBioPortal) showed that 32 of 278 melanoma cases (11.5%) have a point mutation in SNAT4 suggesting an important role in melanoma. These mutations included 9 SNAT4 hotspot mutations, present in 2-4 patients each. Using a SNAT4 homology model to predict loss-of-function mutants, we selected five of these mutations to assess in a SNAT4 transport assay in Xenopus oocytes. Two of the five hotspot mutations (G31E, S76F, G78E, G150E and S371F) were able to completely inactivate SNAT4 alanine transport in oocytes. Further analysis of 52 melanoma patient samples in Oncomine showed SNAT4 downregulation in all patients, suggesting that, in contrast to most amino acid transporters, SNAT4 plays a tumor suppressor role in melanoma.

We are currently examining these mutations in melanoma cell lines to determine their effects on cell growth. We are also examining how SNAT4 is involved in either the import or export of amino acids and the downstream metabolic consequences that may directly inhibit melanoma cell growth. Through this research we will gain a better understanding of the role of SNAT4- mediated amino acid metabolism in preventing melanoma cell growth.

#1008

Leptin-induced NILCO in endometrial cancer is linked to obesity.

Danielle Daley-Brown,1 Gabriela Oprea-Ilies,2 Kiara T. Vann,1 Roland Pattillo,1 James Lillard,1 Ruben Rene Gonzalez-Perez1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

Background: Endometrial cancer (EmCa) Type I is estrogen dependent whereas Type II is estrogen independent and usually associated with endometrial atrophy. Type II is the more aggressive form with a poor prognosis. Obesity is a growing epidemic and is a major risk factor for EmCa. African-American women have higher incidence of obesity, and are more likely to die from EmCa even though the risk of developing EmCa is lower in Caucasian women. Obesity is characterized by high serum leptin levels, which are associated cancer incidence, metastasis and poor prognosis. Leptin's effects in breast cancer are related to the induction of a crosstalk between Notch, IL-1 and leptin (NILCO). It is hypothesize that there is a positive correlation between Type II EmCa, NILCO and obesity.

Methods: The expression of NILCO components was determined by immunohistochemistry in Type I vs Type II EmCa biopsies from obese African American and lean tissue array from Chinese patients. Additionally, the effects of leptin on cell proliferation and NILCO expression were determined in cell lines derived from Type I and Type II EmCa (Ishikawa, Hec1a, An3ca and KLE), respectively.

Results: NILCO components were expressed higher in Type II EmCa, more advanced disease, and correlated with obesity. Also, leptin induction of NILCO was more prominent in Type II EmCa cells.

Conclusions: Obesity signals (i.e., leptin) could induce NILCO in EmCa. Leptin could play a significant role in EmCa progression, especially in Type II EmCa. Therefore, NILCO could represent a novel biomarker for the more aggressive Type II EmCa. These observations could be more relevant for obese EmCa patients.

#1009

Metabolic reprogramming discriminates aggressive vs. slowly growing preneoplastic lesions at early stages of HCC development.

Marta A. Kowalik,1 Giulia Guzzo,2 Andrea Morandi,3 Andrea Perra,1 Silvia Menegon,4 Ionica Masgras,2 Elena Trevisan,2 Maria M. Angioni,1 Francesca Fornari,5 Luca Quagliata,6 Giovanna M. Ledda-Columbano,1 Laura Gramantieri,5 Luigi Terracciano,6 Silvia Giordano,4 Paola Chiarugi,3 Andrea Rasola,2 Amedeo Columbano1. 1 _University of Cagliari, Department of Biomedical Sciences, Cagliari, Italy;_ 2 _University of Padova, Department of Biomedical Sciences, Padova, Italy;_ 3 _University of Florence, Department of Experimental and Clinical Biomedical Sciences, Florence, Italy;_ 4 _University of Torino School of Medicine, Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy;_ 5 _St.Orsola-Malpighi University Hospital, Bologna, Italy;_ 6 _Institute of Pathology, University Hospital, Basel, Switzerland_.

Introduction and aim: Among the several changes underlying metabolic reprogramming of cancer cells, increased glucose utilization and its uncoupling from oxygen availability is a well-established phenomenon and has been recognized as a hallmark of cancer. To what extent these metabolic changes are important for the progression of slow growing tumors and whether a metabolic rewiring occurs in the very early stages of neoplastic progression represent key questions on the significance of these metabolic alterations in cancer. Here, we compared the metabolic features of preneoplastic hepatic lesions with those of advanced hepatocellular carcinomas (HCCs) and of proliferating liver, following partial hepatectomy (PH).

Materials and Methods: Expression levels, activity and modulation of several enzymes with key roles in glycolysis, pentose phosphate pathway (PPP) and oxidative phosphorylation (OXPHOS) were assessed in preneoplastic hepatic lesions and HCC, induced in rats exposed to the Resistant-Hepatocyte (R-H) model. In vitro experiments were performed on HCC cells obtained by perfusion of HCC-bearing rats. Expression of metabolic genes was also investigated in two different cohorts of human patients carrying HCC.

Results and discussion: A switch from OXPHOS to PPP was observed in very early preneoplastic lesions generated 10 weeks after the treatment with DENA. Notably, this metabolic reprogramming was observed only in the most aggressive preneoplastic lesions, characterized by positivity for cytokeratin 19 (CK-19+). PPP induction, shown by a strong increase in the expression and activity of glucose 6-phosphate dehydrogenase (G6PD) was supported both by inhibition of pyruvate kinase activity and by TP53-inducible glycolysis and apoptosis regulator (TIGAR) induction. Importantly, such metabolic rewiring was not observed in normal hepatocytes, undergoing proliferation following 2/3 partial hepatectomy (PH). Activation of the NRF2/KEAP1 pathway and down-regulation of miR-1 accompanied the metabolic reprogramming in CK-19+ preneoplastic lesions. Accordingly, NRF2 silencing decreased G6PD and increased miR-1 expression, consequently inhibiting PPP, while forced expression of miR-1 downregulated G6PD expression in HCC cells. Finally, an inverse correlation between miR-1 and its target gene G6PD was found in human HCC patients.

Conclusion: These results demonstrate that metabolic reprogramming takes place at early stages of hepatocarcinogenesis and is likely the consequence of the concomitant activation of the NRF2-KEAP1 pathway.

#1010

Evaluation of small-molecule FASN inhibitors in preclinical models of colorectal cancer.

Yekaterina Y. Zaytseva,1 Piotr G. Rychahou,1 Tianyan Gao,2 Eun Y. Lee,3 Heidi L. Weiss,1 Timothy S. Heuer,4 George Kemble,4 B. Mark Evers5. 1 _University of Kentucky Markey Cancer Center, Lexington, KY;_ 2 _University of Kentucky Markey Cancer Center and Department of Molecular and Cellular Biochemistry, Lexington, KY;_ 3 _University of Kentucky Markey Cancer Center and Department of Pathology and Laboratory Medicine, Lexington, KY;_ 4 _3-V Biosiences, Menlo Park, CA;_ 5 _University of Kentucky Markey Cancer Center and Department of Surgery, Lexington, KY_.

Fatty Acid Synthase (FASN), a key enzyme of de novo lipid synthesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN activity is associated with decreased survival and increased disease recurrence. Recently, a first-in-class, oral FASN inhibitor (TVB-2640) entered a Phase I clinical trial (3V2640-CLIN-002) in solid tumor patients demonstrating a favorable tolerability profile with no significant adverse events; however, tumor characteristics that would indicate responsiveness to FASN inhibition are not fully understood. The purpose of our study was: (i) to determine the effect of novel, selective and reversible FASN inhibitors on proliferation of primary CRC cell cultures, established CRC cell lines, and CRC patient-derived xenografts (PDXs); and (ii) to identify potential biomarkers associated with CRC responsiveness to FASN inhibition. METHODS. The effect of TVB-3166, TVB-3664, and TVB-3693 (all developed by 3-V Biosciences) on the proliferation of primary cells (established from 1st generation PDX tumors) and CRC cell lines was assessed by cell count; apoptosis was assessed by Cell Death ELISA. In addition, tumor growth was assessed in PDX models established in NOD SCID gamma mice using freshly resected CRC specimens (either primary CRC or metastasis) from our patient population. Once the xenografts grew to ~100 mm3, mice were randomized into two groups (n=5) to receive either vehicle or TVB-3664 (3mg/kg) by gavage daily. Tumor volume and animal weights were measured weekly. Western blot analysis, immunohistochemistry and immunofluorescent staining were used to identify FASN-mediated changes in β-catenin, Akt and AMPK pathways. RESULTS. TVB compounds tested showed similar efficacy in primary and established CRC cells with a wide range of sensitivity to FASN inhibition. The 5 cell lines that were most responsive to FASN inhibition demonstrated a low basal level of pAMPK and pAkt as compared to the 5 least responsive cells. Moreover, we noted that increased FASN protein expression was also associated with increased sensitivity to FASN inhibition. Inhibition of proliferation by TVB compounds was associated with decreased expression of active β-catenin, c-MYC, pAkt, and survivin, while an increase in apoptosis was noted by induction of PARP cleavage. Consistent with our in vitro studies, TVB-3664 treatment significantly reduced tumor volume in vivo with no weight changes or toxicity observed. CONCLUSIONS. Our studies show that the novel FASN inhibitors, as a single agent, significantly inhibit CRC growth both in vitro and in vivo. Importantly, our results suggest that basal activation of AMPK and Akt may be predictive of responsiveness to FASN inhibition and may function as potential biomarkers to allow a more personalized treatment approach.

#1011

Endostatin regulates androgen receptor-mediated metabolic and oxido-reductive pathways in prostate cancer cells.

Joo Hyoung Lee, Minsung Kang, James A. Mobley, Guru Sonpavde, W. Timothy Garvey, Victor M. Darley-Usmar, Selvarangan Ponnazhagan. _University of Alabama at Birmingham, Birmingham, AL_.

Endostatin (ES) has been recognized for decades as an endogenous protein with antiangiogenic function. Recent findings, however, indicate the pleiotropic effects of ES in different tissue and cell types, ensuing further investigation on unidentified molecular mechanisms of action. Previously, we have shown that ES exerts a direct role in suppressing prostate cancer cell proliferation by inhibiting androgen receptor (AR) activity, signifying therapeutic potential of ES for targeting both tumor epithelia and endothelia. Subsequent to this finding, we identified robust glucose influx and significant reduction of intracellular ROS levels in LNCaP cells upon ES treatment. The ES mutants with low AR binding did not promote glucose uptake through GLUT1 augmentation, suggesting that AR-targeted effects of ES include modulation of downstream metabolic pathways. Surprisingly, global proteome analysis showed that the levels of major metabolic enzymes either in glycolytic pathway or TCA cycle were not changed upon ES treatment. Instead, ES markedly increased the levels of G6PD (5-fold), NAMPT (2.5-fold), and NAPRT (5-fold), indicating upregulation of the late-limiting steps in NAD biosynthesis and pentose phosphate pathway (PPP). Further proteome analysis of ES-treated LNCaP cells strongly indicated upregulation of ROS scavenging machinery, including SOD2 (3- fold), CAT (2-folds), GSS (2-fold), GSR (1.8-fold), and POR (4.6-fold). Overall, these data suggest that ES can modulate intracellular ROS levels by augmenting glucose uptake, NAD biosynthesis, and the NADPH levels by shunting metabolic pathways to PPP. Given that basal ROS levels are known to increase upon disease progression to higher grade prostate tumors, ES effect of promoting ROS scavenging machinery can be employed as an adjuvant to re-sensitize prostate cancer cells to ROS-inducing chemo- and radiation therapies.

Abbreviations: CAT: catalase, GLUT1: glucose transporter isoform 1, G6PD: glucose-6-phosphate dehydrogenase, GSS: glutathione synthase, GSR: glutathione reductase, NAMPT: nicotinamide phosphoribosyltransferase, NAPRT: nicotinate phosphoribosyltransferase, POR: NADPH-cytochrome P450 reductase, ROS: reactive oxygen species, SOD2: manganese superoxide dismutase, TCA: tricarboxylic acid

#1012

Systems analysis of colon cancer cell metabolism rewired by p53 and KRAS mutations.

Manuela Salvucci,1 Robert O'Byrne,1 Natalia Niewidok,1 Séan Kilbride,1 Caoimhín G. Concannon,1 Heiko Düssmann,1 Heinrich H. Huber,2 Jochen HM Prehn1. 1 _Centre Systems Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium_.

Introduction. Somatic mutations in proto-oncogenes and tumour-suppressor genes contribute to rewire the already deregulated metabolic network in cancer cells, resulting in uncontrolled proliferation and oncogenesis. In this study, we set out to establish a dynamic mathematical model of bioenergetics and to exploit it to explore the multifaceted cross-talk between bioenergetics, somatic gene mutations in KRAS and p53, and cell proliferation and survival.

Model Development. We have developed an ordinary differential equations-based model of central carbon metabolism in cancer cells which includes glycolysis, pentose phosphate pathway, citric acid cycle and respiratory chain, based on our previous work and published models. The model describes how nutrients (glucose, glutamine, lactate, pyruvate, serine and glycine) from the extracellular micro-environment affect bioenergetics parameters. The resulting model predictions are linked to cell proliferation via a heuristic function. Enzymatic activities regulated by p53 and KRAS mutations were obtained by mining publically available datasets and their regulation by the mutational status was modelled by adapting the corresponding kinetic parameters. To estimate the kinetic parameters, model simulation outputs were fitted to a portfolio of experimental data both generated de novo in house and gathered from the literature in HCT-116 colon cancer cells. Experiments were performed on parental HCT-116 (p53 competent; harbouring a KRAS mutation on exon 2 of codon G13) and three derived mutant cell lines covering all four combinations of p53 and KRAS mutational status to isolate their relative and joint effect on bioenergetics signatures. HCT-116-derived cell lines included: p53 proficient cells with the KRAS allelic mutation silenced by homologous recombination in the presence or absence of p53 knockout by lentiviral shRNA.

Results. The model was calibrated against ATP concentrations measured via single-cell microscopy (ATeam probe and TMRM dye) following pharmacological inhibition of respiratory chain complexes (rotenone, sodium azide and oligomycin) as a function of nutrients availability (glucose, lactate, pyruvate). Modelling results revealed that p53 and KRAS mutations drive a shift in metabolic signatures and L-lactate emerged as a pivotal metabolite to stratify the different phenotypes. Systems analysis revealed that in KRAS mutated cells p53 deficiency leads to an increase in glucose uptake and flux through the pentose phosphate pathway and a decrease in lactate production. Indeed, p53 deficient HCT-116 cells showed a decrease in extracellular lactate with respect to p53 proficient cells in validation experiments.

Conclusions. The computational model developed can be used to benchmark mechanistic hypotheses by which tumour suppressors and/or oncogenic mutations rewire metabolism and to identify putative targets for therapeutic intervention.

#1013

Quantification of NADPH balance in cancer.

Ling Liu,1 Li Chen,1 Greg Ducker,1 Junyoung Park,1 Supriya Shah,2 Kathryn E. Wellen,2 Joshua D. Rabinowitz1. 1 _Princeton University, PRINCETON, NJ;_ 2 _University of Pennsylvania, Philadelphia, PA_.

NADPH is a key cofactor involved in antioxidant defense and reductive biosynthesis. The quantitative contribution of different NADPH pathways, either in normal tissues or in tumors, remains unclear. Here we enable tracing of NADPH production using deuterium-labeled nutrients and mass spectrometry, providing methods that for the first time can track each of the major cytosolic pathways: malic enzyme, isocitrate dehydrogenase, folate metabolism, and the pentose phosphate pathway. This is achieved via a battery of different 2H-tracers. A linear algebra method for deducing the fractional contribution of each pathway, even when a specific tracer for that pathway is not available, will be described. Utility of these methods will be illustrated through examples drawn from both normal cellular physiology and cancer. Specifically, we find that most NADPH in differentiating adipocytes is made by malic enzyme. Examining cancer cells, we find that the pentose phosphate pathway is typically the largest cytosolic NADPH source, but that the collective contribution of alternative pathways is often larger than the pentose phosphate pathway. The specific other pathways involved differ strongly depending on the cancer cell type. We also provide examples where NADPH production routes in both normal and cancer cells vary with nutrient availability, including hypoxia. Given the heightened redox stress of cancer cells, understanding NADPH production routes is likely to illuminate new avenues for therapeutic intervention.

#1014

The anti-Warburg agent BPM 31510 arbitrates fatty acid metabolism in eliciting an anticancer response.

Bianca Jambhekar, Tulin Dadali, Anne R. Diers, Fei Gao, Hannah Rockwell, Emily Chen, Stephane Gesta, Vivek K. Vishnudas, Michael A. Kiebish, Niven R. Narain, Rangaprasad Sarangarajan. _Berg, LLC, Framingham, MA_.

De-regulated lipid homeostasis is a key feature of the altered metabolic phenotype observed in cancer, particularly in the adipose-rich microenvironment of breast tumors. Mitochondria serve as a central hub for lipid metabolism through mitochondrial β-oxidation pathways and biosynthesis through production of citric acid cycle-derived citrate and the activity of ATP citrate lyase, the rate-limiting enzyme in lipid biosynthesis. We have previously demonstrated BPM 31510 ability to effectuate Warburg switch from glycolysis to mitochondrial oxidative phosphorylation resulting in activation of apoptosis in multiple cancers including triple negative breast cancer. The present investigated the role of BPM 31510 in influencing lipid metabolism in cancer cells to arbitrate its anti-cancer effect. Cell viability was assessed in MDA-MB231 and SkBr-3 breast cancer cells exposed to BPM 31510 alone or in combination with fatty metabolism inhibitors including C75, etomoxir, and trimetazidine, inhibitors of fatty acid synthase (FASN), carnitine palmitoyltransferase 1 (CPT-1), and β-oxidation, respectively, as well as a more pleiotropic fatty acid metabolism modulator, metformin. Breast cancer cells were more sensitive to BPM 31510 when treated in combination with C75, etomoxir, and trimetazidine as indicated by a left-shift in the BPM 31510 dose-response curve in both MDA-MB231 and SkBr-3 cells. In contrast, combined treatment with metformin did not alter cytotoxic responses to BPM 31510, indicating specificity for responses in fatty acid metabolism pathway modulation. Interestingly, BPM31510 treatment was associated with a dose- and time-dependent increase in mRNA expression of fatty acid metabolism gene products (FASN, CPT1, ACSL1) and accumulation of the triglyceride backbone, glycerol, in MDA-MB-231 cells. Structural lipidomic analysis used to assess the metabolic fate of BPM 31510 liposomal formulation components demonstrated that these were readily incorporated into prevalent diacyl-glyerol (DAG) and triacyl-glycerol (TAG) species along with de novo fatty acid species such as palmitate. Together, these results demonstrate that BPM 31510 alters endogenous and exogenous lipid homeostasis in breast cancer cells and rationale for potential combination with fatty acid metabolism inhibitors for anti-cancer therapy. The results expands on BPM 31510 anti-cancer mechanism, suggesting a central role in arbitrating the convergence of glycolysis, glucose and fatty acid oxidation pathways within the cancer cell metabolism network.

#1015

Repurposing the FDA-approved drug carbidopa to treat human cancers.

Vadivel Ganapathy, Ellappan Babu, Sabarish Ramachandran, Yangzom D. Bhutia. _Texas Tech University Health Science Center, Lubbock, TX_.

Carbidopa is used in combination with L-DOPA to treat Parkinson's disease; it does not have any therapeutic use by itself in Parkinson's disease, but when used along with L-DOPA prevents the conversion of the latter into dopamine in the periphery by inhibiting aromatic amino acid decarboxylase. Carbidopa however does not cross the blood-brain barrier; thus does not impact on the conversion of L-DOPA into dopamine in the brain. We hypothesized that carbidopa might have potential as an anticancer drug with the following rationale: (a) carbidopa is an amino acid derivative and therefore might block the entry of amino acids into cancer cells via certain amino acid transporters; (b) the stress hormones epinephrine and norepinephrine are known to promote cancer progression, and carbidopa as an inhibitor of aromatic amino acid decarboxylase might interfere with the generation of these tumor-promoting hormones; (c) carbidopa is also an analog of phenylhydrazine, which is an inhibitor of the immunosuppressive enzyme indoleamine-2,3-dioxygenase, a drug target for cancer treatment; carbidopa might inhibit this enzyme and thus enhance the ability of the immune system to recognize cancer cells as foreign and fight against them. Based on this rationale, we examined the efficacy of carbidopa to treat pancreatic and breast cancer. We found carbidopa to be effective in blocking the proliferation of pancreatic cancer cell in vitro. We then examined its efficacy in vivo using xenografts of pancreatic cancer cells in nude mice; again, the drug was effective in decreasing the growth of the xenografted tumor cells into tumors. We also tested its efficacy on proliferation of breast cancer cell lines; we used ZR-75.1, MB-231, and HCC-1937 breast cancer cell lines as models for ER-positive, ER-negative and BRCA-1 mutant breast cancers, respectively. Carbidopa decreased the proliferation of all three cell lines. We then examined its in vivo efficacy against breast cancer using the MMTV-PyMT-transgenic mouse as a model of spontaneous breast cancer. In this model, breast cancer develops initially as an ER-positive subtype but then turns into an ER-negative subtype. Carbidopa markedly decreased the growth of breast cancer in this mouse model. Based on these in vitro and in vivo data, we conclude that carbidopa has promise for use as an anticancer drug. As the drug potentially elicits its anticancer effects by targeting multiple pathways, the anticancer efficacy of the drug is likely to be broad against different types of human cancers. For in vivo studies, we used the drug intraperitoneally at a dose of 1 mg/mouse that approximately translates to a human dose of 300-400 mg/day. As the drug has been shown to have no detectable side effects in humans at doses as high as 400 mg/day, it can be taken to clinical trials readily to test its efficacy in humans as an anticancer drug.

#1016

Variants in autophagy related genes and clinical characteristics in melanoma: a population-based study.

Kirsten A. m. White,1 Li Luo,1 Todd A. Thompson,1 Salina Torres,1 Chien-An A. Hu,1 Nancy E. Thomas,2 Hoda Anton-Culver,3 Stephen B. Gruber,4 Lynn From,5 Klaus J. Busam,6 Irene Orlow,6 Peter A. Kanetsky,7 Lorraine D. Marrett,5 Richard P. Gallagher,8 Roberto Zanetti,9 Stefano Rosso,9 Terry Dwyer,10 Anne E. Cust,11 Allison Venn,12 Colin B. Begg,6 Marianne Berwick,1 Jenna Lillyquist13. 1 _University of New Mexico - Albuquerque, Albuquerque, NM;_ 2 _University of North Carolina, Chapel Hill, NC;_ 3 _University of California, Irvine, Irvine, CA;_ 4 _USC Norris Comprehensive Cancer Center, Los Angeles, CA;_ 5 _Cancer Care Ontario, Toronto, Ontario, Canada;_ 6 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 7 _Moffitt Cancer Center, Tampa, FL;_ 8 _BC Cancer Research Centre, Vancouver, British Columbia, Canada;_ 9 _Piedmont Cancer Registry, Torino, Italy;_ 10 _The George Institute for Global Health, Oxford, United Kingdom;_ 11 _The University of Sydney, Sydney, Australia;_ 12 _University of Tasmania, Hobart, Australia;_ 13 _Mayo Clinic, Rochester, NY_.

Autophagy has been linked with melanoma, but no polymorphisms in autophagy related (ATG) genes have been investigated for association with melanoma prognostic indicators and survival. We examined 5 ATG gene single nucleotide polymorphisms (SNPs) in a large international multicenter population-based case-control study of melanoma. DNA from 911 melanoma patients was genotyped for five SNPs with a known or suspected impact on autophagic flux. While we did not identify an association with survival, a significant association was identified between the minor allele for an ATG16L polymorphism (rs2241880) and a decrease in Breslow thickness (p = 0.03), earlier tumor stage at diagnosis (OR 0.47, 95% CI 0.27-0.81, p = 0.02) and younger age at diagnosis (p = 0.02). In addition, two SNPs in ATG5 (rs2245214 and rs510432) were found to be significantly associated with increased tumor stage of melanoma (OR 1.84 95% CI 1.12-3.02, p=0.05; OR 1.47 95% CI 1.11-1.94, p=0.03). Finally, we identified inverse associations between the minor allele of rs2245214 and melanomas on the scalp or neck (OR 0.20, 95% CI 0.05-0.86, p= 0.03); rs1864182 (OR 0.42, 95% CI 0.21-0.88, p= 0.02) and brisk TILs, and rs510432 (OR 0.55 95% CI 0.34-0.87, p= 0.01) with non-brisk TILs, although they were not globally significant. In summary, our data suggests that ATG SNPs, while not associated with survival, may be associated with Breslow thickness, tumor stage, age at diagnosis, and aggressive histopathological factors. These associations could contribute to our current understanding of the significant role of autophagy in melanoma progression.

#1017

Pro-inflammatory cytokine secretion and gene networks associate with pancreatic cancer induced cachexia.

Daniel Delitto, Sarah M. Judge, Rachel L. Nosacka, Andrea Knowlton, Fernanda G. Rocha, Kevin E. Behrns, Steven J. Hughes, Shannon M. Wallet, Andrew R. Judge, Jose G. Treviño. _University of Florida, Gainesville, FL_.

Introduction: Pancreatic cancer (PC) is associated with a high rate of cachexia, which specifically diminishes quality of life, prohibits effective therapies, and subsequently contributes to morbidity and mortality. Unfortunately, mechanisms underlying this cancer-induced muscle wasting in the human disease remain incompletely described, in part due to limited translational models. Therefore, we hypothesize that the development of more representative models of PC cachexia will allow for development of therapeutic targets for cachexia. To test our hypothesis, we propose to 1) establish the first patient-derived xenograft (PDX) cancer cachexia models to identify the muscle associated with PC induced cachexia and 2) support these results by examining the corresponding skeletal muscle of PC patients whom contributed to the PDX models. Methods: Rectus abdominis muscle was biopsied from surgically resected PC patients and matched non-cancer controls. After surgical harvest of PC specimen, PDX models were derived in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and skeletal muscle was subsequently harvested for histologic investigations on ultrastructural disorganization and qRT-PCR for atrophy-related transcription factors differentially regulated in PC patients. Systemic cytokine expression profiles were analyzed with luminex technology and confirmed by ELISA. Results: Rectus biopsies from patients with resected PC displayed marked muscle fiber atrophy, increased extracellular space, greater variation in fiber size and shape and more centralized nuclei compared to controls. These architectural abnormalities were also present in mice bearing xenografts from corresponding PC patients. Despite the absence of an adaptive immune system, PDX mice demonstrated high levels of systemic TNFα, IL-1β, IL6 and KC (IL8) with a concomitant decrease in anti-inflammatory cytokine IL10 when compared to matched controls. Further, skeletal muscle from both patients with PC and mice bearing PDX tumors demonstrated increased expression of the Forkhead boxO1 (FoxO1) transcription factor and FoxO target gene and E3 ubiquitin ligase, MuRF1, both of which have been directly implicated in the regulation of muscle mass. Conclusions: Preoperative muscle wasting in PC is associated with characteristic architectural abnormalities and elevated FoxO1-MuRF1 levels. Mice bearing PDX demonstrate comparable elevations in circulating pro-inflammatory cytokines, muscle pathology and FoxO1-MuRF1 levels. These results provide a valid translational model of cachexia which suggests a central role for FoxO1 and MuRF1 in PC-associated muscle wasting.

#1018

Structural features of novel dimeric quinacrines that have single-agent antitumor activity determine the mechanism of action: destabilization of mTORC1/lysosomal interaction versus DNA damage.

Vito W. Rebecca,1 Michael Nicastri,1 Noel McGlaughlin,1 Quentin McAfee,1 Gao Zhang,2 Gretchen M. Alicea,2 Shengfu Piao,1 Colin Fennelly,1 Sengottuvelan Murugan,1 Zhi Wei,3 Gordon B. Mills,4 Yiling Lu,4 Meenhard Herlyn,2 Jeffrey D. Winkler,1 Ravi K. Amaravadi1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Wistar Institute, Philadelphia, PA;_ 3 _New Jersey Institute of Technology, Newark, NJ;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

The safety and preliminary activity of hydroxychloroquine in phase I cancer clinical trials have established the feasibility and rationale for targeting the lysosome in cancer. We previously reported a more potent lysosomal inhibitor Lys05, which is a dimeric chloroquine (CQ) linked with a triamine linker. Here we report that high throughput screening of >100 Lys05 derivatives (72-hour viability assay) revealed extending linker length between the CQ motifs markedly enhanced anti-proliferative potency (IC50 <500 nM v. 5 μM Lys05). Longer linker dimeric CQs produced significantly greater autophagy inhibition (2-5 fold improved v. Lys05; mCherry-eGFP-LC3 reporter) and apoptosis (2-8 fold improved v. Lys05) in human pancreatic cancer, melanoma and in KRAS mutant P53-/- mouse pancreatic cells. We then substituted the CQ heterocycle of Lys05 with other antimalarial heterocycles including those found in mefloquine, primaquine and quinacrine. Dimeric quinacrines (DQ's) were exquisitely cytotoxic to cancer cell lines (IC50 9 - 90nM; ≥40 fold improvement v. quinacrine). These agents were renamed DQ221-661, where the first and second digit of each compound indicates the number of carbons flanking the central nitrogen and the third digit reflects whether the central nitrogen in the dimeric molecule is methylated (1) or not (0). Inherent fluorescence of the DQs uncovered a striking pattern of subcellular localization dependent on central nitrogen methylation. All of the methylated DQs localized to the lysosome and inhibited autophagic flux (bafilomycin clamp assay), while all of the unmethylated DQ's localized to the nucleus, produced pH2AX-positive DNA damage, and induced autophagy. Methylated DQs produced lysosomal membrane permeabilization (LMP; galectin-3 puncta), and equal cytotoxicity in ATG5 WT and ATG5-null MEFs, indicating their cytotoxicity is not dependent on functional upstream canonical autophagy. Reverse phase protein array analysis of dimeric CQs Lys05, Lys75 and DQ661 revealed a signature associated with inactivation of mTORC1 (decreased phosphorylation of S6K, 4E-BP1, PRAS40). DQ661 disrupted mTOR/LAMP2 co-localization and induced greater levels of apoptosis compared to BRAF/MEK inhibition in BRAF-MT melanoma cells, gemcitabine in pancreatic cancer cells, or early stage autophagy inhibitors Spautin-1 and SBI-0206965. Unlike quinacrine, which had no effect, or DQ660, which produced a modest but significant growth impairment, DQ661 produced significant tumor regression in a melanoma xenograft model, establishing the therapeutic potential of this compound in cancer. Our data identifies a new class of lysosomal inhibitors, the centrally methylated dimeric quinacrines, devoid of DNA damaging properties, that are capable of concurrently inhibiting autophagy-lysosome function and mTORC1 through LMP.

#1019

Investigation of cabozantinib, a MET and VEGFR2 inhibitor, on tumor metabolism and efficacy in a colorectal cancer patient-derived tumor explant model.

Aaron J. Scott, Rachel Yahn, Stacey Bagby, Kendra Huber, Natalie Serkova, Wells Messersmith, John Arcaroli. _University of Colorado, Denver, CO_.

Background:

Anti-angiogenic therapy is commonly used for the treatment of metastatic colorectal cancer (mCRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. Upregulation of MET in response to anti-VEGF therapy may play an essential role in treatment resistance. Based on this premise, we investigated cabozantinib, an inhibitor of kinases including MET and VEGFR2, in mCRC patient-derived tumor explant (PDTX) models. Based on initial observations of anti-tumor activity, we hypothesized that cabozantinib may alter tumor metabolism concurrent with its antitumor effects, and compared its efficacy with regorafenib in our models.

Material and Methods:

Ten CRC PDTX models were treated with cabozantinib (30 mg/kg daily) or regorafenib (10 mg/kg daily) and treatment responses were determined after 28 days. The tumor growth inhibition index (TGII) was calculated to compare treatment effects on tumor growth between cabozantinib and regorafenib. RNA Seq was used to assess gene expression and pathways modulated by cabozantinib treatment. Proteins involved in metabolism and autophagy were evaluated by immunoblotting at day 3, 7 and 28. The effects of cabozantinib on tumor glucose metabolism were investigated by 18FDG-PET at baseline, 7 and 28 days following treatment. Lastly, combination effects of cabozantinib and an ULK1 inhibitor, an autophagy inhibitor, were evaluated on the HCT116 cell line by a clonogenic assay.

Results:

Cabozantinib (average TGII: 3.202) demonstrated antitumor effects among all 10 CRC explants that compared favorably to regorafenib (average TGII: 48.48). In addition, cabozantinib significantly reduced glucose uptake measured by 18FDG-PET at days 7 and 28. A comprehensive pathway analysis using RNA Seq post-cabozantinib treatment revealed a significant reduction in pyruvate metabolism and the TCA cycle in the most sensitive tumors. Protein analyses showed downregulation of hexokinase 1 and pyruvate dehydrogenase and a marked increase in many components of oxidative phosphorylation and autophagy (LC3 and Beclin) at day 7 and 28 following cabozantinib treatment. The combination of an ULK1 inhibitor and cabozantinib enhanced the activity of cabozantinib in the HCT116 CRC cell line.

Conclusions:

Cabozantinib showed significant antitumor activity compared to regorafenib in our CRC PDTX models. Alterations in glucose metabolism and autophagy were identified in cabozantinib sensitive tumors, suggesting that a shift in cellular metabolism facilitates the survival of tumor cells following treatment. The combination effect of cabozantinib and an ULK1 inhibitor supports the hypothesis that induction of autophagy may be a mechanism of cabozantinib resistance. These findings support further evaluation of cabozantinib in patients with mCRC.

Acknowledgements: Exelixis for providing cabozantinib.

#1020

Autophagy forms part of a metabolic switch during epithelial-to-mesenchymal transition and metastasis in a murine claudin-low breast cancer model.

Ciara H. O'Flanagan, Emily L. Rossi, Stephen D. Hursting. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Epithelial-to-mesenchymal transition (EMT), the process through which epithelial cells gain mesenchymal characteristics including reduced adhesion and enhanced invasion, is a critical step in progression of cancer cells toward metastasis. However, the molecular mechanisms governing this process remain poorly understood. Furthermore, though metastasis is the cause of 90% of cancer deaths, no targeted therapy is currently available for metastatic disease. Metabolic reprogramming is a key feature of most cancer cells, with oxidative phosphorylation often being switched off in favor of glycolytic and synthetic pathways. Autophagy is a catabolic process in which protein complexes, damaged organelles and other macromolecules are lysosomally degraded. Autophagy can promote cancer cell survival, providing protein, lipid, nucleic acid and membrane precursors as well as auxiliary energy during periods of energy stress. The role of metabolism in EMT and metastasis has not been well studied. We hypothesized that the transition to metastasis involves metabolic alterations, including activation of autophagy.

Previously, we derived (from a spontaneous mammary tumor in an MMTV-Wnt transgenic mouse) epithelial-like (E-Wnt) and mesenchymal-like (M-Wnt) cell lines, which recapitulate basal-like and claudin-low and breast tumors, respectively. Here, a metastatic derivative of M-Wnt cells was generated from lung metastases following serial passages in a severe combined immunodeficient (SCID) mouse. metM-Wnt cells were more proliferative, invasive and formed more colonies in soft agar than their parental M-Wnt cell line. Tail vein injection or mammary fat pad orthotopic implantation of metM-Wnt cells resulted in metastatic tumor formation in either the lungs or liver as detected by IVIS imaging and histological analysis. metM-Wnt cells displayed higher rates of glycolysis and oxidative phosphorylation, indicating that these cells are highly energetic. Furthermore, M-Wnt and metM-Wnt cells and tumors were found to have increased autophagic flux, as measured by LC3B expression and cleavage compared to E-Wnt cells and tumors. metM-Wnt cells were more sensitive to autophagy inhibition (either via chloroquine treatment or knockout of Atg5, a key component of the autophagic machinery) than M-Wnt or E-Wnt cells. Conversely, metM-Wnt cells were resistant to treatment with rapamycin, concomitant with sustained activation of mTOR, which controls many metabolic pathways including autophagy and protein synthesis. These results indicate that metabolic alteration is a feature of EMT in claudin-low breast cancer, including increased glycolysis, oxidative phosphorylation and autophagy. Furthermore, metastatic breast cancer cells may be more reliant on autophagy than non-metastatic cells, and autophagy may therefore be a therapeutic target in which to treat metastatic breast cancers.

#1021

Lipidomic analysis: a powerful tool for evaluating lipid metabolism in cultured cancer cells.

Finnur F. Eiríksson,1 Manuela Magnusdottir,1 Skarphedinn Halldorsson,1 Margret Thorsteinsdottir,2 Helga M. Ögmundsdottir1. 1 _Univ. of Iceland Faculty of Medicine, Reykjavik, Iceland;_ 2 _Univ. of Iceland Faculty of Pharmaceutical Sciences, Reykjavik, Iceland_.

Lipids have numerous functions in biological processes, structural as well as regulatory. Cancer cells show differences from healthy normal cells in their metabolism, including lipid metabolism, which contribute to their survival and growth. Overexpression of fatty acid synthase (FASN) is commonly observed in cancer cells and there is also evidence of other lipid pathways being changed in tumor cells. The general aim of this project is to establish a lipidomic based UPLC-QToF method for evaluation of lipid composition in cultured cells of normal and cancerous origin using electrospray quadrupole traveling wave ion mobility Q-ToF mass spectrometry. Cell lines were selected based on their lipid metabolism, including three breast cancer cell lines: SK-Br-3 (overexpresses FASN), T47D (TP53-mutated), MCF7 (estrogen receptor-positive); and one pancreatic cancer cell line, ASPC-1(overexpresses 5-and 12-lipoxygenases). Furthermore, we analyzed the immortalized breast epithelial stem cell line D492 along with its subline, D492M, that has undergone epithelial to mesenchymal transition (EMT). Principal component analysis (PCA) revealed distinct differences in lipid composition between all the cell lines tested, each cell line showed a clear cluster of lipid components and patterns that were reproducible between experiments. The Sk-Br-3 was most clearly separated from the other two breast cancer cell lines. Further analysis revealed significant differences in levels of phosphocholins (PC) that are elevated in Sk-Br-3compared to other cell lines. Further investigations of the data have led to identification of certain PC that are different between cell lines. Significant differences were detected in the lipid composition of D492 and D492M. In conclusion, different levels of lipid-synthesizing enzymes are reflected in distinct lipid profiles in breast cancer cells. The observed differences in lipid profiles of the breast epithelial cell line and its mesenchymal-like subline may provide an insight into membrane changes associated with EMT and help evaluate lipidomes of tumor samples from patients in terms of invasive potential.

#1022

The ketone body β-hydroxybutyrate down regulates c-Myc signaling in a malignant glioma model.

Alex P. Rossi, Helena B. Silva-Nichols, Eric C. Woolf, Adrienne C. Scheck. _Barrow Neurological Institute, Phoenix, AZ_.

Glioblastoma (GB) is the most common primary malignant brain tumor with a dismal median survival of 1.5 years. To address the need for novel therapeutics for the treatment of GB, investigations into high fat, low carbohydrate ketogenic diets (KD) have been explored as potential adjuvant therapeutic modalities. Preclinical studies of the KD in a mouse model of malignant glioma demonstrated that the KD extended median survival when used alone and substantially potentiated both radiotherapy and chemotherapy. However, the underlying mechanisms by which the KD exerts its therapeutic benefits remain poorly understood. A key result of the KD is the production of the ketone bodies β-hydroxybutyrate (ΒHB), acetoacetate, and acetone. Of these, BHB is the most prevalent ketone body produced. In order to elucidate the underlying molecular mechanisms of the KD, we have examined the interactions between BHB and the mouse glioma cell line GL261-Luc2. Previously, we demonstrated the ability of ΒHB to recapitulate the in vivo effects of the KD in tumor cells in an in vitro model. In the present study, we demonstrate that the ketone ΒHB is able to significantly down regulate the master transcription factor c-Myc, which is intimately involved in cell cycle progression, apoptosis, proliferation, DNA damage and repair, and metabolism. Overexpression of c-Myc is tied to a variety of human malignancies. In GB, upregulation of c-Myc signaling has been connected to an increase in tumor glucose metabolism. Furthermore, downregulating c-Myc expression has been demonstrated to confer sensitivity to tumors. We show that doses of both 5mM and 10mM ΒHB significantly reduced the expression of c-Myc in the GL261-Luc2 cell line when evaluated by both immunocytochemistry and western blot. Downregulation of c-Myc may, in part, be a mechanism by which ΒHB and thus the KD exert their radiosensitizing and antiproliferative effects on tumors. We intend to further investigate the role of c-Myc in the KD by investigating downstream targets of c-Myc as well as potential regulators of c-Myc transcription that are affected by BHB.

#1023

Functional genomics reveals dependency on 6-phosphogluconate dehydrogenase in OXPHOS-deficient tumors.

Yuting Sun, Madhavi Bandi, Timothy Lofton, Melinda Smith, Christopher Bristow, Norma Rogers, Chang Edward, Mary Geck Do, Yongying Jiang, Pietro Morlacchi, Florian Muller, Faika Mseeh, Barbara Czako, Wylie Palmer, Carlo Toniatti, Philip Jones, Giulio Draetta, Timothy Heffernan, Joseph Marszalek. _UT MD Anderson Cancer Center, Houston, TX_.

Cancer cells display metabolic properties distinct from normal cells and are therefore believed to be dependent on key metabolic enzymes. The effectiveness of targeting metabolism for cancer therapy has been largely restricted due to metabolic adaptability. We postulate that tumors with metabolic vulnerabilities will be limited in such adaptation, thus providing a unique opportunity for therapeutic intervention. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is a metabolically vulnerable cancer, due to mutations in a key TCA cycle enzyme - fumarate hydratase (FH), thus rendering them deficient in oxidative phosphorylation (OXPHOS). We performed a loss-of-function genetic screen in a FH-null HLRCC model (UOK262 cells) and identified 6-phosphogluconate dehydrogenase (6PGD) - a rate-limiting enzyme in the non-oxidative arm of pentose phosphate pathway - as a critical regulator of cell growth. Inhibition of 6PGD using either dox-inducible shRNA or a small molecule inhibitor dramatically blocks the growth of UOK262 cells, and in contrast, has no effect on 6PGD-deficient NB1 cells. The growth inhibition from knockdown is fully rescued by expressing shRNA-resistant 6PGD. Conversely, ectopic expression of 6PGD in NB1 cells results in a gain of function phenotype. Importantly, in vivo, 6PGD knockdown causes robust regression of established UOK262 xenografts. Mechanistically, 6PGD inhibition causes the accumulation of 6-phosphogluconate, blocks glycolysis, and induces ROS production. Furthermore, in tumors with intact TCA cycle, 6PGD inhibition shows synergism with OXPHOS Complex I inhibitor IACS-10759. Together, our data suggest that 6PGD is a bona fide oncology target and provide a strong rationale for developing small molecule inhibitors that could be used to treat OXPHOS-deficient tumors as single agent, or combined with inhibitors of OXPHOS.

#1024

Expression pattern of ALDH1A1 and HLTF predicts sensitivity to lysosomal autophagy inhibitors in cancer cells.

Shengfu Piao, Arabinda Samanta, Xiaohong Ma, Quentin W. Mcafee, Vito W. Rebecca, Meghan Buckley, Eric J. Brown, Phyllis A. Gimotty, Ravi K. Amaravadi. _University of Pennsylvania, Philadelphia, PA_.

Autophagy inhibition with hydroxychloroquine (HCQ)is cytotoxic to only a subset of cancer cell lines. Clinical trials involving HCQ in cancer patients are underway, but predictive biomarkers to identify patients most likely to respond are unavailable. To identify determinants of sensitivity to HCQ, an mRNA microarray analysis of HCQ-sensitive(S) compared to HCQ- resistant (R) colon and lung cancer cell lines identified differentially expressed genes in HCQ-S cells. Immunoblotting against the 2 most upregulated (Lysozyme (LYZ); aldehyde dehydrogenase 1 (ALDH1A1)) and 2 most down regulated proteins (helicase like transcription factor (HLTF); p-glycoprotein (ABCB1)) was performed in 35 human cancer cell lines. The IC50 and slope of the HCQ dose response curve (72 h MTT) were used to create a composite score to categorize cell lines as -S or -R, and correlated to protein expression of the 4 genes. Classification and regression tree (CART) analysis determined that high expression of the stem cell marker ALDH1A1 or low expression of both ALDH1A1 and HLTF identified 100% of sensitive cell lines. To understand how ALDH1A1 mechanistically interacts with HCQ, the aldefluor assay determined that a more potent dimeric CQ (Lys21) did not impair ALDH1A1 enzymatic function, however knockdown or chemical inhibition of ALDH1A1 impaired cellular uptake of fluorescently tagged Lys21 and conferred resistance to HCQ. Overexpression of ALDH1A1 conferred HCQ sensitivity in HCQ-R cells. To understand the interaction between HLTF and HCQ, we determined that HLTF promoter methylation correlated with silenced expression and sensitivity to HCQ. Forced expression of HLTF or treatment with a demethylating agent conferred resistance to HCQ-S cells. Knockdown of HLTF in HCQ-R cells conferred sensitivity. HCQ produced reactive oxygen species (ROS) irrespective of HLTF status. Cotreatment with a ROS scavenger mitigated HCQ cytotoxicity in HLTF silenced cells. DNA damage (p-H2AX) was observed at 100-fold lower HCQ concentrations in HLTF silenced compared to HLTF expressed cells. Overexpression of HLTF significantly reduced HCQ associated double strand breaks (Rad52 foci). Knockdown of DNA polymerase eta, a low fidelity DNA polymerase involved in translesion synthesis (TLS) completely abrogated HLTF-associated HCQ resistance. In vivo expression of HLTF mitigated the antitumor activity of the dimeric CQ Lys05 in a HCQ-S colon cancer xenograft. These results indicate ALDH1A1 acts as a sink for CQ derivatives to enter the cell, localize to the lysosome, and produce ROS-mediated DNA damage. The DNA damage can be counteracted by intact HLTF/Pol eta-dependent TLS. Analysis of the TCGA found that the ALDH1A1 high and HLTF low phenotype is prevalent across 10 human cancers. In conclusion this study identifies a 2 gene signature that can be translated into an IHC-based assay to identify patients most likely to respond to lysosomal autophagy inhibitors.

#1025

A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces cell autophagy and apoptosis in esophageal squamous cell carcinoma cells.

Wei Li,1 Guixue Hou,2 Dianrong Zhou,3 Xiaomin Lou,2 Yang Xu,4 Siqi Liu,2 Xiaohang Zhao4. 1 _Cancer Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China; _2 _CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China;_ 3 _Graduate School of Southern Medical University, Guangzhou, China;_ 4 _Cancer Hospital, Chinese Academy of Medical Sciences, Beijing, China_.

The protocatechuic acid ethyl ester ethyl-3,4-dihydroxybenzoate (EDHB) is an antioxidant found in the testa of peanut seeds. The cytotoxic effect of EDHB on esophageal squamous cell carcinoma (ESCC) cells was investigated and 46 up- and down-regulated genes involved in several signaling pathways associated with cell cycle regulation and cellular metabolism were identified . Our studies have shown that EDHB can effectively induce autophagy and apoptosis of esophageal cancer cells by BNIP3 and NDRG1 respectively, and AKR1C family were one of the differentially expressed genes after cells treated with EDHB. We also found that the esophageal cancer cells with higher (KYSE 180) and lower (KYSE 510) AKR1C expressing levels were selected, and the cell proliferation was found inhibited more effectively in KYSE 180 than KYSE 510 cells after treated with EDHB. Furthermore, at the same condition, the effective subunits of AKR1C superfamily, AKR1C1/C2, were identified quantitatively by multiple reaction monitoring (MRM) assays. And the involvement of AKR1C1/C2 in the metabolism of the EDHB in ESCC cells and induced cell death. When autophagy was inhibited using 3-methyladenine (3-MA), KYSE 180 cells exhibited higher sensitivity to EDHB and it may act as metabolic substrate of AKR1C1/C2. These results indicate that esophageal cancer patients with high expression of AKR1C1/C2 are more sensitive to EDHB and facilitate its induced autophagy and apoptosis that providing potential guidance for the chemoprevention of ESCC.

#1026

PEG-LPrA2 is a non-toxic adjuvant for triple negative breast cancer.

Courtney D. Dill, Adriana Harbuzariu, Antonio Rampoldi, Crystal C. Lipsey, Viola Lanier, Tia Harmon, Danielle Daley-Brown, Cynthia Tchio, Pierre Candelaria, Ruben Rene Gonzalez-Perez. _Morehouse School of Medicine, Atlanta, GA_.

Background: Triple Negative Breast Cancer (TNBC) is an aggressive cancer with poor prognosis and is difficult to treat. Current standard therapy for the disease includes a combination of chemotherapeutic drugs: doxorubicin (DOX), paclitaxel (TAX), and cyclophosphamide (CTX). These drugs are ineffective as they exhibit shortcomings and several side effects. TNBC patients develop chemoresistance that may be enhanced by leptin, which affects survival, proliferation, and angiogenesis. Our lab developed and tested a novel and specific inhibitor of leptin signaling, LPrA2. A pegylated derivative of LPrA2 (PEG-LPrA2), with enhanced bioavailability, was successfully used in mouse breast cancer models. Preliminary data showed that PEG-LPrA2 was non-toxic in vitro. Therefore, it is hypothesized that PEG-LPrA2 is not toxic in vivo.

Methods: To determine potential toxicity of PEG-LPrA2, in vitro and in vivo assays were performed. In vitro toxicity of PEG-LPrA2 was tested in a human non-malignant mammary epithelial cell line (MCF-10A). MCF-10A cells were cultured in 96 well plates (5,000 cells/ well) and grown to 70-80% confluence. Cells were treated with PEG-LPrA2 for 24 hrs and viability was determined via MTT assay. In vivo toxicity studies were performed in obese mice. Fifty-seven, eight week old C57BL/6J mice (Charles River Laboratories) were divided into 6 groups. Control mice were fed a low fat diet (10% Kcal from fat) and the rest of the mice were fed a high fat diet (60% Kcal from fat) for 11 weeks. Obesity was characterized as body weight (BW) ≥ 25% BW of control mice. Obese mice were divided into six groups (n = 7/each). Mice were injected with 50 µL of either Sc-PEG-LPrA2 (scramble control) or active inhibitor, PEG-LPrA2, (0.1 mM, 1 mM, or 5 mM) two times a week, for eight weeks. Blood chemistries were analyzed. Additionally, heart, liver, and kidney tissue were harvested and examined to determine toxicity. The tissues were probed for OB-R via immuno histochemistry.

Results: The results showed no changes in BW or food intake. Additionally, no evident changes in blood parameters and organ histology were found.

Conclusions: PEG-LPrA2 is non-toxic and could serve as an adjuvant therapy for standard TNBC chemotherapeutics.

#1027

Autophagy as a mediator of increased mammary cancer risk in socially isolated mice.

Leena A. Hilakivi-Clarke, Fabia O. Andrade, Allison Sumis, Kerrie B. Bouker, Rong Hu, Robert Clarke, Katherine L. Cook. _Georgetown Lombardi Comp. Cancer Center, Washington, DC_.

Social isolation in mice is a model of post-traumatic stress disorder (PTSD) which affects 12-20% of newly diagnosed breast cancer patients. Since social isolation increases the risk of breast and other cancers and increases overall and cancer-specific mortality, it is critical to determine the biological pathways that mediate the effects of social isolation on cancer to identify targets to prevent its adverse effects. Among the potential mechanisms is autophagy, which we have found previously to be induced in the mammary glands of socially isolated mice. Others have reported upregulation of p53 and Hk2 in socially isolated animals. Both are genes that induce autophagy. In our study, social isolation increased nuclear p53 levels, expression of the p53-regulated autophagy genes Dram1 and Mdm2 in the mammary glands of mice. Further, social isolation led to an increase in body weight, particularly when mice were fed a high fat diet, but the ability of social isolation to induce autophagy was independent of high fat diet. Here we investigated the effect of social isolation on mammary tumorigenesis in heterozygous Atg7 knockout mice. Social isolation was implemented by housing mice singly and feeding them a high fat diet from weaning onwards. Socially isolated wildtype C57BL6 mice exhibited upregulation of Atg7 and LC3II and downregulation of p62, indicative of increased autophagy that allows cancer cells to survive. Atg7+/- mice, in contrast, exhibited significantly lower LC3II and higher p62 levels compared with group-housed wildtype mice. We also found that socially isolated wildtype mice were at a significantly increased risk of developing estrogen receptor positive (ER+) mammary cancer induced by treating mice with 15 mg medroxyprogesterone acetate (MPA) and three doses of 1 mg 7,12-dimethylbetz(a)anthracene (DMBA) (given on weeks 7, 8 and 9). No increase in mammary tumorigenesis was seen in socially isolated Atg7+/- mice, compared with group-housed Atg7+/- mice. Our results confirm that social isolation induces autophagy and show that increased autophagy in socially isolated mice was causally linked to increased breast cancer risk. Since activation of survival autophagy is known to impair response to cancer treatments, including to chemotherapy and endocrine therapy, it is imperative to determine if cancer patients suffering from PTSD exhibit increased autophagy. Autophagy inhibitors, such as chloroquine, are currently being investigated in the clinic as a 2nd line therapy for metastatic cancer, and therefore available also to be used in cancer patients experiencing PTSD-like symptoms.

#1028

A mouse model of allelic loss of the core autophagy gene BECN1 in ovarian cancer.

Joe R. Delaney, Chandni Patel, Dwayne G. Stupack. _UC San Diego: Moores Cancer Center, La Jolla, CA_.

Autophagy, a process of protein and organelle recycling, can both suppress and promote tumors. Gliomas, KRAS mutant, and BRAF driven tumor models require functional autophagy for maximal tumorigenicity and/or tumor progression. Inhibition of autophagy is able to eliminate many human cancer cell lines. However, allelic loss of the autophagy gene BECN1 is present in the majority of ovarian cancers, and BECN1 loss incurs early tumorigenesis in mouse models free of other oncogenic stimuli. Recent sequencing efforts and analyses, depending on the study, have both supported and refuted the relevance of BECN1 allelic loss in ovarian and breast cancers. To directly address the role of BECN1 allelic loss in ovarian cancer, we crossed BECN1 heterozygous knockout dams with MISIIR-TAg sires. In this model, the MISIIR promoter induces expression of large T antigen specifically in the ovarian and fallopian epithelium, inhibiting p53 activity and driving ovarian cancers in a manner befitting its human analogue. Here we present our pre-publication findings relating to the oncogenesis and progression of this murine BECN1 allelic loss model of ovarian cancer. We compare the in vivo murine results to those of in vitro human cell line models of beclin depletion, which exhibit higher basal dsDNA break repair signaling, mitochondrial stress, and reactive oxygen species generation.

#1029

Rab1A and Rab1B promote esophageal squamous cell carcinoma through activating mTORC1 signaling and inhibiting autophagy.

Xian-Zi Yang, Ren Wang, Xiao-Xing Li, Yang Yang, Hui-Yun Wang, X. F. Steven Zheng. _State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, guangzhou, China_.

Background: Rab1A has recently been shown to act as an oncogene and promote colorectal and liver tumorigenesis. In this study, we aim to understand the potential role of Rab1A and its homolog Rab1B in esophageal squamous cell carcinoma (ESCC) and the underlying molecular mechanisms.

Methods: The expressions of Rab1A and Rab1B in139 paired human ESCC tissues and adjacent normal tissues were evaluated by immunohistochemistry. A series of in vitro and in vivo assays were performed to elucidate the biological functions of Rab1A and Rab1B, and their role in the regulation of mTOR signaling, autophagy and ESCC tumorigenesis.

Results: The level of Rab1A and Rab1B is elevated in ESCC tissues, which is positively correlated with tumor TNM stages and N stages. The overall expression of Rab1A and Rab1B is an independent predictor of patient survival (P<0.01). Down-regulation of Rab1A and Rab1B individually and in combination in ESCC cells inhibits cell proliferation and migration, and enhances autophagy. Mechanistically, Rab1A and Rab1B activate mTORC1 to enhance growth and suppress autophagy.

Conclusion: Our results suggest that Rab1A and Rab1B cooperatively regulate ESCC proliferation and autophagy by activating mTORC1 signaling. They may serve as promising prognostic biomarkers and/or potential molecular targets of ESCC treatments.

#1030

Targeting tumor-stroma metabolic symbiosis for head and neck cancer therapy.

Dhruv Kumar,1 Vikalp Vishwakarma,1 Jacob New,1 Radhika Joshi,1 Fangchen Lin,1 Sumana Dasari,1 Wade Gutierrez,1 Hemant Chavan,1 Ossama Tawfik,1 Shary Shelton,1 Yelizaveta Shnayder,1 Kiran Kakarala,1 Terance Tsue,1 Douglas Girod,1 Bennett Van Houten,2 Partha Kasturi,1 Sufi M. Thomas1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy affecting 40,000 cases in the USA annually. Despite aggressive therapies HNSCC is associated with less than a 50% 5-year survival rate. Limited treatment options and high morbidity necessitate the development of new treatments. HNSCC tumors frequently consist of up to 80% tumor-associated fibroblasts (TAFs). We reported that tumor associated fibroblasts (TAFs) facilitate HNSCC progression. However, the molecular mechanisms that facilitate TAF-mediated tumor growth and metastasis remain unknown. Unlike normal cells, malignant cells often display increased glycolysis, even in the presence of oxygen; a phenomenon known as the Warburg effect. As a consequence, there is an increase in lactic acid (LA) production. It has been reported that HNSCC tumors have high LA levels that correlate with reduced survival. The mechanisms whereby HNSCC tumors survive in highly acidic conditions remain unknown. We previously reported that TAF-secreted hepatocyte growth factor (HGF) facilitates HNSCC progression. Here we demonstrate that TAF-secreted HGF regulates HNSCC glycolysis and increases basic fibroblast growth factor (bFGF) secretion from HNSCC. Further, HNSCC-secreted bFGF regulates mitochondrial oxidative phosphorylation (OXPHOS) in TAFs. Inhibition of c-Met by PF-02341066 or knockdown by siRNA decreases glycolysis and bFGF expression in HNSCC. In addition, inhibition of FGFR by AZD-4547 decreased OXPHOS and HGF expression in TAFs. Thus, HNSCC and TAFs engage in reciprocal signaling through paracrine effects of HGF and bFGF. Combined treatment with AZD-4547 and PF-02341066 significantly inhibited TAF-induced proliferation of HNSCC in vitro compared to the vehicle control treated cells. For the first time, we tested FGFR inhibitor AZD-4547 in combination with c-Met inhibitor PF-02341066 both in vitro and in vivo xenograft models. The combination treatment significantly inhibited proliferation of HNSCC in vitro and effectively reduced HNSCC tumor growth compared to either agent alone in vivo (p<0.001). Our cumulative findings underscore the therapeutic potential of combinatorial treatment with PF-02341066 and AZD-4547 in HNSCC. 

### Metabolic Reprogramming in Cancer

#1031

Fumarate hydratase deficiency redirects glucose metabolism of hypoxic cancer cells into the pentose phosphate pathway.

Luana Schito,1 Sergio Rey,1 Judy Pawling,2 James W. Dennis,2 Bradly G. Wouters,1 Marianne Koritzinsky1. 1 _Princess Margaret Cancer Centre / University Health Network, Toronto, Ontario, Canada;_ 2 _Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada_.

Hypoxia is a common feature of all solid cancers and strongly correlated with poor prognosis. As an adaptive response to hypoxia, cancer cells reprogram their metabolism by increasing glycolysis and reductive carboxylation at the expense of mitochondrial respiration, a phenomenon orchestrated by the transcription factor hypoxia inducible factor (HIF) -1. Mutations in the gene encoding for the mitochondrial enzyme fumarate hydratase (FH), found in the hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome, lead to a similar phenotype despite the presence of O2, a phenomenon due to normoxic stabilization of HIF-1 (pseudohypoxia). Here, we report for the first time that FH loss-of-function (LOF) redirects glucose metabolism into the pentose phosphate pathway (PPP) in non-RCC cells subjected to severe hypoxia (O2< .02%). We show that this metabolic shift favors the buildup of biosynthetic precursors supporting hypoxic cell growth and proliferation.

HCT-116 (colon), HeLa (cervix) and H460 (lung) adenocarcinoma cells were transfected with lentiviral vectors encoding for shRNAs targeting FH. Immunoblot analysis showed that FH LOF did not induce pseudohypoxia in these cells. In contrast, HLRCC-derived UOK262 cells showed accumulation of HIF-1 under normoxia which was reversed upon FH re-introduction. A comprehensive analysis utilizing a RT-qPCR array to profile the mRNA expression of 84 HIF-1 target genes, further confirmed that FH LOF did not result in a pseudohypoxic phenotype in HCT-116 cells. An unbiased analysis of 250 metabolites detected by liquid chromatography-tandem mass spectrometry followed by quantitative enrichment analysis, identified glycolysis and the PPP among the most enriched metabolic pathways in hypoxic FH knockdown cells (P< 5×10-8). Since the PPP provides precursors for synthesis of nucleic acids, we analyzed the effect of FH LOF on cell cycle progression and found an inhibition of hypoxia-induced cell cycle arrest in HCT-116 and HeLa cells.

Our study reveals novel insights into the effect of FH loss-of-function in cancer cells and indicates a stark contrast between the pseudohypoxic phenotype described in kidney cancer cell lines obtained from HLRCC patients (i.e, UOK-262) and a HIF- independent mechanism of metabolic rerouting in colon, lung and cervix cancer cell lines. Our data show that FH LOF promotes an anabolic phenotype in hypoxic cancer cells that could be exploited to enhance the therapeutic response targeting this resistant subset of cancer cells.

#1032

Characterizing the metabolic dependencies of rhabdomyosarcoma and Ewing's sarcoma cells.

Sameer Issaq, Lee Helman. _National Cancer Institute, Bethesda, MD_.

Sarcomas represent a diverse group of malignancies with unique molecular and pathological characteristics. In order to improve sarcoma treatment, a better understanding of the alterations associated with specific sarcoma subtypes is critically important. Renewed interest in the altered metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a novel therapeutic strategy. In this study, we have characterized the dependency of rhabdomyosarcoma (RMS) and Ewing's sarcoma (EWS) cells on key metabolic substrates by examining cell proliferation and bioenergetic properties under varying concentrations of glucose and glutamine. We utilized two alveolar RMS cell lines, one embryonal RMS cell line, and five EWS cell lines. While all cell lines tested were completely growth-inhibited by lack of glucose, individual RMS and EWS cell lines were differentially sensitive to removal of glutamine. Furthermore, glutamine deprivation significantly reduced mitochondrial bioenergetic function, as demonstrated by Seahorse metabolic flux analysis. The observed effects of glutamine deprivation on proliferation and mitochondrial bioenergetics were rescued by the re-introduction of glutamine into the culture media. Our findings suggest that metabolic dependencies of RMS and EWS cells should be further investigated in order to identify metabolic vulnerabilities that could be targeted for potential therapeutic benefit.

#1033

Cigarette smoke and bile acids induce metabolic reprogramming mediated by uncoupling protein-2 (UCP2) during esophageal carcinogenesis.

Yuan Xu, Kate Brown, Deborah R. Depew, Paul L. Feingold, Mahadev Rao, Jeremy Davis, David S. Schrump, R. Taylor Ripley. _NIH, Bestheda, MD_.

Background: In cancer cells, glucose is processed not through mitochondrial respiration, but mainly through glycolysis to form lactate and minimal ATP (Warburg effect). Glutamine is an essential amino acid in cancer cells and the primary fuel for the mitochondria. Uncoupling protein 2 (UCP2) decreases mitochondrial proton membrane potential and reduces reactive oxygen species (ROS). Limited information is available regarding mechanisms that link environmental risk factors such as gastro-esophageal reflux and cigarette smoking to metabolomic alterations in esophageal adenocarcinoma (EAC). The present study was undertaken to examine the effects of cigarette smoke and bile acids on UCP2 expression, metabolic activity, and malignant phenotype of EAC cells.

Methods: Ten EAC lines were screened for UCP2 expression using qRT-PCR and immunoblot techniques. Two EAC lines, Flo-1 and NCI-SB-ESC2 (Esc2), had low and high UCP2 expression, respectively. Flo-1 and Esc2 cells were exposed to normal media with or without cigarette smoke condensate (CSC) and/or the bile acid, deoxycholic acid (DCA) for 5 days. Cell growth, migration, invasion, and clonogenicity were examined using CellTiter 96® Aqueous Kit, scratch, and soft agar clonogenicity assays. Mitochondrial respiration and glycolytic capacity were analyzed using the Seahorse Extracellular Flux Analyzer, and ATP and Lactate Production Kits. Levels of ROS were quantitated using Cellular Reactive Oxygen Species Detection Kit.

Results: Flo-1 cells had low UCP2 protein expression and large spare mitochondrial respiratory capacity, whereas Esc2 cells exhibited high UCP2 protein expression and no spare mitochondrial respiratory capacity. CSC and DCA mediated synergistic increases in lactate levels with decreases in ATP production (Warburg effect) without altering levels of UCP2 or ROS in Flo-1 cells. In contrast, similar treatment did not alter lactate or ATP production, but did synergistically deplete UCP2 and increase ROS levels in Esc2 cells. Whereas CSC and DCA exposure did not alter proliferation, migration, or invasion of Flo-1 cells, these treatments enhanced proliferation, migration, and invasion of Esc2 cells.

Conclusions: Following exposure to CSC and DCA, EAC cells can shift toward glycolysis when spare mitochondrial respiration is present. In contrast, EAC cells with maximal utilization of glycolysis have no ability to increase glycolysis with CSC or DCA exposure. However, in these cells, CSC and DCA down-regulates UCP2 and increases ROS; these metabolic effects coincide with increased malignant phenotype of EAC cells. These differences suggest that low levels of UCP2 may function as a metabolic sensor whereas high levels may serve as a cellular redox trigger effecting ROS. These preliminary insights into mitochondrial function may help guide the development of novel therapeutic strategies for EAC.

#1034

Leptin induces early onset and aggressiveness of pancreatic cancer.

Adriana Harbuzariu,1 Antonio Rampoldi,1 Danielle S. Daley-Brown,1 Tia L. Harmon,1 Derrick J. Beech,1 Gabriela Oprea-Ilies,2 Ruben R. Gonzalez-Perez1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

Background: During the past 30 years, pancreatic cancer (PC) has constantly been the fourth leading cause of cancer death. PC is usually advanced at the time of diagnosis, with an overall five years survival rate of less than 5%. Obesity, characterized by high levels of leptin, is pandemic in United States and a risk factor for PC. Notch abnormal signaling is linked to carcinogenesis, tumor angiogenesis and self renewal capacity of PC stem cells (PCSC). We have previously shown that leptin induces proliferation of PC cell lines, increases Notch expression, PCSC and tumorsphere formation. To test leptin effects in a PC mouse model, a specific and potent signaling inhibitor bound to nanoparticles, IONP-LPrA2 was produced by us.

Hypothesis: High levels of leptin induce early onset and multiplicity of PC xenografts, which is abrogated by IONP-LPrA2 treatment.

Methods: Human PC cell line MiaPaCa-2 was challenged with leptin or IONP-LPrA2. The expression of pluripotency associated genes (STAT3, Oct4, Sox2 and NANOG) was evaluated. MiaPaCa-2 cells forming tumorspheres were treated with leptin or IONP-LPrA2 and inoculated into CD1 nu/nu mice. Untreated tumorspheres were inoculated as controls. A group of mice were injected twice a week with IONP-LPrA2. Tumor onset and growth were assessed. PC xenografts were analyzed for expression of Ki67 (proliferation marker), leptin targeted molecules (Notch), PCSC markers etc.

Results: Leptin induced the expression of PCSC pluripotency markers. Moreover, leptin treatment of tumorspheres induced early tumor onset and multiplicity. Also, leptin increased proliferation, PCSC and oncogenic markers in PC xenografts. IONP-LPrA2 reduced leptin effects. No changes of mice food intake and body weight were observed.

Conclusions: This observation might imply that obese patients are at high risk to develop PC. Inhibition of leptin signaling may be used as preventative or therapeutic strategy for PC, especially in obese patients.

Acknowledgements: this work was supported by DOD W81XWH-13-1-0382; NIH/SBIR 1R41CA183399-01A1; Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR 03034 and 1C06 RR18386).

#1035

Targeting glutamine metabolism with the novel inhibitor JHU-083 inhibits tumor growth and alters the tumor immune microenvironment.

Judson Englert,1 Chih-Hsien Cheng,1 Robert Leone,1 Pavel Majer,2 Rana Rais,1 Barbara Slusher,1 Jonathan Powell1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic_.

Glutamine metabolism plays a critical role in promoting multiple biosynthetic pathways necessary for tumor growth. Therefore we endeavored to create a small molecule inhibitor that would block multiple pathways of glutamine metabolism as opposed to blocking one enzyme. Previous studies employing the non-specific glutamine antagonist 6-diazo-5-oxo-L-norlecuine (DON) have achieved this goal however, with much toxicity. To this end we devised a novel prodrug of DON (JHU-083) with distinct distribution and cellular partitioning properties. When dosed on an equimolar basis, JHU-083 retains the antitumor efficacy of DON but is devoid of the gut toxicity and morbidity/mortality.

To this end Wild-type mice were injected subcutaneously EL4 T-cell lymphoma cells. The tumors were allowed to engraft and treatment was initiated on day 5 with either vehicle, DON or JHU-083. After 7 days of treatment there was complete tumor regression in both the DON and JHU-083 treated mice, while tumors continued to grow in the vehicle treated mice. Importantly, after 7 days of treatment the DON treated mice appeared ill with lethargy and ruffled fur and even death as a result of treatment toxicity. Alternatively, the JHU-083 treated mice remained healthy and vibrant.

Based on these studies in a lymphoma model we next tested JHU-083 in 2 models of colon cancer. To this end we injected wild-type mice subcutaneously with MC38 or CT26 cells. Again, similar to the EL4 lymphoma experiments, we observed that JHU-083 resulted in complete regression of MC38 and CT26 tumors after 4 and 7 days of treatment respectively.

The tumor microenvironment, which promotes immune evasion, is very much influenced by tumor metabolism. Thus, we wanted to determine the effect of inhibiting glutamine metabolism on the tumor immune microenvironment. To this end mice injected with B16 melanoma cells were treated for 3 days with DON during which time the tumors decreased in size. Interestingly, there was also a concomitant decrease in the percentage of Foxp3+ cells and an increase in the CD8:Foxp3+ T cell ratio amongst the TILS.

Overall our studies demonstrate the principle of cellular selectivity based upon demand. That is, a non-selective inhibitor of glutamine metabolism can robustly inhibit tumor growth while demonstrating minimal side effects based on the differential demand of cancer cells versus normal cells. Furthermore, our studies suggest that sequencing anti-metabolic therapy with immunotherapy might prove to be a novel means of broadening and enhancing the role of immunotherapy for the treatment of cancer.

#1036

Cellular phenotypes of a novel series of 3-phosphoglycerate dehydrogenase inhibitors.

Nello Mainolfi, Adam Friedman, Mikel P. Moyer, Vipin Suri, Mark Manfredi. _Raze Therapeutics, Cambridge, MA_.

PHGDH (3-phosphoglycerate dehydrogenase) is the first enzyme branching from glycolysis into the serine synthetic pathway and it oxidizes 3-phosphoglycerate into phospho-hydroxypyruvate using nicotinamide adenine dinucleotide (NAD) as cofactor.[1] Increases in PHGDH expression at both mRNA and protein levels have been observed in nearly 70% of estrogen receptor-negative breast cancers; in addition a fraction of malignant breast and melanoma cells are dependent on elevated expression of 3-phosphoglycerate dehydrogenase (PHGDH).[2] Furthermore, serine starvation has been shown to have a dramatic effect on tumor growth during in vivo mouse xenograft experiments.[3] PHGDH has been a target of interest in the pharma/biotech industry for several years since the initial reports in early 2012 of its relevance in cancer2 where PHGDH amplified and overexpressing cancer cell lines have been shown to possess unique sensitivity to PHGDH knockdown that cannot be rescued by nutritional serine. The mechanisms underlying these studies have been subject to intense investigation but remain unclear. We have been able to successfully identify first in class small molecule inhibitors of this target with nanomolar cellular potency, high degree of selectivity and oral bioavailability. In several cancer cell lines, these compounds inhibited glucose derived serine flux with nanomolar median inhibitory concentrations without significantly affecting glucose derived lactate. These compounds also inhibited glucose derived serine in animal studies and have the potential to be highly useful tools for understanding the role of PHGDH in tumor progression. The data presented here will provide unexpected insights on the role of PHGDH in serine biosynthesis and the dependency of cancer cells on PHGDH catalytic function.

References

[1] Snell, K. Enzymes of serine metabolism in normal, developing and neoplastic rat tissues. Advances in enzyme regulation. 1984; 22:325-400.

[2] Cantor, J.R.; Sabatini, D. M. Cancer Cell Metabolism: One Hallmark, Many Faces, Cancer Discovery. 2012; 2(10):881-898.

[3] Maddocks et al., Serine starvation induces stress and p53-dependent metabolic remodeling in cancer cells Nature, 2013, 493(7433):542-6

#1037

Predicting fluxes in large-scale personalized metabolic networks using cancer phenotype driven flux analysis.

Abhinav Achreja,1 Lifeng Yang,1 Tyler Moss,2 Juan C. Marini,3 Prahlad T. Ram,2 Deepak Nagrath1. 1 _Rice University, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Baylor College of Medicine, Houston, TX_.

Deregulation of cellular metabolism is a well-established cancer hallmark that can initiate and perpetuate several pathological characteristics such as uncontrolled growth, metastasis and drug resistance. Several in vivo and in vitro studies are conducted to identify metabolic adaptations that support cancer progression. Computational techniques like metabolic flux analysis (MFA) have supported these studies by providing detailed insights of cancer cells' metabolic profiles. Here, we describe a novel systems approach to model cancer metabolism using transcriptomic, proteomic and phenotypic information. Our method (1) considers the diversity of cancer patients' gene and protein signatures to build personalized metabolic models (2) makes flux estimations based on interaction between metabolism and observable phenotypes and (3) predicts fluxes in peripheral and cyclic pathways without additional experiments. We employ NetWalker to integrate transcriptomic and proteomic data of patients and cell-lines to reconstruct patient-specific models from a core metabolic network. Our algorithm highlights gene subnetworks by calculating interaction scores from gene and protein expressions and finds links between the subnetworks and core metabolism to provide an integrated network representing a patient-specific model. This network is matched with Human Recon 2 Gene-Protein-Reaction library to build a model with feasible stoichiometry. We performed reconstruction on 4 ovarian cancer cell lines with varying invasive capacities for our novel MFA approach. We hypothesized that cancer cells perform several metabolic tasks to achieve phenotypic functions with limited nutrient availability. This leads to competition between metabolic objectives and their corresponding phenotypes. We show in NCI60 and CCLE cell lines that several phenotypes are competitive via correlation studies and principal component analyses of gene-expression data. Next, we found metabolic objectives related to phenotypes using pathway analysis and Gene Ontology. Our tool, 13C Multiobjective Metabolic Flux Analysis (13C MOMFA) is designed to use personalized metabolic models, along with measurements from metabolic assays and 13 Carbon stable isotope-labeled tracer studies to quantify fluxes. Current MFA techniques are limited to small networks or require multiple tracer experiments to estimate fluxes satisfying goodness-of-fit criteria. We show that 13C MOMFA can estimate fluxes within large-scale metabolic network satisfying statistical criteria and predict fluxes in extended metabolic pathways such as the nitric oxide and urea cycle, serine-glycine one-carbon cycle and pentose phosphate pathway without the need for expensive parallel labeling experiments. Our innovative approach performs a dual role, by providing a platform for personalized therapy and understanding metabolic adaptations driven by malignant phenotypes

#1038

Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer.

Medhi Wangpaichitr,1 Chunjing Wu,2 Ying Ying Li,1 Sumedh Shah,3 Shu-Mei Chen,3 Vy Dinh,1 Macus T. Kuo,4 Lynn G. Feun,3 Niramol Savaraj1. 1 _University of Miami/VA Medical Center, Miami, FL;_ 2 _VA Medical Center, Miami, FL;_ 3 _University of Miami, Miami, FL;_ 4 _MD Anderson Cancer Center, Houston, TX_.

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the KRAS mutation status, share one common parameter which is increased reactive oxygen species (ROS) when compared to their parental cells counterpart. Importantly, we found that CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism (OXMET) for energy and biosynthesis. Thus, alteration in mitochondria which resulted in metabolic changes and higher basal levels of ROS may be one of the major factors contributing to cisplatin resistance. We have investigated mitochondrial morphology in parental vs. CR cells using transmission electron microscopy (TEM). Our data clearly showed that CR cells possessed significantly higher number of mitochondria per total cell area (n=4; p=0.0006). CR cells also consumed 2-4 fold more oxygen when compared to their parental cells. To further confirm that oxidative phosphorylation was utilized more in CR cells, we assayed mitochondrial membrane potential (MMP) using TMRE staining. MMP was significantly higher in CR cells suggesting that CR cells possess highly active mitochondria. Key glycolytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), as well as lactate production were decreased in all CR cell lines which corresponded to the resistance to glycolytic inhibitor, 2DG (n=8; p<0.03). Moreover, CR cells were highly sensitive to glutamine deprivation. Glutamine withdrawal for 72h resulted in 75-80% cell death in CR cells while only 10-15% cell death occurred in parental cells. CR also take up twice as much L-[G-3H] glutamine when compared to its parental counterpart (n=4, p<0.05). Here, using Riluzole (the FDA approved drug that interferes with cystine/glutamate pump and results in reduced intracellular glutathione (GSH) levels), can selectively kill CR cells. ROS levels were increased (2-3X) more in resistant cells after Riluzole treatment while no significant change occurred in parental cells. The ID50 dosage of parental cells were 3-4 fold more than theirs CR cell counterparts and no cytotoxicity were found in normal lung fibroblast (n=8; p<0.05). However, antioxidant, N-acetylcysteine (NAC, 0.1mM), cannot completely abort cell death by Riluzole suggesting other mechanisms are involved. Importantly, Riluzole is also effective in 2 mouse xenograft models (H460CR (KRAS mutant) and SC (wt)). Tumors completely disappeared in wild type mice and were significant reduced in KRAS mutant mice (n=5 per treatment group; p<0.002). We conclude that addiction to glutamine instead of glucose and higher basal ROS levels seen in CR cells can make them hypersensitive to Riluzole. Repurpose this drug should be considered for future treatment of CR lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289) and Woman Cancer Association Fund.

#1039

Metabolic hormones FGF19/15 promote hepatocellular carcinoma through activation of CSCs in fatty liver.

Guozhen Cui,1 Xingkai Liu,1 Harshul Pandit,2 Yingbin Yang,2 Suping Li,2 Lu Cai,2 Robert C Martin,2 Hengjun Zhao,1 Wei Li,1 Yan Li2. 1 _The First Hospital of Jilin University, Chang Chun, China;_ 2 _University of Louisville, Louisville, KY_.

Background: Fibroblast growth factor 19 (FGF19), a member of the fibroblast growth factor (FGF) family, is a hormone that regulate glucose, lipid, and energy homeostasis in human. It has been shown that FGF19 and its specific receptor FGFR4 play an important role in non-alcoholic steatohepatitis (NASH) which is closely related hepatocellular carcinoma (HCC). Recently, we found FGF19 overexpressed in the HCC tissue and serum in patients. The high level of FGF19 was positively related to cancer stem cells (CSCs) specific surface markers such as CD133 and EpCAM. The aim of this study is to identify potential mechanism in which FGF15 (the FGF19's orthologous protein in mouse)/FGFR4 associated abnormal lipid metabolism initiates HCC progression in a NASH-HCC mouse Model. Methods: In vivo study, male C57L/J mice were injected intraperitoneally with N-nitrosodiethylamine(DEN) when 2 weeks old. Both the DEN and control mice were divided into two groups based on receiving high fat diet (HFD) or control diet (CD). Tumor nodules in the liver were monitored by ultrasound. Body weight, glucose tolerance test (GTT) and insulin tolerance test (ITT) were recorded during the experimental period. The liver weights, tumor volume, histology, alanine transaminase (ALT), alpha fetoprotein (AFP), and triglyceride (TG) were determined at 8, 20 and 32weeks. In vitro study, human HCC cell lines (Hep3B and Huh7) as well as mouse HCC cell lines (Hepa1-6 and FL83B,) were exposed to a long-chain mixture of free fatty acid(FFA) at different concentrations (UT, 0.5mM and 1mM FFA) for 24 and 48 hours, respectively. EpCAM and CD133 were determined by flow cytometery in the isolated tumor cells from HCC tissues. FGF19/15, FGFR4, CSCs surface markers and Wnt signaling pathway (β-catenin, GSK3β) were analyzed by RT-PCR and Western blot. FGFR4 signal was blocked to elucidate the role of FGF19/15 during the CSCs activation in vitro. In addition, FGF19, FGFR4 and EpCAM were also analyzed in human HCC samples. Results: Metabolic disorders were observed when DEN mice fed with HFD. HCC incidence and tumor volume were significantly increased in DEN+HFD group compared to that in DEN+CD group. DEN mice fed with HFD showed increased levels of FGF15/FGFR4 and β-catenin as well as the CSCs surface makers. Increased expression of CSCs surface makers were also found in the HCC cell lines when exposure to FFA in vitro. Blockage the FGF19 signal can abolish Wnt/β-catenin pathway and cell stemness features in the FFA environment. Increased levels of FGF19, FGFR4 and EpCAM were found in human HCC samples. Conclusion: Up-regulated FGF19/15 and FGFR4 promoted the development of HCC by active CSCs signaling in metabolic disorder microenvironment. FFA enhanced the CSCs activation was partially dependent on Wnt/β-catenin signaling. FGF19/FGFR4 could be a potential therapeutic target in HCC patients.

#1040

Differential levels of glycogen in breast cancer cell lines: A potential new target.

Joel A. Yates, Megan Altemus, Zhifen Wu, Michelle L. Wynn, Sofia D. Merajver. _Univ. of Michigan, Ann Arbor, MI_.

Cancer cells have been known to alter their metabolic processes in order to survive and proliferate. Normally in muscle and liver, excess glucose is stored within the cells as glycogen. Elevated levels of glycogen have also been found in various cancers, including breast cancers. Recent studies have implicated glycogen metabolism as important in promoting survival of cancer cells, suggesting targeting of glycogen metabolism as a possible treatment to inhibit cancer cell growth. In general, modulation of cancer metabolism is believed to be an attractive adjunct strategy to conventional or targeted therapies. Here we set out to investigate glycogen levels as well as levels of proteins involved in glycogen synthesis and degradation vary across different breast cancer cell lines.

A glucose metabolism qPCR array found differential levels of the alpha subunit of phosphorylase kinase 1, a key enzyme involved in glycogen degradation among three different breast cancer cell lines. Expression levels of glycogen synthesis and degradation enzymes were assessed using qPCR and immunoblot in various breast cancer cell lines. Glycogen levels in these breast cancer cell lines were quantified using an amyloglucosidase reaction coupled with other enzymatic reactions to produce a fluorescent product. It was found that MDA-MB-231, SUM149, and MCF7 cell lines had increased levels of glycogen, between 6.5 and 23.5 μg glycogen per mg protein, whereas SUM190 and normal-like breast epithelial cell line MCF10A had undetectable levels of glycogen. These findings demonstrate that glycogen metabolism can vary widely amongst cancer types, indicating that therapies targeted to disrupt glycogen degradation may produce differential results and that further study of the role of glycogen metabolism in cancer is warranted.

#1041

Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines.

Daniel Sappington, Rosalind Penney, Eric Siegel, Gunnar Boysen. _University of Arkansas for Medical Sciences, Little Rock, AR_.

Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems. The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and BPTES a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry. A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [13C5]glutamine demonstrated that by 12 hrs >50% of excreted glutathione is derived from glutamine. Culturing in glutamine-free medium or treatment with Bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a glutaminase (GLS)-specific inhibitor, reduced cell proliferation and viability, and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity. We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well.

#1042

Development of AEB1102, an engineered human arginase 1 for patients with solid tumors.

Scott W. Rowlinson,1 Susan E. Alters,1 Giulia Agnello,1 Joseph Tyler,1 Ann Lowe,1 Mauri Okamoto-Kearney,1 Dale Johnson,1 Everett Stone,2 George Georgiou,2 David G. Lowe1. 1 _Aeglea Biotherapeutics, Austin, TX;_ 2 _University of Texas, Austin, Austin, TX_.

Introduction: Tumor dependence on specific amino acids for survival and proliferation is well recognized and has been exploited effectively with the use of Asparaginase for the treatment of Acute Lymphoblastic Leukemia. Decades of research have led to an understanding of tumor L-Arginine (L-Arg) dependence, with functional expression of the three enzymes of the L-Arg biosynthetic pathway: Ornithine Transcarbamylase (OTC), Argininosuccinate Synthase (ASS1) and Argininosuccinate Lyase (ASL) being required to convert ornithine to L-Arg. In a variety of tumor types, silencing of one or more of these enzymes disables endogenous L-Arg synthesis forcing a reliance on the extracellular pool of L-Arg for tumor survival and proliferation. This mechanism has been confirmed with Arginine Deiminase (ADI-PEG) and pegylated wild-type Arginase I (BCT-100). Aeglea Biotherapeutics Inc. has developed an alternative approach using a bioengineered human PEGylated Arginase I with enhanced pharmacological properties. Replacement of manganese, the natural metal co-factor in wild-type human Arginase I, with cobalt confers improved catalytic activity and serum stability. The resulting product candidate (AEB1102) displays highly favorable PK/PD properties, and is expected to be naturally tolerated by the human immune system as no protein modifications have been introduced.

Experimental Procedures and Results: Non-clinical dose range finding and GLP toxicology studies were performed with AEB1102 in monkeys and mice. Bioanalytical assays detecting L-Arg and AEB1102 enzyme activity were used to monitor PD/PK in the dose range finding studies with these results subsequently being used to design the toxicology studies. Subsequent toxicology studies identified an NOAEL in both species at a dose that is predicted to translate to significant sustained reduction of L-Arg serum levels with weekly intravenous dosing. To determine tumor types most likely to respond to AEB1102 expression profiling of OTC, ASS1 and ASL was performed using in situ hybridization on multiple tumor histologies. Melanoma was identified as a tumor type likely to respond to AEB1102 owing to a significant reduction in ASS1 expression. Non-clinical in vivo studies using the A375 melanoma xenograft as well as melanoma PDx models confirm sensitivity to AEB1102, weekly dosing resulted in a significant delay in tumor growth and improved survival.

Conclusion: Non-clinical activities required to support the IND submission of AEB1102 for solid tumors were successfully executed, enabling the Phase 1 study to be initiated in October 2015. Translational work profiling the expression of OTC, ASS1 and ASL has identified melanoma as a relevant tumor type to pursue in future clinical studies.

#1043

Targeting ASCT2-mediated glutamine uptake and metabolism in breast cancer.

Michelle van Geldermalsen, Qian Wang, Jeff Holst. _Centenary Institute, Sydney, Australia_.

Although a nonessential amino acid in normal cell growth, the demand for glutamine is dramatically increased throughout malignant transformation to provide catabolic substrates for ATP production and anabolic substrates for macromolecule biosynthesis. To maintain glutamine availability for these metabolic processes, cancer cells overexpress cell surface transporters that function to exchange amino acids across the plasma membrane. One such transporter is ASCT2 (alanine, serine, cysteine-preferring transporter 2; SLC1A5), a sodium-dependent symporter that mediates uptake of small, neutral amino acids, including glutamine.

Blocking ASCT2 to prevent glutamine uptake and glutaminolysis has been shown to successfully prevent tumor cell proliferation in melanoma, non-small cell lung cancer, prostate cancer and acute myeloid leukaemia. We recently showed that in breast cancer, although ASCT2 is highly expressed in most tumor subtypes, only aggressive triple-negative (TN) breast cancer cells require ASCT2-mediated uptake of glutamine to sustain cell growth in vitro and in vivo. Gene expression analysis of xenograft-derived tumor tissue and TN patient samples suggested coordinate regulation of ASCT2 and other glutamine metabolism-related genes, such as glutaminase (GLS) and glutamate-ammonia ligase (GLUL), with global activation of glutaminolytic energy production pathways in these tumors. The metabolism-regulating transcription factors, MYC and ATF4, were significantly correlated with these genes, suggesting a dynamic MYC and ATF4-driven transcriptional program in TN breast cancer. We therefore hypothesized that highly proliferative TN breast cancers that are sensitive to ASCT2 inhibition may have unique metabolic signatures that could be additionally exploited for therapeutic purposes.

We developed a targeted metabolomics approach that combined labelled substrate tracing, liquid chromatography coupled tandem-mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) to analyze intracellular levels of key tricarboxylic acid (TCA) cycle intermediates, glycolytic metabolites, fatty acid precursors, and amino acids in human breast cancer cell lines. These analyses revealed distinct metabolic effects when ASCT2 transporter function was blocked in vitro by pharmacological inhibitors or inducible shRNA knockdown, in combination with CB-839, a GLS inhibitor in Phase I clinical trials. To confirm the clinical utility of these findings, we also determined mRNA and protein expression of glutamine metabolism-related genes in tissue microarrays of TN patient samples. These data suggest a reliance on glutamine availability in TN breast cancers and reinforce the link between increased glutamine metabolism and clinically aggressive breast cancers, thus highlighting the therapeutic potential of targeting the ASCT2 glutamine uptake and metabolism pathway in these patients.

#1044

Glutamine modulates cellular NAD+/NADH homeostasis thereby regulating drug sensitivity.

Lifeng Yang, Abhinav Achreja, Stephen Waling, Joelle Baddour, Joseph Ni, Ryuichi Nakahara, Katherine Stilles, Lisa Chiba, Sun Hye Kim, Juan C. Marini, Anil K. Sood, Deepak Nagrath. _Rice University, Houston, TX_.

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Recently, we uncovered a missing link between glutamine metabolism and chemo-drug resistance. Using isotope tracer and bioenergetic analysis, we found that drug resistant ovarian cancer cells are more glutamine-dependent for their proliferation and colony formation. Notably, the glutamine's importance in maintaining redox balance is prominent in drug resistance by balancing NADPH, NADH and glutathione levels. We found that cisplatin caused mitochondrial dysfunction in glutamine-sensitive ovarian cancer cells, as well as reduced glutaminolysis. However, drug resistant cells lines increased NADPH/NADP+ and NADH/NAD+ ratio by enhancing glutamine's entry into TCA cycle. The overexpression of NAD+ biosynthesis pathway enhanced glutamine's entry into TCA cycle for cancer metastasis, as well as induced chemo-drug resistance. Our findings suggest a novel approach of targeting drug-resistant OVCA by blocking glutamine's entry into the TCA cycle, along with targeting NAD+ biosynthesis pathway. Our insights present a unique opportunity for overcoming the drug resistance limitation in clinical trials in ovarian cancers.

#1045

Targeting NAMPT in Ewing's sarcoma cells.

Cornelia N. Mutz,1 Raphaela Schwentner,1 Eric Bouchard,2 Edgard M. Mejia,3 Anna M. Katschnig,1 Maximilian O. Kauer,1 Dave N.T. Aryee,4 Antje Garten,5 Versha Banerji,2 Heinrich Kovar4. 1 _Children's Cancer Research Institute Vienna, Vienna, Austria;_ 2 _Department of Biochemistry and Medical Genetics, University of Manitoba; Research Institute in Oncology and Hematology, CancerCare Manitoba, Winnipeg, Manitoba, Canada;_ 3 _Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada;_ 4 _Children's Cancer Research Institute Vienna; Department of Pediatrics, Medical University Vienna, Vienna, Austria;_ 5 _Center for Pediatric Research Leipzig, Hospital for Children and Adolescents, University of Leipzig, Leipzig, Germany_.

Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in the generation of the chimeric transcription factor EWS-FLI1.

NAD is a key metabolite of energy metabolism being involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability serving as a donor of ADP-ribose. This study describes targeting NAMPT (nicotinamide phosphoribosyltransferase), the rate-limiting enzyme in salvage generation of NAD, by FK866 in ES cells. Using FK866 has been proposed as a treatment option for various inflammatory diseases as well as cancer, rendering ES cells with high NAMPT expression especially susceptible to FK866-induced cytotoxicity.

Here we report that NAMPT inhibition in ES cells leads to exhaustive NAD depletion, followed by a delayed reduction of ATP levels and concomitantly to apoptosis-mediated cell death. These effects can be reversed by nicotinic acid, a substrate for the NAD salvage generation. However, the use of a doxycycline-inducible shRNA against EWS-FLI1 revealed that the cytotoxic activity of NAMPT inhibition is significantly lowered in the absence of EWS-FLI1. EWS-FLI1-low ES cells have higher viability and lower rates of apoptosis throughout inhibitor treatment compared to cells with high EWS-FLI1 expression. Additionally, changes in mitochondrial respiration and glycolytic rate can be observed when comparing untreated versus EWS-FLI1 knockdown ES cells after NAMPT inhibition. Interestingly, loss of EWS-FLI1 leads to elevated NAD levels and results in alteration of RNA expression of some enzymes involved in the NAD synthesis pathway. These results might explain the high susceptibility of Ewing Sarcoma cells to FK866 treatment.

Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of ES cells and suggests NAMPT inhibition as a potential new treatment approach for Ewing Sarcoma in combination with standard therapies.

Supported by the Austrian Science fund, grant I1225-B19; and the Research Manitoba and CancerCare Manitoba Foundation.

#1046

Statin inhibits malignant mesothelioma cell growth by inactivating YAP1.

Kosuke Tanaka, Taketo Kato, Akihiro Matsushita, Hiromi Furuta, Yuko Murakami, Hirotaka Osada, Yoshitaka Sekido. _Aichi Cancer Center Insutitute, Nagoya, Japan_.

The prognosis of patients with malignant mesothelioma (MM) remains very poor because of highly invasive tumor characteristics and therapeutic resistance of this tumor. Recent studies have shown that MM frequently shows Hippo signaling pathway inactivation, which leads to activation of YAP1 transcriptional co-activator. Although substantial data from clinical trials and epidemiological studies showed promising results for the use of statins in many cancers, few studies have argued about the underlying tumor-suppressive mechanisms of statins. Here, we show that statins act as an inhibitor of YAP1-dependent MM cell growth via mevalonate pathway. In cell culture, statins attenuate migration and invasion of H290 malignant mesothelioma cells with homozygous deletion of NF2, which encodes Merlin, an upstream regulator of Hippo pathway. In contrast, geranylgeranyl diphosphate (GGPP), a down-stream activator of mevalonate pathway, completely rescues H290 cell growth inhibition by statins. We also found that statins accelerate YAP phosphorylation/inactivation, which results in the downregulation of Hippo-pathway targeting gene transcriptions including CTGF, CYR-61 and ANKRD1. Moreover, among 11 MM cell lines, we demonstrated that five out of the six cell lines, which showed significant cell growth inhibition with statins, harbor NF2 and/or LATS2 mutations. Finally, we found that phospho-YAP/YAP ratio was increased in statin-sensitive cells, but not in statin-resistant cells. Together, our findings suggest that mevalonate pathway plays crucial roles in YAP-dependent MM cells by Hippo pathway inactivation, and therefore represents a potential therapeutic target for MM.

#1047

Effect of 1,25-Dihydroxyvitamin D in regulating glutamic-oxaloacetic transaminase 1 (GOT1) and redox balance during breast cancer progression.

Xuanzhu Zhou, Tomasz Wilmanski, Dorothy Teegarden. _Purdue University, West Lafayette, IN_.

Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1α,25 dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes that may prevent breast cancer. Possible pro-oxidative effects of 1,25(OH)2D in breast cancer cells which may selectively promote cell death have been proposed. Glutamic-oxaloacetic transaminase 1 (GOT1) is an essential transaminase in glutamine metabolism. GOT1 converts aspartate into oxaloacetate in the cytoplasm, which is then subsequently converted to malate and pyruvate. NADPH is produced during the last step of this process and increased NADPH/NADP+ ratio has the potential to maintain cellular redox balance. Therefore, it is of interest to study 1,25(OH)2D regulation of GOT1 during cancer progression to understand the role of vitamin D in regulating oxidative stress as a mechanism to prevent breast cancer. We employed untransformed MCF10A human breast epithelial cells (MCF10A), as well as Harvey ras-oncogene or ErbB2 (HER-2/neu) oncogene transformed cells (MCF10A-ras and MCF10A-ErbB2) as cell models for early cancer progression. Results showed that 1,25(OH)2D significantly decreased GOT1 mRNA expression at 48 hours in MCF10A-ras cells and MCF10A-ErbB2 cells by 35 ± 8.8% and 49 ± 10%, respectively, and GOT1 protein expression at 72 hours by 51 ± 4.9% and 51 ± 16%, respectively. The susceptibility of MCF10A-ras cells to hydrogen peroxide induced cell death was significantly increased by 1,25(OH)2D, as determined by measuring viable cell number using MTT assay. We further showed that 96-hour 1,25(OH)2D treatment significantly reduced cellular NADPH/NADP+ ratio by 30 ± 2.8% and GSH/GSSG ratio by 34 ± 2.7% in MCF10A-ras cells. Collectively, these results support that 1,25(OH)2D inhibits GOT1 expression and has pro-oxidative effects in oncogene transformed MCF10A cell lines, which may contribute to its role in preventing breast cancer development.

#1048

**Modulation of lipid metabolism through inhibition of acetyl-CoA carboxylase with ND-646 leads to potent inhibition of breast cancer cell growth** in vitro **and** in vivo **.**

Jennifer L. Rocnik,1 Wenyan Miao,1 Geraldine Harriman,1 Jeremy Greenwood,2 Sathesh Bhat,2 H. James Harwood,1 Rosana Kapeller,1 William F. Westlin1. 1 _Nimbus Therapeutics, Cambridge, MA;_ 2 _Schrodinger, New York, NY_.

Metabolic attenuation is a promising approach to cancer therapy and rate-limiting steps in key biosynthetic pathways are particularly attractive targets. Many cancer types are dependent on fatty acid synthesis as a primary source of energy and for providing lipids for expansion of cell and nuclear membranes in rapidly proliferating cells. The rate-limiting enzyme in fatty acid synthesis, acetyl-CoA carboxylase (ACC), has been shown to be highly expressed in human breast cancer. ACC is thought to be critical for the growth and survival of cancer cells, especially within a tumor microenvironment where exogenous fatty acids might be limited. Effective therapeutic options for triple negative breast cancer are limited and identification of robust targeted agents without overt toxicity for this indication are especially needed. Dual inhibition of the ACC isozymes, ACC1 and ACC2, results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. We have identified ND-646, a potent, selective, allosteric inhibitor of ACC with broad tissue distribution that binds to the ACC biotin carboxylase domain and potently inhibits the dimerization and enzymatic activity of both ACC1 (IC50 = 3.5nM) and ACC2 (IC50 = 4.1nM). Profiling the potency of ND-646 in vitro in a panel of breast cancer cell lines including triple negative and BRCA1 mutant cell lines demonstrated potent inhibition of cell proliferation with IC50s<100nM. The anti-proliferative effects were more pronounced when cells were cultured in media containing delipidated serum. Daily oral dosing of ND-646 at 25 mg/kg BID, 50 mg/kg QD, and 50 mg/kg BID in mice bearing orthotopic triple negative MDA-MB-468 breast cancer xenografts led to tumor growth inhibition of 60-70% that correlated with compound exposure and target engagement in the tumor. Analysis of ND-646 treated tumors demonstrated disruption of tumor tissue architecture and induction of apoptosis and necrosis suggesting a direct effect on cell survival. These results provide further evidence that de novo lipogenesis is an important mediator of breast cancer cell growth and survival, and that selective inhibition of ACC is a viable therapeutic strategy for treatment of breast cancer.

#1049

**Pharmacological characterization of a novel potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with robust** in vivo **efficacy and increased therapeutic index with niacin supplementation.**

Maria Quanz, Claudia Merz, Andreas Bernthaler, Stefan Kaulfuss, Anja Richter, Marcus Bauser, Andrea Haegebarth. _Bayer Pharma AG, Berlin, Germany_.

Nicotinamide adenine dinucleotide (NAD+) is an important metabolite and cofactor for a number of cellular processes including genomic stability and epigenetic regulation. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate limiting enzyme in NAD+ salvaging from nicotinamide. Many tumor cells have an increased need for NAD+ and are therefore highly sensitive to NAMPT inhibition. We have developed a selective and potent small molecule inhibitor, Cmpd1, which inhibits the NAMPT enzyme with sub-nanomolar potency. In cells, Cmpd1 treatment leads to an almost complete reduction of both NAD- and ATP-levels, followed by the induction of cell death. In order to identify sensitive cancer subtypes and associated biomarkers, we analyzed cell sensitivity to NAMPT inhibition in a panel of 240 cell lines. Small cell lung cancer and hematological malignancies were particularly sensitive to NAMPT inhibition. Overall, expression levels of NAMPT and NAD+ consuming enzymes correlated well with sensitivity to NAMPT inhibition.

In order to investigate the effect of niacin on the antiproliferative effect of NAMPT inhibition across the cell panel, we further analyzed the cell sensitivity in the presence of niacin. Cells with a functional Preiss-Handler pathway can generate NAD+ from niacin, independent of NAMPT. We found that the antiproliferative effect of Cmpd1 was neutralized by niacin in 60% of the analyzed cells. The mRNA levels of nicotinate phosphoribosyltransferase 1 (NAPRT1), a central enzyme of the Preiss-Haendler pathway, predicted well the niacin rescue status of the cells. In vivo, niacin supplementation led to an at least 10-fold increase in the maximum tolerated dose of Cmpd1 in mice. Antitumor efficacy of Cmpd1 was abolished by niacin supplementation in xenografts derived from NAPRT1 expressing cells, but not in NAPRT1-deficient models. These data demonstrate that the therapeutic index of NAMPT inhibitors may be increased in NAPRT1-deficient tumors by niacin supplementation.

#1050

Inhibition of the polyamine synthesis pathway is synthetically lethal with loss of argininosuccinate synthase 1 in cancer.

Peter W. Szlosarek,1 Matthew Locke,2 Essam Ghazaly,2 Laura Lattanzio,3 Cristiana Lo Nigro,3 Sarah A. Martin2. 1 _Barts Cancer Institute & St.Bartholomew's Hospital, London, United Kingdom; _2 _Barts Cancer Institute, London, United Kingdom;_ 3 _AO S. Croce e Carle, Cuneo, Italy_.

Argininosuccinate synthetase 1 (ASS1) is the rate-limiting enzyme for arginine biosynthesis. ASS1 expression is lost in a range of different tumour types, including 50% of malignant pleural mesotheliomas. Starving ASS1-deficient cells of arginine with arginine blockers such as ADI-PEG20 can induce selective lethality and has shown great promise in the clinic. However, recent data has shown that ASS1-deficient tumours can become resistant to this therapy although the mechanisms behind this resistance remain unclear. We have generated the first model of ADI-PEG20 resistance in mesothelioma cells whereby we observe re-expression of ASS1, via demethylation of the ASS1 promoter. Through coordinated transcriptomic and metabolomic profiling, we have shown that ASS1-deficient cells have decreased levels of acetylated polyamines, resulting in an increased activation of the polyamine synthesis pathway. Upon arginine deprivation, we observe a decrease in polyamine metabolites in the ASS1-deficient cells only, suggesting that exogenous arginine is required to maintain polyamine biosynthesis in the absence of ASS1. We identify for the first time a compensatory increase in polyamine synthesis gene expression upon ASS1 loss and highlight a synthetic lethal dependence between ASS1-deficiency and polyamine metabolism, which could potentially be exploited for the treatment of ASS1-negative cancers.

#1051

Activation of PI3K pathway in breast cancer associates with tumor phospholipid fatty acid composition.

Nawale Hajjaji,1 Flavie Arbion,2 Alain Fautrel,3 Claire Villalva,4 Lucie Karayan-Tapon,4 Marie-Lise Jourdan2. 1 _Breast International Group, Brussels, Belgium;_ 2 _University Hospital of Tours, Tours, France;_ 3 _H2P2 plateform Inserm U991, Rennes, France;_ 4 _University Hospital of Poitiers, Poitiers, France_.

Background

Ever since the discovery of the PI3K/Akt/mTOR pathway key role in breast cancer resistance, several molecules have been developed to target this pathway. However, mechanisms of resistance restrain their efficacy. An in-depth understanding of the interactions of PI3K pathway is essential to improve its blockade and avoid resistance. Preclinical studies reported that lipid composition of cancer cells altered the activation of PI3K pathway, thus suggesting another possible molecular interaction with lipid metabolism. The aim of this study was to analyze the relationship between the activation of PI3K pathway and tumor lipid composition in breast cancer patients.

Methods

Frozen and formalin-fixed paraffin-embedded tumor specimens were retrieved for 50 French patients with stage I to III primary breast cancer treated uniformly with surgery, adjuvant chemotherapy, radiation therapy and endocrine therapy. The tumor specimens were analyzed for PIK3CA gene mutation, PTEN protein expression by immunohistochemistry, and pAktS473, pAktT308, mTOR, pS6K, and S6RP by protein array. Tumor total fatty acid (FA) and phospholipid FA compositions were analysed using mass spectroscopy. Pearson test was used for correlation analyses.

Results

Mean age was 53 years. All tumors were ER or PR positive and HER-2 negative, and 76% were node positive. PI3K mutation was observed in 14% of cases, and 34% were PTEN negative or had low PTEN expression. Mean tumor satured fatty acid (SFA) level was 33.78 mol%, monounsaturated FA (MUFA) level was 46.46 mol%, n-6 polyunsaturated FA (PUFA) level was 18.08 mol%, and n-3 PUFA level was 1.68 mol%. Large and high grade tumors had a higher SFA content. MUFA rich tumors were frequently low grade. There was a positive correlation between d18:1/C20:0 ceramide level and Akt, mTOR and p70S6K expression. mTOR expression also correlated with 18:0 lysophosphatidylserine (lysoPS), 36:1 PS, 38:3 PS, 38:5 PS, 40:5 PS, 40:6 PS, 34:0 phosphatidylcholine (PC), 34:1 PC, and 38:3 PC.

Conclusions

Our results show that SFA associated with a more aggressive tumor phenotype and that PI3K pathway activation significantly correlated with fatty acid composition of specific tumor phospholipids. These results suggest a potential role of tumor lipid metabolism in PI3K pathway activation.

#1052

Stearoyl-CoA-Desaturase (SCD1) regulates lung cancer stemness via stabilization and nuclear localization of YAP/TAZ.

Alessia Noto,1 Maria Elena Pisanu,2 Claudia De Vitis,3 Giovanni Sorrentino,4 Giannino Del Sal,4 Alfredo Budillon,1 Gennaro Ciliberto,1 Rita Mancini3. 1 _Istituto Tumori Pascale, Napoli, Italy;_ 2 _Magna Graecia University, Catanzaro, Italy;_ 3 _University of Rome La Sapienza, Rome, Italy;_ 4 _University of Trieste, Trieste, Italy_.

One of the most common traits of cancer is the radical change of cellular metabolism. A distinctive aspect of this altered metabolic status is the presence of a larger pool of monounsaturated fatty acids (MUFA), the precursors of the components of cell membranes necessary to sustained the rapidly dividing cancer cells. MUFA are largely generated from saturated fatty acids by the action of the two Stearoyl-CoA desaturases isoforms, namely SCD1 and SCD5. Recent evidences suggest that the major isoform SCD1 plays a role in several cancers.

Our group has previously reported, using primary adenocarcinoma cell lines from malignant pleural effusions, that SCD1 inhibition selectively kills ALDH positive cells, a marker of cancer stem cells, causes spheroid 3D cultures collapse in vitro and impairs their growth in vivo. These results suggest that SCD1 may be a critical target in lung cancer tumor-initiating cells and further studies are needed to assess the role of this enzyme in lung cancer. An increasing number of literature points to the Hippo (and their effectors YAP/ TAZ) and to the Wnt/β-catenin signaling pathways as key factors in the development of cancer. Notably, SCD1 has been shown to be involved in the secretion and maturation of active wnt ligands, contributing to the activation of the β-catenin signaling.

We report here, using primary cell cultures from adenocarcinoma of the lung, that YAP and TAZ are required for the generation of 3D cultures, as silencing of both genes completely abrogated the capability of generating lung cancer spheroids. Moreover, simultaneously silencing of YAP and TAZ reduced the mRNA levels of OCT4, Nanog and ALDH. We demonstrated that both silencing of SCD1 and its pharmacological inhibition determined a decrease in the expression and nuclear localization of both YAP and TAZ. Moreover, when SCD1 was inhibited, YAP and TAZ transcriptional activity was reduced. We also demonstrated that SCD1 blockade induced a dramatic reduction of the Axin2 mRNA level, one of the β-catenin target genes and a decreased in the β-catenin transcriptional activity, without affecting β-catenin protein expression. YAP and TAZ downregulation induced by SCD1 blockade could be rescued by the addition of exogenous wnt3a ligand, thus indicating that the active β-catenin signaling is necessary for YAP and TAZ stabilization. Ultimately, we observed that the ubiquitin ligase β-TrcP is involved in the SCD1 mediated degradation of YAP and TAZ.

These data, overall, demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway and β-catenin pathway in lung cancer 3D spheroid cultures, pointing to lipid metabolism as regulator of cancer stem features. Moreover, these results can be the starting point for further studies focused on the regulation of SCD1 in cancer stem cells, suggesting a new perspective for improving chemotherapeutic responses in cancer treatment, centered on SCD1 inhibition.

#1053

Redirecting abiraterone metabolism to biochemically fine tune prostate cancer therapy.

zhenfei li, Mohammad Alyamani, Nima Sharifi. _Cleveland Clinic Lerner Research Institute, cleveland, OH_.

Prostate cancer is the most diagnosed cancer in men and Abiraterone is used for castration resistant prostate cancer therapy by suppressing androgen production. In patients, abiraterone is converted to D4A, a novel metabolite with more potent anti-tumor activity and structural similarities to endogenous steroidal 5α-reductase substrates, such as testosterone. Here, we show that D4A is further metabolized by the related steroidogenic enzymes to at least 3 5α-reduced and 3 5β-reduced metabolites. The initial 5α-reduced metabolite, 3-keto-5α-abi, is more abundant than D4A in patients taking abiraterone, and is an androgen receptor (AR) agonist, which promotes prostate cancer progression. In a clinical trial with patients taking abiraterone alone, followed by abiraterone plus dutasteride (a 5α-reductase inhibitor), 3-keto-5α-abi and downstream metabolites are depleted by dutasteride, while D4A concentrations rise, effectively blocking production of a tumor promoting metabolite and permitting D4A accumulation. Our findings suggest a previously unappreciated and biochemically specific method of clinically fine tuning abiraterone metabolism to optimize therapy.

#1054

SNAI1 regulates the hexosamine biosynthetic pathway to promote tumorigenesis and oncogene-induced senescence escape in lung cancer.

Kekoa Taparra,1 Hailun Wang,1 Katriana Nugent,1 Reem Malek,1 Jen Groves,1 Gokben Yildirir,1 Brian Simons,1 Dean Felsher,2 Natasha Zachara,1 Phuoc Tran1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Stanford University School of Medicine, Stanford, CA_.

Lung cancer is the deadliest and most common malignancy worldwide. Tumor invasion and metastasis influence the dismal 16% overall 5-year survival. Evidence suggests malignancies commandeer epithelial plasticity programs like the epithelial-mesenchymal transition (EMT) to promote tumorigenesis, tumor progression, and metastasis. Furthermore, EMT has recently been linked to alterations in cancer metabolism, which can engender adaptation to environmental stress while supporting the macromolecular demand of rapid proliferation. Thus it is important to better understand the emerging roles of EMT in regulating cancer metabolism. SNAI1 is a master EMT regulator and is aberrantly overexpressed in many cancers including lung cancer. To investigate the role of SNAI1 in tumorigenesis, we created a novel inducible autochthonous mouse model. Our model reveals SNAI1 overexpressing mutant Kras mice generate identifiable tumors twice as fast (median = 14 weeks) as mice overexpressing mutant Kras alone (median = 30 weeks). To identify possible mechanisms of SNAI1-accelerated tumorigenesis, we used mRNA expression profiling. We identified significantly altered gene signatures related to metabolic reprogramming. Specifically, SNAI1 upregulates the hexosamine biosynthetic pathway (HBP) in mutant Kras-driven mouse lung tumors. SNAI1 elevates protein and mRNA of the rate limiting step (GFPT2) and final step (UAP1) of this pathway. This SNAI1-driven promotion of the HBP was recapitulated in vitro using human non-small cell lung cancer (NSCLC) SNAI1 isogenic cell lines. NSCLC mRNA expression data from The Cancer Genome Atlas (TCGA) also showed SNAI1 expression in patient tumor samples correlate with a significant tendency towards co-expression with HBP genes (Fisher's exact test, p<0.05). Functionally, the final metabolite generated by the HBP, UDP-GlcNAc, is an essential nucleotide sugar found in a plethora of N- and O-linked glycosylations, regulating protein stability and activity. Here we show SNAI1 promotes flux through the HBP to promote increased total levels of O-GlcNAcylated proteins in lung tumors in vivo. In so doing, modulation of the HBP and specific glycosylations may impact cell death and senescence, suggesting a novel targetable EMT-HBP axis. Numerous oncogenes, including c-Myc and SNAI1 itself, are known to be stabilized by O-GlcNAcylation. This link is becoming of increasing importance in cancer biology, as half of the body of literature on O-GlcNAcylation and cancer has been published in the past three years. Together, our data suggest heightened protein O-GlcNAcylation, mediated by SNAI1 and the HBP, serves as a novel means by which EMT contributes to mutant Kras-induced lung tumorigenesis and tumor progression.

#1055

CPT1A-mediated lipid catabolism modulates growth, AR expression and hypoxia survival of prostate cancer.

Isabel R. Schlaepfer, Gagan Deep, Rajesh Agarwal, Zhiyong Zhang, Maren Salzmann-Sullivan, Lih-Jen Su, Thomas Flaig. _University of Colorado Anschutz Medical Campus, Aurora, CO_.

Background: Androgen deprivation results in hypoxia in prostate cancer (PCa) cells and subsequently enhances the transcriptional activity of AR leading to more aggressive tumors. Concomitant with hypoxia, a significant accumulation of lipids has been observed but its significance remains unclear. Recent data indicate that lipid oxidation via carnitine palmitoyltransferase (CPT1) results in decreased growth and apoptosis, underscoring the role of lipid as a fuel for PCa growth. Since androgen deprivation ultimately increases the activity of AR, we hypothesized that dual targeting AR and lipid catabolism axes represents an attractive therapeutic approach.

Methods: To address the role of lipid use by PCa cells we have used two experimental approaches that lead to lipid accumulation: 1- Use of clinically relevant drugs etomoxir and ranolazine: Etomoxir works by inhibiting CPT1, the rate-limiting step in fat oxidation. Ranolazine is FDA-approved for angina and blocks lipid oxidation in the mitochondria. 2- Hypoxia exposure followed by re-oxygenation to mimic the dynamics of the tumor environment and induce lipid accumulation. Enzalutamide was used by itself or in combination with etomoxir to treat PCa cells, as well as CPT1-edited cells generated in our lab. Drug effects on metabolism and AR expression were studied by growth assays, LCMS, westerns, qRTPCR, and mouse xenograft studies.

Results: CPT1 is abundant in androgen-sensitive cells and tumors. Treatment with etomoxir alone significantly decreased cell viability and AR, including the ARv7 variant. Combinatorial treatment with enzalutamide synergistically enhanced this effect on viability. Systemic treatment with ranolazine alone in nude mice bearing enzalutamide-resistant cells resulted in decreased xenograft growth over 21 days, underscoring the therapeutic potential of blocking lipid catabolism to decrease CRPC tumor growth. Lipids accumulated under hypoxia (especially arachidonic acid) were used for growth following re-oxygenation. Pharmacologic inhibition of lipid use or CPT1-knockdown significantly compromised this growth following re-oxygenation, underscoring the role of lipids in supporting aggressive growth. This was observed in sensitive and CRPC models.

Conclusions: Lipid accumulation and oxidation is a cyclical phenomenon that modulates AR content and PCa growth. Combination of fat burning inhibitors and enzalutamide may offer a novel approach to anti-AR resistance in PCa, requiring less anti-androgen for more effective therapy.

#1056

Inhibition of hexose monophosphate pathway promotes castration resistant prostate cancer.

Akash K. Kaushik,1 Ali Shojaie,2 Katrin Panzitt,1 Rajni Sonavane,1 Harene Venghatakrishnan,3 Mohan Manikkam,1 Alexander Zaslavsky,3 Vasanta Putluri,1 Vihas Vasu,4 Yiqing Zhang,1 Ayesha Khan,5 Stacy Lloyd,1 Adam Szafran,1 Subhamoy Dasgupta,1 David Bader,1 Fabio Stossi,1 Hangwen Li,3 Susmita Samanta,1 Xuhong Cao,3 Efrosini Tsouko,5 Shixia Huang,1 Daniel Frigo,5 Lawrence Chan,1 Dean Edwards,1 Benny Kaipparettu,1 Nicholas Mitsiades,1 Nancy Weigel,1 Michael Mancini,1 Michael Ittmann,1 Arul Chinnaiyan,3 Nagireddy Putluri,1 Ganesh Palapattu,3 George Michailidis,3 Arun Sreekumar1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Washington, Seattle, WA;_ 3 _University of Michigan, Ann Arbor, MI;_ 4 _The Maharaja Sayajirao University of Baroda, Vadodra, India;_ 5 _University of Houston, Houston, TX_.

Prostate Cancer (PCa) is the second highest cause of cancer-related death in men in the US. PCa is androgen dependent when organ-confined and is conventionally treated using surgery or using a combination of anti-androgens and radiation therapy. However, in about 30% of the patients tumor recurs and are initially administered androgen deprivation therapy (ADT). Majority of the patients become resistant to ADT and develop hormone-refractory disease also termed castration-resistant prostate cancer (CRPC), which is lethal. Currently, the molecular and biochemical alterations driving CRPC are not well understood. Using a novel network-based integrative approach, we show distinct alterations in the Hexosamine Biosynthetic Pathway (HBP) to be critical for sustaining the castrate resistant state. Our data suggests expression of key HBP enzymes to be significantly elevated in androgen dependent (AD) PCa while interestingly enough, relatively diminished in CRPC. Genetic loss of function experiments for these HBP enzymes in CRPC-like cells had tumor promoting effect both in vitro and in vivo. This was mediated by alterations in either PI3K-AKT pathway or SP1-ChREBP (SP1- carbohydrate response element binding protein) network in CRPC cells containing full length androgen receptor (AR) or its splice variant AR-V7, respectively. Strikingly, addition of HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) or glucosamine (GlcN) to CRPC-like cells attenuated tumor cell proliferation, both in vitro and in animal studies. Interestingly, these metabolites demonstrated additive efficacy when combined with enzalutamide in vitro. These findings are particularly significant given that the CRPC-like cells tested, inclusive of those containing AR-V7 variant, are inherently resistant to enzalutamide. These observations demonstrate the therapeutic value of targeting altered HBP in CRPC.

#1057

Bioluminescent assays for investigating cellular energy metabolism.

Donna M. Leippe, Mary Sobol, Mike Valley, Natasha Karassina, Sarah Duellman, Jolanta Vidugiriene, Jim Cali. _Promega Corporation, Madison, WI_.

Cellular energy metabolism is recognized to have a central role in many cell processes and is an increasingly important area of study in many research fields such as cancer, immunology, obesity, diabetes and neurology. To facilitate these studies, we have taken advantage of key features of bioluminescence, such as increased sensitivity and wide assay windows, to develop assays for measuring energy metabolites. These assays are easy-to-use plate-based assays with streamlined protocols that require minimal cell sample processing. The assays can be used to measure consumption of major fuel sources such as glucose and glutamine from the cell medium, as well as lactate and glutamate secretion. The sensitivity (1-5pmol/sample), broad linear range (0.1-100 uM) and wide dynamic range (maximum signal-to-background > 100 fold) allow all four metabolites to be measured from the same sample of medium and over time, with earlier detection from fewer cells compared to other methods. The assays are also sensitive enough to detect intracellular levels of glutamine, glutamate and lactate and changes in those levels. Additional assays have been developed to measure glucose uptake and NAD/NADH levels in cells. Analysis of these metabolites provides information regarding the energetic state of the cell. In this poster we provide examples demonstrating the utility of these assays to detect a glycolytic shift in cancer cells, monitor cell growth and differentiation with associated changes in glucose uptake and glycolysis, and evaluate insulin sensitivity in primary adipocytes.

#1058

Targeting pentose phosphate pathway (PPP) represents a novel therapeutic strategy for hepatocellular carcinoma (HCC) treatment.

Ming Jing Xu,1 Kit Ho Lai,1 Shu Hai Lin,2 Pui Wah Tse,1 David Kung Chun Chiu,1 Hui Yu Koh,1 Cheuk Ting Law,1 Chun Ming Wong,1 Zong Wei Cai,2 Carmen Chak Lui Wong,1 Irene Oi Lin Ng1. 1 _The University of Hong Kong, Hong Kong;_ 2 _Hong Kong Baptist University, Hong Kong_.

Background and objectives: Hepatocellular carcinoma (HCC) is the primary liver malignancy with an extremely low survival rate. HCC progression is frequently associated with accelerated glucose consumption that confers growth advantage to tumor cell through two essential metabolic pathways - glycolysis and the pentose phosphate pathway (PPP). Glycolysis utilizes glucose for ATP production, while the PPP converts glycolytic intermediates to generate NADPH, the cellular antioxidant, and ribose-5-phophate, the nucleotide precursor. Although glycolysis has been extensively studied in HCC, how PPP supports HCC growth remains largely unknown. Our study aims at delineating the clinical significance, regulation and functions of PPP in HCC development and explore the therapeutic potential of PPP inhibitors for HCC therapy.

Methods: The expression and abundance of the the PPP genes were examined in 16 pairs of human HCC tumors and adjacent non-tumors by transcriptome sequencing. Level of reactive oxygen species (ROS) and glucose uptake were measured by CM-H2DCFDA and 2-NBDG stainings, respectively. Metabolomics study was performed with CE-TOF-MS analysis. Metabolic flux analysis was conducted using UPLC-MS/MS.

Results: Transcriptome sequencing data showed that enzymes in the PPP were frequently upregulated in HCC. Transketolase (TKT), the gene encodes the reversible enzyme connecting PPP and glycolysis, is the most abundant and most overexpressed PPP gene in HCC. Overexpression of TKT was confirmed in an expanded sample cohort at the mRNA and protein level. Meanwhile, TKT overexpression was significantly correlated with venous invasion, microsatellite formation, tumor size and absence of tumor encapsulation. Notably, TKT expression was controlled by NRF2/KEAP1/BACH1 pathway, which is a major transcription regulator for redox homeostasis. CHIP assay revealed that NRF2 and BACH1 competitively bound to the same antioxidant responsive elements (ARE) in TKT. Functionally, knockdown of TKT in HCC cells retarded cell growth, attenuated glucose uptake and NADPH production, increased intracellular ROS, and induced oxidative stress-associated cell cycle delay. In line with these findings, knockdown of TKT greatly suppressed tumor growth in vivo. Metabolomics and metabolic flux analysis revealed that loss of TKT disrupted the PPP and subsequently reduced NADPH production. Intriguingly, genetic knockdown and pharmacological inhibition of TKT enhanced the efficacy of Sorafenib, the only FDA-approved drug for HCC treatment, both in vitro and in vivo.

Conclusion: Our study suggested the clinical significance of TKT in HCC and illustrated the anti-oxidative role of TKT in HCC progression. We also proposed that disrupting the metabolic machinery by TKT inhibition might be a novel therapeutic strategy for HCC treatment.

#1059

Pancreatic cancer cells acclimatize to low pH by increasing glutamine metabolism.

Jaime Abrego, Venugopal Gunda, Pankaj K. Singh, Gennifer Goode. _University of Nebraska Medical Center, Omaha, NE_.

Pancreatic cancer (PC) is the fourth leading cause of cancer related deaths in the United States. PC has a five-year survival rate of only 6%; this is due to the lack of specific symptoms at the earliest stages of disease progression. Currently, surgery is the only treatment option with a reasonable hope of cure; however, due to late detection of the disease, only 15-25% of patients are eligible for surgery. Pancreatic tumors are characterized by increased glucose uptake, high glycolysis rate, and reduced flux to the TCA cycle. This metabolic phenotype, also known as the Warburg effect, is typical of rapidly dividing tumor cells and forms the basis of imaging by utilizing [18F]-FDG-PET (glucose analog). The consequence of this metabolic behavior is the continuous acidification of the tumor microenvironment. Acidification of the tumor microenvironment (TME) and its effects in cancer cell metabolism are not well defined. In this study we analyzed metabolic adaptations of PC cells experiencing physiological pH 7.4 versus cells cultured in 6.8~7.0 pH that is similar to the pH range observed in the pancreatic TME. Using high performance liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis to determine the metabolite levels of PC cells cultured in these TME conditions, we observed a marked reduction in glycolysis metabolites in cells cultured at low pH. We observed reduced glucose uptake, as well as, reduced lactate secretion in low pH culture conditions. Furthermore, we observed that cells in low pH microenvironment were able to survive upon glucose deprivation, but not upon glutamine deprivation. In contrast, cells in normal physiologic pH culture could not survive upon glucose deprivation. Based on the up regulation of metabolites in the glutaminolysis pathway identified through LC/MS/MS analysis, and the increase in metabolic enzyme mRNA levels, as well as the increased sensitivity to inhibitors of this pathway, we conclude that glutamine metabolism is essential for survival of pancreatic cancer cells under low pH conditions. Furthermore, due to the increased levels of ATP and sensitivity of low-pH cultured cells to oligomycin, we conclude that oxidative phosphorylation is essential for the maintenance of cellular homeostasis at low pH. Such changes result in reduced Warburg Effect, denoted by reduced lactate release. This is the first study to establish glutamine dependence of PC cells under low pH conditions. Our results may provide novel therapeutic opportunities for targeting metabolic adaptations in pancreatic cancer cells in response to changes in the microenvironment.

#1060

Targeting serine hydroxymethyltransferase 2 (SHMT2) suppresses hepatocarcinoma.

Chern Chiuh Woo, Way Cherng Chen, Teck Hock Philip Lee. _Singapore Bioimaging Consortium, Singapore, Singapore_.

Glycolytic intermediate 3-phosphoglycerate branched from glycolysis pathway to fuel serine-glycine biosynthesis pathway. Several studies have shown that glycine consumption is strongly correlated to the proliferation rate of cancer cells. Therefore targeting serine-glycine biosynthesis pathway is a promising strategy due to the fact that many of its genes are up-regulated in cancer cells. These genes include PHGDH, PSAT1 and GLDC. Here, we demonstrate that targeting serine hydroxymethyltransferase 2 (SHMT2), also a member of serine-glycine biosynthesis pathway, is able to suppress hepatocarcinoma both in vitro and in vivo. SHMT2 simultaneously converts serine to glycine and tetrahydrofolate to 5,10-methylenetetrahydrofolate. We found that this gene is up-regulated in Hep3B, HepG2 and Huh-7 liver cancer cells versus THLE2 normal liver cells. Knockdown of SHMT2 effectively suppressed Huh-7 cell growth and tumorigenicity. No tumor was formed after inoculation of SHMT2-knockdown Huh-7 cells in nude mice as compared to 100% tumor formation with control Huh-7 cells. The data from tetracycline-inducible SHMT2-knockdown xenograft mouse model showed that SHMT2 inhibition could reduce tumor growth and tumor incidence as well as delayed tumor onset. We also found that SHMT2-knockdown Huh-7 cells were more vulnerable to the cytotoxicity of doxorubicin, a conventional chemotherapeutic agent, as compared to the control Huh-7 cells. On the other hand, overexpression of SHMT2, however, could not transform THLE2 cells into malignancy suggesting that SHMT2 might not a key driver in liver tumorigenesis. SHMT2 activity is correlated to cancer phenotype as shown by the NMR data via metabolic tracing with 13C-glycine. Supplementation of glycine, rather than 5,10-methylenetetrahydrofolate, could reverse the reduced tumorigenicity caused by SHMT2-knockdown. Taken together, our data support that SHMT2 is a potential target in the treatment of hepatocarcinoma.

### MicroRNAs as Biomarkers and Therapeutics

#1061

MicroRNA profiling by a next generation sequencing approach in urine and plasma samples: from genomics to diagnostics and prognostics of bladder cancer.

Barbara Pardini,1 Francesca Cordero,2 Alessandra Allione,1 Clara Viberti,1 Alessio Naccarati,1 Marco Oderda,3 Mirko Preto,3 Marco Allasia,3 Maddalena Arigoni,4 Federica Riccardo,4 Raffaele Calogero,4 Carlotta Sacerdote,5 Giuseppe Matullo1. 1 _Human Genetics Foundation, Turin, Italy;_ 2 _Department of Computer Science, University of Turin, Turin, Italy;_ 3 _Molinette Hospital, University of Turin, Turin, Italy;_ 4 _Molecular Biotechnology Center, Department of Biotechnology and Health Sciences, University of Turin, Turin, Italy;_ 5 _Center for Cancer Prevention (CPO-Piemonte), Turin, Italy_.

Bladder cancer (BC) is one of the leading causes of cancer-related death worldwide.

Most BCs are non-muscle invasive (NMIBC), which generally have a good prognosis but frequently recur or progress to muscle invasion (MIBC) within 5 years. By contrast, MIBC has a poor prognosis and treatment requires a multidisciplinary approach with radical surgery, radiotherapy, and chemotherapy. BC is among the most expensive cancer per patient because it requires frequent surveillance and repeated treatments over many years.

Reliable predictors of disease and progression for BC are lacking so developing novel noninvasive diagnostics is imperative to both improve patient outcomes and decrease health costs.

MicroRNAs (miRNAs) are aberrantly expressed in many cancers, including BC, and may be isolated from various biological specimens, including plasma and urine.

Next generation sequencing (NGS) technology provides novel information about miRNA expression and is likely to become the gold standard method for comprehensive miRNA analysis in cancer genomics. So far, only few studies investigated miRNA signatures in BC by NGS and only on tissues. To investigate miRNA signatures in surrogate tissues may be a useful alternative to reduce invasiveness of biopsies, allowing repetitive samplings during follow-up and reducing health care costs for detection, monitoring of progression and treatment.

We aim to identify specific miRNA signatures in urine and plasma samples from 20 NMIBC, 20 MIBC patients and 40 healthy controls using a NGS approach able to accurately distinguish BC patients and predict disease outcome. The measurement of miRNA levels in both urine and plasma samples from the same patients will allow us to compare the levels of promising biomarkers in two surrogate tissues. Urine is the best and closest surrogate tissue for BC, since it is in direct contact with the tissue of tumor origin; while plasma is one of the best surrogate tissue for distant metastasis and advanced cancer detection.

In plasma samples, 5 miRNAs were differentially expressed among cases and controls (miR-222, miR-146b, miR-126, miR221 upregulated and miR-5096 downregulated). However, after repetition of the analyses stratifying cases for NMIBC (the most represented group in BC cases), only 2 of the previously described miRNAs (miR-222 and miR-126) resulted dysregulated among NMIBC and controls.

In urine, 31 miRNAs were differentially expressed among BC cases and controls (adjusted p-value ranging from 0.0007 to 0.05). On the other hand, 26 and 31 miRNAs resulted differentially expressed among NMIBC and controls and MIBC and controls, respectively (for NMIBC: adjusted p-value from 0.0009 to 0.04; for MIBC adjusted p-value from 7.2x10-9 to 0.04).

Interestingly, miRNAs differentially expressed among cases and controls were able to discriminate not only BC cases from controls, but also NMIBC and MIBC.

#1062

Overexpression of miRNAs 181a and 222 play a role in triple negative breast cancer, and are targeted by entinostat.

Armina A. Kazi,1 Alexa Giammarino,1 Nicholas Musacchio,1 Gauri Sabnis,2 Amanda Schech,2 Angela Brodie2. 1 _Loyola University Maryland, Baltimore, MD;_ 2 _University of Maryland, Baltmore, Baltimore, MD_.

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer in women. Clinically, this subtype is characterized by high recurrence rates, poor prognosis, and lack of targeted therapies. Therefore, there is considerable need to identify TNBC-specific biomarkers that can serve as a diagnositc markers and therapeutic targets. MIcroRNAs (miRNAs), may be such biomarkers. miRNAs are short, non-coding regulatory RNA molecules that modulate the expression of specific proteins by binding to target messenger RNAs (mRNAs) and causing either degradation of the mRNAs or inhibition of their translation to protein. Thus, they play an important role in a variety of normal cellular processes (e.g., differentiation, cell growth, cell death, etc.), and in diseases, such as cancer. MiRNAs have been implicated in breast cancer, but there is not consistent agreement as to which miRNAs are involved in TNBCs, nor have molecular targeting drugs been identified that could treat TNBCs by affecting miRNAs. Previous studies by our lab and others indicate that miRNAs 181a and 222 are involved in estrogen-receptor independence, cancer stem cells, and drug resistance (ex. letrozole resistance). Thus, in this study, the expression of miRNAs 181 and 222 in TNBCs, the effect of inhibiting each miRNA on cell viability and cancer stem cells, and the effect of histone deactylase inhibitor entinostat on miRNA expression are explored. RT-PCR analysis of miRNA expression in both HS578T and BT547 TNBCs and MCF-7 cells (represents the least aggressive subtype), and in representative breast cancer patient biopsy samples indicates that both miRNA 181a and 222 are upregulated by at least 15-fold in TNBCs compared to MCF-7 (least aggressive subtype). Specific inhibition of miRNA 181a via siRNA/miRNA inhibitor in HS578T cells significantly decreased cell viability by at least 80%, as determined by MTT assay, and cancer stem cells by 50%, as determined by mammosphere assays. In addition, inhibition of miRNA 181a produced morphological changes in HS578T cells, in which cells lost their protrusions, became more round, and grew in colonies. Lastly, treatment of HS578T cells or of patient derived xenografts with entinostat, which is currently being explored as a TNBC-targeting drug, decreased miRNA 181a and 222 expression and produced similar results as miRNA inhibition on cell viability, cancer stem cells, and morphology. Overall, these results suggest that miRNAs 181a and 222 are overexpressed in TNBCs and may play a role in regulation of cell viability and cancer stem cells. They also indicate that HDAC inhibitor, entinostat, may be an effective treatment for TNBCs through their action on miRNAs 181a and 222.

#1063

miR-195 represses the tumorigenesis of non-small cell lung cancer and synergizes with microtubule targeting agents.

Xiaojie Yu,1 Zhenze Zhao,1 Xiuye Ma,1 Liqin Du,2 Alexander Pertsemlidis1. 1 _UTHSCSA, San Antonio, TX;_ 2 _Texas State University, San Marcos, TX_.

MicroRNAs (miRNAs) play important roles in nearly all cellular physiological and pathological pathways. Dysregulated miRNAs have been shown to contribute to tumorigenesis of many cancers, including lung cancer. The let-7 miRNA family, among the first miRNAs discovered, suppresses the growth of non-small cell lung cancer (NSCLC). Other miRNAs have been shown to regulate the response of cancer cells to anticancer agents, such as the microtubule-targeting agent (MTA) paclitaxel. These findings highlight the potential application of miRNAs as the next generation of therapeutic agents, either alone or in combination with chemotherapy.

A high throughput screen was performed in NSCLC cells. miRNAs that both inhibited NSCLC cell growth and sensitized NSCLC cells to paclitaxel were validated by short- and long-term assays for cell viability. The targets of miRNAs were identified by expression profiling following miRNA transfection, validated by luciferase reporter assay, and confirmed by functional assays.

We show that miR-195 causes G1 phase arrest of NSCLC cells by targeting CCND3, and leads to apoptosis and reduction of cell migration and invasion by targeting BIRC5. Conversely, the inhibition of cell growth by miR-195 is compromised by overexpressing CCND3 or BIRC5; the inhibition of cell migration and invasion by miR-195 is abrogated by overexpressing BIRC5. We also show that miR-195 synergizes with MTAs paclitaxel and eribulin to inhibit the growth of NSCLC cells by regulating CHEK1. Analysis of miRNA and gene expression profiles from The Cancer Genome Atlas (TCGA) shows that miR-195 expression is negatively correlated with BIRC5 and CHEK1 but not CCND3. Clinical data from TCGA show that miR-195 is significantly down-regulated in lung tumors compared to adjacent normal tissues and that its down-regulation in lung cancer patients is associated with worse survival. The TCGA data also show that BIRC5 and CHEK1 are significantly up-regulated in lung tumors and that their up-regulation is associated with worse survival. CCND3, however, is slightly downregulated in lung tumors and its expression is not correlated with patient survival. Intriguingly, we also show that miR-195 up-regulates the oncogene MYC, inhibition of which sensitizes NSCLC cells to miR-195. We show that Myc, reported to transcriptionally repress let-7c, also represses miR-195, and further identify a feedback loop between let-7c and miR-195 in which each inhibits the other.

We identify miR-195 as a tumor suppressor and chemotherapy sensitizer in NSCLC mediated by its repression of CCND3, BIRC5 and CHEK1. We also uncover a feedback network between miR-195, Myc and let-7c. The ability of miR-195 and Myc to regulate each other indicates a mechanism by which NSCLC cells can become more sensitive to miR-195. The mutual inhibition between miR-195 and let-7c suggests an interesting regulatory loop between cancer-related miRNAs that deserves further study.

#1064

MicroRNA-based diagnosis and therapy in NRF2-stabilized tumor.

Jun Inoue,1 Naoto Fujiwara,2 Shinsuke Yamamoto,1 Tatsuyuki Kawano,2 Johji Inazawa1. 1 _Department of Molecular Cytogenetics, Tokyo Medical & Dental Univ., Tokyo, Japan; _2 _Department of Esophageal and General Surgery, Tokyo Medical & Dental Univ., Tokyo, Japan_.

NRF2 is a master transcriptional regulator that integrates cellular stress responses and is negatively regulated by KEAP1 at the posttranslational level. In human cancers, aberrantly stabilized NRF2, either by mutation of NRF2 or KEAP1, plays a vital role in chemoresistance and tumor cell growth through the transcriptional activation of target genes, suggesting that targeted inhibition of NRF2 is a potential therapy for NRF2-stabilized tumors. Some tumor-suppressing microRNAs (miRNAs) target multiple oncogenes concurrently and therefore may be useful as cancer therapeutic agents. Further, such miRNAs may be useful to address chemotherapeutic resistance in cancer, which remains a primary clinical challenge in need of solutions. Thus, cytoprotective processes upregulated in cancer cells that are resistant to chemotherapy are a logical target for investigation.

By using a reporter-coupled miRNA library screening, we have recently identified four miRNAs (miR-507, -634, -450a, and -129-5p) that negatively regulate the NRF2 activity by directly targeting. Importantly, downregulation of these miRNAs, in addition to the somatic mutation of NRF2 or KEAP1, is associated with stabilized NRF2 and poor prognosis in esophageal squamous cell carcinoma (ESCC). Interestingly, we found that overexpression of miR-634 activates the mitochondrial apoptotic pathway by direct concurrent targeting of genes associated with mitochondrial homeostasis, antiapoptosis, and autophagy, together with NRF2-mediated antioxidant ability. In particular, we showed how enforced expression of miR-634 enhanced chemotherapy-induced cytotoxicity in a model of ESCC, where resistance to chemotherapy remains clinically problematic. Our findings illustrate how reversing miRNA-mediated cytoprotective processes may offer a broadly useful approach to improving cancer therapy.

#1065

Reduced expression of microRNA-200 family associated with aggressiveness and poorer patient outcome of clear cell renal cell carcinomas.

Masahiro Gotoh,1 Eri Arai,2 Akio Matsuda,3 Hiroyuki Fujimoto,4 Kenji Matsumoto,3 Yae Kanai2. 1 _Division of Molecular Pathology, National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Department of Pathology, Keio University School of Medicine, Tokyo, Japan;_ 3 _Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan;_ 4 _Department of Urology, National Cancer Center Hospital, Tokyo, Japan_.

[Introduction] The aim of this study was to clarify the significance of alterations of microRNA (miRNA) expression during renal carcinogenesis.

[Materials and methods] Expression profiles of miRNA were examined using the SurePrint G3 miRNA Expression microarray in paired samples of cancerous tissue (T) and non-cancerous renal cortex tissue (N) from 95 patients with clear cell renal cell carcinomas (RCCs). The expression levels of miRNA and mRNA were quantified by reverse transcription PCR (RT-PCR) using TaqMan system. The MetaCore pathway analysis by GeneGo was performed using miRNAs of which expression levels were significantly altered during renal carcinogenesis.

[Results] The expression levels of 39 and 169 miRNAs were increased (T/N>=2) and reduced (T/N<0.5) in T samples relative to the corresponding N samples, respectively. MetaCore pathway analysis revealed that such expression alterations of the 208 miRNAs were significantly accumulated in four pathways, which included "Development microRNA-dependent inhibition of epithelial-mesenchymal transition (EMT) pathway" (P=8.695×10-15). The expression levels of any of 5 EMT-related miRNAs, i.e. miR-200a, miR-200c, miR-141, miR-429 and miR-200b, called as miR-200 family, were reduced in 93 (98%) of the examined 95 RCCs. Quantitative RT-PCR analysis verified that the expression levels of miR-200 family were significantly reduced in T samples relative to the corresponding N samples. Additionally, the mRNA expression levels of the ZEB1 and ZEB2 genes, which were downstream targets of miR-200 family, were significantly increased, and their target CDH1 gene expression was further decrease in T samples. The expression levels of miR-200 family in T samples were significantly correlated with clinicopathological parameters reflecting tumor aggressiveness, such as invasive macroscopic configuration, higher histological grade, vascular involvement, renal vein tumor thrombus, invasive growth pattern, tumor necrosis, distant metastasis and higher histological TNM stage. Moreover, the expression levels of miR-200 family were significantly correlated with both of cancer-free and overall survival rates of patients with clear cell RCCs.

[Conclusions] These data suggested that reduced expression of miR-200 family may participate in the malignant progression resulting in poorer patient outcome of clear cell RCCs, via regulation of the EMT pathway.

#1066

Synthetic miR-22 inhibits alternative non homologous end-joining DNA repair and increases sensitivity to PARP-inhibition in multiple myeloma cells.

Daniele Caracciolo, Eugenio Morelli, Maria Teresa Di Martino, Daniela Talarico, Cirino Botta, Nicola Amodio, Cinzia Federico, Emanuela Altomare, Lavinia Biamonte, Maria Angelica Stamato, Pierosandro Tagliaferri, Pierfrancesco Tassone. _UMG of Catanzaro, Catanzaro, Italy_.

Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by deep genomic instability. Alternative non-homologous end-joining (A-NHEJ) DNA repair is up-regulated in a significant fraction of cancers and it is more error-prone than the major LIG4-dependent NHEJ repair pathway (classic-NHEJ). Principal components of A-NHEJ (PARP1, POLQ and LIG3) represent therefore a new class of cancer cell-specific therapeutic targets.

There is Increasing evidence that microRNAs take actively part in the regulation of the DNA damage/repair network. Based on this rationale, we aimed to elucidate the role of miRNAs in the regulation of A-NHEJ-driven genomic instability in MM.

We first examined the prognostic role of DNA Ligases involved in NHEJ pathways interrogating the public MM gene expression dataset GSE9782, which revealed higher expression of LIG3 in patients with shorter survival; conversely LIG4 expression was not associated with worse prognosis, thus supporting the idea that LIG3 has a pivotal role in genomic instability and progression of MM. Next, we aimed to identify LIG3-targeting miRNAs by an integrated approach that predicted miR-22 as negative regulator of LIG3 in MM patients. On this basis we evaluated miR-22 and LIG3 expression in a panel of MM cell lines and primary samples compared to plasma cells from healthy donors. We detected significant down-regulation of miR-22 and up-regulation of LIG3, strictly correlated to disease progression. Subsequently, we induced miR-22 overexpression (by transduction or transfection) in a panel of MM cell lines and we found cell growth inhibition, apoptosis induction and cell cycle arrest. Importantly, transfection of miR-22 mimics reduced the viability of primary MM cells but not of HD-PBMCs. We next examined the effects of enforced overexpression of miR-22 on DNA-damage response and we observed an increase of the DNA damage marker yH2AX, reduction of LIG3 at mRNA and protein level, and decrease of LIG3-3'UTR luciferase activity, indicating specific 3' UTR targeting. Importantly, we observed inhibition of A-NHEJ repair by a functional assay based on the plasmid EJ2-GFP.

Finally, miR-22 mimics potentiated cytotoxic effects of PARP-inhibitor Olaparib (Selleckchem), both in vitro and in vivo, confirming that co-inhibition of LIG3 and PARP-1 have a synergistic effect on A-NHEJ dependent MM cells.

Taken together, our preliminary findings indicate that miR-22 inhibits A-NHEJ by specific targeting of LIG-3 and modulates sensitivity to PARP inhibition. Further investigations could shed new light on A-NHEJ in MM pathogenesis and define a role for miR-22 as therapeutic agent in this still incurable disease.

Acknowledgement: This work has been supported by Italian Association for Cancer Research (AIRC). PI: PT. "Special Program Molecular Clinical Oncology - 5 per mille" n.9980, 2010/15

#1067

A paradoxical observation for its upregulation and the prognostic significance about miR-7 in esophageal squamous cell carcinoma.

Kano Masayuki, Yasunori Matsumoto, Isamu Hoshino, Kentaro Murakami, Yasunori Akutsu, Hisahiro Matsubara. _Chiba University, Chiba-City, Japan_.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers of the gastrointestinal tract. However, molecular indicators of the origin of cellular deregulation in ESCC have not been identified. MicroRNAs (miRNAs), noncoding RNAs 21-25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor-suppressive function in ESCC was performed using the miRNA expression signatures. miRNA signatures from ESCC identified miRNAs that are downregulated in common, and these miRNAs are candidate tumor suppressors. Gain-of-function analysis revealed that 3 transfectants (miR-145, miR-133a and miR-133b) inhibit cell proliferation and cell invasion. These miRNAs, which have conserved sequences in the 3'UTR of FSCN1, inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR-145 target sites and 1 miR-133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor-suppressive miRNAs, miR-145, miR-133a and miR-133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.

On the other hands, miRNAs upregulated in cancer is the candidates of tumor marker. The miRNA expression signatures we perform for ESCC revealed seven upregulated miRNAs in ESCC. In the upregulated miRNAs of miRNA expression signatures, miR-7 downregulation is the independent poor prognostic factor for ESCC patients paradoxically.miR-7 expression is reported upregulated or downregulated variously in the same carcinoma (eg. Gastric adenocarcinoma). The correlation between patients' prognosis and miR-7 expression has not yet reported in Pubmed research in Dec. 2015. And the target genes of miR-7 is not clarified.

#1068

The promise of miR-205 in HER2+ breast cancer: predicting response to Trastuzumab and overcoming resistance.

Claudia Piovan, Elvira D'Ippolito, Ilaria Plantamura, Patrizia Casalini, Elda Tagliabue, Marilena V. Iorio. _Istituto Nazionale Tumori, Milan, Italy_.

HER2, member of HER family, is overexpressed in up to 30% of breast cancers and often associated to a more aggressive phenotype, resistance to chemotherapeutic agents and increased risk of metastasis. Anti-HER2 targeted therapies significantly increased the overall survival of HER2 breast cancer patients, however tumor cells frequently develop alternative resistance mechanisms and the molecular bases of this phenomenon are still far to be fully elucidated. A crucial issue is then represented by the urgent need of biomarkers able to predict the response to treatment, and certainly of new therapeutic tools to counteract the resistance mechanisms. The oncosuppressive role of miR-205 in the pathogenesis of breast cancer has been demonstrated, as well as a possible cross-regulation between miR-205 and HER signaling. MiR-205 is in fact negatively regulated by HER2 and is involved in the regulation of HER2-mediated proliferation by directly targeting HER3, preferential partner of HER2. We have investigated miR-205 role on trastuzumab resistance by using human HER2 overexpressing breast cancer cell lines and patient-derived xenograft (PDX) models. Here we show that HER3 silencing, either mediated by miR-205 or by a siRNA specific against HER3, is able to improve the responsiveness to Trastuzumab, reducing tumor cell growth and colony formation capability. This effect is mediated by impairment of Akt-mediated pathway.Combination of Trastuzumab treatment and in vivo delivery of miR-205-conjugated lipid nanoparticles in PDX models is currently on going. Finally, expression analysis of miR-205 in breast cancer patients treated in adjuvant with trastuzumab has provided new insights about its possible role as predictive biomarker of response. In summary, here we show that miR-205 improves the responsiveness to Trastuzumab treatment at least partially though HER3 silencing, and associated to outcome of patients treated in adjuvant setting, thus representing a potential adjuvant tool and predictive biomarker.

#1069

MiR-21 may serve as a predictive biomarker of response in the assessment of efficacy of HSP-90 inhibition in gastrointestinal (GI) cancers.

Andrea Lampis,1 Luciano Cascione,2 Rosemary Burke,1 Paul Clarke,1 Michele Simbolo,3 Aldo Scarpa,3 Else Bosma,1 Sijia Yu,1 Rebecca Cole,1 Mark Stubbs,1 Swee Sharp,1 Rob Van Montfort,1 Jens C. Hahne,1 Matteo Fassan,4 Paul Workman,1 Nicola Valeri,1 Chiara Braconi1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Institute of Oncology Research, Bellinzona, Switzerland;_ 3 _University of Verona, Verona, Italy;_ 4 _University of Padua, Padua, Italy_.

Background and Aims: MicroRNAs mediate drug resistance and are often deregulated in cancer with miR-21 upregulation being shared across different gastrointestinal (GI) cancers. In this study we aim to identify small drugs for which miR-21 may be considered a biomarker of sensitivity.

Methods: High throughput screening technologies (HST) was applied in RKO colorectal cancer (CRC) cells engineered to knock out miR-21 locus (miR-21KO) and parental isogenic wild type (WT) cells, as well as 6 Biliary Tract Cancer (BTC) cell lines that were NGSed for a panel of 64 genes. Library included 484 small molecules that were screened at 3 doses against each cell line in triplicate. Transient miR-21 inhibition was achieved by reverse transfection. Stable doxycycline-activated miR-21 expressing clones of BTC and CRC cells were generated with doxycycline-inducible TRIPZ lentiviral vectors.

Results: Twenty four drugs reduced cell viability compared to DMSO at a greater extent in miR-21KO RKO in comparison to WT cells. Enrichment in HSP-90 inhibitors (including 17-DMAG, 17-AAG and AUY-922) was noticed, suggesting that miR-21 may be involved in resistance to HSP-90 inhibition. HST in BTC cells showed enrichment of HSP-90 inhibitors independently on mutational status. However IC50 to AUY-922 was correlated to baseline miR-21 expression in BTC cells. Transient inhibition of miR-21 enhanced sensitivity to AUY-922 in BTC cells. AUY-922 IC50 was 35nM for WT and 17nM for miR-21KO RKO cells. Knock-out of miR-21 in DLD-1 cells did not change sensitivity to AUY-922 in line with the lower baseline levels of miR-21 in DLD-1 wild type cells. However, the reduced levels of baseline miR-21 made them more sensitive to AUY-922 than RKO. Doxycycline-activated overexpression of miR-21 in miR-21KO DLD-1 cells conferred resistance to AUY-922. When co-cultured with non-infected miR-21KO DLD-1 cells, miR-21 overexpressing miR-21KO DLD-1 cells were able to drive cell growth in presence of AUY-922. To similar extent CCLP with enforced expression of miR-21 were more resistant compared to control cells; inactivation of the induction of miR-21 overexpression recovered sensitivity to the drug. HSP Array blot of AUY-922-treated cells with induced expression of miR-21 showed reduction of the co-chaperone HSP-40 protein expression compared to control cells.

Conclusions: Our data suggest the development of studies looking at the biomarker potential of miR-21 to guide treatment with HSP-90 inhibitors in GI cancers, as well as pursuing the combination with miR-21 inhibitors that may enhance the effect of the drug and avoid toxicity by enabling dose reduction.

#1070

miR-579-3p is a novel master regulator of melanoma progression and drug resistance in metastatic melanoma.

Luigi Fattore,1 Mario Acunzo,2 Giulia Romano,2 Alessandro Laganà,3 Debora Malpicci,4 Maria Elena Pisanu,4 Antonio Maria Grimaldi,1 Franco Fulciniti,5 Carlo Maria Croce,2 Rita Mancini,6 Paolo Antonio Ascierto,1 Gennaro Ciliberto1. 1 _Istituto Tumori Pascale, Napoli, Italy;_ 2 _Department of Molecular Virology, Immunology and Medical Genetics, Columbus, OH;_ 3 _3 Department of Genetics and Genomic Sciences Icahn School of Medicine at Mount Sinai, New York, NY;_ 4 _Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi di Catanzaro "Magna Graecia", Catanzaro, Italy;_ 5 _Istituto Cantonale di Patologia, Locarno, Switzerland;_ 6 _Dipartimento di Medicina Clinica e Molecolare, Sapienza Università di Roma, Roma, Italy_.

Introduction:

Malignant melanoma is the most aggressive form of skin cancer presenting in approximately 50% of cases activating mutations in the BRAF oncogene. For these patients new therapeutic options are represented by the combination of BRAF inhibitors and MEK-inhibitors. However, a major limitation is the occurrence of resistance related in the majority of cases to mutations causing either reactivation of the MAPK/ERK pathway or of the PI3K/PTEN/AKT pathway. This complex scenario is worsened by the absence, in a significant proportion of cases, of new mutations. This suggests the involvement also of epigenetic or post-transcriptional changes at the basis of resistance. We have focused our attention over the past years to adaptive changes occurring in melanoma cells upon exposure to BRAF and/or MEK inhibitors in order to identify new therapeutic targets. Here we hypothesize that miRNAs deregulation upon cell exposure to kinase inhibitors may contribute to the development of drug resistance.

Experimental procedures:

The involvement of specific miRNAs in the establishment of drug resistance in melanoma was investigated through the online software miRò while target predictions were performed with TargetScan and microrna.org. Impact on melanoma cell growth, migration and establishment of drug resistance was assessed through enforced-transient miR overexpression. Tumor biopsies were obtained from patients before therapy with kinase inhibitors or after relapse and used for RNA extraction.

Results:

We identified by bioinformatics analysis a novel miRNA, miR-579-3p, relevant for BRAF mutated melanoma progression and found that it targets both BRAF, the relevant oncoprotein in these melanomas, and MDM2. miR-579-3p expression levels are high in nevi, heavily decline in stage III-IV melanomas and further decrease in melanomacells selected in vitro for resistance to BRAF and/or MEK inhibitors. Through in vitro studies we obtained evidence that mir-579-3p is able to inhibit melanoma cell growth and migration and to induce apoptosis alone or in combination with BRAF and/or MEKi. Moreover we found that miR-579-3p overexpression impairs the establishment of resistance to BRAFi in human melanoma cells. Finally we found this miR to be down-regulated in tumor samples obtained from patients who developed resistance to target therapies and its expression to be inversely correlated to the expression levels of its target genes, BRAF and MDM2.

Conclusions:

Our results strongly demonstrate that miR-579-3p is a novel oncosuppressor miRNA, controlling melanoma progression and development of drug resistance and which could be used as a new therapeutic target for intervention.

#1071

Mir-145 based magnetic nanoformulation for pancreatic cancer therapy.

Saini Setua, Sheema Khan, Murali Mohan Yallapu, Mohammed Sikander, Stephen W. Behrman, Meena Jaggi, Subhash C. Chauhan. _University of Tennessee Health Science Center, Memphis, TN_.

Background: Pancreatic cancer (PanCa) is the fourth leading cause of cancer related deaths in the USA, with a 5-year survival rate of less than 5%. MicroRNAs have been identified as attractive targets for therapeutic intervention. The functional significance of lost microRNAs have been reported in several human malignancies, including PanCa. Therefore, restoring lost miRNA function can provide a potential therapeutic benefit. Prior work has identified microRNA-145 (miR-145) as a tumor suppressor miRNA in pancreatic cancer. The restoration of miR-145 downregulates a number of oncogenes including mucin MUC13, a glycoprotein that is aberrantly expressed in PanCa, and efficiently inhibits tumor growth in mice. The main challenge for successful translation of microRNAs into clinical practice remains an effective in vivo delivery system. The focus of this study was to develop and assess the efficacy of a miR-145 based nanoparticle formulation for PanCa treatment.

Methods: Magnetic nanoparticle (MNP) based nanoformulation of miR-145 (miR-145-MNPF) was developed for the intracellular delivery and sustained release of miR-145. The positively charged polyethyleneimine molecules were used to increase the loading efficiency of miR-145. MUC13 expressing pancreatic ductal adenocarcinoma cell lines (HPAF-II and AsPC-1) were used for the study. Following transfection of miR-145-MNPF, Western blotting and immunofluorescence techniques were used to investigate the effects of miR-145 restoration on number of proteins including MUC13. Additionally, functional studies of the effects of miR-145 restitution using miR-145-MNPF included cell proliferation, colony formation, cell migration, and cell invasion assays.

Results: miR-145 expression was progressively suppressed over the course of development from PanIN I-III to late stage poorly differentiated PDAC. Treatment of cells with miR-145-MNPF led to efficient intracellular delivery of miR-145 mimics as observed through prussian blue staining. This led to the simultaneous upregulation of miR-145 levels in cells as confirmed by qRT-PCR. miR-145 restitution resulted in significant downregulation of target oncogenes including MUC13, HER2, P-AKT and p53 as observed through Western blotting and immunofluorescence techniques. miR-145-MNPF inhibited cell proliferation, clonogenicity, migration, and invasion of PanCa cells. MNPF mediated restitution of miR-145 effectively sensitizes PanCa cells for paclitaxel and TRAIL therapy.

Conclusions: 1) MNP based delivery systems can be efficiently used for microRNA replacement therapy in order to restore lost microRNAs in cancer. 2) miR-145-MNPF efficiently restores miR-145 in pancreatic cancer cells and inhibits growth and invasion of PanCa. 3) miR-145 restitution using miR-145-MNPF may offer a potential therapeutic strategy for pancreatic cancer treatment alone or in combination with other therapies.

#1072

miR-671-5p and miR-638 serve as novel biomarkers for early breast cancer detection.

Xiaohui Tan,1 Woojin Lee,1 Xiaoling Wu,2 Weaam Alshenawy,1 Danielle Soberman,1 Katayoon Rezaei,1 Sana Tabbara,1 Christine Teal,1 Robert S. Siegel,1 Rachel F. Brem,1 Sidney W. Fu1. 1 _George Washington University, Washington, DC;_ 2 _Chengdu Military General Hospital, Chengdu, China_.

Breast cancer progression involves stepwise transition from atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Percutaneous core needle biopsy (CNB) is the standard procedure after an abnormal mammography finding. However, a CNB diagnosis with either ADH or DCIS is often non-conclusive. A definitive diagnosis relies on surgical excision for further pathological analysis to differentiate simple ADH (sADH) from ADH coexisted with advanced lesions such as DCIS and/or IDC (cADH). Therefore, development of reliable molecular biomarkers is essential to avoid unnecessary surgical excision. microRNAs (miRNAs) are single-stranded non-coding RNAs that play an important role in breast cancer progression. A series of miRNAs that are differentially expressed during stepwise transition of breast carcinogenesis were identified earlier, of which miR-638 and miR-671-5p were functionally characterized. The expression of these miRNAs was decreased gradually from ADH, DCIS, to IDC. We hypothesize that these miRNAs may serve as valuable biomarkers following abnormal mammogram and CNB procedure. Using an improved microdissection protocol, we isolated normal, hyperplasia, DCIS, and/or IDC lesions from FFPE tissue of CNB. We also collected patients' blood samples before CNB procedure. In addition, we employed the Human 21T breast epithelial cell lines, which were originally derived from the same patient diagnosed with metastatic breast cancer, including H16N2 (normal mammary epithelial), 21PT (ADH), 21NT (DCIS) and 21MT-1 (IDC) for in vitro model study. Real-time qRT-PCR assay was performed for examination of miRNA expression. Cell proliferation and invasion capability were examined by MTT and Transwell assays, respectively after transfection of candidate miRNAs. EMT markers were evaluated by Western blot and Immunofluorescence assays. In clinical samples, we found a synergistic expression pattern between miR-671-5p and miR-638 in cADH but not in sADH lesions. Interestingly, decreased miR-671-5p expression was detected in cADHs, but not in sADHs in both FFPE and serum samples. To explore the potential function of the two miRNAs in the transition from sADH to cADH, we performed further analysis in the Human 21T breast epithelial cell lines. Forced expression of miR-671-5p significantly inhibited cell proliferation in H16N2, 21PT, 21NT and 21MT-1 series cell lines. Overexpression of miR-671-5p attenuated invasion in 21PT, 21NT, and 21MT-1 cell lines but not in H16N2 cell line. Further, miR-671-5p overexpression resulted in significant suppression of mesenchymal marker, vimentin, and promoted the expression of epithelial marker, E-cadherin, in 21PT, 21NT, and 21MT-1cell lines. Our data suggest that miR-671-5p and miR-638 expression in serum and/or CNB tissue may server as a novel companion screening tool following an abnormal mammogram and a subsequent ADH diagnosis by CNB.

#1073

Modulation of aromatase inhibitor resistance by miRNAs in breast cancer.

Reiner Hoppe,1 Ping Fan,2 Florian Büttner,3 Stefan Winter,1 Heather Cunliffe,4 V. Craig Jordan,2 Hiltrud B. Brauch3. 1 _Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, University of Tuebingen, Germany;_ 2 _Department of Breast Medical Oncology, MD Anderson Cancer Center, University of Texas, Houston, TX;_ 3 _Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, University of Tuebingen, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 4 _Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand_.

The blockade of E2 signaling by either tamoxifen or aromatase inhibitors (AI) is the standard treatment of patients with estrogen receptor (ER)-positive breast cancer. However, acquired resistance to antiestrogen therapies remains a big clinical challenge. The in vitro breast cancer models MCF-7:5C and MCF-7:2A mimic clinical AI resistance in that they grow under tamoxifen, but can rapidly regress with E2 due to the reconfiguration of survival signaling known as E2-inducible apoptosis. This principle is currently explored in clinical trials in order to develop strategies to overcome endocrine resistance. To better understand the biology of AI resistance we previously ascertained a wide range of alterations of stress related pathways including the accumulation of endoplasmic reticulum stress, oxidative stress, and inflammatory stress that occur prior to E2-induced apoptosis (Ariazi et al. 2011; Fan et al. 2013, 2015; Sweeney et al. 2014). In this current work we investigated miRNA expression profiles of the 5C and 2A models (both compared to MCF-7:WS8 reference) in order to further elucidate the molecular scenario that characterizes the AI resistance phenotypes and their susceptibility to E2-induced apoptosis. Using Affymetrix GeneChip miRNA2.0 arrays we identified 184 miRNAs differentially expressed between 2A and 5C (FC > 1.5 or < 1/1.5, P < 0.05). Of these, 30 miRNAs were specific for 2A, 99 for 5C, and 55 overlapped. Common miRNAs include upregulated oncogenic miRNAs of the miR-17-92 cluster (Chr. 13q31) and the paralogous 106a-363 cluster (Chr. X), as well as downregulated tumor suppressive miRNAs such as miR-342-5p. Thirty-four miRNAs that cluster at Chr. 14q32 were overexpressed in 5C cells and highly correlate with the downregulated miR-99a and miR-125b (Chr. 21q21). At the clinical level based on data from The Cancer Genome Atlas (miRNA-Seq v. 3.1.17.0), low expression of miR-31 was associated with poor (HR = 3.0, 95% CI: 1.9-4.8; Padj = 8.7E-5), and low miR-222 expression was associated with better outcome (HR = 0.3, 95% CI: 0.1-0.6; Padj. = 4.4E-3). As miR-31 expression is high and miR-222 expression is low in 5C cells compared to the MCF-7:WS8 reference, we suggest protective roles. Functions of miRNAs were deciphered via analyses of their predicted targets (CLIP-confirmed). Using KEGG and GO databases functional enrichment analyses of 5C and 2A specific miRNA sets revealed pathways associated with cell proliferation for both models including insulin, mTOR, and ErbB signaling as well as immune response and metabolism associated pathways. While the 2A specific miRNA set revealed additional metabolic pathways, the 5C specific miRNA set points to pathways involved in apoptosis. Thus, we confirmed the biological processes inherent to AI resistance and provide critical evidence for miRNA profiles as an important regulatory principle in these AI resistance models.

#1074

A novel synthetic biotinylated microRNA-1207-3p duplex targets the 3'UTR of FNDC1 and inhibits proliferation and migration of prostate cancer cells.

Dibash K. Das,1 J David Warren,2 Olorunseun O. Ogunwobi1. 1 _CUNY Graduate Center/Hunter College, New York, NY;_ 2 _Weill Cornell Medical College, Cornell University, New York, NY_.

Prostate cancer (PCa) is the second leading cause of cancer death in US men and the dysregulation of microRNAs contributes to its development. Recently we discovered that miR-1207-3p, encoded at the PVT1 gene locus on the 8q24 human chromosomal region, is significantly underexpressed in PCa cell lines and directly targets the 3' untranslated region (UTR) of Fibronectin type III domain containing 1 (FNDC1). Moreover, using an oligonucleotide mimic and inhibitor of miR-1207-3p, we established that miR-1207-3p regulates the proliferation and migration of prostate epithelial cells. To discover the molecular targets of miR-1207-3p unbiasedly, a RNA pull-down assay needed to be performed, yet no biotinylated miR-1207-3p mimic was commercially available. Consequently, we designed and synthesized a novel synthetic biotinylated miR-1207-3p duplex, and a novel synthetic biotinylated scramble duplex (patents pending). Prior to using the novel duplexes for molecular target discovery, we compared their effects to that of the previously used commercially available miR-1207-3p mimic and scramble control on the 3'UTR of FNDC1, and PCa cell proliferation and migration. Effects were assessed using the E006AA mildly tumorigenic PCa cell line and its aggressively tumorigenic derivative, E006AA-hT. Using these two PCa cell lines transiently transfected with 3'UTR of FNDC1-containing Luc-Pair™ Duo-Luciferase expressing vector, we found that the biotinylated miR-1207-3p duplex directly targets the 3'UTR of FNDC1 in comparison to the biotinylated scramble duplex. MTT assays revealed that the biotinylated miR-1207-3p duplex significantly decreased the proliferation of both PCa cell lines in comparison to the control biotinylated scramble duplex. Interestingly, the biotinylated miR-1207-3p duplex further decreased the proliferation of the aggressively tumorigenic E006AA-hT by an extra 25%, compared to the decrease in the proliferation of the indolent E006AA, suggesting that it may have some selectivity for aggressive PCa cells. Wound healing assays revealed that the biotinylated miR-1207-3p duplex significantly inhibited migration of both PCa cell lines by about 60% in comparison to the control biotinylated scramble duplex. Notably, the mimic of miR-1207-3p had no effects on the proliferation or migration of these cell lines when compared to scramble controls. In conclusion, to our knowledge, we have designed and synthesized the first synthetic biotinylated miR-1207-3p duplex. Importantly, unlike the mimic of miR-1207-3p, this novel synthetic biotinylated miR-1207-3p duplex significantly inhibits proliferation and migration of PCa cells including those aggressively tumorigenic. Consequently, the novel synthetic biotinylated miR-1207-3p duplex is a useful new tool for miR-1207-3p molecular target discovery, and potentially as a novel therapeutic strategy in PCa.

#1075

Chemically modified gamma PNAs targeting oncomiR-210 as a potential anticancer therapy.

Anisha Gupta, Yanfeng Liu, Elias E. Quijano, W Mark Saltzman, Peter M. Glazer. _Yale University School of Medicine, New Haven, CT_.

The specific aims of this study are to test the hypothesis that new generation PNA molecules targeting miR-210 delivered using nanoparticles will have therapeutic effects in xenograft mouse models and in human cancer cell lines.

MicroRNAs (miRs) are a class of small non-coding RNAs (22-23nt) that regulate the translation and stability of mRNA during post-transcriptional events. miR profiling experiments reveal that certain miRs are widely over-expressed in several cancers, and inhibiting these miRs may offer a promising anticancer therapy. Little is known about how they work to cause cancer. This warrants further investigation to understand the relationship between miRNAs and cancer, but also to capitalize on fact that oncomiRs may be potent therapeutic targets. In particular, miR-210 exhibit differential expression levels in cancers, especially in hypoxic cancer cells, resulting in a demonstrated capability to affect cellular transformation and carcinogenesis by acting as an oncomiR.

Many antisense molecules such as locked nucleic acids (LNA) have been developed that can target specific miRs, but they are few in number and often require further optimization for cellular delivery, off target toxicity, tumor selectivity and enzymatic resistance properties. Unlike most nucleic acids, PNA is a synthetic DNA/RNA mimic in which the phosphodiester backbone is substituted with a neutral N-(2-aminoethyl) glycine backbone. PNA has been shown to bind single-stranded targets sequence-specifically, with high binding affinity, and is not susceptible to protease or nuclease degradation. These salient features make PNAs excellent candidates for targeting miRs. Here, we used a new class of conformationally preorganized gamma peptide nucleic acids (γPNAs), which possess superior properties in terms of solubility, binding affinity, biocompatibility, and in vivo delivery in order to target miR-210. For cellular delivery, we used FDA approved poly(lactic-co-glycolic acid) (PLGA) nanoparticles. γPNA encapsulated in PLGA nanoparticles were evaluated in human cancer cell lines in vitro as well as in xenograft mouse model in vivo. Our results show that nanoparticles containing γPNAs targeting miR-210 cause significant delay in tumor growth in a xenograft mouse model. Further, histopathological analyses also support the enhanced antimiR-210 activity of γPNA. Hence our work will provides a) a framework for a antimiR-210 based therapeutics at the clinical level using γPNA; b) a promising clinically translatable system for selectively targeting cancer using a PLGA based nanoparticle system which can be scaled up easily; c) a step forward for personalized therapy for cancers by inhibiting specific miRs. In addition, use of hydrophilic chains (MP) at the γ position will improve the pharmacokinetic properties of PNA for preclinical and clinical studies for oncomiR-based therapeutics.

#1076

Plasma microRNAs associated with overall survival in patients with hepatocellular carcinoma treated with galunisertib (LY2157299 monohydrate), an inhibitor of transforming growth factor-β receptor1.

Shawn T. Estrem,1 Michael Man,1 Xuekui Zhang,1 Duytrac Nguyen,1 Danni Yu,1 Michael M. Lahn,1 Ann Cleverly,2 Durisala Desaiah,1 Sandrine Faivre,3 Gianluigi Gianneli,4 Karim A. Benhadji5. 1 _Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN;_ 2 _Eli Lilly and Company, Erl Wood, ELCL, United Kingdom;_ 3 _Hopital Beaujon, Clichy, France;_ 4 _Internal Medicine, Immunology, Infectious Diseases, University of Bari Medical School, Bari, Italy;_ 5 _Lilly France, Paris, France_.

Background: Galunisertib, a selective transforming growth factor-β receptor1 inhibitor, is being investigated in clinical trials for hepatocellular carcinoma (HCC). MicroRNAs (miRs) are small (~22 nucleotide) non-coding RNAs that regulate expression of targeted genes, and are secreted by cells into blood. miRs are differentially expressed during HCC progression, differentiation, and response to therapy. We hypothesize that circulating miRs may be useful to identify patients who benefit from galunisertib treatment.

Patients and Methods: Plasma samples from HCC patients (n = 105) treated with galunisertib were analyzed for miR expression. Patients were enrolled in part A, of a multi-part single-arm study in 2nd-line HCC (phase II trial NCT01246986/JBAK). All patients had elevated alpha-fetoprotein (AFP) levels at baseline (AFP ≥1.5 x ULN). The median OS of this cohort of patients was 7.2 mo. Eighty percent of patients received prior sorafenib treatment. Plasma samples were collected during patient screening, cycle 1 day 1 (pretreatment), and cycle 2 day 14. The Exiqon RT microRNA PCR Human panel I+II was used to measure 752 miRs. Expression levels of detectable miRs and their association with overall survival (OS) were investigated.

Results: Low plasma levels of miR-665 (HR = 0.50, p = 0.001, q = 0.13), miR-320d (HR = 0.44, p = 0.001, q = 0.13), miR-320a (HR = 0.47, p = 0.001, q = 0.13), and miR-130b-3p (HR = 0.44, p = 0.002, q = 0.13) are associated with better survival. Whereas, low plasma levels of miR-451a (HR = 2.0, p = 0.002, q = 0.13), miR-let7g-5p (HR = 2.3, p = 0.002, q= 0.13), and miR-18a-3p (HR = 2.0, p = 0.002, q = 0.13) are associated with poor survival.

To assess within patient baseline biological variation of miR expression, a comparison of the expression of 369 miRs in patients (n = 42) with 2 pre-treatment samples was performed. The proportion of miRs attaining statistical significance was smaller than what we would expect by chance.

Conclusions: Circulating miRs may serve as easily accessible markers to identify HCC patients who may benefit from galunisertib treatment, which requires confirmation in randomized controlled study. Given the low intra-patient variability measured at baseline for most of the miRs, circulating miRs may represent reliable molecular markers with prognostic and/or predictive utility.

#1077

Targeting oncogenic miR-17-92 primary transcripts by LNA gapmeRs in multiple myeloma: Molecular findings and therapeutic potential.

Eugenio Morelli,1 Lavinia Biamonte,1 Cinzia Federico,1 Maria Teresa Di Martino,1 Nicola Amodio,1 Maria Eugenia Gallo Cantafio,1 Niels M. Frandsen,2 Maria Rita Pitari,1 Daniele Caracciolo,1 Annamaria Gullà,1 Marco Rossi,1 Pierosandro Tagliaferri,1 Pierfrancesco Tassone1. 1 _UMG of Catanzaro, Catanzaro, Italy;_ 2 _Exiqon A/S, Vedbaek, Denmark_.

There is emerging evidence that miR-17-92 plays a crucial role in c-Myc driven tumorigenesis of multiple myeloma (MM). We attempted to antagonize its full-oncogenic activity by targeting primary transcripts (pri-miR-17-92) with RNase H-triggering antisense oligonucleotides (LNA gapmeRs). Specifically, 7 different molecules were generated and screened for their ability to inhibit pri-miR-17-92 expression. The most effective molecule, henceforth named miR17-92-i-PT, was selected for furher investigation. As assessed by qRT-PCR, transfection of MM cells with miR17-92-i-PT resulted in downregulation of both pri-miR-17-92 and all 6 mature miRNA transcripts. Importantly, miR17-92-i-PT inhibited MM cell proliferation more effectively than inhibitors targeting each cluster's miRNA. Lack of relevant off-target effects was demonstrated by specifically-designed negative (LNA mixmeRs) and positive (not-phosphorothioated LNA gapmeRs) control oligos, and a dominant-negative genome-edited cell line by CRISPR/CAS9 technology is currently under development. Importantly, treatment of MM cells with naked miR17-92-i-PT resulted in miR-17-92 downregulation. Low micromolar concentrations of miR17-92-i-PT significantly affected survival of MM patient plasma cells (n=14) and MM cell lines (n=15), while the viability of CD138+ cells from MGUS patients (n=3) or PBMCs from healthy donors (n=3) was not impaired. Further, a synergistic in vitro activity with Bortezomib,Carfilzomib or Melphalan was demonstrated. Molecular perturbations in MM cells exposed to miR17-92-i-PT were firstly investigated by gene expression profiling (HTA 2.0, Affimetrix). Ingenuity pathway analysis (IPA) of differentially expressed genes highlighted alteration of relevant molecular functions, including "cell cycle", "DNA replication, recombination, and repair", and "cell death and survival", which were validated by functional assays. Moreover, impairment of BRD4 activity was indicated by upstream regulator analysis (Zscore<2). Indeed, Hexim1 -the endogenous antagonist of BRD4- was upregulated upon treatment with miR17-92-i-PT and is currently being validated as a direct target of miR-18a, miR-19a and miR-19b cluster members. Consistent with a BET bromodomain inhibitor-like activity, miR17-92-i-PT downregulated c-Myc, thus indicating a novel feedback loop between miR-17-92 and c-Myc. Finally, we found a significant in vivo anti-tumor activity of systemically delivered naked miR17-92-i-PT against human MM xenografts in SCID/NOD mice. Different animal models were utilized, including SCID-hu and mouse orthotopic models, besides classic subcutaneous xenografts. Lack of systemic toxicity was demonstrated both in mice (balb/c) and in monkeys (cynomolgus monkeys). Overall, our results provide the rational framework for development of miR17-92-i-PT-based therapies in MM.

#1078

Micro RNAs as promising therapeutic targets for anti-metastatic therapy in colorectal cancer.

Takashi Kawai,1 Takeshi Nagasaka,1 Yoshiko Mori,1 Tomokazu Fuji,1 Fumitaka Taniguchi,1 Keisuke Kimura,1 Toshiaki Toshima,1 Kazuya Yasui,1 Yuzo Umeda,1 Hiroshi Tazawa,1 Ajay Goel,2 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Baylor Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX_.

Background: Colorectal cancer (CRC) is the third most common cancer and is a leading cause of cancer related death worldwide and arises by accumulation of genetic and epigenetic alterations. Recently, it has been demonstrated that microRNAs (miRNAs) play critical roles in tumor progression in various cancers. In this study, we tried to find miRNAs associated with distant metastasis and poor outcome and evaluate those miRNAs as therapeutic target for advanced CRC.

Methods: MiRNA array was done on CRC specimens derived from tumors with various gene status (KRAS/BRAF/microsatellite instability [MSI] status) with reference of their normal mucosal specimens. The array analysis revealed that a set of miRNAs was specifically down-regulated in CRC with BRAF V600E mutation without MSI, considered to be the poorer outcome. To examine whether the set of miRNAs causes distant metastasis or not, we investigated malignant potential of cell lines transfected and knock-downed the set of miRNAs by siRNA. Expression status of ZEB2 and Epithelial-Mesenchymal Transition (EMT) markers, E-cadherin, were evaluated. In addition, we analyzed expression level of the set of miRNAs in a cohort of 67 stage IV CRCs (TNM staging system by UICC 7th) by quantitative reverse transcription PCR using a comparative Ct method and examined association of target-miRNA fold change (tumor/normal tissue) and their clinocopathilogical findings. Patient survival analysis were performed by Cox proportional hazard model and Kaplan-Meier analysis.

Results: In vitro, cell lines transfected the set of miRNAs significantly reduced their malignant potentials. In contrast cell lines knock-downed the set of miRNAs by siRNA obviously increased their malignant potentials. Finally, to confirm our results obtained from in vitro assays, we analyzed expression level of the set of miRNAs in a cohort of stage IV CRCs. Of 67 patients with stage IV CRCs, 15 patients showed KRAS mutation, 4 patients showed BRAF mutation. CRCs with the lower expression level of the set of miRNAs showed poor outcome compared with those with the higher expression level by Cox proportional hazard model and Kaplan-Meier analysis.

Conclusions: Our data indicate that miRNAs associated with BRAF mutant CRCs is promising prognostic biomarkers and therapeutic targets for anti-metastatic therapy in CRC.

#1079

Pharmacokinetics and biodistribution of LNA-i-miR-221 in NOD.SCID mice and Cynomolgus monkeys.

Maria Teresa Di Martino,1 Maria Eugenia Gallo Cantafio,1 Boye S. Nielsen,2 Mariamena Arbitrio,3 Annamaria Gullà,1 Chiara Mignogna,1 Massimo Breda,4 Niels M. Frandsen,5 Pierosandro Tagliaferri,1 Pierfrancesco Tassone1. 1 _Magna Graecia University, Catanzaro, Italy;_ 2 _Bioneer A/S, Hørsholm, Denmark;_ 3 _ISN-CNR, Catanzaro, Italy;_ 4 _Aptuit, S.r.l., Verona, Italy;_ 5 _Exiqon, Vedbaek, Denmark_.

miRNAs therapeutics is based on the use of oligonucleotide engineered to efficiently replace or sequester endogenous miRNAs. miR-221 is upregulated in several tumors, including multiple myeloma (MM), where it promotes cancer cell proliferation mainly via targeting the negative regulators of G1/S cell cycle progression p27 and/or p57. We previously demonstrated that direct miR-221 inhibition by the use of an original LNA inhibitor, LNA-i-miR-221, induces significant anti-MM activity and upregulates canonical targets in vitro and in vivo.To assess pharmacokinetics (PK)/pharmacodynamics of LNA-i-miR-221, we set-up, and report here, novel assays for oligo quantification in plasma, urine and tissues of NOD.SCID mice and Cynomolgus monkeys. We investigated the LNA-i-miR-221 PKs by UHPLC-MS/MS, following solid-phase extraction, in plasma and urine, and tissue uptake by in-situ hybridization (ISH). In particular, PK profile was calculated by UHPLC-MS/MS after single dose i.p injection of LNA-i-miR-221 (25 mg/kg) in mouse and the Tmax was identified at 1.5 hours with Cmax of 1110 ng/mL. Three hours after bolus injection, the plasma concentration was about 60% reduced and the molecule was not detectable at later time-points. The plasma exposure of the oligo showed an AUClast of 2330 h*ng/mL. Similarly, in monkeys the LNA-i-miR-221 plasma concentration following a single i.v. bolus injection (8.75 mg/kg) peaked at a Tmax of ½ hour, with a Cmax of 23600 ng/mL. The plasma exposure of the oligo showed an AUClast of 49300h*ng/mL, C0 of 61400 ng/mL and the t1/2 was 12.8 hours. Urine PK evaluation was performed in monkey on samples collected from 2 up to 48 hours. The excreted LNA-i-miR-221 was quantified equal to 0.68 mg with a total urinary recovery of approximately 2.68% of the amount injected (25,375 mg). Specifically, the urinary concentration/time-points plot shows the excretion of 9394,5 ng/mL (1.63% of recovery) in the interval of 2-6 hours. In addition, murine tissues analyzed by ISH revealed strong signals and long-lasting accumulation of the oligo in mice vital organs together with upregulation of the main canonical miR-221 target p27, as assessed by IHC. In addition, ISH analysis excluded that the LNA-i-miR-221 crosses the blood-brain barrier. All together PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary excretion of the intact molecule, with a very long tissue half-life and biological activity. Moreover, we report preliminary toxicity analysis in non-human primates. No changes in the monkeys' behavior, body weight or food consumption were observed in treated animals.In conclusion our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a favorable PK, long-lasting tissue bioavailability and good safety profile in mice and in monkeys, providing the rationale for translation of this novel miR-221 inhibitor in early clinical investigation

#1080

Kras downregulation by nanoparticle delivery of siRNA reduces tumor growth in mice.

Matthew Strand, Hua Pan, Xiuli Zhang, Julie Grossman, Peter Goedegebuure, Timothy Fleming, William E. Gillanders, Samuel A. Wickline, Ryan C. Fields. _Washington University in St. Louis, Saint Louis, MO_.

Introduction

Small-interfering RNA (siRNA) is an attractive option for delivering precision therapy to cancer patients because of the potential for highly-specific control of gene expression. Attempts to utilize siRNA in vivo have been hampered by its short circulating half-life, limited cellular uptake, and cellular confinement within endosomes. To overcome these barriers, we packaged siRNA within serum-stable, cell-penetrating, and endosomolytic peptide-based nanoparticles (NPs) to deliver siRNA to human and mouse pancreas and colorectal cancers. In a small pilot study, twice weekly administration of KRAS siRNA-NP to mice bearing human colorectal cancers significantly reduced tumor growth compared to controls.

Methods

NP uptake was assessed in cell lines utilizing fluorescent siRNA-NPs in combination with fluorescence microscopy and flow cytometry. Cell lines were incubated with KRAS-siRNA NP and KRAS knockdown was assessed by quantitative PCR. Mice bearing tumors derived from these cell lines were injected intravenously with fluorescent NP, and localization and uptake were assessed with an in vivo imaging system (IVIS) and flow cytometry. Immune deficient mice were inoculated subcutaneously with a human colorectal cancer and half were treated for 4 weeks with twice weekly IV injections of KRAS siRNA-NP (1 nmol KRAS siRNA per dose).

Results

On average, fluorescent siRNA was detected in more than 93% of human and mouse pancreas and colorectal cancer cells (n=5 cell lines) after 24 hours of incubation. Exposure of colorectal cancer cells to KRAS siRNA-NP in vitro produced a conservatively-estimated 70% downregulation of KRAS. Tumors derived from all cell lines were strongly fluorescent 2 hours after IV injection of fluorescent NP and signal persisted beyond 30 hours. The liver and kidneys also expressed fluorescent signal by IVIS. 86% of tumor cells derived from tumors exposed to fluorescent-NP by IV injection expressed fluorescent signal 24 hours post-injection. Only 18% and 14% of liver and kidney cells, respectively, were fluorescent. Compared to control mice, tumor size was reduced in mice bearing colorectal cancer that received KRAS-siRNA NP IV injections. Eighteen days after initiation of treatment, tumor volumes in the control and treatment groups were 229.5 mm³ and 460.25 mm³ respectively (p < 0.05). Hematologic, liver and kidney function tests revealed no differences between the treatment and control groups.

Conclusions

Peptide NPs are rapidly and efficiently taken up by human and mouse pancreas and colorectal cancer cells both in vitro and in vivo. Incubation of cancer cells with peptide NP-packaged KRAS-siRNA reduces KRAS expression in vitro. Treatment of tumor-bearing mice with KRAS-siRNA NP leads to a statistically significant reduction in tumor growth compared to control mice. This delivery platform overcomes traditional limitations of siRNA and may be generalizable to target other putative drivers of tumor progression.

#1081

**Activity of microRNA replacement reagent, MRX34, in multiple myeloma** in vivo **model.**

Woo June Jung,1 Youngil Koh,2 Sinil Kim,3 Hyejoo Park,1 Sung-Soo Yoon1. 1 _Seoul National Univ. Cancer Research Inst., Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Mirna Therapeutics, Austin, TX_.

Background: Despite recent progress in multiple myeloma (MM) treatment with the advent of new targeted anticancer agents such as Bortezomib, Lenalidomide and, Thalidomide MM still remains incurable. To improve clinical outcome of MM patients, many research have been trying to develop novel targeted drugs. Recent studies demonstrated that microRNA Replacement reagent could be a promising therapeutic strategy in patients with hematologic malignancies. Hence, we tried to examine the efficacy of MRX34, a microRNA-34 replacement reagent, in MM animal model.

Methods: We examined the anti-tumor activity of a MRX34 against 3MM cell lines (LP1, MOLP-8, and U266), and investigated the survival effects through which this novel agent shows anti-MM activity. For an animal study, we used NRG (NOD-Rag1null IL2rgnull, CB-17 SCID) mice that were injected with MM cells via tail vein. MOLP-8, LP1 and U266 cells were injected Intravenous 106 cells of NRG. When tumors were measurable, mice were assigned to 3 treatment groups receiving the vehicle alone (control), MRX34 0.3mg/kg treatment group and, MRX34 1mg/kg treatment group were given Intravenous 5 times a week.

Results: Median survival of mice injected with LP1, MOLP-8 and U266 cell lines were 28days, 29 days and 34days respectively without any treatment. When mice were treated with MRX-34, median survival was 33days, 37days and 42days respectively. There was a significant survival advantage with MRX-34 treatment in MOLP-8 and U266 cell lines. For monoclonal proteins, mice treated with MRX34 group are suppressed M-protein level almost 30% less than M-protein.

Summary / Conclusion: Our data indicated that MRX34 had beneficial effects in multiple myeloma animal model. Mice treated with MRX34 showed M-protein suppression and survival advantage. Our results warrant further clinical studies using this novel agent in MM.

#1082

High-throughput,purification-free, multiplexed profiling of circulating miRNA for discovery,validation, and diagnostics.

Jessica Tytell, Issac Stoner, Michael Tackett, Graeme Doran, Conor Rafferty, Andreas Windemuth, Daniel Pregibon. _Abcam, Inc., Boston, MA_.

To address the needs for circulating miRNA biomarker validation, we developed the Multiplexed Circulating microRNA assay. This assay enables the detection of up to 68 microRNA targets per sample in 96-well format with readout on standard flow cytometers and analysis with an included bioinformatics software package. The Circulating microRNA assay combines particle-based multiplexing, using patented Firefly hydrogel particles, with single-step RT-PCR signal amplification using universal primers. Thus, the Circulating microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the Circulating microRNA assay are displayed and interpreted using our included Firefly Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid discovery and validation of biomarkers, we have generated fixed panels for Oncology, Cardiology, Neurology, Immunology, and Liver Toxicology. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating and tumor microRNA profiles using the Firefly Circulating microRNA Assay Fixed Panels.

Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and validation of microRNA biomarker signatures from fluid specimens.

#1083

Temporal trends in microRNAs during subchronic aflatoxin dosing and modulation by the chemopreventive oleane triterpenoid, CDDO-Im.

Merricka C. Livingstone,1 Bill D. Roebuck,2 Natalie M. Johnson,3 Thomas W. Kensler,4 John D. Groopman1. 1 _Johns Hopkins University School of Public Health, Baltimore, MD;_ 2 _Dartmouth Medical School, Hanover, NH;_ 3 _Texas A &M School of Public Health, College Station, TX; _4 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Liver cancer is the second leading cause of cancer death worldwide: risk factors include the viruses, HBV and HCV, and the environmental carcinogen aflatoxin B1 (AFB1). Chemoprevention strategies that activate genes controlled by the KEAP1-NRF2 pathway, such as CDDO-Im, afford complete protection against AFB1-induced hepatocarcinogenesis in rats. The opportunity to deploy these types of agents in high-risk populations would be advanced by the validation of biomarkers reflecting their efficacy. MicroRNAs (miRNAs) affecting gene expression are important in normal physiology and have been shown to be frequently dysregulated in cancer. It is our hypothesis that specific rat liver miRNAs are biomarkers that temporally track with AFB1-induced liver cancer, and are modulated in animals that receive complete protection by CDDO-Im.

MiRNAs were isolated from archived liver tissue of rats (Johnson et al., CaPR, 2014) that were dosed with AFB1 (200 μg) for 28 consecutive days. Two additional groups received either vehicle only or AFB1 \+ CDDO-Im (30 μmol). After RNA isolation (miRCURY tissue, Exiqon), samples were profiled by RNA sequencing (Illumina). Fourteen miRNAs were selected based on dynamic range and level of detection for validation by RT-qPCR (TaqMan microRNA assay).

MiRNA profiling from animals at the end of the carcinogenic 28-day dosing regimen revealed an increased total number of miRNAs due to AFB1 exposure alone (n≈500) compared to control. Those miRNAs displaying a greater than 10 fold increase in expression between control and AFB1 treatment groups were examined in detail: 541-5p, 34a-5p, 127-3p, 205, 434-3p, 429, 411-5p, 181c-3p, 200b-3p, 221-3p. RNA-seq data showed that these were all upregulated in the AFB1-treated samples. Co-treatment with CDDO-Im shifted expression levels back to control. The miRNAs 192-3p, 92a-3p, 26b-3p, and 375-3p were expressed consistently in all treatment groups and were selected for normalization. Expression levels of the 14 candidate miRNAs were then examined in liver samples obtained from rats after 7, 14 and 21 doses AFB1 and compared to levels determined at week 4 (28 doses AFB1). Quantitative analysis demonstrated an increase in expression of the panel of miRNAs due to AFB1 exposure over the dosing period, potentially indicating a role in early carcinogenesis. MicroRNA expression showed a varying trend over time in animals treated with AFB1 plus CDDO-Im. Three invariant miRNAs (miR-375-3p, 92a-3p, 192-3p) showed little change during the 28-day exposure period.

In conclusion, we have identified a panel of miRNAs that are upregulated and track with AFB1 exposure. These changes are abrogated by treatment with CDDO-Im and thus reflect the protective efficacy of the intervention. Supported by T32ES007141-31A1 and CA197222.

#1084

Clinical significance of p63/miR-138 deregulation in tongue squamous cell carcinoma.

Zehang Zhuang, Yun Wang, Cheng Wang, Nan Xie, Yue Wu, Jinsong Hou, Xiqiang Liu, Hongzhang Huang. _Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, China_.

BACKGROUND and AIMS: p63 is critical for the development and maintenance of stratified epithelial tissues. The role of p63 in tumorigenesis remains poorly defined. This study aims to determine the levels of p63 and miR-138 expression both in vitro and in vivo, and further to analyze their correlation with the clinicopathological parameters and prognosis in patients with tongue squamous cell carcinoma (TSCC).

METHODS: Levels of p63 and miR-138 in TSCC and paired para-cancerous tissues were investigated by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. The correlations of p63 and miR-138 expression with the clinico- pathological parameters and prognosis of patients with TSCC were analyzed using SPSS 13.0 software package. Expression of p63 and miR-138 in TSCC cell lines (n=5) was measured using immunoblot analysis and qRT-PCR, respectively. p63 was expressed transgenically, or knocked down with shRNAs in SCC9, SCC25, SCC15 and UM1 cell lines, and effects on cell invasion and migration were evaluated.

RESULTS: Elevated level of p63 and reduced level of miR-138 was observed in TSCC tissues in comparison with paired para-cancerous tissues (p < 0.05). In particular, a more elevated expression for p63 appeared in the invasive tumor front compared with superficial or central parts of the primary tissues (p < 0.05). A negative correlation between p63 and miR-138 expression can be observed in the TSCC tissues. Statistical analysis revealed that p63 and miR-138 expression level are associated with cell differentiation, cervical lymph node metastasis status and clinical stage as well in patients with TSCC. COX regression analysis shows that p63 over-expression was correlated with reduced overall survival. P63 can be served as an independent prognostic factor for patients with TSCC. These observations were confirmed in vitro, in which ΔNp63 is the predominant p63 isoform expressed in the TSCC cell lines, and a negative correlation between p63 and miR-138 expression can be observed (p < 0.05). Ectopic transfection of p63 led to reduce miR-138 expression and enhanced cell invasion and migration. On the contrary, knockdown of p63 enhanced miR-138 expression and suppressed cell invasion and migration. Furthermore, transfection of miR-138 partly reversed cell invasion and migration induced by p63 overexpression.

CONCLUSION: The dysregulation of p63 and miR-138 are common molecular events in TSCC progression. These findings suggest that p63 and miR-138 may collaboratively play a role in tongue carcinogenesis.

#1085

Serum miR-30e and miR-223 are promising non-invasive biomarkers for hepatocellular carcinoma.

Ratna B. Ray,1 Sourav Bhattacharya,1 Robert Steele,1 Adrian M. Di Bisceglie,1 Sounak chakraborty2. 1 _St. Louis University, St. Louis, MO;_ 2 _University of Missouri-Columbia, Columbia, MO_.

Hepatocellular carcinoma (HCC) is the world's third most common cancer and causes 600,000 deaths annually. Early detection is the key for improving the survival of HCC patients. Current diagnostic markers, such as alpha fetoprotein or fibroscan have their own limitations. Investigating serum microRNA as biomarkers for early diagnosis of cancer has become an important tool in clinical research. In this study, we tested the hypothesis that serum microRNAs (miRNAs) can be used as a potential biomarker for HCC. We analyzed serum miRNA using reverse transcriptase-quantitative PCR (qRT-PCR) from serum samples. Our study revealed that two miRNAs, miR-30e and miR-223, were expressed at significantly lower levels (P < 0.003) in the sera of HCC patients as compared to healthy volunteers. Further, expression of these miRNA was compared between sera from chronic liver disease (CLD) and HCC patients. miR-30e and miR-223 expression was significantly lower in HCC sera as compared to sera from CLD patients. Both miRNA expression levels were lower in HCC liver biopsy specimens as compared to normal liver RNA. Taken together, our results indicated that serum miR-30e and miR-223 are useful biomarkers of HCC irrespective of etiology.

#1086

Elevated level of circulating microRNA-99a correlates with serous epithelial ovarian cancer and can be used as a potential biomarker.

Akihiko Yoshimura. _Osaka University Hospital, Suita, Osaka, Japan_.

[Objective] There is a critical need for improved diagnostic markers for ovarian high grade serous cancer (HGSC). Emerging evidence have shown that microRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and these are present in circulating microvesicles such as exosomes. The purpose of this study was to identify which miRNAs are highly produced from HGSCs and to analyze whether circulating miRNA can discriminate patients with HGSC from healthy volunteers.

[Methods] Secreted exosomes from serous ovarian cancer cell lines were collected and exosomal miRNAs were extracted. miRNA microarray was performed and several elevated miRNAs specific to ovarian cancer cells were picked-up. Among these, we focused on miR-99a in this study. Sera were collected from 9 consecutive patients with HGSC and 5 healthy volunteers. Expression level of miR-99a was determined by miRNA RT-qPCR.

[Results] miRNA microarray revealed several miRNAs were highly expressed in exosomes in patients with HGSC. In patients, serum miR-99a levels were significantly increased (7.2 fold) compared with healthy controls. Receiver operating curve (ROC) analysis for miR-99a showed that at the cut-off of 2.1 times compared with control, the sensitivity and specificity of this marker were 77.8 % and 100 %, respectively for detecting HGSC (AUC = 0.84). Serum miR-99a levels decreased after surgery by 0.2 fold, indicating that serum miR-99a was originated from ovarian cancer.

[Conclusion] Elevated serum miR-99a were detected in sera of patients with HGSC and reflected the disease condition. This miRNA profiling in serum can be used as a diagnostic biomarker, extending its utility to screening asymptomatic populations.

#1087

Circulating microRNAs as metastasis biomarkers in oral cavity and oropharyngeal squamous cell carcinomas.

Flavia Maziero Andreghetto,1 Mariana Barbosa de Souza,2 Raquel Ajub Moyses,3 Tatiana Natasha Toporcov,4 Fabio Daumas Nunes,5 Marcos Brasilino de Carvalho,2 Patricia Severino1. 1 _Hospital Israelita Albert Einstein, Sao Paulo, Brazil;_ 2 _Laboratorio de Biologia Molecular, Hospital Heliopolis, Sao Paulo, Brazil;_ 3 _Disciplina de Cirurgia de Cabeça e Pescoço, Hospital das Clínicas da Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil;_ 4 _Departamento de Epidemiologia, Faculdade Saúde Pública, Universidade de Sao Paulo, Sao Paulo, Brazil;_ 5 _Departamento de Estomatologia e Patologia, Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo, Brazil_.

MicroRNAs are small non-coding RNA molecules with roles in gene expression regulation and implications in cancer initiation and progression. Cell-free microRNAs detected in body fluids, and in particular in plasma, are of clinical interest due to their possible use as biomarkers. Despite the fact that their reliable quantification depends on several technical parameters, there is great clinical interest in this approach due to its simplicity and low risk to the patient. Squamous cell carcinoma of the head and neck is one of the most common cancer types worldwide, with survival rates of about 50% in 5 years. Tobacco and alcohol consumption are the most important risk factors and, currently, the presence of cervical lymph node metastases remain the strongest prognostic factor for this cancer type. In this study we identified plasma microRNAs associated with squamous cell carcinomas of the oral cavity and oropharynx as well as with the presence of cervical lymph node metastases. We evaluated the expression of 179 microRNAs in plasma from 45 patients (28 presenting cervical lymph node metastases at diagnosis and 17 with no lymph node metastasis) and 15 healthy controls. Samples were matched according to age, sex, drinking and smoking habits. Among microRNAs mostly expressed in tumor samples, some had been already detected in oral squamous cell carcinoma tissues, such as miR-210, while others, such as miR-573, are still poorly studied. Comparisons between oral and oropharyngeal carcinomas yielded several differences, in accordance with molecular distinctions between these cancer types. MicroRNAs possibly involved in the metastatic process were identified when comparing plasma samples from individuals presenting lymph node metastasis at diagnosis and plasma from patients who were metastasis-free. Among these molecules, miR-192 and miR-574 were identified as more expressed in plasma from individuals with metastatic oropharyngeal cancer. We conclude that plasma microRNAs associated with specific clinical conditions or cancer sub-sites may help in the diagnosis and prognosis assessment of head and neck squamous cell carcinomas.

#1088

Efficient detection of germ cell cancer related miRNAs in serum using the TaqMan miRNA ABC purification bead kit in combination with the Taqman advanced miRNA assays.

Leendert HJ Looijenga, Ton Van Agthoven, Ad Gillis. _Erasmus Medical Ctr., Rotterdam, Netherlands_.

Malignant germ cell tumors, also known as germ cell cancers (GCC), are the most frequent cancer in young Caucasian males, with an increasing incidence during the last decades. They are clinically and histologically subdivided into seminomas and nonseminomas. The last can be composed of different elements, including embryonal carcinoma (the stem cell component), teratoma (TE) (somatic lineage) as well as yolk sac tumor (YST) and choriocarcinoma (CHC) (extra-embryonic lineages). The currently used serum markers, informative for both diagnosis as well as follow-up are AFP and hCG, predominantly indicative for the presence of the YST and CHC components. Recently, we reported that a selected set of miRNAs are highly expressed in all histological elements of GCC, with the exception of TE, including the miR 371-3 cluster. In addition to the presence in the primary cancers, they can also be identified in the serum of patients with such a malignancy, outperforming the classical markers AFP and hCG (Gillis and Rijlaarsdam et al. Molec. Oncol. 2013; Rijlaarsdam et al. Andrology, 2015). Here we demonstrate the power of the TaqMan Advanced miRNA Assays (PN 478007, Applied Biosystems) in combination with the use of the TaqMan miRNA ABC Purification Kit (PN 4473087, Applied Biosystems), starting with 50 µL human serum. The protocol includes polyadenylation of the miRNA targets at the 3' end, followed by an adapter ligation at the 5' end. Universal forward and reverse primers are used for amplification, followed by detection using the Applied Biosystems 7500 RealTime PCR System. Quality controls were added using dilution experiments of the cell lines, also included as inter-plate control, as well as for cDNA synthesis and RT-PCR efficiency. The non-human spike-ins ath-miR159a and cel-miR-39-3p were added as exogenous controls, while hsa-miR-30b-5p was used as endogenous control for normalization (reference). In total 30 GCC-related sera as well as 10 normal controls proved the informativity as well as efficiency of the developed pipeline in a standardized laboratory setting.

#1089

Cardiac glycosides affect miRNA expression profiles in renal cell carcinoma cell lines.

Elke Nolte,1 Sven Wach,1 Izabella T. da Silva,2 Arif Ekici,1 Frieder Müller-Uri,3 Wolfgang Kreis,3 Julio Vera-Gonzales,1 Bernd Wullich,1 Helge Taubert,1 Xin Lai1. 1 _Univ. Hospital Erlangen, Erlangen, Germany;_ 2 _Universidade Federal de Santa Catarina, Florianópolis, Brazil;_ 3 _Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany_.

Background: Renal Cell Carcinoma (RCC) is the ninth most common cancer worldwide, with about 338,000 new cases diagnosed in 2012. Especially metastatic renal cell carcinoma has still despite surgical cytoreduction, VEGF-tyrosine kinase inhibitors, mTOR inhibitors and first promising results of immunotherapies a very poor prognosis what implies an urgent need for additional therapies. Cardiac glycosides have come into the focus as potential cancer treatment option. They can have anti-proliferative and apoptotic characteristics in several cancer cell lines but affect only in a much lesser extent normal cell lines. MicroRNAs are small non-coding RNAs of 17-25 nucleotides length that widely exist and they play an important regulating role in many normal and pathophysiological cellular processes, as cell proliferation, differentiation, induction of apoptosis, tumorigenesis and tumor progression.

Material and Methods: Four renal cell carcinoma cell lines were treated with three different cardiac glycosides in the IC50 concentrations during 72h. For the investigation of miRNA expression miRNA microarray expression analyses were performed. In silico target gene and pathway enrichment analyses were performed.

Results: An overlap between all treatments and all cell lines could be detected for two mature miRNAs and two premature miRNAs. Pathway enrichment analysis identified MAPK and axon guidance pathways. Moreover, we looked for genes predicted to be regulated by the deregulated miRNAs in all cell lines at all treatments in the MAPK and the axon guidance pathways.

Conclusion: The identification and characterization of deregulated miRNAs after cardiac glycoside treatment could give insight in their gene regulation and thus help to clarify the molecular basis of their anti-proliferative and apoptotic effects.

#1090

microRNA regulation of Nrf2 and the antioxidant response in breast cancer cells following redox therapy.

Francesca Mascia, Kaytee L. Pokrzywinski, V. Ashutosh Rao. _Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD_.

Mitoquinone (MitoQ), a redox-active mitochondrially targeted chemotherapy, activates autophagy through the production of reactive oxygen species (ROS) in breast cancer cells. Studies have also shown a concomitant activation of Nrf2 and the antioxidant response with MitoQ treatment. Nrf2 (nuclear factor (erythroid-derived 2)-like 2) is a transcription factor that regulates the antioxidant response element. Drugs that stimulate Nrf2 are ideal for the treatment of diseases induced by oxidative stress; however, over-activation of Nrf2 may promote tumor growth and diminish the effects of redox active chemotherapies. Therefore, understanding the relationship between autophagy and oxidative stress is crucial for determining the fate of cancer cells exposed to redox-active oncology agents. The aim of this study was to evaluate microRNA (miRNA) as a link between oxidative stress and autophagy through modulation of Nrf2 activity. microRNA are endogenous small (18-22 base pairs long) double stranded RNA that bind to the 3' UTR of target mRNA. miRNA function through translational repression via mRNA recycling and degradation. To this aim we set out on finding miRNA that suppress the antioxidant response, modulated by Nrf2, in cancer cells after redox therapy with the goal of inducing a death response. After 24 hours of non-target pool or Nrf2 silencing, using small interfering RNAs, MDA-MB-231 cells were treated with DMSO or MitoQ for 18 hours. RNA was extracted and miRNAs were identified using Illumina RNA sequencing. DEseq revealed 97 miRNAs that were differentially expressed at α < 0.05. Biologically significant miRNAs were then distinguished by using a Log2 fold change cut off of -1.0 < and < 1.0, which identified 39 microRNAs. Since miRNA should be high when their target is low we were predominantly interested in the 15 miRNAs that were down-regulated (DMSO:MitoQ). 7 of the 15 down-regulated miRNAs were also down-regulated in the presence of Nrf2 silencing in both the DMSO and MitoQ treatments. We are currently investigating these 7 miRNAs in terms of the specific effect they have on the Nrf2 pathway during redox therapy. 

### MicroRNAs as Oncogenes and Tumor Suppressors

#1091

miR-125 family of miRNAs mediates prostate cancer cell proliferation and migration.

Sinead T. Aherne,1 Fiona O'Neill,2 Stephen F. Madden,3 Martin Clynes,2 Conor C. Lynch4. 1 _Dublin City University & H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland;_ 3 _Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland;_ 4 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL_.

Background: Prostate cancer is the second most common malignancy in American men and causes ∼27540 deaths per annum. microRNAs (miRNAs) are a class of short non-coding RNAs that regulate the expression of protein-coding genes via either mRNA degradation or translational repression. These small molecules are known to be aberrantly expressed in prostate cancer (PCa) and possess both tumor suppressive and oncogenic properties. miR-125 is a highly conserved miRNA family comprised of three homolog members hsa-miR-125a, hsa-miR-125b-1, and hsa-miR-125b-2. Members of this family have been attributed both disease suppressing and promoting functions in certain contexts however their role in PCa has yet to be fully elucidated.

Methods: miRNA expression was evaluated in a publicly available dataset (GSE21032) comprised of 28 normal, 98 primary PCa and 13 metastasis samples. miR-125 expression was assessed in a panel of 8 prostate cell lines comprising PrEC prostate epithelial cells, RWPE-1, BPH1, DU145, LNCaP, C4-2B, PC3, and PC3-2M cell lines using the Qiagen miScript system. MirWalk was used to identify predicted targets of the miRNAs. miR-125 expression was transiently inhibited in LNCaP, C4-2B, PC3, and PC3-2M cell lines using miScript miRNA inhibitors and miRNA and target mRNA expression, cell proliferation and migration were measured post-transfection.

Results: Analysis of human data revealed miR-125a-3p was significantly up-regulated in metastasis samples compared to both primary PCa and normal tissues (log fold change (FC) 1.743, p-value 9.01E-08 logFC=1.536 p-value=8.80E-08 respectively). Next, we validated miR-125 family member expression in prostate cancer cell lines compared to PrEC and RWPE-1 prostate epithelial cells. Most notably, we observed that miR-125b-2 expression was significantly higher in all prostate cancer cell lines. Inhibition of miR-125b-2, significantly reduced the proliferation of the LNCaP, C4-2B, and PC3-2M cell lines. Moreover, inhibition of this miRNA significantly impaired prostate cancer cell migration by 43.8% (p-value=0.003). Using MirWalk, we examined potential targets for regulation by miR-125b-2 and identified the potent tumor suppressor Quaking family as top hit. Our results show that inhibition of miR-125b-2 significantly enhanced the expression of Quaking family members (1.79 fold).

Conclusions: Our results demonstrate that the miR-125 family of miRNAs plays a role in regulating prostate cancer cell proliferation and migration, potentially by mitigating Quaking activity.

#1092

miR-603 acts as a tumor suppressor in triple-negative breast cancer and inhibits cell proliferation, invasiveness and tumorigenesis by targeting elongation factor 2-kinase (eEF2K).

Recep Bayraktar, Martin Pichler Pichler, Cristina Ivan, George Calin, Bulent Ozpolat. _UT MD Anderson Cancer Center, Houston, TX_.

Triple Negative Breast Cancer (TNBC) is characterized by heterogeneous disease due to its unique molecular profile, aggressiveness, distinct metastatic patterns and lack of molecular targets such as ER, PR and HER2/neu for effective targeted therapies including tamoxifen and herceptin reduced sensitivity to chemotherapies, representing unmet therapeutic challenge. Eukaryotic elongation factor-2 kinase (eEF2K) is an atypical calcium/calmodulin dependent serine/tyrosine kinase that regulates protein synthesis through phosphorylation of EF2. Recently, we reported that eEF2K promotes TNBC cell proliferation, colony formation, migration/invasion, angiogeneis and tumorigenesis and drug resistance by inducing PI3K/Akt, mTOR, IGF1R, Src/FAK, C-myc,, Bcl-2, CyclinD1, and VEGF (Tekedereli 2012). We also demonstrated that in vivo therapeutic targeting of eEF2K by systemically administered liposomal eEF2KsiRNA nanoparticles significantly inhibited growth of highly aggressive MDA-MB-231 tumor xenografts in and enhanced the efficacy of standard chemoagents (ie, doxorubicin, paclitaxel), representing a potential molecular target in TNBC. However, currently the transcriptional regulation of eEF2K is unknown. Here, we report that eEF2K expression is highly upregulated in TNBC cells and associated with poor patient survival and clinical outcome in these patients.. Thorough extensive analysis of databases and predictive algorithms, and in vitro and in vivo correlation studies we identified that miR-603 inversely correlated with eEF2K and demonstrated miR603 as the first microRNA that directly binds to the 3'-UTR of eEF2K gene and suppresses its expression, though gene reporter assays. Introduction of mutations to the miR-603 binding site completely reversed eEF2K expression in gene reporter assays. We also showed that ectopic expression of miR-603 in TNBC cell lines resulted in the inhibition of cell growth, colony formation, migration and invasion of TNBC cells, recapitulating the effects of eEF2K inhibition by siRNA. Systemic administration of miR-603 mimics incorporated in liposomal nanoparticles (0.3 mg/kg once a week for 4 weeks) in an orthotopic xenograft TNBC mouse model led to significant inhibition of eEF2K protein expression and tumor growth. Analysis of the tumors after the end of study reveled that miR-603-induced downregulation of eEF2K expression was correlated with Src and Akt. In conclusion, our findings suggest for the first time that miR-603 function as a tumor suppressor and loss of its expression contributes to upregulation of eEF2K, tumor growth and progression of TNBC and miR-603 based gene targeting strategy may be used to control eEF2K in TNBC, representing a novel molecularly targeted therapy.

#1093

The melanoma-enriched microRNA miR-4731 regulates genes involved in cell cycle and the melanosome.

Mitchell S. Stark,1 Lisa Tom,1 Glen M. Boyle,2 Vanessa F. Bonazzi,3 Adrian C. Herington,3 Pamela M. Pollock,3 Nicholas K. Hayward2. 1 _University of Queensland, Brisbane, Australia;_ 2 _QIMR Berghofer Medical Research Institute, Brisbane, Australia;_ 3 _Institute of Health and Biomedical Innovation, Brisbane, Australia_.

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanomas from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines. Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) were predicted to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the 'cell cycle' and 'melanosome' pathways. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as result of miR-4731 overexpression. We therefore speculate that loss of miR-4731 expression supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels together with members of the cell cycle pathway, which warrants further investigation.

#1095

S100A4 is post-transcriptionally inhibited by miR-505-5p and miR-520c-3p in colorectal.

Giridhar Mudduluru,1 Katharina Ilm,2 Steffen Fuchs,2 Ulrike Stein1. 1 _Experimental and Clinical Research Center, Charité University Medicine Berlin and Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association and German Cancer Consortium (DKTK), Berlin, Germany;_ 2 _Experimental and Clinical Research Center, Charité University Medicine Berlin and Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany_.

The calcium binding protein S100A4 induces epithelial mesenchymal transition (EMT), migration, invasion, angiogenesis and metastasis. Its induced expression is reported in several cancer types and correlates with poor prognosis. S100A4 is transcriptionally regulated by Sp1, AP1 and CCAAT/enhancer binding protein β transcription factors and its regulation epigenetically controlled by CpG hyper methylation. However, apart from S100A4 functional and transcriptional regulatory aspects, post-transcriptional regulation is not yet elucidated. In this study, we show that specific microRNAs (miR) target the 3'UTR of S100A4. Luciferase-reporter assays with wild-type and mutated miR-505-5p and miR-520c-3p seed sequences of S100A4-3'UTR confirmed the specificity of targeting. Similarly, overexpression of these miRs reduced the S100A4 and inhibition of these miRs function increased the S100A4 protein significantly. Moreover, co-transfection of either S100A4 expressing vector or siRNA-S100A4 along with the miR-505-5p and miR-520c-3p significantly altered the S100A4 mediated migration and invasion in colorectal cancer (CRC) cell lines (p=0.05). Both these miRs were down-regulated in CRC cell lines and 5 Aza treatment increased these miRs expression and reduced the S100A4 protein amounts. Moreover, in a panel of CRC resected patient tissues (n=59), miR-520c-3p significantly down regulated (p=0.03). miR-520c (located in Chr 19, in a big cluster) upstream region was checked for methylation status using methylation specific PCR and combined bisulfite restriction analysis (COBRA) in CRC cell lines, metastatic and non-metastatic CRC resected tissues. We found the upstream promoter region hyper methylated irrespective of metastasis status of patients and in all CRC cell lines checked. 5 Aza treatment of S100A4 less or no expressing cells line Colo206 and Colo320 showed induced miR-520c expression and reduced S100A4 protein in Colo320. Colo206 cells did not show any alternations in expression of S100A4. Taken together, our findings demonstrate that S100A4 is post-transcriptionally regulated by tumor suppressor genes miR-505c and miR-520c and especially miR-520c expression is epigenetically silenced in CRC.

#1096

microRNA-193a-3p acts as a tumor suppressor in BRAF-mutated colorectal cancer.

Hidekazu Takahashi,1 Masanobu Takahashi,1 Shin Takahashi,2 Hideki Shimodaira,2 Chikashi Ishioka2. 1 _Tohoku University Hospital, Sendai, Japan;_ 2 _IDAC, Tohoku University, Sendai, Japan_.

The aim of this study was to identify microRNAs (miRNAs) specifically dysregulated in BRAF-mutant colorectal cancer, a malignant subtype of colorectal cancer. The genome-wide miRNA expression analysis in a screening set (n = 30) identified 22 miRNAs specifically dysregulated in BRAF-mutant cancers. Among the top 5 dysregulated miRNAs, the validation analysis using another set of patients (n = 34) confirmed miR-31 and miR-135b as significantly up-regulated miRNAs and miR-193a-3p as a significantly down-regulated one. Moreover, functional assays supported its tumor-suppressive role in colorectal cancer. Finally, the expression status of miR-193a-3p correlated with clinical outcome of patients treated with anti-EGFR antibodies. Taken together, these results show a novel functional role of miR-193a-3p as a tumor suppressive miRNA in colorectal cancer, particularly with BRAF mutations. Further studies are needed to clarify the precise molecular mechanisms as to how this miRNA is involved in colorectal tumorigenesis, which could help further develop a therapeutic strategy for colorectal cancer.

#1097

The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression.

Jyotika Varshney,1 Nicholas J. Slipek,1 John Osborne,1 Adrian Chang,2 Anne E. Sarver,1 Ingrid Cornax,1 Gerry M. O' Sullivan,1 Subbaya Subramanian,1 David A. Largaespada1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Macalester College, Saint Paul, MN_.

Osteosarcoma is the most common primary bone malignancy that affects adolescents. Around 30% of patients with localized osteosarcoma and 70% of patients with metastasis will experience treatment failure within 5 years of diagnosis. The complex biology of osteosarcoma and astounding genetic heterogeneity has made it challenging to identify effective new gene targets and therapeutic agents. Our studies found an overall overexpression of a microRNA cluster, miR-17-92 microRNAs in human primary osteosarcoma compared to normal bone. We learned that upregulation of this miR-17-92 cluster simultaneously silences a suite of key tumor suppressors. Using data from a novel spontaneous osteosarcoma mouse model and genetic screen, we discovered miR-17-92 targets, such as PTEN, PTRPD and SRGAP2, which are potential tumor suppressor genes. Specifically blocking miR-17-92 function in osteosarcoma cells reduced their migration and ability to grow larger tumors in immunodeficient mice compared to the controls. Also, knockdown of miR-17-92 cluster microRNAs led to increase in the levels of PTPRD and SRGAP2 in cells as well as tumors; further suggesting that miR-17-92 is targeting these genes. We also performed gain-of-function of miR-17-92 studies in a poorly aggressive osteosarcoma cell line and found that overexpression of miR-17-92 leads to ability to grow in an anchorage independent manner and form tumors in immunodeficient mice, both features that are lacking in the parental line. Ongoing RNA sequencing studies on miR-17-92 target transcripts in osteosarcoma cells, and functional analyses of miR-17-92 deletion mutants, will be presented. In an attempt to target miR-17-92 miRNA expression, we have tested small molecules. Our data suggests that triptolide, a diterpenoid epoxide, inhibits MYC expression and downregulates miR-17-92 miRNAs resulting in upregulation of several tumor suppressor driver proteins including PTEN, PTPRD, and SRGAP2. Together, our data suggests that upregulation of miR-17-92 miRNAs contributes to osteosarcoma progression and triptolide inhibits miR-17-92 expression. These data have implications for how sarcomas develop in general and suggest a new way to treat cancer by targeting microRNAs using small molecules.

#1098

MiR-203 suppresses cutaneous squamous cell carcinoma growth and targets the myc oncogene.

Warangkana Lohcharoenkal,1 Masako Harada,1 Jakob Lovén,2 Florian Meisgen,1 Ning Xu Landén,1 Lingyun Zhang,1 Liisa Nissinen,3 Veli-Matti Kähäri,4 Mona Ståhle,1 Enikö Sonkoly,1 Dan Grander,1 Marie Arsenian-Henriksson,1 Andor Pivarcsi1. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _Whitehead Institute for Biomedical Research, Cambridge, MA;_ 3 _MediCity Research Laboratory, Turku, Finland;_ 4 _University of Turku and Turku University Hospital, Turku, Finland_.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer in man and accounts for approximately 20% of non-melanoma skin cancers. Although most cSCC are benign, poorly differentiated cSCC poses a significant risk of metastasis and death. To date, little is known about the difference in molecular background between low-risk and high risk cSCC. MicroRNAs are short regulatory RNAs that can regulate gene expression and cellular functions. Here we demonstrate for the first time that the expression of miR-203 in cSCC correlates with tumor differentiation grade, being down-regulated in poorly but not in moderately or well differentiated cSCC. In vitro, miR-203 causes a delay in G1 to S phase transition and suppresses cell proliferation in human cSCC cells. Furthermore, miR-203 suppresses scratch-wound closure, cell migration, cell invasion, colony forming ability and angiogenesis-inducing capacity of cSCC cells. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 reveals dramatic changes in gene networks related to carcinogenesis, with significant suppression of genes with known oncogenic functions (e.g. PCNA, EGFR, HGF). Using luciferase reporter assays and site-specific mutagenesis, we identify c-MYC as a novel target of miR-203. Highlighting the importance of c-MYC within miR-203-regulated gene network, in rescue experiments overexpression of c-MYC reverses miR-203-induced growth arrest in cSCC. In vivo, overexpression of miR-203 in cSCC cell lines result in reduced xenograft tumor volume and decreased vessel density. Together our data show that miR-203 acts a tumor suppressor in cSCC, affecting several oncogenic and angiogenic mechanisms. Importantly, its restoration may provide therapeutic benefit in particular in poorly differentiated cSCC.

#1099

Up-regulated miR-96-5p inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma.

Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Itoh. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer death worldwide, especially in Asia and sub-Saharan Africa. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and can act as tumor suppressors or oncogenes. To identify miRNA involved in HCC, we performed a genome-wide miRNA gene expression analysis using the human miRNA microarray (Agilent) in paired tumor and non-tumor tissues from patients with primary HCC. The array-based miRNA expression profiles were validated by quantitative PCR. We identified miR-96-5p as the most significantly upregulated miRNA in HCC tumors. Although miR-96-5p has been recognized as an oncogenic miRNA, its role in the pathogenesis of HCC remains unknown. Our study showed that the inhibition of miR-96-5p reduced proliferation of HCC cells and induced apoptosis. Furthermore, we found that the forkhead box O1 (FOXO1) gene was a target of miR-96-5p. FOXO1, a transcription factor, plays an essential role in cell fate decisions. FOXO1 can induce apoptosis through mitochondria-dependent and mitochondria-independent pathways. Collectively, our results suggested that miR-96-5p may function as an oncogenic miRNA through inhibiting apoptosis by targeting FOXO1 in HCC.

#1100

Targeting ITGA3/ITGB1 signaling by tumor-suppressive microRNA-223 inhibits cancer cell migration and invasion in prostate cancer.

Ichiro Fukumoto,1 Akira Kurozumi,1 Yusuke Goto,1 Ryosuke Matsushita,2 Mayuko Kato,1 Rika Nishikawa,1 Shinichi Sakamoto,1 Tomohiko Ichikawa,1 Naohiko Seki1. 1 _Chiba University Graduate School of Medicine, Chiba, Japan;_ 2 _Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan_.

BACKGROUNDS:

Most patients initially respond to androgen-deprivation therapy, but eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). Several clinical trials for CRPC have shown limited benefits, eventually resulting in disease progression and metastasis. Our recent study of microRNA (miRNA) expression signature in CRPC revealed that miR-223 expression was significantly reduced in PCa tissues, suggesting miR-223 functions as a tumor suppressive miRNA in PCa cells. The aim of this study was to investigate the functional significance of miR-223 and to identify its regulated oncogenic genes in PCa.

METHODS:

Expression levels of miR-223 were evaluated in PCa clinical specimens and PCa cell lines (PC3 and PC3M) by quantitative real-time PCR methods. Gain-of-function studies (cell proliferation, migration and invasion assays) were performed using transfection of mature miR-223 into cancer cells. Genome-wide gene expression analysis and in silico analysis were applied to investigate molecular targets regulated by miR-223 in PCa cells. A luciferase reporter assay was carried out to determine whether 3' UTR of target genes have actual biding sites for miR-223. To investigate the functional role of target genes in PCa cells, loss-of-function studies by silencing of these genes were performed.

RESULTS:

The expression levels of miR-223 were significantly reduced in PCa clinical specimens and cell lines compared to non-cancerous prostate tissues. Restoration of miR-223 significantly inhibits cancer cell migration and invasion in PCa cells. Integrin alpha-3 (ITGA3) and integrin beta-1 (ITGB1) were identified as direct target genes of miR-223 by microarray, in silico studies, and luciferase reporter assays. ITGA3 and ITGB1 interact with multiple extracellular matrix proteins and mediate several survival signaling into the cancer cells. Knockdown of ITGA3 and ITGB1 significantly inhibited cancer cell migration and invasion in PCa cells by regulating downstream signaling.

CONCLUSIONS:

Downregulation of miR-223 was a frequent event of PCa cells and it functions as a tumor suppressor via targeting ITGA3 and ITGB1. Overexpression of ITGA3 or ITGB1 might be contributed to PCa oncogenesis and metastasis. Elucidation of tumor-suppressive miR-223 regulated molecular pathways and targets could provide new information on potential therapeutic strategies in PCa.

#1101

The role of miR-601 in prostate cancer progression.

Jessica L. Fleming, Erica Hlavin Bell, Kathryn Andrews, Arnab Chakravarti. _The Ohio State University, Columbus, OH_.

Prostate cancer (PCa) is the second most common cancer among men worldwide. In order to advance treatment options for these men, it is crucial to understand the molecular underpinning behind this cancer. Previously, our group identified miR-601 as a predictive biomarker in PCa. Few studies to date have functionally validated molecular biomarkers and currently nothing is known about the function of miR-601 in PCa. Based on our previous publication, we hypothesized that miR-601 is playing a role in PCa progression. In order to assess this, in vitro experiments were conducted in three PCa cell lines, DU-145, PC-3, and LNCaP. miR-601 was over-expressed and knocked down in each cell line and assays were performed evaluating cell proliferation, colony formation, and apoptosis. Additionally, to provide more information regarding miR-601-associated pathways, we identified putative gene targets of miR-601 using in silico prediction programs, microrna.org and TargetScan.org and evaluated top gene targets in vitro. The results of our experiments indicate that miR-601 is playing a role of a tumor suppressor. Over-expression of miR-601 in cell lines resulted in a significant reduction in cell viability. This was confirmed both by MTS as well as colony formation assays. We looked into the mechanism behind the reduction in cell viability and found that cells over-expressing miR-601 had reduced levels of full-length caspase-3, signifying that these cells are undergoing apoptosis. Two putative gene targets of miR-601 were identified and investigated in vitro, SIRT1, a histone deacetylase known to be both an oncogene and tumor suppressor, and BCL2L2, an anti-apoptotic gene known to be an oncogene. SIRT1 and BCL2L2 had strong scores on both online prediction programs as likely targets of miR-601. Our in vitro results confirmed this. We saw reduced mRNA and protein expression of these targets in cells over-expressing miR-601. Our data thus far suggest that miR-601 is acting as a tumor suppressor in PCa. Targeted therapies for miR-601 and/or its targets may be promising in the treatment of PCa, however additional work is needed to warrant this.

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

#1102

miRNAs are key mediators of crosstalk between estrogen and progesterone regulating breast cancer invasiveness.

Thomas B. McFall. _Wayne State University, Detroit, MI_.

Even though estrogen receptor (ER)-positive breast tumors are exquisitely sensitive to anti-estrogen therapy, the disease is virtually incurable when it has progressed to ER+ distal metastasis. Therefore a better understanding of regulatory processes in ER+ breast tumor invasiveness is important. Invasive ER+ breast tumors frequently express high levels of the progesterone receptor (PR) isoform A (PR-A), relative to PR-B. We have previously reported that E2 strongly represses in vitro invasiveness below concentrations of E2 corresponding to median plasma and breast tissue levels post-menopause. Progesterone, at concentrations corresponding to postmenopausal levels, completely abrogates inhibition of invasiveness by E2. The ability of progestins to rescue invasiveness from estrogen regulation is exclusively mediated by PR-A. In contrast, at the high concentrations associated with luteal phase and pregnancy levels, progesterone induces invasiveness independent of E2 through PR-B. Interventions that target PR-A are limited by the fact that functional PR-A is needed to protect against E2-induced uterine dysplasia and hyperplasia. In order to attenuate PR-A dependent invasiveness, it is necessary to identify and target mediators of specific cross-talk between E2/ER and progestin/PR-A in the context of hormonal regulation of invasiveness. As micro-RNAs (miRs) are known to play a major role in the actions of E2, we investigated two possible modalities for miRs to mediate this cross talk: (1) Progesterone/PR-A could induce expression of critical miRs that attenuate suppressive effects of E2 on invasiveness; (2) E2-regulated miRs that directly influence invasiveness may be counter-regulated by progesterone/PR-A. Using microarray analysis and quantitative PCR in several ER+ cell lines, we identified five miRs (miR-6805, miR-584, miR-1228, miR-501, and miR-668) that were activated by progestin exclusively by PR-A, but were not regulated by E2. Whereas these miRs could serve as a signature of hyperactive PR-A, loss-of-function studies using miR inhibitors indicated that they did not play a functional role in the ability of PR-A to regulate invasiveness. An additional ten miRs were regulated by E2/ER (activated or repressed), in a manner that was counter-regulated by progestin/PR-A. Two of these miRs (miR-92a, and miR-26b) were identified using miR inhibitors to have a direct role in mediating the ability of progesterone to rescue invasiveness from E2 regulation. In conclusion, this study has provided a potential diagnostic miR signature of hyper-active PR-A and has also identified two miRs that could potentially be targeted to attenuate breast tumor invasiveness supported by progesterone/PR-A.

#1103

Tumor suppressive role of a novel microRNA at frequently deleted chromosomal locus 8p21 in prostate cancer.

Nathan Bucay, Kiran Sekhon, Shahana Majid, Varahram Shahryari, Soichiro Yamamura, Z. Laura Tabatabai, Kirsten Greene, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng, Sharanjot Saini. _UCSF VA Medical Center, San Francisco, CA_.

Background: A frequent genomic alteration in prostate cancer (PCa) is the loss of chromosome (chr) 8p21 in approximately 30%-50% of cases. Genomic deletions of this region increase significantly with tumor grade and are associated with tumor progression and poor prognosis suggesting its role in PCa progression. A common region of loss of heterozygosity (LOH) has been mapped to the chr8p21 locus that harbors prostate-specific NKX3.1 homeobox gene. Recent genomic studies suggest that this region harbors alternative tumor suppressor genes apart from NKX3.1 that have largely remained elusive. We propose a novel, paradigm shifting hypothesis that this frequently deleted locus is associated with a cluster of microRNA genes- miR-3622a/b- that are lost in prostate cancer and play an important mechanistic role in PCa progression and metastasis. We previously demonstrated that miR-3622a plays a crucial role in PCa epithelial-to-mesenchymal transition by direct targeting of ZEB1 and SNAI2. Here we demonstrate the role of miR-3622b in prostate cancer.

Methods: miRNA expression profiling was performed in microdissected prostate cancer tissues and matched adjacent normal regions by real-time PCR. To assess the functional significance of miR-3622b in PCa, we overexpressed miR-3622b/ control miRNA (miR-CON) in PCa cell lines (Du145, PC3, LNCaP) followed by functional assays.We also examined the therapeutic potential of synthetic miR-3622b mimics in vivo in a prostate cancer xenograft mouse model.

Results: Expression analyses in a cohort of prostate cancer clinical specimens showed that miR-3622b expression is frequently lost in prostate cancer. Further, loss of miR-3622b expression significantly correlated with poor survival outcome and tumor progression. miR-3622b overexpression significantly decreased cellular viability and clonogenicity in PCa cell lines supporting an anti-proliferative role for this microRNA. miR-3622b overexpression also led to significant reduction of invasiveness and migration in PCa cell lines. In vivo studies demonstrated that miR-3622b overexpression induced regression of established prostate tumor xenografts.

Conclusions: Collectively, these data suggest that miR-3622b plays a tumor suppressive role in PCa. We propose that frequent loss of miR-3622a/b at chr8p21 region by genetic and epigenetic mechanisms promotes prostate cancer progression and metastasis. This study supports a novel concept that connects a long standing observation of frequent loss of a chromosomal region with a novel miRNA cluster in prostate cancer.

#1104

MicroRNA miR-31 regulates oral squamous cell carcinoma cell migration by targeting critical enzyme of peroxisomal lipid metabolism.

Yi-Shiuan Lai,1 Hsuan Liu,2 Ting-Wen Chen,3 Shu-Jen Chen,4 Hua-Chien Chen,4 Bertrand Chin-Ming Tan5. 1 _Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan County, Taiwan;_ 2 _Department of Cell and Molecular Biology, Chang Gung University, Taoyuan County, Taiwan;_ 3 _Molecular Medicine Research Center, Bioinformatics Center, Chang Gung University, Taoyuan County, Taiwan;_ 4 _ACT Genomics, Co., Ltd., Taipei City, Taiwan;_ 5 _Department of Biomedical Science, Chang Gung University, Taoyuan County, Taiwan_.

MicroRNAs (miRNAs) are small endogenous non-coding RNAs that play important roles in a variety of cellular processes, such as growth, differentiation and metabolic homeostasis. Dysregulation of microRNA has been linked to the development of various types of human diseases, including cancer. Our previous study has identified that miR-31 is significantly up-regulated in oral squamous cell carcinoma (OSCC) tissues. Further gain- and loss-of-function analyses revealed that miR-31 promotes OSCC by enhancing the migration and invasiveness of OSCC cells but not cell proliferation. However, the exact role of miR-31 in OSCC neoplastic development, especially metastasis, has not been explored. Computational microRNA target prediction and pathway analysis showed an enrichment of putative targets in lipid metabolism pathway. In this study, we aimed to investigate the role of miR-31 in the regulation of lipid metabolism and to establish the relationship between lipid metabolism and tumor pathogenesis in OSCC cells.

First, we demonstrated that ACOX1, the rate-limiting enzyme in peroxisomal β-oxidation, is the direct target of miR-31. We further analyzed publicly available mRNA and miRNA deep sequencing datasets and found that the expression levels of miR-31 inversely correlate with those of ACOX1. Furthermore, we discovered that depletion of miR-31 significantly decreased the intensity of cellular lipid droplets (LDs), and knockdown of ACOX1 conversely showed an increase of LD formation. These data suggested that miR-31 may regulate lipid metabolism by targeting ACOX1. In cultured OSCC cells, the migration capability and invasiveness were significantly increased after knockdown of ACOX1. We further confirmed that ACOX1 depletion enhances ERK1/2 expression and phosphorylation and increases the expression levels of MMP9 in OSCC cells. Collectively, our findings suggest that the elevated expression of miR-31 in OSCC cells directly contributes to the down-regulated expression of ACOX1, subsequently leading to accumulation of lipid metabolites and elevated OSCC migration and invasion through ERK pathway.

#1105

Dual tumor suppressors miR-139-5p/-3p derived from pre-miR-139 via targeting matrix metalloprotease 11 (MMP11) in bladder cancer.

Masaya Yonemori,1 Naohiko Seki,2 Ryosuke Matsusihta,1 Kazutaka Miyamoto,1 Hirofumi Yoshino,1 Yusuke Goto,2 Mayuko Kato,2 Akira Kurozumi,2 Msayuki Nakagawa,1 Hideki Enokida1. 1 _Kagoshima University, Kagoshima, Japan;_ 2 _Chiba University, Chiba, Japan_.

Introduction & Objective: The molecular mechanisms of recurrence and muscle invasion process of bladder cancer (BC) are not well understood. Therefore, understanding the molecular mechanisms of metastatic pathways underlying advanced BC using currently available genomic approaches might improve therapies for and prevention of the disease. Our recent study of microRNA (miRNA) expression signature of BC by deep-sequencing revealed that miR-139-5p (guide strand) and miR-139-3p (passenger strand) were significantly downregulated in BC tissues. In miRNA biogenesis, the passenger strand of miRNA duplex is discarded, while the guide strand is retained to direct recruitment of the RISC to target mRNAs. We hypothesis that these miRNAs act as tumor suppressors in BC. The aim of the present study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells.

Material & Methods: Expression levels of miR-139-5p/-3p and its candidate target genes were validated in two BC cell lines (T24 and BOY) and BC clinical specimens (62 BCs and 23 normal bladder epitheliums: NBEs) by real-time PCR methods. Gain-of-function study, cell proliferation, migration, and invasion analyses were performed by using miR-139-5p/-3p transfected BC cell lines. In silico database and genome-wide gene expression analyses were applied for identification of miR-139-5p/-3p targets. Loss-of-function study for its target gene was performed by using siRNAs. Luciferase reporter analyses were employed to validate direct binding between the target gene and miR-139-5p/-3p. Overall patient survival between high and low expression of the target gene was analyzed by the Kaplan-Meier method.

Results: The expression levels of miR-139-5p and miR-139-3p were significantly reduced in tumor tissues and BC cell lines compared with NBEs (P< 0.0001). Restoration of miR-139-5p or miR-139-3p in cancer cells revealed that both miRNAs inhibited cancer cell migration and invasion. Our data demonstrated that the gene coding for matrix metalloprotease 11 (MMP11) was a direct target of miR-139-5p/-3p regulation. Silencing of MMP11 inhibited cell migration and invasion in BC cells. Moreover, Kaplan-Meier analysis revealed that the patients with high MMP11 expression had lower overall survival probabilities than those with low expression (P=0.043).

Conclusions: Aberrant expressed novel miRNAs were identified by deep sequencing of BC miRNA signature. Two mature miRNAs, miR-139-5p and miR-139-3p derived from pre-miR-139 act as tumor-suppressors in BC cells. To the best of our knowledge, this is the first report demonstrating that dual tumor-suppressive miR-139-5p/-3p directly regulate MMP11 in BC cells. The identification of novel target gene regulated by the dual tumor-suppressors may lead to a better understanding of BC and the development of new therapeutic strategies to treat this disease.

#1106

Mir-223-5p works as an oncomir in vulvar carcinoma by TP63 regulation.

Rafael Rocha,1 Beatriz De Melo Maia,2 Iara S. Rodrigues,2 Nayra S. Amaral,2 Hui Ling,3 George A. Calin,3 Fernando A. Soares2. 1 _Federal University of São Paulo, Gynecologic Department, São Paulo, Brazil;_ 2 _AC Camargo Cancer Center, São Paulo, Brazil;_ 3 _MD Anderson Cancer Center, Houston, TX_.

Recently, miR-223-5p was associated with metastasis in HPV negative vulvar samples and in several other tumor types. In the present study, we hypothesized that this microRNA would have a potential impact on in vitro vulvar cancer behavior. We artificially mimicked microRNA expression in vulvar cancer cell line, SW962, derived from lymph node metastasis of vulvar carcinoma, and performed in vitro assays as follows in the result section. Impaired cell proliferation (p<0.01) and migration (p<0.001) were observed when miR-223-5p was overexpressed. In contrast, increased invasive potential of the cells was observed (p<0.004). By Luciferase assay we provide evidence that miR-223-5p targets TP63 through its 3'UTR direct ligation (p<0.05). We also demonstrate that this microRNA is capable to decrease levels of p63 at both mRNA and protein levels (p<0.001, and p<0.0001; respectively). We further demonstrated that low p63 protein expression in samples from patients was correlated with deeper tumor (p<0.0345) invasion and lower overall survival (p<0.0494). Our study highlights the complexity surrounding p63 biological mechanisms and the importance of this protein as a prognostic factor in vulvar cancer. We also point out miR-223-5p overexpression as a putative pathological mechanism of tumor invasion and a promising therapeutic target. Also, it is plausible that the evaluation of p63 expression in vulvar cancer at the biopsy level may bring important contribution on prognostic establishment and in elaborating better surgical approaches for vulvar cancer patients.

#1107

miR-29b acts as an oncogene or tumor suppressor gene depending on the external stimulus.

Erik Vassella, Stephanie Langsch. _University of Bern, Bern, Switzerland_.

microRNAs (miRNAs), short regulatory sequences at the posttranscriptional level, are important mediators of signaling pathways that act as backups of transcriptional control. To identify miRNAs implicated in epidermal growth factor receptor (EGFR) signaling, transformed bronchial epithelial BEAS-2B cells were retrovirally transduced with KRASG12V and alterations in miRNA expression were assessed by microarray analysis. Here we show that miR-29b is significantly induced by mutant KRAS in bronchial epithelial and non-small cell lung cancer (NSCLC) cell lines as well as in primary NSCLC tissue. In agreement with these results, inhibitors of EGFR and MEK resulted in reduced levels of miR-29b while inhibitors of PI3K had no effect. KRASG12V-transduced BEAS-2B cells were significantly more protected from extrinsic apoptosis than control transduced cells, but co-transduction of cells with KRASG12V and anti-miR-29b constructs sensitized cells to apoptosis indicating that miR-29b is a mediator of KRAS-induced resistance to apoptosis. Protection from extrinsic apoptosis was due to enhanced nuclear factor κB (NF-ĸB) activity. The ubiquitin-editing enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) is a negative regulator of NF-ĸB signaling. Enhanced NF-ĸB activity elicited by miR-29b was due to targeting TNFAIP3/A20. Overexpression of miR-29b-refractory TNFAIP3 restored NF-ĸB activity as well as extrinsic apoptosis, demonstrating that TNFAIP3 is a relevant target of miR-29b. Interestingly, miR-29b conferred sensitivity to cisplatin-induced intrinsic apoptosis by targeting Mcl-1. Thus, miR-29b tips the balance from extrinsic apoptosis towards intrinsic apoptosis. Our results indicate that miR-29b can act either as an oncogene or tumor suppressor gene depending on the external stimulus.

#1108

MicroRNA-211 enhances the oncogenicity of oral carcinoma through targeting TCF12 and up-regulating FAM213A expression.

Yi-Fen Chen,1 Kuo-Wei Chang,2 Shu-Chun Lin1. 1 _Institute of Oral Biology, Taipei, Taiwan;_ 2 _National Yang-Ming Univ. Department of Dentistry, Taipei, Taiwan_.

To address the oncogenic roles of miR-211, we generated K14-EGFP-miR-211 transgenic mouse lines. Upon the 4-nitroquinoline 1-oxide (4NQO) induction, the transgenic mice exhibited more extensive and severe tumorigenesis in tongue compared to the controls. 4NQO up-regulated miR-211 expression in oral squamous cell carcinoma (OSCC) cells. miR-211 directly targeted transcription factor 12 (TCF12), which mediated suppressor activities and was drastically down-regulated in OSCC tumors. GeneChip analysis, bioinformatic algorithm, reporter assay and ChIP assay pinpointed that Family with Sequence Similarity 213, Member A (FAM213A), an anti-oxidative molecule, was transcriptionally repressed by TCF12. Knockdown of FAM213A decreased oncogenic activity and ALDH1-positive cell population, and increased oxygen stress in OSCC cells. The expression of TCF12 and FAM213A was inversely correlated in head and neck carcinoma samples according to The Cancer Genome Atlas. OSCC patients carrying tumors with high FAM213A expression tended to have worse survival. Furthermore, 4NQO treatment down-regulated TCF12 and up-regulated FAM213A through modulating miR-211. The transgenic mouse model recaptures the molecular and histopathological changes as seen in human OSCC pathogenesis. This study highlights that miR-211 responds to oncogenic stimuli to promote the neoplastic process of OSCC by targeting TCF12 and up-regulating FAM213A.

#1109

AAGUGC-microRNAs are an integral part of an oncogenic signaling network driving non-small cell lung cancer proliferation.

Yan Zhou,1 Oliver Frings,1 Erik Fredlund,1 Jorrit Boekel,2 Rui M. Branca,1 Lukas M. Orre1. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _Science For Life Laboratory, Stockholm, Sweden_.

microRNA dysregulation is a common feature of cancer cells, but the complex roles of microRNAs in cancer are not fully elucidated. Here we used functional genomics to identify oncogenic microRNAs in non-small cell lung cancer (NSCLC) and to evaluate their impact on response to EGFR targeting therapy. Our data demonstrates that microRNAs with an AAGUGC-motif in their seed-sequence increase both cancer cell proliferation and sensitivity to EGFR inhibitors.

Using miR-372 as a prototypic AAGUGC-miRNA we set out to identify target mRNAs and an explanation to the discovered phenotype. Global transcriptomics, proteomics and target prediction, resulted in the identification of more than 500 candidate target mRNAs, including several tumor suppressors involved in the G1/S transition. The clinical relevance of our findings were evaluated by analysis of public domain data of NSCLC patients, revealing that almost 200 of the candidate target mRNAs showed a negative correlation to AAGUGC-miRNAs. In addition, the analysis of NSCLC clinical data supported the link between this microRNA seed-family and cancer cell proliferation and indicated that high expression of this type of microRNAs is associated with shorter relapse free survival.

Expanding the analysis to include additional types of cancer revealed large differences in expression of AAGUGC-miRNA both within and between cancer types. In general the results from the analysis of NSCLC was replicated in other cancer types, where expression of AAGUGC-miRNAs was associated with decreased mRNA levels of tumor suppressor targets and increased expression of genes involved in proliferation.

In conclusion we propose that AAGUGC-microRNAs are an integral part of an oncogenic signaling network, and that these findings have potential therapeutic implications, especially in selecting patients for EGFR-targeting therapy.

#1110

Inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer.

Shinya Sato, Keisuke Katsushima, Keiko Shinjo, Satoru Takahashi, Yutaka Kondo. _Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan_.

Targeting androgen receptor (AR) by pharmacological intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters epigenetic status of tumor-associated genes, including microRNAs (miRNAs), and affects the behavior of cancers. Here we examined molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment of OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 is not due to downregulation of transcriptional activity of the AR gene, but due to post-transcriptional regulation, namely miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in three prostate cancer cell lines, miR-320a , of which expression was significantly correlated with prognosis of prostate cancers (P=0.0185), was the most closely associated with AR expression. miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR320a significantly abrogated the growth suppression by OBP-801 treatment. Fluorescent in situ hybridization analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells but rarely expressed in prostate cancer cells. In AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased the miR-320 expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent drug as an AR targeting therapy in AR positive prostate cancer regardless of androgen dependency.

#1111

Reprogramming the breast cancer microenvironment using microRNAs that target DNA repair.

Cristina Espinosa-Diez, RaeAnna Wilson, Nathan Kanner, Rebecca Ruhl, Christina M. Hipfinger, Sudarshan Anand. _OHSU, Portland, OR_.

Preclinical and clinical studies have revealed that tumor endothelium is abnormal, resistant to genotoxic stress, and as such, functions as a key determinant of therapeutic responses to radiation and chemotherapy. While it is well established that radiation and chemotherapy cause DNA damage in tumor vasculature, the molecular mechanisms leading to subsequent cell cycle arrest, apoptosis or senescence in vascular cells are poorly understood. Therefore, identifying and understanding factor(s) that mediate DNA damage responses in tumor endothelial cells will provide potential targets for sensitizing tumor vasculature to radiation and other DNA damaging agents and improve their therapeutic efficacy in cancer.

Recent data indicates that microRNAs (miRs) are potent regulators of DNA damage responses in the tumor microenvironment. miRs are short 20-22 nucleotide (nt) RNA molecules that regulate gene expression by binding to partially complementary sites in target mRNAs. Since miRs mediate several physiological processes in endothelial cells, we hypothesized that miRs regulate endothelial (EC) DNA damage responses. We used an expression screen to identify miRs induced by radiation, cisplatin or hydrogen peroxide in human ECs and identified seven specific miRs unique to intrinsic EC apoptosis pathways regulated by genotoxic stress. In vitro gain-of-function assays show that three of them, miR-21, miR-99b and miR-494 lead to endothelial senescence by impairing telomerase function and inhibit sprouting angiogenesis in vitro, in a 3D assay. Strikingly, we observed that these three miRs each target every member of the MRN (Mre11a-Rad50 and NBS1) complex, a critical part of the cellular DNA repair machinery. MRN complex plays a vital role in DNA ds break repair, replication, and telomere maintenance. Pulldown of a mutant RNA Induced Silencing Complex (RISC) from cells transfected the miR mimics enriched for the MRN mRNAs suggesting direct miRNA-MRN complex mRNA binding. Consistent with these results, knockdown of the MRN complex recapitulated the effects of the miRs, reproducing the senescence phenotype, angiogenesis inhibition and also impaired telomerase activity.

Since MRE-11a is upregulated in human breast cancer patients, we asked if there was any differential expression of miR-494 in either the tumor ECs or tumor cells. Interestingly, ISH of a breast cancer tissue array revealed a significant reduction in tumor miR-494 levels compared with the adjacent normal tissue. Furthermore, ectopic expression of miR-494 diminished breast cancer cell proliferation in 2D and 3D.

Our observations indicate that miR-494 behaves as a tumor suppressor microRNA by targeting the MRN complex, inducing senescence, cell cycle arrest and decreases angiogenesis. Therefore, we propose that restoration of these miRs targeting the MRN complex in breast cancer is likely to synergize with DNA damaging agents and decrease tumor burden.

#1112

miR-550a-x fuctions as a tumor suppressor by inhibiting the oncogenic YAP1 in human cancers.

Min Ho Choe,1 Joon Kim,2 Young-Hoon Han,1 Jae-Sung Kim1. 1 _Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea;_ 2 _Laboratory of Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea_.

Although Yes-associated protein 1 (YAP1), an essential downstream effector of hippo pathway, is a potent oncogene and amplified in human cancers, the overexpression mechanism is unclear. Herein, this study shows that miR-550a-x directly inhibits the oncogenic YAP1 and functions as a tumor suppressor in various human cancer cells. Ectopic overexpression of miRNA-550a-x suppressed proliferation and survival of various cancer cells via inducing apoptosis. miR-550a-x also had a capacity to suppress cell migration, epithelial-mesenchymal transition and tumor sphere formation. Using bioinformatics and microarray analysis, we found YAP1 as a putative target gene. miR-550a-x decreased the mRNA and protein levels of YAP1 in various cancer cells and significantly repressed YAP1 activity with its downstream target genes including CTGF and CYR61. The expression level of miRNA-550a-x were inversely correlated with the YAP1 expression in normal and colorectal cancer samples. Therefore, our data suggest that miRNA-550X functions as a tumor suppressor by targeting YAP1, which may provide a novel therapeutic option for cancer treatment.

#1113

Mir-194-3p suppresses tumorigenic potential of glioblastoma stem cells by targeting the canonical NF-kB pathway.

Rajbir Singh, Kamalakannan Palanichamy, John R. Jacob, Arnab Chakravarti. _The Ohio State University, Columbus, OH_.

Glioblastoma (GBM) is the most common tumor of the central nervous system, with its deadly nature owing to high rates of invasion and angiogenesis. A key element in its pathogenesis is a small subpopulation of neoplastic cells called glioma stem cells (GSCs) that are responsible for tumor initiation, maintenance and relapse. Approaches to target GSCs have mostly proved futile due to their chemo- and radio-resistance potential. Given that microRNAs carry a potential to affect both normal and stem cells, we employed a microRNA-based targeted approach to identify the subset of microRNAs that could distinctively affect the stemness of GSCs. Our study tests the hypothesis that GSCs will show an altered miRNA expression upon their differentiation, and their profiling may lead to identification of specific microRNAs that regulate the process. Our initial efforts resulted in identification of hsa-miR-194-3p as one of the potential candidates, which was consistently downregulated in multiple GSCs that we investigated. Stable cell lines overexpressing hsa-miR-194-3p showed a decreased proliferation rate and reduced sphere-forming ability. In silico analysis revealed TAB2, an upstream activator of NF-κB pathway to be the potential target of hsa-miR-194-3p, which was confirmed by luciferase assay and knockdown approaches. The regulatory role of hsa-miR-194-3p was substantiated by its ability to inhibit the NF-κB pathway and the corresponding downstream targets including IL-6 and STAT3. In vivo studies revealed a reduced tumor forming ability when the cell lines overexpressing hsa-miR-194-3p were injected intracranially in NOD/SCID mice, compared with the non-target control. Our results indicate that hsa-miR-194-3p blocked the canonical NF-κB signaling and negatively affected tumor formation. We have carried out this study this in a unique model system expecting it to uncover another layer of complexity that may add a new facet to meet the challenge imposed by the cancer stem cell paradigm. In conclusion, we accentuate the anti-tumorigenic role of hsa-miR-194-3p as a connecting link to both stemness and NF-κB signaling.

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

#1114

Downregulated miR-337-5p is associated with unfavorable features in acute myeloid leukemia and may play a role in modulating the extracellular signal-regulated kinase pathway.

Rosalind Lie, Chi Keung Cheng, Natalie Pui Ha Chan, Rosalina Ip, Nelson Chan, Joyce Cheung, Margaret Ng. _The Chinese University of Hong Kong, Hong Kong_.

We previously revealed that a homozygous deletion T polymorphism in the 3'-untranslated region of the Nucleophosmin (NPM1) gene is associated with adverse outcomes and can independently predict survivals in patients with de novo acute myeloid leukemia (AML). This polymorphism creates an illegitimate binding site for miR-337-5p, which is expressed in different AML subtypes. We speculate that the delT-carrying NPM1 mRNA competes with as yet unidentified oncogenes for miR-337-5p binding, thereby activating their functions in trans and perturbing normal microRNA regulation. In this study, we aim to investigate the clinicopathological and biological significance of miR-337-5p expression in AML. Using real-time RT-PCR, we measured miR-337-5p expression in bone marrow of 156 adult patients with de novo AML excluding acute promyelocytic leukemia. Patients were dichotomized into low and high expression group at the median level for clinicopathological correlations. miR-337-5p levels were significantly reduced in the low expression patient group than the normal controls (n=27) (P<0.001). Low miR-337-5p expression correlated with higher white blood cell counts (P=0.024), lactate dehydrogenase levels (P=0.023), bone marrow blast counts (P=0.001) and FLT3-ITD (P=0.0092) mutation. Using TargetScan, DIANA, PITA and miRDB, three putative oncogenes involved in the extracellular signal-regulated kinase (ERK) pathway, namely RAP2B, MAPK1 and WNK1, were consistently indicated as the potential targets of miR-337-5p. Overexpression of miR-337-5p in OCI-AML3 cells, which have barely detectable miR-337-5p levels, downregulated the expression of all targets. Concordantly, luciferase reporter assay confirmed that miR-337-5p bound to the 3'-untranslated region of these targets and inhibited translation. The ERK signaling pathway is frequently activated in AML and confers pro-survival functions. We demonstrated that overexpression of miR-337-5p in OCI-AML3 cells significantly inhibited proliferation in both WST-1 and colony-forming assay. Together, our findings suggested that reduced miR-337-5p is associated with unfavorable clinicopathological and molecular features in AML. Restoration of miR-337-5p suppressed cell proliferation, potentially by downregulation of genes implicated in the mitogenic ERK signaling pathway.

#1115

Serum miR152 as a novel prognostic biomarker and potential tumor suppressor for early stage non-small cell lung cancer.

Taro Oba, Yuanqing Ye, Liren Zhang, Jing Lin, Emanuela Gentile, Jing Wang, Yang Zhao, Jiang Gu, Ignacio Wistuba, Jack A. Roth, Xifeng Wu, Lin Ji. _MD Anderson Cancer Center, Houston, TX_.

Cancer-derived circulating miRNAs represent a promising new class of circulating biomarkers for cancer detection, prognosis, and therapeutic targets. We analyzed the differential c-miRNA expression levels of ~100 candidate serum miRNAs by quantitative RT-PCR in 174 Caucasian, early stage lung cancer patients and 184 Caucasian healthy controls. Serum miR152 exhibited significant differential expression between cases and controls (P = 0.014). Low levels of serum miR152 expression were consistently associated with an increased risk of lung cancer. The down-regulated miR152 expression was also detected across a panel of 40 NSCLC and normal bronchial epithelial cell lines and in 250 primary tumors by miRNA expression profiling on microarrays. The tumor-derived origin of the c-miR150 was confirmed by an increased level of serum miR152 with increased tumor volumes three weeks after tumor cell inoculation in a human H1299 tumor xenograft mouse model by qRT-PCR assay. Ectopic expression of miR-152 suppressed tumor cell proliferation, colony formation, and cell migration/invasion in NSCLC H1299, H460, and A549 cells transfected by miR-152 expression plasmids. Inhibition of miR-152 expression by a miR-152 inhibitor promoted tumor cell proliferation in HCC15 cells. Overexpression of miR152 in H1299, H460 and A549 cells significantly down-regulated the constitutive expression of the DNMT1 miR-152 target gene and protein, suggesting its potential role in the epigenetic regulation of lung cancer pathogenesis. These results suggest that serum miR-152 may serve as a novel negative predictor for lung cancer risk, recurrence, and survival in early stage NSCLC and function as a potential tumor suppressor miRNA by epigenetic down-regulation of oncogenesis.(This study is partially supported by NIH/NCI grants Lung SPORE 5P50CA070907 and R01CA176568, a CPRIT Grant and a MDACC Moonshot Program Grant)

#1116

Contribution of miR-199a to subtype switching of the SW13 adrenocortical carcinoma cell line.

Tracy Gerona, McKale Davis, Elizabeth Hull. _Midwestern University, Glendale, AZ_.

The human adrenocortical carcinoma SW13 cell line has two subtypes that are phenotypically distinct. The SW13- subtype is highly proliferative and epithelial-like, while the SW13+ subtype is slow growing, motile, and mesenchymal-like. These differences are mediated in part by the post-transcriptional regulation of BRM, which is expressed in SW13+ cells, and not detectable in SW13- cells. While treatment with histone deacetylase inhibitors (HDACi) can force cells to transition from SW13- to SW13+, the epigenetic mechanisms involved in the subtype switch are unknown. Recently, it was shown that miR-199a is a potent negative regulator of BRM expression in many epithelial tumor cell lines. Given this, we examined the expression of miR-199a in the two SW13 subtypes, and indeed found that miR-199a expression was significantly higher in the BRM-deficient SW13- cells than the BRM-expressing SW13+ cells. To begin to further assess the involvement of miR-199a in the SW13 subtype switch, SW13+ and SW13- cells were treated with either a miR-199a mimic or miR-199a inhibitor, respectively and assessed for differences in BRM mRNA and protein expression, as well changes in cell-type morphology. Our results indicate that although miR-199a can mediate differences in BRM expression, miR-199a alone is not sufficient to induce a full subtype conversion as evidenced by retention of subtype morphology following miR-199a inhibition and over-expression. We are currently investigating the role of miR-199a in HDACi mediated subtype conversion, as well as the expression of other miRNA and their targets that are differentially expressed between the two SW13 subtypes.

#1117

Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a player in hematopoiesis and AML.

Sophia E. Maharry, Christopher J. Walker, Sandya Liyanarachchi, Sujay Mehta, James S. Blachly, Denis C. Guttridge, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Background: Over 15 genome-wide association studies (GWASs) have identified a narrow region in chromosome 6q23.3 as a major hematopoietic quantitative trait locus (QTL) for many hematologic traits. The associated phenotypes have yet to be fully explained by genes in the region, including MYB. We noted a newly annotated microRNA within the QTL, miR-3662, and sought to characterize its role in the QTL by exploring its function in normal and malignant hematopoiesis, specifically acute myeloid leukemia (AML).

Results: To assess the association between the QTL-defining SNPs and miR-3662 abundance, we genotyped 10 GWAS SNPs in 200 blood samples from non-leukemic individuals. The risk alleles of all 10 SNPs associated with higher miR-3662 abundance. Homozygosity for rs66650371, a three base pair (bp) deletion, showed the strongest association (P<.0001). Reinserting the three bp in cells homozygous for rs66650371 with CRISPR/Cas9 technology lowered miR-3662 abundance by 20%, suggesting rs66650371 directly affects miR-3662 transcription. MiR-3662 was nearly absent in primitive hematopoietic progenitor cells (HPCs) cells, whereafter its abundance increased throughout differentiation. MiR-3662 overexpression increased cell growth and colony formation of HPCs, particularly of the erythroid lineage.

To explore the role of miR-3662 in malignant hematopoiesis, we overexpressed miR-3662 in AML cell lines (Kasumi1, KG1a, MV4-11) and primary AML patient blasts (n=12). Overexpression of miR-3662 reduced cell growth and viability in vitro, and significantly reduced tumor size in a murine xenograft model (scramble vs. miR-3662; n=9 mice/group).

We used targeted RNA sequencing to identify differentially expressed genes between forced miR-3662-expresssing- and control AML patient blasts using a panel of 361 candidate genes (TruSeq RNA platform, Illumina). IKBKB was identified as a promising target, and direct regulation of IKBKB's 3′-UTR by miR-3662 was confirmed via luciferase assay. MiR-3662 overexpression lowered IKBKB mRNA and protein expression in both HPCs and AML patient blasts. As IKBKB promotes activation and nuclear localization of NF-κB, overexpression of miR-3662 led to decreased NF-κB transactivation potential via luciferase assay. Confocal microscopy of HPCs and AML cells, as well as subcellular fraction immunoblots, revealed decreased NF-κB nuclear localization upon miR-3662 overexpression. Re-introduction of IKBKB partly rescued miR-3662's phenotype by increasing nuclear localization of NF-κB, decreasing growth of HPCs, and increasing viability of AML cells.

Conclusion: The risk alleles of the 6q23.3 region associated with higher miR-3662 abundance, and miR-3662 abundance was functionally linked to rs66650371. We characterized miR-3662 as an agent in normal and malignant hematopoiesis, and demonstrated that miR-3662 reduces NF-κB activity by targeting IKBKB.

#1118

The role of Epstein-Barr virus-encoded miRNAs in ATM regulating DNA damage response in nasopharyngeal carcinoma.

RAYMOND Wai-Ming LUNG,1 Tom Pok-Man Hau,1 Wing-Po Chak,1 Joanna Hung-Man Tong,1 Ken Hung-On Yu,1 Sai-Wah Tsao,2 Kevin Yuk-Lap Yip,1 Ka-Fai To,1 Kwok-Wai Lo1. 1 _The Chinese University of Hong Kong, Hong Kong SAR, China;_ 2 _The University of Hong Kong, Hong Kong SAR, China_.

Nasopharyngeal carcinoma (NPC) is an aggressive epithelial malignancy that has remarkably high incidence and mortality rates in Hong Kong and south China. It is well-known to be associated with Epstein-Barr virus (EBV) infection. Previous work in our team revealed that EBV-encoded miRNAs derived from the BARTs (miR-BARTs) were abundantly expressed in primary NPCs. They function in controlling apoptotic and immune responses in the infected cells during NPC development.

Most recently, we attempted to use in silico method to predict cellular targets of miR-BARTs. Results suggested that there were multiple putative miR-BART binding sites on the 3'UTRs of an important DNA double-strand breaks (DSBs) repair gene, Ataxia-Telangiectasia-Mutant (ATM). The down-regulation of ATM mRNA and protein had also been demonstrated in our local primary NPC samples. Although the interaction of miR-BARTs on each suggested putative binding site had not been completely validated, our preliminary data indicated at least three EBV-encoded miRNAs (BART5-5p, BART9-3p and BART14-3p) were involved in repressing ATM expression in NPCs. They could work either alone or cooperatively to control ATM expression in transient assays. Notably, ectopic expression of these three miR-BARTs could successfully suppress γ irradiation-induced ATM activity in two EBV-negative cell lines, NP69 and HeLa.

It had been reported that ATM null cells were defective to repair the DSB lesions and sensitive to the Poly(ADP-ribose) polymerase (PARP) inhibitor treatment in the presence of DNA-damaging agents. In contrast, the DSBs are effectively repaired in normal cells to retain genetic integrity. We believed that EBV infection, via miR-BARTs to reduce ATM activity and disrupt Homologous Recombination (HR) repair function, made the NPC cells sensitize to ionizing radiation and DNA-damaging agent treatment. Hence, the knowledge generated from this project is not only enhance our understanding of EBV biology, but also opens an avenue for the development of effective NPC therapies.

#1119

Tumor suppressing dual-action miRNA: Targeting Warburg effect and androgen receptor function in CRPC.

Jey Sabith Ebron,1 Jagjit Singh,1 Kavleen Sikand,1 Eswar Shankar,2 Sanjay Gupta,2 Girish C. Shukla1. 1 _Cleveland State University, Cleveland, OH;_ 2 _Case Western University, Cleveland, OH_.

The ability of prostate cancer cells to alter their metabolism in a manner distinct from benign cells and energy reprogramming is a major concern in treatment of castration resistant prostate cancer (CRPC). On the other hand, development of resistance to next-generation drugs directed against the AR signaling axis (Abiraterone acetate and Enzalutamide) confers a grim prognosis. Hence, targeting aerobic glycolysis of tumor cells as well as AR signaling axis in CRPC is highly desirable. Here, we report the role of a dual-action miRNA which targets the Androgen Receptor function as well as action of key factors involved in tumor energy metabolism. In addition, the miRNA also targets the key anti-apoptotic factors and enhance the apoptosis in cultured cells. Furthermore, together with Enzalutamide, the miRNA shows a potent synergistic anti-tumor activity in 22RV1 mouse xenografts. We will present the detailed results showing the therapeutic potential of dual-action miRNA in the treatment of CRPC.

#1120

Differential expression of human cytomegalovirus microRNA in triple-negative breast cancer tumors.

Tia Hudson,1 Scott Harrison,1 Dukka K C,1 Jan Prins,2 Perpetua M. Muganda1. 1 _North Carolina A &T University, Greensboro, NC; _2 _University of North Carolina, Chapel Hill, NC_.

Triple-negative breast cancer (TNBC) accounts for 15-20% of the breast cancer cases.

These tumors are heterogeneous and aggressive, fail to respond to targeted therapy, have poor prognosis, and a high risk of relapse. The molecular basis of this breast cancer subtype is currently unknown. Viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human papillomavirus (HPV), are major suspects in the etiology of breast cancer. Since miRNAs have been implicated in the pathogenesis of breast cancer, including triple-negative breast cancer, we hypothesized that viral miRNAs may play a role in this aggressive disease. The objective of this project, therefore, was to determine the identity, prevalence, sequence variation, and differential expression of viral miRNAs in TNBC tumors as compared to control samples. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Five novel putative HCMV miRNAs were found to be differentially expressed in TNBC tumors as compared to controls. Two of the putative miRNAs were differentially expressed in 60-79% of the TNBC tumors as compared to 0% of normal controls. Two additional putative miRNAs were expressed in 67-88% of TNBC tumors as compared to 25-31% of normal controls. One putative miRNA was expressed in 67% of TNBC tumors as compared to 88% of normal samples, while 1 putative miRNA in addition to HCMV-US25-1-3p displayed no significant differences. These putative HCMV miRNAs localize within genomic regions of HCMV that encode UL56 DNA packaging terminase subunit 2, single-stranded DNA-binding protein, noncoding RNA4.9, and enveloped glycoprotein M. Ongoing work has so far computationally validated one of these miRNAs as a novel HCMV miRNA. This is the first report on the differential expression of HCMV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 60-80% of TNBC tumors specifically express the same HCMV miRNA. Our findings suggest that these differentially expressed HCMV miRNAs may potentially play a role in the pathogenesis of TNBC. 

### Oncogene Function, Regulation, and Targeting

#1121

Validation of FOXR2 and ARHGAP36 as oncogenes in medulloblastoma.

Pauline Jackson, Alex Larsson, Paramita Das, David Largaespada. _University of MN, Minneapolis, MN_.

We are working to validate two putative oncogenes in medulloblastoma (MB), ARHGAP36 and FOXR2. These genes were identified as MB drivers using a Sleeping Beauty mutagenesis screen in mice and are overexpressed in subsets of human MB. We are using gain (GOF) and loss of function (LOF) studies both in vitro and in vivo to understand the roles of FOXR2 and ARHGAP36 in MB genesis. In GOF studies, both genes individually drove anchorage independent growth in a mouse cerebellar progenitor cell line (C17.2) and a human MB cell line (ONS76). When overexpressed individually in C17.2 cells, both FOXR2 and ARHGAP36 drove tumor formation in the flank of NU/J mice. We are also using an orthotopic injection model with C17.2 cells overexpressing either ARHGAP36 or FOXR2 individually to determine if these genes drive tumor formation in a more relevant setting. RNA sequencing and reverse-phase protein array analyses were also performed on WT and C17 cells overexpressing each oncogene to identify their effects in an unbiased manner. We are working to validate those effects using RT-PCR and Western analysis. In LOF studies, the CRISPR/Cas system is being used to create mutations in either the ARHGAP36 or FOXR2 locus in human MB cell lines (ONS76, MED8A, Daoy). This has been accomplished with ARHGAP36 in ONS76 cells, and we are currently characterizing those cells using proliferation, soft agar colony formation, and tumor formation assays. Lastly, we are working to create GOF and LOF mouse models of both FOXR2 and ARHGAP36 to characterize their oncogenic potential in vivo. We identified ARHGAP36 and FOXR2 as putative oncogenes in MB and our findings may elucidate novel targets for therapeutic efforts aimed at treating patients with MB.

#1122

FGFR1 can act as an oncogene or a tumor suppressor depending on the molecular context.

Álvaro Quintanal-Villalonga,1 Laura Ojeda-Márquez,1 Ángela Marrugal,1 Rocío Suárez,1 Irene Ferrer,1 Amancio Carnero,1 Sonia Molina-Pinelo,1 Luis Paz-Ares2. 1 _Instituto de Biomedicina de Sevilla (IBIS) (HUVR, CSIC, Universidad de Sevilla), Sevilla, Spain;_ 2 _Medical Oncology Department, Hospital Universitario Doce de Octubre & Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain_.

FGFR family of proteins has been extensively related to oncogenic properties in several types of tumors, including lung cancer. Among its members, FGFR1 has been related to squamous cell lung carcinoma, where FGFR1 amplification is present in 20% of patients. However, its role in lung cancer has not yet been thoroughly described.

Several lung cancer cell lines with different genetic backgrounds were transfected with plasmids to either overexpress or silence FGFR1. Then several tumorigenic abilities were tested in vitro and in vivo. mRNA from Paraffin-embedded tissue of a cohort of lung cancer patients was extracted and FGFR1 expression levels were measured and related to clinical characteristics.

FGFR1 increases oncogenic properties in SCC cell lines, but exerts anti-oncogenic effects in several lung ADC cell lines, suggesting a tumor suppressor role under certain circumstances. This is a consequence of differentially expressed genes among these two histologies. According to this, analysis of FGFR1 mRNA expression of a cohort of lung cancer patients revealed that high FGFR1 mRNA expression was associated to a shorter overall survival (OS) and progression free survival (PFS) in lung SCC patients. However, a trend for longer OS was observed for the ADC patients with higher FGFR1 mRNA expression.

FGFR1 has the potential to be either a tumor suppressor or an oncogene and the molecular context is a determining factor to define its final role in tumorigenesis.

#1123

The role of proto-oncogene PELP1 in breast cancer stem cell maintenance and therapy resistance.

Suryavathi Viswanadhapalli, Monica Mann, Gangadhara Reddy Sareddy, Xaionan Lix, Hariprasad Vankayalapati, Ratna K. Vadlamudi. _UT Health Science Center at San Antonio, San Antonio, TX_.

BACKGROUND: Cancer stem cells (CSCs) are known to evade hormonal therapy and regrowth of tumor cells from cancer stem cells is a major clinical problem. Histone methyltransferase G9a/EHMT2 plays a critical role in stem cell maintenance. Proline, glutamic acid, and leucine rich protein (PELP1) is a proto-oncogene, functions as a coregulator of nuclear receptors, and is prognostically linked to shorter breast cancer survival. Recent studies from our lab discovered that PELP1 interacts with G9a/EHMT2. The objective of this study is to develop small molecular inhibitors that block PELP1 interactions with G9a/EHMT2 .

METHODS: We isolated CD44high/CD24low CSCs from three breast cancer cell lines (ZR75, MCF7, T47D) using FACS. Cells were analyzed for spheroid formation, morphological changes, immunofluorescence for differentiation markers, protein (Western) and RNA (RT-qPCR) analysis. Expression of differentiation markers K19 and K14 and stem cell markers CD133, CD44, Id1, Nestin, Musashi-1, SOX2, Notch2, and OCT1 was determined. Effect of inhibitors on the cell viability of breast cancer cells was determined using cell titer glow assays. Xenograft studies were used to determine the in vivo efficacy of the inhibitors.

RESULTS: Using yeast based genetic screen, we identified a small peptide inhibitor (PIP1) that interferes PELP1 interaction with G9/EHMT2. Utilizing Hit-Ligand interaction site with the PELP1 hot spot residues based on 3D alignment and shape, we have identified 61 potential hits from Ligand-Based screening using a 10,000 Diverse Set. Screening of these 61 potential hits using MTT based cell viability assays identified three small organic molecule inhibitors (peptidomimetics) as leads. All three peptidomimetics showed activity similar to PELP1 peptide inhibitor 1 (PIP1) in MTT assays and in biochemical assays disrupted PELP1 interaction with G9a/EHMT2. Peptidomimetic treatment inhibited the proliferation of tamoxifen and letrozole resistant cells. In mechanistic studies, we found that knockdown of PELP1 inhibited stem cell maintenance. In FACS analysis of ZR75, ZR75-PELP1 and ZR75-PELP1KD cells, the percentage of CD44high/CD24low cells correlated with PELP1 status. In mammosphere assays, PELP1 targeting peptidomimetics significantly inhibited the formation of mammospheres. Further, in self-renewal assays, peptidomimetic-treated cells had decreased self-renewal capacity. In xenograft assays using ZR-75 cells, PELP1 inhibitor significantly reduced the tumor growth.

CONCLUSIONS: Collectively, our studies have discovered an essential role for PELP1 in breast cancer stem cell maintenance and identified the PELP1-G9a/EHMT2 axis as a potential therapeutic target for reducing stemness. Further, the novel small molecule inhibitors of PELP1 could be used for therapeutic targeting of breast cancer stem cells and therapy resistance.

#1124

Expression of the lipocalin 2 oncogene in the development of smoking-associated and Kras mutant lung adenocarcinoma.

Joshua Ochieng, Sayuri Nunomura, Wenhua Lang, Jinsong Wei, Li Xu, Christina McDowell, Edwin Ostrin, Ralph Arlinghaus, Junya Fujimoto, Humam Kadara. _UT MD Anderson Cancer Center, Houston, TX_.

There are more than 90 million smokers in the United States who are at elevated risk for lung adenocarcinoma (LUAD), the most common lung cancer subtype. LUADs in smokers frequently (greater than 25%) harbor activating mutations in the Kras oncogene. Kras mutant LUAD exhibits very poor prognosis warranting the need for new strategies to prevent and treat this fatal lung cancer subtype. Limiting these advances is a poor understanding of the earliest events that drive Kras mutant LUAD development in smokers and that would constitute targets for early treatment. The retinoic acid-inducible G-protein coupled receptor Gprc5a is preferentially expressed in normal lung and mice with knockout of the gene (Gprc5a-/-) develop late onset lung tumors spontaneously. In our data aimed at understanding smoking-associated lung cancer pathogenesis, we found that Gprc5a-/- mice, in contrast to wild type (WT) littermates, not only developed LUADs after tobacco carcinogen exposure but also that these lesions harbored somatic Kras mutations, the same variants thought to act as drivers of human LUAD in smokers. In the present study we construed that pinpointing early events preceding lung oncogenesis in Gprc5a-/- mice will help us comprehend how smoke-associated LUADs with Kras mutations evolve. Towards this, we performed RNA-sequencing, using the Ion Proton platform, of visually normal airway epithelia in Gprc5a-/- mice prior to tumor development compared to epithelia in WT mice. We noted distinctively marked up-regulation of the Lcn2 oncogene in normal airways of Gprc5a-/- mice. Functional pathways and gene set enrichment analyses revealed that Lcn2 up-regulation was associated with activation of mitogen activated protein kinase- and interleukin-associated pathways and with gene sets attributable to cigarette smoke. We then analyzed Lcn2 expression in murine cell lines we had derived and found that Lcn2 was significantly elevated in both Gprc5a-/- LUAD and airway cell lines compared to WT airway cells. Moreover, immunohistochemical expression of Lcn2 protein was elevated, compared to normal lung tissue, in early lesions (hyperplasias and adenomas) and in the LUADs of tobacco carcinogen exposed Gprc5a-/- mice. Of note, we also found that alternatively activated M2 macrophages surrounding all lesions were positive for Lcn2. We then analyzed human LCN2 expression in publicly available array datasets and found that the gene was significantly elevated in human KRAS mutant LUADs compared to wild type LUADs or normal lung (all P < 0.05). Our preliminary findings suggest that elevated Lcn2 expression, in lung (airway and tumor) epithelia and in the protumorigenic inflammatory environment, may be implicated in the early development of smoking-associated Kras mutant LUAD. Efforts are underway to further probe this supposition in mice with knockout of Lcn2.

#1125

FOXM1 and FOXQ1 are novel oncogenes in colorectal cancer and can be therapeutically targeted through miRNA-based therapeutics.

Wenhao Weng,1 Yoshinaga Okugawa,2 Shusuke Toden,1 Ajay Goel1. 1 _Baylor Research Institute, Dallas, TX;_ 2 _Mie University Graduate School of Medicine, Japan_.

OBJECTIVES: The development of colorectal cancer (CRC) is a highly complex process which needs coordinated regulation in the gene regulatory network. Forkhead box (FOX) proteins constitute a large family of transcriptional regulators. Accumulating evidence indicates that several FOX family members are critical regulators of tumor progression. However, the role of FOX proteins in CRC remains vastly unknown. miRNAs are single stranded small non-coding RNAs which can regulate a variety of biological processes in various human diseases including cancer.

AIMS: Herein, we aimed to identify the expression patterns of FOX family members in CRC, followed by extensive characterization for their functional and mechanistic role in this malignancy. In addition, we sought to identify specific miRNAs that are responsible for dysregulated expression of FOX proteins in CRC.

METHODS: The web-based database ONCOMINE was used to identify most highly expressed FOX family members in CRC. A cohort of 178 frozen tissues including 134 primary CRC tissues, and 44 matched normal mucosa tissues were collected to analyze the expression levels of FOX genes. For functional analysis, cell proliferation, migration and invasion assays were performed following siRNA knockdown of specific FOX proteins in CRC cells. The potential FOX-targeting miRNAs were initially identified using in-silico approaches, followed by luciferase reporter assays to validate their binding to downstream target genes.

RESULTS: We systematically analyzed the expression profiles of FOX family members across 24 studies in cancer and normal tissues, and identified FOXM1, FOXQ1 and FOXA2 as the most highly expressed FOX genes in CRC. The over-expression of FOXM1 and FOXQ1 in CRC was confirmed in a cohort of CRC and matched adjacent normal tissues. Furthermore, the analysis of 134 CRC samples revealed that high FOXM1 and FOXQ1 expression resulted in worse overall survival. We next identified miR-342 as a potential regulator of both FOXM1 and FOXQ1, and subsequently validated its direct binding to the 3'UTR regions of both genes. Furthermore, the expression of miR-342 in CRC tissues inversely correlated with both FOXM1 and FOXQ1 expression. In-vitro functional analysis revealed that suppression of FOXM1 or overexpression of miR-342 inhibited CRC cell proliferation, while FOXM1 and FOXQ1 knockdown or miR-342 overexpression suppressed migration and invasion capacity of CRC cell lines. The stable overexpression of miR-342 reduced FOXM1 and FOXQ1 expression and significantly inhibited tumor growth in CRC xenografts in nude mice.

CONCLUSIONS: FOXM1 and FOXQ1 promote cancer progression and act as potential prognostic factors in colon cancer. The downregulation of miR-342 in tumor tissues may contribute to the CRC development through inhibition of FOXM1 and FOXQ1 expression, suggesting that miR-342 could be used as a therapeutic target in colorectal cancer.

#1126

Importance of the REM (Ras exchange) motif for membrane interactions by RasGRP3.

Agnes Czikora, Noemi Kedei, Peter M. Blumberg. _National Cancer Institute, LCBG, Bethesda, MD_.

RasGRP comprises a family of guanine nucleotide exchange factors that activate small GTPases, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP3 is a complex protein with REM (Ras exchange motif), GEF (catalytic domain), EF-hand, C1, SuPT, and PT (plasma membrane-targeting) domains, each of which can affect the cellular membranes to which RasGRP1/3 localizes. The C1 domain was the first membrane localizing domain identified in RasGRP1/3. It binds to the lipid second messenger diacylglycerol (DAG) and has the potential to mediate translocation of RasGRP1/3. The catalytic GEF domain and REM domain are both required for membrane localization of RasGRP1, affecting both endomembrane and plasma membrane targeting of RasGRP1. The objective of this study was to explore the importance of these two domains in translocation of RasGRP3. For these studies, we prepared full length RasGRP1/3, RasGRP1/3 C1 domains, and truncated RasGRP1/3 green fluorescent protein labeled constructs to transfect human prostate adenocarcinoma (LNCaP) cells. In the LNCaP cells, which are a PTEN mutant cell line with markedly elevated phosphoinositide levels, RasGRP1 translocates to the plasma membrane in response to PMA. In contrast, the full length RasGRP3 does not translocate to the plasma membrane even though it has a PT (plasma membrane-targeting) domain. In parallel, we examined the translocation of the C1 domain of RasGRP1/3 and of the truncated RasGRP1/3 constructs. The C1 domain of RasGRP3 was insufficient for targeting RasGRP3 to the plasma membrane. However, truncated RasGRP3s, which contain the PT domain and are missing the REM motif, showed stronger translocation. To better understand differences in the translocation pattern of these two proteins we compared RasGRP1 ("template") and RasGRP3 ("target") domains using Swiss-Model (homology or comparative modelling methods) and amino acid sequence alignment (NCBI/ BLAST). The percentage of identities showed remarkable differences in the structure of the REM and SuPT-PT domains. Based on these findings we prepared chimeras where we exchanged either the N or C terminus of RasGRP1/3. Exchange of the REM motif between RasGRP1 and RasGRP3 enhanced the translocation of RasGRP3, while it does not affect the translocation of RasGRP1. Exchange of C termini (SuPT-PT domain) of both proteins did not cause any changes in plasma membrane translocation. Our results clearly show that the N terminus (REM motif) of RasGRP3 is a critical element contributing to the difference between RasGRP1 and RasGRP3 in their ability to translocate to the plasma membrane. This difference in membrane translocation, in turn, should imply important differences in their regulation and, potentially, in their ligand selectivity in the living cell.

#1127

KIT/D816V induces SRC-mediated tyrosine phosphorylation of MITF and altered transcription program in melanoma.

Bengt Phung,1 Julhash U. Kazi,1 Alicia Lundby,2 Kristin Bergsteinsdottir,3 Jianmin Sun,1 Colin R. Goding,4 Göran Jönsson,5 Jesper V. Olsen,2 Eiríkur Steingrímsson,3 Lars Rönnstrand1. 1 _Div. of Translational Cancer Research, Dept of Laboratory Medicine, Lund University, Lund, Sweden;_ 2 _NNF Center for Protein Research, University of Copenhagen, Copenhagen, Denmark;_ 3 _Department of Biochemistry and Molecular Biology, University of Iceland, Reykjavik, Iceland;_ 4 _Ludwig Institute for Cancer Research, University of Oxford, Oxford, United Kingdom;_ 5 _Melanoma Genomics, Div. of Oncology and Pathology, Dept. of Clin. Sciences, Lund, Sweden_.

The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KITD816V has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KITD816V induces tyrosine phosphorylation of the microphthalmia-associated transcription factor (MITF) through a triple protein complex formation between KIT, MITF and SRC family kinases. In turn, phosphorylated MITF activates target genes that are involved in melanoma proliferation, cell cycle progression, suppression of senescence, survival and invasion. By blocking the triple protein complex formation, thus preventing MITF phosphorylation, the cells become hypersensitive to SRC inhibitors. We have therefore delineated the mechanism behind the oncogenic effects of KITD816V in melanoma and provide a rationale for testing SRC family kinase inhibitors to treat KITD816V-transformed melanomas.

#1128

Role of STAT3 in pediatric sarcoma cell lines.

Seethalakshmi Hariharan, Doris A. Phelps, Peter J. Houghton. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Signal transducer and activator of transcription-3 (STAT3) is a transcription factor that plays a major role in embryonic development, cell growth, differentiation and apoptosis. STAT3 becomes activated in the cytosol via phosphorylation at tyrosine-705 by JAK-1/2 kinases, in response to stimulation of either cytokine receptors or receptor tyrosine kinases. Phospho-STAT3 forms dimers, which then translocate into the nucleus to function as a transcription factor. Various pro-survival and anti-apoptotic genes including VEGF, Bcl-xL, Bcl-2 and survivin have been identified as STAT3 gene targets. Constitutive activation of STAT3 is observed in various cancers including breast, lung, pancreatic cancers and sarcomas. Since our studies indicate that STAT3 is upregulated in a number of pediatric sarcomas as well, we set out to understand the functional significance of STAT3 activation in these tumors in the context of tumor cell proliferation, survival, migration and resistance to apoptosis.

High basal expression levels of phospho-STAT3 (Tyr705) were detected in a number of rhabdomyosarcoma, Ewing's sarcoma and osteosarcoma xenografts and cell lines. Interleukin-6 (IL-6) was capable of inducing STAT3 (Tyr705) phosphorylation in these cell lines in a time-dependent manner. On the other hand, stimulation with insulin-like growth factors-1 and 2 or IGF-1/2 resulted only in a weak induction in STAT3 phosphorylation during early timepoints. Treatment with a potent JAK-1/2 inhibitor, ruxolitinib suggested that STAT3 phosphorylation in these cell lines is dependent on JAK-1/2 activities.

To understand the role of STAT3 in tumorigenesis, STAT3 was either knocked down using siRNAs or STAT3 phosphorylation was attenuated using ruxolitinib in sarcoma cell lines that express constitutively activated STAT3. Neither complete knockdown of STAT3, nor inhibition of STAT3 phosphorylation by ruxolitinib inhibited proliferation or decreased survival. These results indicate that activation of STAT3 may not have a significant role in tumor cell proliferation and survival under culture conditions. However, STAT3 inhibition resulted in decreased cell migration implicating STAT3 in regulation of tumor cell migration and invasion.

Overall, our data demonstrates a role for STAT3 activation in sarcoma cell migration and invasion and we speculate that targeting STAT3 might serve as a useful therapeutic approach to control tumor invasion and metastasis. Supported by USPHS grant CA165995.

#1129

EGFR-mediated activation of RET in ASCL1+ lung adenocarcinoma.

Kaustubh N. Bhinge, Yang Lin, Hamed Rahi, Marie Christine Aubry, Aaron Mansfield, Irina Kovtun, Stephen Murphy, Ping Yang, Dennis Wigle, Joanne (Eunhee) Yi, Aqsa Nasir, Simone Terra, Julian Molina, George Vasmatzis, Farhad Kosari. _Mayo Clinic, Rochester, MN_.

Lung cancer accounts for about 27% of the cancer related deaths in the USA annually. Pathologically, it is a very complex disease broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLCs are further classified into squamous cell carcinoma, large cell carcinoma and adenocarcinoma. Achaete-scute homolog 1 (ASCL1), an important transcription factor essential in the development of neuroendocrine cells (NE) in lungs is shown to be specifically expressed in lung NE cancers and 10-20% of adenocarcinomas (AD) with NE differentiation (NED), thus suggesting a role in the pathogenesis of these tumors. Our previous study showed that ASCL1 is a regulator of the RET oncogene in AD with NED. RET is a receptor tyrosine kinase with two isoforms in humans: RET9 (short) and RET51 (long). We performed survival analysis to study implications of RET isoforms in ASCL1+ tumors and found that elevated expression of the long RET mRNA was associated with poor survival. Subsequent in vitro experiments demonstrated that treatment with EGF robustly induced phosphorylation of RET in HCC1833 and H1755 cell lines which have high endogenous levels of ASCL1 and RET but not in VMRC-LCD cell line which has high level of ASCL1 but low level of RET. EGF induced phosphorylation of RET was diminished by gefitinib and by EGFR siRNA. Immunoprecipitation results indicated direct binding between EGFR and RET in presence of EGF. Furthermore, a high throughput drug screening found 8 EGFR inhibitors that were 10 - 250 fold more cytotoxic in ASCL1+ compared with ASCL1- AD cells. These results implicate EGFR as a key regulator of RET activation in ASCL1+ AD and suggest that EGFR inhibitors may be therapeutic for this population of patients.

#1130

Subcellular localization of the cell polarity protein Scribble defines its oncogenic activity in hepatocellular carcinoma.

Shan Wan,1 Anne-Sophie Meyer,1 Sofia Weiler,1 Teresa Lutz,1 Stephanie Roessler,1 Peter Schirmacher,1 Federico Pinna,2 Kai Breuhahn1. 1 _Univ. Hospital Inst. of Pathology, Heidelberg, Germany;_ 2 _Institute of Pathology, University Hospital RWTH Aachen, Aachen, Germany_.

Background:

Disturbance of hepatocyte polarity is frequently observed in liver cancer tissues; however, it is still controversially discussed if this phenotype represents the cause or the result of tumorigenesis. Due to their multi-functional properties, hepatocytes are characterized by distinct luminal, lateral and basal domains that are defined by specific protein complex. These cell protein complexes such as the apical Crumbs- (CRB3, Pals1, Patj), the lateral Par- (Par3, Par6, aPKC) and the basolateral Scribble-complex (Scrib, Dlg, Lgl) are essential for the formation of hepatocellular polarity. So far, it is unknown if and which protein complex constituents are involved in liver tumor development and how these factors induce possible downstream effector mechanisms.

Methods:

To identify complex proteins that affect hepatocellular polarity, a siRNA screen targeting all relevant polarity factors was performed in HCC cells that form bile canaliculi in vitro (HepG2). Localization of Scribble in HCC cell lines and human HCC tissues was analyzed by western immunoblotting and immunofluorescence. Expression vectors containing wildtype Scribble and an isoform lacking polarity complex binding capacity were cloned (Scrib-wt, Scrib-P305L) and stably transfected in HCC cells. The impact of these isoforms on PI3K/AKT signaling pathway activity and cell viability were analyzed. In addition, the interaction of Scrib-wt, Scrib-P305L with phosphatases regulating the PI3K/AKT pathway was tested by Co-IP analysis.

Results:

The siRNA screen revealed that Scribble strongly affects hepatocellular polarity. Scribble located near the membrane in polarized cells (e.g., HepG2, HuH-1) but in the cytoplasm in non-polarized cells (HLE, HLF). Scribble overexpression and cytoplasmic accumulation correlates with poor overall survival of HCC patients. In vitro, the inhibition of Scribble reduced and overexpression of cytoplasmic Scribble (Scrib-P305L) induced AKT phosphorylation. In addition, Scrib-P305L supported HCC cell viability in an AKT-dependent manner. Immunofluorescence revealed co-localization of Scribble with the AKT phosphatases PTEN and PHLPP1. Co-IP studies revealed a physical interaction between PHLPP1 and both Scribbles isoforms (wildtype and P305L mutant).

Conclusion/Outlook:

Our data demonstrate that the cytoplasmic accumulation of Scribble in HCC cells promotes tumor cell growth in an AKT-dependent manner probably through the interaction with AKT phosphatases. These results indicate that the disturbance of cell polarity (e.g. by Scribble overexpression) can foster oncogenic effects and therefore must be regarded as initiating event in hepatocarcinogenesis.

#1131

Uncovering oncogenic KRAS allele specific phenotypes using an isogenic MEF cell line system.

Nicole Fer, Brian Smith, Leslie Garvey, William Burgan, Katie Powell, Kanika Sharma, Andrew Waters, Xiaolin Wu, Dan Soppet, Robert Stephens, Dwight Nissley, Matthew Holderfield, Rachel K. Bagni. _NCI RAS Initiative, CRTP, FNLCR, Frederick, MD_.

The RAS Initiative at the Frederick National Laboratory has generated an isogenic mouse embryonic fibroblast (MEFs) cell line panel containing single transgene alleles using the RAS-less MEF system developed by Matthias Drosten and Mariano Barbacid (CNIO). These MEFs are H, N and K-RAS null, growth is dependent on exogenous RAS or MAPK pathway activation. The panel includes: KRAS 4A WT, KRAS 4B WT, KRAS 4B G12C, G12D, G12V, G13D, Q61L, Q61R, HRAS WT, NRAS WT or BRAF V600E. The workflow to generate the lines requires cre-lox removal of endogenous KRAS and the cells arrest in the G1 phase of the cycle. Proliferation is resumed through the delivery of the transgene to the cells using lentiviral transduction. Clonal cell lines are derived from the initial pools and are thoroughly characterized including confirmation of endogenous KRAS gene removal, identification of the transgene insertion site(s), calculation of proliferation rates and doubling times, analysis of signaling pathways, response to tool compounds and exome sequenced to exclude lines with mutations in onco-relevant genes. This panel is a unique resource that can be used to ask allele and isoform specific questions (gene expression profiles, GTP-loading, etc.) and screen for RAS inhibitors as well as determine novel compound specificity. This panel is available to the academic RAS research community.

#1132

Regulation of CT120 gene as a new target of c-myc in head and neck squamous cell carcinoma (HNSCC).

Onur Baykara, Elif Baltaci, Betul Seyhan, Nejat Dalay, Nur Buyru. _Istanbul University Cerrahpasa Medical Faculty, Dept. of Medical Biology and Genetics, Istanbul, Turkey_.

HNSCC is the sixth most common form of cancer and represents the third common cause of cancer-related deaths worldwide. Therefore, there is a need to understand the molecular mechanisms involved in the pathogenesis of HNSCC.

The c-myc protein belongs to the myc family of transcription factors and plays a fundamental role in cell cycle progression, apoptosis and cellular transformation. In the human genome, the MYC gene is located on chromosome 8 and its protein product regulates expression of 15% of all genes through binding to the Enhancer box (E-box) sequences. An E-box is a DNA sequence found in the promoter regions of eukaryotic genes that acts as a protein binding site to regulate gene expression. The CANNTG consensus sequence of the E-box is known as the canonical E-boxes. There are several other non-canonical E-box motifs such as the GATGTG, CATGCG, CACGCG, CACGAG, CGCGAG and CAACGTG sequences.

Amplification of the MYC gene is observed in many different types of cancers including HNSCC. CT120 is a novel gene which encodes a human trans-membrane plasma protein. It has been shown that up-regulation of CT120 is associated with lung and head and neck carcinogenesis. In this study we investigated the promoter region of the CT120 gene and identified 6 E-boxes. To test whether c-myc binds to these E-boxes we performed chromatin immunoprecipitation (ChIP) assays. We demonstrated that c-myc binds strongly to the E-boxes 2, 3 and 4. It is well known that the expression rates of the genes are under the control of epigenetic modifications. Methylation of the CpG islands which are found in the promoter regions is one of the most studied epigenetic modifications. We identified two putative CpG islands in the promoter of the CT120 gene. To investigate the possible epigenetic regulation of CT120 and the effect of the promoter methylation to the binding of c-myc, we also investigated the methylation levels of the two islands in the CT120 promoter by MS-PCR. We observed a slight decrease (3.1%) in the methylation level of the first CpG-rich region. In 56% (28/50) of the tumor tissues methylation was decreased compared to the non-cancerous tissues. However, there was no statistically significant correlation between the promoter methylation and CT120 mRNA level. Depending on these data we investigated the expression levels of the c-myc and CT120 in the 50 HNSCC and normal tissue samples by qRT-PCR and observed a direct correlation between the overexpression of the c-myc and CT120 genes. Overexpression of CT120 and c-myc were observed in 29 (58%) and 34 (68%) of the tumors, respectively, compared to non-cancerous tissues. A total of 24 (48%) patients out of 50 showed a concurrent pattern of either up- or down regulation.

Our results indicate that the CT120 gene is a target of c-myc and that both proteins may participate in the progression of HNSCC.

#1133

Prevalence of activated NOTCH receptor in solid tumors and chronic lymphocytic leukemia.

Gerard Oakley, Lisa Riegle, Karim Benhadji, Andrew Schade. _Eli Lilly and Company, Indianapolis, IN_.

Recent reports identified high expression of Notch 1 receptor in various tumors including adenoid cystic carcinomas (ACC). Notch 1 receptor is activated by gamma secretase cleavage to exert transcriptional control of processes including cellular differentiation and proliferation. Notch is also known to be activated in approximately 60% of T-cell acute lymphoblastic leukemias. We investigated the prevalence of Notch 1, 2 and 3 activation in different tumors by immunohistochemistry (IHC) designed separately for Notch 1, 2 and 3. Our proprietary antibodies recognize the post-gamma secretase cleavage activated Notch intracellular domain (NICD) fragments of Notch 1, Notch 2 and Notch 3, allowing direct measurement of activation of these receptors, in addition to their expression level by the tumor cells.

In total we tested 48 ACCs of the salivary glands, 19 breast cancer, 35 chronic lymphocytic leukemias (CLL), 7 glioblastomas, and 35 non-small cell lung carcinomas (NSCLC) We found that at least 65% (31 out of 48) of ACCs were positive for Notch 1 activation using our assay. Of other tumor types, 4 out of 7 glioblastomas (57%), 14 out of 35 CLLs (40%), 7 out of 35 NSCLC (20%) and 2 out of 19 breast cancer cases (11%) were positive for Notch 1. Within NSCLC, 5 of the 16 tested squamous cell carcinomas were positive for Notch 1 activation (31%). Notch 2 and Notch 3 showed only rare activation in these tumors. We conclude that high levels of Notch 1 activation are identifiable in different tumor types including high prevalence in ACC, CLL and glioma. These tumors may benefit from therapeutic Notch pathway inhibition.

#1134

**Association of ZC3H8 with nuclear bodies and its role in promoting tumor cell behavior** in vitro **and** in vivo **.**

John A. Schmidt, Keith G. Danielson, Jani L. Swiatek, Emily R. Duffner, Janice E. Knepper. _Villanova University, Villanova, PA_.

The Zinc Finger CCCH-Type Containing 8 (zc3h8 or Fliz1) gene is overexpressed in many mouse and human breast cancer cell lines that we examined, although the exact function of this gene and the ZC3H8 protein are largely unknown. To characterize ZC3H8 protein, we localized it to distinct sub-nuclear domains that also contain markers for promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. Furthermore, when cells are treated with a casein kinase-2 (CK2) inhibitor, ZC3H8 mislocalizes out of the nuclear bodies and diffuses into the nucleoplasm. CK2 is a known activator of transcription and a potential drug target. ZC3H8 contains a putative CK2 phosphorylation site at position T32, and may be regulated by this kinase. Based on this localization and other preliminary data, we hypothesize that ZC3H8 has a role in transcriptional regulation or RNA modification at these nuclear bodies. This hypothesis is supported by published studies that have found a role for ZC3H8 in the Little Elongation Complex (LEC), which transcribes snRNA involved in RNA splicing in nuclear bodies. Since many tumors and cancer cell lines overexpress zc3h8, we used stably expressed shRNA to knock down expression in two independent mouse breast cancer cell lines for in vitro and in vivo studies. Cells with reduced zc3h8 expression have decreased rates of proliferation and migration compared to overexpressing cells when tested using MTS and wound healing assays. Decreased rates of migration in wound healing assays are reversed by overexpression of the known migration-associated gene MEMO1. Additionally, cells with reduced expression of zc3h8 failed to form colonies in soft agar or pass through an extracellular matrix coating a transwell chamber- a phenotype that can be rescued using recombinant human zc3h8. Conversely, untransformed mouse mammary COMMA-1D cells gain the ability to form colonies in soft agar when transfected to stably overexpress zc3h8. Our in vitro studies suggest that zc3h8 expression has a role in tumorigenesis. These observations have been supported with in vivo studies in mice. We implanted cells knocked down for zc3h8 and control cells into the mammary glands of syngeneic female mice. Mice injected with cells that have reduced expression of zc3h8 form substantially smaller tumors by weight compared to control. When taken together, these data show that zc3h8 has a role in the formation and progression of cancer through its association with nuclear bodies involved in transcriptional regulation and RNA modification.

#1135

FYN expression potentiates FLT3-ITD-induced mitogenic signaling in acute myeloid leukemia.

Rohit A. Chougule, Julhash U. Kazi, Lars Rönnstrand. _Lund University, Lund, Sweden_.

FYN is a non-receptor tyrosine kinase belonging to the SRC family of kinases. SRC family kinases are frequently over-expressed in human cancers, and play key roles in cancer cell invasion, metastasis, proliferation, survival and many other biological processes. SRC has long been recognized as an important oncogene; but little attention has been given to its other family members such as FYN. In this report, we have studied the role of FYN in FLT3 signaling with respect to acute myeloid leukemia (AML). FLT3 is a type III receptor tyrosine kinase which is found to be mutated in around 30% of AML cases. We observed that FYN displays a strong association with wild-type FLT3 as well as oncogenic FLT3-ITD. While association with wild-type FLT3 is dependent on ligand stimulation, it is constitutively associated with FLT3-ITD. In addition, a kinase-dead FLT3 mutant was unable to associate with FYN suggesting that FLT3 activation is required for association with FYN. A FYN SH2-domain mutant lacking a critical arginine residue was unable to associate with FLT3 indicating that FYN associates with FLT3 through its SH2 domain. A phopho-peptide fishing experiment identified multiple FYN binding sites in FLT3, which partially overlap with the known SRC binding sites but also differ from these sites. To understand the role of FYN in FLT3 signaling, we have generated Ba/F3-FLT3/FYN or Ba/F3-FLT3/empty vector cell lines. We observed that expression of FYN results in enhanced phosphorylation of AKT, ERK1/2 and p38 phosphorylation in response to ligand stimulation. Ba/F3-FLT3-ITD cells overexpressing FYN also showed higher STAT5 activation. Furthermore, FYN expression led to a significant increase in FLT3-ITD-dependent cell proliferation but did not alter apoptosis induced by cytokine starvation. Finally, we showed that FYN expression was deregulated in AML patient samples and that higher expression of FYN in combination with FLT3-ITD expression correlates with poor prognosis in AML. Taken together our data suggest that FYN cooperates with oncogenic FLT3-ITD which results in poor prognosis in AML and therefore inhibition of FYN in combination with FLT3 inhibition will most likely be beneficial for AML patients.

#1136

Cooperative functional roles of RNA binding proteins LIN28B and IMP1 in the pathogenesis of colorectal cancer.

Priya Chatterji,1 Kathryn Hamilton,1 Sarah Andres,1 Rei Mizuno,1 Philip Hicks,1 Arjun Jeganathan,1 Monte M. Winslow,2 Antoni Castells,3 Miriam Cuatrecasas,4 Blair Madison,5 Anil Rustgi1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Stanford University, CA;_ 3 _Hospital Clínic, CIBERehd, IDIBAPS, Spain;_ 4 _University of Barcelona, Spain;_ 5 _Washington University School of Medicine, MO_.

Introduction: RNA binding proteins and miRNAs have emerged as crucial regulators of intestinal homeostasis by controlling the stability and translation of target mRNAs. LIN28B, an mRNA binding protein, plays a critical role in regulating growth and proliferation in the intestinal epithelium. Previous work in our lab revealed that LIN28B promotes growth and tumorigenesis of the intestinal epithelium via suppression of mature let-7 miRNAs.LIN28B suppression of let-7 promotes upregulation of let-7 targets, including IMP1 (Insulin-like growth factor II mRNA-binding protein 1). Indeed, previous studies from our lab have shown that transgenic mice expressing LIN28B from the mouse Vil1 promoter (Vil-Lin28b mice) have an increase in IMP1 protein levels that is increased further with conditional knockout of let-7. Mechanistically, Let-7 isoforms have been known to physically and functionally interact with IMP1; however, the specific role of IMP1 in Lin28b-let 7-mediated tumorigenesis remains unknown. The current study tested the hypothesis that IMP1 maybe required for LIN28B-mediated tumorigenesis and that LIN28B and IMP1 may cooperatively promote a tumor-initiating phenotype.

Methods: We evaluated LIN28B and IMP1 expression and localization in colorectal cancer patient samples using tissue microarrays and clinical outcomes. We used intestinal epithelial cell lines with LIN28B overexpression and CRISPR-Cas9 mediated knockout of IMP1 to study the functional consequences on migration, invasion and proliferation.

Results: LIN28B expression correlates with expression of IMP1 in colorectal cancer patient samples. Individually, LIN28B and IMP1 expression intensity each was associated with worse prognosis in stage II colon cancer. Knock-down of IMP1 in Lin28B overexpressing cells decreased migration of colorectal cancer cell lines, suggesting a relationship of IMP1 for the tumorigenic effects of LIN28B. Furthermore, our RNA target analyses show that Lin28b and IMP1 both bind to target mRNAs in the WNT and adherens junction pathways.

Conclusions: These data implicate a novel role for the Lin28b-let7-IMP1 axis in colorectal cancer and support an emerging paradigm for a critical and cooperative role of RNA-binding proteins in intestinal homeostasis and cancer.

We are using the cell lines generated for orthotopic xenograft studies to see the effects of IMP1 loss and Lin28b overexpression on tumor growth and dissemination. Furthermore, we are generating mice with intestinal epithelium specific Lin28b overexpression-IMP1 loss to study the effects in vivo.

#1137

Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells.

Hitoshi Akiyama, Koichi Nakashiro, Norihiko Tokuzen, Hiroshi Tanaka, Satoshi Hino, Hiroyuki Hamakawa. _Ehime University Graduate School of Medicine, Toon, Japan_.

Whole-exome sequencing studies showed the mutational landscape of head and neck squamous cell carcinoma including oral cavity. Recent several reports also demonstrated the somatic mutations of oncogenes and tumor suppressor genes in oral squamous cell carcinoma (OSCC), and identified the mutations of HRAS, BRAF, FGFR3, SMAD4, KIT, PTEN, NOTCH1, AKT1, CTNNB1, and PTPN11 to predict disease-free survival of OSCC patients. In this study, we have attempted to elucidate somatic mutations of these genes and their functions in human OSCC cells. We isolated and cultured tumor cells from the resected primary tumor (KT-T), metastatic lymph node (KT-N), and cancerous pleural effusion (KT-M) in the same patient with OSCC. KT-T cells showed epithelial-like shape, whereas both KT-N and KT-M cells indicated the morphological changes to fibroblast-like spindle shape. Genomic DNA was extracted from these cells, and then we performed the mutational analysis by ultra-deep targeted sequencing using Haloplex Cancer Research Panel. All types of KT cells had the mutation of TP53 (R141P, C135S, and E182*) and PIK3CA (H1047R), and an additional active mutation of HRAS (Q61R) was detected in only metastatic KT-N and KT-M cells. Subsequently, we transfected all KT cells with synthetic small interfering RNA (siRNA) specific for PIK3CA (siPIK3CA) and/or HRAS (siHRAS) at the concentration of 10 nM complexed with Lipofectamine RNAiMAX. We confirmed the knockdown effects of these siRNAs in KT cells by quantitative RT-PCR. After transfection of these siRNAs for 72 hours, the growth of KT cells was evaluated by WST-8 assay. Knockdown of PIK3CA or HRAS expression significantly suppressed the growth of KT-T cells only, whereas double knockdown of PIK3CA and HRAS inhibited the growth of KT-N and KT-M cells as well. Next, we investigated the function of mutant HRAS (Q61R) in KT-T cells. Overexpression of mutant HRAS did not change the growth rate and morphology of KT-T cells. Finally, we examined the effects of BEZ-235 (dual PI3K/mTOR inhibitor) and trametinib (MEK1/2 inhibitor) on the growth of KT-T, KT-N, and KT-M cells. BEZ-235 (100 nM) almost completely suppressed the growth of these cells. Trametinib (2 μM) also reduced the growth rate by 16.9% in KT-T, 13.5% in KT-N, and 45.7% in KT-M cells. Furthermore, BEZ-235 combined with trametinib inhibited the cell growth more effectively even at low concentrations. These results suggest that constitutive active mutations of HRAS and PIK3CA support the growth of human OSCC cells and these signaling pathways may be useful therapeutic targets for the patients with OSCC.

#1138

Prognostic impact of BRAF-V600E expression and correlation with mutation status in cutaneous melanoma.

Emilia Hugdahl, May Britt Kalvenes, Hanne Puntervill, Rita Ladstein, Lars A. Akslen. _University of Bergen, Bergen, Norway_.

Around 50% of primary cutaneous melanomas harbour BRAF mutations, but their prognostic impact has not been clear. Recently, a BRAF-V600E mutation-specific antibody has become available for immunohistochemistry (IHC). Here, we investigated for the first time the prognostic impact of BRAF-V600E protein expression in primary melanoma. In a patient series of 248 nodular melanomas, BRAF-V600E and total BRAF expression were assessed by IHC using tissue microarray sections of paraffin-embedded archival tissue. Mutation status was assessed by real-time PCR. Positive BRAF-V600E expression was present in 86 (35%) of the cases and was significantly associated with increased tumor thickness, presence of tumor ulceration and reduced survival. Further, BRAF-V600E expression was an independent prognostic factor by multivariate survival analysis. There was 88% concordance between BRAF-V600E expression and mutation status. Our findings indicate that BRAF-V600E expression is a novel prognostic marker in primary melanoma.

#1139

Translational control of MYC protein stability as a target of small molecules.

Naohiko Ikegaki, Luqman Baloch, Christopher Chu, Joshua Lomahan, Jamie Harris, Bill Chiu. _Univ. of Illinois at Chiacgo, Chicago, IL_.

It is well established that MYC family proteins drive tumor growth and sustain the malignant phenotype. However, MYC family proteins have gained the reputation of being "un-druggable" by small molecule inhibitors. This is because MYC proteins lack intrinsic enzymatic activities or appropriate molecular grooves to which small molecules may bind and inhibit their function. Nonetheless, previous studies suggest that MYC family proteins (MYC, MYCN, MYCL) can be "targetable" indirectly by accelerating their turnover rate at different biochemical pathways that regulate the steady state level of the proteins. These include transcriptional-inhibition of the corresponding genes and acceleration of their proteasome-dependent degradation. The goal of this study is to gain a better understanding of how the MYC family protein turnover is regulated, which in turn might help develop future new drugs against "MYC family-driven" cancers. We have used neuroblastoma, a childhood cancer, as a disease model and identified several small molecules that rapidly destabilize MYC and MYCN proteins in MYC-driven neuroblastoma cells. The small molecules include FCCP, OSU-03012 and Salinomycin. Preliminary data suggest that the common target of these compounds is mitochondria. Recently, we have also found Halofuginone, an FDA approved orphan drug for Scleroderma treatment exhibits a similar MYC-destabilizing activity. At submicromolar concentrations and less than one hour of drug-treatment, Halofuginone rapidly causes significant down-regulation of MYC proteins in neuroblastoma cells. Halofuginone is known to activate the eIF2α pathway, which controls translation. Activation of the eIF2α pathway is initiated by the activation of eIF2akinases (GCN2, HRI, PERK, PKR) via autophosphorylation by various stress-related stimuli such as reactive oxygen species and misfolded proteins. The activated eIF2αkinases then inactivates eIF2α by phosphorylation, which subsequently halts translation globally. Interestingly, FCCP, OSU-03012 and Salinomycin treatments also caused eIF2α phosphorylation. These observations suggest that activation of the eIF2α pathway results in translational block of the MYC family protein expression in tumor cells. Because half-lives of MYC family proteins are very short, a sudden attenuation in protein translation can cause rapid down-regulation of the proteins. Hence, activation of the eIF2α pathway by small molecules may prove effective for rapid down-regulation of MYC family proteins in MYC-driven cancer cells.

#1140

CD24 exists in the nucleus and drives cancer growth.

Jason Duex, Changho Lee, Charles Owens, Ana Chauca-Diaz, Dan Theodorescu. _University of Colorado Anschutz Medical Campus, Aurora, CO_.

CD24 is an oncoprotein whose high level of expression in patient tumors yields a poor prognosis. This observation has been shown in breast, prostate, pancreas, ovary, colorectal, and bladder cancers. In vitro, elevated expression of CD24 has been shown to drive cancer cell proliferation while in vivo, it promotes tumor growth and metastasis.

Cancer stem cells (CSC), in many different tissue types, are denoted by the presence or absence of CD24 cell surface expression. Most prominent among these tissue types is the finding that sorting breast cancer cells for a CD24neg population yields a CSC cohort. Using bladder cancer cells we discovered that a CD24neg sorted population exhibits a 2 fold increase in anchorage independent growth relative to parental cells and a 5 fold increase relative to cells depleted of CD24 by RNAi (shCD24). In vivo we determined that CD24neg cells were dramatically more successful than shCD24 cells at establishing metastases (6/10 vs 0/10 mice). Strikingly, we found that treatment of CD24neg cells with CD24 siRNA eliminated their increased growth potential. These results strongly suggest a significant biological role for non-surface CD24. Therefore, we set out to determine the precise locations of CD24 within cells and assess any possible biological relevance.

Cellular fractionation experiments revealed the existence of CD24 in the nuclear fraction in 3 different bladder lines as well as lines from breast, lung, colon and prostate cancer cells. Furthermore, this nuclear signal was the same in both CD24neg cells and parental cells. Using immunofluorescence we observed CD24 signal colocalizing with DAPI signal. Again, this nuclear signal remained relatively unchanged in CD24neg cells compared to parental cells. We also found that blocking nuclear import (Ivermectin) lead to a decrease in nuclear CD24 signal and blocking nuclear export (Leptomycin B) lead to an increase in CD24 signal in the nucleus, suggesting cells actively regulate nuclear CD24 levels. To assess the potential functional significance of nuclear CD24 we expressed a mutant which localizes exclusively to the nucleus (NLS-CD24). Comparing cells expressing NLS-CD24 to those expressing either wild-type CD24 (Wt-CD24) or a control construct with a scrambled amino acid sequence in place of NLS (Scr-CD24) revealed that NLS-CD24 expressing cells exhibit a 2-3 fold increase in anchorage independent growth over both Wt-CD24 and Scr-CD24. Similarly, growth rates of subcutaneous tumors in mice expressing NLS-CD24 were 3 times higher than those expressing Wt-CD24.

Taken together, we discovered and validated a nuclear cohort of CD24 and found that nuclear specific expression of CD24 promotes greater growth phenotypes both in vitro and in vivo. Thus, it is important that cancer models and therapeutic approaches involving CD24 expression consider this nuclear cohort.

#1141

The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells.

Guanghua Wan, Scott A. Ness, Ashwani Rajput. _University of New Mexico Health Sciences Center, Albuquerque, NM_.

INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and Inositol polyphosphate-4-phosphatase, type II (INPP4B) are two important enzymes maintaining homeostasis of phosphoinositides in cells. Class IA PI3K produces Phosphatidylinositol-3,4,5-bisphosphate (PIP3) by phosphorylation of Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at D3 position, while INPP4B dephosphorylate Phosphatidylinositol-4,5-bisphosphate (PI(3,4)P2) at the D4 position to generate Phosphatidylinositol 3-phosphate (PI(3)P). All of these phospholipids are important second messengers. PI3K activates its downstream substrate Protein Kinase B (Akt) by PIP3. INPP4B, however, blocks Akt activities in epithelial cells through the turnover of PI(3,4)P2. PI3K/Akt pathway is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. Gain of function mutations in the catalytic subunit p110α of PI3K occur in up to 1/3 of human colorectal cancers (CRC). The kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α. Our previous report showed that this mutation in p110α results in an increase in cellular levels of PIP3 and in turn an enhancement of Akt activity. Since the H1047R mutation and INPP4B have an opposite effect on Akt activities, the interesting question is if there is crosstalk between the two or if H1047R mutation can affect cellular INPP4B expression. We hypothesize that H1047R mutated p110α down-regulates INPP4B and therefore results in increased Akt activity.

METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous protein expression level of INPP4B was detected by immune-blotting analysis in parental, WT and MUT HCT116 cells. To further investigate the effect on INPP4B of the H1047R mutation, we conducted RNA-sequencing for profiling, quantifying RNA transcripts in WT and MUT HCT116 cells, and compared RNA transcript level of INPP4B between the two isogenic cell lines of HCT116 cells.

RESULTS: 1) Endogenous expression level of INPP4B protein in HCT116WT cells was over 10-times higher than in HCT116MUT cells. 2) INPP4B RNA was down regulated by the H1047R mutation in p110α (PIK3CA) of Class 1A PI3K.

CONCLUSION: Our results suggested that hyperactivity of Akt in H1047R mutation bearing cells may be also related to low level of cellular INPP4B, and that the H1047R mutated p110α may increase cell migration and cancer metastasis through down regulation of tumor suppressor gene INPP4B. Although further studies are needed to reveal the mechanism of H1047R mutated PI3K mediated INPP4B-regulation, INPP4B expression level may be a potential maker to predict PI3K mutation induced CRC metastasis.

#1142

E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma by promoting ubiquitination-mediated degradation of RARa.

Qian Zhou, Meidan Ying, Bo Yang, Qiaojun He. _Zhejiang Univ. Inst. of Pharm. & Toxicol., Hangzhou, Zhejiang, China_.

Objective: All-trans retinoic acid (ATRA) is a widely-used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA induced osteogenic differentiation of osteosarcoma cells.

Methods: Osteosarcoma cell lines were used as our research models. (1) Protein levels were detected by Western blotting; (2) The interactions between E2F1 and RARalpha were determined by immunofluorescence, immunoprecipitation and GST pull-down assay; (3) The mRNA levels were determined by RT-PCR; (4) Related proteins and genes were investigated via transfection; (5) RARE activity were detected by luciferase reporter assay. (6) Alkaline phosphatase activity was assessed by colorimetric assay using BCIP/NBT Alkaline Phosphatase Color Development kit. Results: We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to down-regulate retinoic acid receptor (RAR), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RAR and promotes its ubiquitination-mediated degradation; as a consequence, RAR-mediated differentiation is inhibited in osteosarcoma.

Conclusion: E2F1 may serve as a potent biomarker as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients.

#1143

Pro-proliferative function of SIRT3 in a human melanoma xenograft mouse model.

Jasmine George, Minakshi Nihal, Mary A. Ndiaye, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Melanoma is one of the most aggressive forms of skin cancer and is often lethal, if not treated early. Therefore, it is necessary to try to develop novel target-based strategies to combat this neoplasm. SIRT3 is a nicotinamide adenine dinucleotide (NAD+) -dependent mitochondrial protein deacetylase involved in metabolism- and aging- related disorders, including certain cancers. Interestingly, studies have suggested both tumor suppressor as well as tumor promoter roles of SIRT3 in cancer. However, its role in melanoma is not well-established. Previously, we have shown that SIRT3 was upregulated in human melanoma cells and its lentiviral knockdown resulted in anti-proliferative effects in human melanoma cells. We also found that SIRT3 knockdown resulted in the G1-phase arrest of the cell cycle, induction of senescence, and decrease in cell migration. This study was designed to determine the relevance of our in vitro findings to the in vivo situation, in human melanoma xenograft models. Employing immunodeficient mice (Crl:NU-Foxn1nu, homozygous; Strain 088), we first determined the effect of SIRT3-knockdown on melanoma tumorigenesis. The mice were subcutaneously implanted with shNS-SK-MEL-2 (control) and shSIRT3-SK-MEL-2 (SIRT3-knockdown) melanoma cells, followed by assessing tumorigenesis of melanoma cells. We found that compared to shNS-SK-MEL-2 cells, shSIRT3-SK-MEL-2 cells showed a significantly decreased tumorigenic potential in these mice, in terms of average tumor volume (measured weekly) and average tumor weight (measured at termination of the study). Further, the Kaplan-Meier analysis showed that SIRT3 knockdown resulted in a significant survival advantage, in terms of reaching the cutoff tumor size (2.0 cm in one dimension). In an additional strategy, we determined the tumorigenicity of SIRT3 overexpressing melanoma cells (Hs294T-SIRT3) in NU/NU mice. Our data demonstrated that compared to control Hs294T-pcDNA cells, the SIRT3 overexpressing Hs294T-SIRT3 cells demonstrated a significantly enhanced tumorigenic potential (average tumor volume and tumor weight) in NU/NU mice. Overall, our study provides evidence supporting a pro-proliferative role of SIRT3 in melanoma and suggests that SIRT3 needs to be further evaluated as a potential druggable therapeutic target for melanoma management. However, detailed mechanistic and in vivo studies in relevant melanoma models are needed to further validate our findings.

#1144

Detection of a novel ALK fusion variant in lung adenocarcinoma using a comprehensive genomic analysis.

Kazuhiko Shien,1 Dennis Ruder,1 Ecaterina E. Ileana,1 Vassiliki A. Papadimitrakopoulou,1 Garrett M. Frampton,2 Carmen Behrens,1 Neda Kalhor,1 J. Jack Lee,1 Ximing Tang,1 Roy S. Herbst,3 Ignacio I. Wistuba,1 Julie G. Izzo1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Foundation Medicine Inc., Cambridge, MA;_ 3 _Yale Cancer Center, Yale School of Medicine, New Haven, CT_.

Background: Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in a subset of non-small cell lung cancer (NSCLC), mainly as a result of chromosomal translocation. In ALK-rearranged NSCLC, the growth of tumors is strongly addicted to ALK activity, and suppression of ALK is a potent therapeutic strategy. To date, 6 known fusion partners of ALK onco-kinase have been reported in NSCLC, although the sensitivity to ALK inhibitor according to fusion variant except for EML4-ALK is still unclear. In this study, we reviewed clinical trial samples analyzed by comprehensive genomic profiling approach to detect unknown actionable ALK rearrangements.

Materials and methods: We reviewed gene alteration and expression profile of clinical samples in preliminary molecular studies from the ongoing BATTLE-2, phase II clinical trial of targeted drugs in chemo-refractory NSCLC.

Results: In 187 NSCLC samples, 3 samples harbored ALK fusion gene: 2 EML4-ALK and 1 novel ALK fusion variant PRKAR1A-ALK in a lung adenocarcinoma of male former smoker patient. The ALK gene expression levels of ALK-rearranged samples were significantly higher compared to other samples. Interestingly, the patient harboring PRKAR1A-ALK was treated with ALK inhibitor crizotinib and showed long time stable disease.

Conclusions: Our findings suggest that PRKAR1A-ALK fusion could be a novel oncogenic driver in lung adenocarcinoma and an indicator of ALK inhibitor response. (NHI-NCI CA155196; 2P50CA070907-16A1)

#1145

Zhx2 promotes survival of B-cell acute lymphoblastic leukemia cell lines.

Namit Sharma, Huayun Hou, Michael D. Wilson, Catherine Jane McGlade. _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Zhx2, a zinc finger containing transcription factor is frequently overexpressed in B cell malignancies including B cell acute lymphoblastic leukemia (B-ALL), however its role in development of B cell malignancies remains unknown. Previously, dysregulated activity of transcription factors due to mutation e.g. Myc or overexpression e.g. Sox11 has been shown to perturb B cell differentiation leading to malignant phenotypes, resistance to chemotherapeutics and genetic heterogeneity. Zhx2 is required for normal neuronal and erythropoietic differentiation and has been identified as a tumor suppressor in hepatocellular carcinoma. Furthermore, analysis of public databases shows that Zhx2 is highly overexpressed in several cell lines representing B cell malignancies including B cell acute lymphoblastic leukemia (B-ALL) and B cell lymphoma. Consistently, Zhx2 is also overexpressed in patient samples derived from different subtypes of B cell malignancies. Quantification of Zhx2 mRNA expression obtained from human donors showed a marked upregulation during transition from common lymphoid progenitor to Pro B-cell stage suggesting potential role in B cell differentiation. Here we report that Zhx2 is overexpressed in Philadelphia chromosome positive (Ph+) B-ALL and T-ALL cell lines as well as Ph- B-ALL cell line. Zhx2 silencing in BV-173 (Ph+ B-ALL) and CMLT1 (Ph+ T-ALL) showed enhanced apoptosis with low doses of Dasatinib, a dual Src/Abl kinase inhibitor. Furthermore, global gene promoter and gene expression analysis on Zhx2 silenced cells identified putative Zhx2 regulated genes including CREB and YY1. Both of these transcription factors have roles in B cell differentiation, survival, proliferation and are commonly deregulated in B cell malignancies. We propose that Zhx2 regulates a B cell differentiation program and that overexpression of Zhx2 could lead to blocked differentiation of B cell progenitors promoting the malignant phenotype. Therefore, Zhx2 could be exploited as a chemo-therapeutic target in B cell malignancies.

#1146

**Enhancer of zeste homolog 2 (EZH2) inhibition in metastatic melanoma promotes tumor regression** in vivo **.**

Deepanwita Sengupta, Nathan Avaritt, Sara C. Shalin, Alan J. Tackett. _UAMS, Little Rock, AR_.

Melanoma is a deadly variety of skin cancer, which is responsible for three-fourths of skin cancer-related deaths, but constitutes less than 5% of all skin cancer cases. It has been estimated that in 2015, about 73,870 new cases of melanoma will be diagnosed in United States alone, of which 9,940 are expected to die. According to the ACS, melanoma is also the fastest growing cancer, which makes it essential to investigate the pathogenesis involved in the advanced stage of melanoma, and design effective therapeutics for its treatment.

Drugs targeting epigenetic modifiers like EZH2 are becoming increasingly popular because of the fact that most epigenetic modifications and their effects can be reversed using these drugs. There has been no study till date investigating the role of EZH2 in melanoma. Our laboratory is investigating the role of EZH2 in the pathogenesis involved in the advanced stage of melanoma, to enable the design of effective therapeutic strategies for the treatment of melanoma. Here we investigate the relationship between EZH2 and tumor suppressors E-cadherin and RUNX3 in melanoma using matched melanoma cell lines (WM115 and WM266-4), formalin-fixed-paraffin-embedded (FFPE) patient tissue samples and in vivo xenograft model. The studies performed in cell lines and FFPE tissue samples suggested EZH2-mediated silencing of E-cadherin and RUNX3 by promoting H3K27-trimethylation of the E-CADHERIN and RUNX3 promoters in WM266-4 cells (metastatic; high EZH2; low E-cadherin) relative to WM115 cells (primary; low EZH2; high E-cadherin). Furthermore, we observed that EZH2 inhibition restores E-cadherin and RUNX3 expression in metastatic melanoma cells by reducing EZH2 and H3K27me3 occupancy at E-CADHERIN and RUNX3 promoters respectively. In addition, we found that inhibition of EZH2 besides inducing apoptosis and inhibiting cellular proliferation, also adversely affected invasion of metastatic melanoma. E-cadherin depletion had an opposite effect, suggesting that EZH2 mediated E-cadherin silencing promotes invasion in metastatic melanoma. In vivo, treatment of WM266-4 xenografts with EZH2 inhibitor resulted in significant reduction of tumor growth. In combination with Dabrafenib, this effect was highly synergistic. Analysis of harvested tumors indicated increased E-cadherin and RUNX3 levels in EZH2 inhibited group compared to vehicle, due to reduced H3K27me3 enrichment by EZH2 at E-cadherin and RUNX3 promoters, validating our in vitro finding. Tumor regression by combination therapy was also accompanied by significant increased pro-apoptotic proteins like Bak, Bax and decreased anti-apoptotic proteins Bcl-xL and Bcl-2.

Hence, our findings suggest strong potential of EZH2 inhibitors as promising melanoma drugs either by itself (monotherapy), or in conjunction with other chemotherapeutics (combination therapy).

#1147

CDCA5 is a novel therapeutic target molecule in oral squamous cell carcinoma.

Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroshi Tanaka, Kazuki Iwamoto, Hitoshi Akiyama, Hiroyuki Hamakawa. _Ehime University Graduate School of Medicine, Toon. Ehime, Japan_.

Molecularly targeted drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. We attempted to find appropriate molecular targets for OSCC, and identified cell division cycle associated 5 (CDCA5) as a cancer-related gene which was overexpressed in 9 human OSCC cells tested by microarray analysis. CDCA5 was initially identified as a substrate of anaphase-promoting complex and as a regulator of sister chromatid cohesion in HeLa cells. In addition, CDCA5 has been reported to be overexpressed in the majority of human lung cancers and urothelial cancers and to play a critical role in carcinogenesis. However, function of CDCA5 in OSCC remains still unknown. In this study, we investigated the expression and function of CDCA5 in OSCC. First, we confirmed the expression level of CDCA5 in 4 human OSCC cell lines and an immortalized non-neoplastic human keratinocyte cell line, HaCaT, by quantitative RT-PCR (qRT-PCR) and Western blotting. The expression levels of CDCA5 mRNA and protein were markedly elevated in all human OSCC cell lines compared to HaCaT cells. Next, we tested the effect of synthetic small interfering RNAs specific for CDCA5 (siCDCA5) on the growth of human OSCC cells. The RNA interference effect of siCDCA5 was confirmed by Western blotting. Transfection of siCDCA5 suppressed the expression of its target protein in all types of cells. The growth inhibitory effect of siCDCA5 in OSCC cells was evaluated by WST-8 assay. The Knockdown of CDCA5 significantly inhibited the growth of OSCC cells in vitro. Subsequently, we assessed the effect of siCDCA5 on the in vivo growth of human OSCC cells, green fluorescent protein (GFP)-SAS. We administered siCDCA5/atelocollagen complexes into the subcutaneous spaces around the xenograft tumors every 3 days. We found that these complexes significantly reduced the size of subcutaneously xenografted GFP-SAS tumors, compared to the control group treated with synthetic siRNA specific for GFP (siGFP)/atelocollagen complexes. Next, we analyzed the influence of siCDCA5 on the cell cycle of OSCC cells by flow cytometry. Suppression of CDCA5 resulted in the decrease in percentage of cells in G0/G1 phase and the increase in G2 phase. This indicated that the anti-proliferative effect of siCDCA5 was due to G2 arrest. Finally, we investigated the clinical significance of CDCA5 expression in OSCC. We examined the expression of CDCA5 in OSCC tissues by immunohistochemistry, and then found a significant association between CDCA5 expression levels and overall survival. These results suggest that CDCA5 functions as a critical gene supporting OSCC progression and that targeting CDCA5 may be a useful therapeutic strategy for OSCC.

#1148

A muscle specific protein "myoferlin" modulates IL-6/STAT3 signaling by chaperoning activated STAT3 to nucleus.

Arti Yadav, Bhavna Kumar, Theodoros N. Teknos, Pawan Kumar. _The Ohio State University, Columbus, OH_.

Recently, we made a provocative and novel discovery in our laboratory that a muscle-specific protein, myoferlin, is markedly upregulated in head and neck cancer cells. Myoferlin is a member of ferlin family of membrane proteins and most of the studies related to myoferlin's biological and molecular function(s) were carried out on muscle cells, as it was assumed that myoferlin is predominantly present only in the muscle cells. Recent studies have shown that myoferlin is also expressed in endothelial and cancer cells and it modulates VEGFR-2 and EGFR stability and function. Based on these reports, we hypothesized that myoferlin might be regulating IL-6 signaling by modulating IL-6R stabilization and recycling. However, in our immunoprecipitation (IP) experiments, we did not observe myoferlin binding with IL-6R. Instead, we observed that myoferlin is bound to EHD2 protein in the resting cells and when the cells are treated with IL-6, myoferlin dissociates from EHD2 and binds to activated STAT3. Interestingly, myoferlin knock-down did not affect STAT3 phosphorylation, but completely blocked STAT3 translocation to nucleus and expression of its target proteins including Snail and Nanog. In addition, inhibition of STAT3 phosphorylation by phosphorylation defective STAT3 mutants or JAK inhibitor blocked STAT3 binding to myoferlin and nuclear translocation. Myoferlin knockdown significantly decreased IL-6-mediated tumor cell migration, tumorsphere formation and ALDH positive cancer stem cell population, in vitro. Furthermore, myoferlin knockdown significantly decreased IL-6-meditated tumor growth and tumor metastasis, in vivo. Based on these results, we have proposed a novel model for the role of myoferlin in chaperoning phosphorylated STAT3 to the nucleus.

#1149

Interaction with NHERF1 enhances protein stability of G protein-coupled estrogen receptor.

Ran Meng, Ying Xiong, Yuan Zhao, Yan Wang, Tao Tao, Qiqi Wang, Hua Liu, Songlin Wang, Qiong Qin, Junfang Zheng, Junqi He. _Capital Medical Univ., Beijing, China_.

G Protein-Coupled Estrogen Receptor (GPER), also called GPR30, has been demonstrated to play important roles in a variety of physiological and pathological responses, especially in cancer cells proliferation, migration and invasion. Despite increasing evidences have showed clinical correlations between GPER expression level and poor prognosis in human cancer specimens, little is known regarding the regulation of GPER expression level, especially in protein level. Our previous studies have shown that PDZ protein NHERF1 can interact with ACTN4 (FASEB J. 2015) or PTEN (Endocrinology. 2011) and prevent their degradation. In this study, NHERF1 was demonstrated to interact with GPER in HEK293 cells and breast cancer cells MCF7 and T47D. GST Pull-down, Co-IP and immunocytochemistry analysis revealed that this interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal of GPER. Mutation of the last c-terminal amino acid of GPER V375A could abolish the interaction of NHERF1 with GPER. Change of NHERF1 expression level by overexpression or knockdown affects the overall protein level of GPER. NHERF1 overexpression improved the GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in an interaction dependent manner. Further analysis of public TCGA data revealed that NHERF1 expression level positively associated with activation of GPER downstream genes in invasive breast cancer, in which GPER mRNA level was similar with that of normal breast tissue. Our findings identify NHERF1 as a novel binding partner for GPER and uncover a novel role of NHERF1 in promoting GPER protein stability by inhibiting ubiquitin-proteasome degradation of the receptor. Considering the importance of GPER in breast cancer progression, these findings may help elucidate mechanisms associated with breast cancer invasion and metastasis.

#1150

Targeting MET Exon 14 mutations with the selective small molecule inhibitor Savolitinib.

Evan Barry, Elizabeth Maloney, Ryan Henry, Alexandra Borodovsky, Edwin Clark, Melanie Frigault, Michael Zinda, Celina D'Cruz. _AstraZeneca, Waltham, MA_.

Alterations in the MET oncogene occurs across a broad range of tumor indications. Amplification or mutations in MET lead to increased activity of downstream pathways including PI3K and MAPK, eventually resulting in tumor formation. Several small molecule inhibitors are currently in clinical trials, including the selective inhibitor Savolitinib (HMP-504, Volitinib, AZD6094), which shows single digit nanomolar activity in MET-amplified cell lines. Newly emerging data suggest mutations in MET causing complete skipping of Exon 14 occur in approximately 4% of non-small cell lung cancer (NSCLC), and are more rare in other indications [1, 2]. MET exon 14 skipping mutations were shown to be mutually exclusive from EGFRm, ALK and KRAS and can occur in the context of MET gene amplification [3]. Exon 14 harbors the CBL binding site (Y1003), which is critical for receptor degradation after binding of its ligand, HGF, and suppression of downstream signaling events. Clinical trial results with less potent, pan RTK inhibitors Crizotinib (31nM GI50 vs 3nM for Savolitinib) and Cabozantinib show promising early results, but fall short in long term responses. Therefore, better therapies targeting MET are needed. Human cell line models with Exon 14 deletions are rare. Therefore, we used engineered cell lines to test the effect of Savolitinib on these mutations. To do this, we expressed MET-Y1003F mutants in NIH-3T3 and HEK293T cells. We found that Savolitinib potently inhibited phospho-MET in both models expressing this mutant (100% phospho-MET inhibition). In addition, we tested whether or not Savolitinib could inhibit HGF-dependent signaling and growth of a NSCLC cell line, NCI-H596. In the presence of FBS (10%), Savolitinib had no effect on the growth rate of these cells, however was highly efficient at blocking HGF-dependent growth in the absence of FBS. To test the effect of this mutation in the background of amplification, we also tested the gastric cancer cell line Hs746T, which harbors exon 14 skipping in addition to MET amplification. Savolitinib was highly efficacious at blocking the growth of this cell line. Future studies are aimed at looking at the in vivo effect of Savolitinib targeting exon 14 mutants. These data provide a platform of evidence for using Savolitinib to target exon 14 mutant MET in patients.

1.

Paik, P.K., et al., Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping. Cancer Discov, 2015. 5(8): p. 842-9.

2.

Frampton, G.M., et al., Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discov, 2015. 5(8): p. 850-9.

3.

Cancer Genome Atlas Research, N., Comprehensive molecular profiling of lung adenocarcinoma. Nature, 2014. 511(7511): p. 543-50.

### Oncogenes and Tumor Suppressor Genes and Pathways

#1151

IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer.

Zhen Lu,1 Aaron Orozco,1 Weiqun Mao,1 Yan Wang,1 Margie Nicole Sutton,1 Hailing Yang,1 Douglas A. Levine,2 Robert C. Bast Jr.1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Memorial Sloan Kettering Cancer Cente, New York City, NY_.

Ovarian cancer is the most lethal gynecologic malignancy. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at "second look" operations. Previously, we reported that more than 80% of persistent, dormant drug resistant ovarian cancers express ARHI and are undergoing autophagy. Autophagy can protect or kill tumor cells depending upon the context. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers. Re-expression of ARHI in cell culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced after 6 weeks of induction, xenografts grow promptly. Our current experiments were designed to determine whether survival of autophagic cells in vivo requires permissive levels of VEGF, IL-8 and IGF-1 in the tumor microenvironment. In cell culture, co-incubation of VEGF, IGF-1 and IL-8 with SKOv3-ARHI cells, increased AKT activity, reduced ARHI-mediated autophagy formation and decreased autophagic cell death. SKOv3-ARHI cells grown subcutaneously in nude mice exhibited higher levels of AKT activity and lower levels of autophagy than cells grown in cell culture. When cytoplasmic and nuclear fractions were obtained from xenografts, the nuclear fractions showed increased phospho-AKT, and decreased levels of a transcription factor EB (TFEB) and low mRNA expression of LC3, suggesting survival factors in tumor environment may stimulate AKT, downregulate expression of TFEB and inhibit formation of autophagy. Treatment with anti-VEGF, anti-IL-8 and anti-IGF-1 antibodies, individually and in combination, inhibited survival factors-mediated AKT activation and slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability to induce autophagy and to inhibit AKT. These preclinical studies support that the possibility that treatment with a combination of anti-VEGF, anti-IL8 and anti-IGF-1 antibodies could prevent outgrowth of dormant cells in patients with ovarian cancer. Furthermore, these results suggest that survival factors are required to limit autophagic death.

#1152

MUC1: A metabolic regulator in triple-negative breast cancer.

Gennifer D. Goode,1 Nina Chaika,1 Vinee Purohit,2 Venugopal Gunda,1 Pankaj Singh1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _‎University of Michigan Health System, Ann Harbor, MI_.

Breast cancer, the second leading cause of cancer deaths in women, is the most common cancer among North American women, accounting for nearly 1 in 3 cancer cases diagnosed in the U.S. women. Triple negative breast cancer (TNBC) subtype accounts for approximately 15-25% of all breast cancer cases and has an increased incidence of metastasis, high recurrence within 1-3 years and a high mortality rate. Therefore, identifying factors that facilitate tumor growth and metastases have the potential to serve as novel molecular targets for breast cancer therapy. Mucin1 (MUC1), a glycoprotein associated with chemoresistance, is aberrantly overexpressed in TNBC and facilitates growth and metastasis of TNBC cells. This occurrence can be partially attributed to MUC1 interaction with hypoxia-inducible factor alpha (HIF1α), a key regulator of glycolysis. As metastasis is the leading cause of cancer related deaths, this process relies on the ability of tumor cells to adapt to the changing nutrient levels in the surrounding stroma. In the present study we examined how signaling through MUC1 facilitates metabolic phenotype in TNBC; thus facilitating tumor growth and metastasis. Our results indicate that MUC1 expression facilitates glucose and glutamine uptake in a panel of TNBC cell lines. Furthermore, metabolomic analysis through liquid chromatography-coupled tandem mass spectrometry approach revealed that MUC1 expression modulates metabolite levels in glycolysis, pentose phosphate pathway and TCA cycle in TNBC cells. Thus, our results support the notion that MUC1 serves as a metabolic regulator in TNBC, facilitating metabolic reprogramming that influences growth of TNBC.

#1153

Hand-1 overexpression inhibits medulloblastoma cell invasion and metastatic ability via Oct-3/4 / β-catenin interaction.

Swapna Asuthkar,1 Maheedhara R. Guda,1 Andrew J. Tsung,2 Reuben Antony,1 Dzung H. Dinh,1 Kiran K. Velpula1. 1 _University of Illinois College of Med. at Peoria, Peoria, IL;_ 2 _Illinois Neurological Institute, Peoria, IL_.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Treatment of medulloblastoma includes surgery, radiation and chemotherapy. Despite long-term survival for the majority of children, 50% of medulloblastomas recur. Recently, medulloblastomas are classified into four distinct molecular subgroups, viz., WNT, SHH, group 3, and group 4 based on constitutive activation of WNT/β-catenin, SHH, and NOTCH signaling pathways that regulate cancer stem cell pluripotency, tumor growth, progression and metastasis. Previously, we investigated the molecular machinery underlying uPAR and WNT/β-catenin interdependent signaling pathways in medulloblastoma stem cells. Further, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. Complementing to this we demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand-1. Here, we show that Hand-1 expression is observed to be downregulated in all the molecular subgroups of medulloblastoma. We also showed that the pluripotency marker, Oct-3/4 (encoded by the Pou5f1 gene) interacts with β-catenin and Hand-1 that ultimately regulated the uPAR expression. Our preliminary study demonstrated that Oct-3/4 preferentially cooperated with β-catenin in medulloblastoma to maintain tumor progression. Immunohistostaining of clinical specimens also confirmed our aforementioned results. Taken together, our studies show that overexpression of Hand-1 in medulloblastoma cells may effectively inhibit uPAR-induced invasion and metastatic ability, thereby regulating Oct-3/4/β-catenin balance.

#1154

Elevated nuclear GSK3β promotes more aggressive disease in AML.

James J. Ignatz-Hoover, Nathan Mackowski, David N. Wald. _Case Western Reserve University, Cleveland, OH_.

Acute myeloid leukemia (AML) is a devastating disease with a poor overall 5-yr survival rate and an antiquated and poorly tolerated therapy regimen. Despite an increasing knowledge of the molecular drivers of AML, new therapeutic targets are needed. GSK3β is a key signaling molecule that plays a role in several key oncogenic pathways including the β-catenin and NF-κB pathways, key pathways in AML and other cancers. Upregulation of GSK3β is correlated with poor prognosis in several cancers. We and others have shown GSK3β inhibition promotes AML differentiation as well as death of AML cells while sparing normal cells, suggesting GSK3β plays an important role in AML.

Here we show by western blotting and confocal microscopy that GSK3β is upregulated and more nuclear in AML cells. We utilize flow cytometry to show AML cells express elevated GSK3β compared to normal marrow lineages (Average upregulation= 6 fold). We transform mouse hematopoietic progenitor cells with the MLL-AF9 oncogene and observe GSK3β upregulation as a course of AML leukemogenesis. These data suggest that elevated and nuclear GSK3β is characteristic of AML.

To probe biological effects of GSK3β, we knocked down endogenous GSK3β and utilize a targetted, tetracycline-inducible GSK3β rescue model to show nuclear GSK3β specifically can promote AML growth. Nuclear GSK3β increases colony formation of OCI-AML3 and HL60 cell lines more potently than cytoplasmic (cyto) GSK3β in the same lines (OCI-AML3- Nuclear: 118% Cyto: 93% of control colony growth HL60- Nuclear: 117% Cyto: 100% of control colony growth, Nuclear difference- p<0.01 in both lines). Utilizing a mouse xenograft model and inducing GSK3β via doxycycline water, we show inducing nuclear GSK3β shortens mouse survival compared to uninduced controls, while inducing cytoplasmic GSK3β promotes no change from controls (HL60- Nuclear: Uninduced= 35.4 days mean survival, Induced= 28.75 days mean survival Cyto: Uninduced= 38 days mean survival, Induced= 38.6 days mean survival, Induced nuclear GSK3β difference p<0.01 by log rank test).

At a molecular level, when compared to cells expressing cytoplasmic GSK3β, nuclear GSK3β more potently promotes NF-κB DNA binding and NF-κB mediated transcription as measured by EMSA assay and luciferase assay (Nuclear- 18 fold increase Cyto- 3 fold increase). Nuclear GSK3β more potently promotes activation of the NF-κB subunit p65, as measured by pS536, than cytoplasmic GSK3β. Nuclear GSK3β also promotes nuclear localization of p65. These data suggest that nuclear GSK3β can promote NF-κB activation in AML.

Finally, we utilize imaging cytometry to show GSK3β is upregulated and more nuclear in clinical AML patient samples (N=80) compared to normal bone marrow controls (N=5). We also show nuclear localization of GSK3β and p65 correlate linearly in AML patient samples (N=80 R2= 0.44721 P<.01). These data suggest elevated nuclear GSK3β is observed in clinical AML and may be related to NF-κB activation.

#1155

Inhibitory effect of CYP1B1 on antitumor activities induced by dysregulated CDC20 and DAPK1 in renal cell carcinoma.

Yozo Mitsui,1 Inik Chang,2 Shinichiro Fukuhar,3 Hiroshi Hirata,1 Ryan Kenji Wong,1 Soichiro Yamamura,1 Varahram Shahryari,1 Guoren Deng,1 Saini Sharanjot,1 Shahana Majid,1 Hiroaki Shiina,4 Rajvir Dahiya,1 Yuichiro Tanaka1. 1 _Department of Urology, San Francisco Veterans Affairs Medical Center and University of California S, San Francisco, CA;_ 2 _Department of Oral Biology, Yonsei University College of Density, Democratic People's Republic of Korea;_ 3 _Department of Urology, Osaka University Graduate School of Medicine, Japan;_ 4 _Department of Urology Shimane University Faculty of Medicine, Japan_.

Introduction and Objective: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC. Methods: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses.

Results: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p<0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples.

Conclusions: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.

#1156

Downregulation of glycine decarboxylase contributes to paclitaxel resistance in ovarian cancer cells.

Jin Young Kim, Sojin Shin, Eunyoung HA, Soondo Cha, Chi-Heum Cho. _Keimyung Univ. Dongsan Medical Ctr., Daegu, Republic of Korea_.

Glycine decarboxylase (GLDC) is a metabolic oncogene that links glycine metabolism with tumorigenesis. In humans, GLDC is part of a multienzyme complex that couples the decarboxylation of glycine to the biosynthesis of serine. The role of GLDC in relation to chemoresistance is unclear. We designed this study to determine the role of GLDC in chemoresistance in ovarian cancer cells. First, we found GLDC is downregulated in paclitaxel resistant ovarian cancer cells (OVCAR3/PTX). Prompted by decreased glucose transport in OVCAR3/PTX cells, we transplanted OVCAR3 and OVCAR3/PTX onto the back of the NOD-scid IL2Rgammanull mice and found that the OVCAR3/PTX tumor grew less rapidly than paclitaxel sensitive ovarian cancer cells (OVCAR3). We also found that GLDC is downregulated in OVCAR3/PTX tumor. In vitro study revealed that the inhibitory effect of siGLDC on proliferation was significantly greater in OVCAR3 than in OVCAR3/PTX. We also found that knockdown of GLDC in OVCAR3 cells rendered ovarian cancer cells PTX-resistant. Finally, we determined GLDC expressions in patients with chemosensitive and chemoresistant serous type ovarian cancer and found that GLDC expression was markedly downregulated in chemoresistant cancer patients. These results suggest GLDC is associated with ovarian cancer chemoresistance.

#1157

Single nucleotide polymorphisms in ARHGAP17 are associated with pancreatic intraepithelial neoplasia in 2401 autopsied elderly patients.

Yoko Matsuda,1 Masashi Tanaka,1 Atsuko Seki,1 Keisuke Nonaka,1 Makoto Nishimura,1 Toshiyuki Ishiwata,2 Hisashi Yoshimura,3 Kaiyo Takubo,1 Tomio Arai1. 1 _Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, Japan;_ 2 _Nippon Medical School, Tokyo, Japan;_ 3 _Nippon Veterinary and Life Science University, Tokyo, Japan_.

Background: Aging, obesity, smoking, alcohol consumption, diabetes, and pancreatitis have been reported to be the risk factors for pancreatic cancer. There is much evidence showing that single nucleotide polymorphisms (SNPs), especially those in BCAR1, PDX1, ZNRF3, or TERT, influence one's predisposition to pancreatic cancers and survival. However, the association between SNP and pancreatic precancerous lesions is not yet clarified. In the present study, we clarified the clinicopathological features of precancerous lesions of the pancreas by examining autopsied elderly cases. Methods: We analyzed serial autopsy cases (men, 1326; women, 1075; mean age, 80.6) of pancreatic intraepithelial neoplasia (PanIN) and performed a comparative analysis based on the sex, age, and body mass index (BMI) of the patients. The SNPs were analyzed using HumanExome BeadChip. Results: The occurrence of PanIN increased with advanced age. Approximately, 60% of the elderly patients showed low-grade PanIN. The autopsy cases showed that the occurrence of low-grade PanIN was positively correlated with the female sex, advanced age, and increase in BMI. The SNPs of the ARHGAP17 gene, p.G782D (rs28365822) and rs1106576, were risk factors for PanIN (Table, P<0.0001, *adjusted with age and gender). Discussion: A high incidence of low-grade PanIN in the elderly might be related to the increased risk of pancreatic cancer in the elderly. Further studies are needed to clarify the role of SNPs of ARHGAP17 in the carcinogenesis of pancreatic cancer.

Association between SNPs in ARHGAP17 and pancreatic intraepithelial neoplasias

---

Genotype | PanIN (-) | |

PanIN (+) | |  | P value | |

unadjusted | adjusted*

rs28365822 | |

(%) | |  | Chi square | Dominant-Fisher | Logistic regression | Odds ratio | (95% confidence interval )

CC | 1028 | (99.1) | 1192 | (96.7) | <0.0001 | 0.00004 | 0.0002 | 3.93 | 4.05

TC | 9 | (0.9) | 41 | (3.3) | |  | |

(1.90-8.12) | (1.94-8.47)

TT | 0 | (0) | 0 | (0) | |  | |

|

Genotype | PanIN (-) | |

PanIN (+) | |  | P value | |

unadjusted | adjusted*

rs1106576 | |

(%) | |  | Chi square | Dominant-Fisher | Logistic regression | Odds ratio | (95% confidence interval )

TT | 1027 | (99.1) | 1192 | (96.7) | <0.0001 | 0.00004 | 0.0002 | 3.93 | 4.05

TC | 9 | (0.9) | 41 | (3.3) | |  | |

(1.90-8.12) | (1.94-8.47)

CC | 0 | (0) | 0 | (0) | |  | |

|

#1158

Differential expression and functional impact of the alternatively spliced transcripts of Extracellular matrix protein 1 in esophageal squamous cell carcinoma.

Valen Zhuoyou YU,1 Josephine Mun-Yee Ko,1 Simon Law,1 Li Dong Wang,2 Maria Li Lung1. 1 _The University of Hong Kong, HONG KONG, Hong Kong;_ 2 _Zhengzhou University, Zhengzhou, China_.

Background: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in China, with five-year survival rates in Hong Kong being of ~14%. An oligonucleotide microarray was utilized to identify differentially-expressed genes in ESCC using 31 pairs of matched normal/tumor samples from Hong Kong and Henan, China. Extracellular matrix protein 1 (ECM1) was among the top down-regulated genes, indicating a potential tumor-suppressive role. Public cDNA microarray data also shows significant down-regulation of ECM1 expression in ESCC. Interestingly, this gene has been extensively studied in breast cancer and thyroid cancer for its oncogenic role. There are three alternatively-spliced variants of ECM1. Therefore, this gene was selected for further variant expression and functional analysis in ESCC.

Results: RNA sequencing analysis of matched normal/ESCC samples showed that ECM1b (NM_022664) is the dominant expressed variant in esophagus, followed by ECM1a (NM_004425). In tumor samples, the expression of ECM1a is down-regulated and that of ECM1b is down-regulated or lost, as compared to normal counterparts. Expression of ECM1c (NM_001202858) is not detected in either normal or tumor samples. Quantitative PCR using variant-specific primer sets confirms the findings of microarray and RNA sequencing analyses. When re-expressed in ESCC cell lines after lentiviral transduction, ECM1b expression shows a trend of tumor suppression in the orthotopic ESCC mouse tumorigenicity model; ECM1a shows no tumor-suppressive effect in either. ECM1b expression also shows a metastasis inhibitory effect in a tail vein experimental metastasis model. Immunohistochemical staining shows E-cadherin up-regulation in orthotopic tumors re-expressing ECM1b.

Conclusions: These results suggest differential roles of variants of ECM1 in ESCC. Unlike the cases in other types of cancer, both expression of ECM1a and ECM1b is down-regulated in ESCC compared to normal esophageal tissues. ECM1b represents the dominantly expressed variants in esophagus epithelium, and its expression is highly reduced in esophageal carcinoma. ECM1b plays a potential suppressive role in tumorigenesis and metastasis.

Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for funding support (HKU 774413M to M.L.L).

#1159

Downregulation of TMPRSS11B in squamous cell carcinoma.

Suburu Amano, Takeshi Iwaya, Satoshi Nishizuka, Kohei Kume, Chie Ito, Yuji Akiyama, Yoshihiro Shioi, Fumitaka Endo, Akira Sasaki. _Iwate Medical University, Morioka, Japan_.

[Background] Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers. The molecular mechanism of ESCC development has not been fully elucidated. [Aim] The aim of this study is to identify responsible genes for ESCC development. [Materials and Methods] Firstly, we performed transcriptome sequence (RNA-seq) analysis of surgically resected tumor and corresponding normal tissues form 3 ESCC patients with different tumor locus, histrogical differentiation, and TNM stage.by RNA-seq analysis: (Case 1) upper thoracic esophagus, well differentiated SCC, TNM, Stage I; (Case 2) middle thoracic esophagus, poorly differentiated SCC, T3N0M0, Stage II; and (Case 3) lower thoracic esophagus, moderately differentiated SCC, T3N1M0, Stage III. Next, we validated the expression status of the candidate gene in 64 clinical samples and 10 ESCC cell lines (KYSE150, KYSE270, KYSE410, KYSE450, KYSE510, TE1, TE6, TE8, TE9, and TE10) using quantitative real-time PCR (qRT-PCR). [Results] In RNA-seq analysis, 34 genes showed more than 30-fold decrease in tumor samples compared to normal tissues, and the most downregulated gene was transmembrane protease serine 11B (TMPRSS11B). Amon these 34 genes, other 5 genes of TMPRSS11 family (TMPRSS11A, TMPRSS11BNL, TMPRSS11D, TMPRSS11E, and TMPRSS11F) were included. qRT-PCR analysis revealed that TMPRSS11B expression in ESCC samples were lower than those in normal samples (p < 0.001). Downregulation of TMPRSS11B in tumor samples compared to corresponding normal samples were observed in 51 cases out of 64 (79.7%). Patients with expression levels of TMPRSS11B that were below the median value were assigned to the low expression group (n=32), whereas those with expression values above the median were assigned to the high expression group (n=32). Significant difference was not observed between two groups regarding clinicopathological data, including age, gender, TNM stage, and lymphatic/venous invasion. TMPRSS11B expression was not detected in all of ESCC cell lines and noncancerous esophageal epithelial cell line, HET1A. [Conclusion] Our results suggest that downreguration of TMPRSS11B expression occurs in early stage of the ESCC oncogenesis and that TMPRSS11B has tumor suppressive roles in ESCC development.

#1160

FRY Inhibits progression and metastasis of breast cancer through activating Hippo signaling pathway.

Hao Zhang,1 Fan Fei,2 Xushen Chen,1 Zi Ceng,2 Xiaojiang Tang,2 Helmut Zarbl,3 Xuefeng Ren1. 1 _SUNY at Buffalo, Buffalo, NY;_ 2 _Guangdong Medical Laboratory Animal Center, China;_ 3 _Rutgers University, Piscataway, NJ_.

Background: Recently, we reported that using quantitative trait locus (QTL) mapping in differentially susceptible rat strains, we identified Fry, the mammalian ortholog of the furry gene in Drosophila melanoganster, as a candidate mammary carcinoma susceptibility gene (Mcs30). Preliminary studies of rat Fry gene suggest that Fry may play a key role in epithelial-mesenchymal transition (EMT), tumor progression and metastasis.

Methods: In the present study, we modified and established multiple breast cancer cell lines with enhanced expression of either wildtype or mutated human FRY gene. We examined the anti-tumorigenic and anti-metastatic effects of enhanced FRY expression on these cell lines in vitro and in vivo. We also utilized RNA-seq and proteomic approach to dissect FRY-regulated signaling molecules and pathways.

Results: Ectopic expression of wild-type FRY significantly diminished tumorigenicity and metastasis of breast cancer cells both in vitro and in animal studies, particularly in the triple negative breast cancer cell line, MDA-MB-231 cells. Relative to the rapidly tumors growth of MDA-MB-231 cells in vitro and in nude mice assay, the growth of MDA-MB-231 cells ectopically expressing the FRY significantly reduced. In comparison, the anti-tumorigenic effects of mutated FRY were compromised but remained significant. Further, we showed that FRY's effects in suppressing proliferation, invasion, and metastasis of breast cancer cells were partially through the activation of Hippo signaling pathway.

Conclusions: These results provide direct evidence in supporting the important role of FRY in breast cancer progression and metastasis, and first time reveal a connection between FRY and the Hippo signaling pathway.

#1161

A new, therapeutically actionable target for the VHL E3 ubiquitin ligase in renal cell carcinoma.

Elshad Hasanov,1 Guang Chen,1 Pratim Chowdhury,1 Justin Weldon,1 Eric Jonasch,2 Subrata Sen,2 Cheryl Lyn Walker,1 Ruhee Dere1. 1 _Texas A &M Health Science Center, Institute of Biosciences and Technology, Houston, TX; _2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Loss of function of the von Hippel Lindau (VHL) tumor suppressor is a nearly ubiquitous feature of clear cell renal cell carcinoma (ccRCC). The current therapeutic strategies in RCC were developed around knowledge that the VHL E3 ubiquitin ligase targets hypoxia inducible factors (HIFs) for degradation. AURKA is a well-established mitotic kinase and an emerging target in numerous cancers arising from its critical roles in tumor growth and survival. More recently AURKA was discovered to also have important non-mitotic functions in G1/G0 cells, where it activates HDAC6 to regulate microtubule stability. Here we report that AURKA is a novel target for the VHL E3 ligase, which plays an important role in modulating the non-mitotic activity of AURKA.

Methods: In vitro and in vivo ubiquitination assays were performed to establish AURKA as a direct target of VHL. Immunoblot analyses were used to determine AURKA levels in human RCC patient material and VHL deficient cell lines. Immunofluorescence was utilized to assess the impact of VHL-mediated AURKA ubiquitination on microtubule targets using primary cilia.

Results: In this study, we show that AURKA is a novel target for VHL's E3-ligase activity. VHL directly regulates AURKA expression in non-mitotic, quiescent cells by promoting AURKA degradation via the 26S proteasome. The ubiquitination assays showed enhanced AURKA ubiquitination in the presence of VHL. We found that VHL mediates AURKA degradation via a multi-monoubiquitin chain linkage, in contrast to the more traditional and abundant K48-linkage of proteins targeted for proteasome-mediated degradation. Biochemical studies revealed that unlike HIFα recognition by VHL, which requires proline hydroxylation, VHL interacted with, and degraded AURKA independent of its hydroxylation status, suggesting an alternate mechanism for recognition of AURKA. Importantly, using primary cilia as a biomarker for stabile microtubules and activity of AURKA/HDAC6, we showed that the loss of primary cilia observed in VHL-null cells was rescued by alisertib (AURKA inhibitor) and rocilinostat (HDAC6 inhibitor).

Conclusion: In conclusion, the biochemical evidence presented here identifies AURKA as a novel target of VHL's E3 ubiquitin ligase activity. We demonstrate a previously unknown mechanism for VHL mediated multi-mono ubiquitination of AURKA in regulating its non-mitotic functions, important in enabling the G0/G1 transition of cells and maintaining the cilia-centrosome cycle. Importantly, as an alternative to the current HIF related anti-angiogenic therapies, identifying AURKA as a novel VHL target lays the foundation for new therapeutic avenues in RCC, targeted specifically at the epithelial defects associated with VHL-null renal cell carcinoma.

#1162

Multiple genes on chromosome 17q25 were involved in sporadic esophageal squamous cell carcinoma development.

Takeshi Iwaya,1 Suburu Amano,1 Fumitaka Endo,1 Yuji Akiyama,1 Yoshihiro Shioi,1 Kohei Kume,1 Satoshi Nishizuka,1 Chie Ito,1 Akira Sasaki,1 Koshi Mimori2. 1 _Department of Surgery, Iwate Medical University, Morioka, Japan;_ 2 _Department of Surgery, Kyushu University, Beppu Hospital, Beppu, Japan_.

[Background] Tylosis is an autosomal dominant skin disorder that is associated with the early onset of esophageal squamous cell carcinoma (ESCC) in several families. The tylosis esophageal cancer (TOC) gene locus has been mapped to chromosome 17q25.1 using linkage analyses. This region is also frequently lost in sporadic ESCC. [Materials and Methods] Gene expression profiles on 17q25 in tumor samples from 3 ESCC patients were analyzed using RNA-seq. We validated the expression status of candidate genes in samples from 90 ESCC patients using qRT-PCR. Direct sequence, microsatellite LOH analysis, and methylation assay were also performed in the candidate gene, ST6GALNAC1. Mutation profiles of genes on 17q were also evaluated by exome sequence analysis from 144 ESCC patients. [Result] EVPL and ST6GALNAC1 were found to be significantly downregulated in tumor samples using RNA-seq. Significant downregulation of ST6GALNAC1 and EVPL expression in cancer tissues was confirmed by qRT-PCR in 90 ESCC samples. Although direct sequence of ST6GALNAC1 demonstrated missense mutations in 7 out of 46 (15%) cases, these mutations were not tumor specific. ST6GALNAC1 expression was significantly upregulated in 5-aza-dC treatment groups compared with control in the 5 ESCC cell lines. LOH in ST6GALNAC1 gene locus were identified 18 of 27 informative cases (67%). On chromosome 17q, exome sequence revealed that recurrent mutations were observed only in ZNF750 (14.5%) on 7q25.3. Recently, missense mutations in RHBDF2 were identified tylosis familys. However, RHBDF2 mutation was not observed in sporadic ESC samples by exome sequence analysis. [Conclusion] Our results suggest that ST6GALNAC1 is a putative tumor suppressor gene for ESCC. Furthermore, recent studies on tylosis families and our results on sporadic ESC suggest that multiple genes on chromosome 17q25 are involved in ESCC development.

#1163

Estrogen receptor beta (ESR2)-mediated tumor suppression of glioblastoma involves modulation of cell cycle and DNA damage response pathways.

Ratna K. Vadlamudi, Gangadhara Reddy Sareddy, Xiaonan Li, Jinyou Liu, Lauren Garcia, Andrew Brenner. _UT Health Science Center at San Antonio, San Antonio, TX_.

Purpose: Glioblastomas (GBM) are deadly brain tumors, have greater incidence in males than females. Epidemiological evidence supports a role of estrogen in tumor suppression. Estrogen signaling is mediated by estrogen receptor alpha (ESR1) and beta (ESR2). Our earlier studies showed that GBM preferentially express ESR2. However, mechanisms by which E2-ESR2 signaling promote suppression of GBM is poorly understood. The objective of this study is to determine the mechanisms by which ESR2 promote tumor suppression.

Experimental design: Established and patient derived primary GBM cells overexpressing or under expressing ESR2 were generated using lentiviral vectors. Three different ESR2 agonists (LY500307, Liquiritigenin, and Erb041) were used in the study and transcription changes elicited by ESR2 were profiled using RNA-seq. Cell cycle was analyzed using FACS analysis. In vivo efficacy of ESR2 agonists was tested in an orthotopic model using syngeneic murine GBM model (GL26), tumor growth was measured using bioluminescence, and survival was recorded. The synergy experiments using 119 FDA approved drugs was done using cell viability assay.

Results: ESR2 agonist treatment or ESR2 overexpression significantly reduced the viability and colony formation and induced apoptosis of various GBM cell lines with minimal effect on normal astrocytes. Further, ESR2 agonist (LY500307 5mg/kg/day) treatment significantly improved survival of GL26 tumor-bearing mice. RNA-seq studies using ESR2 overexpression or ligands treatment revealed downregulation of a number of genes involved in DNA repair, DNA damage response and cell cycle. The canonical pathways modulated by ESR2 included cell cycle, DNA damage, p53 signaling, and checkpoint regulation. In addition, pathways related to molecular mechanism of cancer, ILK signaling, Wnt signaling and glioma invasion were also altered. ESR2 agonist treatment significantly increased the percentage of cells in G2/M phase in U87, U251 and LN229 GBM cells when compared to vehicle further supporting the RNA-seq pathway analysis of cell cycle and G2/M DNA damage checkpoint regulation. Using combinational screening of 119 FDA-approved drugs combined with a low dose of ESR2 agonists (below the IC50 dose), we discovered synergism of ESR2 agonist treatment with several DNA-damaging agents. We independently validated synergy using additional ESR2 agonists with currently used chemotherapy drugs in GBM including temozolomide.

Conclusions: Our results demonstrate that ESR2 as tumor suppressor for GBM and ESR2 agonists have potential as a therapeutic agent for GBM. ERβ agonists' ability to suppress pathways involved in DNA repair can be exploited to promote apoptosis of GBM cells.

#1164

Semaphorin 3C promotes de novo steroidogenesis in androgen-deprived prostate cancer cells.

Parvin Yenki, Hans Adomat, Chi Wing Cheng, Christopher Ong. _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Introduction: This project aims to study the biological role of Semaphorin 3C (SEMA3C) in castration resistant prostate cancer (CRPC) progression. We have found that SEMA3C is a key driver of prostate cancer (PCa) growth through activation of epidermal growth factor receptor (EGFR) and cMet receptor tyrosine kinase (RTK) pathways. RTK signalling activation has been shown to induce de novo steroidogenesis, therefore we hypothesize that SEMA3C-mediated RTK signalling may contribute to CRPC progression through promotion of androgen biosynthesis in androgen-deprived PCa cells.

Methods: LNCaP and 22Rv1 cells stably overexpressing SEMA3C were compared to mock transfected cells with respect to their in vitro steroidogenesis, secreted hormone levels were measured by Liquid chromatography-mass spectrometry (LC-MS) technique. Also the expression of steroidogenic enzymes and regulatory proteins were tested by quantitative PCR and western blotting. De novo steroidogenesis was detected by pulsing cells with C14-labeled acetate as a steroid precursor; production of intermediates was measured by scintillation.

Results: Steroid analysis showed that SEMA3C overexpression can significantly elevate testosterone and dihydrotestosterone secretion by prostate cancer cells. SEMA3C is also able to upregulate STAR, CYP17A1, HSD17β3, AKR1C3 and SRD5A1 expression. Steroid analysis of SEMA3C-overexpressing LNCaP cells incubated with C14-labeled acetate resulted in the increase of the radioisotope-labeled testosterone and pregnenolone.

Conclusions: These findings indicate that SEMA3C can promote steroid biosynthesis in androgen deprived prostate cancer cells, thereby SEMA3C signaling pathway can contribute to maintain active AR signaling in CRPC cells. This research line will be followed to measure intratumoral androgen level by inducing subcutaneously xenografted tumors in athymic nude mice. Tumor homogenates' steroids will then be extracted and run on LC-MS system.

#1165

TRPM8 is avidly targeted for degradation in prostate cancer.

Swapna Asuthkar, Eleonora Zakharian. _University of Illinois College of Med. at Peoria, Peoria, IL_.

Transient receptor potential melastatin 8 (TRPM8) is a cold-sensing receptor and a hallmark Ca2+ channel of the prostate epithelium. The TRPM8 mRNA is overexpressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Thus, androgens, which are at the crossroads of several signaling pathways, appear to be associated with TRPM8-mediated Ca2+ signaling. Although, TRPM8 mRNA levels are upregulated during prostate cancer progression, this upregulation is not adequately translated to the TRPM8 protein levels. We identified that the lower TRPM8 protein abundance and activation in LNCaP cells is associated with increased ubiquitination and loss of TRPM8 from the plasma membrane (PM). The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of the initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the PM of LNCaP cells. Our data indicate that PMTRPM8 plays a protective role in prostate cancer progression accompanied by enhanced activation of p53 and Caspase-9. In addition, we found that the promoter region of trpm8 possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. Our findings support previous studies that suggest the fine balance between AR and p53 expression that regulates androgen-dependent growth of prostate cancer. Thus, we hypothesize that TRPM8 activity significantly contributes to anti-tumor defense mechanism and may serve as a novel therapeutic target in prostate cancer.

#1166

The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) invasion and migration.

Hsin-Han Hou, Chong-Jen Yu. _National Taiwan University Hospital, Taipei, Taiwan_.

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and enzymatic activity of ADAM15 extracellular domain activates MMP-9 and promotes NSCLC migration and invasion. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC invasion and migration. Overexpression of ADAM15 i6 promoted CL1-0 cell invasion and migration according to the transwell and wound healing assay. Ablation of Ras protein activator (RASA)1 and protein tyrosine kinase (PTK)6 attenuated the ADAM15 i6-promoted invasion and migration respectively. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC.

#1167

Cytoplasmic p21WAF1/CIP1 increases metastatic melanoma survival upon chemotherapy.

Gabriela Nana Colaneri, Adriana Taveira Cruz, Débora CP Silva, Roberta Sessa Stilhano, Sang Won Han, Miriam G. Jasiulionis. _Federal Univ. of São Paulo, São Paulo, Brazil_.

Initially described as a potent cyclin-dependent kinase inhibitor and thus a negative regulator of cell cycle, p21waf1/cip1 is a protein whose expression is often altered in cancer. Paradoxically, its upregulation has been correlated with aggressiveness and poor prognosis. p21waf1/cip1 fits the definition of antagonistic duality, since its cytoplasmic functions differ from its nuclear tasks. Our group had previously developed a murine melanoma progression model, which culminated in the establishment of cell lines representing different stages in melanoma genesis. Surprisingly, we detected overexpression of p21waf1/cip1 protein in a metastatic melanoma cell line (4C11+), which exhibits highly aggressive behavior. Despite its aberrant overexpression has already been described in melanoma, including metastases, little is known about the impact of its ectopic overexpression in melanoma aggressiveness. We performed viral-mediated overexpression and downregulation of p21waf1/cip1 in the non-metastatic melanoma lineage (4C11-) which does not express this protein and in the metastatic melanoma cell line (4C11+), respectively, and assessed for its impact on melanoma survival upon Dacarbazine treatment. Flow cytometry analyses were conducted to investigate p21waf1/cip1 cell cycle control in these lineages. Through Western blot analysis, we also evaluated the expression of phospho-Akt and phospho-p21 as well as p21waf1/cip1 subcellular localization, in an attempt to investigate the mechanisms underlying ectopic location of p21waf1/cip1 and thus its oncogenic activities. Our research shows that upregulated cytoplasmic p21waf1/cip1 increases metastatic melanoma survival upon Dacarbazine treatment, which can be reverted by p21waf1/cip1 silencing. We also detected high levels of phospho-Akt(Ser473) and phospho-p21(Thr145), which are determinant in promoting p21waf1/cip1 cytoplasmic retention. Using Wortmannin, a PI3K inhibitor that prevents Akt activation, cytoplasmic p21waf1/cip1 level was substantially diminished. Importantly, we identified high levels of p21waf1/cip1 and phospho-p21waf1/cip1 in patients-derived metastatic melanoma cells. We provided new insights about molecular pathways involved in Dacarbazine-associated resistance in melanomas, pointing p21waf1/cip1 and PI3K/Akt as key contributors in this process. Altogether, our findings contribute to not only design new therapeutic approaches against metastatic melanoma, but also help to elucidate the contradictory and complex role exerted by p21waf1/cip1 in cancer.

Supported by FAPESP and CNPq

#1168

The codon 389 polymorphism of PICT-1 is a risk factor for human cervical cancer.

Masafumi Yoshimoto,1 Aoi Tokuda,2 Shoko Takahashi,2 Yuji Yaginuma3. 1 _Graduate School of Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan;_ 2 _School of Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan;_ 3 _Department of Oncology, Graduate School of Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan_.

The uterine cervical cancers profoundly involve in the infection with human papillomavirus (HPV), and cause the deaths of more than 250,000 women all over the world. To date, the carcinogenesis of uterine cervical cancers was not fully understood, then we have to investigate more precise genetic and molecular events to develop early diagnosis and identify new therapeutic targets. The PICT-1 (Protein Interacting with Carboxyl Terminus-1), one of the nucleolar proteins, has tumor suppressor functions, but its association with uterine cervical carcinogenesis has been unknown. So we investigate PICT-1 gene mutations, polymorphism on codon 389 (rs184994), and protein expression levels to investigate the association with pathogenesis of uterine cervical cancers. Firstly we found missense mutations of functionally important regions of PICT-1 in one of seven uterine cervical cancer cell lines (14%) and one of 10 surgical samples (10%). Next we focused on codon 389 polymorphism (Gln to Arg) which located in important regions of PICT-1 function. In result there was a statistically significant association between PICT-1 codon 389 polymorphism and the risk of uterine cervical cancers: A/G (odds ratio 6.48, 95% confidence interval 1.98-21.22, P = 0.0012), G/G (odds ratio 3.15, 95% confidence interval 1.03-9.61, P = 0.0403), and A/G or G/G (odds ratio 4.44, 95% confidence interval 1.62-12.23, P = 0.0026). We also identified PICT-1 protein level was reduced in uterine cervical cancer cell lines by Western blotting and cervical cancer tissues by immunohistochemistry staining (IHC). In IHC analysis, expression of PICT-1 in adjacent normal cervix was found in nucleus, especially nucleolus, but in differentiating cells layers located upper parabasal cell layer, PICT-1 also expressed in cytoplasm and gradually reduced the expression of nucleolus. In patient tissues, compared to normal epithelium, we found reduction of PICT-1 expression in most samples found even in uterine cervical cancers at early invasive lesions, and weaker PICT-1 expression was observed in more invasive lesions. Interestingly, we also detected high expressed and/or re-expressed PICT-1 in cytoplasm and/or nucleolus of keratinized lesions of cervical cancers. Our results indicate that disruption of PICT-1 by gene mutation and/or reduction of protein level may associate with the pathogenesis of uterine cervical cancers, and its codon 389 polymorphism may increase the risk of uterine cervical cancers. And also we suggested that PICT-1 located in cytoplasm may be important to squamous cell differentiation, and we speculated the further study of association between PICT-1 and cell differentiation would be of value to the field of the differentiation-inducing therapy for many hard-to-treat patients of many cancers, including uterine cervical cancers.

#1169

Somatic MED12 mutations in hematological malignancies.

Tuomas Heikkinen,1 Kati Kämpjärvi,1 Sirpa Leppä,1 Peter Hokland,2 Heikki Kuusanmäki,1 Satu Mustjoki,1 Marjatta Sinisalo,3 Caroline Heckman,4 Mika Kontro,1 Pia Vahteristo1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Århus University Hospital, Århus, Denmark;_ 3 _University of Tampere, Tampere, Finland;_ 4 _Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland_.

Somatic mutations in exons 1 and 2 of the Mediator complex subunit 12 (MED12) have been identified in 70% of uterine leiomyomas, 7-20% of uterine leiomyosarcomas, 59% of breast fibroadenomas and 67% of breast phyllodes tumors. In addition to female hormone-dependent tumors we have recently found the same specific missense and small in-frame insertion and deletion mutations in approximately 5% of chronic lymphocytic leukemias (CLL). In CLL the presence of MED12 mutations was also associated with markers of poor prognosis (IgVH (-), Zap70 (+), and Zap70-methylation). The surprising finding of the same mutations in a completely different tumor type prompted us to further screen other hematological malignancies for MED12 mutations.

We have thus far collected samples of 107 T-cell acute lymphoblastic leukemias (T-ALL), 21 large granular lymphocyte leukemias (LGL), 33 acute myeloid leukemias (AML), 154 diffuse large B cell lymphomas (DLBCL), and 6 six follicular lymphomas (FL). Also a set of 30 additional CLLs was collected. The MED12 mutation status was determined by direct Sanger sequencing of exons 1 and 2 of the gene. A c.107T>G, p.L36R mutation was found in a single DLBCL case and c.100-8T>A, p.E33_D34insPQ in one AML sample. A novel variant of uncertain significance, c.-3A>G, was detected on 5'UTR of one FL sample. One T-ALL patient harbored a nonsense mutation affecting the last codon of exon 1 c.97G>T, p.E33X. No mutations were identified in the LGL or CLL samples. We are also analyzing approximately 100 new AML samples and 100 multiple myeloma samples.

MED12 mutations are present, in addition to CLL, also in other hematological malignancies. The frequency, however, seems to be low and, as the number of samples in the preliminary analysis is limited, screening of larger sample sets is needed. The c.107T>G, p.L36R and c.100-8T>A, p.E33_D34insPQ mutations have previously been detected recurrently in uterine leiomyomas and in CLL. Further studies are required for evaluation of the effects of c.-3A>G and c.97G>T, p.E33X mutations. The latter mutation affects the last codon of exon 1, which is also a mutational hotspot in CLL, with recurrent missense mutations. MED12 exon 1 and 2 missense mutations disrupt the interactions between the Mediator complex and Cyclin C and cause loss of CDK8 kinase activity. Studies to determine the impact of the c.97G>T, p.E33X mutation on MED12 function are ongoing.

#1170

EphA3 maintains radioresistance in head and neck cancers.

Myung Woul Han,1 Song Hee Kim,1 Hyo Won Chang,2 Hae Yun Nam,2 Myungjin Lee,2 Gui Chul Kim,2 Yoon sun Lee,2 Mi Ra Kim3. 1 _Korea/University of Ulsan, Ulsan, Republic of Korea;_ 2 _Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 3 _Inje University College of Medicine, Haeundae Paik Hospital, Busan, Republic of Korea_.

Radiotherapy is a well-established therapeutic modality used in treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after treatment by radiation. Thus, special emphasis has been given to the discovery of effective radiosensitizers. EphA3 receptors functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains to be elucidated. Here, we established the stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and identified that EphA3 was overexpressed predominantly in the AMC HN3R cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we identified that EphA3 was overexpressed in recurred laryngeal cancer specimen after radiation therapy. And we found that EphA3 mediated the tumor invasiveness and migration and EMT (epithelial mesenchymal transition) related protein expression in AMC HN3R cancer cell line. To investigate the role of EphA3 in modulating the radiosensitivity, we observed the change of survival fraction after transfection EphA3 siRNA. And we found that inhibition of EphA3 enhances radiosensitivity in AMC HN 3R cell line. In conclusion, these results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and can play a crucial role in development radioresistance in head and neck cancers through regulation of EMT pathway.

#1171

LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop.

Richard Ottman, Lisa Ritchey, Ryan Herchan, Alexia Bossan, Ratna Chakrabarti. _Univ. of Central Florida, Orlando, FL_.

Increased activation of the GPCR CXCR4/CXCL12 signaling axis is commonly noted in metastatic prostate cancer and shows an association with poor prognosis. CXCR4, and its ligand, CXCL12/SDF-1a, are recognized as powerful mediators for tumor microenvironment remodeling and chemoprotection. LIM kinase 1 (LIMK1), a serine/threonine kinase, is an actin and microtubule modulatory protein that is also overexpressed in a variety of cancers including prostate cancer. Earlier, we showed that LIMK1 is involved in cell invasion and inhibition of LIMK1 expression reverted the invasive phenotype of PC3 prostate cancer cells. Activation of CXCR4/CXCL12 axis promotes activation of LIMK1 through RhoGTPAse pathway and promotes docetaxel resistance. Here we show that LIMK1 expression and functions have a positive correlation with CXCR4 expression and subcellular localization. We used metastatic prostate cancer cell lines PC3 and M12 and the non-tumorigenic prostate cell lines BPH and P69 to monitor expression and membrane localization of CXCR4 using western blots, flow cytometry and immunofluorescence analysis. Our analysis showed that cells with higher expression of LIMK1 (PC3) overexpress CXCR4, and that cell lines with low levels of LIMK1 (BPH and P69) express relatively lower amounts of CXCR4. Flow cytometric analysis showed a stronger surface staining of CXCR4 in the LIMK1 overexpressing PC3 cells compared to BPH cells. Next, we studied the effect of shRNA-mediated inhibition of LIMK1 expression on CXCR4 expression in PC3 or M12 cells. Western blot and immunofluorescence analysis showed noticeable reduction in CXCR4 levels, and its surface localization, as noted by flow cytometry, in LIMK1 shRNA transfected PC3 and M12 cells. To assess if the kinase activity of LIMK1 is essential for CXCR4 expression and surface localization, we treated these cells with LIMK1 specific kinase inhibitor BMS-5 and determined expression of CXCR4. Western blots and immunofluorescence analysis showed reduced expression of CXCR4 in treated cells compared to vehicle treated cells. Our observation indicates an indirect association between LIMK1 and CXCR4 expression and shows for the first time that expression of LIMK1 is required for the maintenance of CXCR4 expression. Our study demonstrates a novel positive feed back regulatory mechanism between LIMK1 and CXCR4 and suggest a possible combination treatment option through inhibition of LIMK1 expression and/or its kinase activity for improving chemosensitivity of prostate cancer cells with overactive CXCR4/CXCL12 axis.

#1172

Expression of notch1, numb, and musashi1 in non-small cell lung cancer.

Hajime Kikuchi,1 Jun Sakakibara-Konishi,1 Yasuyuki Ikezawa,1 Taichi Takashina,1 Hidenori Mizugaki,1 Eiki Kikuchi,1 Junko Kikuchi,1 Naofumi Shinagawa,1 Satoshi Oizumi,1 Yasuhiro Hida,2 Kichizo Kaga,2 Ichiro Kinoshita,3 Hirotoshi Dosaka-Akita,3 Masaharu Nishimura1. 1 _First Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan;_ 2 _Department of Cardiovascular and Thoracic Surgery, Hokkaido University School of Medicine, Sapporo, Japan;_ 3 _Department of Medical Oncology, Hokkaido University School of Medicine, Sapporo, Japan_.

Background: Deregulation of Notch pathway is associated with carcinogenesis, and Notch signaling is identified as tumor activator in non-small cell lung cancer(NSCLC). The function of Musashi1 has been found to activate Notch signaling through the translational repression of Numb, which represses an intracellular Notch signaling. In NSCLC, the association between Numb or Musashi1 expression and clinicopathological factors or prognosis has remained unclear. In this study, we evaluated the expression of Notch1, Numb, and Musashi1 in NSCLC. Methods: A total of 135 surgically resected NSCLCs were immunohistochemically assessed for Notch1, Numb, and Musashi1 expression. Only cytoplasm and nuclear staining was considered in the evaluation of activated Notch1 expression. The immunoscores were determined, and then its correlation with either the clinicopathological variables or the survival outcomes was analyzed. Results: Immunohistochemical reactivity for Numb, and Musashi1 was detected in the cytoplasm and nuclear of the tumor cells. Notch1 expression was associated with several clinicopathological factors, including pathological histology (p=0.002), and differentiation of tumor (p=0.024). Numb expression was also associated with several clinicopathological factors, including sex (p=0.037), smoking habit (p=0.033), pathological histology (p=0.002), and differentiation of tumor (p=0.015). No correlations were noted between Musashi1 expression and any of the variables. Analysis of cellular biological expression demonstrated a link between low Numb expression and high Notch expression. Multivariate Cox regression analysis showed that positive Numb expression was an independent favorable prognostic factor. Conclusions: We demonstrate that Numb expression was associated with better prognosis in NSCLC. Numb might have the function as tumor suppressor in NSCLC.

#1174

AZD4547 and AZD5363 synergistically inhibit prostate cancer progression by modulating MAPK and AKT activation.

Shu Feng, Michael Ittmann. _Baylor College Of Medicine, Houston, TX_.

Prostate cancer (PCa) is the second most common cancer and the second most common cause of cancer mortality in men in the United States. Multiple approved therapies target the androgen signaling axis in this metastatic disease, however, after advancing to the androgen-independent stage, PCa is unresponsive to androgen ablation therapy and is refractory to further curative treatment. Recent advances have demonstrated that two intracellular signaling pathways promote prostate cancer progression, one is the Fibroblast Growth Factor (FGF) pathway included multiple FGFs and 4 kinds of FGF receptors (FGFR), which remains activated in the cancer cells, the other is the oncogenic PI3K/AKT pathway, which is constantly active due to the loss of an important tumor suppressor gene. Targeted inhibition of FGF and AKT signaling pathways has shown promising both in vitro and in vivo studies. However, FGFR or AKT inhibitor, as a single agent, would result in a reciprocal feedback loop. Therefore, it is imperative that both pathways be targeted simultaneously in the treatment of advanced prostate cancer. In this study, we evaluated the effect of combined treatment of FGFR inhibitor AZD4547 and AKT inhibitor AZD5363 on PCa progression. Proliferation assay shows that AZD4547 just weakly, or not inhibits PCa cell proliferation, AZD5363 significantly inhibits PCa cell proliferation; the combined treatment of AZD4547 and AZD5363 shows similar inhibition effect to AZD5363. Both AZD4547 and AZD5363 inhibit PCa cell anchorage-independent growth and invasion, which are synergistically decrease by the combined treatment. AZD4547 significantly inhibits phosporylation of FGFR1 and FGFR4 in PCa cells; AZD5363 increases FGFR1 and FGFR4 phosphorylation, which was significantly repressed in combined treatment. The activation of FGFR2α, MEK1/2, and Erk1/2 in PCa cells was remarkably inhibited by AZD4547 but increased by AZD5363, and synergistically inhibited by the combined treatment. AKT was hyperphosphorylated by AZD5363 and inhibited by AZD4547. The phosporylation of AKT downstream molecules PRAS40, GSK3β and S6, was slightly decreased by AZD4547 but strongly inhibited by AZD5363 and the combined treatment. Both AZD4547 and AZD5363 suppress STAT3 phosphorylation in PCa cells and the combind treatment shows synergistic inhibition effect. Both AZD4547 and AZD5363 promote AR and PTEN expression at both RNA and protein levels in VCaP and 22RV1cells, and the combination treatment remains the promotion effect. In LNCaP cells, AZD5363, but not AZD4547, stimulates AR mRNA and protein expression, which keep increased in combined treatment. This study provides a preclinical proof of concept that combination of a FGFR inhibitor with an AKT inhibitor has a profound potential to treat PCa.

Acknowledgement: This project was supported by the DOD Prostate Cancer Program, PCRP-IDEA-PC120481 (Ittmann), the Prostate Cancer Foundation (Ittmann)

#1175

Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival.

Rizwan Ahmad,1 Zhimin Chen,2 Balawant Kumar,1 Xi Chen,3 Mary kay Washington,4 Punita Dhawan,1 Amar B. Singh1. 1 _UNMC, Omaha, NE;_ 2 _Zhengzhou University, China;_ 3 _University of Miami Miller, Miami, FL;_ 4 _Vanderbilt University, Nashville, TN_.

Dysregulation of colonocyte differentiation, imposing a crypt progenitor phenotype, characterize colorectal cancer (CRC) progression. Thus, an improved understanding of the molecular processes that regulate colonocyte differentiation can help identify novel therapeutic targets and biomarkers. In this regard, we found claudin-3, a tight junction integral protein, to be the highest expressed cell-cell adhesion moiety in the normal colonic epithelium and particularly concentrated amongst terminally differentiated colonocytes at the crypt top. We therefore postulated a key role for claudin-3 in maintaining colonocyte differentiation and negative association with colon tumorigenesis. In accordance, claudin-3 expression was low in 10 out of 14 CRC cells lines tested and undetectable in poorly differentiated and highly tumorigenic HCT116 and SW620 cell lines. In further analysis, using mRNA and protein expression, and utilizing samples from a large patient cohort (<250 CRC specimen), we found claudin-3 expression to be significantly suppressed (p<0.001 versus normal) in cancer tissues versus normal mucosa. The colon tissues from the established mouse models of colon cancer (APCmin mice and Azoxymethane (AOM)-DSS-induced colon cancer) demonstrated similar tumor specific decrease in claudin-3 expression. Interestingly, claudin-3 negative tumors retained E-cadherin expression. Most notably, we found a significant and positive correlation (p<0.001) of the greater levels of claudin-3 expression with patient survival. Genetic manipulation studies using colon cancer cells further supported a causal role of claudin-3 in upholding colonocyte differentiation, by modulating ZEB-1 protein synthesis and Wnt-signaling activation (Topflash promoter activity, p<0.05; claudin-3 siRNA versus control cells), and inhibiting cancer cell mobility and invasive ability. Importantly, homozygous deletion of claudin-3 expression in mice similarly inhibited colonocyte differentiation (down-regulated P-27 expression and up-regulated Vimentin expression), and promoted colon cancer growth and invasion when subjected to the AOM-DSS-induced mouse model of colorectal cancer (p<0.001 versus WT littermates). Pharmacological manipulations further revealed epigenetic regulation as potential mechanism for the inhibition of caludin-3 expression in CRC. Taken together, our data reveal a tissue-specific and inverse causal association of claudin-3 expression with CRC progression and patient survival, and highlights key importance of claudin-3 in maintaining colonocyte differentiation in Wnt-ZEB1-dependent manner.

#1176

The role of KIF3A in the suppression of canonical Wnt signaling through the KIF3A and β-arrestin complex, independent of the ciliary mechanism, in non-small cell lung cancer (NSCLC).

Minsuh Kim,1 Yong-Ah Suh,1 Ju-hee Oh,1 Bo Ra Lee,1 Joon Kim,2 Se Jin Jang3. 1 _Asan institute for life sciences, Seoul, Republic of Korea;_ 2 _Graduate School of Medical Science & Engineering, KAIST, Daejeon, Republic of Korea; _3 _Department of Pathology, Asan Medical Center, Seoul, Republic of Korea_.

Aberrant Wnt/β-catenin signaling is implicated in the progression of several human cancers, including non-small-cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway com-ponents are uncommon in NSCLC, and epigenetic mechanisms controlling the Wnt/β-catenin path-way remain unclear. Here, we show that KIF3A, a member of the kinesin-2 motor family, plays a key role in suppressing Wnt/β-catenin signaling in NSCLC cells. Knockdown of KIF3A increases both β-catenin levels and transcriptional activity, with a concomitant promotion of malignant phenotypes, such as enhanced proliferation and migration, and upregulation of stemness markers. KIF3A binds to β-arrestin, and KIF3A depletion allows β-arrestin to form a complex with DVL2 and AXIN, result-ing in β-catenin stabilization. Although primary cilia, of which the biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis does not enhance β-catenin activity in NSCLC cells. A correlation between KIF3A loss and worse NSCLC prognosis as well as upregulation of β-catenin and Cyclin D1 further suggests that KIF3A is a suppressor of Wnt/β-catenin signaling and tumorigenesis in NSCLC.

#1177

The role of nischarin in the breast tumor microenvironment.

Mazvita Maziveyi, Shengli Dong, Suresh K. Alahari. _LSUHSC, New Orleans, LA_.

Women with denser breasts, have a higher risk of developing breast cancer. Breast density reflects the amount of connective tissue and collagen present. During metastasis, tumor cells move through tracks of extracellular matrix (ECM). A stiff and ordered tissue microenvironment is necessary for tumor cell invasiveness. Matrix stiffness is mediated by ECM fiber organization and cell-matrix adhesions. Our lab is investigating a tumor suppressor that contributes to ECM organization. The protein Nischarin interacts with a number of signaling proteins such as integrin α5, PAK1, LIMK1, LKB1, MLCK, ERK and Rac1 to prevent cancer cell migration. Our data shows that spontaneous breast tumors from wild-type (WT) animals have reduced ECM fiber expression and organization compared to those from homozygous Nischarin Null mice. Furthermore, we have found Nischarin to alter integrin and focal adhesion signaling, therefore affecting cell-matrix adhesions. We hypothesize that the absence of Nischarin in the breast tumor leads to increased matrix stiffness, through increased fiber organization, and altered cell-matrix adhesions. Since no breast tumor suppressors have been linked to collagen and fibronectin ECM remodeling, we present a novel role for Nischarin in organizing the ECM. By integrating cell signaling with optical and computational instrumentation, we will assess the role of Nischarin in the bidirectional cellular/matrix crosstalk, which will add to our understanding of the metastatic process in breast cancer.

#1178

Runx1 is obligatory for mammary epithelial cell morphology and phenotype.

Deli Hong, Terri Messier, Andrew Fritz, Gillian Browne, Janet Stein, Jane Lian, Gary Stein. _University of Vermont, Burlington, VT_.

Introduction: Runx1 belongs to the Runx family of transcription factors. Translocation of Runx1 is well established as a causative factor of leukemia. However the recognition of Runx1 expression in epithelial lining cells of glands suggests that Runx1 functions in maintaining the integrity of that cell layer. Recent studies have identified Runx1 as one of the top genes mutated in breast cancers. Further Runx1 levels are decreased in high-grade primary breast tumors compared to low/mid-grade tumors. Based on these findings, we raise the hypothesis that Runx1 is a tumor suppressor that functions to maintain the normal epithelial phenotype and that loss of Runx1 could promote epithelial to mesenchymal (EMT). Methods and results: We used two human breast cancer cell models systems, the MCF10A to MCF7/T47D and to MDA-MB-231 and the MCF10A progression model from normal-like mammary epithelial cells (MCF10A) to tumorigenic (MCF10AT1) and to metastatic (MCF10CA1a) cells. We observed that the level of Runx1 is decreased in tumorigenic cells. To understand Runx1 function, Runx1 was depleted by shRNA in mammary epithelial cells that resulting in disrupted/altered cellular morphology and suppressed expression of the epithelial gene E-cadherin. To reinforce this observation, EMT was induced by addition of TGFB1 or by growth factor depletion in MCF10A cells. A striking Runx1 decrease resulted suggesting that Runx1 has a crucial role in preventing EMT. Furthermore our analysis of breast tumors and survival data supports the above finding that loss of Runx1 promotes tumor progression.

Conclusion and Impact: Our results highlight an essential role for Runx1 in maintaining normal epithelial phenotype and preventing epithelial to mesenchymal transition as well as tumor progression in breast cancer. Identifying this Runx1 mechanism provides the basis for a novel strategy to treat early stage breast cancer.

#1179

Combined genetic and genealogic studies uncover a large BAP1 cancer syndrome kindred, tracing back nine generations to a common ancestor from the 1700s.

Michele Carbone,1 Erin G. Flores,1 Mitsuru Emi,1 Giovanni Gaudino,1 Sandra Pastorino,1 Haining Yang,1 Todd Johnson,2 Tatsuhiko Tsunoda,2 Mary Hesdorffer,3 Harvey I. Pass4. 1 _University of Hawaii Cancer Center, Honolulu, HI;_ 2 _RIKEN Center for Integrative Medical Sciences, Yokohama City, Kanagawa, Japan;_ 3 _MARF, Washington, DC;_ 4 _NYU, New York, NY_.

Germline BAP1 mutations cause a cancer syndrome characterized by high incidence of mesothelioma (MM), uveal melanoma and other cancers, and by very high penetrance, as all individuals carrying BAP1 mutations developed at least one, and usually several, malignancies throughout their lives. Through screening MM patients with histories of multiple cancers, we found four supposedly unrelated patients that shared an identical germline BAP1 mutation. We investigated whether this BAP1 mutation occurred in a 'hot-spot' for "de novo" mutations or whether these four MM patients shared a common ancestor. Using molecular genomics analyses we found that they are related. By genealogic studies we traced their ancestor to a couple that emigrated from Germany to North America in the early 1700's; we traced the subsequent migration of their descendants, who are now living in at least three different US States. Our findings demonstrate that BAP1 mutations are transmitted among subsequent generations over the course of centuries. This knowledge and methodology is being used to identify additional branches of the family carrying BAP1 mutations. Our study shows that the application of modern genomic analyses, coupled with "classical" family histories collected by the treating physician, and with genealogical searches, offer a powerful strategy to identify high-risk germline BAP1 mutation carriers that will benefit from genetic counseling and early detection cancer screening.

#1180

The TCA cycle transferase DLST is critical for MYC-mediated leukemogenesis.

Nicole Anderson,1 Dun Li,1 H.L. Peng,2 Marc Mansour,2 Fabrice Laroche,1 Evisa Gjini,2 Daniel Helman,2 Itrat Harrold,1 Le Meng,1 Takaomi Sanda,3 Adam Amsterdam,4 Donna Neuberg,2 Travis Denton,5 Anurag Singh,1 A. Thomas Look,2 Hui Feng1. 1 _Boston University School of Medicine, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _National University of Singapore, Singapore, Singapore;_ 4 _Massachusetts Institute of Technology, Cambridge, MA;_ 5 _Washington State University, Spokane, MA_.

The proto-oncogene MYC has been implicated in the pathogenesis of many human cancers, including hematological and solid malignancies1. In the majority of T-ALL cases, MYC is aberrantly expressed downstream of activated NOTCH1 mutations. Despite the pivotal role of MYC in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and many other cancers, the mechanisms underlying MYC-mediated tumorigenesis remain inadequately understood2. Here we utilized a well-characterized zebrafish model of Myc-induced T-ALL for genetic studies to identify novel genes contributing to disease onset. We found that heterozygous inactivation of a tricarboxylic acid (TCA) cycle enzyme, dihydrolipoamide S-succinyltransferase (Dlst), significantly delayed tumor onset. Concordant with our zebrafish results, RNAi knockdown of DLST led to decreased cell viability and induction of apoptosis in human T-ALL cell lines. DLST is the E2 transferase of the -ketoglutarate dehydrogenase complex (KGDHC), which converts -ketoglutarate (-KG) to succinyl-CoA in the TCA cycle3. Polar metabolomics profiling revealed that the TCA cycle was disrupted by DLST knockdown in human T-ALL cells, as demonstrated by an accumulation of -KG and its precursor glutamine, as well as a loss of succinyl-CoA. Addition of succinate, the downstream TCA cycle intermediate, to human T-ALL cells was sufficient to rescue defects in cell viability caused by DLST inactivation. Taken together, our studies identify DLST as an important mediator of MYC-driven leukemogenesis and provide compelling evidence for the metabolic dependence of T-ALL cells on the TCA cycle.

1. Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene 1999 May 13; 18(19): 3004-3016.

2. Sharma VM, Calvo JA, Draheim KM, Cunningham LA, Hermance N, Beverly L, et al. Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc. Molecular and cellular biology 2006 Nov; 26(21): 8022-8031.

3. Sheu KF, Blass JP. The alpha-ketoglutarate dehydrogenase complex. Ann N Y Acad Sci 1999; 893: 61-78.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Differentiation Therapy

#1182

Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma.

Angela L. Gaviglio, Gerard C. Blobe. _Duke University, Durham, NC_.

Neuroblastoma is the most common cancer in infancy. Current therapies are only modestly effective; patients with high-risk disease have less than a 50% chance of survival. High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble proteins, including heparan sulfate proteoglycans (HSPGs), which act to promote neuroblast differentiation.

Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent pro-differentiating factor in neuroblastoma. Using microarray analysis, HBEGF mRNA expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from high-stage disease having decreased stroma and HBEGF. Soluble HBEGF increased neuroblast differentiation and decreased proliferation, while HBEGF knockdown decreased neuroblast differentiation. In patient samples, HBEGF expression correlates with SOX10, a neural crest differentiation marker, and the cell cycle inhibitor, CDKN1A. Alternatively, HBEGF negatively correlates with ASCL1, a primitive neuroectodermal marker, and the cell cycle promoter CCND1. HSPGs, including TβRIII, GPC1, GPC3, and SDC3 further promote the differentiating effects of HBEGF treatment by forming a complex with the epidermal growth factor receptor (EGFR) via glycosaminoglycan modifications, leading to activation of the Erk1/2 pathway and upregulation of the inhibitor of DNA binding transcription factor, whose expression correlates with HBEGF in patient samples. Inhibition of EGFR with erlotinib, lapatinib, and gefitinib diminish HBEGF-induced differentiation.

These data identify a novel member of the pro-differentiating secretome and support the use of heparan sulfate mimetics in neuroblastoma, while cautioning against the use of EGFR inhibitors for neuroblastoma treatment.

#1183

PPARgamma agonist promotes adipocytic differentiation and potentiates the activity of trabectedin in myxoid liposarcoma.

Ezia Bello,1 Roberta Frapolli,1 Simonetta Andrea Licandro,1 Silvia Brich,2 Laura Carrassa,1 Roberta Sanfilippo,2 Alessandro Gronchi,2 Paolo Casali,2 Silvana Pilotti,2 Maurizio D'Incalci1. 1 _IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy;_ 2 _Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy_.

Trabectedin (ET-743, Yondelis) is a marine alkaloid isolated from the tunicate Ecteinascidia turbinata, approved in Europe and US for the 2nd line therapy of soft tissue sarcomas. It is particularly effective against myxoid liposarcoma, a specific histological subtype within the family of adults soft tissue sarcomas. More than 90% of usual myxoid/round cell liposarcomas (MRCLS) are characterized by the chromosomal translocation t(12;16) (q13; p11), which produces the FUS-CHOP oncogene. Different chimera subtypes seem to respond differently to trabectedin in clinical setting. Trabectedin was able to remove FUS-CHOP type I, II and III from its own target genes but it causes adipocytic maturation and tumor regression only in type I and II, not in type III MRCLS (Di Giandomenico et al, Oncogene, 2014 Oct 30; 33(44): 5201-10). Pioglitazone is a PPARgammaagonist with pro-differentiation effects in different cancer types included liposarcomas (Tontonoz P. et al, Proc Natl Acad Sci U S A. 1997 Jan 7;94(1): 237-41).

The aim of the study is to evaluate the efficacy of the combination of trabectedin with pioglitazone in type III myxoid liposarcoma xenografts.

ML006 and ML004 type III MRCLS xenografts mice were treated with trabectedin 0.15 mg/kg iv every seven days for three times or pioglitazone 150 mg/kg po daily for 28 days or with their combination. For antitumor activity evaluation, tumor growth was measured with a caliper and the tumor weights (1 mm3 = 1 mg) were calculated with the formula: length × (width)2/2. Fifteen days after the last dose of trabectedin and 4 h after the last dose of pioglitazone tumors were collected from a separate group of mice to perform histological and molecular analyses.

Trabectedin and pioglitazone as single agents have a comparable antitumor activity on these models with T/C around 40-50% with no tumor regression observed. In the combination groups an impressive and long lasting tumor regression was observed in ML 006 xenograft, with an observed optimal T/C of 17 %. In ML004 MRCLS model the combination of trabectedin and pioglitazone induced a minimal regression followed by a long lasting tumor stabilization, resulting in a T/C of 23%. The histological analyses showed adipocytic maturation in tumors treated with pioglitazone alone or in combination but not in samples from mice treated with trabectedin alone.

In conclusion, the combination of trabectedin with pioglitazone was able to induce tumor regression and adipocytic maturation in type III myxoid liposarcoma xenografts that were only marginally sensitive to trabectedin alone. Chromatin Immunoprecipitation, gene expression profile and Western Blot analysis are on-going to unravel the molecular mechanism behind the potentiation of the antitumor efficacy observed with this combination.

#1185

Translational study of the enhancement of cytarabine toxicity to AML blasts by a differentiation agent combination.

Xuening Wang,1 Jonathan Harrison,2 George P. Studzinski1. 1 _Rutgers New Jersey Medical School, Newark, NJ;_ 2 _University of Missouri, Columbia, MO_.

Cytarabine (AraC) is an important component of AML therapy, but like other DNA damaging therapeutic agents it is rarely curative. Exploratory studies suggest that therapy outcomes may be improved by combining AraC with other compounds. In this context, we have recently reported that the addition of a vitamin D-based differentiating agent combination following AraC damage to AML cells in established culture, increases the cell kill. Here we present data obtained with AML blasts cells freshly obtained from patients with this disease which demonstrate that HL60 and U937 cell lines provide a good model to supplement studies of the mechanisms underlying this potential therapeutic regimen. Importantly, in contrast to malignant cells, mononuclear cells isolated from normal human bone marrow do not show the enhancement of AraC-induced cell death by combinations of differentiation agents. The cells were exposed to AraC at concentrations which produced approximately 30-40% cell kill, followed by a combination of 100 nM 1alfa-hydroxyvitamin D2 (D2) and 10 uM carnosic acid (CA), which together serve as a powerful differentiating agent combination (D2/CA) for AML cells, but are minimally toxic alone. Increased cell death, measured by Annexin V/Propidium Iodide, compared to AraC alone, was selective for malignant cells, occurred by both apoptosis and necrosis, and was associated with increased levels of vitamin D receptor (VDR), DNA damage, and higher levels of DNA damage response marker P-H2AX. The knock-down of VDR with siRNA markedly reduced the potentiating effect of D2/CA on the AraC- induced cell death and the associated cellular changes. Since several caspases showed increased expression, and a caspase 3-specific inhibitor markedly reduced the enhancement of cell death, we measured the levels of the pro-apoptotic member of the Bcl family, Bim, which prominently increased following the addition of D2/CA to cells previously exposed to AraC. The increased Bim expression occurred at both mRNA and protein levels. Consistent with a role in the enhancement of cell death in this system, knock down of Bim by siRNA abrogated the effect of adding D2/CA to AraC-treated cells. Thus, our data indicate that in AML cells with pre-existing DNA damage activated VDR can interact with apoptotic pathways, and rather than promoting differentiation, leads to increased leukemia cell kill. (Supported by grant R01CA044722-25 from the National Cancer Institute, NIH, and grant 10A049 from American Institute for Cancer Research, both to GPS; JSH received support from the Nellie B. Smith Endowment at the University of Missouri and the Ellis Fischel Cancer Research Fund at the University of Missouri).

#1186

Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway.

Eric J. Norris, Amy Dorszynski, Aaron Lucander, Darla Destephanis, Ram Ganapathi, Mahrukh Ganapathi. _Carolinas Healthcare System, Charlotte, NC_.

Pharmacologic inhibition or molecular down-regulation of topoisomerase 2β (TOP2β) potentiates all trans retinoic acid (ATRA)-induced differentiation and apoptosis in acute myeloid leukemia (AML) cells. Since ATRA-induced myeloid differentiation involves activation of the MAPK/ERK signaling pathway and aberrant activation of MAPK/ERK is frequently observed in AML we tested the role of this pathway in modulating the cellular effects of ATRA when TOP2β is inhibited with the bisdioxopiperazines, ICRF 187 (dexrazoxane, Zinecard™) or ICRF193. Treatment of HL-60 AML (M2) cells with ATRA increased the percentage of differentiated cells, as assessed by the nitroblue tetrazolium reduction assay, and enhanced levels of phosphorylated ERK. Paradoxically, while combination treatment with ATRA/ICRF187 enhanced differentiation (P<0.05), ERK phosphorylation was attenuated compared to treatment with ATRA alone. Moreover, pretreatment with the MEK inhibitor PD98059, which completely blocks ATRA-induced differentiation and ERK phosphorylation, only partially blocked (46% reduction) differentiation induced by ATRA/ICRF187 (P<0.01) and had no effect on ATRA/ICRF187-induced apoptosis. In agreement with this observation, combination treatment with ATRA/ICRF187 induced apoptosis (P<0.05) in KG-1 (M1) AML cells and primary AML cells from patients, that do not phosphorylate ERK or differentiate in response to ATRA treatment. In summary, our data suggest that inhibition of TOP2β sensitizes AML cells to ATRA induced differentiation and apoptosis primarily via an ERK-independent mechanism. Further, combination treatment ATRA and ICRF187 may be clinically relevant to overcome ATRA resistance in AML patients with a deregulated MAPK/ERK signaling pathway.

#1187

Discovery of new AML and MDS patient subsets sensitive to the highly selective RARα agonist SY-1425 (tamibarotene) through super-enhancer analysis.

Michael R. McKeown,1 Emily Lee,1 Chris Fiore,1 Matthew L. Eaton,1 Jeremy Lopez,1 Ryan Corces-Zimmerman,2 Ravindra Majeti,2 Christian Fritz,1 Eric Olson1. 1 _Syros Pharmaceuticals, Cambridge, MA;_ 2 _Stanford University, Palo Alto, CA_.

Super-enhancers (SEs) are large, highly active chromatin regions that regulate key cell identity genes including oncogenes in malignant cells. Using our gene control platform, we identified SEs genome-wide in 60 primary AML patient samples to enable the discovery of novel tumor vulnerabilities. One of the SEs that showed a highly asymmetric distribution amongst patient samples was associated with the gene encoding the retinoic acid receptor alpha (RARα). We have discovered that the presence of a RARA SE is predictive for response of non- APL AML cell lines to the highly selective RARα agonist SY-1425. SY-1425 is a clinical stage RARα agonist with improved PK, potency, and selectivity over existing pan-retinoic acid agonists.

SY-1425 has sub-nanomolar potency on RARα, with 200-2000 fold selectivity over other RAR family members. SY-1425 treatment of AML cell lines containing a RARA SE results in 0.2-2 nanomolar EC50 anti-proliferative effects, whereas proliferation of cell lines without SEs are not affected. The RARA enhancer level (as measured by H3K27ac ChIP-seq) is well correlated with mRNA levels for RARα. Two patient derived xenograft models with high RARα mRNA show prolonged survival and tumor cell differentiation response to SY-1425, whereas two models with low RARα mRNA do not show a treatment effect. In contrast, treatment with the non-selective agonist ATRA fails to show a survival benefit in a RARα mRNA high model, presumably due to its lower agonist activity and its inferior PK.

RARα is a nuclear hormone receptor that acts as a transcriptional repressor when unliganded and as a transcriptional activator when bound by an agonist. SY-1425 treatment of SE containing AML cells shows a transcriptional response very similar to retinoid treatment of APL cells. Analysis of ex vivo treated primary AML cells as well as analysis of bone marrow and blood samples from SY-1425 treated patient derived xenograft models support an anti-proliferative and differentiation induction effect in cells containing the RARA SE but not in cells lacking the RARA SE. In addition to AML, a sub-population of MDS patient samples also contain elevated RARα mRNA levels, suggesting the potential utility of SY-1425 in this closely related indication.

In summary, using our gene control platform, we have identified subsets of non-APL AML and MDS who may respond to the selective RARa agonist, SY-1425. A phase 2 clinical study with SY-1425 is being planned in patients identified with a novel biomarker based on these findings.

#1188

Forced induction of differentiation in osteosarcoma tumor initiating cells.

Margaret E. White, Emma Viktoria Marie Hyddmark, Ali Zarezadeh, Elham Nasri, Maria V. Guijarro, Padraic P. Levings, Glyn Palmer, Steven C. Ghivizzani, C. Parker Gibbs. _University of Florida, Gainesville, FL_.

Osteosarcoma (OS) is a highly malignant bone cancer that is defined histologically by the secretion of immature osteoid. The chemotherapeutic regimen has not changed in the last 40 years, and similarly the five-year survival rate has remained a dismal ~60%, largely due to metastatic spread to the lungs. OS is believed to originate from osteogenic committed progenitors, involving disruption of extracellular matrix synthesis in favor of proliferation.

In previous work, our lab developed a method to selectively identify Tumor Initiating Cells (TICs) in OS xenografts based on their ability to activate a transcriptional reporter comprised of a human OCT4 promoter linked to the coding sequence for Green Fluorescent Protein (GFP). Clonally derived, stably transfected OS Oct4/GFP+ cells are capable of initiating and maintaining the growth of heterogeneous tumors and driving disease progression. The loss of GFP expression and tumorigenic capacity occurs during tumor formation in response to cues within the tumor microenvironment. These conditions appear to reprogram the GFP+ cells, resulting in loss of reporter activity, reduced proliferation, and adoption of a specialized secretory phenotype as an adaptive survival mechanism: a phenotypic change similar to that seen in normal physiological differentiation. During skeletal formation, Bone Morphogenetic Proteins (BMP) play key roles in osteogenic maturation. Because the OS lineage of origin suggests an innate sensitivity to BMP proteins, we hypothesize that BMP stimulation will force the induction of differentiation in OS TICs and will impair the ability of these cells to initiate and maintain tumor growth.

To test our hypothesis, we investigated the expression of BMP receptors in two primary OS cell lines, using Mesenchymal Stem Cells and Human Fetal Osteoblasts as positive controls. Expression of BMP Receptors II, 1A, and 1B was confirmed using flow cytometry indicating that the OS cells could potentially respond to BMP stimulation. The functionality of the BMP signaling pathway was subsequently investigated after stimulation with BMP heterodimers, BMP2/7 and BMP4/7. Immunoblotting showed OS TICs are capable of activating canonical and non-canonical BMP signaling pathways after BMP stimulation, suggesting a functional and intact signaling network potentially able to induce differentiation. However, only cells treated with BMP4/7 showed changes in cell cycle distribution as visualized by DAPI. Furthermore, BMP4/7-stimulated TICs showed reduced tumor formation in our xenograft mouse model

Our data suggests an inhibition of OS tumorigenicity due to a BMP4/7 induced differentiation based response. Currently, we are investigating if forced expression of BMPs using an OS specific recombinant Adeno-Associated Virus can inhibit tumor growth in vivo. Such a differentiation-based therapy could become a more effective and safer alternative to cytotoxic chemotherapy.

#1189

Combination treatments with retinoic acid and the synthetic retinoid ST1926 in 2D and 3D breast cancer models overcome retinoic acid resistance and eradicate breast cancer stem/progenitor cells.

Patrick Aouad,1 Melody Saikali,1 Rana Abdel-Samad,1 Leeanna El--Houjeiri,1 Claudio Pisano,2 Rabih Talhouk,1 Nadine Darwiche1. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _Biogem, Research Institute, Ariano Irpino, Italy_.

Despite recent advances in breast cancer therapy, achieving complete remission in metastatic breast cancer patients remains a challenge. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is acquired resistance. Therefore, synthetic retinoids such as ST1926 emerged as potent anti-cancer agents. Here, we investigated the anti-tumor activities of ATRA, ST1926, and their combination treatments in 2D and 3D human breast cancer models and their targeting of breast cancer stem cells (CSCs)/progenitor cells. We have shown that in 2D culture models, MCF-7 and MDA-MB-231 cells are resistant to ATRA while being sensitive to ST1926 at sub-micromolar (µM) concentrations in an irreversible manner. Importantly, ST1926 had no effect on the 'normal-like' MCF-10A breast epithelial cells. ST1926 induced massive apoptosis in MCF-7 cells and it resulted in S-phase arrest and necrosis in the triple negative and metastatic MDA-MB-231 cells. Furthermore, ST1926 caused early DNA damage, increased the expression of the tumor suppressors p53 and p21, downregulated the Wnt/β-catenin pathway, and modulated the expression levels of the different retinoid receptors. Interestingly, combination treatments as low as 0.1 µM ST1926 and 0.5 µM ATRA synergistically inhibited the proliferation in 2D models of MCF-7 and MDA-MB-231 cells, independently of retinoid receptor signaling, while sparing the normal breast epithelial cells. Anchorage-independent growth of MCF-7 and MDA-MB-231 cells was examined using the soft agar colony formation assay where sub-µM concentrations of ST1926 or µM concentrations of ATRA were shown to reduce the size and the number of breast cancer colonies. ST1926 drastically induced cell death in 3D Matrigel 'on-top' assay cultures of breast cancer cells while the lumen of the normal-like breast epithelial cell line S1 was maintained. Finally, treatment with 0.01 µM ST1926 alone or 0.001 µM ST1926 in combination with 0.1 µM ATRA abrogated sphere formation and the self-renewal ability of breast CSCs in the 3D sphere formation assay. In summary, ST1926, ATRA, and their combination treatments were shown to display more potent anti-tumor properties in 3D versus 2D human breast cancer models. Our results also demonstrate the therapeutic potential of ST1926 in sensitizing breast cancer cells to ATRA and in targeting the population of breast CSCs. As 3D culture models are more representative of the tumor microenvironment and serve as valid tools in drug discovery, our results highlight the promising use of ATRA/ST1926 combinations in metastatic and triple negative breast cancers.

#1190

Pretreatment with 1,25 dihydroxyvitamin D3 modulates p73 levels and activity to increase pro-apoptotic effects of cisplatin in bladder cancer.

Brittany L. Bunch,1 Candace S. Johnson,1 Donald L. Trump2. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Inova Schar Cancer Institute, Falls Church, VA_.

Cisplatin-based combination chemotherapy is the standard treatment for advanced bladder cancer. While there is a substantial initial response rate and occasional complete response, long term control is uncommon. There is a substantial need to develop new therapeutic approaches to bladder cancer. 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D, enhances the anti-tumor effects of cisplatin in preclinical bladder cancer models. We sought to evaluate the mechanism of 1,25D3 potentiation of cisplatin cytotoxicity through in vitro studies in bladder cancer cell lines. Our studies suggest that enhanced cytotoxicity is mediated through modulation of p73. In a clonogenic assay, the surviving fraction after treatment with 1,25D3 (0.1 uM) for 24 hrs followed by cisplatin (0.1 ug/ml) for 48 hrs increases from 64% to 97% in T24 cells transfected with shRNA targeting p73. The ratio of the pro-apoptotic TAp73 isoform to the anti-apoptotic ΔNp73 isoform is important in determining cellular response to cisplatin. Our studies demonstrate that pretreatment with 1,25D3 followed by cisplatin significantly increases the ratio of TA/ΔNp73 mRNA transcripts 2-fold and increases BAX, the transcriptional target of TAp73, approximately 3-fold. Protein levels of TAp73 and BAX are also increased 2- and 3-fold, respectively, as determined by western blot. Protein stability of TAp73 was determined following cycloheximide treatment for 1, 2, and 4 hrs. After 4 hrs of cycloheximide treatment, protein levels of TAp73 decrease by approximately 50% in all treatment groups except the 1,25D3 and cisplatin group, in which the level of TAp73 protein was maintained. This suggests an increase in TAp73 protein stability after treatment. Using a TransAm p53 binding assay after p53 immunodepletion, 1,25D3 and cisplatin treatment increases DNA binding of TAp73 approximately 2-fold. Protein levels of TAp73 upstream activators, c-Abl and p38, also increase approximately 2-fold after 1,25D3 treatment, suggesting a mechanism of action for increased stability and activation of TAp73. Lastly, pharmacologic inhibition of p38 activation using SB203580 prevents the synergistic effects of 1,25D3 and cisplatin. Taken together, these data suggest that the mechanism of synergy for 1,25D3 and cisplatin combination therapy is through an increase in TAp73 stability and pro-apoptotic transcriptional activity. These findings suggest that 1,25D3 may have potential to be used in combination with cisplatin to increase the apoptotic response. Further studies are being performed to confirm the effects of treatment in an in vivo model, looking both at short term molecular response and long term therapeutic response to treatment. Supported by NCI grants CA067267 and CA016056.

#1191

Anti-tumor effect of selective progesterone receptor modulator (ulipristal acetate) on endometrial cancer cell lines.

Ha-Young Lee,1 A Ra Ko,2 Dong-Woo Kang,3 Yu-Seon Kim,4 Su-Min Kim,1 Tae-Wook Kang,4 Shin-Wha Lee,5 Yong-Man Kim5. 1 _University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Samsung Jeil Women's Clinic, Pusan, Republic of Korea;_ 3 _Institute for Innovative Cancer Research, Asan Medical Center, Seoul, Republic of Korea;_ 4 _Asan Institute for Life Science, Seoul, Republic of Korea;_ 5 _Department of Obstetrics and Gynecology, College of Medicine, University of Ulsan, Asan Medical Center, Seoul, Republic of Korea_.

Background: Endometrial cancer is the most common malignancy of the female reproductive tract and the third most common cause of death from women's cancer. Risk factors of endometrial cancer include high levels of estrogen, obesity, diabetes mellitus, breast cancer, long term use of tamoxifen, late menopause, high blood pressure, and increasing age. Although the exact mechanisms in endometrial carcinogenesis due to chronic exposure are unclear, it is though that the pro-proliferative and DNA-damaging effects of estrogen and its metabolites, related with and insufficient counterbalance by progesterone, promote the hyper-proliferation and transformation of cells. Selective progesterone receptor modulator (SPRM) is an agent that acts on the progesterone receptor. Ulipristal acetate (UA) is one of SPRM and other SPRM, such as Mifepristone, showed inhibitory effect on various endometrial cancer cell lines. UA has been available as an emergency contraception and a treatment for uterine leiomyoma. The effect of UA on leiomyoma is decreasing neovascularization, proliferation, and viability.

Objective: This study was undertaken to investigate antitumor effect of UA and to explore the effect of combination treatment with chemotherapy drug in endometrial cancer.

Method: Inhibitory concentration (IC), proliferation assay, and migration assay were performed using Ishikawa, HEC-1-A, HEC-1-B, and/or patient-derived primary cancer cells (PCC). Different concentration of UA were treated in endometrial cancer cell lines and PCCs for the each assay. The CellTiter-Glo assay were performed to investigate IC50 in cell lines and PCCs. Crystal violet staining and wound healing assay were performed to examine cell proliferation and migration.

Results: UA inhibited cell viability of endometrial cancer cell lines and PCCs at high concentration, most abviously 10uM in dose-dependent manner. In combination treatment assay with paclitaxel, UA showed synergistic anti-tumor effect with low-dose of paclitaxel on cell lines and PCCs.

Conclusions: We showed that combination treatment of UA and paclitaxel effectively than single treatment by low dosage. In summary, we are actively exploring new effective applicable drugs for endometrial cancer, and our data suggest that UA may have therapeutic value with chemo-combination treatment of patients with endometrial cancer.

### Growth Factor Receptors and Surface Antigens as Therapeutic Targets

#1192

Preclinical evaluation of HER3 mutations and rational combinations with AKT inhibition in enhancing anti-tumor activity of HER3 inhibition in gastric cancer.

Gopi Ganji,1 Sherry Qin,2 Crystal Qin,2 Neil Clarke,3 Carolyn Buser-Doepner,1 Christopher Matheny,1 Rakesh Kumar,1 Biju Mangatt1. 1 _GlaxoSmithKline, Collegeville, PA;_ 2 _GlaxoSmithKline, Shanghai, China;_ 3 _GlaxoSmithKline, Stevenage, United Kingdom_.

Molecular activating events involving the ERBB RTK family members (EGFR (ERBB1), HER2 (ERBB2), HER3 (ERBB3), HER4 (ERBB4)) drive oncogenesis by inducing proliferation, invasion and survival primarily through RAS/MAPK and PI3K/AKT signaling pathways in several cancers. The clinical successes of HER2 directed therapies are well known in breast cancer and more recently, in gastric cancer. However, resistance develops invariably and gastric cancer continues to be a largely unmet disease necessitating novel therapeutic interventions. Recent reports have highlighted HER3 as an emerging target as it is frequently overexpressed, mutated, preferentially dimerizes with HER2 to activate signaling and is induced as a result of de novo or acquired resistance to PI3K-AKT, MAPK and RTK pathway inhibitors. We explored the utility of an ADCC and CDC enhanced potent anti-HER3 therapeutic antibody (GSK2849330) as a single agent or in combination with a selective small molecule AKT inhibitor (GSK2110183) in patient-derived xenograft (PDX) models of gastric cancer.

A panel of 15 HER3 mutant and 4 HER3 wildtype PDX models was screened in vivo for responses to GSK2849330 as measured by % tumor growth inhibition (%TGI). Several models were characterized for other molecular alterations (e.g. HER2, PTEN, HER3, etc.) and represented various subsets of gastric cancer. As a single agent administered at 25mg/kg IP BIW, GSK2849330 was modestly effective (TGI ≥ 50%) in 2/15 mutant and 2/4 wildtype models. While context is likely to matter to drive dependence on HER3, no obvious predictive markers were observed. Furthermore, we evaluated the effect of combination therapy with an AKT inhibitor (GSK2110183) administered at 60mg/kg PO QD in a HER3 wildtype, PTEN deficient model. This resulted in significant durable tumor growth inhibition (~94% TGI) with improved survival and noticeable tumor regression in a few mice in the combination treatment group relative to either single agent groups. Tumor samples collected at the end of the study showed pronounced pharmacodynamic modulation of p-AKT and p-HER3, demonstrating on target activity of these agents.

Taken together, our findings suggest that modest anti-tumor activity was elicited by GSK2849330 as monotherapy in select gastric PDX models with no clear associations between response and HER3 mutations or other known markers. However, robust durable activity was observed upon combination with GSK2110183. To our knowledge, this is the first in vivo evidence supporting the rational combination of a selective AKT inhibitor (GSK2110183) and an anti-HER3 therapeutic antibody (GSK2849330), both of which are actively undergoing clinical trials and warrant further investigation in gastric cancer.

#1193

Treatment of breast cancer xenografts with paclitaxel enriches for cancer stem cells that can be targeted by a ROR1-specific antibody.

Suping Zhang, Grace Liu, Sam H. Lai, Han Zhang, Emanuela M. Ghia, Bing Cui, Rongrong Wu, George Widhopf, Jian Yu, Richard Schwab, Karen Messer, Barbara Parker, Thomas J. Kipps. _University of California, San Diego, La Jolla, CA_.

Although initially responsive to hormone therapy and chemotherapy, many patients with metastatic breast cancer develop resistant and potentially fatal disease. One of the mechanisms of resistance is the emergence of post-treatment survival of a small number of breast cancer stem cells (CSC), which are relatively resistant to chemotherapy, have self-renewal capacity, and can repopulate the tumor and spread to distal sites. New therapies are required to eliminate breast CSC that could be used alone or in combination with standard therapy to mitigate the risk of developing relapsed disease.

In this study, treatment of immune-deficient mice bearing breast-cancer patient-derived xenografts (PDX) with paclitaxel reduced tumor volumes, but enhanced the proportion of tumor cells that expressed ROR1, an RTK-like orphan receptor that is ordinarily expressed during embryogenesis. We found that breast cancer tumors with high levels of ROR1 expression were enriched for stem-cell gene-expression signatures. Moreover, cells isolated from breast cancer PDX that were positive for CSC markers such as aldehyde dehydrogenase 1 (ALDH1) were enriched for ROR1 expression, and ROR1+-cells isolated from breast cancer PDX had a greater capacity to form spheroids in vitro or re-engraft immune-deficient mice, than did ROR1-negative (ROR1Neg) cells isolated from the same tumor population. Treatment of breast cancer cells with a humanized anti-ROR1 mAb, UC-961, suppressed expression of the polycomb-ring-finger oncogene Bmi-1, and genes encoding proteins implicated in the epithelial-mesenchymal transition. UC-961 also impaired the capacity of breast-cancer cells to form spheroids or to re-engraft immune-deficient mice. Finally, combined therapy with UC-961 and paclitaxel appeared more effective in reducing re-engraftment potential than either agent alone (Table1). Collectively, this study implies that patients with advanced breast cancer may benefit from anti-CSC therapy, either alone or in combination with conventional anti-cancer drugs.

#1194

Discovery and preclinical development of a highly potent NaPi2b-targeted antibody-drug conjugate (ADC) with significant activity in patient-derived non-small cell lung cancer (NSCLC) xenograft models.

Natalya Bodyak, Alex Yurkovetskiy, Mao Yin, Dmitry Gumerov, Reddy Bollu, Patrick Conlon, Venu R. Gurijala, Dennis McGillicuddy, Cheri Stevenson, Elena Ter-Ovanesyan, Peter U. Park, Laura Poling, Winnie Lee, Michael DeVit, Dongmei Xiao, LiuLiang Qin, Timothy B. Lowinger, Donald A. Bergstrom. _Mersana Therapeutics, Cambridge, MA_.

The type II sodium-dependent potassium transporter NaPi2b (SLC34A2) is highly expressed in non-squamous NSCLC and non-mucinous ovarian cancer (OC) with restricted normal tissue expression, suggesting it may be a suitable ADC target for these indications. XMT-1536 is a novel, highly potent anti-NaPi2b ADC comprised of an average of 15 auristatin molecules conjugated to XMT-1535, a novel humanized anti-NaPi2b antibody, via the Dolaflexin ADC platform. The auristatin payload is enzymatically cleaved upon ADC trafficking to the endosome/lysosome compartment, releasing a cytotoxic auristatin derivative that is capable of bystander effect killing. In cell binding assays, XMT-1535 antibody binds to OC cells with low nanomolar affinity, which is unaffected by conjugation of the Dolaflexin drug conjugate. In vitro cytotoxicity assays show picomolar potency of XMT-1536 in OVCAR3 (OC; 32,000 NaPi2b molecules/cell; IC50 2 pM), TOV21G (OC; 10,000 NaPi2b molecules/cell; IC50 40 pM), and HCC-4006 (NSCLC; 52,000 NaPi2b molecules/cell; IC50 130 pM). In each cell line, XMT-1536 is 1-2 logs more potent than a non-binding Dolaflexin ADC control, consistent with target-dependent cytotoxic effect. XMT-1536 was tested in mouse xenograft models of OC and NSCLC. In the OVCAR3 OC model, XMT-1536 induced partial tumor regressions after a single dose of 3 mg/kg (0.21 mg/kg payload equivalent dose), and complete tumor regressions after a single dose of 5 mg/kg (0.36 mg/kg payload dose) or 3 weekly doses of 3 mg/kg. In contrast, a non-binding Dolaflexin ADC with comparable drug loading was inactive after 3 weekly administrations of 3 mg/kg, consistent with the anti-tumor activity of XMT-1536 being mediated through binding to the NaPi2b target. XMT-1536 was also tested in a patient-derived model of KRAS mutant NSCLC, where 3 weekly doses of 3 mg/kg led to significant tumor growth delay and regressions in some animals. Evaluation of XMT-1536 in additional patient derived xenograft models is on-going and will be updated at the meeting. XMT-1535 is cross-reactive with cynomolgous monkey NaPi2b, allowing an informative evaluation of whether XMT-1536 retains good tolerability in non-human primate. XMT-1536 was administered to cynomolgous monkeys in an exploratory single dose study up to 5 mg/kg ADC (4294 μg/m2 auristatin payload equivalents), with no observed target-mediated toxicity and limited adverse findings. Of note, there was no evidence of bone marrow toxicity, which has been observed generally for cleavable auristatin ADCs, and specifically for a recently published auristatin-based NaPi2b ADC (Lin et al., Clinical Cancer Research, 2015). Based on these data XMT-1536 is advancing to early clinical development for the treatment of NaPi2b-expressing tumors.

#1195

SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma.

Tim Lewis, Devra J. Olson, Kristine A. Gordon, Sharsti L. Sandall, Jamie Miyamoto, Lori Westendorf, Germein Linares, Chris Leiske, Heather Kostner, Ivan Stone, Martha Anderson, Albina Nesterova, Mechthild Jonas, Che-Leung Law. _Seattle Genetics, Inc., Bothell, WA_.

Multiple myeloma (MM) is a hematologic malignancy of transformed plasma cells. In spite of recent advances, MM remains an incurable disease, underscoring the need to develop new targeted biological therapeutics to augment existing treatments. In this study we describe SGN-CD352A, a potent new CD352-targeting antibody-drug conjugate (ADC) under development for the treatment of MM. CD352, or SLAMF6 (Signaling Lymphocyte Activation Molecule family member 6), is a type 1 membrane protein in the SLAM family of immunoreceptors. Like other SLAM family members, CD352 is a positive regulator of natural killer (NK) cell functions. CD352 is also a tumor antigen expressed on B cell malignancies such as MM, chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). We observed CD352 expression on the surface of malignant plasma cells in 87% (13/15) of human multiple myeloma patient samples examined by flow cytometry. Monoclonal antibodies (mAbs) specific for human CD352 were produced and a lead antibody was selected based on affinity, endocytic internalization rate, and tumor cell cytotoxic activity as an ADC. SGN-CD352A is a humanized anti-CD352 engineered cysteine (ec) mAb (h20F3ec) to which two molecules of pyrrolobenzodiazepine (PBD) dimer, a potent DNA damaging cytotoxic drug, have been conjugated. Upon binding CD352 at the MM cell surface, SGN-CD352A undergoes rapid clathrin-dependent endocytosis (< 2 hours) and traffics to lysosomal vesicles. PBD dimers released from SGN-CD352A in lysosomes induce a dose dependent DNA damage signaling response in MM cells, activating ATM and ATR kinases, and caspase 3/7 dependent apoptotic cell death results within 48 hours. SGN-CD352A demonstrated potent cytotoxic activity (EC50 values 6.6 - 52.6 pM) against a panel of human myeloma and lymphoma cell lines, with efficient cancer cell killing observed at CD352 expression levels as low as 3,500 receptors per cell. In contrast, SGN-CD352A had no effect on the viability of resting human T lymphocytes and minimal cytotoxic activity on B lymphocytes. We evaluated the in vivo antitumor activity of SGN-CD352A in disseminated MM cell line mouse xenograft models. In the MM.1R xenograft model, a single intraperitoneal dose of 30 μg/kg SGN-CD352A produced durable complete remissions in 10/10 mice. Similarly, in the U-266 xenograft model, a single dose of 100 μg/kg SGN-CD352A produced durable complete remissions in 9/10 mice and significant (P <0.0001) tumor delay at a single dose of 30 μg/kg. Neither unconjugated h20F3ec mAb nor a non-binding control PBD dimer ADC produced remissions in these MM xenograft models, demonstrating that targeted delivery of PBD dimer drug through CD352 binding is required for activity. In summary, CD352 is a newly validated multiple myeloma tumor antigen and the novel PBD dimer ADC SGN-CD352A shows potent antitumor activity against cell line models of MM at clinically relevant doses.

#1196

Combination of neuregulin with EGFR activation signatures predict activity of the anti-ErbB3 antibody KTN3379 in SCCHN.

Gwenda F. Ligon,1 Jay S. Lillquist,1 Scott B. Seibel,1 Jerry Wallweber,2 Veronique Neumeister,3 David L. Rimm,3 Theresa M. LaVallee,1 Diego Alvarado1. 1 _Kolltan Pharmaceuticals, Inc., New Haven, CT;_ 2 _Monogram Biosciences, Inc., South San Francisco, CA;_ 3 _Yale University, New Haven, CT_.

ErbB3, the kinase-dead member of the EGFR/ErbB family, has been implicated as an oncogenic driver in a number of solid tumor types, including squamous cell carcinoma of the head and neck (SCCHN). However, clinical trials have demonstrated limited responses with certain single agent anti-ErbB3-directed therapies, suggesting that multiple ErbB inhibition and/or biomarker-directed patient selection may be beneficial. The anti-EGFR antibody cetuximab has demonstrated improved overall survival in SCCHN, but the overall response rate is relatively low. Several preclinical studies have suggested that pan-ErbB inhibition may result in improved clinical benefit and additionally, may overcome cetuximab resistance. Here we show that ErbB3 and its ligand neuregulin (NRG) are widely expressed in SCCHN, and that ErbB2, but not EGFR, drives ErbB3 activation in the presence of NRG. Although NRG is required for ErbB3 activation, it is not sufficient to fully predict activity of the anti-ErbB3 antibody KTN3379 in SCCHN models. Metadata analysis of human SCCHN tumors revealed that NRG expression is significantly associated with expression of EGFR ligands amphiregulin (AREG) (P<0.0001; R2 = 0.33) and transforming growth factor α (TGFα) (P<0.0001; R2 = 0.25) but not other EGFR ligands. We demonstrate using NRG-positive cell lines that KTN3379 antitumor activity correlates with the level of secreted AREG and TGFα. Using a proximity-based assay for EGFR homodimerization as a surrogate assay for ligand (AREG or TGFα)-induced EGFR activation, we show that high EGFR homodimer levels correlate with KTN3379 antitumor activity in NRG-positive SCCHN cell lines and primary tumor models in vitro and in vivo. These preclinical studies provide the molecular rationale for combination treatment with KTN3379 and cetuximab in SCCHN patients. We propose that an enrichment strategy for NRG expression and EGFR activation signature may result in better clinical activity in response to treatment with KTN3379 and cetuximab in SCCHN patients.

#1197

Outstanding preclinical efficacy of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 in patient-derived xenograft solid tumors.

Loreley Calvet, Anne-Marie Lefebvre, Celine Nicolazzi, Lydia Blot, Corinne Thomas, Yves Baudat, Beatrice Cameron, Carlos Garcia-Echeverria, Jean-Francois Mayaux, Veronique Blanc, Cecile Combeau, Souad Naimi, Sukhvinder Sidhu. _sanofi, Vitry-sur-seine, France_.

Lysosome-associated membrane protein 1 (LAMP1) is involved in the maintenance of lysosome membrane integrity and phagolysosome formation. LAMP1 shows very limited expression at the cell surface in normal tissues while, a moderate to high expression was found in a number of solid tumors. We generated SAR428926, an antibody drug conjugate (ADC) comprising an anti-LAMP1 humanized monoclonal antibody conjugated via a cleavable SPDB linker, to the maytansinoid derivative DM4, a tubulin interfering molecule.

Here we report the annotation of LAMP1 expression in a large panel of human tumors and correlate it to the in vivo efficacy of SAR428926 in a panel of over 50 human patient-derived xenograft (PDX) solid tumors.

Indications of interest were identified by immunohistochemistry (IHC) conducted on a large panel of frozen tumor samples using the murine version of the therapeutic anti-human LAMP1 antibody. The prevalence of tumor samples with positive membrane staining was 49% in breast invasive lobular or ductal carcinoma, and in particular in a TNBC subset (87%), 25% in gastric adenocarcinoma, 52% in colon/rectum adenocarcinoma, 22% in lung adenocarcinoma, 20% in lung squamous cell carcinoma, 25% in prostate adenocarcinoma and 31% in ovary adenocarcinoma.

In vivo efficacy of SAR428926 was evaluated in PDX models engrafted subcutaneously into immunocompromised SCID mice. PDX models retain the molecular diversity, cellular heterogeneity, and histology typically seen in patient tumors and offer a distinct advantage over cell line models. The PDX models used in the study, which include colon, breast, lung, prostate, gastric and ovarian cancer, were selected by IHC based on LAMP1 expression levels distribution and the frequency of LAMP1 membrane positive cells, which varied between 5% and 100% of the tumor cells, allowing coverage of a wide variety of cases.

Outstanding efficacy with complete regressions was observed at 5 mg/kg iv single administration, a dose that gives an exposure tolerated in toxicology species. Efficacy was also observed at lower doses of 2.5 mg/kg and 1.25 mg/kg after single administration. Our results show that while the presence of the antigen is required as there is no efficacy in LAMP1-negative PDX, there is no linear correlation between the level of antigen expression and the antitumor activity across the panel of PDX tested. In addition to membrane LAMP1 expression, a key driver for efficacy was the intrinsic sensitivity of the PDX models to DM4.

These results highlight the potential of ADCs that target human cancers expressing membrane LAMP1 at the surface of tumor cells. These encouraging preclinical data have prompted us to initiate IND-enabling studies with the goal to progress anti LAMP1-ADC to the clinic in patients with solid tumors expressing LAMP1 at the cell surface. The phase I study started in October 2015.

#1198

Characterization of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 expressed at the surface of tumor cells.

Yves Baudat, Beatrice Cameron, Tarik Dabdoubi, Anne-Marie Lefebvre, Ana Merino-Trigo, Corinne Thomas, Veronique Pecheux, Bruno Genet, Loreley Calvet, Lydia Blot, Magali Mathieu, Laurence Gauzy, Laurence Berthou-Soulie, Catherine Prades, Celine Amara, Manoel Nunes, Christophe Henry, Cecile Combeau, Francis Blanche, Jean-Francois Mayaux, Carlos Garcia-Echeverria, Souad Naimi, Veronique Blanc. _SANOFI, vitry sur Seine, France_.

Lysosome-associated membrane protein 1 (LAMP1), one of the most abundant protein expressed at the membrane of lysosomes, is a type I transmembrane protein involved in the maintenance of lysosome membrane integrity and phagolysosome formation. In activated lymphocytes LAMP1 is described as a marker of degranulation.

Unexpectedly, by immunizing mice with a colon patient-derived xenograft (PDX) followed by a screening of monoclonal antibodies (mAb) by immunohistochemistry (IHC) for selection of antibodies that specifically stain tumor plasma membrane and de-orphaning by Immunoprecipitation-Mass spectrometry, we identified LAMP1 as the target of several antibodies. One of them, Ab-1, showed binding to the luminal domain of human LAMP1 with nM affinity. Crystal structure of its Fab with LAMP1 extracellular domain, showed that the epitope was non-linear, not a glycotope and spanned between position 29 to195.

LAMP1 expression was further documented by IHC with Ab-1, showing limited cell surface expression in normal tissues while moderate to high plasma membrane expression was found in a number of breast, including TNBC, colorectal, gastric, prostate, lung and ovary tumors.

The humanized Ab-1 mAb, humAb-1, was shown to display rapid cycling after binding to LAMP1 at the surface of colo205 cell line, allowing internalization and processing of a number of LAMP1/antibody complex 10 folds higher than the number of LAMP1 molecules at the cell surface.

humAb-1 was conjugated to DM4 maytansinoid derivative using an SPDB cleavable linker to generate a new antibody-drug conjugate, SAR428926, for the treatment of patients with cell surface LAMP1-positive tumors.

Conjugated and naked antibody displayed similar affinities for LAMP1. SAR428926 killed tumor cell lines (engineered to express cell surface LAMP1) in the sub-nM range. In contrast, no target-mediated cytotoxicity was observed when SAR428926 was incubated with normal cells, including resting or activated lymphocytes.

PDX models reflecting the pattern and level of LAMP1 expression at the surface of human tumors were selected to evaluate SAR428926 in vivo efficacy. Outstanding in vivo activity was observed in different indications, including TNBC, lung and colon PDXs, with complete regressions after a single administration at 5 mg/kg.

These encouraging preclinical data have prompted the initiation of IND-enabling studies with the goal to progress humAb-1-ADC to the clinic in patients with tumors expressing LAMP1 at the cell surface. The First-In-Human trial has been initiated in October 2015.

#1199

A network biology screen reveals ligand-receptor pathway connections and resistance mechanisms to RTK-directed therapies in cancer cells.

Kristina Masson, Andreas Raue, Gavin MacBeath. _Merrimack Pharmaceuticals, Cambridge, MA_.

Purpose: Although there are many well-defined signaling pathways in tumor cells that provide druggable targets, emergence of growth factor-mediated resistance and parallel pathway compensation frequently occurs, limiting the effectiveness of these treatment strategies. The purpose of this study was to model cancer cell responses to combinations of growth factors (ligands) and targeted investigational therapies in order to identify which signaling pathways are mechanistically related. The results presented here demonstrate the use of network biology-based phenotypic screening and modeling to reveal unexpected behaviors, identify positive and negative biomarkers, and guide novel treatment strategies.

Methods: To assess the effect of ligands and RTK-directed antibodies in combination with targeted investigational therapies in cancer cells, we conducted a high throughput viability screen in 3D cultures. A library of 87 small molecules targeting different components of signaling and metabolic pathways was used in combination with growth factors and therapeutic antibodies. The therapeutic antibodies included in the screen were MM-121 (anti-ErbB3), MM-131 (anti-Met/EpCAM), MM-141 (anti-IGF-1R/ErbB3), and MM-151 (anti-EGFR). Using these data, we inferred a pathway connectivity model to identify pathway crosstalk and novel and effective combination strategies, which were then further evaluated in vitro and in mouse xenograft models.

Results: Overall, we found that ligand-mediated resistance varies depending on the cancer type. For example, HGF and EGF play particularly strong roles in gastric and colorectal cancer cells, respectively. Interestingly, a few cases were found in which a ligand reduced cell viability compared to control, but this had no effect on drug response. In most cases where a ligand rendered cells insensitive to a certain drug treatment, a combination with the appropriately matched therapeutic antibody re-sensitized cells to the drug, suggesting that a combination strategy could potentially be used to overcome ligand-mediated resistance. Among the findings that were observed in multiple cell lines, MM-131 combined well with EGFR-directed agents in HGF responsive tumor cells, and MM-141 combined well with metformin in colorectal and lung cancer cell lines. These findings were further validated in vivo.

Conclusions: Growth factors for EGFR, Met, ErbB3, and IGF-1R decrease sensitivity to a wide range of targeted investigational agents in cancer cell lines. These effects, however, are not general, but instead are dependent on the type of cancer cell being treated. Insights from this screen may help guide the future clinical development of MM-121, MM-131, MM-141 and MM-151.

#1200

HER-2 isoform interaction in mammary carcinoma onset and progression.

Arianna Palladini,1 Massimiliano Dall'Ora,1 Tania Balboni,1 Giordano Nicoletti,2 Marianna Ianzano,1 Roberta Laranga,1 Lorena Landuzzi,2 Veronica Giusti,1 Alessia Lamolinara,3 Carla De Giovanni,1 Augusto Amici,4 Serenella M. Pupa,5 Manuela Iezzi,3 Patrizia Nanni,1 Pier-Luigi Lollini1. 1 _Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy;_ 2 _Rizzoli Orthopedic Institute, Bologna, Italy;_ 3 _Aging Research Centre, "Gabriele d'Annunzio" University, Chieti, Italy;_ 4 _University of Camerino, Camerino, Italy;_ 5 _Istituto Nazionale Tumori, Milano, Italy_.

Human breast cancer cells express full-length HER-2 along with proteins resulting from mutation, alternative splicing, alternative initiation of translation and post-translational modification. The Delta16 splice variant, lacking exon 16, has the properties of an activated oncogene, but it could also play beneficial roles in the response to targeted therapeutic agents. To study mammary carcinogenesis in a mouse model that mimics human coexpression of full-length HER-2 and Delta16 isoforms, we produced hybrid mice bearing heterozygous copies of both human transgenes (F1 mice), and we compared them to parental mice (Delta16 and HER-2 transgenic mice, respectively). Tumor onset in F1 and Delta16 mice was much faster than in HER-2 mice (30 vs >80 weeks), but the growth of established tumors and metastatic spread were not enhanced. Each mammary carcinoma of F1 mice expressed both isoforms at variable ratios. Most (~80%) expressed high levels of Delta16 and low levels of full length HER-2, a few (~5%) expressed full-length HER-2 and little, if any, Delta 16, and the remainder (~15%) coexpressed at high level both isoforms. The study of tumor vascularization showed that full-length HER-2 tumors mainly contained few large vessels or vascular lacunae, whereas Delta16 tumors were perfused by numerous endothelium-lined small vessels. F1 tumors with high full-length HER-2 expression made few large vessels, whereas tumors with low full-length HER-2 and high Delta16 contained numerous small vessels and expressed high levels of both VEGF and VEGFR2. Administration of trastuzumab to young F1 mice effectively prevented mammary carcinoma onset in ~85% of mice at 1 year of age. The preventive effect of trastuzumab was stronger in F1 mice than in both parental strain. To analyze the intrinsic sensitivity of F1 mammary carcinoma cells to targeted drugs in 3D, we established cell lines expressing different HER-2 isoform ratios. High Delta16 expression caused high sensitivity to HER-2 inhibitors (trastuzumab, neratinib, lapatinib), whereas high full-length HER-2 was associated with a lower sensitivity. Interestingly, high levels of both isoforms was associated with resistance to trastuzumab, but sensitivity to small molecule inhibitors. In conclusion, the coexpression of full-length HER-2 and Delta16 controls several determinants of mammary carcinoma development, progression and sensitivity to targeted agents, as revealed by the study of F1 mice.

#1201

Anti-B7-H3 antibody-drug conjugates as potential therapeutics for solid cancer.

Deryk Loo,1 Juniper A. Scribner,1 Thomas Son,1 Jeff Hooley,1 Timothy Hotaling,1 Michael Chiechi,1 Pam Li,1 Anushka De Costa,1 Yan Chen,1 Ann Easton,1 Francine Z. Chen,1 Bhaswati Barat,2 Valentina Ciccarone,2 James Tamura,2 Mark Kubik,2 Scott Koenig,2 Syd Johnson,2 Paul A. Moore,2 Ezio Bonvini2. 1 _MacroGenics, Inc., South San Francisco, CA;_ 2 _MacroGenics, Inc., Rockville, MD_.

Introduction: Monoclonal antibodies (mAbs) were generated via a target-unbiased approach based on intact cell immunization with cell lines, fetal progenitor cells, and cancer stem cells. An immunohistochemical screen for cancer-specific candidates identified a panel of anti-B7-H3 (CD276) mAbs with highly differential tumor-versus-normal tissue binding. B7-H3 expression was observed in tumor epithelium as well as tumor-associated vasculature and stroma. Consistent with our findings, B7-H3 has been reported to be overexpressed in a growing number of solid cancers, including breast, lung, pancreatic, prostate, kidney, and colon cancer, as well as melanoma and glioblastoma. Furthermore, overexpression of B7-H3 has been correlated with disease severity and poor outcome in a number of these cancer types. A humanized version of an anti-B7-H3 mAb engineered with an enhanced Fc domain (enoblituzumab or MGA271) and a humanized Dual-Affinity Re-Targeting (DART®) protein that recognizes both B7-H3 and CD3 and redirects T cells to kill B7-H3-expressing cells (MGD009) are being investigated in Phase 1 clinical studies. In this nonclinical study, we evaluated the therapeutic potential of anti-B7-H3 antibody-drug conjugates (ADCs) toward B7-H3-expressing solid cancers.

Methods: A panel of anti-B7-H3 mAbs was screened for internalization and a subset of mAbs that were efficiently internalized by tumor cells was identified. These mAbs were converted to ADCs via chemical conjugation; in vitro and in vivo activity studies were then conducted with a range of tumor cell lines representing human cancer types that overexpress B7-H3.

Results: The anti-B7-H3 ADCs exhibited specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro, including breast, lung, ovarian, pancreatic, and prostate cancer lines, with IC50 values generally in the sub-nM range. Cytotoxicity was not observed with cell lines lacking B7-H3 expression. The anti-B7-H3 ADCs exhibited potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human breast, lung, and ovarian tumor xenografts.

Conclusion: Anti-B7-H3 ADCs exhibited dose-dependent cytotoxicity in vitro and potent antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing cancer types that overexpress B7-H3. Our findings demonstrate that ADCs targeting B7-H3 may serve as potential therapeutics for B7-H3-expressing solid cancers.

#1202

Target identification for a new type of ADC.

James R. Prudent, Chad Hall, David J. Marshall, Scott Harried, John Murphy. _Centrose, Madison, WI_.

Purpose:

To explore the mechanism of action of a potent new type of antibody drug conjugate (ADC) called Extracellular Drug Conjugates or EDCs.

Experimental Design:

EDCs are a new type of ADC that have similar activity and components, yet EDCs are different in the following ways; 1) EDCs do require internalization, 2) EDCs are not pro-drugs but instead require the antibody and payload to remain permanently linked and 3) EDCs target two proteins in unison, requiring long non-cleavable linkers to span distances between bound antibody to the payload binding site. To define the MOA of the EDCs, high definition phase contrast imaging and protein marker experiments were conducted. In order to verify the targets of a set of EDCs consisting of Na,K-ATPase specific payloads, siRNA knockdown experiments were designed and tested.

Results:

First, we observed that after 24 hour exposure to each of the EDCs (or the free payload), cells rounded up, partially detached from the substrate, swelled, and lost plasma membrane integrity - morphology resembling necrosis. These observations were consistent with no induction apoptosis or autophagy. Second, our siRNA studies determined that alpha 1 of the Na,K-ATPase was a lethal target. Consistent with this observation on all tested cells lines tested, the EC50 values of each EDC decreased with decrease in alpha 1 expression. Additionally, when the corresponding antibody target expression decreased, overall activity of each EDC went down. Alpha 1 expression was also found to change the effects of the free payload, indicating that the EDCs and the free payload share the same target. Yet unlike the EDCs, payload alone was not affected by the expression of any antibody target used in the study. We also observed that siRNA knockdown of the Na,K-ATPase beta 1 or beta 3 subunits alone did not affect EDC or payload activity, yet a significant decrease in activity could be observed when both subunits were knocked down simultaneously. These results are also consistent with microarray data analysis using siRNAs against all 8 alpha and beta subunits which show that only alpha-1, beta-1, and beta-3 are expressed in the cell types tested.

Conclusions:

These results show that when payloads targeting the Na,K-ATPase are attached to certain antibodies via long flexible linkers, the resulting EDC activity specifically target the alpha 1 subunit of the Na,K-ATPase, of which we determined to be a lethal target. These results also show that EDC's activity is dependent on the expression of the corresponding antibody target. In addition, the mechanisms of EDCs are similar to that of the free payload and appear to be necrosis like. These results support the continuing efforts to identify the detailed mechanism of the new ADCs which may lead to identifying patients most likely to respond to EDC based therapies.

#1203

Targeting activated tumor vasculature with human Granzyme B variants engineered for improved stability and activity.

Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. _UT MD Anderson Cancer Center, Houston, TX_.

Angiogenesis is a critical process in numerous diseases, and targeting tumor neovasculature has therapeutic value in controlling tumor progression and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) and its receptors VEGFR-1 and VEGFR-2 have been implicated as central mediators of normal angiogenesis and tumor neovascularization. VEGF121 is a naturally-occurring VEGF-A splice variant that binds to these receptors, which are over-expressed on the endothelium of activated tumor vasculature or neovasculature but not normal vasculature. Granzyme B (GrB) plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade through both caspase-dependent and -independent mechanisms. The fusion protein GrB/VEGF121 expressed in mammalian cells and purified to homogeniety was lethal to tumor and endothelial cells in vitro in a manner that correlated closely to total VEGFR-2 expression. IC50 levels were found to be in the nanomolar range. GrB/VEGF121 internalized rapidly into VEGFR-2 expressing cells while the internalization into VEGFR-1 expressing cells was significantly reduced. We engineered GrB variants for improved protein production and for resistance to serpin B9 (PI-9), a natural inhibitor of Granzyme B found at varying levels in cells and plasma, and compared these GrB/VEGF121 constructs directly to unmodified GrB/VEGF121. Variants took into account the impact of the mutation on substrate specificity, and the ability of the mutated GrB to cleave its various cellular substrates. As expected, mutation of the active site 195serine to alanine (S195A) resulted in a complete loss of GrB enzymatic activity as well as in vitro cytotoxicity against VEGFR-1+ and VEGFR-2+ cells. Enzymatic activity in PBS of the K27E, R28A ("EA-GV") double mutant was identical to that of GrB/VEGF121. In 50% human serum, however, the enzymatic activity of GrB/VEGF121 steadily declined to less than 10% over 24 hours while the enzymatic activity of EA-GV remained over 40% during that time period. This construct retained higher cytotoxic activity after pre-incubation in serum for 4h, compared to GrB/VEGF121. C-GrB/VEGF121, without an unpaired cysteine, resulted in reduced aggregation and a vastly improved yield with otherwise similar serum stability and in vitro cytotoxicity as unmodified GrB/VEGF121. The pharmacokinetic profile for C-GrB/VEGF121 indicated a t1/2ɑ of 6.6 min and t1/2β of 33.3 h. A limited profile for EA-GV indicates a longer terminal-phase half-life for this construct compared to C-GrB/VEGF121. Our studies suggest that these novel GrB variants may significantly improve in vivo half-life. Further studies are underway to determine whether these modifications impact production of soluble material and in vivo therapeutic efficacy against established tumor xenograft models. Research conducted, in part, by the Clayton Foundation for Research.

#1204

A construction platform for hexavalent agonists targeting receptors of the tumor necrosis factor superfamily: Where death meets co-stimulation.

Christian Merz, Christian Gieffers, Michael Kluge, David M. Richards, Tim Schnyder, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. _Apogenix GmbH, Heidelberg, Germany_.

Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells. Their importance in many locations and phases of the anti-tumor immune response is now broadly appreciated and several TNFR agonists are currently in preclinical and clinical development. Importantly, signaling through many TNFRSF members, such as CD40, CD27, OX40, 4-1BB, HVEM and GITR, is potentially associated with an enhanced anti tumor response via co-stimulation of immune cells.

Apogenix has established a development platform for a novel class of TNFRSF-agonists for the treatment of cancer. Unlike their natural homotrimeric counterparts, the Apogenix recombinant TNFSF proteins consist of one single polypeptide chain composed of three receptor-binding domain-forming protomers. These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) are mimics of the three-dimensional organization of the natural TNFSF-cytokine and can be used to engineer fully human fusion-proteins from a modular toolbox. For example, fusing an IgG1 Fc-domain to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist as the Fc-domain acts as a dimerization scaffold for two trivalent scTNFSF-RBDs. As a result of this molecular design, each drug molecule is capable of clustering six receptors in a spatially well-defined manner. Consequently, TNFSF receptor signaling following treatment with the Apogenix scTNFSF-RBD-Fc in vivo is independent of secondary clustering through Fc-γ receptors that is required for many anti-TNFRSF agonistic antibodies (e.g., anti-TRAILR2 or -CD40).

Following up the scTRAIL-RBD-Fc prototype, this engineering concept has now been successfully translated to CD40L and CD27L resulting in hexavalent agonists suitable for further development. Expression of the drug candidates in CHO suspension cells followed by an AFC and SEC-based lab-scale purification process resulted in homogenous aggregate-free protein lots. The purified proteins bind their respective target-receptors with high affinity. In vivo stability/PK studies have been performed in addition to in vitro experiments with primary human and mouse lymphoid and myeloid cell populations. Specifically, it was shown that scCD27L-RBD-Fc was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells. Importantly, binding significantly increased T cell expansion following activation. Treatment with scCD40L-RBD-Fc induced differentiation of B cells and enhanced primary human monocyte differentiation into DCs or M1 macrophages.

Encouraged by the promising results obtained with TRAIL, CD40L, and CD27L, Apogenix is currently expanding the TNFRSF-agonist pipeline to target additional cell populations, locations and phases of the immune response in order to develop novel therapies to treat cancer and other conditions.

#1205

HER3 is a functional molecular target in HPV-associated head and neck cancer.

Toni Michel Brand,1 Stefan Hartmann,2 Neil Bhola,1 Noah D. Peyser,1 Hua Li,1 Yan Zeng,1 Max V. Randall,1 Sourav Bandyopadhyay,1 Jennifer R. Grandis1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University Hospital Würzburg, Würzburg, Germany_.

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a rising incidence of more than 600,000 new cases each year. Human papillomavirus (HPV)16 plays an etiologic role in a growing subset of HNSCCs, where viral expression of the E6 and E7 oncoproteins are necessary for tumor growth and maintenance. Although patients with HPV(+) tumors have a more favorable prognosis compared with HPV(-) patients, there are currently no HPV-selective therapies. Recent studies have identified differential receptor tyrosine kinase (RTK) profiles in HPV(+) vs. HPV(-) tumors. One such RTK, HER3, is overexpressed and highly associated with phosphoinositide 3-kinase (PI3K) in HPV(+) tumors.

Methods: In the current study, HER3 was investigated as a molecular target in HPV(+) HNSCC cell lines and patient-derived xenografts (PDXs). HNSCC dependency on HER3 was evaluated with both anti-HER3 siRNAs and the clinically advanced anti-HER3 monoclonal antibody, KTN3379. Furthermore, the anti-growth effects of combinatorial HER3 and PI3K inhibition was examined in HPV(+) HNSCC models.

Results: HER3 was overexpressed and activated in HPV(+) HNSCC models. Further investigation indicated that HPV(+) HNSCC cells were reliant on HER3 for cellular proliferation and PI3K pathway signaling, whereas little effect was observed in HPV(-) cell lines. Intriguingly, HPV oncoproteins directly regulated HER3, where knockdown of E6 and E7 resulted in loss of total HER3 protein expression and reduced PI3K pathway signaling (p-AKT, p-p70S6K, p-rpS6) in HPV(+) cell lines. Conversely, overexpression of E6 and E7 increased total HER3 protein expression (5-7 fold) and PI3K pathway signaling in HPV(-) cell lines. Since HER3 signals via the PI3K pathway to mediate cellular proliferation and survival, we sought to identify if HER3 can mediate response to PI3K pathway inhibitors. Stimulation of HPV(+) cell lines with neuregulin-1, the cognate ligand of HER3, resulted in complete resistance to the p110α inhibitor, BYL719, and prevented BYL719 induced inhibition of AKT. Neuregulin-1 stimulation of HPV(-) cell lines had no effect on their response to BYL719, and did not rescue AKT activation. Furthermore, treatment of HPV(+) cell lines with BYL719 resulted in enhanced HER3 expression and activation (20-50 fold), indicating that HER3 may mediate resistance to PI3K inhibitors in HPV(+) cells. Finally, dual targeting of HER3 and PI3K synergistically inhibited HPV(+) cell line proliferation (by 50-70%) and resulted in sustained knockdown of PI3K pathway signaling. While HPV(-) cell lines were responsive to PI3K inhibition, HER3 blockade did not provide additional benefit. HPV(+) PDX models are currently being evaluated for response to dual kinase inhibition.

Conclusions: These studies identify HER3 as a potential therapeutic target, and provide a rationale for the clinical evaluation of combined HER3 and PI3K inhibition in HPV(+) HNSCCs.

#1206

Preclinical evaluation of targeting Notch-3 in breast cancer.

Sara A. Hurvitz,1 Erika von Euw,1 Neil O'Brien,1 Dylan Conklin,1 Chuhong Hu,1 Jiaying Zhuo,1 Alice Zhao,1 Frank Calzone,1 Hsiao-Wang Chen,1 Judy Dering,1 Ken Geles,2 Puja Sapra,2 Dennis J. Slamon1. 1 _UCLA, Santa Monica, CA;_ 2 _Oncology-Rinat R &D Group, Pfizer, Inc, Pearl River, NY_.

Introduction: Notch-3 overexpression has been implicated in the development of breast cancer (BC) and is associated with poor outcomes. A critical challenge to eliminating treatment resistance in breast cancer likely relates to the presence of cancer stem cells (CSCs) that maintain the ability to differentiate and divide indefinitely. We postulate that targeted eradication of CSCs is possible using a Notch3 antibody drug conjugate (ADC) without irreversibly reducing stem cell viability in vital normal tissues. PF-06650808, is an ADC comprised of a humanized anti-Notch-3 antibody linked to an auristatin-based cytotoxic agent. To better understand the therapeutic index of targeting Notch-3, we evaluated PF-06650808 across a large panel of BC lines and normal cells and correlated response with Notch-3 levels. PF-06650808 was also evaluated in a murine BC xenograft model.

Methods: Response to PF-06650808 and control ADC was evaluated across a panel of BC and normal cell lines by a 2D proliferation assay. Notch-3 mRNA expression was measured by flow cytometry (FC) and RPPA. MDA-MB-468 (triple negative BC, TNBC) tumor bearing mice were randomized into 4 arms of 8 mice and treated with 3 mg/kg PF-06650808 or control-ADC (days 0, 4, 8 & 12), 10 mg/kg docetaxel (q week) or vehicle control.

Results: High expression of Notch-3 was detected in multiple BC cell lines by RPPA and FC. BC cell lines with elevated levels of Notch-3 were sensitive to PF-06650808 (HCC1187, MDA-MB-468, HCC1143, HCC70, EFM-19, HCC202). Responders were also enriched for TNBC. All normal cell lines were resistant to PF-06650808 ADC. When treated with a control ADC against a non-relevant target, all cell lines exhibited IC50s between 5-50ug/ml, indicating that the sub-0.5ug/ml responses seen with the Notch-3 ADC were target-dependent. Durable complete tumor regressions were observed in PF-06650808-treated mice bearing MDA-MB-468 TNBC cell line xenografts.

Conclusions: Sensitivity to a novel anti-Notch-3-ADC is associated with high expression of Notch-3 in BC cell lines. Normal cells are resistant to PF-06650808, possibly predicting a better therapeutic index than seen with other Notch inhibitors. Xenograft studies evaluating the in vivo efficacy of PF-06650808 in a panel of xenografts with varying levels of Notch-3 expression will be presented. Ongoing experiments are exploring the potency of PF-06650808 on CSCs. Our data will help identify breast cancer subtypes most likely to respond to a Notch3-ADC based on high tumor/normal target concentration as well as its effects on CSCs.

#1207

Preclinical development of 2nd generation HER2-directed antibody-drug conjugates.

Gail D. Lewis Phillips, Guangmin Li, Jun Guo, Jeffrey Lau, Shang-Fan Yu, Thomas Pillow, Byoung-Chul Lee, Jack Sadowsky, Melissa Schutten, Carter Fields, Mark X. Sliwkowski. _Genentech, Inc., South San Francisco, CA_.

The HER2 receptor tyrosine kinase is amplified in approximately 20% of human breast cancer and is associated with poor clinical outcome. The humanized antibodies trastuzumab and pertuzumab are approved for use in both early and metastatic HER2-positive breast cancer, and are most often given with chemotherapy. Antibody-drug conjugates (ADCs) are anti-tumor agents designed to deliver potent cytotoxic drugs selectively to target-expressing tumor cells. Trastuzumab emtansine is a HER2-directed ADC comprised of trastuzumab covalently linked to the microtubule inhibitor DM1, through the stable MCC linker. Trastuzumab emtansine is approved for use in HER2-positive metastatic breast cancer as a single agent in patients who have received prior trastuzumab and a taxane. We are now exploring new HER2-directed ADCs ('2nd generation ADCs') with different mechanisms of action (MOA) than trastuzumab emtansine by investigating ADCs utilizing DNA-damaging agents, such as pyrrolobenzodiazepine (PBD) dimers and cyclopropylbenzindole (CBI) dimers, as the cytotoxic drug components. These agents have been conjugated to either trastuzumab or the humanized anti-HER2 antibody 7C2 (hu7C2) using both uncleavable and cleavable linkers. As free drugs and ADCs, the PBDs and CBIs show similar or greater potency in cell proliferation assays in vitro compared to DM1 and trastuzumab emtansine. However, unlike DM1, these agents are not strong substrates of Pgp/MDR1. Moreover, the PBDs and CBIs are active on non-dividing cells, whereas microtubule inhibitors such as DM1 do not affect non-dividing cells. Robust anti-tumor activity was observed in vivo in the fo5 HER2 transgenic tumor transplant model with the 2nd generation ADCs. Efficacious doses resulting in tumor stasis or regression ranged from 0.25-3 mg/kg administered as a single injection. In contrast, doses of trastuzumab emtansine required for stasis/regression in this model are 10 and 15 mg/kg, respectively. Efficacious doses were well-tolerated in the mouse xenograft models. Further tolerability studies of the 2nd generation ADCs were performed in rats. As rats are a non-binding species for trastuzumab and hu7C2, these studies assessed antigen-independent toxicities. Maximum tolerated doses for the different ADCs ranged from 2.5-15 mg/kg administered as a single injection, compared to 46 mg/kg for trastuzumab emtansine (Poon et al., 2013), likely reflecting both the different MOA and greater potency of the cytotoxic agents utilized in the 2nd generation HER2-directed ADCs. Overall, our findings demonstrate robust in vitro and in vivo activity of HER2 ADCs comprised of DNA-active agents, allowing for further development of a HER2-directed ADC distinct from trastuzumab emtansine.

#1208

Combination effect of afatinib and BI836845, a humanized IGF ligand-neutralizing antibody, on EGFR-TKI-resistant NSCLC cells.

Tohru Ohmori,1 Toshimitsu Yamaoka,1 Satoru Arata,2 Motoi Ohba,1 Yasunori Murata,3 Yasunari Kishida,3 Sojiro Kusumoto,3 Masanao Nakashima,3 Takashi Hirose,4 Tsukasa Ohnishi,3 Kazuto Nishio5. 1 _Inst Mol Oncol, Showa Univ, Tokyo, Japan;_ 2 _Cent Biotechnol, Showa Univ, Tokyo, Japan;_ 3 _Div Resp Med and Allergol, Dept Int Med, Showa Univ, Tokyo, Japan;_ 4 _Natl Hosp Org, Tokyo Natl Hosp, Tokyo, Japan;_ 5 _Dept Genome Biol, Kinki Univ Faculty Med, Osaka, Japan_.

Insulin-like growth factor (IGF) signaling is thought to have a role in the cancer progression and survival, and many carcinomas are known to overexpress IGF-1 receptor (IGF1R). Furthermore, it was clarified that IGF1R signaling contributes to EGFR-TKI resistance in NSCLC. IGF1R is thought to be a therapeutic target for cancer chemotherapy. Previously, we screened the combination effect of afatinib and various anticancer drugs on a panel of NSCLC cell lines including gefitinib-resistant and afatinib-resistant PC-9 cells which we established. As the result, the combination of afatinib and small molecule IGF1R-TKIs (BMS754807, NVP-ADW742) has shown the most cytotoxicity on wide variety cancer cells. However, these IGF1R-TKIs have cross-reactivity to insulin receptor and, as a result, are known to cause hyperglycemia. Moreover, a phase III trial of the combination chemotherapy between erlotinib and IGF1R specific antibody, figitumumab, in advanced nonadenocarcinoma NSCLC patients (ADVIGO 1018)was closed early because of futility and an increased incidence of serious adverse events.

BI836845 is a humanized IGF ligand-neutralizing antibody which is neutralizing both IGF-1 and IGF-2. The proliferation of several cell lines was reported to be potently inhibited by BI 836845 with lower than 100 μg/ml EC50 values. This antibody is thought to be a substitute approach to inhibit IGF1R signaling pathway other than previous IGF1R-TKIs. We evaluated the combination effect of afatinib and BI836845 on the same NSCLC panel by MTS assay. BI836845 had only a limited cytotoxicity by itself. The combination of afatinib and BI836845 had additive or synergistic cytotoxicity on most of the NSCLC cell lines including EGFR-TKIs-resistant cells except the cells overexpressing c-Met or expressing K-ras mutation. This combination significantly inhibited both EGFR and IGF1R autophosphorylation and their downstream signaling, especially Akt/mTOR pathway, as compared with each singular usage. These combination effects were attenuated when BI836845 combined with erlotinib. Since BI836845 cross-react to rodent IGFs, we examined pharmacological activity of this combination using SCID mice xenograft mode. The mice bearing tumor were given 6 mg/kg afatinib (5 times/week, p.o.) and 200 mg/kg BI836845 (1 time/week, i.p.) for 8 weeks. This combination synergistically inhibited afatinib-resistant tumor (PC-9Afa1, PC-9Afa2) and gefitinib-resistant tumor (PC-9ZD, expressed T790M mutant EGFR). This BI836845 administration completely inhibited IGFs activity in the mice serum without alteration of blood sugar level and insulin concentration. Though growth rate of the treated mice was slightly reduced, body weight loss and other serious adverse events were not observed.

From these observations, this combination chemotherapy is thought to be a potential therapeutic strategy for NSCLC.

#1209

Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models.

Michael D. Curley, Gege Tan, Isabel Yannatos, Adam Camblin, Sergio Iadevaia, Chrystal Louis, Alexey Lugovskoy. _Merrimack Pharmaceuticals, Inc., Cambridge, MA_.

Insulin-like growth factor receptor 1 (IGF-1R) signaling has been implicated in the pathogenesis of ovarian cancer. However, clinical trials evaluating monospecific IGF-1R inhibitors have demonstrated limited clinical efficacy. Our data indicate that ErbB3, a member of the ErbB receptor tyrosine kinase family, can activate pro-survival AKT signaling in response to IGF-1R blockade and may represent a potential escape route in the development of resistance to therapy. Istiratumab (MM-141), an IGF-1R and ErbB3 directed bispecific antibody, inhibits ligand activation of these signaling pathways and degrades IGF-1R and ErbB3 receptor-containing complexes, leading to inhibition of downstream pro-survival signaling. Here we tested the activity of istiratumab, alone and in combination with chemotherapy, in in vitro and in vivo models of ovarian cancer.

Anti-proliferative activity of istiratumab monotherapy was evaluated in a panel of ovarian cancer cell lines in vitro. The effects of istiratumab and the ligands IGF-1 and heregulin on IGF-1R- and ErbB3-mediated survival signaling were tested by ELISA and immunoblotting. Co-treatment assays with istiratumab and chemotherapy investigated mechanisms of synergy and additivity. Anti-tumor activity of istiratumab, alone and in combination with chemotherapy, was tested in in vivo ovarian xenograft tumor models.

Our results indicated that istiratumab monotherapy inhibits ovarian cancer cell line proliferation in vitro. In addition, istiratumab blocked ligand-mediated resistance to chemotherapy. Co-treatment of istiratumab, ligands or chemotherapy indicated a strong correlation between drug activity and IGF-1R expression. Furthermore, co-treatment of chemotherapies and ligands potentiated AKT activation, which was inhibited by istiratumab. In vivo studies showed that istiratumab potentiates the activity of chemotherapy in ovarian xenograft tumor models.

Our findings demonstrate that co-inhibition of IGF-1R and ErbB3 signaling with istiratumab can potentiate standard of care chemotherapies in ovarian tumor models and warrant further investigation of istiratumab as a potential therapy for ovarian cancer patients.

#1210

Preclinical pharmacology and repeated dose toxicity of the novel agonistic TWEAK receptor binding antibody BAY-356.

Sandra Berndt, Christian Votsmeier, Ruprecht Zierz, Jakob Walter, Anna-Lena Frisk, Stefanie Hammer, Heiner Apeler, Bertolt Kreft. _Bayer Pharma AG, Berlin, Germany_.

TWEAK receptor (TWEAKR, FN14) is a member of the tumor necrosis factor receptor superfamiliy and is highly expressed in a variety of human solid tumor types, and its overexpression is associated with poor prognosis and metastasis. To explore targeting of TWEAKR for cancer therapy we have generated the novel, anti-TWEAKR antibody BAY-356. Its potent agonistic activity leads to TWEAKR hyperactivation and subsequent induction of cell death in vitro and tumor growth inhibition in vivo.

BAY-356 is a fully human aglycosylated antibody (Kd ~ 10nM) that binds to a novel epitope within the TWEAKR ectodomain of various species as determined by BiaCore. In vitro, BAY-356 showed strong agonistic activity on TWEAKR-positive tumor cells, including activation of NFκB- and STAT1 pathways, increase of TWEAKR protein expression, increased IL-8 secretion, caspase 3/7 activation, and proliferation inhibition in a dose-dependent manner. BAY-356 inhibited tumor growth in several TWEAKR-positive tumor models (NCI-H1975, WiDr, ScaBER, and HN10321) with growth inhibition rates of 49-71% when treated with 3-10 mg/kg BAY-356 twice weekly for up to 3 weeks. The activity of BAY-356 was independent of ADCC activation. In a preventative syngeneic CT26-tumor model in Balb/c mice, BAY-356 induced complete responses. Anti-tumor activity of BAY-356 was associated with high tumor levels of TNF alpha protein.

To investigate the toxicity of BAY-356, a repeated dose-toxicity study was performed in Cynomolgus monkeys. Animals were dosed with 10, 20, and 40 mg/kg by weekly intravenous injection for 4 weeks. Compound-related clinical findings consisted of an increase of the serum markers amylase and lipase from 10 mg/kg onwards, urea and creatinine from 20 mg/kg onwards and the transaminases ALT and GDPH at 40 mg/kg. Histopathological evaluation revealed focal ductular epithelial hyperplasia with periductular fibrosis in the exocrine pancreas (at 10 & 20 mg/kg), renal tubular hyperplasia and degeneration, Bowman capsule hyperplasia, and glomerulosclerosis in the kidney starting at 10 mg/kg and bile duct hyperplasia in liver at 20 mg/kg and higher. The HNSTD was set as the highest tested dose of 40 mg/kg. Immunohistochemical analysis of TWEAKR expression in these organs demonstrated a dose dependent induction and increase when compared to untreated controls which correlated with the histopathological findings.

From these data it can be concluded that hyperactivation of TWEAKR signaling by BAY-356 leading to strong anti-tumor efficacy in various mouse models is invariably accompanied by target-mediated side-effects originating from enhanced TWEAKR induction in in particular in kidneys, pancreas, and liver of sensitive species such as Cynomolgus monkeys.

#1211

Killing cancer one cell at a time: Development and characterization of a novel antibody drug conjugate.

Michael Xiao,1 Roger Chu,1 Jayson Pagaduan,2 Richard A. Robison,1 Kim L. O'Neill1. 1 _Brigham Young University, Provo, UT;_ 2 _Johns Hopkins University, Baltimore, MD_.

Thymidine Kinase 1 (TK1) is a protein that we have established to be present on the surface of cancer cells and absent on the surface of normal cells. Various methods have been used to confirm the presence of TK1 using localization methods such as scanning electron microscopy gold labeling, fluorescence imaging, and flow cytometry analysis. In this project, we explored the potential of using the TK1 marker as a therapeutic target by conjugating an anti-TK1 antibody to the toxin saporin, a ribosome inhibiting protein derived from the seeds of Saponaria officinalis. The toxin is widely used in the biological and neurological fields due to its stability and adaptability. An additional benefit to using this particular toxin is the inability of saporin to transverse the cell membrane without an antibody or other agent mediating internalization into the cell. We expressed a cysteine mutated form of saporin in the BL21(DE3) E. coli strain. Saporin from the expressed protein was subsequently isolated with a cation exchange cellulose column before verification on a non-reducing SDS-PAGE gel. A single band was observed at approximately 30 kD, indicating purity of the protein. After thiolation of the amine groups in the anti-TK1 antibody, the cysteine residues on the mutated saporin toxin was linked to the antibody through the formation of disulfide bonds. Further purification of the conjugate was achieved by eliminating unconjugated saporin and antibody, and high molecular weight aggregates using a Sephacryl S-200 column. Over 90% cancer cell death was observed in the Burkitt's Lymphoma Raji cell line when exposed to conjugate concentrations of 10 nM. Complete cell death was observed with exposure to 100 nM concentrations. In addition, there was a significant difference between the saporin conjugated treated and untreated samples (p < 0.001). Further tests are underway to determine the effectiveness of the conjugate in other cancer cell lines. These promising results suggest the use of saporin in an effective antibody drug conjugate therapeutic that could influence the way we treat cancer: a drug that will selectively target cancer cells.

#1212

Effect of metformin and insulin-/IGF1-receptor inhibitor combination drug therapy in triple negative breast cancer.

Lei Xue, Laura Camacho, Sushma Kothapalli, Sao Jiralerspong. _Baylor College of Medicine, Houston, TX_.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor survival outcomes. Chemotherapy is the only approved treatment, as there are no targeted therapies. Metformin is an anti-diabetic drug for Type 2 Diabetes. It has been shown to have anti-tumor effects by lowering serum levels of the mitogen insulin, and by pleiotropic effects on cancer cell signaling pathways. BMS-754807 is a potent and reversible inhibitor of the insulin receptor (IR)/insulin-like growth factor 1 receptor (IGF-1R) family kinases that has been shown to have activity in a subset of TNBC. The aim of this study is to evaluate the hypothesis that the combination of metformin and BMS-754807 is more effective than single drug treatment in TNBC.

Experimental design and methods: We treated TNBC cell lines with metformin and BMS-754807 alone and in combination and tested cell viability using MTS assays. Then we used CompuSyn software to analyze the dose-effect of drug combinations for additivity, synergism, or antagonism. We also examined the mechanism by performing Western blots of candidate pathways.

Results: 5 out of the 9 cell lines tested (56%) showed synergism of metformin and BMS-754807 in the drug combination assays. Western blots showed significant inhibition of IR/IGF-1R and PI3K/Akt pathways with the drug combination.

Conclusion: We conclude that the combination of metformin and BMS-754807 is more effective than single drug in a significant proportion of triple negative breast cancer cell lines. This combination may represent effective targeted therapy for a subset of TNBC. Further studies to evaluate this possibility are underway.

#1213

Thymidine kinase 1 is on the cell surface of prostate cancer cells.

Evita G. Weagel, Roger P. Chu, Wei Meng, Rachel A. Brog, Michelle H. Townsend, Richard A. Robison, Kim L. O'Neill. _Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT_.

Thymidine Kinase 1 (TK1) is a salvage pathway enzyme that assists in DNA repair and is characteristically expressed in the cytosol, but it also presents at an abnormally high level in the serum of cancer patients. We developed a unique mouse-anti-human TK1 antibody (A72), and used it in conjunction with Flow Cytometry, Scanning Electron Microscopy (SEM), and Confocal Microscopy, to explore the presence of TK1 on the cell surface of two prostate cancer cell lines, namely PC3 and DU145. Flow Cytometry was performed using a standard protocol. Briefly, cells were stained with A72 conjugated to FITC. Anti-NF-ĸB-FITC and a non-specific IgG-FITC were used as negative controls. Anti-Na+/K+-ATPase-FITC was used as positive control. Cells were then analyzed using a Flow Cytometer. SEM was carried out by first incubating cells with anti-Na+/K+-ATPase and anti-TK1 conjugated to biotin. After probing with streptavidin-gold solution, cells were imaged to examine the presence of gold particles on cell surface. Our data demonstrated TK1-positive populations after flow cytometry analysis, and the presence of gold particles on the membrane of prostate cancer cells. The presence of TK1 on plasma membrane of PC3 and DU145 was further confirmed by Confocal Microscopy. Cells were first stained with A72-FITC and a red plasma membrane stain, then consecutively imaged with a Confocal Microscope. Results showed that A72-FITC associate with the cells in the same region that the membrane had been stained red, concluding that the A72 binds to the cell membrane. These data strongly suggest that TK1 is located on the surface of prostate cancer cells, and indicate that TK1 may be used as a new immunotherapy target for prostate cancer treatment.

#1214

Development of anti-5T4 antibody-drug conjugates, ZV05-ADCs for targeted cancer therapy in different type of cancers.

Zhaohui Li,1 Zhangming Xie,1 Qian Zhang,1 Jie Fang,1 Liqin Wu,1 Hedi Fang,1 Hong Zhang,2 Tong Zhu,2 Gang Chen,2 David Miao,2 Sheldon Cao1. 1 _Zova Biotherapeutics, Inc., Hangzhou, China;_ 2 _Concortis Biosystem, San Diego, CA_.

5T4 is an N-glycosylated transmembrane 72 kDa glycoprotein expressed in a number of carcinomas, and has been explored as a target for cancer therapy in different type of cancers, including NSCLC, RCC, colorectal, ovarian, pancreatic and gastric cancers. The expression of 5T4 in normal tissue is very limited, but widespread in malignant tumors throughout their development, making it an attractive target for antibody-drug conjugate (ADC). In this aspect, a 5T4-MMAF ADC from Pfizer is under clinical investigation in phase I/II trials.

We have developed novel anti-5T4 ADCs, using our proprietary conjugation technologies. A highly potent anti-5T4 antibody ZV05 was conjugated to a variety of internally developed linkers and toxins and purified to populations containing defined DAR (Drug-Antibody Ratio) numbers. The ADCs were studied both in vitro and in vivo for their activity against a variety of tumors. The results showed that the lead ZV05 ADCs were potent towards a bunch of cancer cell lines that express 5T4, and also dramatically inhibited tumor growth in several models in vivo. In the xenograft studies, a single dose of the lead ZV05 ADCs completely wiped out the tumors and kept the tumor volume down for the whole period of studies. ZV05 ADCs are currently under further evaluation to select a candidate for clinical treatment of 5T4-positive cancers.

#1215

Utilization of podoplanin as a chemotherapeutic target for oral squamous cell carcinoma.

Edward P. Retzbach,1 Harini Krishnan,1 Jhon A. Ochoa-Alvarez,1 Yongquan Shen,1 Evan Nevel,1 David J. Kephart,1 Evelyne Kalyoussef,2 Soly Baredes,2 Mahnaz Fatahzadeh,3 Maria I. Rameriz,4 Kingsley Yin,1 Mary Ann Young,1 Lisa Deluca-Rapone,1 Alan J. Shienbaum,1 Lasse D. Jensen,5 Gary S. Goldberg1. 1 _Rowan University School of Medicine, Stratford, NJ;_ 2 _Rutgers New Jersey Medical School, Newark, NJ;_ 3 _Rutgers School for Dental Medcine, Newark, NJ;_ 4 _Boston Univ, Dept Med, Sch Med, Ctr Pulm, Boston, MA;_ 5 _Linköping University, Linköping, Sweden_.

Oral cancer is diagnosed in over 300 thousand people, and kills over 100 thousand people, around the world each year. Current treatments rely on radiation and surgery procedures that often decrease the quality of life for oral cancer survivors. There is a clear need to improve treatments for these patients. Over 90% of oral cancers are formed by oral squamous cell carcinoma (OSCC). Most OSCC cells express the transmembrane receptor podoplanin (PDPN), which has emerged as a promising target for OSCC treatment. The PDPN receptor promotes tumor cell invasion and metastasis which leads to the vast majority of cancer deaths. Here, we describe efforts to target PDPN intracellularly and extracellularly in order to prevent and treat oral cancer. PDPN contains intracellular serine residues that can be phosphorylated by protein kinases including CDK5 and PKA to decrease cell migration. Additionally, the extracellular portion of PDPN can be targeted with Maackia amurensis seed lectin (MASL) to inhibit tumor cell migration and viability. Previous studies suggest that PDPN induces RhoA GTPase activity to promote cell migration. However, we show here that MASL dynamically binds PDPN to downregulate Cdc42 GTPase activity, but not RhoA or Rac1 GTPase activity. These data suggest that MASL suppresses Cdc42 GTPase activity to disrupt cell polarity and inhibit cell migration. Taken together, these data indicate that PDPN can serve as a functionally relevant target to prevent and combat oral cancer.

#1216

Assays for the selection and functional characterization of antibody-drug conjugates at the National Research Council of Canada.

Maria L. Jaramillo, Luc Meury, Normand Jolicoeur, Myriam Banville, Limei Tao, Maureen O'Connor McCourt. _National Research Council of Canada, Montréal, Quebec, Canada_.

One of the most promising and fastest growing classes of cancer therapeutics builds on the molecular targeting abilities of antibodies by combining them with drugs to generate highly specific antibody-drug conjugates (ADCs). However, the development of ADCs requires time-consuming selection of the antibody for every target and cancer type. Screening technologies based on the use of conjugated secondary antibodies provide a fast and efficient surrogate assay from which to identify which antibodies are best internalized and suitable for immunoconjugate development into ADCs.

As part of its integrated antibody development initiative, NRC has isolated and characterized anti- mouse Fc and anti-human Fc monoclonal antibodies to serve as very selective detective reagents for various IgG isoforms . We have shown that these secondary antibodies are species specific, selective and of high affinity . Furthermore, they exhibit high specific potency and low background toxicity after conjugating them to drugs (DM1, MMAE) or immunotoxins(saporin) in cell viability studies. By combining this methodology with our proprietary mRNA and DNA expression database for the selection of appropriate cell lines, we plan on screening thousands of NRC antibodies generated against variety of cancer associated cell surface targets for ADC development.

In addition to secondary conjugate cytotoxicity assays, we have developed a suite of assays based on cellular accumulation, endosomal routing and activation (intracellular drug release) of antibody drug conjugates. These assays can all contribute to our mechanistic understanding of ADCs under development. Combined with our strength in biologic production and characterization, this expertise promotes the integration and advancement of NRC's capabilities and strengths in the area of Antibody-Drug Conjugates (ADCs), and can be used to establish strategic collaborations with other Canadian or international partners to develop external or internal NRC antibodies into novel ADC biologics.

#1217

Preclinical evaluation of a next-generation, EGFR targeting ADC that promotes regression in KRAS or BRAF mutant tumors.

Lei Huang,1 Bob Veneziale,1 Mark Frigerio,2 George Badescu,2 Xiaoming Li,1 Qiping Zhao,1 Jesse Bahn,1 Jennifer Souratha,1 Ryan Osgood,1 Chunmei Zhao,1 Kim Phan,1 Jessica Cowell,1 Sanna Rosengren,1 Jason Parise,1 Martin Pabst,2 Mathew Bird,2 William McDowell,2 Gina Wei,1 Curtis Thompson,1 Antony Godwin,2 Michael Shepard,1 Christopher Thanos1. 1 _Halozyme Therapeutics, San Diego, CA;_ 2 _Abzena, Antitope Limited & PolyTherics Limited, Cambridge, United Kingdom_.

Cancers with downstream activating KRAS or BRAF mutations in the EGFR pathway are resistant to EGFR targeting agents such as cetuximab and correspond to a significant unmet need. We hypothesized that an anti-EGFR ADC could be effective against KRAS or BRAF mutated tumors due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, a mAb was engineered with increased tumor microenvironment (TME) specificity for EGFR. The lead mAb demonstrated undetectable in vivo binding to human donor foreskins grafted onto nude mice, while binding to human A431 tumor xenografts with similar intensity to cetuximab (P < 0.005, detected using DyLight-755 conjugated versions of each mAb, measured with a Caliper IVIS system). The lead mAb was further optimized and conjugated to the potent cytotoxic drug MMAE using a novel bis-alkylating conjugation linker, which covalently re-bridged the inter-chain disulfide bonds, creating a stable and defined ADC. The resulting ADC, HTI-1511, incorporated a vc-PAB cleavable moiety and a short linear PEG (24 ethylene glycol units) in a side-chain configuration. Analytical HIC revealed that HTI-1511 possessed a nearly homogenous drug:antibody ratio (DAR) of 4 (>99.7%). Approximately 70% of this compound was rapidly internalized by human tumor cells grown in vitro over 4 hours, overlapping the internalization kinetics of the unconjugated mAb. HTI-1511 was evaluated for efficacy against two human EGFR overexpressing tumor models, MDA-MB-231M (triple-negative breast cancer, KRAS-G13D) and HT-29 (colorectal cancer, BRAF-V600E), and dosed at 5, 10, and 15 mg/kg, (qw, IV). A clear dose dependent anti-tumor response was observed with complete tumor regressions observed at the 15 mg/kg dose in both models, which were resistant to treatment by cetuximab. In addition, HTI-1511 was well-tolerated at 2 and 8 mg/kg in a cynomolgus monkey toxicity study (n=3 per group), with limited dermal findings that were comparable with the vehicle control group. No adverse findings were observed at either dose. HTI-1511 showed a high degree of circulating stability in cynomolgus monkeys, and lacked in vivo degradation and instability that was observed in a control ADC conjugated using maleimide chemistry. HTI-1511 demonstrated significantly attenuated binding to FcγRIIa, FcγIIb, FcγIIIa 158V, and FcγIIIa 158F receptors, but not attenuated binding to FcγR1, in a FACS based assay format specific for each receptor, suggesting that HTI-1511 might have improved tolerability due to lack of binding by FcγRII-III receptors, possibly due steric hindrance from the PEG side chain. Thus, HTI-1511 holds promise as a potentially safe and effective treatment of EGFR overexpressing tumors with KRAS or BRAF mutations.

#1218

A novel synergistic antibody pair targeting non-overlapping epitopes of MET effectively inhibits MET-driven cancer models.

Michael M. Grandal, Thomas T. Poulsen, Klaus Koefoed, Karsten W. Eriksen, Anna Dahlman, Paolo Conrotto, Thomas Bouquin, Helle Jacobsen, Ivan D. Horak, Michael Kragh, Johan Lantto, Mikkel W. Pedersen. _Symphogen A/S, Ballerup, Denmark_.

The receptor tyrosine kinase MET (Hepatocyte Growth Factor Receptor, HGFR) has been associated with development and progression of a range of human tumors due to its regulation of cell proliferation, migration, invasion, and angiogenesis. A subset of human tumors, particularly of lung or gastric origin, appear to have a primary dependency on MET, which is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents.

Sym015 is a mixture of two humanized monoclonal antibodies against non-overlapping epitopes on MET. The specific pair of antibodies was identified in a high-throughput cell based screen searching for antibody mixtures with superior growth inhibitory activity against four MET-dependent cell lines. Both antibodies bind the SEMA domain of MET with high affinity. Synergistic activity was confirmed by dose-response curves in several MET-dependent cell lines and cell line and patient-derived xenograft models. Mechanistically, Sym015 blocks HGF binding to MET, induces MET internalization and degradation effectively diminishing MET oncogenic signaling. Sym015 also induces higher levels of antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro compared to individual mAbs.

In conclusion, Sym015, a novel monoclonal antibody mixture against MET, shows strong inhibitory activity against MET driven cell lines and xenografts due to a combined effect of MET degradation and secondary effector functions. These data support initiation of clinical trials.

#1219

Sym015, a novel antibody mixture targeting non-overlapping epitopes of MET, effectively inhibits growth of MET dependent tumors and overcomes resistance to a single monoclonal antibody.

Thomas T. Poulsen, Michael M. Grandal, Helle J. Jacobsen, Dorte S. Hansen, Trine Lindsted, Mikkel W. Pedersen, Ivan D. Horak, Michael Kragh, Johan Lantto. _Symphogen A/S, Ballerup, Denmark_.

The tyrosine kinase receptor MET is involved in progression of a variety of human cancers and constitutes a promising therapeutic target. Particularly, subsets of tumors originating from lung or gastric tissues appear to be truly MET dependent. MET dependency is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, kinase activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents in lung and colorectal cancers.

Sym015, a novel antibody mixture comprising two monoclonal antibodies targeting non-overlapping epitopes on the SEMA domain of MET, was shown to effectively inhibit cell growth in vitro through effective MET degradation. In the present study, we screened a large panel of highly annotated human cancer cell lines for sensitivity to Sym015 in order to identify potential markers of response. Sym015 effectively inhibited growth of cell lines with MET-amplification, MET-exon 14 deletion, and autocrine HGF production, including MET-amplified cell lines with acquired resistance to EGFR targeting agents.

To validate the in vitro findings, a range of cell line- and patient-derived xenograft models with MET amplification or Exon 14 deletion were tested for sensitivity to Sym015 and an analogue of the clinical stage anti-MET monoclonal antibody emibetuzumab (LY2875358). Sym015 effectively inhibited growth of tumors with autocrine HGF production, MET-amplification, and/or Exon 14 deletion, and had superior activity compared to the emibetuzumab analogue in many of the models. Importantly, tumors with a partial response to the emibetuzumab analogue were strongly inhibited by subsequent treatment with Sym015 in two MET-amplified models, one of which also harbors a MET-exon 14 deletion.

In summary, our findings demonstrate a potent antitumor effect of Sym015 in MET-dependent models. The data thus strongly support initiation of clinical trials for patients with MET-amplification and Exon 14 deletions.

#1220

A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions.

Marc Isaac Damelin,1 Alex Bankovich,2 Jeff Bernstein,2 Justin Lucas,1 Liang Chen,1 Sam Williams,2 Albert Park,2 Jorge Aguilar,2 Elana Ernstoff,1 Manoj Charati,1 Russell Dushin,1 Amy Jackson-Fisher,1 Monette Aujay,2 Christina Lee,2 Hanna Ramoth,2 Milly Milton,2 Johannes Hampl,2 Sasha Lazetic,2 Virginia Pulito,1 Douglas Armellino,1 Edward Rosfjord,1 Magali Guffroy,1 Hadi Falahatpisheh,1 Lindsay King,1 Frank Barletta,1 Robert Stull,2 Marybeth Pysz,2 Paul Escarpe,2 David Liu,2 Orit Foord,2 Brenda Gibson,1 Eric Powell,1 Christopher O'Donnell,1 Xiaohua Xin,1 Hans Peter Gerber,1 Puja Sapra,1 Scott Dylla2. 1 _Pfizer, Inc., Pearl River, NY;_ 2 _Stemcentrx, Inc., South San Francisco, CA_.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor regrowth and metastasis. Here we identify Protein Tyrosine Kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase, as an antigen that is enriched on TICs in low-passage patient-derived xenografts (PDX) of TNBC, NSCLC and other tumor types. An anti-PTK7 antibody-drug conjugate (ADC) was generated from a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker and the Aur0101 auristatin microtubule inhibitor. The anti-PTK7 ADC induced sustained regressions of TNBC, NSCLC and ovarian cancer PDX, with improved activity over standard-of-care chemotherapy, and reduced the frequency of TICs as determined by serial transplantation experiments. Moreover, the ADC may have additional mechanisms of action, including an anti-angiogenic effect, that promote anti-tumor immune responses. Together these preclinical results indicate the potential of the anti-PTK7 ADC to improve the long-term survival of cancer patients. The ADC is currently being tested in a Phase 1 clinical trial, from which interim results will be presented.

#1221

Sigma-2 receptor/progesterone receptor membrane component 1 is a potential therapeutic target that modulates expression of HER2 in gastric cancer cell lines.

Balaqis Al Naamani, Maiya Al Bahri, Shadya Al-Sinawi, Ikhlas Ahmed. _Sultan Qaboos University, Al Khod, Oman_.

Background: Gastric cancer (GC) is the third leading cause of death from cancer globally. Sigma-2 Receptor/Progesterone Receptor Membrane Component 1 (S2R/Pgrmc1) is a cytochrome-related protein and it is upregulated in multiple types of cancer. S2R/Pgrmc1 binds to several proteins including epidermal growth factor receptor 1 (EGFR) and regulates the EGFR signaling in lung and breast cancer cells, as we previously showed. Human Epidermal Growth Factor Receptor 2 (HER2), a member of EGFRs family, has been evaluated as a potential therapeutic target for GC due to an identified link between expression of HER2 and severity of GC. Several HER2 inhibitors are currently under investigation in Phase I and II clinical trials. Most of them inhibit ligand binding or kinase activity of HER2. Here, we suggest that HER2 is a novel molecular target of S2R/Pgrmc1, thus, inhibiting S2R/Pgrmc1 can be a potential therapeutic strategy for GC.

Results: We found that the protein expression of S2R/Pgrmc1 is elevated in human gastric cancer tissue array and in multiple gastric cancer cell lines such as AGS, NCI-N87, and Hs 746T. Approximately 60% of GC cells have moderate to strong staining of S2R/Pgrmc1 as compared to weak staining noted in normal gastric cells, suggesting an association of S2R/Pgrmc1 with pathogenesis of GC. We also found that S2R/Pgrmc1 was inhibited by AG205, an inhibitor of PGRMC1, in concentration-dependent manner in NCI-N87 GC cell line. Consistently, AG205 inhibited proliferation of NCI-N87 cells in concentration- and time-dependent manner (p-value = 0.025). HER2 is a prognostic marker in GC and overexpression of protein level of HER2 is associated with tumor growth. We found that HER2 expression was decreased in AG205 treated NCI-N87 in concentration-dependent manner. Furthermore, we showed that AG205 enhanced the sensitivity of Herceptin, an anti-HER2 antibody that inhibits tumor growth. Akt, a downstream target of HER2, is overexpressed in GC, and regulates tumorigenic properties of cancer cells including tumor growth and survival. Here, we demonstrated that concomitant treatment of the GC cells with Herceptin and AG205 significantly decreased Akt protein level as compared to control cells or cell treated with Herceptin alone. Consistently, the viability of the cells was significantly lower in combination treatment group (p-value = 0.009), as compared to groups treated with Herceptin or AG205 alone.

Conclusion: We have demonstrated that S2R/Pgrmc1 promotes proliferation of GC cell line and regulates the expression of HER2. Our results suggest that targeting S2RPgrmc1 could be a potential therapeutic target for gastric cancer. 

### Novel Antitumor Agents and Epigenetics

#1222

Fluorinated N, N-diarylureas treatment as effective strategy to target colorectal cancer stem cells.

Piotr Rychahou,1 Dasha Kenlan,2 Vitaliy M. Sviripa,3 David S. Watt,3 B. Mark Evers1. 1 _University of Kentucky Markey Cancer Center and Department of Surgery, Lexington, KY;_ 2 _University of Kentucky Markey Cancer Center, Lexington, KY;_ 3 _University of Kentucky Department of Molecular and Cellular Biochemistry, Lexington, KY_.

Colorectal (CRC) cancer is the second leading cause of cancer deaths in the US; treatment of metastatic CRCs is limited due to drug resistance and eventual relapse. Cancer stem cells and PI3K mutation (i.e., pik3ca mutation) have been implicated in the relapse and lack of treatment response for many cancers including CRCs; therefore, it is critical to develop a therapeutic strategy that eliminates both the fast-growing cancer cells and the more resistant cancer stem cells. The purpose of the present study was to evaluate the anticancer activity of recently-described, novel AMPK activators, fluorinated N, N-diarylureas (FNDs), against CRC metastatic cell lines, pik3ca mutant cell lines, and CRC stem cells. METHODS. i) First, to assess AMPK activation, metastatic CRC cell lines (HT29 and KM20 cells), HCT116 pik3ca wild-type (WT) and mutant (MUT) cell lines, and CRC stem cell lines (from Celprogen) were treated with varying concentrations of 8 FNDs (4a, 4b, 4h, 4j, 4k, 4z, 4aa, 4bb). AMPK activation and activation of the upstream regulator, LKB1, was assessed by western blot. ii) Next, we determined the effect of the FNDs and, for comparison, metformin (an AMPK activator) on induction of cell cycle suppression and induction of apoptosis as assessed by cyclin D1 expression and PARP cleavage, respectively; β-actin antibody was used as a loading control. RESULTS. i) Treatment with the FNDs resulted in AMPK activation in HT29 and KM20 cells and CRC stem cells at concentrations as low as 10 µM with a peak activation at 12 h. We observed LKB1-independent AMPK kinase activation. Decreased LKB1 phosphorylation after FND treatment was dose dependent (i.e., decreased by ~50% at 10 µM and undetectable at 50 µM). ii) All 8 FNDs (at a 25 µM dosage) completely suppressed cyclin D1 expression in HT29 and KM20 cells; a similar cyclin D1 suppression was noted with metformin at a 500x higher dosage (i.e., 10mM). Five of the 8 FNDs (4b, 4j, 4z, 4aa, 4bb) increased PARP cleavage in HT29 and KM20 cells. We used these 5 FNDs (at a dosage of 50 µM each) to treat the CRC stem cell lines; cyclin D1 suppression was noted for all 5 FNDs but strong induction of apoptosis was only identified using the 4b FND. Finally, we treated HCT116 pik3ca WT and MUT cell lines with 4b FND and found that cyclin D1 expression was completely suppressed at 40 µM and induction of apoptosis was noted at a dosage of 20 µM. CONCLUSIONS. We demonstrate cell cycle suppression and apoptosis in CRC cells and stem cells using the recently-developed FND compounds with the 4b compound as the most effective. These compounds appear to have considerable promise as a targeted cancer stem cell-specific agents and offer a potentially novel strategy for the treatment of CRC metastases.

#1223

NMS-P293, a novel potent and selective PARP-1 inhibitor with high antitumor efficacy and tolerability.

Alessia Montagnoli, Sonia Rainoldi, Antonella Ciavolella, Dario Ballinari, Francesco Caprera, Lucio Ceriani, Rosita Lupi, Marina Ciomei, Eduard Felder, Antonella Isacchi, Daniele Donati, Arturo Galvani, Gianluca Papeo. _Nerviano Medical Sciences, Nerviano, Italy_.

Poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology. Inhibition of PARP-1 is synthetically lethal with loss of function of the BRCA1 and BRCA2 tumor suppressor genes, as well with additional DNA repair defects. Tumor cells harboring defects in DNA repair pathways can thus be selectively targeted with PARP-1 inhibitors, and several such compounds are at different stages of clinical investigation in diverse tumor types, either as single agents or in combination regimens. However none of these agents selectively inhibits PARP-1 within the PARP family of enzymes: all drugs currently in clinical development, as well as the vast majority of preclinical compounds described to date potently cross-inhibit PARP-2, due to the high sequence homology between the two enzymes. Although PARP-2 is reported to be involved in DNA single-strand break repair, its contribution to total cellular levels of DNA damage-induced PARP activity is minimal (5-10%). Gene ablation studies show that loss of both PARP-1 and -2 function is incompatible with normal embryonic development, while PARP-2 single knockout mice show a variety of defects, including impaired erythropoiesis, thymopoiesis adipogenesis and spermatogenesis, increased neuronal loss after ischemic damage and higher risk of pancreatitis following chemical insult. We therefore reasoned that a potent and highly selective PARP-1 inhibitor might represent a significant advancement over currently available agents targeting both PARP-1 and -2, since sparing of PARP-2 inhibition potentially limits on target side-effects and offers greater opportunity for combination with other chemotherapeutic agents.

Here we describe the preclinical characterization of NMS-P293, a novel highly potent PARP-1 inhibitor possessing >200-fold selectivity versus PARP-2. In cells, NMS-P293 inhibits hydrogen peroxide induced poly ADP-ribose (PAR) synthesis with an IC50 in the single digit nanomolar range, confirming expected mechanism of action in cells.

NMS-P293 is selectively active on tumor cell lines defective in the HR repair pathway, such as pTEN and BRCA mutated lines, while sparing DNA repair proficient cells and normal myelocytes. NMS-P293 possesses favorable ADME properties including a low efflux ratio, high cross-species metabolic stability, low clearance and nearly complete oral bioavailability in rodents and non rodents. Oral administration to mice bearing BRCA mutated breast cancer xenografts resulted in complete tumor regressions and cures.

The highly favorable preclinical characteristics of NMS-P293 make this compound a promising candidate for further development.

#1224

Insights into the mechanism of action of NVP-HDM201, a differentiated and versatile Next-Generation small-molecule inhibitor of Mdm2, under evaluation in phase I clinical trials.

Stéphane Ferretti,1 Ramona Rebmann,1 Marjorie Berger,1 Francesca Santacroce,1 Geneviève Albrecht,1 Kerstin Pollehn,1 Dario Sterker,1 Markus Wartmann,1 Andreas Hueber,1 Marion Wiesmann,1 Michael R. Jensen,1 Francesco Hofmann,1 William R. Sellers,2 Philipp Holzer,1 Sébastien Jeay1. 1 _Novartis Institutes for Biomedical Research, Basel, Switzerland;_ 2 _Novartis Institutes for Biomedical Research, Cambridge, MA_.

Activation of p53 by blocking p53-Mdm2 interaction using small-molecule inhibitors is being pursued as a promising cancer therapeutic strategy in p53 wild-type tumors. Here, we report the identification of NVP-HDM201, a novel, highly potent and selective inhibitor of the p53-Mdm2 interaction, with optimized drug-like properties allowing a versatile use with regard to route of administration, dose and scheduling. We determined the pharmacokinetics, pharmacodynamics and efficacy relationship of NVP-HDM201 with various dosing schedules in xenograft bearing mouse and rat models. NVP-HDM201 administered either daily at a low dose or once at a high dose revealed a differentiated engagement of the p53 molecular response. In contrast to the daily low dose treatment regimen, the single high dose NVP-HDM201 regimen resulted in a rapid and dramatic induction of p53-dependent PUMA expression and apoptosis. This was consistent with the finding that a single high dose NVP-HDM201 treatment, administered orally or intravenously, resulted in a robust and sustained tumor regression. Overall, both daily and once every 3 weeks dosing regimen showed comparable long term efficacy in preclinical studies. The ongoing clinical trial is currently designed to compare both dosing regimens with regard to efficacy and tolerability.

#1225

NVP-HDM201: cellular and in vivo profile of a novel highly potent and selective PPI inhibitor of p53-Mdm2.

Sébastien Jeay,1 Patrick Chène,1 Stéphane Ferretti,1 Pascal Furet,1 Bjoern Gruenenfelder,1 Vito Guagnano,1 Nelson Guerreiro,1 Ensar Halilovic,2 Francesco Hofmann,1 Joerg Kallen,1 Michelle Léonard,1 Robert Mah,1 Keiichi Masuya,1 Rita Ramos,1 Caroline Rynn,1 Stephan Ruetz,1 Thérèse Stachyra-Valat,1 Stefan Stutz,1 Andrea Vaupel,1 Jens Wuerthner,1 Philipp Holzer1. 1 _Novartis Institutes for BioMedical Research, Basel, Switzerland;_ 2 _Novartis Institutes for BioMedical Research, Cambridge, MA_.

Stabilization of p53 protein by preventing its interaction with the negative regulator Mdm2 leads to selective induction of the p53 pathway, thus offering a promising cancer therapeutic strategy in p53 wild-type tumors. In the present study, we show the identification of NVP-HDM201, a novel, highly optimized, and selective inhibitor of the p53-Mdm2 interaction. NVP-HDM201 activates p53 in human cells and induces robust p53-dependent cell cycle arrest and apoptosis, selectively in p53 wild-type tumor cells. Its activity and selectivity has been tested and confirmed across a large panel of cancer cell lines from the Cancer Cell Line Encyclopedia. In vivo, NVP-HDM201 shows a dose-proportional pharmacokinetic (PK) profile and a clear PK/PD relationship, resulting in tumor growth inhibition and regression in SJSA-1 tumor-bearing rats at well-tolerated oral (p.o.) doses. The validation and understanding of its mechanism of action, the overall favorable drug-like properties and the characterization of its on-target toxicological profile in preclinical species strongly supported the initiation of Phase I clinical trials with NVP-HDM201 in pre-selected patients with p53 wild-type tumors.

#1226

Highly specific ATR inhibitors as a therapeutic approach for a broad spectrum of cancers.

Laura R. Butler,1 Ryan L. Ragland,2 Hank J. Breslin,1 Tina Gill,1 Erin George,2 Fiona Simpkins,2 Eric J. Brown,2 Oren Gilad1. 1 _Atrin Pharmaceuticals, Doylestown, PA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments.

Atrin Pharmaceuticals has synthesized a novel series of small molecules that inhibit ATR at low nanomolar concentrations in cultured cells. These compounds have the highest known potency for inhibiting ATR and maintain >800-fold lower cellular activity towards other kinases of the same family (ATM, DNA-PKcs and mTOR), which are substantially off-targeted by previously reported ATR inhibitors. Atrin's lead compound (ATRN-119), as a single agent, selectively kills cancer cells subjected to oncogenic stress, alternative lengthening of telomeres (ALT) or loss of double strand break (DSB) homologous recombination repair mechanism (BRCA1 or BRCA2 deficiency). Furthermore, ATRN-119 cytotoxicity is synergistically enhanced when combined with conventional chemotherapeutics, such as etoposide, cisplatin and olaparib. Therefore, this inhibitor can be used in combination with other therapeutics to potentiate its anti-tumor activity.

In vivo studies of RAS oncogene driven HCT116 p53-null flank tumors in mice demonstrate the ability of our lead compound to slow tumor progression, with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived ovarian tumors show a significant reduction in progression after 5 weeks treatment (100 mg/kg BID) of ATRN-119, and display no toxicity or significant weight loss. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian cancer, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies.

#1227

New small molecules targeting MYC:MAX interactions that inhibits tumor cell growth in a MYC-dependent manner.

Alina Castell, Karin Ridderstråle, Qinzi Yan, Fan Zhang, Per Hydbring, Marcela Franco, Jacob Goodwin, Inga Müller, Siti Mariam Zakaria, Lars Johansson, Lars-Gunnar Larsson. _Karolinska Institutet, Stockholm, Sweden_.

Deregulated expression of MYC family oncogenes MYC, MYCN and MYCL (here collectively referred to as MYC) occurs in many types of human tumors, and is often associated with aggressive tumor development and poor prognosis. In mouse tumor models, inactivation of MYC often leads to tumor regression with well-tolerated side effects, suggesting that MYC is a potential and suitable target for anti-cancer therapy. At present there are no drugs targeting MYC in the clinic, and transcription factors like MYC are considered difficult to target. However, MYC function is strictly dependent on interaction with cofactors, and targeting such interaction may be a plausible way to combat Myc. We have used cell-based screens utilizing Bimolecular Fluorescence Complementation (BiFC) and split Gaussia luciferase (G-Luc) to identify small molecule inhibitors of the interaction between MYC and its essential partner MAX. Several potent MYC:MAX interaction inhibitors have been identified in these screens and have been validated by other protein-protein interactions (PPI) assays such as in situ Proximity Ligation Assay (isPLA), Fluorescence Resonance Energy Transfer (FRET), Surface Plasmon Resonance (SPR) and coimmunoprecipitations. In general the identified molecules fall into two categories: those that inhibit MYC:MAX interactions not only in cells but also in vitro using purified MYC and MAX, and those that inhibit the interaction in cells but not in vitro, suggesting an indirect mode of action. Molecules of both categories inhibit cell growth and viability of a variety of tumor cells in culture with high efficacy in a MYC-dependent manner. So far, one of the molecules have been utilized for cancer treatment in mouse tumor models and found to significantly inhibit tumor growth and improve survival. These molecules have the potential to become important tools in the studies of MYC function in cells and in vivo as well as potentially basis for drug development for treatment of MYC-driven tumors. We have thus provided proof of principle that our screening systems are able to identify potent PPI inhibitors and we are at present setting up similar systems to screen for inhibitors of other MYC:cofactor interactions of relevance for specific tumor types.

#1228

Indolo-pyrido-isoquinolin based alkaloid inhibits epithelial-mesenchymal transition and stemness via activation of p53-miR34a axis.

Arumugam Nagalingam,1 Dimiter. B Avtanski,1 Joesph Tomaszewski,2 Risbood Prabhakar,3 Michael Difillippantonio,4 Brian Mears,1 Neeraj Saxena,5 Sanjay Malhotra,6 Sanjay Malhotra,6 Dipali Sharma1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _National Institutes of Health, Baltimore, WA;_ 3 _National Institutes of Health, MD;_ 4 _National Institutes of Health, WA;_ 5 _University of maryland, Baltimore, MD;_ 6 _National Institutes of Health, Bethesda, WA_.

The tumor suppressor p53 plays a critical role in suppressing cancer growth and progression and is the most frequently mutated and functionally inactivated gene in all human malignancies. Owing to its widespread alteration/inactivation in cancer, p53 is an attractive target for the development of new targeted therapies. We synthesized several indolo-pyrido-isoquinolin based alkaloids to restore/activate p53 function and examined their therapeutic efficacy using NCI-60 screening. Here, we provide molecular evidence that one of these compounds, 11-Methoxy-2,3,4,13-tetrahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ylium-bromide (termed P18 or NSC-768219) inhibits growth and clonogenic potential of cancer cells. P18 treatment results in downregulation of mesenchymal markers and concurrent upregulation of epithelial markers as well as inhibition of migration and invasion. Experimental epithelial-mesenchymal-transition (EMT) induced by exposure to TGFβ/TNFα is also completely reversed by P18. Importantly, P18 also inhibits mammosphere-formation along with a reduction in the expression of stemness factors, Oct4, Nanog and Sox2. We show that P18 induces expression, phosphorylation and accumulation of p53 in cancer cells. P18-mediated induction of p53 leads to increased nuclear localization and elevated expression of p53 target genes. Using isogenic cancer cells differing only in p53 status, we show that the alteration of mesenchymal and epithelial genes, inhibition of migration and invasion of cancer cells mediated by P18 is p53-dependent. Furthermore, P18 increases miR-34a expression in p53-dependent manner and inhibition of mammosphere-formation by P18 is further enhanced by miR-34a mimic. Collectively, these data provide evidence that p53-miR-34a activation by P18 may represent a promising therapeutic strategy for the inhibition of growth and progression of cancer.

#1229

The next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibit distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources.

Chia-Ping H. Yang,1 Changwei Wang,2 Iwao Ojima,2 Susan Band Horwitz1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _State University of New York at Stony Brook, Stony Brook, NY_.

Next-generation taxanes are known to be active against taxane-resistant cell lines that overexpress P-glycoprotein (P-gp) and/or βIII-tubulin, both of which are associated with drug resistance. In this study, we examined the effect of two potent next-generation taxanes, SB-T-1214 and SB-CST-10202 (C-seco-taxane), on growth of the ovarian cancer cell line Hey and its drug-resistant daughter cell lines that overexpress different levels of P-gp and express increased levels of βIII-tubulin. We found that SB-T-1214 was ~6-9-fold more potent than SB-CST-10202 against these drug resistant cells. Since the taxanes bind to both tubulin and P-gp, the relative binding affinity of the taxanes to these two cellular targets may influence the effectiveness of the taxanes in resistant cells. To determine the effects of these two taxanes on binding of Taxol® to P-gp and tubulin, a tritium-labeled Taxol® analog, 2-(m-azidobenzoyl)taxol (2-m-AzTax), was used. We have previously demonstrated that 2-m-AzTax photolabels a peptide (amino acids 217-231) in β-tubulin. To study the relative binding affinities of SB-T-1214 and SB-CST-10202, tubulins from bovine brain (BBT) and chicken erythrocytes (CET) were specifically photolabeled with [³H]2-m-AzTax, in the presence and absence of either Taxol®, Taxotere®, SB-T-1214 or SB-CST-10202. β-tubulin isotype content from BBT and CET is different, the former contains βI-, βII-, βIII- and βIV-tubulins, but the latter has only βVI. Taxol® had a minimal inhibitory effect (~3%) on BBT, but a strong inhibitory effect (99%) on CET. Taxotere® appeared to have a stronger inhibitory effect than Taxol® on photolabeling (31% and 99% for BBT and CET, respectively). The inhibitory effects elicited by the two next-generation taxanes on photolabeling were distinct for β-tubulins from these two sources. SB-T-1214 and SB-CST-10202 inhibited BBT photolabeling by 38% and 32%, respectively. Interestingly, these two drugs caused a 41% and only 6% inhibition, respectively, on photolabeling of CET that does not contain βIII-tubulin. We are currently analyzing the effect of SB-T-1214 on binding of [³H]2-m-AzTax to different tubulin isotypes resolved by isoelectrofocusing. To study photolabeling of P-gp, plasma membranes from a multidrug resistant (MDR) cell line SKVLB1 that expresses very high levels of P-gp were prepared. SB-T-1214 and SB-CST-10202 inhibited photolabeling of P-gp markedly (> 80% inhibition), suggesting that these two taxanes are good substrates for P-gp. They also caused an increase in steady state [³H]2-m-AzTax accumulation in the MDR cell line SKVLB1. Since drug binding to tubulin and P-gp is the primary event that occurs in the cell following drug administration, our data on taxane binding affinity to the two targets will help in understanding the effectiveness of the next-generation taxanes in taxane-resistance.

#1230

BGB-283: a novel RAF Dimer inhibitor, displays potent antitumor activity in HCC patient derived xenograft models.

Zhiyu Tang, Yong Liu, Beibei Jiang, Yajuan Gao, Wenfeng Gong, Xing Wang, Dan Su, Fenglong Yu, Ye Liu, Min Wei, Lai Wang. _BeiGene, Beijing, China_.

Liver cancer has the third highest mortality rate among all cancers in China and hepatocellular carcinoma (HCC) is the most common type of liver cancer. The mitogen-activated protein kinase (MAPK) signaling pathway is often constitutively active in HCC. The growth of HCC requires the formation of new blood vessels. and VEGF is critical in this process. Sorafenib, a multikinase inhibitor that targets both RAF and VEGF receptor, is to date the only approved drug to treat advanced stages of HCC. Despite sorafenib extended the survival in patients with HCC, its clinical benefits remain modest and drug resistance is common. BGB283 is a second generation RAF inhibitor with unique RAF dimer and VEGFR inhibition activity. Thus, there is good rationale to test BGB-283 in HCC. In this study, we compared the in vitro and in vivo activities of BGB-283 and sorafinib in human HCC cell lines and patient derived HCC xenograft models.

In the biochemical assays, BGB-283 demonstrated great potency for BRAF-V600E, BRAF-WT, CRAF(IC50 = 32, 69 and 6.5 nM, respectively) and for VEGFR family enzymes, VEGFR1, VEGFR2 and VEGFR3 (IC50 = 25, 14 and 58 nM, respectively). In the cellular assays, the anti-proliferative effect of BGB-283 and sorafinib was evaluated in several HCC cell lines. A head-to-head comparison of in vivo anti-tumor activities of BGB-283 and sorafinib were also evaluated in human primary HCC xenograft mouse models. The patient derived xenograft (PDX) HCC models were established in house using HCC patient surgical samples. Sorafinib was not active in 2 out of 7 models. Oral administration of BGB-283 resulted in significant tumor growth inhibition in all 7 models and was significantly more efficacious than sorafinib in 4 models and similar in the other 3 models.

In conclusion, BGB-283 is a unique RAF dimer inhibitor with VEGFR inhibition activity. BGB-283 has also demonstrated better anti-tumor activity than sorafinib in HCC PDX models, suggesting BGB-283 could be a promising drug candidate for treating HCC patients.

#1231

Novel chromene analogs as small-molecule microtubule destabilizers for the treatment of chemo-resistant ovarian cancer.

Arpita Kulshrestha, Safaa A. Ibrahim, Gajendra K. Katara, Renukadevi Patil, Shivaputra Patil, Kenneth D. Beaman. _Rosalind Franklin University of Medicine and Science, North Chicago, IL_.

Ovarian cancer is the most lethal gynecological malignancy with a 5-year survival rate of less than 40%, primarily because of treatment failure due to emergence of chemo-resistance to standard agents. This indicates a dire need for new treatments to improve survival rates. The tubulin dynamics is a promising target for new chemotherapeutic agents. In continuation of our efforts, using medicinal chemistry approach, we recently identified small molecular inhibitors of tubulin polymerization called chromenes, as new ovarian anti-cancer agents. A set of chromene compounds were screened for anti-cancer effect on cisplatin sensitive (A2780, SKOV-3, TOV112D) and cisplatin resistant (OVCAR-3, A2780-cisR, TOV112D-cisR) ovarian cancer cells. In vitro cell viability was assessed by fluorescence based Alamar Blue assay. Our preliminary studies identified SP-6-27 (IC50 range: 0.102± 0.006 - 2.24±0.056 µM) and SP-6-37 (IC50 range: 0.258± 0.159 - 3.61±0.049 µM) as most potent chromene analogs. Both compounds exhibited a potent anticancer activity towards both cisplatin sensitive (SP-6-27 IC50: 0.14± 0.03 µM; SP-6-37 IC50: 1.35± 0.88 µM) and cisplatin resistant (SP-6-27 IC50: 0.81± 1.2 µM; SP-6-37 IC50: 2.09± 1.7 µM) cell lines. The compounds exhibited least cytotoxicity towards normal ovarian epithelial cells (SP-6-27- IC50: 83.35 ± 9.47 µM; SP-6-37- IC50: 75.77 ± 5.37 µM) . Additionally, the analysis of apoptotic changes as determined by Annexin-V assay revealed an enhanced apoptosis in both cisplatin sensitive and resistant cells upon treatment with compounds SP-6-27 or SP-6-37. Together, the data demonstrates that ovarian cancer cell lines respond sensitively to SP-6-27 and SP-6-37, demonstrating that the chromene scaffold is effective in suppressing ovarian cancer cell growth. The findings indicate that the novel chromene analogs are a new class of chemotherapeutic agents that will offer advantages for the treatment of ovarian cancer.

#1232

A-type proanthocyanidins selectively target acute myeloid leukemia cells in vitro and in vivo.

Laura M. Bystrom,1 Luis Andres Lara-Martinez,1 Bernardo Gomel,1 Burak Isal,1 Hongliang Zong,1 Sabrina Martinez,1 Catherine Neto,2 Stefano Rivella,3 Monica L. Guzman1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _University of Massachusetts-Dartmouth, North Dartmouth, MA;_ 3 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. A-type proanthocyanidins (A-PACs) are a unique class of compounds found in cranberries (Vaccinium macrocarpon Ait.) that we have found to be effective against several leukemia cell lines and primary AML samples in vitro. Moreover, A-PACs possess a unique ether bond and have ortho-hydroxyl phenolic groups that have the potential to bind to iron, alter redox status, and other biological effects.

We found that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), whereas normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction consisting of A-PACs that ranged from 2 to 7 degrees of polymerization were more effective than individual A-PACs. This information led us to investigate the anti-leukemia effects of A-PACs in AML patient-derived xenografts (AML-PDX) and to further investigate the mechanisms associated with these compounds.

AML-PDX mice (n=15), were treated for 2.5 weeks via intraperitoneal injections of A-PACs (25 or 50 mg/kg dose every 3 days) or a vehicle control (PBS every 3 days). Mice were sacrificed and leukemia engraftment was evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, oxidative stress, cytokine response, and survival pathways by gene expression analysis or flow cytometry.

Administration of A-PACs to AML-PDX tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 12.51% (95% CI: 4.9, 20.11; n=5), whereas the mice treated with the 50 mg/kg and 25 mg/kg A-PACs showed a level of engraftment of 5.2% (95% CI: 1.5, 8.9; n=5) and 5.4% (95% CI: 2.3, 8.5; n=5), respectively. These results indicated more than a 50% reduction in engraftment, which was better or equal to the effects we observed in mice treated with high-dose cytarabine, a standard care drug. Moreover, no toxic effects were observed in the mice. It was also found that both cells and mice treated with A-PACs lead to the production of specific subset of cytokines. Global gene expression data showed consistent upregulation of some of these cytokines, and also upregulation of NF-κB and enzymes indicative of oxidative stress.

The results indicate that A-PACs not only target primary AML cells in vitro, but are also effective in vivo by a potentially novel mechanism. Further elucidation of this mechanism may uncover new vulnerabilities of this disease.

#1233

In vitro **and** in vivo **pharmacological study of EB-3D: a novel choline kinase inhibitor for breast cancer treatment.**

Elena Mariotto,1 Roberta Bortolozzi,1 Roberto Ronca,2 Luisa C. López-Cara,3 Benedetta Accordi,1 Valentina Serafin,1 Giuseppe Basso,1 Giampietro Viola1. 1 _University of Padova, Padova, Italy;_ 2 _University of Brescia, Brescia, Italy;_ 3 _University of Granada, Granada, Spain_.

Choline kinase (ChoK) catalyzes the phosphorylation of choline to phosphocholine (PCho) in the first step of the Kennedy pathway for phosphatidylcholine (PtdCho) byosyntesis. PtdCho is the most abundant phospholipid in eukaryotic cell membrane and plays a crucial role for cell division and lipid second messengers production. In mammalian cells, only the ChoKα isoform is fundamental in sustaining PtdCho synthesis, indeed ChoKβ alone cannot compensate this activity.

A large number of studies have shown that ChoKα is overexpressed and hyperactivated in many cancers and that correlates not only with increased cancer cell proliferation but also with malignancy, making it a potential prognostic marker. The cholinic phenotype, characterized by increased total choline-containing compound (tCho) and ChoKα upregulation renders therefore ChoKα an attractive new therapeutic target. Indeed, the feasibility of ChoKα inhibition as antitumoral therapy is being pursued through the development of chemical inhibitors of its enzymatic activity.

For this reason we evaluated both in vitro and in vivo the activity of EB-3D (1,1'-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino) pyridinium) bromide), a new choline kinase inhibitor endowed with high antiproliferative activity in two human breast cancer cell lines, MCF-7 and MDA-MB-231. EB-3D treatment induced a strong decrease in cell proliferation due to G1/G0 arrest of the cell cycle in a concentration-dependent manner in both tested cell lines, whereas only a slight increase in apoptotic cells was observed. In addition EB-3D strongly synergized with drugs commonly used in protocols for breast cancer.

Reverse-phase protein array (RPPA) data revealed the activation of AMPK and the dephosporylation of mTORC1 downstream targets such as 4E-BP1(S65), p70S6K(T389) and RPS6(S235/236), suggesting that ChoKα inhibition may affect protein synthesis.

To further examine the antitumorigenic potential of EB-3D in vivo, a syngeneic orthotopical EO771-C57BL/6 mouse model of breast cancer was used. Preliminary results indicated that the compound significantly reduced the tumor size, with no apparent sign of toxicity.

The evaluation of EB-3D as in vivo anti-metastatic adjuvant is ongoing, since cell migration and cell invasion ability of the highly metastatic MDA-MB-231 cell line was impaired after in vitro treatment.

Taken together our results suggest that EB-3D have a potent cytostatic effect and it could be a promising new anticancer agent, worthy of further development.

#1234

Galeterone-induced degradation of the androgen receptor involves inhibition of deubiquitinating enzymes.

Daniel T. Dransfield, Nivedita Namdev, Douglas B. Jacoby, Karen Ferrante. _Tokai Pharmaceuticals, Boston, MA_.

Galeterone is a highly selective oral small molecule drug candidate that disrupts androgen receptor (AR) signaling through degradation of the AR, is a potent CYP17 lyase inhibitor, and possesses AR antagonist activity. Galeterone-induced AR degradation was observed in models having either full-length AR or known constitutively active truncated forms of the AR receptor that lack the ligand binding domain (LBD), AR-V7 and AR567es, suggesting the LBD of the AR is not required for galeterone-dependent AR degradation. Further, galeterone-induced AR degradation activity is blocked by co-administration of the proteasome inhibitor MG132. Along these lines, galeterone-induced AR degradation can be blocked by selective knock-down of the E3 ligases, Mdm2 and CHIP. We utilized a series of biochemical and cell-based in vitro methodologies to further elucidate and characterize additional signaling molecules participating in the proteasomal-dependent mechanism of galeterone-induced AR degradation. We screened a panel of 22 deubiquitinating enzymes (DUBs) in vitro and demonstrated that galeterone selectively inhibited enzymatic activity of two of the DUBs, USP12 and USP46. Separately, we used surface plasmon resonance to demonstrate a dose-dependent direct binding of galeterone to each of USP12 and USP46, alone or when pre-complexed with UAF1. USP12 is a co-activator of the AR, and selective knock-down of this DUB has been shown to increase AR degradation. USP12/USP46 have also been linked to regulation of phosphatases (PHLPPs) through ubiquitination. PHLPPs dephosphorylate AKT, providing an important regulatory mechanism for controlling the PI3K/AKT pathway. It is known that galeterone induces an increase in pAKT and pMdm2, the latter being a substrate of activated AKT. This suggests that inhibition of USP12/USP46 may regulate pAKT levels through enhanced degradation of PHLPPs via increased ubiquitination. These data support a differentiating mechanism of galeterone from other AR targeting agents whereby inhibition of USP12/USP46 leads to enhanced AR degradation.

#1235

A long pentraxin-3-derived small molecule FGF trap for the treatment of multiple myeloma.

Arianna Giacomini, Roberto Ronca, Marco Presta. _University of Brescia, Brescia, Italy_.

Despite advances in systemic and supportive therapies, multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factor-2 (FGF2) plays a pivotal role in MM acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells and fibroblasts. In keeping with the pivotal role of the FGF system in MM, the recurrent chromosomal translocation t(4;14) identified in MM patients is associated with upregulation of FGF receptor-3 (FGFR3). Agents able to hamper FGF signaling are therefore of interest as a novel approach for the treatment of MM. The soluble pattern recognition receptor long pentraxin-3 (PTX3) owns an unique N-terminal amino acid domain containing the pentapeptide ARPCA able to bind FGFs with high affinity and selectivity and to prevent their binding to FGFRs and their activation. Molecular modeling based on ARPCA structure and small molecule library fishing have allowed the identification of a novel orally active 480 Da FGF trap named NSC12. NSC12 has been shown to hamper tumor growth and vascularization in FGF-driven human lung and prostate tumor models.

Here, the therapeutic potential of NSC12 for MM treatment was tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation. NSC12 blocked the proliferation of all human MM cell lines with an IC50 ≅ 3 µM. Western blot and immunofluorescent analyses showed that NSC12 inhibited the activation of FGFR3 signaling, hampered nuclear translocation of NF-kB and strongly downregulated the anti-apoptotic protein mcl-1, leading to an apoptotic response. In addition, co-treatment with the proteasome inhibitor Bortezomib strongly increased KMS-11 cell death. In vivo, oral delivery of NSC12 significantly blocked the growth of KMS-11 (286.9 ± 22.4 vs 459.8 ± 27.4 mm3, p<0.001) and RPMI8226 (141.5 ± 17.7 vs 245.9 ± 46.4 mm3, p<0.001) xenografts injected subcutaneously in immune-compromised NOD/SCID mice. Analysis of KMS-11 xenografts from NSC12-treated mice showed a significant reduction of CD31-positive tumor vascularization and a strong increase of TUNEL-positive apoptotic cells compared to xenografts from vehicle-treated mice.

Altogether, these data suggest that NSC12 affects MM tumor growth through a direct cytotoxic activity on MM cells and by inhibiting FGF-mediated paracrine/autocrine crosstalk in tumor stroma/microenvironment. Thus, NSC12 may represent a novel "two compartment" inhibitor in MM by hampering FGF-dependent angiogenic/oncogenic signaling. Finally, combinations of NSC12 with conventional therapies, such as Bortezomib, may enhance cytotoxicity and overcome drug resistance, thereby improving MM patient outcome.

#1236

ONC201 sensitivity profiling indicates pronounced sensitivity in lymphoid, prostate, colon and brain tumors.

Rohinton Tarapore,1 Mathew Garnett,2 Ultan McDermott,2 Cyril Benes,3 Joshua Allen1. 1 _Oncoceutics Inc, Philadelphia, PA;_ 2 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom;_ 3 _Massachusetts General Hospital, Boston, MA_.

ONC201 is a first-in-class small molecule inducer of the integrated stress response that is currently in phase II clinical trials in select advanced cancers and exhibits a benign safety profile and promising early clinical results. The efficacy of this novel agent has been demonstrated in a variety of preclinical advanced cancer models in multiple indications with an exceptional safety profile that has translated well to the clinic. To better characterize the differential sensitivity of tumor cells to ONC201 we performed in vitro efficacy assays in the Genomic of Drug Sensitivity in Cancer collection of cell lines (>1,000 cell lines), in addition to profiling in NCI60 panel. Sensitivity profiling was assessed by cell viability assays using dose responses curves at concentrations up to 20uM and at 72 hours post-treatment. Ranking the sensitivity dataset by tumor type revealed that lymphoma, colon, prostate and brain cancers were highly responsive to ONC201. The strongest predictor of sensitivity to ONC201 was tumor type, with lymphoma being the strong predictor. Within lymphomas, diffuse histiocytic lymphoma was the most sensitive subtype. Mutations that are frequent in lymphoma were not associated with ONC201 sensitivity.

Recent mechanistic studies have implicated the ER stress response in the early stage mechanism of ONC201 that triggers its downstream antitumor effects. The mutation-agnostic efficacy that is pronounced in lymphomas, prostate, colon, and brain cancers is in accordance with the recent findings that ONC201 induces the integrated stress response through a novel target to trigger is downstream late apoptotic effects. Cancers of the prostate and brain (glioblastoma multiforme) are among some of the solid tumors that are susceptible to induction of apoptosis via the integrated stress response, as they have relatively high basal activation of this pathway due to ER stress. Confirmatory studies revealed that brain cancer cell lines and ex vivo samples indeed possess pronounced sensitivity with nanomolar GI50s, unlike most other tumor types, which is particularly encouraging given the systemic concentrations observed in the first-in-man study. Together, these studies suggest specific advanced cancer indications, such as non-Hodgkin's lymphoma, multiple myeloma, and glioblastoma as promising lead indications for this novel agent that are being evaluated in phase II clinical trials.

#1237

Anti-cancer activity of a stable, orally active angiotensin-(1-7) analog.

Patricia E. Gallagher,1 Kenneth A. Gruber,2 Michael Callahan,2 E. Ann Tallant1. 1 _Wake Forest University School of Medicine, Winston-Salem, NC;_ 2 _University of Missouri, Columbia, MO_.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, peptide hormone that reduces tumor growth through effects on proliferation, inflammation, angiogenesis, fibrosis and metastasis. Ang-(1-7) mediates biological responses by activating mas, a unique G protein-coupled receptor, thereby providing specific targeted actions when used as a therapeutic agent. Unfortunately, Ang-(1-7) has unfavorable pharmacokinetic properties, resulting in a plasma half-life of about 30 min. In this study, TCAng05, a modified cyclic analog of Ang-(1-7), was compared to the parent compound Ang-(1-7) for efficacy, stability, and oral bioavailability. Ang-(1-7) or TCAng05 was incubated with human A549 lung or MDA-MB-231 breast cancer cells for 6 to 10 days, to assess the effect on tumor cell growth. A single dose of TCAng05 significantly reduced the growth of both MDA-MB-231 and A549 cells, while daily doses of Ang-(1-7) were required due to rapid degradation. Growth inhibition by either compound was blocked by the Ang-(1-7) receptor antagonist D-Ala7-Ang-(1-7) [Dala], showing a receptor-mediated response. Ang-(1-7) was rapidly degraded following incubation in rat plasma with a half-life of about 30 min, while TCAng05 was stable under the same conditions for approximately 50 h. The oral efficacy of TCAng05 to inhibit tumor growth in vivo was assessed by injecting 4T1 cells into the mammary fat pad of BALB/C mice. Once the tumors reached 100 mm3, the mice received daily gavage of TCAng05 at 12 µg/kg/day (low), 60 µg/kg/day (medium) or 300 µg/kg/day (high). Tumors in mice with no treatment continued to grow until sacrifice at day 19, reaching a size of 671.2 ± 127.2 mm3. Oral treatment with TCAng05 at the medium or high dose reduced tumor volume, by 56.8% and 43.6%, respectively, to final tumor volumes of 289.7 ± 64.2 mm3 and 378.3 ± 151.2 mm3. Tumor weight was also reduced by treatment with the medium or high dose of oral TCAng05. TCAng05 had no effect on mouse weight, heart or kidney weight, indicating that oral administration of the Ang-(1-7) analog was well-tolerated. TCAng05 caused a dose-dependent decrease in Ki67 immunoreactivity in tumor tissue sections from mice treated with the analog, suggesting reduced tumor cell proliferation. TCAng05 administration to mice with breast tumors also caused a significant decrease in tumor blood vessel density, as measured by CD34 labeling, suggesting that the analog inhibits angiogenesis to decrease tumor size. Collagen staining with Picrosirius red was markedly decreased in human breast tumor xenografts by oral administration of TCAng05, demonstrating that the analog reduces tumor associated fibrosis. These results indicate that oral administration of TCAng05 to mice with breast tumors reduced tumor size, by decreasing tumor cell proliferation, angiogenesis and fibrosis, as previously observed with the native Ang-(1-7), and suggest that the modified analog may serve as an effective, orally active, targeted chemotherapeutic.

#1238

A novel Ras inhibitor potently and selectively suppresses lung tumor cell growth by blocking Ras-Raf binding.

Bing Zhu,1 Xi Chen,1 Veronica Ramirez-Alcantara,1 Kevin Lee,1 Jacob Valiyaveettil,1 Joshua Canzoneri,1 Sara Sigler,1 Kristy Berry,1 Ashley Lindsey,1 Adam Keeton,1 Michael R. Boyd,2 Gary A. Piazza1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _ADT Pharmaceuticals Inc., Gulf Shores, AL_.

Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Screening using a phenotypic assay designed to select for Ras inhibitors and iterative chemical synthesis, identified a preclinical drug development candidate with attractive drug-like properties designated as DC070-547. Here we evaluated the sensitivity of non-small cell lung cancer (NSCLC) cell lines to DC070-547. The compound potently suppresses the growth of human A549, H460, HOP62, H1299, H1975 lung tumor cells having high levels of activated Ras with low nanomolar IC50 values. By contrast, normal human airway epithelial cells (NHAECs) and H322 lung tumor cells with low levels of activated Ras displayed IC50 values in the mid micromolar range. The Ras selectivity of DC070-547 was confirmed by ectopic expression of mutant H-Ras-G12V (H-Rasm) in H322 cells using a retroviral construct, in which the IC50 value of DC070-547 for growth inhibition was reduced approximately 2,500 fold compared with vector control cells. To study the mechanism of action, Ras activation status was measured by Ras-Raf pull-down assays using GTP-bound Ras from cell lysates with GST-Raf1-RBD/GSH sepharose and followed by western blotting with Ras antibodies. Direct binding to Ras was apparent by the ability of DC070-547 to inhibit Ras-Raf binding at concentrations that inhibit Ras-dependent tumor cell growth. Pull-down assays also showed a dose-dependent inhibition of Ras-GTP levels in intact H-Rasm-H322 cells treated with DC070-547 at the same concentration range. Western blot analysis of Ras-immunoprecipitated proteins revealed that DC070-547 also attenuated high levels of EGFR phosphorylation at Y1068 in H-Rasm-H322 cells but not in control cells. These results demonstrate novel Ras inhibitory activities of DC070-547 in NSCLC cell lines. DC070-547 and its pipeline analogs are being evaluated for antitumor efficacy in preclinical mouse models of lung cancers.

#1239

NVP-HDM201: Biochemical and biophysical profile of a novel highly potent and selective PPI inhibitor of p53-Mdm2.

Thérèse Stachyra-Valat, Frédéric Baysang, Anne-Cécile D'Alessandro, Erdmann Dirk, Pascal Furet, Vito Guagnano, Joerg Kallen, Lukas Leder, Robert Mah, Keiichi Masuya, Stefan Stutz, Andrea Vaupel, Francesco Hofmann, Patrick Chène, Sébastien Jeay, Philipp Holzer. _Novartis Institutes for Biomedical Research, Basel, Switzerland_.

An effective strategy to restore p53 activity in cancer cells containing wild type p53 is to inhibit the Mdm2-p53 protein-protein interaction (PPI). NVP-HDM201 is a novel PPI inhibitor currently under evaluation in a Phase I clinical trial. It binds to the p53 binding-site of the Mdm2 protein, disrupting the interaction of the two proteins and leading to the activation of the p53 pathway.

NVP-HDM201 belongs to a novel chemical series with a distinct biophysical and biochemical profile. Affinity constant of NVP-HDM201 for Mdm2 is in the picomolar range, with a selectivity ratio greater than a 10000-fold vs. Mdm4. Analysis of its binding mode provides evidence for a distinct set of critical interactions between the small molecule and its target, as compared with our other Mdm2 inhibitor NVP-CGM097, and explains as to why NVP-HDM201 binds equally to human, mouse, rat and dog Mdm2. Characterization of its binding kinetics indicates that the optimized interactions of NVP-HDM201 with Mdm2 protein are responsible for the increased stabilization of the complex resulting in high potency against Mdm2. This feature, together with favorable physicochemical and drug-like properties, supported the selection of NVP-HDM201 for clinical development.

#1240

Targeting epigenetic regulation in clear cell renal cell carcinoma.

Anthony J. Apostoli,1 Nazleen Lobo,2 Panagiotis Prinos,3 Dalia Barsyte-Lovejoy,3 Cheryl Arrow Smith,3 Laurie Ailles1. 1 _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;_ 2 _Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada;_ 3 _Structural Genomics Consortium, Toronto, Ontario, Canada_.

Clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell cancer, is highly resistant to traditional radiation and chemotherapy. Recently, targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved; thus, treatment options are scarce for patients bearing this disease. The majority of ccRCC cases (~90%) are characterized by bi-allelic loss of the von Hippel Lindau (VHL) gene, followed by mutations in the epigenetic regulators PBRM1 (23%), SETD2 (8.4%), BAP1 (7.1%), and KDM5C (4.7%). The latter four genes implicate an integral role for chromatin remodeling in ccRCC, and thus signify a new realm of exploration and therapeutic targeting for this disease. Given the limited number of commercially available ccRCC cell lines, and reports that they may not accurately reflect primary tumors at the molecular level, our lab developed a novel method to generate primary patient-derived ccRCC (VHLmut) cells and matched normal renal proximal tubular epithelial (VHLwt) cells from human surgical specimens with high efficiency. This involved fluorescence-activated cell sorting of primary tumor single cell suspensions using the cell surface marker carbonic anhydrase IX (CA9), a HIF target which is upregulated upon VHL loss, to establish CA9+ VHLmut and CA9− VHLwt cells. Transcriptional profiling of VHLmut and VHLwt pairs found gene signatures that corresponded with patient matched primary ccRCC tumors and adjacent normal tissues in The Cancer Genome Atlas, demonstrating that these cells represent novel models for interrogation of ccRCC biology and testing of therapeutic strategies. Here, in the current study, these newly established cells were utilized to screen a focused library of 30 well-characterized drug-like compounds targeting specific components of chromatin remodeling complexes to determine the effect on cell growth in culture. At Day 0, 786-0 (−VHL) cells and VHLmut cells corresponding to four patient primary ccRCC tumors were plated at 2,500 cells/well in 96-well black clear-bottom plates (n=5). Cells were then treated with the library of chromatin-targeting drugs on Day 1 and monitored until control-treated cells were 90-95% confluent. At endpoint, cells were fixed and stained with DRAQ5, a DNA intercalating dye that stains DNA stoichiometrically, for visualization on the LI-COR Odyssey CLx Imaging System. Across all five samples, there was a significant reduction in growth observed among cells treated with drugs targeting: i) BET bromodomains (BRD2, BRD3, BRD4 and BRDT), ii) EZH1/2, and iii) JMJD3, UTX and JARID1B. Ongoing work will determine mechanisms of action and whether these targets are also affected in matching VHLwt cells in order to identify compounds that may translate well in the clinic to treat patients with ccRCC.

#1241

Podophyllotoxin acetate (PA) as an anti-cancer drug candidate regulates various malignancy traits of non-small cell lung cancer.

Jeong Hyun Cho, Wan Gi Hong, Sang-Gu Hwang, Hong-Duck Um, Jong Kuk Park. _KIRAMS, Seoul, Republic of Korea_.

Here, we reported that podophyllotoxin acetate (PA) showed various aniti-cancer effects: induction of several cell death pathways, inhibition of γ-ionizing radiation (IR)-induced cancer invasion, chemo-sensitizer effect with topoisomerase (TOP) inhibitors against non-small cell lung cancer (NSCLC) cell lines as follows. (1) Stimulation of cell death via various mechanisms. First, PA dose-dependently induced cell cycle arrest at G2/M phase, as shown by accumulation of the mitosis-related proteins, p21, survivin and aurora B. This arrest was due to the PA-induced inhibition of microtubule polymerization that related to DNA damage (reflected by accumulation of γ-H2AX) resulting induction of intrinsic/extrinsic damage apoptotic pathways. PA also activated the pro-apoptotic ER stress pathway, as evidenced by increased expression levels of BiP, CHOP, IRE1-a, phospho-PERK, and phospho-JNK. Next, PA induced autophagy that reflected the expression of beclin-1, Atg3, Atg5 and Atg7, and the cleavage of LC3. (2) Inhibition of IR-induced cancer invasion. IR increased the invasion/migration of A549 cells, and this effect was decreased by PA treatment that the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). In this study, we also identified IR-induced new signaling pathway, EGFR - p38/ERK - CREB-1/STAT3 - EMT pathway, that was inhibited by PA. (3) Chemo-sensitizer effect conjugated with TOP inhibitors - etoposide (Eto) and camptothecin (Cpt). Combination Index (CI) values of the combinations showed synergistic effects between PA and topoisomerase inhibitors. Combination with PA and Eto/Cpt promotes disruption of dynamics of actin filaments, which subsequently enhanced apoptotic cell death that accompanied to increased phosphorylation of p38. We also observed these combinations also inhibited activation of CREB-1, synergistically. Therefore, this study indicated that PA functions as a chemosensitizer by enhancing apoptosis through activation of p38/caspases apoptotic axis and suppression of CREB-1.Taken together; these results suggest PA is a new anti-cancer drug candidate that could control several malignancy traits of cancer including uncontrolled cell cycle and IR-induced invasion.

#1242

Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer.

Elena Shagisultanova,1 Vladislav Korobeynikov,2 Anna Nikonova,2 Jonathan Chernoff,3 Virginia Borges,1 Erica Golemis2. 1 _Young Women's Breast Cancer Translational Program, University of Colorado Denver, Aurora, CO;_ 2 _Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA_.

Estrogen receptor alpha (ERα) signaling plays a critical role in the development and progression of hormone receptor-positive (HR+) breast cancer. One mechanism of tumor progression of HR+ breast cancer is phosphorylation of ERα by non-receptor protein kinases, leading to ligand-independent activation of ERα. Mitotic kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast cancer and can phosphorylate ERα on serine 305, increasing the ability of ERα to induce transcription of target genes implicated in tumorigenesis. In addition, PAK1 phosphorylates and activates AURKA and its functional partners, suggesting possible synergistic activity in tumors overexpressing both kinases. We tested the hypothesis that combined inhibition of AURKA and PAK1 would synergistically suppress tumor growth of HR+ breast cancer cells in vitro and in vivo. Treatment of T47D HR+ breast cancer cell line with the PAK1 inhibitor FRAX1036, combined with AURKA inhibitor alisertib, improved cell killing in comparison to single agents. FRAX1036 and alisertib were then evaluated in an orthotopic xenograft model using BT474 HR+ breast cancer cell line. Ten million BT474 cells were implanted into the mammary fat pads of 6-week old NSG mice simultaneously with implantation of estrogen pellets to facilitate growth of xenografts. Mice received 21 days of treatment with FRAX1036 20mg/kg daily and alisertib 10 or 15 mg/kg BID, alone or in combination. We observed exceptional inhibition of tumor growth in the combination treatment groups. Combination of FRAX1036 20mg/kg daily with alisertib 15mg/kg BID was the most effective, with partial or near-complete responses that continued through 21 days of treatment. This combination resulted in significantly better inhibition of tumor growth than alisertib 15mg/kg BID alone or FRAX1036 20mg/kg daily alone (p=0.003 and p=1.29e-6, respectively). Similarly, combination treatment of FRAX1036 20mg/kg daily with alisertib 10mg/kg BID was significantly better then alisertib alone or FRAX1036 alone in the corresponding doses (p=0.002 and p=3.52e-7, respectively). Analysis of H&E stained sections and Ki-67 proliferation index of BT474 xenograft tumors indicated a highly significant decrease in viable tumor cells and replacement by stroma after combination treatment compared to treatment with single agents or with control. Caspase-3 IHC analysis showed significantly greater proportion of apoptotic cells per tumor area in the group of mice treated with FRAX1036 and Alisertib 15mg/kg BID combination comparing to control (p=0.008). We conclude that combining FRAX1036 and alisertib is a novel and promising approach in HR+ breast cancer.

#1243

Inside of multiple mechanism of adaption towards the proteasome inhibitors in multiple myeloma.

Andrej Besse,1 Lenka Besse,1 Marianne Kraus,1 Jurgen Bader,1 Mario Novkovic,2 Fedor Kryukov,3 Pavel Nemec,4 Tobias Silzle,1 Christoph Driessen1. 1 _Hematology/Oncology, Kantonsspital St. Gallen, Switzerland;_ 2 _Institute of Immunobiology, Kantonsspital St Gallen, Switzerland;_ 3 _Department of Hematooncology, Faculty of Medicine University of Ostrava and University Hospital Ostrava, Czech Republic;_ 4 _Department of Biology, Faculty of Medicine, Masaryk University, Czech Republic_.

Malignant plasma cells drive massive overproduction of monoclonal immunoglobulin in multiple myeloma (MM). Hence MM cells critically rely on a proper balance between protein biosynthesis and folding, and degradation of unfolded/ misfolded protein orchestrated by the unfolded protein response (UPR). Inhibitors of the proteasome, the terminal protease along the UPR pathway, are highly effective against MM. Adaptive resistance to current proteasome inhibitors (PI) (Bortezomib-Btz, Carfilzomib-Cfz) is a major cause of treatment failure. Btz is a peptide boronate-type reversible inhibitor, whereas Cfz is of epoxyketone-type and binds irreversibly to the same proteasomal B5 active site target. Next generation PI are likewise B5-directed and either of the peptide boronate (ixazomib, dalanzomib), or the epoxyketone type (oprozomib). PI resistance is currently understood via two alternative concepts, the load/capacity concept characterized by proteasome upregulation in conjunction with downregulation of the UPR via reduced IRE/XBP-1 signaling, and the mutation concept basing on B5 active site mutations that prevent PI binding. Only IRE1/XBP1 downregulation have been confirmed in patient-derived material. We generated an in vitro model where AMO-1 myeloma cells (IC50: 3.1nM for Btz, 4.9nM Cfz) were adapted to Btz (AMOaBtz; IC50: 298.7nM Btz, 43.2nM Cfz) or to Cfz, (AMOaCfz; IC50: 27.9nM Btz, 411.5nM Cfz). AMOaBtz cells showed preferential resistance against ixazomib and dalanzomib, while AMOaCfz cells were preferentially resistant to oprozomib and other epoxyketone-type experimental inhibitors. AMOaBtz and AMOaCfz likewise downregulated IRE/XBP-1, but only AMOaBtz contained an acquired proteasome mutation (A310G in PSMB5), however this had only a modest influence on Btz binding and proteasome inhibition. Global gene expression analysis revealed 1430 differentially expressed genes in AMOaBtz/ AMOaCfz (FC>2, p<0.05). Analysis of uniquely upregulated genes in AMOaCfz identified the multi-drug resistance gene ABCB1 (MDR1) that was found 120-fold upregulated by qPCR. Inhibition of ABCB1 with Verapamil or Reserpin strongly increased Cfz sensitivity, confirming functional significance. Genes responsible for immunoglobulin production were selectively downregulated in AMOaBtz, and paraprotein production (IgA) was abolished as measured by ELISA. In conclusion, AMOaCfz is the first model combining the key features found in PI-resistant patient cells (lack of PSMB5 mutation and low IRE1/XBP1 activity). The comparison of AMOaCfz and AMOaBtz suggests the presence of class-preferential adaptation mechanisms, which may be overcome by using alternative type proteasome inhibitors or mMDR1 inhibitors.

#1244

A novel microtubule-disrupting indole-based chalcone that induces caspase-independent cell death in glioblastoma and melanoma cells.

Shengnan Du,1 Jeffrey G. Sarver,2 Christopher J. Trabbic,2 Paul W. Erhardt,2 Jean H. Overmeyer,1 William A. Maltese1. 1 _University of Toledo College of Medicine, Toledo, OH;_ 2 _University of Toledo College of Pharmacy, Toledo, OH_.

Indolyl-pyridinyl-propenones (a subclass of synthetic indole-based chalcones) have been shown to induce a novel form of cell death termed 'methuosis' in glioblastoma and other tumor cell lines. This form of cell death is caspase-independent and involves massive vacuolization of macropinosome and endosome compartments. In the course of structure-activity relationship studies identifying a lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (abbreviated 5-MOMIPP), we discovered that changing the methoxy substitution from the 5-position to the 6-position on the indole ring markedly alters the biological activity of the compound. 6-MOMIPP exhibits potent anti-proliferative and cytotoxic activity in glioblastoma, melanoma and other cancer cell lines at a concentration of 100 nM, nearly two orders of magnitude lower than the maximally effective concentration of 5-MOMIPP. Instead of inducing methuosis, 6-MOMIPP (at > 500 nM) immediately disrupts microtubules, as confirmed by immunofluorescence microscopy. By 24 h the majority of the cells are rounded and arrested in mitosis, and by 48 h cell viability is greatly reduced. The few remaining live cells contain multiple abnormal nuclei. Extensive cell death occurs before activation of caspases 3, 7 and 9 can be detected. Scintillation proximity assays show that 6-MOMIPP competes with 3H-colchicine, but not 3H-vinblastine, for binding to tubulin. Studies aimed at testing the toxicity of 6-MOMIPP in normal cells are in progress, but work with a closely related microtubule-active indole-based chalcone indicates that toxicity is substantially reduced in quiescent human fibroblasts and rat neuronal progenitor cells. Although drugs targeted to microtubules (e.g., Vinca alkaloids and taxanes) are widely used as anti-neoplastic agents, they have shown little efficacy in treating primary or metastatic brain tumors, partly due to limited penetrance of the blood-brain barrier (BBB). Preliminary pharmacokinetic studies in mice show that 6-MOMIPP injected IP can readily cross the BBB, attaining brain concentrations nearly equal to plasma concentrations within 30 min to 1 h. Therefore, these preliminary findings suggest that 6-MOMIPP merits further preclinical evaluation as a potential therapeutic agent for primary and metastatic brain tumors. Supported by NIH grant CA115495 and by the Helen and Harold McMaster Endowment.

#1245

RRx-001 is effective in temozolomide-sensitive and resistant GBM.

Veronica Steri,1 Bryan Oronsky,2 Jan Scicinski,2 Gabriele Bergers1. 1 _Department of Neurological Surgery, Brain Tumor Research Center and UCSF Comprehensive Cancer Center, University of California, Helen Diller Family Cancer Research Center, San Francisco, CA;_ 2 _EpicentRx, Mountain View, CA_.

Glioblastoma Multiforme (GBM) is one of the most aggressive malignancies in humans, being highly invasive and drug-resistant, limiting the effectiveness of surgery, chemotherapy and radiotherapy. Concurrent Temozolomide (TMZ) and radiation is the standard of care for newly diagnosed GBM patients, however, patients respond poorly and prognosis remains dismal. TMZ response is limited to patients with hypermethylation of the methylguanine methyltransferase (MGMT) promoter. Despite recent advances in therapeutic strategies, treating GBM is still an unsolved clinical challenge requiring the investigation of novel approaches with better responses over approved therapies. RRx-001 is a novel ROS-mediated systemically non-toxic chemo-sensitizing epigenetic agent with vascular normalizing properties under investigation in Phase II clinical trials in brain metastases and GBM.

We assessed whether RRx-001 is an effective approach for treating GBM by testing its effects alone and in combination with TMZ both in vitro as well as in GBM mouse models. RRx-001 induced cytotoxicity in human TMZ-sensitive as well as resistant patient-derived GBM cell lines. In addition, its effect in TMZ-sensitive GBM cells was greater than the one induced by TMZ alone.

We then asked whether the in vitro effects of RRx-001 translated to therapeutic efficacy in vivo. To this end, we injected mice intracranially with a TMZ-resistant murine mesenchymal GBM cells, and treated tumor-bearing mice with RRx-001 alone and in combination with TMZ. RRx-001 treatment alone modestly prolonged survival compared to control cohorts, whereas the combination of RRx-001 and TMZ substantially prolonged survival compared to controls and either treatment alone. Moreover, histological evaluation of GBM tumors revealed that combination of RRx-001 and TMZ induced vessel hyperdilation and apoptosis compared to RRx-001 alone. Thus, in vivo RRx-001 combined to TMZ may also indirectly regulate tumor growth by modifying the tumor stroma.

These data suggest that TMZ may still affect the microenvironment in TMZ-resistant tumors and that RRx-001 enhances these effects by both directly and indirectly restricting GBM growth, making it a promising therapeutic agent alone and in combination with TMZ.

#1246

Development of STAT3 dual-targeting strategies for the treatment of pancreatic cancer.

Melissa L. Fishel,1 Michelle L. Grimard,1 Mark R. Kelley,1 David A. Rosa,2 Andrew Shouksmith,2 Gary Tin,2 Ji Park,2 Patrick T. Gunning2. 1 _Indiana University, Indianapolis, IN;_ 2 _University of Toronto Mississauga, Toronto, Ontario, Canada_.

Pancreatic cancer remains a largely incurable disease, with patients facing the worst 5-year survival rate of any cancer. The challenge is to identify the molecular effectors that regulate the survival of pancreatic ductal adenocarcinoma (PDAC) cells, to devise molecular-targeted strategies that are effective in the metastatic setting, and overcome the protective role of the tumor-associated fibrosis and stroma. Strategies targeting multiple molecular effectors in PDAC are likely going to make a bigger impact. Thus, we are identifying molecular targets or synthetic lethal pairs that regulate critical pro-survival or pro-invasive pathways in PDAC. Constitutively activated Signal Transducer and Activator of Transcription 3 (STAT3) protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. To better model the tumor and its microenvironment, we utilized ex vivo 3-Dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAFs). We can quantitate the inhibitory effect on both the tumor and CAFs as they are labeled with different fluorescent markers. In this co-culture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. We screened several STAT3 small molecule inhibitors, derived from the SH-4-54 class of STAT3 inhibitors, and found inhibition of pancreatic cancer cell proliferation in the low µM range. Our inhibitors bind the STAT3 protein potently, as shown by SPR, and demonstrate no effect in a kinome screen. In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in the 3D co-culture model. We have previously reported that Ref-1 (redox factor-1) regulates STAT3 activity through its redox function and blocking STAT3 through phosphorylation and redox inhibition synergizes for PDAC cell killing. In our 3D co-culture system, Ref-1 inhibitor, APX3330 decreases tumor area and intensity in a dose-dependent manner. The addition of APX3330 to STAT3 pathway inhibition via Ruxolitinib (Rux, Jak 2 inhibitor) or direct STAT3 inhibitor potentiated the killing effect in the tumor. However, the combination treatment did not appear to sensitize CAF cells, suggesting that targeting of Ref-1 and the STAT3 pathway is more specifically targeting tumor cells. Utilizing APX3330, Rux, and our lead STAT3 inhibitors, we evaluated the effects of Ref-1/STAT3 inhibition in PDAC low passage patient-derived cell lines. The activity of STAT3 and specificity of lead compounds was assessed by immunoblotting for levels of phosphorylated proteins including STAT3 (Y705) and STAT5 (Y694) in 3D culture. These studies establish the rationale for the development of STAT3 dual-targeting strategies for the treatment of pancreatic cancer and suggest that Ref-1 and STAT3 may be a synthetic lethal pair.

#1247

ERK1/2 inhibitors with distinct modes of inhibition confer different response profiles in KRAS mutant cancers.

Joanne M. Munck, Brent Graham, Juan Castro, Aurelie Courtin, Sandra Muench, Sharna Rich, Harpreet K. Saini, Jessica Brothwood, John Lyons, Neil T. Thompson, Nicola G. Wallis, Chris Murray. _Astex Pharmaceuticals, Cambridge, United Kingdom_.

The clinical benefit of targeting the MAPK pathway is demonstrated by the success of RAF and MEK inhibitors in the treatment of melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms that result in reactivation of ERK signalling. Additionally, many BRAF- and KRAS-mutant cancers exhibit de novo resistance. As the direct targeting of ERK may overcome the limitations of RAF or MEK inhibitors, there are several ERK1/2 inhibitors in development. SCH772984 (SCH, Merck-Schering) is an example of an ERK1/2 inhibitor that in addition to inhibiting ERK catalytic activity also inhibits the phosphorylation of residues within the ERK activation loop. Several ERK1/2 inhibitors have been described that inhibit ERK catalytic activity without modulating ERK phosphorylation e.g GDC-0994 (GDC, Genentech). Here we aim to characterise the biological relevance of the different modes of ERK inhibition.

We investigated the biochemical mechanism by which SCH inhibits ERK phosphorylation and demonstrated that it prevents the phosphorylation of ERK by MEK (T202/Y204), but does not affect MEK activity.

We characterised the effects of the distinct modes of ERK inhibition on ERK substrates, cellular pERK levels, pathway feedback activation and sensitivities of cell lines with different genetic backgrounds.

In BRAFV600E-mutant cells e.g A375 (melanoma) and Colo205 (colorectal), 16 hr treatment with SCH and GDC significantly inhibited the phosphorylation of RSK (ERK substrate). SCH significantly inhibited ERK phosphorylation, whereas GDC did not modulate pERK levels. Neither compound induced pathway feedback activation. In proliferation assays A375 and Colo205 cells had remarkably similar sensitivities to SCH (EC50 40 nM and 30 nM, respectively) and GDC (EC50 = 140 nM in both).

In contrast to BRAF-mutant cells, SCH and GDC treatment conferred pMEK induction in the KRAS-mutant cell line HCT116 (while still significantly inhibiting RSK phosphorylation). This is likely to be attributable to the feedback-driven C-RAF activation commonly associated with MAPK pathway inhibition in KRAS-mutant cells. In addition to pMEK, GDC also conferred an induction of pERK. However, SCH overcame pathway feedback and inhibited the ERK phosphorylation. The inhibition of pathway feedback conferred by SCH was associated with increased sensitivity of HCT116 cells to SCH (EC50 = 30 nM) compared to GDC (EC50 = 1.1 µM). In a cell panel screen comprising 25 BRAF-mutant and 63 KRAS-mutant cell lines, SCH and GDC had similar activity in BRAF-mutant cell lines. However, although KRAS-mutant cell lines were sensitive to SCH, they were largely resistant to GDC. These data suggest that due to their ability to overcome MAPK inhibitor-induced pathway feedback, ERK inhibitors that also inhibit the phosphorylation of ERK would be more effective in KRAS-mutant cancer than those that inhibit ERK activity alone.

#1248

Ibrutinib inhibits platelet aggregation through inhibition of mitochondrial respiratory chain function and suppression of Syk activation.

Jie Huang, Helen Pelicano, Feng Li, Michael J. Keating, Peng Huang. _MD Anderson Cancer Center, Houston, TX_.

Ibrutinib represents a therapeutic improvement in hematologic cancers, but it is associated with bleeding-related adverse events. To minimize such adverse events and improve the therapeutic utility of this drug, it is important to understand the underlying mechanisms responsible for the adverse events. In this study, we observed that platelets isolate from the blood samples of chronic lymphocytic leukemia (CLL) treated with Ibrutinib exhibited deficiencies in collagen-mediated aggregation. Mechanistic studies identified the mitochondrial respiratory chain as a potential mediator of ibrutinib-induced impairment of platelet function. A short-term treatment of platelets isolated from human blood with ibrutinib for less than one hour led to reactive oxygen species (ROS) generation associated with inhibition of mitochondrial respiration, as evident by a reduction in oxygen consumption rate measured by a Seahorse metabolic analyser. Long-term incubation of megakaryocytes with ibrutinib markedly suppressed the expression of mitochondrial electron transport chain components in platelets and a reduction in mitochondrial respiration. Interference with the respiratory chain by rotenone also resulted in similar reduction in collagen-induced platelet aggregation. Interestingly, ROS generation upon collagen stimulation induced tyrosine phosphorylation of Syk (spleen tyrosine kinase) to activate platelet, while impairment of respiratory chain induced by ibrutinib diminished collagen-evoked activation of the Syk pathway. These data suggest that ibrutinib suppresses mitochondrial respiration, leading to defects in collagen-induced platelet aggregation, potentially through inhibiting the activation of the Syk pathway.

#1249

PRN1371, an irreversible, covalent inhibitor of FGFR1, 2, 3 and 4 is highly efficacious in preclinical tumor models.

Eleni Venetsanakos, Michael Bradshaw, David M. Goldstein, Kwan Leung, Dane Karr, Yan Xing, Jacob LaStant, Philip Nunn, Jin Shu, Abha Bommireddi, Steven G. Gourlay, Jens Oliver Funk, Ken A. Brameld. _Principia Biopharma, South San Francisco, CA_.

Introduction:

Multiple human cancers harbor alterations in FGFRs that drive tumor growth, including mutations, translocations and amplifications. We developed a covalent, irreversible, highly selective FGFR1, 2, 3 and 4 inhibitor, PRN1371, by targeting a cysteine residue within the kinase domain. This approach enables highly selective and sustained inhibition of FGFR which extends well beyond circulating drug concentrations. PRN1371 is currently in a phase 1 clinical trial for the treatment of solid tumors.

Materials and Methods:

FGFR1-4 enzymatic activities were measured using the Caliper microfluidics Labchip system. Inhibition of proliferation was assessed over 3 days using Cell-Titer-Glo. SNU16 and RT4 xenograft tumor models were used to investigate pharmacodynamics and efficacy. For tumor inoculation, cancer cells were implanted into the rear flank of immunocompromised mice. Once tumor volume reached a mean average of 175mm3, mice were randomized and treated with FGFR inhibitor.

Results:

PRN1371 is a novel investigative drug that is a potent ATP competitive, highly selective inhibitor of the FGFR family of protein kinases. PRN1371 binds irreversibly to FGFR1, 2, 3 and 4, leading to long duration of target inhibition. The IC50 values for FGFR1, 2, 3 and 4 are 0.7, 1.3, 4.1 and 19.2 nM, respectively. The IC50 values are 55.0, 12.0, 1.2 and 2.3 nM for FGFR3-V555M, FGFR2-N549H, FGFR3-G697C and FGFR3-K650E, respectively. PRN1371 exhibited no significant activity against 247 other protein kinases. PRN1371 demonstrates potent inhibition of cell proliferation in multiple tumor cell lines driven by dysregulated FGFR signaling. In RT4, RT112, SNU16, AN3-CA, LI7, JHH7, and OPM2 cells, EC50's are 4.0, 4.1, 2.6, 43.3, 33.1, 231 and 14.0 nM, respectively.

PRN1371 is rapidly and well-absorbed. Bioavailability is dose and species-dependent.

PRN1371 demonstrates potent tumor growth inhibition and regression in FGFR3-fusion expressing RT4 and FGFR2-amplified SNU16 xenograft models, when dosed either continuously, or intermittently, and is generally well-tolerated. PRN1371 is also shown to have significant anti-tumor activity against a panel of PDX tumor xenografts of various lineages harboring different FGFR pathway alterations.

The phase 1 clinical trial will evaluate ascending doses of PRN1371 in a conventional "three plus three" design. Expansion cohorts of selected tumor types will then be investigated at the recommended dose level.

Conclusion:

PRN1371 is a potent, highly selective, irreversible inhibitor exhibiting sustained inhibition of FGFR 1, 2, 3 and 4. A Phase 1 clinical trial of PRN1371 for the treatment of solid tumors is currently ongoing.

#1250

**Antitumor activities of CD161, a structurally novel and orally bioavailable BET inhibitor, in leukemia and triple negative breast cancer cells** in vitro **and** in vivo **.**

Longchuan Bai, Yujun Zhao, Liu Liu, Chao-Yie Yang, Donna McEachern, Jeanne Stuckey, Jennifer Meagher, Bo Wen, Duxin Sun, Shaomeng Wang. _University of Michigan, Ann Arbor, MI_.

Bromodomain and extraterminal (BET) proteins, such as bromodomain-containing protein 2 (BRD2), BRD3 and BRD4, bind lysine acetylated proteins and regulate gene transcription. Several BET inhibitors are currently under clinical development for hematological malignancies and solid tumors. We have recently developed a novel class of BET inhibitor, CD161, which binds the bromodomain 1 (BD1) and BD2 domains of BRD2, BRD3, BRD4 and BRDT with Ki less than 10 nM. Co-crystal structure of CD161 and BRD4 BD2 at 2.5 Å resolution confirmed that the docked CD161 occupies the acetyl-lysine binding pocket of BD2. CD161 has >1000-fold selectivity over other bromodomain containing proteins including CREBBP, ATAD2B, TRIM24 and ASH1L. In human leukemia cell lines MV4-11 and MOLM-13, CD161 strongly inhibits cell growth and induces apoptosis, with IC50s <100 nM. Furthermore, CD161 exerts potent growth-inhibitory activities in a panel of breast cancer cell lines representative of triple-negative breast cancer (TNBC), with IC50s in the sub micromolar range. At 20-50 mg/kg daily oral dosing for 2 weeks, CD161 significantly inhibited the growth of MV4-11 and MDA-MB-231 tumors (TGI: 55-80%) in mouse xenograft models and had no significant effect on body weights. Pharmacokinetics studies show that CD161 has a T1/2 of 2-3 hours and oral bioavailability (F%) of 93% in male Sprague−Dawley rats. Transcriptional profiling revealed that CD161 causes broad transcriptional changes in TNBC cell lines. Our preliminary results suggest the therapeutic potential of CD161 in human leukemia and TNBC.

#1251

Significant growth suppressive effects of TOPK and MELK inhibitors in kidney cancer.

Taigo Kato,1 Hiroyuki Inoue,1 Seiya Imoto,2 Yoshinori Tamada,2 Takashi Miyamoto,3 Yo Matsuo,3 Yusuke Nakamura,1 Jae-Hyun Park1. 1 _The University of Chicago, Chicago, IL;_ 2 _The University of Tokyo, Japan;_ 3 _OncoTherapy Science Inc., Kanagawa, Japan_.

Kidney cancer is the seventh most common cancer and the tenth most common cause of cancer death for men, and it is the tenth most common cause of cancer for women. The 5-year disease-specific survival has improved from about 50% in 1975-1977 to 65% in 2000-2005, although that of patients at an advanced stage still remains poor with less than 10%; 30% of patients who underwent surgery of localized kidney cancer develop distant metastasis and have limited therapeutic options such as tyrosine kinase inhibitor (TKI) and mammalian target of rapamycin (mTOR) inhibitors. In addition, the clinical effects of these drugs are limited and patients often discontinue these drugs due to severe side effects including hand-foot syndrome, liver dysfunction and interstitial pneumonia. Therefore, development of more effective therapy for kidney cancer is eagerly expected. T-lymphokine-activated killer cell-originated protein kinase (TOPK) and maternal embryonic leucine zipper kinase (MELK) are promising molecular targets that play critical roles in cancer cell proliferation and maintenance of their stemness. In this study, we investigated growth promotive effect of TOPK and MELK in kidney cancer and found a complex feedback mechanism of these two molecules. Blockade of either of these two kinases effectively caused downregulation of forkhead box protein M1 (FOXM1) activity which is known as an oncogenic transcriptional factor in various types of cancer cell. On the other hand, knockdown and overexpression of FOXM1 caused downregulation and upregulation of both TOPK and MELK mRNA levels, respectively, suggesting that FOXM1 is a critical transcriptional factor for these two molecular targets. Small molecular compound inhibitors against TOPK (OTS514) and MELK (OTS167) effectively suppressed the kidney cancer cell growth and the combination of these two compounds additively induced the very strong growth suppressive effect on kidney cancer cells. Collectively, our results suggest that both TOPK and MELK are promising molecular targets for kidney cancer treatment and that dual blockade of OTS514 and OTS167 should produce synergistic anti-tumor effects with low risk of side effects.

### Novel Molecular Targets

#1252

TIMELESS is a KSR1-like effector of Ras-driven colon tumorigenesis.

Beth K. Clymer,1 Kurt W. Fisher,1 David L. Kelly,1 Michael A. White,2 Robert E. Lewis1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _UT Southwestern, Dallas, TX_.

Multiple studies have revealed that Ras-driven tumors acquire unique vulnerabilities by adapting cellular mechanisms that promote uncontrolled proliferation and suppress apoptosis. Targeting these vulnerabilities provide opportunities to develop novel, efficacious cancer therapeutics that lack the harmful side effects accompanying current therapies. RNA interference (RNAi) of the molecular scaffold Kinase Suppressor of Ras 1 (KSR1), which modulates ERK activation downstream of oncogenic Ras, selectively kills malignant, Ras-driven cancer cells, but does not kill immortalized, non-transformed human colon epithelial cells (HCECs). With the exception of a minor hair follicle defect, KSR1-/- mice are fertile and phenotypically normal, suggesting that KSR1 is not required for normal cell survival and that Ras-driven and KSR1-dependent pathways may yield valuable new targets for therapeutic development.

To identify targets, like KSR1, that are required for cancer cell survival but not normal cell survival, we used a gene expression-based signature screening approach termed Functional Signature Ontology (FUSION, Potts et al. Sci. Signaling 2013) to screen 15,172 genes in the K-RasD13-bearing human colorectal cancer cell line HCT116. We quantified the functional similarity between KSR1 and each individual gene screened using Euclidean Distance and Pearson Correlation similarity metrics. Additional metrics were added to identify the best targets for biological validation including off-target analysis (siRNA seed sequences), cell viability evaluation, expression analysis, and enrichment analysis. Initial biological validation is completed by assessing cell viability following transient depletion of a screen hit in anchorage-independent and normal culture conditions in HCECs and HCT116s.

Due to the similarity between the gene expression signatures, Timeless Circadian Clock (TIMELESS) was identified as being KSR1-like and a potential target. We found that transient TIMELESS depletion decreases cell viability in HCT116 cells under anchorage-independent conditions (47% decrease, p < 0.0001, N=4). In normal culture conditions, TIMELESS depletion decreases cell viability in HCT116 cells, but not HCECs (HCEC 14% decrease, p > 0.05; HCT116 49% decrease, p < 0.0001, N=6). TIMELESS is upregulated at the RNA level in colon tumors compared to normal colon tissue (~2.2 fold, p < 0.0001) (TCGA) and is upregulated at the protein level in three human colon cancer cell lines (HCT116, SW480, SW620) bearing activated Ras compared to HCECs (4-7 fold).

Our data indicate that the FUSION screen provides a platform for identifying novel therapeutic targets and demonstrates the potential to identify oncogene-specific vulnerabilities in an unbiased manner. TIMELESS overexpression represents a vulnerability in Ras-driven tumors that will reveal novel and selective targets found in Ras-driven cancers that can be used in the development of selective therapeutics.

#1253

Inhibition of FN14 to suppress KRAS-driven lung cancer migration and survival.

Vashti M. Carson,1 Seungchan Kim,1 Landon J. Inge,2 Timothy G. Whitsett1. 1 _TGen, Phoenix, AZ;_ 2 _Norton Thoracic Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ_.

Lung cancer remains the leading cause of cancer related mortality throughout the world, and is estimated to kill more than one million people this year. Mutations in KRAS occur in about 1/3 of all lung adenocarcinomas, the most common histologic subtype of lung cancer. Mutations in KRAS are correlated with poor patient prognosis, advanced disease, therapeutic resistance and metastatic outcomes. There is an unmet clinical need to target KRAS-driven NSCLC towards abating the high mortality rate of this disease. We hypothesize that the tumor necrosis factor receptor, FN14 is induced and sustained by mutant KRAS signaling, and represents a therapeutic target in KRAS-driven NSCLC. Employing The Cancer Genome Atlas (TCGA) data, we showed that within a subgroup of adenocarcinoma patients harboring mutant KRAS, elevated FN14 expression conferred significantly worse survival outcomes compared to those with lower FN14 expression. Alone, elevated expression of FN14 conferred a poor prognosis in adenocarcinoma patients compared to lower expression of FN14. In lung cancer cell lines driven by mutant KRAS, elevated FN14 protein expression was observed. The ectopic induction of mutant KRAS in normal rat lung epithelial cells was sufficient to induce FN14 protein expression, an induction dependent on ERK signaling. In an animal model of mutant KRAS-driven NSCLC, FN14 protein was highly expressed and correlated with metastasis in vivo. TWEAK, the only known ligand for FN14, induced migration in NSCLC cell lines harboring mutant KRAS. Conversely, depletion of FN14 expression through siRNA suppressed KRAS-driven cell migration in vitro. We further demonstrated using a clonogenic assay that depletion of FN14 by siRNA was sufficient to reduce KRAS-driven NSCLC cell survival and significantly enhanced the cell killing effects of standard-of-care treatments such as radiation or cisplatin. These data suggest that NSCLC tumors harboring mutant KRAS may be vulnerable to inhibition of FN14 signaling. The recent development of FN14-targeted therapeutics provides an avenue towards clinical utility. As no therapeutics exist to target KRAS directly, a therapeutic strategy targeting FN14 might impact survival in KRAS-driven tumors and reduce the high mortality rate in this subgroup.

#1254

Small molecule targeting of the kRAS mid-G-quadruplex for potential pancreatic cancer therapeutics.

Rhianna K. Morgan, Tracy A. Brooks. _University of Mississippi, University, MS_.

Pancreatic cancer is disproportionally lethal (7% of all cancer deaths), as compared to its incidence (3% of cancer incidences) within the United States. One of the most important genetic alterations in pancreatic cancer is within the kRAS oncogene. Over 60% of all pancreatic cancers, and up to 95% of pancreatic ductal adenocarcinomas, harbor mutations in this oncogene. While approaches to therapeutically modulate kRAS activity have not yielded clinical agents, downregulation of expression shows promise in preclinical models. To date, no molecular target has allowed for small molecule mediated downregulation of kRAS expression; however, the promoter region holds promise for a new approach. The kRAS promoter has three distinct guanine-rich regions capable of forming higher order non-B-DNA structures, G-quadruplexes (G4s). In particular the middle (mid) of these three regions forms a stable, inducible, and transcriptionally silencing G4 structures, whereas the more distal (far) region forms no such structure, and the more proximal (near) region has little to no biological function. The structure of this mid-region G4 has been elucidated by electromobility shift assay, DMS footprinting, and circular dichroism (CD). Small molecules capable of stabilizing this structure have been screened from the NCI Diversity Set III by the FRET melt assay, confirmed by CD, and examined for their downregulation of kRAS promoter activity. From the 1600 compounds, the indoloquinolone NSC 317605 is the lead molecule identified. It is particularly interesting as it prefers the mid-, as opposed to the near-, G4 and significantly reduces kRAS promoter activity. This is in contrast to the ellipticine NSC 176327, which favors the near-, over the mid-, G4 and does not decrease kRAS promoter activity. The effect of each compound on cell viability on kRAS transcription is under examination in a panel of pancreatic cancer cells. These works support previous findings that the mid-, and not the near-G4, harbors the potential to transcriptionally downregulate kRAS, and identify a new scaffold for future development.

Brooks Lab startup funds, DoD, Rhianna Morgan GSC Grant, University of Mississippi School of Pharmacy

#1255

Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein.

Lisa Maria Mustachio,1 Yun Lu,1 Laura J. Tafe,2 Angeline S. Andrew,2 Vincent Memoli,2 Jaime Rodriguez-Canales,3 Pamela A. Villalobos,3 Ignacio Wistuba,3 Jun Yu,3 Jack J. Lee,3 Fadzai Chinyengetere,1 David J. Sekula,3 Xi Liu,3 Sarah J. Freemantle,1 Ethan Dmitrovsky3. 1 _Geisel School of Medicine at Dartmouth, Hanover, NH;_ 2 _Dartmouth-Hitchcock Medical Center, Lebanon, NH;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

KRAS is frequently mutated in lung cancers. Innovative strategies are needed to combat KRAS mutant lung cancers because these tumors are often resistant to therapy. This study reports that loss of the deubiquitinase USP18 leads to destabilization of the KRAS oncogenic protein in panels of murine and human lung cancer cells. In marked contrast, engineered gain of USP18 expression using retroviral vectors introduced into the same panel of murine and human lung cancer cells stabilized KRAS protein. Loss of USP18 expression in KRAS mutant-expressing lung cancer cells inhibited their growth while gain of USP18 expression opposed this effect. We sought to identify mechanisms engaged in this KRAS protein destabilization. Intriguingly, immunoprecipitation assays established that KRAS conjugates with the ubiquitin-like protein ISG15. This leads to KRAS protein destabilization. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown was shown to destabilize mutant KRAS more prominently than wild-type KRAS protein. To independently confirm that KRAS directly complexes with ISG15, site-directed mutagenesis was performed to render the C-terminal domain of KRAS lysine-less. This led to stabilization of KRAS, despite knockdown of USP18. These studies were confirmed and extended in the in vivo setting of KRAS-driven lung cancers in KRASLA2/+ mice within the FVB strain background. We crossed these mice with USP18 null (USP18-/-) mice to obtain KRASLA2/+;USP18-/- compound mice. Strikingly, these compound mice had statistically significantly (P < 0.05) reduced lung cancers as compared to parental KRASLA2/+ mice. To establish the biological importance of this finding, USP18 immunohistochemical expression was compared in these engineered mice versus a second engineered mouse lung cancer model that was not driven by KRAS, but by aberrant cyclin E expression. USP18 immunohistochemical expression was substantially higher in the KRAS-driven than cyclin E-dependent lung cancers. To explore the translational relevance of this work, lung cancer tissue arrays were examined in 551 lung cancers where a clinical database was available. USP18 immunohistochemical expression was significantly (P < 0.001) higher in KRAS mutant adenocarcinomas as compared to wild-type cases. There was also a significant increase in USP18 expression in adenocarcinoma versus squamous cell carcinoma cases. Taken together, these studies broaden the role of USP18 as an antineoplastic target to combat lung cancers that harbor KRAS mutations.

#1256

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides effective therapeutic targets in cancer.

Amanda Taylor,1 Clare Adams,2 Xiao-yong Zhang,1 Victoria Gennaro,1 Justin Cotney,3 Gerald Shadel,3 Craig Cameron,4 Christine Eischen,2 Steven McMahon1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _Yale University School of Medicine, New Haven, CT;_ 4 _Pennsylvania State University, University Park, PA_.

The MYC transcription factor is the most dramatically overexpressed gene product in human cancer. However, defining the MYC-driven transcriptome critical to malignant transformation has remained a challenge, in part because MYC controls several thousand genes. Consequently, little progress has been made in the identification of sensitive nodes downstream of MYC that might be targeted therapeutically in order to interrupt its oncogenic signaling. To overcome this impediment, we performed a screen for downstream MYC effector pathways that are selectively linked to MYC's functional properties. Via this strategy, we identified the mitochondrial RNA polymerase (POLRMT) as a direct downstream target of MYC. POLRMT induction by MYC regulates transcription of the mitochondrial genome, as well as mitochondrial DNA replication. Recent studies have shown that POLRMT also interacts with a mitochondrial ribosomal RNA methyltransferase TFB1M to regulate mitochondrial ribosome assembly. Notably, blocking induction of POLRMT (or TFB1M) by MYC converts the cellular response to MYC activation from enhanced cell cycle progression to apoptotic cell death. Of immediate clinical relevance, compounds blocking mitochondrial gene expression at any of several steps cause acute synthetic lethality with oncogenic levels of MYC. Among these compounds, the mitochondrial targeting antibiotic Tigecycline is FDA-approved and well-tolerated in patients. Tigecycline eliminates tumor formation and dramatically improves survival in our animal model of MYC-driven lymphoma. Evidence presented here demonstrates that uncoupling of MYC's nuclear and mitochondrial transcriptomes exposes a point of exquisite vulnerability in tumor cells. Although targeting MYC as a therapeutic strategy has proven to be challenging in the past, our identification of a new node of sensitivity in MYC-driven cancers offers a potential for broad therapeutic implications.

#1257

A MYC-Aurka protein complex represents an actionable target in p53 altered liver cancer.

Dauch Daniel,1 Ramona Rudalska,1 Giacomo Cossa,2 Jean Charles Nault,3 Sandrine Imbeaud,4 Nisar P. Malek,1 Thomas Longerich,5 Stefan Laufer,6 Antti Poso,1 Jessica Zucman-Rossi,3 Martin Eilers,2 Lars Zender1. 1 _University Hospital Tübingen, Tübingen, Germany;_ 2 _University of Würzburg, Würzburg, Germany;_ 3 _INSERM, Paris, France;_ 4 _Inserm, Paris, France;_ 5 _University Hospital Aachen, Aachen, Germany;_ 6 _University of Tübingen, Tübingen, Germany_.

Myc oncoproteins are causally involved in the genesis of a large fraction of human tumors. Three closely related Myc proteins, c-myc, N-myc and L-myc, have been implicated in cancer, whereas c-myc was identified as an important oncogenic driver in frequent solid tumors.Applying transposon-based mosaic liver cancer mouse models and direct in vivo shRNA screening technology, we found that bypassing a latent p19Arf- and Aurka mediated G2/M cell cycle arrest is required for development of p53 altered liver carcinomas with activated Ras/MAPK signalling. The resulting tumors depend on high MYC levels for survival. A direct interaction of Aurka and MYC was identified in p53 altered liver cancer cells, which could be efficiently disrupted by conformation changing Aurka inhibitors, resulting in MYC degradation and cell death specifically of p53 altered human and murine hepatoma cells. Treatment studies in mouse models of p53 deficient therapy resistant liver cancer revealed marked therapeutic efficacy of conformation changing Aurka inhibitors, thus suggesting a new therapeutic strategy against this major lethal cancer.

#1258

Therapeutic potential of the cyclin- dependent kinase inhibitor on c-Myc overexpressing esophageal adenocarcinoma.

Sazzad Hassan, Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz, Urs V. Holzen. _Indiana University School of Medicine-South Bend, South Bend, IN_.

Introduction: Esophageal adenocarcinoma (EAC) is now the fastest growing cancer in the western world and most EAC patients present with widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. The transcription factor proto-oncogene c-Myc is a potent activator of tumorigenesis. Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype. c-Myc amplification has been shown to be frequent in esophageal adenocarcinoma and has been implicated in Barrett's carcinogenesis. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. In this study, we therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a cyclin-dependent kinase (CDK) inhibitor flavopiridol. Methods: Western blot analysis was done to see the expression of c-Myc and apoptotic signaling pathways in a panel of nine esophageal cancer cell lines. c-Myc overexpression and knockdown were performed using both genetic and novel chemical approaches. Cell viability assays were performed in 96-well plates using the colorimetric WST-1 reagent. Esophageal cancer tumors growth was measured in xenograft and a novel peritoneal disseminated metastatic survival model of immunodeficient mice. Results: Western blot analysis revealed frequent overexpression of c-Myc in EAC cell lines. In this panel of esophageal cancer cell lines tested more than 70% of EAC cell lines showed overexpression of c-Myc. When we tested these cell lines for their ability to form xenograft tumor and peritoneal dissemination, c-Myc overexpression correlated with accelerated EAC tumor growth in xenograft and peritoneal disseminated metastatic survival model of NOD/SCID mice. The xenograft tumor growth rate and formation rate of peritoneal cancer after injection of 5 million cells were highest in OE19 EAC cell line which showed the highest c-Myc expression. In addition, median animal survival with peritoneal dissemination was lowest for OE19 (55 days) whereas OACM5.1 C EAC cell line which had the lowest c-Myc expression didn't form any peritoneal tumor. EAC cell lines with elevated c-Myc expression are preferentially more sensitive to induction of apoptosis by CDK inhibitor flavopiridol compared to EAC cell lines with lower c-Myc expression. When we tested the role of c-Myc expression by upregulation/downregulation in this apoptotic effect we found that this effect is very much dependent on c-Myc expression. Conclusion: These results indicate that CDK inhibitor alone or in combination with other cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing EAC.

#1259

**MutT homolog 1 (MTH1) is upregulated in glioblastoma multiforme: its inhibition by siRNA or crizotinib results in impaired cell migration and tumor growth** in vivo **.**

Marco Timmer, Sara Gerdi Kannampuzha, Gabriele Röhn, Roland Goldbrunner. _University Hospital Cologne, Cologne, Germany_.

Introduction:

MutT homolog1 (MTH1) is required for the survival of cancer cells. These are characterized by irregular redox formation resulting in the production of reactive oxygen species which damage the deoxynucleoside triphosphate pool and subsequently the DNA. MTH1 inhibits the incorporation of oxidized bases into the DNA thus avoiding apoptosis of cancer cells. Our aim was to analyze the pathophysiological role of MTH1 in glioma.

Materials and Methods:

MTH1 expression was analyzed using quantitative PCR and Western Blot analysis in human glioma tissue (n=80). We compared peritumoral tissue with grade II, III, and IV gliomas. Moreover, we compared primary to secondary GBM and initial tumors with recurrent tumors after temozolomide treatment. U87 cell migration was tested after siRNA knock-down of MTH1 and treatment with the MTH1 inhibitor crizotinib by scratch assays. After orthotopic implantation of MTH1-siRNA treated U87 into rats, tumor size was measured after 8 days.

Results:

In RT-PCR and Western blot we found a significantly higher expression of MTH1 in primary and secondary glioblastoma than in lower grade brain tumor. Immunofluorescence staining of histological sections of glioblastoma showed also a higher level of MTH1 than in lower grade tumors. By nucleofection, we transfected siRNA MTH1 into the U-87 glioblastoma cells and a slower migration was investigated applying migration assay. Furthermore, treatment with the drug crizotinib inhibited MTH1 and decreased U-87 cell migration. The xenograft study

showed a slower tumor growth when U87 cells were transfected with MTH1 siRNA compared to normal U87.

Conclusion:

These results show that MTH1 plays an essential role in migration and proliferation of glioma cells resulting in clinically observed malignization of glioma and disease progression in recurrent glioblastoma. Moreover, MTH1 can be targeted by Crizotinib indicating a role for potential future therapies.

#1260

Polymerase kappa determines the sensitivity of MTH1 inhibitors to cisplatin-resistant cell.

Kumar Sanjiv,1 Helge Gad,1 Sean Rudd,1 Rachel Hurley,2 Patric Herr,1 José Manuel Calderón Montaño,1 Oliver Mortusewicz,1 Tobias Koolmeister,1 Sylvain Jaques,1 Estefanía Burgos Morón,1 Andreas Hoglund,1 Te-Chang Lee,3 Martin Scobie,1 Scott Kaufmann,2 John Weroha,2 Ulrika Warpman Berglund,1 Andrea Wahner Hendrickson,2 Thomas Helleday1. 1 _SciLifeLab, Karolinska Institutet, Stockholm, Sweden;_ 2 _Medical Oncology, Mayo Clinic College of Medicine, Rochester, MN;_ 3 _Institute of Biomedical Sciences, Taipei, Taiwan_.

Resistance is one the main reason for overall decrease in survival of cancer patient treated with cisplatin in different types of cancer. Cisplatin kills cancer cells by various mechanisms, but mainly through formation of inter- and intra stand crosslinks of DNA. Different types of translesion polymerase including Polymerase kappa (POLK) are involved in repair of DNA lesions. We observed high expression levels of POLK in cisplatin resistant bladder and ovarian cancer cells compared to parental cells. Due to its low proof-reading activity POLK can incorporate 8-oxo-dGTP into DNA. The MTH1 protein (Nudix hydrolase- NUDT1) sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Recently we have generated MTH1 inhibitors that damage the DNA and induce cancer specific cell death through incorporation of more oxidized dNTPs. We found cisplatin resistant bladder cancer cells (NTUB1/P) were more sensitive to MTH1 inhibitors in comparison to parental NTUB1 cells. As POLK is involved in incorporation of 8-oxo-dGTP into DNA, we hypothesized that high expression levels of POLK in cisplatin resistant cells make them more sensitive to MTH1 inhibitors as more 8-oxo-dGTP would be incorporated into DNA, resulting in more DNA damage and cell death in comparison to parental cells. Indeed, we observes higher induction of cleaved-PARP, γH2AX, cleaved-Caspase 3 and more annexin v positive cells in cisplatin resistant NTUB1/P cells in comparison to parental NTUB1 cells upon treatment with MTH1 inhibitors. MTH1 inhibitor also significantly delays the NTUB1/P xenograft tumor growth in comparison to vehicle treatment in immunosuppressive mice. Knocking down POLK in cisplatin resistant NTUB1/P cells by siRNA resulted in decreased incorporation of 8-oxo-dGTP and sensitivity to MTH1 inhibitors compared to non target control cells. Overexpression of POLK in NTUB1 and NTUB1/P cells results in further sensitization to MTH1 inhibitors. In conclusion elevated levels of POLK in cisplatin resistance cells determines increased sensitivity towards MTH1 inhibitors. Thus MTH1 inhibitors can be a potential promising therapy for the treatment of cisplatin resistant tumors in patients.

#1261

Nintedanib induces antitumor effects in triple-negative breast cancer cells by targeting SHP-1/p-STAT3-mediated signaling pathway.

Chun-Yu Liu,1 Chia-Han Lee,1 Chun-Teng Huang,2 Pei-Yi Chu,3 Wan-Lun Wang,1 Ling-Ming Tseng,1 Chung-Wai Shiau,4 Kuen-Feng Chen5. 1 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 2 _Yang-Ming Branch of Taipei City Hospital, Taipei, Taiwan;_ 3 _Show Chwan Memorial Hospital, Changhua City, Taiwan;_ 4 _National Yang-Ming University, Taipei, Taiwan;_ 5 _National Taiwan University Hospital, Taipei, Taiwan_.

Background:

Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive subtype that currently lack of specific targeted therapy. Previous studies have suggested that disturbing the oncogenic STAT3 signaling by activating a protein tyrosine phosphatase SHP-1 as a possible anti-cancer strategy. Our previous study also found that nintedanib (BIBF1120), a multi-kinase inhibitor that blocks mainly anti-angiogenic pathways and has been approved for idiopathic pulmonary fibrosis and for non-small cell lung cancer (in Europe), inhibits hepatocellular carcinoma cell growth independent of its anti-angiokinase activity (J Hepatology 2015). In this study, we tested nintedanib in TNBC cells and examined the underlying molecular mechanism by which nintedanib shows anti-TNBC effects.

Methods:

TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC-1395) were used for in vitro studies. Cell viability was examined by MTT assay. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. Purified SHP-1 proteins or cells expressing deletion N-SH2 domain or D61A point mutants (SHP-1 mutants with constant open form conformations) were used to investigate the potential effects of nintedanib on SHP-1. In vivo efficacy of nintedanib was tested in nude mice bearing orthotopic breast cancer cells xenografts.

Results:

MTT assays revealed nintedanib induced anti-proliferation in the 3 TNBC cell lines. Sub-G1 flow cytometric and western blot analysis showed that nintedanib induced apoptosis in association with downregulation of p-STAT3 and its downstream proteins such as cyclin D1 in both dose and time-dependent manners. Overexpression of STAT3 suppressed nintedanib-mediated apoptosis in MDA-MB-231 cells. Nintedanib further increased SHP-1 activity in purified SHP-1 proteins, suggesting that nintedanib may directly affects SHP-1 activity for p-STAT3 inhibition. Moreover, either specific SHP-1 activity inhibitor or SHP-1 siRNA reduced the apoptotic effects of nintedanib on MDA-MB-231 and MDA-MB-468 cells, validating the role of SHP-1 activation in nintedanib-induced apoptosis. Furthermore, nintedanib-induced apoptosis was attenuated in TNBC cells expressing SHP-1 mutants with constant open form conformations, including deletion mutants of N-SH2 domain or D61A mutants, suggesting that the auto-inhibition structure of SHP-1 mediated the effect of nintedanib. Importantly, intraperitoneal injection of nintedanib showed antitumor activity, increased SHP-1 activity and p-STAT3 downregulation in MDA-MB-231 xenograft tumors.

Conclusions

Our results suggest that nintedanib induced anti-tumor effect by SHP-1 dependent STAT3 inhibition in human TNBC cells. These findings may provide new signaling pathway insight into the inhibition of TNBC growth by nintedanib.

#1262

Primate-specific estrogen-induced long noncoding RNAs as targets for breast cancer treatment.

Donghong Ju,1 Daniel Xie,1 Juan Cai,1 Anton Scott Goustin,1 Mary A. Kosir,2 Leonard Lipovich1. 1 _Wayne State University, Detroit, MI;_ 2 _Barbara Ann Karmanos Cancer Institute, Detroit, MI_.

Purpose: Estrogen is a proliferative hormone that regulates the growth of estrogen receptor positive cells, including breast cancer cells. Although estrogen is a global regulator of protein-coding genes as well as non-coding RNA (ncRNA) genes, its effects on long ncRNA (lncRNA) genes, the most abundant class of human ncRNAs, remain poorly understood. We previously used custom microarrays and Taqman qRTPCR to identify 127 estrogen-responsive lncRNAs, and demonstrated a direct role for 25 of these in breast cancer cell viability and proliferation: estrogen-induced lncRNAs cause cell proliferation (reversed upon siRNA knockdown of these lncRNAs) and estrogen-repressed lncRNAs cause cell death (induced upon overexpression of those lncRNAs).

Furthermore, since over 60% of human lncRNAs, including CR593775, are primate-specific, eight additional primate-specific lncRNAs were used in functional assays to determine whether primate-specific lncRNAs are functional in breast cancer.

Methods/Results: We ablated two new estrogen-induced primate-specific lncRNAs with two independent siRNAs each and validated the knockdowns by qRTPCR. Post-transfection MTT and crystal violet cell proliferation assays revealed that for one lncRNA, both knockdowns reduced cell viability and proliferation. We also used Stellaris RNA fluorescent in situ hybridization (FISH) to determine that the primate-specific estrogen-repressed cell death inducer lncRNA BC041455 is cytoplasmic in MCF7 cells. In addition, CR593775, our top estrogen-induced lncRNA, does not regulate the viability and proliferation of nonmalignant MCF10A breast cells as compared to breast cancer cells that are estrogen receptor positive (T47D, MCF7) and negative (MDA-MB-231).

Conclusion: The primate-specific estrogen-induced lncRNAs are cytoplasmic targets for breast cancer treatment that leads to reduced proliferation and cell viability.

#1263

UCHL1 modulates uterine papillary serous cancer progression through interaction with cyclin B1.

Suet Ying Kwan, Samuel C. Mok, Rosemarie E. Schmandt, Kwong-Kwok Wong, Karen H. Lu. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction:

Uterine papillary serous carcinoma (UPSC) comprises less than 10% of all endometrial cancers but is the most aggressive subtype of endometrial carcinoma. Few prospective studies focus solely on this rare subtype, making it difficult to determine optimal treatment strategies. The purpose of this study was to identify novel therapeutic targets that could aid in the management of UPSC.

Methods:

Clinical and RNA sequencing data from The Cancer Genome Atlas (TCGA) was used to identify differentially expressed genes between the more benign endometrioid endometrial carcinoma (EEC) and UPSC that also correlated with overall patient survival. Ubiquitin C-terminal hydrolase 1 (UCHL1) upregulation and survival correlation was validated by immunohistochemical staining of an independent cohort of paraffin samples. UCHL1 silencing by siRNA in UPSC cell lines ARK1, ARK2 and HEC50 was performed to measure changes in cell proliferation, migration, and apoptosis. Luciferase-expressing ARK1 cells transduced with lentiviral doxycycline-inducible control shRNA or anti-UCHL1 shRNA were injected intraperitoneally into nude mice and monitored by in vivo bioluminescent imaging over time. The effect of UCHL1 silencing on cyclin B1 RNA and protein levels was measured, and the effect on cell cycle progression was analyzed by flow cytometry. ARK1 and ARK2 cells were treated with a drug combination of the UCHL1 inhibitor LDN-57444 and paclitaxel; synergistic effect was evaluated using CompuSyn.

Results:

RNA and protein expression of UCHL1 was significantly higher in UPSC than EEC. Kaplan-Meier analysis and cox regression of TCGA samples and an independent cohort of patient samples confirmed that UCHL1 expression correlated significantly with poorer overall survival in UPSC. siRNA-mediated silencing did not affect cell migration or apoptosis, but reduced cell proliferation in vitro. Following intraperitoneal injection of ARK1 cells in nude mice, control shRNA tumours exhibited significantly higher bioluminescence by week 10 than UCHL1-knockdown tumours. UCHL1 RNA expression correlated positively with cyclin B1 protein levels in the TCGA data set. UCHL1 silencing reduced cyclin B1 protein levels in ARK1, ARK2 and HEC50; this change was not due to reduced RNA expression. Cell cycle analysis after UCHL1 silencing showed a reduction in the percentage of cells in the S and G2/M phases, and inhibition by LDN-57444 delayed the entry of synchronized cells into G2/M. Combination treatment in vitro with LDN-57444 and paclitaxel was synergistic across a range of dose levels.

Conclusions:

These findings indicate that UCHL1 contributes to the aggressiveness of UPSC by stabilizing cyclin B1 and increasing tumor cell proliferation. Inhibition of UCHL1 alone or in combination with paclitaxel may be useful in the management of UPSC.

#1264

Targeting nuclear receptors and their coregulators in triple-negative breast cancer.

Ya-Fang Chang, Yuhao Wang, Geoffrey Greene. _The University of Chicago, Chicago, IL_.

Metastasis and chemoresistance remain significant challenges in treating triple-negative breast cancer (TNBC). Nuclear receptors (NRs) are ligand-inducible transcriptional factors that play essential roles in key biological processes from embryonic development to differentiation, regulation of gene expression, and homeostasis, and their dysregulation has been implicated in many different pathologies including cancer. Using MDA-MB-231 cells as a model, we performed a siRNA screening to determine the importance of NRs and their co-regulators in cancer cell proliferation and death of TNBC. A total of 84 siRNA pools, each targeting a different gene, were evaluated. To further validate the candidate genes, we transfected MDA-MB-231 cells with each of the four individual siRNAs comprising each pool. The effect of candidate genes on the invasion and chemoresistance of TNBC cell lines was also assayed in vitro. Among 4 candidate genes, we focused in greater depth on steroid receptor coactivator-3 (NCOA3), which is a member of the p160 steroid receptor coactivators and found to be overexpressed in breast cancer. We found that although NCOA3 knockdown moderately inhibited the tumor growth of TNBC cells (MDA-MB-231, BT549, and HS578), the addition of siNCOA3 and paclitaxel significantly decreased cell proliferation and increased caspase-3/7 activity. In transwell invasion assays, NCOA3 knockdown also significantly decreased the invasion of TNBC cells. These results suggest that targeting NCOA3 is worth investigating for the treatment of TNBCs.

#1265

Dysregulated tyrosine kinase Tyro3 signaling in acute myeloid leukemia.

Fatma Eryildiz,1 Jeffrey W. Tyner2. 1 _OHSU Institute of Environmental Health, Portland, OR;_ 2 _OHSU Knight Cancer Institute, Portland, OR_.

The undesirable outcomes of classical chemotherapy approaches in the treatment of cancer pathogenesis has directed scientists to find more targeted and less toxic therapies. Numerous studies of targeted therapies in cancer have demonstrated that finding target genes that are responsible for cancer pathogenesis can lead to novel targeted cancer therapies. Among these studies, tyrosine kinases have been prominent as effective gene targets. We have employed this approach to study the hematologic malignancy, Acute Myeloid Leukemia (AML).

In this study, we aimed to understand how human tyrosine kinase members, as functional drivers of leukemogenesis, promote tumor progression by activating downstream molecules. For this purpose, an RNAi assisted silencing assay was designed in which every individual tyrosine kinase member was silenced and impact on primary AML cell viability was assessed. Bioinformatic interpretation of the data revealed that TYRO3 is one of the genes showing significant sensitivity to silencing in 7.64% of AML cases tested (n=484). This data is compatible with previous studies showing aberrant expression of TAM family member tyrosine kinases in several human cancers. In addition, immunoblotting was performed to assess TYRO3 protein expression in several AML cell lines. Several AML lines demonstrated higher expression levels of TYRO3. In a strong correlation with this finding, TYRO3 siRNA was able to partially silence the TYRO3 expression especially in those cell lines that have higher expression levels of TYRO3 and due to this silencing we observed decreased cell viability relative to non-specific siRNA suggesting that high expression of TYRO3 provides a cell survival advantage in AML.

Even though signaling pathways of other TAM family members and their interactions are described in detail, TYRO3 downstream signaling pathway still remains quite undetermined which makes it an intriguing player in cell survival. There are not many small molecules designed to inhibit TAM tyrosine kinases, but our study shows that successful targeted inhibition of this family of receptor tyrosine kinases may represent a promising avenue to provide opportunities to improve therapeutic approaches for patients with AML.

[1] Druker, Brian J. "Imatinib as a paradigm of targeted therapies." Advances in cancer research 91 (2004): 1-30.

#1267

Identification and characterization of oncogenic methyltransferase ESOC1 as a diagnostic and therapeutic target for esophageal cancer.

Yataro Daigo,1 Atsushi Takano,1 Yusuke Nakamura2. 1 _Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 2 _Department of Medicine and Surgery, University of Chicago, Chicago, IL_.

Esophageal cancer is one of the most lethal malignancies of the digestive tract, and at the time of diagnosis most of the patients are at advanced stages. In spite of the use of current surgical techniques combined with various treatment modalities, such as radiotherapy and chemotherapy, the prognosis of esophageal cancer still remains poor. Therefore, further development of new anti-cancer drugs for this disease with minimum risk of adverse event is urgently awaited. To screen oncoantigens which could be used for the development of new diagnostic biomarkers as well as molecular targeted drugs and/or immunotherapy, we have established a screening system as follows; i) To identify overexpressed genes in esophageal cancers by genome-wide screening using the microarray representing 27,648 genes and pure populations of tumor cells taken from cancer tissues by laser microdissection, ii) To verify the candidate genes for their very low or absent expression in normal tissues by northern-blotting, iii) To validate the clinicopathological significance of their expression with tissue microarray covering hundreds of archived esophageal cancers, iv) To verify whether the target genes are essential for the growth or survival of cancer cells by RNAi and cell growth assays. During this process, we selected dozens of druggable oncoproteins with various enzymatic activities, and identified overexpression of metyltransferase ESOC1 (esophageal cancer-associated oncoprotein 1) in the majority of esophageal cancers and its scarce expression in normal tissues except testis. Immunohistochemical staining using tumor tissue microarrays consisting of 251 esophageal cancers demonstrated that ESOC1 expression was associated with poor prognosis (P = 0.0029 by log-rank test), and multivariate analysis confirmed its independent prognostic value (P = 0.0082). Suppression of ESOC1 expression with siRNA effectively suppressed the growth of cancer cells. ESOC1 has a SAM dependent methyltransferase domain, and we confirmed its methyltransferase activity for some small non-coding RNAs that were overexpressed in esophageal cancers. In addition, we confirmed that the exogenous expression of ESOC1 increased the growth activity of HEK293T cells, but the mutant ESOC1 did not have methyltransferase activity, and did not confer the growth promoting activity. Since ESOC1 plays a significant role in the cell growth and malignant nature of tumors, we suggest that ESOC1 is likely to be a prognostic biomarker and a potential therapeutic target for developing new anti-cancer drugs and cancer vaccines.

#1268

Externalized cytokeratin 8 : A relevant target at the crossroads of microenvironment and intracellular signaling.

Marie Alexandra Albaret,1 Claudine Vermot-Desroches,2 Arnaud Paré,1 Jean-Xavier Roca-Martinez,1 Jad Essely,1 Laetitia Gerossier,1 Johan Brière,1 Nathalie Pion,1 Lucie Malet,1 Boris Vuillermoz,2 Carine Rousset,2 Hichem-Claude Mertani,1 Pierre Saintigny,1 Jean-Jacques Diaz1. 1 _Cancer Research Center of Lyon, Lyon cedex 08, France;_ 2 _iDD biotech, Lyon, France_.

Accumulating evidence suggests the unusual presence of proteins at the external leaflet of plasma membrane of cancer cells while, in normal cells, these proteins are expressed in the intracellular compartments. They lack transmembrane domains and signal sequences that allow delivery to the plasma membrane and their role is not fully understood. As such, aberrantly externalized proteins represent a promising source of targets for cancer therapy. In order to find new targets for the treatment of metastatic colorectal cancer (CRC), we performed a comparative dynamic proteomic analysis of Isreco-1, a cell line characterized by a high invasiveness capacity. Among a set of proteins associated with invasion, we identified CK8, an intermediate filament protein of epithelial cells cytoskeleton involved in the maintenance of cell integrity of normal glandular epithelial cells. CK8 was shown to be expressed both intracellularly and associated at the plasma membrane of highly invasive Isreco-1 line (KRAS mutant) as well as in two other cell lines i.e. HCT116 (KRAS mutant) and HT29 (KRAS wild-type), by immunofluorescence. Using a biochemical approach, we demonstrated in all 3 cell lines, that plasma membrane-associated CK8 was strongly anchored to the plasma membrane, and subsequently referred as externalized CK8 (eCK8). eCK8 was co-localized with plasminogen and urokinase-type plasminogen activator (uPA) at the plasma membrane of Isreco-1 cells, consistent with its previously reported role as a plasminogen receptor. Among several murine monoclonal antibodies (MAbs) targeting specifically eCK8, one was able to reduce plasminogen induced invasion in vitro (Isreco-1) as well as tumor growth in vivo (Isreco-1 and HCT-116 subcutaneous xenograft models). It was specific of an extra-cellular portion of eCK8. Besides the inhibition of tumor growth, tumors were strikingly necrotic and characterized by apoptosis activation. No difference in terms of proliferation and microvascular density was observed. Altogether, our results suggest that in colorectal cancer, CK8 may be externalized and strongly anchored to the plasma membrane in highly invasive cells. eCK8 co-localize with plasminogen and uPA. Targeting a specific portion of eCK8 was associated with decreased tumor growth and apoptosis activation. We suggest that this particular domain of eCK8 is essential for bidirectional signaling, toward the tumor cell microenvironment by favouring the invasion process and the intracellular compartment by inhibiting apoptosis. Because the combination of cell invasion and inhibition of apoptosis are key mechanisms involved during metastasis, externalization of CK8 may represent a key event of the metastatic process and a relevant target in CRC, including in tumors with KRAS mutation.

#1269

Enhanced cytotoxicity in primary human metastatic melanoma cells via inhibition of the transient receptor potential melastatin-2 (TRPM2) channel.

David W. Koh, Steven D. Blake, Daniel P. Powell. _Ohio Northern University, Ada, OH_.

The transient receptor potential melastatin-2 (TRPM2) cation channel was previously identified as a potential target in various cancers, where its pharmacologic inhibition caused increased DNA damage and the selective eradication of cancer cells. In this study, we utilized several human primary metastatic melanoma cell lines, in which we analyzed TRPM2 and its isoforms, and evaluated the ability of TRPM2 inhibition to modulate cell death.

As TRPM2 is known to exist as a plasma membrane cation channel in noncancerous cells, we investigated the localization of TRPM2 in melanoma cells. In three lines of primary human metastatic melanoma cells, TRPM2 was localized in the nucleus, as compared to an extra-nuclear localization in noncancerous primary epidermal keratinocyes. Because TRPM2 exists in several isoforms in cancer cells, we investigated the cellular localization of these isoforms. Subcellular fractionations demonstrated that only full-length TRPM2 was localized to the nucleus in primary human metastatic melanoma cells, whereas two smaller isoforms were localized exclusively in the cytoplasmic fraction.

Treatment with the TRPM2 inhibitor, clotrimazole, caused decreased proliferation in all three lines of primary human metastatic melanoma cells. Further, treatment with the DNA alkylating agent, temozolomide (TMZ), led to increased levels of cell death in melanoma cells pretreated with clotrimazole, but not in noncancerous primary epidermal keratinocyes after pretreatment. Similar increases in cell death were observed after RNAi silencing of TRPM2 followed by TMZ treatment.

Taken together, this study demonstrated that TRPM2 inhibition selectively increases cell death in primary human metastatic melanoma cells. Further, the data suggests that, similar to our previous results in breast adenocarcinoma cells, full-length TRPM2 appears to have a novel role in melanoma cell growth and survival. The results therefore suggest that TRPM2 is a potential target in melanoma, where its inhibition may cause the selective eradication of metastatic melanoma.

#1270

Polysialyltransferase ST8SiaII as a target for neuroblastoma dissemination.

Sara M. Elkashef, Rida F. Saeed, Goreti Ribeiro Morais, Xiaoxiao Guo, Marcella Sini, Virginie F. Viprey, Mark Sutherland, Paul M. Loadman, Laurence H. Patterson, Steven D. Shnyder, Robert A. Falconer. _University of Bradford, Bradford, United Kingdom_.

Polysialic acid (polySia) is expressed on the surface of NCAM (neuronal cell adhesion molecule) on neuroendocrine tumours, notably neuroblastoma and small cell lung cancer, where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor prognosis and aggressive disease in neuroblastoma patients in the clinic[1]. SiRNA knockdown of polysialyltransferase (polyST) ST8SiaII, the enzyme primarily responsible for polySia synthesis in tumours, abrogates tumour cell migration and invasion. Besides brain regions with persistent neuronal plasticity, polySia is essentially absent from the body post-embryogenesis. PolyST is thus a selective and largely unexplored therapeutic target for neuroblastoma dissemination [1].

We have established a highly sensitive HPLC-based polyST inhibition assay, amenable to high-throughput screening. We report our efforts to further optimise this cell-free assay, and include details of our novel methodology to quantify cell-surface polySia expression. Having demonstrated in vitro that inhibition of polyST by a small molecule leads to a reduction in tumour cell migration [2], we designed and synthesised ST8SiaII inhibitors. Using isogenic cell lines (C6-STX: polySia+/polyST+ and C6-WT: polySia-/polyST-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y, IMR-32) these compounds were evaluated for their ability to reduce polySia expression, to modulate tumour cell migration and invasion in vitro. We have identified novel agents which significantly reduce polySia expression, tumour cell migration and invasion. These effects were only found in cell lines expressing ST8SiaII and polySia. Specificity of agents for polySTs over other members of the sialyltransferase (ST) family (i.e. α-2,3-ST and α-2,6-ST) was subsequently investigated using lectin differential labelling probes. Agents did not inhibit sialyltransferase activity.

We have investigated effects of agents on key intracellular signalling pathways. We demonstrated the effects of polyST inhibition on the dynamics of FAK and on ERK1/2, AKT, CREB and VEGFR-3 signalling. Furthemore, we have explored the behaviour of polySia-expressing cells under hypoxic conditions. Our data suggest that polySia is associated with a resistant phenotype, with C6-STX polySia-expressing cells demonstrating a survival advantage and additionally maintaining their migratory capacity under hypoxia (compared to WT cells, where migration is dramatically reduced).

In summary, we have demonstrated that polyST inhibition dramatically decreases cell-surface polysialylation, migration and invasion in vitro, under both normoxic and hypoxic conditions. This work paves the way for development of a novel therapeutic for the treatment of neuroblastoma.

[1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939; [2] Al-Saraireh YMJ et al., PLoS ONE, 2013, 8:e73366.

#1271

The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC).

Rossella Marullo,1 Haelee Ahn,1 Mariano Cardenas,1 Ari Melnick,1 Fengtian Xue,2 Leandro Cerchietti1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _University of Maryland, Baltimore, MD_.

BCL6 is a transcription factor that promotes lymphomagenesis by repressing target genes involved in DNA damage sensing and response. In NSCLC patients, BCL6 is amplified in 40% of Squamous Cell Carcinomas (197/501, TCGA) and in 2.2% of Adenocarcinomas (5/230, TCGA), suggesting a potential oncogenic role for BCL6 in lung cancer.

To determine whether BCL6 plays a role in sustaining NSCLC, we first measured the expression of BCL6 in a panel of 15 NSCLC cell lines and found BCL6 expression in 15/15 cell lines. Furthermore, in 7/15 cells lines (46%) we found BCL6 gene amplification. To gain insight into BCL6 function, we engineered two NSCLC cell lines with BCL6 amplification (H1299 and H838) to stably express BCL6-shRNA. BCL6 silencing resulted in 3-5 fold mRNA increase of key DNA damage response genes such as ATR, Chek1, p21 and p53. BCL6-mediated repression of DNA damage response genes has functional effects as BCL6 silencing resulted into i) G1 cell-cycle arrest as determine by flow cytometry analysis, ii) 1.5-2 fold increase in cell doubling time, iii) 40-60% reduction in colony formation. These results suggest that a major role of BCL6 in lung cancer could be to enable proliferation under genotoxic stress, a function that BCL6 exerts physiologically in B-cells during antibody generation. Under these premises, BCL6 inhibition should preferentially affect the proliferation of cells with elevated genomic instability. Thus, we measured the amount of chromosomal aberration harbored by the proliferating fractions of BCL6-shRNA cells compared to the isogenic parental cells using micronuclei assay. BCL6 silencing resulted in 30-40% reduction of micronuclei amount in the proliferating fraction of cells, supporting our hypothesis. Moreover, exposing NSCLC cells to DNA damaging agents leads to 2-3 fold increase in BCL6 expression regardless the presence of BCL6 amplification.

In lymphomas, BCL6 represses genes by recruiting co-repressors to form "repressosome" complexes in gene promoters and enhancers. In particular, repression of DNA damage response genes is achieved by the recruitment of three co-repressors (SMRT, N-CoR, and BCOR) to the BTB-domain of BCL6. To determine whether the BTB-domain of BCL6 is mediating the repression of DNA damage genes in NSCLC, we employed FX1085, a small molecule that specifically interacts with the BCL6 BTB domain and prevents the formation of the repressosome complex. Exposure to FX1085 resulted in 3-10 fold increase in ATR, Chek1, p21 and p53 mRNA levels and prevented the proliferation of NSCLC cells with elevated genomic instability. Moreover, FX1085 affected the survival of 6/15 NSCLC cell lines (40%) at GI50 doses comparable to those effective in lymphoma cells, indicating that BCL6 maybe a suitable therapeutic target in NSCLC.

Overall, our data indicate a role for BCL6 in sustaining NSCLC genomic instability and suggest that this protein may be a potential therapeutic target in this disease.

#1272

Apelin/Apj pathway for targeting ovarian tumor microenvironment.

Bharat Kumar Devapatla, Pharavee Jaiprasart, Samrita Dogra, Jihee Ha, Sukyung Woo. _University of Oklahoma, Oklahoma City, OK_.

Introduction: Ovarian cancer generates unique tumor microenvironment (TME) that promotes and enhances tumor progression and metastasis. Current therapy option for modulating ovarian TME is antiangiogenic therapy but its clinical benefit is limited; thus, alternative drug targets are needed for therapeutic durability. Apelin and its cognate receptor APJ is known to have roles in glucose metabolism, cardiovascular functions, and angiogenesis. We found that apelin/APJ are highly overexpressed in human ovarian tumors, but their functional role is unclear. We also found a significant upregulation of apelin/APJ in tumors that progressed after antiangiogenic therapy in preclinical ovarian cancer models. Our objective is to determine the functional roles and mechanisms of apelin/APJ pathway on promoting ovarian tumor angiogenesis and progression.

Methods: We examined autocrine and paracrine functions of apelin/APJ signaling on cell proliferation, migration, and tube formation using human ovarian cancer cells (SKOV3Apln and SKOV3Apj) and endothelial cells (HUVECApj). Phosphoproteome array was performed to evaluate the apelin/APJ downstream signaling. We characterized the effect of apelin or APJ overexpression on tumor development and response to VEGFR inhibitor in vivo. To confirm the clinical relevance of apelin/APJ in ovarian TME, we measured soluble apelin levels in patients' ascites and APJ expressions in human ovarian tumors using an immunohistochemistry.

Results: Apelin (10-100 ng/ml) induced mitogenic and chemoattractant effects on both cancer and endothelial cells. These effects were more prominent under hypoxic condition, reflecting the significance of apelin/APJ axis in TME. The aplein-mediated proliferative and migratory effects were inhibited by a pharmacological APJ inhibitor (ML-221) in a dose-dependent manner. High APJ expression resulted in enhanced pro-angiogenic activity in HUVEC. Overexpression of apelin/APJ also led to reduced response to antiangiogenic treatment in both HUVEC and cancer cells. Apelin signaling induces phosphorylation of AKT, STAT, CREB, PRAS 40, and AMPKα2 in SKOV3 cells. Our xenograft study indicates APJ-overexpressing tumors showed reduced response to sorafenib treatment compared to those control tumors. We found that APJ is mainly expressed in tumors and some extent in stromal component of human ovarian tumors. The median soluble apelin levels in ascites were 187 pg/ml (6.3 - 4,000 pg/ml).

Conclusions: Our results suggest that apelin/APJ may play an important role in promoting angiogenesis and progression in ovarian cancer and serve as an attractive pathway targeting ovarian TME.

#1273

A novel molecular target for lung cancer.

Edwin J. Velazquez,1 Evita G. Weagel,1 Wei Meng,1 Michelle H. Townsend,1 Alex Cummonck,1 Craig Chandler,1 Michael R. Downey,2 Richard Robison,1 Kim L. O'Neill1. 1 _Brigham Young University, Provo, UT;_ 2 _Intermountain Histopathology Central Laboratory, Utah Valley Medical Center, Provo, UT_.

The purpose of this study is to provide further evidence that Thymidine Kinase I (TK1) is selectively expressed on the surface of lung cancer cells, and therefore could be utilized as a potential molecular target. TK1 is an enzyme in the pyrimidine salvage pathway whose expression is closely correlated with cell proliferation and cell turnover. It has previously been shown that upregulation of TK1 is an early event in the development of most cancers. Additionally TK1 serum levels in cancer patients correlate with tumor progression and cancer aggressiveness. Moreover TK1 levels in the original tumors have also been shown to correlate directly with tumor recurrence thus making TK1 a useful prognostic marker in clinical oncology. Recent studies in our lab have provided evidence that TK1 is localized on the plasma membrane in lung cancer cells. Using scanning electron microscopy (SEM), flow cytometry, confocal microscopy and tissue staining, we confirmed the presence of TK1 on the cell surface of H460 cells. Using normal lymphocytes as a comparative control we found TK1 expression on lung cancer cells to be significantly increased compared to control cells. For this investigation, we used three custom designed monoclonal antibodies against human TK1 conjugated with FITC, namely CB1, A72 and A74. Using flow cytometry we confirmed the presence of TK1 on the cell surface, our data shows that H460 cells stained positive for TK1 (12% for A72, 19% for A74, and 29% for CB1). Furthermore, confocal microscopy indicated a positive fluorescent signal for A72, A74 and CB1, suggesting the presence of TK1 on the plasma membrane. SEM images of lung cancer cells and normal lymphocytes stained with TK1 antibodies and gold labeling reported a positive gold signal on H460 cells with relatively no signal from human lymphocytes. Immunohistochemistry staining of carcinoma tissue array panels compared with normal tissue array panels indicated a statistical significant difference of expression levels of TK1 between normal lung tissue and lung carcinoma (P value <0.05). Positive staining was considered if more than 5% of the cells were stained. There was negligible staining in normal lung tissue. Using these techniques we confirmed that TK1 is selectively expressed on the surface of H460 cells and not on normal cells. These results suggest that TK1 could be used as a possible molecular target in lung cancer therapy. Further studies are currently ongoing to monitor TK1 expression and progression to help to elucidate the mechanisms behind it's over expression in cancer cells and to track its progression and movement from the cytosol to the cell surface.

#1274

Characterization of UROC1 protein as a novel prognostic biomarker and a therapeutic target for oral cancer.

Thang Manh Phung,1 Atsushi Takano,2 Yoshihiro Yoshitake,3 Masanori Shinohara,3 Yohinori Murakami,2 Yataro Daigo1. 1 _Shiga University of Medical Science, Otsu, Japan;_ 2 _The Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 3 _Kumamoto University, Kumamoto, Japan_.

Oral cavity cancer (OCC) is the most common cause of cancer-related death worldwide and shows poor clinical outcome after standard therapies. Therefore, new prognostic biomarkers and therapeutic targets for OCC are urgently awaited. This study aims to identify novel prognostic biomarkers or therapeutic targets for OCC. To screen the new prognostic biomarkers and therapeutic targets for OCC, we selected from our original gene expression database up-regulated in oral cancer 1 (UROC1) that appeared to encode cytoplasmic protein as a candidate. Immunohistochemical analysis using tissue microarray covering 99 OCC tissues confirmed that UROC1 protein was expressed in 67 cases (68%) of 99 OCC tissues, but not in normal oral epithelia. High levels of UROC1 expression was significantly associated with poorer prognosis for OCC patients (P = 0.0299, log-rank test). In addition, knockdown of endogenous UROC1 expression by siRNAs significantly inhibited the growth and invasion of OCC cells, whereas the induction of exogenous UROC1 expression enhanced the growth and invasive ability of OCC cells. In addition, flow cytometric analysis and various apoptosis assays of these tumor cells transfected with siRNAs for UROC1 revealed a significant induction of apoptosis. Assays using phosphor-antibody array and lysates from cells transfected with siRNAs or plasmid for UROC1 and subsequent functional analyses revealed that UROC1 could regulate the growth, survival, and invasion of OCC cells probably through activation of insulin-like growth factor 1 receptor (IGF1R) pathway. These data suggest that UROC1 could be a novel prognostic biomarker and a therapeutic target for OCC.

#1275

Suppression of platelet aggregation and tumor metastasis by anti-podoplanin mAbs recognizing a novel CLEC-2 binding domain.

Takaya Sekiguchi, Ai Takemoto, Naoya Fujita. _Japanese Foundation for Cancer Research, TOKYO, Japan_.

Tumor-induced platelet aggregation promotes tumor growth and hematogenous metastasis by secreting growth factors from platelets, by avoiding immune system and shear stress, and by stimulating the formation of tumor emboli in microvasculature. Thus, tumor-platelet interaction could be a promising target for cancer therapy. We previously identified a sialoglycoprotein podoplanin, also known as Aggrus, as a key factor in tumor-induced platelet aggregation enhancing tumor metastasis. Podoplanin is expressed in various cancers and reported to be a candidate for cancer stem cell marker. Podoplanin-induced platelet aggregation depends on binding to its platelet receptor CLEC-2 . Here, we report the discovery of a novel platelet aggregation-stimulating domain PLAG4 in human podoplanin critical for the binding to CLEC-2. Mutant analyses indicated that PLAG4 exhibits a predominant CLEC-2 binding ability and platelet-aggregating function when compared to the previously reported PLAG3. Moreover, mutation of both PLAG3 and PLAG4 domains could almost completely attenuate its CLEC-2 binding ability and its platelet-aggregation activity. To validate the inhibitory effect against PLAG4 domain, we established anti-PLAG4-neutralizing monoclonal antibodies (mAbs), PG4D1 and PG4D2. Both mAbs recognized PLAG4 domain and exhibited a high affinity to podoplanin. We confirmed those neutralizing ability against PLAG4-CLEC-2 interaction and platelet aggregation. Remarkably, the administration of each mAbs significantly suppressed pulmonary metastasis of podoplanin expressing tumor.

Taken together, we suggest that PLAG4 is the novel CLEC-2 binding and platelet aggregation-stimulating domain on podoplanin and has a role in podoplanin-mediated metastasis formation. Blocking the podoplanin-CLEC-2 interaction targeting PLAG3 or PLAG4 would be a promising strategy for suppressing hematogenous metastasis.

#1276

Pannexin 1 regulates rhabdomyosarcoma tumor growth: a potential novel therapeutic target.

Xiao Xiang, Stephanie Langlois, Marie-Eve St-Pierre, Jessica Barre, Tammy Le Pham, Kyle N. Cowan. _University of Ottawa, Ottawa, Ontario, Canada_.

Rhabdomyosarcoma (RMS) is a skeletal muscle-derived soft tissue sarcoma for which a novel therapeutic strategy is greatly needed. RMS cells have lost the ability to terminally differentiate thus proliferating indefinitely. Promoting their redifferentiation to skeletal muscle tissue is thus a promising therapeutic approach. We have recently shown that levels of Pannexin 1 (Panx1) are below detectable limits in undifferentiated myoblasts, but become highly upregulated during their differentiation and indeed promote this process. We thus hypothesize that the levels of Panx1 are deregulated in RMS and that restoration of Panx1 levels may induce differentiation of RMS cells thereby alleviating their malignant properties. Briefly, our results demonstrate that both Panx1 transcript and protein are at very low levels in, both embryonal and alveolar RMS tumor specimens and patient-derived cell lines similar to that seen in fetal skeletal muscle tissue and undifferentiated myoblasts, respectively. In vitro functional analyses have revealed that while re-introduction of Panx1 in RMS cell lines led to their partial differentiation, it significantly inhibited their rate of proliferation and migration. Moreover, re-expression of Panx1 in RMS cell lines prevented both spheroid formation and growth, and induced RMS cell apoptosis. Based on this in vitro data, pre-clinical orthotopic xenograft studies are currently underway to assess the ability of Panx1 to inhibit tumor onset and growth, and induce tumor regression in vivo. Our preliminary results thus far have shown that Panx1 over-expression significantly suppresses RMS xenograft tumor growth. Taken together, these results indicate that restoration of Panx1 levels in RMS reduces its malignant properties. This, together with further investigation into the tumor-suppressive mechanism by which Panx1 mediates its effects, will establish Panx1 as a novel therapeutic target for RMS.

#1277

Neural crest-like gene FOXD1 plays a role in melanoma cell migration and invasion.

Huizi Wu,1 Lionel Larribere,1 Kasia Weina,1 Nathalie Knappe,1 Christoffer Gebhardt,2 Viktor Umansky,1 Jochen Utikal2. 1 _Skin cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Mannheim, Germany_.

Melanoma is a highly aggressive skin cancer arising from transformed melanocytes. Despite tremendous efforts and considerable progress in clinical treatment, melanoma remains a deadly disease characterized by abundant treatment resistance due to high phenotype plasticity of melanoma cells. In order to evade therapy, melanoma cells can undergo phenotype switching from a differentiated state towards a dedifferentiated and more stem cell-like state. Recently, some melanoma cells with neural crest-like characteristics were shown to be able to sustain tumor growth and to give rise to distant metastases.

Here, we use the technique of directed differentiation of human induced pluripotent stem cells (hiPSCs) to characterize the human neural crest cell population in vitro and to identify potential genes that are reactivated during melanoma progression. We differentiated hiPSCs towards melanocytes via the neural crest stage. Expression analyses revealed an overexpression of the Forkhead Box D1 (FOXD1) transcription factor in neural crest cells and importantly, increased FOXD1 expression was also found in melanoma cells compared to normal human melanocytes. Interestingly, in invasive and less proliferative melanoma cells, FOXD1 expression was elevated compared to highly proliferative and less motile cells, indicating a potential role of this factor in motility acquisition. To gain deeper insight into the expression pattern of FOXD1 in clinical samples, a protein expression analysis using tissue microarrays was performed determining FOXD1 expression in melanocytic nevi, primary and metastatic melanoma samples. Surprisingly, in benign melanocytic nevi, FOXD1 was dominantly localized in the nucleus whereas a shift towards a cytoplasmic localization was observed in both, primary and metastatic melanomas. Loss of FOXD1 gene in melanoma cells also revealed the importance of this gene in melanoma cell migration and invasion. Further characterization of FOXD1 and its role in melanoma progression may help to understand the phenotypic plasticity of melanoma cells, which is required for overcoming resistance in anti-melanoma therapy.

In conclusion, FOXD1 is overexpressed and aberrantly localized in malignant melanoma cells compared to melanocytes. Our data indicate its function in melanoma migration and invasion. FOXD1 might be an interesting target in melanoma therapy.

#1278

Popdc proteins: novel targets in inhibiting breast cancer cell migration and proliferation.

Johanna N. Amunjela, Steven J. Tucker. _University of Aberdeen, Aberdeen, United Kingdom_.

Tumour growth and metastasis are major challenges in cancer treatment and metastasis is the primary cause of death in more than 90% of cancer patients with solid tumours (Gupta & Massagué 2006). This necessitates identification and validation of clinically relevant anticancer therapeutic targets that result in maximum clinical efficacy with minimal side-effects when manipulated by drugs. Our research project has identified Popeye domain containing (Popdc) proteins as promising potential therapeutic targets in the treatment of breast cancer.

Popdc proteins are a class of membrane-tethered proteins encoded by the three members of the Popdc gene family Popdc1, Popdc2 and Popdc3. Their suppression has been correlated with disease progression and poor clinical outcomes in various cancers including breast cancer, gastric cancer, lung cancer, hepatocellular carcinoma and colorectal cancer (Kim et al., 2010, Luo et al., 2012, Williams et al., 2011, Wang et al., 2011). Loss of Popdc protein expression has been correlated with increased cancer cell migration, invasion, metastasis, drug-resistance and poor patient survival (Luo et al., 2012, Williams et al., 2011, Han et al., 2014). In breast cancer, Popdc1 suppression has been reported in breast carcinoma cells while upregulation of Popdc2 has been linked to the mechanism by which arsenic trioxide mediates breast cancer cell death (Williams et al., 2011, Wang et al., 2011).

Our use of various assays including single-cell migration assays, cell population migration assays, cell proliferation assays and fluorescent imaging strongly implicate Popdc proteins as drivers of cancer cell migration and proliferation. Expression patterns of Popdc proteins revealed high expression levels in non-malignant (HMepC) breast cells and low expression in breast cancer (MCF7) cells. This suggests that loss of Popdc protein expression correlates with an increasingly malignant phenotype. Furthermore, knocking down Popdc1, Popdc2 and Popdc3 in MCF7 breast cancer cells significantly promoted cell migration both at single cell and at cell population level while Popdc2 knockdown significantly promoted unstimulated MCF7 cell division. Taken together, this strong data set suggests that Popdc proteins play significant roles in the migration and proliferation of different cancer cell lines and that they indeed represent promising potential therapeutic targets in reducing breast cancer cell division and migration.

#1279

Sigma-2 receptor-mediated cell death is altered by cell density in human SK-N-SH neuroblastoma 2-D and 3-D cultures.

Cheri Z. Liu, Vira Behnam, Wayne D. Bowen. _Brown University, Providence, RI_.

Sigma-2 receptors are highly expressed in tumor cell lines compared to normal cells and are more highly upregulated in proliferative than in quiescent cells. Sigma-2 receptor agonists induce apoptotic cell death. Here, we investigate the effect of cell confluence at the time of harvest, cell plating density, and 3-D cell aggregation on the cytotoxic potency of the irreversible sigma-2 receptor partial agonist CM572. Sensitivity to sigma-2 receptor agonists has in practice appeared to vary with cell confluency, an effect likely due to proliferative state. To examine 2-D SK-N-SH cell culture sensitivity to CM572, we varied two parameters: 1) cell confluence at the time of harvest (cells harvested upon reaching 60% confluency or cells harvested after remaining at 100% confluency for 3 days) and 2) cell plating density (low: 7,000, medium: 15,000, or high: 30,000 cells/well). Cells were treated with CM572 24 h after plating. Using MTT assay, CM572 EC50 values for cells harvested at 60% confluence were similar regardless of cell plating densities (μM): 4.92 ± 2.24, 7.6 ± 1.68, and 6.06 ± 1.30 for cells plated at low, medium, and high densities, respectively. In contrast, CM572 EC50 values for cells harvested at 100% confluence for 3 days were significantly dependent on plating density (µM): 17.62 ±7.62, 35.33 ± 35.3, and >50 for cells plated at low, medium, and high densities, respectively. [3H]DTG Scatchard analysis of cells harvested at 60% confluence vs. 100% confluence for 3 days revealed no significant difference in sigma-2 receptor number: Bmax = 212.3 ± 34.22 and 173.0 ± 4.31 fmol bound/mg protein, respectively. The effect of CM572 on cell aggregation and cytotoxicity was additionally studied in 3-D SK-N-SH cultures. Cells were seeded into rod-shaped agarose wells, to which they could not adhere but only self-aggregate over a period of 24 h. Subsequent treatment with CM572 over 24-72 h significantly reduced 3-D aggregation, which was defined as an increase in the length of the 3-D culture rod after aggregation. However, treatment of these 3-D cultures with up to 70 μM CM572 for 24-72 h resulted in cytotoxicity levels of less than 10%, as measured by the LDH assay. This indicates a significant decrease in CM572 potency compared to the 2-D EC50. Therefore, sensitivity to CM572 decreases both after cells are confluent for 3 days at 100% in 2-D culture and in cells that form aggregates in 3-D culture. These results show that sensitivity of SK-N-SH cell cultures to CM572 is dependent on cell density, suggesting that CM572 may cause cell death in a cell-cycle dependent manner. This may be related to SK-N-SH cells transitioning from a proliferative to quiescent state when they are confluent in 2-D culture or form aggregates in 3-D culture. Taken together, these results suggest that cytotoxic sigma-2 receptor ligands such as CM572 may be more potent in proliferating cancer cells and/or cells leaving tumor masses in the process of metastasis.

#1280

The intersection of the PI3K/mTOR and HIPPO pathways: a potential therapeutic target for medulloblastoma.

Jennifer S. Ronecker, Paul Lee, Sudeepta Sridhara, Michael LaBagnara, Raj Murali, Meena Jhanwar-Uniyal. _Department of Neurosurgery, New York Medical College, Valhalla, NY_.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor; it is genetically defined into 4 subgroups which defines the clinical course of the disease. Despite current treatment modalities such as surgical resection, radiation, and chemotherapy, there are still significant morbidities and adverse side-effects. Recently defined genetic pathways could possibly offer a better management and treatment of patients with MB. The HIPPO signaling pathway has recently been shown to intersect with PI3K and Sonic Hedgehog (SHH) pathways to modulate mechanistic target of rapamycin (mTOR), an important regulator in cell growth and migration. The aim of this study is to elucidate the interaction of Hippo and Akt/mTOR signaling pathways with SHH pathways in MB, and define the potential downstream targets of therapy by using combined inhibitors. In order to achieve our goals, we evaluated the expression of activated AKT/mTOR in MB tumors using immunohistochemistry (IHC), along with the expression of transcriptional co-activators yes-associated protein (YAP), a down-stream effector of the HIPPO pathway. Scratch and chemotactic migration were used to assess the motility of MB cells following treatment with inhibitors of the aforementioned pathways. Cell cycle analysis and proliferation were measured using EdU and MTT techniques, respectively. YAP-SiRNA treatment was used to inhibit HIPPO pathway. MicroRNA-29, which mediates the mTOR pathway by modulating PTEN levels, was investigated. Results demonstrated: 1. A significant number of MBs expressed activated pAKTSer473 and pmTORSer2448, along with YAP, the downstream effector of the HIPPO pathway; 2. Treatment with inhibitors of PI3K/AKT (LY294002) and mTOR (rapamycin), given with EGF, significantly reduced cellular motility; 3. SHH inhibitor cyclopamine given with LY294002 or rapamycin significantly reduced cellular motility; 4. Cell proliferation was suppressed by inhibition of the HIPPO pathway using 3 unique 27mer duplexes of YAP1 siRNA treatments; 5. Combined treatment of cyclopamine with either LY294002 or rapamycin caused maximum suppression of cellular growth. In conclusion, these results demonstrate that the Hippo and PI3K/Akt/mTOR pathways are over-expressed in MB, and that the combined targeting of mTOR/HIPPO pathway with SHH inhibitor may provide alternative strategies for the treatment of MB, and offers a targeted therapy for improved prognosis.

#1281

Prostaglandin D synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ.

Masakazu Yashiro, Tatsunari Fukuoka, Haruhito Kinoshita, Go Masuda, Kishu Kitayama, Yuichiro Miki, Tamami Morisaki, Tsuyoshi Hasegawa, Kosei Hirakawa. _Surgical Oncology, Osaka City Univ. Medical School, Osaka, Japan_.

Background & Aims: The antitumor activity of prostaglandin (PG) D2 has been demonstrated against some types of cancer, including gastric cancer. However, exogenous PGD2 is not useful from a clinical point of view because it is rapidly metabolized in vivo. The aim of this study was to clarify the antitumor efficacy of an alternative, PGD2 synthase (PGDS), on gastric cancer cells. This is the first report showing that PG synthase is a promising agent for the treatment of gastric carcinomas that express peroxisome proliferator-activated receptor γ (PPARγ).

Methods: The effects of PGD2 and PGDS on the proliferation of gastric cancer were examined in vivo and in vitro for five human gastric cancer cell lines. The expression levels of PGD2 receptors and PPARγ were evaluated by RT-PCR. The effects of PGD2 and PGDS on the expression of c-myc and cyclin D1 were examined by Western blotting in the presence or absence of a PPARγ antagonist.

Results: PPARγ was expressed in gastric cancer cell lines, but PGD2 receptors were not. PGD2 and PGDS significantly decreased the proliferation of gastric cancer cells that highly expressed PPARγ. PGDS increased the PGD2 production of gastric cancer cells. A PPARγ antagonist significantly suppressed the growth-inhibitory effects of PGD2 and PGDS. Expression of c-myc and cyclin D1 was significantly decreased by PGD2; this inhibitory effect was suppressed by the PPARγ antagonist. Both PGD2 and PGDS significantly decreased subcutaneous tumor growth in vivo. Tumor volume after PGDS treatment was significantly less than after PGD2 treatment. PGD2 decreased the proliferation of gastric cancer cells through PPARγ signaling.

Conclusion: PGDS and PGD2 decreased the proliferation of gastric cancer via the PPARγ pathway. PGDS is a promising therapeutic agent for gastric cancer and PPARγ might be a predictive biomarker for PGDS treatment in gastric cancer. 

### Regulation of Anticancer Drug Effects

#1282

Combination of PI3K targeting with cetuximab and irradiation: a preclinical study on an orthotopic xenograft model of head and neck cancer.

Gérard Milano, Alexandre Bozec, Nathalie Ebran. _Ctr. Antoine Lacassagne, Nice, France_.

The PI3-kinase (PI3K)/AKT/mTOR signaling pathway is of particular interest in head and neck squamous cell carcinoma (HNSCC). Its activation has been identified as an important mechanism implicated in tumor progression and resistance to epidermal growth factor receptor (EGFR) inhibitors. The anti-EGFR monoclonal antibody cetuximab (Cet), radiotherapy (RT) and their combination are widely used in the therapeutic management of patients with HNSCC. Inhibition of the PI3K/AKT/mTOR signaling pathway has been shown to potentialize the effects of anti-EGFR agents and RT. We conducted a preliminary in vitro study demonstrating synergistic effects of Cet followed by the PI3K inhibitor BKM120 (Buparlisib) in both PI3KCA wild-type and mutated cells. The aim of the present study was to investigate the effects of combining BKM120 with Cet and RT on an orthotopic xenograft model of HNSCC.

In the present study we have used an orthotopic (floor of the mouth) model of human HNSCC to measure the effects of a treatment including BKM120 (40 mg/kg, 5 days/week, p.o.), Cet (2,5 mg/kg/day, 1 day/week, i.p.) and RT (6 Gy/dose, 3 days/week), administered alone or in combination. Investigations were performed using the human PI3KCA-mutated (H1047R) HNSCC cell line, CAL33, injected into the mouth floor of nude mice. The tumor growth was followed by IVIS imagery on days 3, 7 and 12 and tumor size was checked with a caliper ruler on day 13 after animal euthanasia.

As compared with the control, the BKM120-Cet and the BKM120-Cet-RT combinations led to the highest tumor inhibition and induced almost complete tumor growth arrest (p < 0.001). The addition of BKM120 and Cet to RT significantly enhanced the impact of RT alone on tumor growth (p < 0.001). The highest inhibitory effects of treatments on cell proliferation (Ki67 labelling), MAPK (pERK labelling) and PI3K/AKT/mTOR (pS6R labelling) signaling pathways were found with the BKM120-Cet association. RT alone (p=0.10) tended to activate the MAPK pathway but the association of BKM120 and Cet to RT inhibited the RT-induced activation of the MAPK pathway (p=0.03).

In conclusion, in this orthotopic HNSCC model, the combination of BKM120 with Cet and RT produced synergistic effects on tumor growth. These results could serve as a strong preclinical basis for innovative treatments combining PI3K inhibition with anti-EGFR therapies and RT in patients with HNSCC.

#1283

Identification and activity of novel GLUT1 inhibitors in hepatocellular carcinoma.

Bhaskar Bhattacharya,1 Sanamerjit S. Mann,1 Min Ji Han,1 Sarah HH Low,1 Gim Hwa Tan,1 Barry E. McGuinness,2 Sarah C. Trewick,2 Phillip M. Cowley,2 Alan Wise,2 Richie Soong1. 1 _Cancer Science Inst. of Singapore, Singapore, Singapore;_ 2 _IOMET Pharma, Edinburgh, United Kingdom_.

Hepatocellular carcinoma (HCC) is an endemic disease globally, with a 5-year survival rate of 15%. Current treatment of late-stage HCC involves the use of chemotherapy and sorafenib, a multi-kinase inhibitor. Metabolic studies have indicated HCC has a high glycolysis rate, and is surrounded by a hypoglycemic microenvironment. Given the need for therapeutic options for HCC and its glycolytic nature, this study explored the activity of novel GLUT1 inhibitors in varied glucose concentrations. To assess target inhibition, [3H] deoxy-D-glucose (2DG) uptake assays were performed in HEK293 cells stably over-expressing GLUT1-4 individually. Six HCC cell lines were cultured in media with standard high glucose (HG:25mM) and low glucose/lactic acidosis (LGLA: 5mM glucose/20mM lactic acid) to mimic the hypoglycemic microenvironment. IC50s were assessed using the SRB assay, and other phenotypes using standard methods. Four novel GLUT1 inhibitors (IOM1-4) from the same chemical series were identified from diversity screening. The compounds exhibited differential potencies to GLUT proteins at the sub-µM level. The inhibitors reduced the uptake of 2DG, the extrusion of lactate, and increased apoptosis. In HCC cells, IC50s were lowest with IOM1 and 2 and highest with IOM4 (Table 1). HEP3B cells displayed exceptional sensitivity to the inhibitors. The ranking of potency according to the IC50s correlated with the 2DG uptake studies. There was no correlation between GLUT protein levels and IC50s. Significant differences in sensitivity to inhibitors were observed in cells cultured in HG and LGLA conditions. Cells cultured in LGLA had consistently lower glucose uptake compared those in HG conditions. In LGLA conditions, cells with low levels of reactive oxygen species (ROS) were more sensitive to GLUT1 inhibitors compared to cells with high ROS levels. In conclusion, GLUT1 inhibition can be influenced by microenvironment glucose levels and could be a strategy for HCC treatment.

IC50 values (uM) for respective inhibitors in respective HCC cell lines

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|  | |

CELL LINES | |  | |

Inhibitor | Culture | C3A | HEP3B | HUH7 | PLC | SKHEP1 | SNU449

IOM1 | HG | >5.0 | 0.8±0.04 | 0.3±0.06 | >5.0 | 1.4±0.45 | 1.7±0.38

IOM1 | LGLA | >5.0 | 0.2±0.13 | 4.0±1.20 | >5.0 | 0.1±0.02 | 1.6±0.28

IOM2 | HG | 4.7±0.44 | <0.01 | 1.0±0.56 | 2.4±0.69 | 0.3±0.05 | 0.9±0.42

IOM2 | LGLA | >5.0 | 0.1±0.08 | 0.2±0.09 | 3.2±1.29 | 0.1±0.02 | 1.3±0.42

IOM3 | HG | >5.0 | 0.2±0.04 | 0.9±0.22 | 2.3±0.25 | 1.1±0.64 | 1.6±0.40

IOM3 | LGLA | >5.0 | <0.01 | 2.7±1.29 | >5.0 | 0.4±0.09 | 2.3±1.13

IOM4 | HG | >5.0 | 2.8±0.35 | 4.3±1.49 | 4.6±1.40 | 4.7±0.85 | >5.0

IOM4 | LGLA | >5.0 | 1.4±0.49 | 3.8±1.10 | >5.0 | 3.1±0.56 | >5.0

#1284

Lurbinectedin reduces tumor-associated macrophages and the production of inflammatory cytokines, chemokines, and angiogenic factors in preclinical models.

Paola Allavena,1 Cristina Belgiovine,1 Manuela Liguori,1 Ezia Bello,2 Roberta Frapolli,2 Carlos M. Galmarini,3 Maurizio D'Incalci2. 1 _Istituto Clinico Humanitas IRCCS, Rozzano (Milan), Italy;_ 2 _IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy;_ 3 _Pharmamar SA, Colmenar Viejo, Spain_.

Lurbinectedin, currently undergoing clinical evaluation in ovarian, breast and small-cell lung cancer patients, inhibits active transcription. The drug is structurally related to trabectedin containing the same pentacyclic skeleton of the fused tetrahydroisoquinoline rings, but differing by the presence of a tetrahydro-B-carboline replacing the additional tetrahydroisoquinoline of trabectedin. We investigated whether lurbinectedin has the ability to regulate the inflammatory tumor microenvironment in vitro and in vivo. Human purified monocytes were highly sensitive to lurbinectedin (IC50: 5-10 nM) and, after drug treatment, underwent caspase-8-dependent apoptosis. Furthermore, in vitro, lurbinectedin significantly inhibited the production of selected inflammatory chemokines (CCL2, CXCL8) and VEGF by stimulated monocytes and liposarcoma cell lines. Administration of lurbinectedin to mice bearing a murine fibrosarcoma -resistant to this compound in vitro- resulted in significant anti-tumor activity (T/C value around 50%). The analysis of immune cells of blood spleen and tumor tissues during treatment with lurbinectedin revealed a significant and selective decrease in the subset of monocytes and macrophages, including tumor-associated macrophages (TAM). A gene expression analysis of monocytes treated with lurbinectedin indicated a modulation of the transcriptional program in LPS-stimulated human monocytes. Overall, these results indicate that lurbinectedin affects the inflammatory micro-environment, with a selective apoptotic-inducing effect on mononuclear phagocytes and a specific inhibition of production of inflammatory cytokines.

#1285

Tumor associated macrophages can process antibody-drug conjugates and contribute to antitumor activity in preclinical xenograft models.

Fu Li, Michelle Ulrich, Mechthild Jonas, Germein Linares, Xinqun Zhang, Lori Westendorf, Dennis Benjamin, Che-Leung Law. _Seattle Genetics, Bothell, WA_.

The primary mechanism of antibody-drug conjugates (ADCs) is the targeted delivery of a cytotoxic payload via cancer antigen mediated internalization. However, stromal components in the tumor microenvironment may also play a role in ADC penetration, distribution, and processing. Here, we study the potential roles of Fc-FcγR interaction between tumor-associated macrophages (TAMs) and ADCs in the antitumor activity observed in xenograft models.

In the CD30+ L428 Hodgkin lymphoma (HL) model, the anti-CD30 ADC (cAC10-vcMMAE) and a non-binding control ADC (h00-vcMMAE) showed similar antitumor activity over a two-week period post treatment initiation. The antitumor activity was correlated with payload release, as h00-vcMMAE produced intratumoral MMAE concentration comparable to cAC10-vcMMAE in this time frame. Histopathology analysis of L-428 xenografts revealed high abundance of TAMs, leading to the hypothesis that TAMs can internalize and process ADCs for payload release. Using immunohistochemistry and flow cytometry, we confirmed that h00-vcMMAE bound to macrophage cell lines and TAMs. We further examined the presence of TAMs in additional xenograft models and correlated that to the antitumor activity of non-binding h00-vcMMAE. High levels of TAMs were observed in the KM-H2 HL and BR620 breast cancer models that were sensitive to h00-vcMMAE. In contrast, xenograft models with much fewer TAMs (DOHH2, SU-DHL8, and Karpas-299) did not respond to h00-vcMMAE treatment. Interestingly, h00-vcMMAF, releasing a membrane non-permeable payload, had no activity on the L-428 tumor model. These data suggest TAM-processed drug can mediate bystander tumor killing when the released payload is membrane permeable.

To evaluate whether Fc-FcγR interaction plays a role in the ADC uptake by TAMs, we mutated the Fc region of h00-vcMMAE to decrease FcγR binding affinity (E233P:L234V:L235, G1V1). h00G1V1-vcMMAE lost its cytotoxicity activity in FcγR+ THP-1 monocytes. Furthermore, h00G1V1-vcMMAE could no longer mediate tumor regression or growth delay in three xenograft models that are sensitive to h00-vcMMAE treatment. These results suggest ADC-FcγR interaction is required for ADC processing by TAMs in these xenografts.

These results suggest that TAMs can contribute to ADC processing through FcγR interaction in preclinical tumor models. Moreover, TAM-processed membrane permeable payload can mediate bystander tumor cell killing and contribute to ADC activity. Although this phenomenon may be an additional mechanism of ADCs in vivo, whether TAMs play a role in patients' response to ADCs requires further correlated studies in clinical trials.

#1286

Cytokine response of stromal cells to prostate cancer chemotherapeutics.

Dominique Gales, Clayton C. Yates, Temesgen Samuel. _Tuskegee University, Tuskegee, AL_.

Non-responsiveness to chemotherapeutic drugs presents a significant challenge to the effective treatment of advanced prostate cancer. Exploration of the therapeutic response in prostate cancer has largely focused on the epithelial tumor cells. However, evidence suggests that the tumor microenvironment may modulate response to chemotherapeutics. The tumor microenvironment consists of immune, fibroblastic, and vascular cells, as well as extracellular matrix (ECM) and stromal-derived soluble factors, all of which contribute to interactions within the tumor microenvironment. It has been demonstrated that tumor cells cross-talk with stromal cells, to establish a protective niche at primary and distant sites and to facilitate survival of cancer cells. Such tumor-stroma cell cross-talk often involves secreted factors, such as cytokines. Previous studies have illustrated the importance of cytokines and cytokine receptors as modulators of drug response. In this study, we sought to identify cytokine factors expressed in response to drugs clinically used to control prostate cancer. To obtain the expression profiles, we conducted a human cytokine and chemokine array, which consisted of 84 specific genes. Analysis of the prostate cancer and stromal cells treated with Docetaxel (4nM), Enzalutamide (30μM) and Bicalutamide (30μM) revealed differential expression patterns. The effect of chemotherapeutics on the expression of the cytokine IL-16 and its cognate receptors CD9 and CCR5 in prostate cancer and stromal cell lines was further examined. We found IL-16 expression increased several fold following chemotherapeutic treatment in stromal cell lines compared to prostate cancer cell lines. To validate gene expression, we examined the expression of IL-16 and its receptors by RT-PCR. All experiments were performed in triplicates. Student t- tests were used to determine the statistical significance between groups. IL-16 expression increased following exposure to chemotherapeutics in stromal cell lines. CCR5 expression showed a decrease in PC3 prostate cancer cells but no significant changes were observed in LNCaP cells. CD9 expression showed an increase in both LNCaP and PC3 cell lines. Collectively, these results indicate that chemotherapy of prostate cancer may alter the expression of IL-16 and its receptors. We hypothesize that IL-16 is a soluble stromal factor that is expressed in response to anticancer drugs, and may have paracrine activity on neighboring cells that express the cognate receptors.

#1287

The dual IAP antagonist, ASTX660, increases the anti-tumor activity of paclitaxel in preclinical models of triple-negative breast cancer in vivo.

Tomoko Smyth,1 Yoko Nakatsuru,2 Ryoto Fujita,2 George Ward,1 Luke Bevan,1 Jon Lewis,1 Nicola Wallis,1 John Lyons1. 1 _Astex Pharmaceuticals, Cambridge, United Kingdom;_ 2 _Taiho Pharmaceuticals, Tsukuba, Japan_.

Background: Paclitaxel-mediated secretion of inflammatory mediators, including TNFα, potentially creates paracrine and autocrine signaling loops that can reduce paclitaxel-induced apoptosis with resultant cancer cell survival (paclitaxel resistance). One mechanism of cancer cell survival is the expression of inhibitor of apoptosis proteins (IAPs): Cellular IAP (cIAP) is involved in inflammatory pro-survival NF-κB activation, blocking the activation of effector caspases 3 and 7, while X-linked IAP (XIAP) directly binds the effector caspases 3, 7 and 9, inhibiting the full activation of the apoptotic pathway. Antagonism of IAPs in the setting of paclitaxel treatment may enhance the cancer cell death by switching the chemotherapy-induced inflammatory TNFα signaling from pro-survival to apoptosis. ASTX660 is an orally bioavailable dual antagonist of cIAP and XIAP, currently being investigated in a single-agent Phase 1/2 clinical trial in patients with advanced solid tumors and lymphomas (NCT02503423). Here, we characterize the activity of ASTX660 in triple-negative breast cancer (TNBC) preclinical models as a single agent and in combination with paclitaxel.

Results: We first investigated the viability of 21 TNBC cell lines treated with ASTX660 in vitro and found that 43% were sensitive either to ASTX660 alone (MDA-MB-231 and HCC38) or in the presence of exogenous TNFα (HCC1806, Hs578T, BT549, HCC1395, DU4475, MDA-MB-453 and mouse EMT6). In HCC1806 xenografts in mice, both ASTX660 (daily oral treatment) and paclitaxel (weekly intravenous treatment) as single agents caused moderate tumor growth inhibition but not regression. ASTX660 and paclitaxel combination treatment, however, caused regression and all tumors achieved a partial response by Day 19 of treatment. An increase in circulating acute-phase-proteins in animals was also observed on paclitaxel treatment.

Conclusions: Our study suggests that paclitaxel treatment causes an inflammatory response which could qualitatively change the tumor environment. Inhibition of IAPs in such inflammatory environment induced by paclitaxel may lead to rapid apoptotic cell death. Thus, addition of IAP antagonist may be a promising approach to enhance the TNBC response to paclitaxel therapy.

#1288

Targeting the Hippo pathway in NF-2 deficient papillary kidney cancers.

Carole Sourbier, Penny Liao, Christopher J. Ricketts, Darmood Wei, Youfeng Yang, Sarah M. Baranes, Toshiki Kijima, Louis S. Krane, Myriem Boufraqech, Lernik Ohanjanian, Ben Gibbs, Ming-Hui Wei, Len Neckers, Cathy D. Vocke, W. Marston Linehan. _National Cancer Inst., Bethesda, MD_.

About 10% of papillary renal cell carcinomas (PRCC) bear mutations in NF2 or SAV1, two members of the Hippo pathway, which controls cell growth by regulating the transcriptional activity of YES1-Associated Protein YAP1. NF2 or SAV1 mutations lead to aberrant YAP1 activation resulting in cell proliferation and anti-apoptotic signals. The src family kinase YES1 is a known regulator of YAP1 transcriptional activity in the context of β-catenin-dependent tumors. Dasatinib and saracatinib are 2 src inhibitors that have shown to also have potent inhibitory effects on YES1. We hypothesized that inhibition of the Hippo pathway might provide a valuable therapeutic approach for patients with PRCC tumors bearing NF2 mutation, and that inhibiting YES1 might inhibit YAP1 in PRCC. Our data demonstrate that inhibition of the Hippo pathway is lethal to NF2-deficient PRCC cell lines, and that inhibiting YES1 with dasatinib or saracatinib effectively inhibits YAP1 and its downstream targets BIRC5 (survivin), CCND1 (cyclin D1) and CTGF (CTGF). Further, dasatinib proves to have an anti-tumor effect in vivo and was cytotoxic in vivo and in vitro, causing G0-G1 cell cycle arrest in NF2-deficient PRCC cell lines. Thus, inhibiting YES1 and the subsequent transcriptional activity of YAP1 with dasatinib or saracatinib might be a viable therapeutic approach for NF2-deficient PRCC tumors.

#1289

The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells.

Stéfano M. Cirigliano,1 María Inés Díaz Bessone,1 Carolina Flumian,1 Damián E. Berardi,1 Silvio Perea,2 Elisa Bal De Kier Joffé,1 Hernán Farina,3 Laura Todaro,1 Alejandro Urtreger1. 1 _Instituto de Oncología Ángel H. Roffo, Ciudad Autónoma de Buenos Aires, Argentina;_ 2 _Center for genetic engineering and biotechnology, Havana, Cuba;_ 3 _National University of Quilmes, Bernal, Argentina_.

Lung cancer is the leading cause of cancer deaths worldwide and despite significant progress, current therapies are limited in efficacy. The CK2 Ser/Thr kinase has been historically linked with cancer. It is involved in cell proliferation, survival and apoptosis by modulating diverse signaling pathways, including Wnt and NF-κB among the most relevant.

CIGB-300 is an antitumor peptide with a novel mechanism of action, capable of binding to CK2 substrates thus preventing the enzyme activity. Previously, we have determined that CIGB-300 induces apoptosis through caspase-3 activation in different lung cancer cell lines. Moreover, CIGB-300 strongly inhibited RelA/NF-κB (p65) nuclear translocation, even in the presence of a phorbol-ester activating stimulus.

NF-κB activation is known to reduce chemotherapy efficiency in different malignancies, including lung cancer. Based on this evidence, we hypothesize that supplementing cisplatin with CIGB-300 would improve the treatment efficiency.

Indeed, we observed by Western blot that nuclear p65 levels were highly increased after treating human NCI-H125 cells with cisplatin. Moreover, when cells were treated with cisplatin plus CIGB-300, NF-κB activation was completely abolished. Therefore, the CIGB-300 effect on NF-κB signaling pathway prevails over cisplatin.

These promising results on NF-κB inhibition led us to evaluate the combined treatment in chemoresistant setting. For this purpose we developed a cisplatin resistant A549 lung cancer cell line (A549-Rcisp) by the chronic administration of cisplatin during six months. A549-Rcisp viability was 40% higher than parental cells, confirming the cisplatin-acquired resistance. Remarkably, cisplatin resistant cells showed a significant increase in CIGB-300 sensitivity as compared to the parental cell line (p<0.01; Student's t-test). More on, only A549-Rcisp showed an increased p65 nuclear level after cisplatin treatment, suggesting that both cisplatin resistance and CIGB-300 sensitization might be linked to the NF-kB transcription factor.

Given that NF-κB dimer stability is regulated by the proteasome-selective proteolysis of its inhibitory proteins, we studied the effect of CIGB-300 on this process. Surprisingly, we observed a significant increase on protease activities associated with the proteasome after 30 minutes of CIGB-300 treatment. Thus, proteasome complex is a newly identified target of CIGB-300 that could be relevant for its mechanism of action and deserves further exploration in order to determine the association with the observed perturbation of different signaling pathways.

#1290

Actinomycin D enhanced immunotoxin RG7787 killing of cancer cells.

Xiu Fen Liu,1 Laiman Xiang,1 Marco Prunotto,2 Gerhard Niederfellner,3 Ira Pastan1. 1 _NIH, Bethesda, MD;_ 2 _Roche, Basel, Switzerland;_ 3 _Roche, Penzberg, Germany_.

Immunotoxins are recombinant fusion proteins that contain an antibody fragment directed against a tumor selective surface antigen attached to a protein toxin. RG7787 is a newly developed, mesothelin-targeted immunotoxin designed to be less immunogenic and better tolerated than the previous clinical candidate SS1P. The targeting moiety of RG7787 consists of a humanized anti-mesothelin Fab, while its effector moiety is a truncated portion of Pseudomonas exotoxin A (PE) consisting essentially only of the catalytic domain III fused via a furin cleavable linker to the Fab fragment. The domain III variant used in RG7787 contains mutations that silence all known human B-cell and some T-cell epitopes. RG7787 targets and kills mesothelin-positive tumor cells, which are prevalent in mesothelioma, ovarian, lung, and pancreatic cancers. Safety and immunogenicity of RG7787 are being assessed in these tumor indications in an ongoing phase I trial. In order to further enhance the anti-tumoral activity of RG7787, we screened for clinically used drugs that can synergize with RG7787 in vitro and/or in vivo. Actinomycin D (Act D) is a potent transcription inhibitor and is used for treating a variety of cancers, including gestational trophoblastic neoplasia, Wilms tumor, rhabdo¬myosarcoma, and Ewing's sarcoma. In this study we found that the combination of Act D with RG7787 greatly stimulated cell killing of mesothelin positive cell lines, such as KB31, KLM1, and Hay. The combination also produced complete regressions of KLM1 pancreatic tumor xenografts in mice. To investigate the mechanism of killing we used an apoptosis RNA array and found that both Act D and RG7787 stimulate apoptotic stress responsive genes including TNFα, TRAILR2 (DR5), NFκB, GAD45A and TP53. The combination also increased cleavage and activation of the pro-apoptotic proteins caspase-3, -9, and PARP. Taken together our data indicate that combining RG7787 and Act D reduces the threshold for activation of apoptosis in cancer cells.

#1291

Applicability of integrative tumor response assays for recurrent colorectal cancer.

Yong Sik Yoon, Jin Cheon Kim. _University of Ulsan College of Medicine, Seoul, Republic of Korea_.

Introduction

Drug response assays use autologous viable tumors to evaluate susceptibility to specific agents in vitro. Histoculture drug reponse assay (HDRA) is a representative methyl thiazolyl-diphenyl-tetrazolium bromide (MTT) assay, which has the advantage of more correctly reflecting the in vivo microenvironment. Many studies showed 66-92% of accuracy rates for clinical correlation of chemotherapy in solid tumors including colorectal cancer (CRC). However, in recurrent cases of metastatic CRC, there is no usable assay to predict second-line chemo-sensitivity. Thus, authors invented novel technique, the integrative tumor response assay (ITRA), can identify drug responses for both first- and second-line chemotherapy simultaneously. We investigated the chemo-sensitivity to clinically used regimens using ITRA of samples from patients with metastatic CRC, and compared their chemo-sensitivity with the observed clinical response.

Methods

A total of 129 patients with metastatic CRC were prospectively enrolled. First-stage HDRAs were performed using 5-fluorouracil with leucovorin and oxaliplatin (FX) or irinotecan (FR). Second-stage HDRAs (ITRA) were done for survived cells after first-stage HDRA, using FX, FR, and their combinations with bevacizumab and cetuximab. The inhibition rate of tumor growth (IR) cut-off value for a positive response was determined to be ≥ 30%. For clinical validation, treatment responses of chemotherapy were evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST). The primary endpoint of this study was a correlation between the ITRA results and the clinical response in the response rate (RR). This was defined as positive for the effect of chemotherapy for tumor responses between complete response (CR) and partial response (PR).

Results

Among 129 used samples, ITRA failure was 9 (7%), due to deficient volume of samples. Out of 79 evaluated first-line chemotherapeutic regimes, RR was 53% (42/79). The correlation rate of HDRA for first-line chemotherapy was 70.9% (56/79), with a 71.4% (30/42) of sensitivity and 70.3% (26/37) of specificity. For 42 cases completing second-line chemotherapy, RR was 43% (18/42). The accuracy rate of ITRA for second-line chemotherapy was 61.9% (26/42), with a 44.4% (8/18) of sensitivity and 75% (18/24) of specificity.

Conclusions

ITRA might be further developed to be a feasible and useful technique for predicting therapy efficacy and selecting the appropriate anticancer regimen for individual patients despite its relatively low accuracy.

#1292

Camalexin, an indole phytoalexin from Arabidopsis thaliana, displays activity against ovarian cancer stem cells.

Roman Mezencev, John F. McDonald. _Georgia Institute of Technology, Atlanta, GA_.

Ovarian cancer is the most lethal of all gynecological cancers. Current treatment of advanced ovarian cancers, which includes debulking surgery and chemotherapy, is initially effective in majority of patients; however, 70-90% of them eventually develop disease recurrence with limited treatment options. This recurrence can be attributed to therapy-resistant cancer stem cells (CSC), which represent a subset of all malignant cells that form the bulk of ovarian cancers. CSC may remain as a minimal residual disease after the bulk of ovarian tumor mass has been removed by surgery and/or chemotherapy; therefore, there is considerable current interest in the development of new therapies that can effectively target this insidious subpopulation of tumor cells. Here we report on the anticancer activity of camalexin, a major indole phytoalexin of Arabidopsis thaliana, against ovarian cancer stem cells. We demonstrate that camalexin inhibits proliferation and induces cell death in ovarian cancer stem cells that exhibit various molecular mechanisms of anticancer drug resistance. Furthermore, we present the mode of action of this prospective anticancer agent identified by combined approach that included in silico predictions, comprehensive analysis of whole-genome gene expression data and target-specific assays. Lastly, we demonstrate the relevance of the identified mode of action in context of ovarian cancer cell signaling.

#1293

JQ1 sensitivity of patient-derived xenograft models of cholangiocarcinoma.

Aubrey L. Miller,1 Patrick L. Garcia,1 Tracy L. Gamblin,1 Leona N. Council,1 Xiangqin Cui,1 James E. Bradner,2 Eddy S. Yang,1 Karina J. Yoon1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Cholangiocarcinoma (CCA) is a lethal malignancy arising from cholangiocytes in any part of the biliary tree. The incidence of CCA has been on the rise worldwide, and the prognosis and clinical outcome have remained essentially unchanged for 30 years. The majority of patients are diagnosed at late stage, and surgery continues to be the only cure. Patients receive systemic chemotherapy with the first-line combination therapy comprising gemcitabine and cisplatin. Median survival for these patients is <12 months, emphasizing the need to improve current treatment. A step toward improving outcome is the pre-clinical evaluation of novel therapeutics. Recently the bromodomain and extra-terminal domain (BET) inhibitor JQ1 has been shown to suppress tumor growth in preclinical models of multiple tumor types. The therapeutic effect of JQ1 has been attributed, at least in part, to its ability to inhibit c-Myc expression. 95% of CCA tumors express c-Myc and its down-regulation has been shown to reduce the invasive potential of CCA cells, suggesting that c-Myc contributes to CCA phenotype. Our lab recently evaluated the efficacy of the BET inhibitor JQ1 in CCA patient-derived xenograft mouse models. JQ1 suppressed CCA tumor growth in 2 of the 3 models.

To determine gene products whose upregulation or downregulation is responsible for the differences in sensitivity to JQ1 among our CCA models, we generated expression profiles of tumors from vehicle control and JQ1 treated mice using NanoString technology (nCounter PanCancer Pathways panel). Our data demonstrate that JQ1 inhibited the expression of c-Myc to a greater extent in the sensitive models than in the insensitive model. Expression array data showed further that gene products involved in cell cycle and DNA repair pathways were also decreased by JQ1. Of particular interest were two transcriptional targets of c-Myc, Chk1 and BRCA2, each of which is involved in DNA damage response. Immunohistochemistry staining confirmed expression profile analyses. We conclude that the inhibition of cell cycle and DNA repair genes may contribute to the mechanism of action of JQ1 in CCA tumors.

#1294

Sensitization of malignant melanomas to TRAIL-induced apoptosis by quercetin.

Katherine Turner,1 Daniel Lindner,2 Michael Kalafatis1. 1 _Cleveland State University, Cleveland, OH;_ 2 _Taussig Cancer Institute, Cleveland, OH_.

Skin cancer is among the most commonly-diagnosed cancers with malignant melanoma being associated with the highest rate of metastasis and mortality. In its early stage, melanoma is easily cured, but the prognosis associated with metastatic malignant melanoma remains very poor and is one of the most treatment-refractory malignancies. We propose the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) as a potential therapeutic for malignant melanoma. TRAIL induces apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to TRAIL, specifically certain melanomas, caused by lack of TRAIL receptors or upregulation of antiapoptotic proteins. Here we analyze the naturally-occurring flavonoid quercetin as a potential cotreatment with TRAIL to overcome the intrinsic resistance of melanoma. Found in a wide variety of sources from onions and apples to red wine, quercetin is a good candidate for TRAIL cotreatment due to its ability to upregulate TRAIL receptors and downregulate antiapoptotic proteins. We have evaluated our cotreatment of TRAIL plus quercetin on four malignant melanoma cells lines which harbor mutations in the MAPK pathway, namely A375 and WM164 (BRAF mutant), SK-Mel-2 (NRAS mutant) and MeWo (BRAF WT, NRAS WT). Numerous cell-based assays were utilized including antiproliferative SRB assay, apoptotic AnnexinV/PI assay and western blot analysis probing for key proteins of the apoptotic cascade. Out of the four melanoma cell lines evaluated, A375 and SK-Mel-2 were sensitive to TRAIL dose-dependently; whereas, MeWo and WM164 were resistant to TRAIL-induced apoptosis, even at the highest tested treatment concentration of 1µg/ml. Quercetin, as a single agent, was able to induce apoptosis in a dose-dependent manner in all four melanoma cell lines. To determine if the cotreatment, TRAIL plus quercetin, is able to sensitize melanoma cells to TRAIL-induced apoptosis, we treated resistant cell lines, MeWo and WM164 with 250 ng/ml TRAIL plus sub-cytotoxic concentrations of quercetin, 25 and 50 µM. Quercetin was able to sensitize both MeWo and WM164 to TRAIL-induced apoptosis marked by the fragmentation of PARP, a hallmark of apoptosis, and the activation of executioner caspases 3, 6 and 7. Specifically, quercetin was able to sensitize resistant melanomas to undergo TRAIL-induced apoptosis as evidenced by the cleavage of procaspase 8 to caspase 8, a marker for the initiation of TRAIL-induced apoptosis. Quercetin also promoted the TRAIL-mediated activation of the intrinsic pathway of apoptosis marked by the release of cytochrome C from the mitochondria. These preliminary data demonstrate that quercetin is a good potential cotreatment for TRAIL; however, further research is needed to reveal the mechanism of quercetin sensitization of TRAIL-resistant melanomas and its role in TRAIL receptor and antiapoptotic protein expression.

#1295

Evaluation of the efficacy of TRAIL plus quercetin as a potential breast carcinoma therapeutic.

Jasmine M. Manouchehri,1 Michael Kalafatis,1 Daniel Lindner2. 1 _Cleveland State University, Cleveland, OH;_ 2 _The Cleveland Clinic, Cleveland, OH_.

Breast cancer is the most commonly diagnosed cancer in women in the United States. There is a continued need for the development of selective and specific treatment options for all types of breast cancer, including hormone-dependent and triple-negative subtypes. Recombinant human Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (rhTRAIL), the optimized form of the endogenous death ligand, shows potential as an effective anti-cancer therapeutic due to its ability to induce apoptosis in cancer independent of wild-type p53 function, while displaying minimal toxicity to normal cells. However, a majority of breast cancer cell lines exhibit resistance to TRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors 4 and 5. To overcome TRAIL resistance, a cotreatment option has been explored utilizing the natural compound Quercetin (Q). Q is a flavonol found in certain fruits, vegetables, and teas. As a single agent, Q has been shown to have antiproliferative and pro-apoptotic effects on a variety of cancer cell lines. The aim of this study is to examine the capacity of Q to enhance TRAIL's pro-apoptotic and antiproliferative effects on breast cancer cells. Sulphorhodamine B (SRB) assays were performed on hormone dependent (MCF-7) and triple negative (BT-20) breast cancer cell lines to determine if the cotreatment of Q and TRAIL hinders cell growth. Growth for both MCF-7 and BT-20 cells was substantially inhibited by single agent Q treatments (12.5 μM; ~20%, 25 μM; ~40%, 50 μM; ~60%) but not by single agent TRAIL treatment (100 ng/mL; <20%). Moreover, BT-20 and MCF-7 cell growth was further inhibited by cotreatment with both agents; for example, the cotreatment of 50 μM Q and 100 ng/mL TRAIL inhibited BT-20 and MCF-7 cell growth by 80% and 90%, respectively. As a single agent, Q was able to induce apoptosis in a dose-dependent manner in both breast cancer cells; thereafter, Q's ability to sensitize breast cancer cells to TRAIL-induced apoptosis was examined by western blot analysis. Compared to single agent treatments, the combination of Q and TRAIL enhanced the induction of apoptosis as indicated by increased PARP cleavage (a hallmark of apoptosis) and the activation of the executioner caspases 3 and 7. Furthermore, the cotreatment of breast cancer cells with Q and TRAIL enhanced the activation of the extrinsic and intrinsic apoptotic pathways, as assessed by the activation of caspase 8 and the release of cytochrome c from the mitochondria, respectively. Overall, these findings suggest that the cotreatment of Q and rhTRAIL possesses the potential to be an anti-breast cancer therapeutic by enhancing pro-apoptotic and anti-proliferative effects in hormone dependent and triple-negative breast cancer cells.

#1296

Vascular shutdown anti-tumor effect of tetra-arsenic oxide on cervical cancer cells.

Yong-Wan Kim. _Yonsei University, Incheon, Republic of Korea_.

Background: Tetra arsenic oxide (As4O6) is a novel arsenic compound that exhibits potent anti-vascular activity and significantly suppresses solid tumor growth. The present study was conducted to investigate the vascular shutdown effects in HPV E6/E7-expressing tumors bearing mice.

Methods: After tumors reached 300-350 mm3 in volume, mice were randomly divided into four groups and treated intraperitoneally with three different dose of As4O6 at 1-week interval. Tumor blood perfusion and vascularity were visualized using Hoechst 33342, CD31 staining respectively from 1 hour to 1 week after As4O6 treatment. Histopathological features were observed following hematoxylin and eosin staining. Apoptosis, TNF-a, caspase activity and tumor regression were used to determine the mechanisms for vascular shutdown effects.

Results: As4O6 significantly suppressed tumor growth compared with control group. This tumor growth inhibition led to massive necrosis and vascular shutdown in tumor tissue, and also tumor tissue necrosis and apoptosis. Mice treated with As4O6 had a reduction in tumor vessel dimensions compared with the control animals, with CD31 stained microvessels still being apparent at the outer edge of the tumor. Additionally, in the control group, vessel area and diameter increased over time, whereas in the tumors from the As4O6-treated group, vessel area and diameter remained stable or decreased. Apoptotic cell populations induced by As4O6 were increased by double staining the TC-1 cells with annexin V and propidium iodide.

Conclusions: The results suggest that As4O6 has potential anticancer activity that is exerted by vascular shutdown in C57BL/6 mice transplanted with TC-1 cells. The study is novel in the observation of recovery of disturbed vascular function.

#1297

CDK4/CDK6 inhibition in childhood B-acute lymphoblastic leukemia: a new strategy to mediate glucocorticoid sensitivity.

Roberta Bortolozzi, Elena Mattiuzzo, Elena Mariotto, Benedetta Accordi, Luca Trentin, Giuseppe Basso, Giampietro Viola. _University of Padova, Padova, Italy_.

Unrestrained cell proliferation and cell cycle deregulation are common features in almost all human cancers. Among the CDKs that tightly control cell cycle progression, cyclin D-dependent kinases, CDK4 and CDK6 are considered important oncogenic drivers in many cancers. Although many reports successfully described CDK4/CDK6 inhibitors against a broad range of carcinomas, few studies have been performed on leukemia.

Deletion and methylation of CDK4/CDK6 inhibitor CDKN2A, are frequently observed in B-acute lymphoblastic leukemia (B-ALL) and gene expression analysis performed in a cohort of childhood patients showed that cyclin D1, CDK4 and CDK6 are highly expressed. Moreover, Reverse Phase Protein Arrays (RPPA) analysis showed that cyclin D1 expression is higher in High Risk-MRD patients. These results suggest specific inhibition of cyclin D/CDK4/CDK6 axis as an attractive strategy to improve the effects of standard chemotherapy on B-ALL patients.

The aim of this study was to evaluate the effect of dual inhibition of CDK4/CDK6 in B-ALL. To this purpose we treated two B-ALL dexamethasone resistant cell lines, SEM and RCH-ACV, and two B-ALL dexamethasone sensitive cell lines, RS4;11 and NALM6, with ribociclib, a highly specific CDK4/6 inhibitor.

As expected, treatment with ribociclib induced a strong cell cycle arrest in G1 phase in a time-dose dependent manner along with a dose-dependent decrease in phosphorylated Rb and increase of the expression of cell cycle inhibitors p21 and p27. Moreover, a strong dose-dependent reduction of clonogenic potential was observed in SEM cell line, by CFU assay. No significant reduction in cell viability was observed.

However, ribociclib exposure strongly synergized (CI<1) with dexamethasone in SEM and RCH-ACV resistant cell lines with a strong decrease of proliferation and a significant increase of apoptotic cell death. Immunoblot analysis showed a decrease in phosphorylated Rb, the activation of caspase-9 and the cleavage of the effector caspase-3 starting from 48 h of co-treatment, in agreement with the appearance of annexin-V positive cells.

The synergistic effect of ribociclib-dexamethasone combination was confirmed on primary cultures derived from pediatric patients affected by B-ALL.

We are actually investigating on the mechanism of this synergistic activity, and the effect of CDK4, CDK6 and cyclin D1 silencing will be presented. Preliminary experiments show a modest increase in glucocorticoid receptor expression after ribociclib treatment or CDK6 silencing in RCH-ACV cells.

Our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanisms of glucocorticoid resistance.

#1298

CD40L-CD40 signaling on B-cell lymphoma response to BTK inhibitors.

Zhijian Sun, Lusong Luo. _BeiGene(Beijing) Co., Ltd., Beijing, China_.

The aberrant activation of B cells receptor (BCR) signaling plays an essential role in the pathogenesis of B-cell lymphomas. Targeting BCR signaling by inhibiting Bruton's tyrosine kinase (BTK) has been shown to be a successful strategy in treating B-cell lymphomas including mantle-cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Ibrutinib, an oral, covalent BTK inhibitor achieved high overall response rate (ORR) and prolonged duration of responses in CLL and MCL patients. In contrast, ibrutinib was reported to have much lower response rate in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) and the responses in DLBCL was restricted to the activated B-cell (ABC) subtype. These differences highlight the need to understand the bypass signaling/pathway to BTK inhibition. In this study, we identified CD40L-CD40 pathway as one of the potential bypass pathways to BTK inhibitory therapy. We showed that CD40 was consistently expressed in mature B cells, whilst CD40L expression levels were heterogenic in tissue microarray (TMA) with 150 lymphoma samples. In these samples, circa 57% of lymphoma tissues have medium or strong level of CD40L expression in TMA, indicating the existence of CD40L-CD40 signaling in human lymphoma tissues. Consistent with the response rate of ibrutinib in patients, in 68 non-GCB type of DLBCL, in which aberrant activation of NF-kB pathway is important for the pathogenesis, about 60% samples showed CD40L expression. Co-culture of Rec-1 or TMD8 cells with HEK293/CD40L attenuated the anti-proliferation activity of ibrutinib in vitro. This observation was also confirmed with recombinant soluble CD40L protein. Detailed mechanistic analysis revealed that CD40L-CD40 signaling activated the NF-κB and MAPK pathway and offset the inhibition of ibrutinib. Additionally, activating NF-κB pathway with PKC agonist abolished the cell killing effects of ibrutinib. These data suggest CD40L may be used as a potential biomarker for studying clinical responses to BTK inhibitors. Additionally, our results support combination strategies of BTK inhibitors with agents blocking CD40 or downstream NF-κB and MAPK pathway in patient population where CD40L-CD40 pathway is activated.

#1299

Selinexor, a selective inhibitor of nuclear export (SINE) compound shows enhanced anti-tumor activity when combined with either venetoclax or bendamustine in diffuse large B cell lymphoma (DLBCL) mouse models.

Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. _Karyopharm Therapeutics Inc, Newton, MA_.

Introduction: Selinexor (sel) is a small molecule inhibitor of CRM1/XPO1, the primary nuclear exporter of over 200 proteins. As such, it affects multiple cellular pathways and has been shown to be broadly synergistic with various drugs in multiple indications. We have previously shown that sel has marked activity in double-hit DLBCL in pre-clinical models and in a small cohort of patients with heavily pre-treated relapsed / refractory double or triple-hit DLBCL. The goal of this study was to test whether combination of sel with either venetoclax (ven), a selective BCL2 inhibitor or bendamustine (benda), a DNA damaging agent, can further enhance the anti-tumor effect of sel in DLBCL.

Methods: The effects of sel, benda and ven as single agents and the effects of sel in combination with either benda or ven on cell viability were tested on DLBCL cell lines including Toledo, DoHH2 and SUDHL6 using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots, respectively. Mice were implanted with either Toledo or DoHH2 cells. Toledo inoculated mice were treated with either sel or ven alone or in combination and DoHH2 inoculated mice were treated with sel, ven or benda alone or with sel-benda or sel- ven combinations. %Tumor growth inhibition (%TGI) and overall survival were determined. Xenografts were harvested and analyzed by Immunohistochemestry (IHC).

Results: Sel-ven and sel-benda were highly effective both in-vitro and in-vivo. Using MTT assay, we showed that each of the drugs have low IC50 values (nanomolar) and they function synergistically/ additively when combined. In-vivo, in the sel-benda model, treatment with each drug showed %TGI of 37% (sel) and 86% (benda) but in combination %TGI was 107%. Western and IHC analyses showed that sel reduces the expression of key DNA Damage Response (DDR) proteins presumably disabling the cells from over coming the damage induced by benda. In the sel-ven model, in both Toledo and DoHH2 mouse models, individual drugs had little effect on %TGI (Toledo: sel 4%, ABT 28%; DoHH2: sel 37%, ABT 24%) but when combined, treatment showed a synergistic effect (Toledo: 109%; DoHH2: 93%). Moreover, combination treatment of Toledo-derived large xenografts resulted in 30% tumor volume shrinkage that was sustained until the end of the study. Interestingly, BCL2 protein levels were reduced by each drug and to a further extent in the combination-treated group suggesting that the synergistic effect is induced via BCL2.

Conclusion: Selinexor is an excellent candidate partner for combination therapies in DLBCL. It shows enhanced antitumor effect with both bendamustine and venetoclax modulating DDR and BCL2 pathway activity, respectively. These data provide rational support for study of sel-ven and sel-benda combination in clinical trials.

#1300

Fatty acid synthase (FASN) inhibition negatively affects cell proliferation and the metastatic capability of prostate cancer cells.

Clayton Wright, Anand Krishnan V. Iyer, Neelam Azad. _Hampton University, Hampton, VA_.

Purpose

Fatty acid synthase (FASN) is a key metabolic enzyme that catalyzes the synthesis of fatty acids (FA). Recent data has shown that several common cancers express elevated levels of fatty acid synthase (FASN) in comparison to normal tissue; with prostate cancer being one. This information has implicated FASN as a possible oncogene and marker for cancer malignancy involving invasion and migration. Three of the most common prostate cancer cell lines (DU145, PC3, and LNCaP) all express elevated FASN levels with LNCaP cells exhibiting the highest levels of FASN among the three cell lines. This study aimed to investigate the effects of a novel FASN inhibitor (1B) designed in our lab on three different prostate cancer cell lines and compare its cytotoxic and phenotypic effects on cell proliferation and viability, apoptosis, lipid levels, and expression and regulation of key cellular proteins involved in FA synthesis to the well-known FASN inhibitor Orlistat and FASN siRNA.

Experimental Methods

DU145, PC3, and LNCaP prostate cancer cell lines were used in all experiments. Cells were exposed to Orlistat and 1B doses of 50 and 100μM for 24h and 48h time points. Cell viability was assessed using the MTT assay, and cell proliferation was assessed using the CyQUANT cell proliferation kit. Western blotting was used to analyz e the relevant protein expression with the immune complexes detected by chemiluminescence. Apoptosis analysis by microscopy was done using the Hoechst dye stain. The percentage of cells having stained nuclei indicating apoptosis was scored by fluorescence microscopy.

Results

Cell proliferation and viability data indicates a dose-dependent decrease in all three cell lines for both Orlistat and 1B. Apoptosis analysis also revealed an increase in cell death in all three cell lines when treated with either drug. Most interestingly, there was a more significant decrease in cell viability and proliferation in LNCaP cells in comparison to DU145 and PC3 cells; an expected result considering that LNCaP cells express higher levels of FASN. Similar data was also seen when utilizing siRNA to FASN.

Conclusions

We conclude that FASN inhibition (by both drug treatment and siRNA) negatively affects cellular viability, proliferation, and apoptosis. Further experimental analysis will determine whether this inhibition also negatively affects cell cycle progression, related protein expression levels, and the ability of these prostatic cells to undergo invasion and migration; a hallmark of prostate cancer progression.

#1301

Anti-invasive and antimetastatic effects of ω3-polyunsaturated fatty acids through inhibition of NF-kB/matrix metalloproteinases in ovarian cancer cells.

Soyeon Jeong,1 Kaipeng Jing,1 Soyeon Shin,1 Soyeon Kim,1 Young-Joo Jeon,2 Jun-Young Heo,2 Gi-Ryang Kweon,1 Seung-Kiel Park,2 Jong-Il Park,2 Kyu Lim3. 1 _Dept. of Biochemistry, School of Medicine, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea;_ 2 _Dept. of Biochemistry, School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 3 _Dept. of Biochemistry, School of Medicine, Cancer Research Institute, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea_.

Ovarian cancer is leading cause of gene cological cancer death in the United States. It is reported that about 14,000 patients will die to ovarian cancer in United States, 2015. ω3- and ω6-PUFAs are considered as essential fatty acids because they cannot be synthesized in mammals. The ω3-desaturase (fat-1) converts ω6-PUFAs to ω3-PUFAs. Cancer cells and transgenic mice stably expressing fat-1 geneare useful models to study the anti-cancer effects of ω3-PUFAs. Here, we show anti-invasive and anti-metastatic action of ω3-PUFA in ovarian cancer.DHA significantly inhibited cell invasion and wound healing activity of human ovarian PA-1 cells. DHA also abolishedthe induction of matrix-metalloproteinases (MMPs) and cyclooxygenase (COX2) as well as the transactivity of NF-κB in PA-1 cells. When ID8 cells were subcutaneously implanted into fat-1 transgenic mice, the tumor size and volume were smallin comparison to wild-type mice. The growth inhibition of tumor was also confirmed with the increase in the level of TUNEL-positive cells. Additionally, when ID8 cells were injected via tail vein of fat1 mice, the lung metastasis was remarkably inhibited in fat-1 transgenic mice. Furthermore, the proliferation of fat-1-stably expressing PA-1 (f-PA-1) cells was more attenuated than that of cells expressing control vectors. The invasion and NF-κB/MMPs promoter activities were also decreased in f-PA-1 cells. These results suggest that ω3-PUFAs inhibit invasion and metastasis of ovarian cancer cells through inhibition of NF-κB/MMPs. Therefore, ω3-PUFAs may contribute an effective and safe therapeutic approach for the chemoprevention and treatment of human ovarian cancer. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932)]

#1302

A comprehensive approach to delineate FGFR2 targeted therapy response in diffuse gastric cancer.

Jiamin Chen, Hojoon Lee, Hanlee Ji. _Stanford University Medical School, Stanford, CA_.

Dysregulation of fibroblast growth factor receptors (FGFRs) signaling has been associated with tumorigenesis and progression in various cancers. Preclinical models have shown that FGFR2 signaling activation due to FGFR2 amplification is essential driver for specific gastric cancers. Cancer cell lines or patient-derived gastric cancer xenograft models with FGFR2 amplification are highly sensitive to AZD4547, which is a small-molecule tyrosine kinase (TRK) inhibitor that selectively targets FGFR1-3. However, recent clinical trial studies failed to demonstrate the therapeutic potential of AZD4547 in patients with FGFR2 amplification. For this study, we identified critical genetic and molecular features that determine the response to FGFR2 inhibitors in gastric cancer.

To systematically delineate the pathways that modulate response to FGFR2 inhibitors, we identified CRISPR-library-based gene knockouts that result in resistance to AZD4547 in Kato III cells, a gastric cancer cell line with high sensitivity to this drug. Although majority of transduced Kato III cells undergo cell senescence upon AZD4547 treatment, a small population of resistant cells rendered by CRISPR knockout is enriched. Subsequent sequencing and pathway analysis reveal the alternative activated pathways that contribute to the cell growth in lieu of FGFR2 inhibition.

Furthermore, we applied the elastic-net regression analysis to identify genetic features associated with the drug sensitivity to FGFR2 inhibition in cancer cell lines. The cell line drug response data are derived from the Cancer Therapeutic Response Portal (CTRP v2) and the Cancer Cell Line Encyclopedia (CCLE). Notably, few cancer cell lines have high FGFR2 copy numbers (CN>4). The elastic-net algorithm simultaneously integrates mutation, copy number, gene expression data, and forms multiple linear regression to learn the coefficient weights associated with FGFR2 inhibitor sensitivity. Using the method, we identify the whole spectrum of genetic and molecular features that are potential drug response markers.

In summary, our study provides new insight into genetic and molecular features that implicate in the cellular response to FGFR2 inhibitors, which can facilitate the development of more effective strategies to treat patients with FGFR2 amplification.

#1303

In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer.

Maitreyee K. Jathal,1 Thomas M. Steele,1 Salma Siddiqui,2 Benjamin A. Mooso,1 Leandro S. D'Abronzo,1 Christiana M. Drake,1 Paramita M. Ghosh1. 1 _UC Davis, Sacramento, CA;_ 2 _VA Northern California Healthcare System, Mather, CA_.

Background: Prostate cancers (PCa) rely on androgenic ligands and the androgen receptor (AR) for their growth and survival, making AR inhibition a predominant therapeutic strategy for these tumors. Some prostate tumors however fail this therapy due to 'bypass' mechanisms that emerge as a result of prolonged AR targeting. This in vivo study attempted to assess the expression and activation of the epidermal growth factor receptor (EGFR) family (whose role is well-documented in PCa) in response to androgen deprivation therapy (ADT).

Methods: Nude mice were implanted (s.c.) with CWR22 tumors (human-patient-derived, androgen-dependent 'AD') and its castration-resistant ('CR') subline CWR22-Rv1 (relapsed CWR22). Androgen deprivation (i.e. AR inhibition) was achieved by surgical or 'sham' castration of mice. Tumors were analyzed (immunohistochemistry/immunoblot) for EGFR/ErbB2/ErbB3/AR proteins and proliferative/apoptotic markers.

Results: Castration caused significant tumor regression in AD but not CR tumors. in vitro viability assays demonstrated that castration (mimicked by using charcoal-stripped serum, 'css') did not slow down CR cells to the same degree as it did AD cells. At baseline, intratumoral EGFR protein was unchanged in R22 tumors, ErbB2 levels decreased and ErbB3 protein increased in Rv1 tumors. Castration increased ErbB3 but not EGFR or ErbB2 proteins in CWR22 tumors. Phosphorylated forms of these receptors were generally difficult to detect but there was more phosphorylated ErbB3 protein in Rv1 tumors. Downstream of the EGFR family, there was less phosphorylated Erk but not Akt protein in CWR22-Rv1 tumors. Castration decreased Erk protein in AD tumors but increased it in CR tumors. Immunohistochemical quantification revealed that cytoplasmic EGFR and ErbB3 proteins were elevated in CR tumors but reduced in AD tumors. Castration greatly decreased Ki-67 staining in AD but not in CR tumors while the number of TUNEL-positive nuclei and intensity of PARP staining decreased in castrated CR but not in AD tumors. ErbB3 and AR proteins were significantly correlated with DNA damage and proliferation in CWR22 tumors but only nuclear AR levels and proliferation were significantly correlated in CR tumors.

Conclusions: We conclude that androgen deprivation therapy may alter EGFR and ErbB3 protein levels and localization in androgen-dependent and castration-resistant tumors. The EGFR family is typically activated at the cell surface hence their presence and activity there, in response to castration, may initiate signalling pathways encouraging tumor cell proliferation and survival.

#1304

**The effect of FoxM1 inhibition with thiostrepton on ovarian cancer immune response and sensitivity to chemotherapy** in vitro **.**

Jill Madden,1 Pingping Fang,1 Bernard Herrman,1 Mary Markiewicz,2 Ryan Moulder,3 Laird Forrest,3 Jeremy Chien1. 1 _Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS;_ 2 _Department of Microbiology, University of Kansas Medical Center, Kansas City, KS;_ 3 _Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS_.

Background: An ideal anti-cancer agent will target a pathway that is altered in cancer, produce a selective cytotoxic response in cancer cells, induce immunogenic cell death, and enhance tumor immunity. FoxM1 inhibitor, thiostrepton, may fit this profile of an ideal anti-cancer agent. FoxM1 is activated in the majority of high-grade serous carcinomas, regulates DNA repair genes, and enhances cell proliferation, metastasis, and chemotherapy resistance. We have previously shown that loss-of-function and gain-of-function TP53 mutations contribute to FoxM1 overexpression, thus the p53-FoxM1 axis represents a novel therapeutic target. However, the therapeutic potential of FoxM1 inhibition by thiostrepton has not been fully investigated in ovarian cancer.

Hypothesis: We hypothesize that FoxM1 inhibition by thiostrepton increases cancer cell sensitivity to chemotherapy and enhances anti-tumor immunity.

Results: Thiostrepton downregulates FoxM1 expression, induces cytotoxic effects across various ovarian cancer cell lines, and shows synergistic activities with cisplatin. In congruence with the Connectivity Map data, qRT-PCR results of A2780 cells treated with thiostrepton (10 µM) show upregulation of pro-apoptotic genes DDIT3, GADD45A and DDIT4, and downregulation of DNA repair and anti-apoptotic genes FANCF, BRCA2, BRCC3, and BCL2. These results suggest that the synergism observed may be a consequence of DNA damage induction by cisplatin combined with inhibition of DNA repair by thiostrepton, making this a lethal duo to cancer cells. Comet assay results from mouse ovarian cancer cells, ID8, support this hypothesis by demonstrating an increase in DNA damage following the combined treatment of thiostrepton and carboplatin or thiostrepton and PARP inhibitor olaparib. Western blots also show increased p-H2AX levels in these combined treatments. In addition to the cytotoxic effect, thiostrepton can alter the cancer cell immune response by affecting the expression of immunomodulatory genes. This is supported by the ENCODE data, which indicates FoxM1 binds to the cis-regulatory region of PD-L1 (programmed cell death ligand 1), a target of ongoing immunotherapy clinical trials. Flow cytometry and Western blots depict increased PD-L1 expression in ID8 cells treated with thiostrepton (5 µM). In these same cells, flow cytometry showed an increase in RAE1 and 4-1BB ligands, which may lead to increased recognition by natural killer T-cells, as well as enhanced externalization of calreticulin, a hallmark of immunogenic cell death.

Conclusion: These studies demonstrate the novel effect of thiostrepton on immunogenic cancer cell death and an effect on anti-tumor immunity. These results suggest thiostrepton as an ideal anti-cancer agent with the ability to target the DNA repair response and the immune response to enhance the chemotherapeutic effects of cisplatin and olaparib.

#1305

Metabolism studies of SH7139, a small molecule drug targeting B-cell malignancies overexpressing HLA-DR10, confirm its prodrug function.

Monique Cosman Balhorn, Rod Balhorn. _SHAL Technologies, Inc., Livermore, CA_.

SH7139 is a small molecule drug developed to target B-cell malignancies overexpressing HLA-DR10. It contains a DOTA metal chelating ring and three recognition ligands (Ct, Cb, and Dv) that are linked together using a scaffold composed of lysines and mini-polyethylene glycols. The amide bonds within the scaffold have D-configurations to make them resistant to hydrolysis by native proteolytic enzymes. However, two of the ligands contain bonds and chemical groups that can be cleaved or modified by metabolic enzymes to produce known cytotoxic compounds. LCMS was used to identify the metabolites of SH7139 produced in cryopreserved hepatocytes, Burkitt's (Raji) lymphoma cells and the liver microsomes of several mammalian species. Following uptake of SH7139 by human, beagle and rat hepatocytes, which occurs slowly, the cleavage of an amide bond within the Ct ligand produced 2-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxyaniline (M8) and a large fragment containing the remainder of SH7139 (M2). Phase II metabolism led to rapid glucuronidation of M8 to facilitate its excretion. This result may explain the lack of SH7139 liver toxicity in animals studied to date. Raji lymphoma cells produce the same two Phase I metabolites, but glucuronidation of M8 is not observed. Thus, M8 is free to inhibit multiple activities required for tumor cell survival, including the processing of misfolded proteins, lipogenesis and sumoylation. Experiments performed with microsomes isolated from Raji cells and human, Cynomologus monkey, beagle, rat and mouse livers identified eight additional metabolites. Removal of a single methyl group from intact SH7139 produced M7. Hydrolysis of the azo group within the SH7139 ligand Dv in both M2 and M7 led to the production of small and large fragments in each case: dimethylphenylenediamine (M9) and M1 and methylphenylenediamine (M10) and M5, respectively. M9 and the phenylenediamine that is usually produced by the further metabolism of M9 and M10 have been shown by others to inhibit ATP production in mitochondria. A final set of demethylation, amide cleavage, oxidation, reduction and glucuronidation reactions involving M7 and M2 produced the remaining metabolite M6. While metabolic processing of the Cb ligand did not produce a biologically reactive metabolite, other experiments have found that the Cb ligand in intact SH7139 inhibits the hydrolysis of GTP by MgcRacGAP-Rac1 and the completion of cytokinesis. Collectively, these results confirm that SH7139 functions similar to both an antibody drug conjugate (ADC) and a pro-drug. In contrast to an ADC, however, the same SH7139 ligand that participates in the targeting function is also involved, either directly or following metabolic processing, in tumor cell killing. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843.

#1306

Combined efficacy of trifluridine and SN-38 in a 5-FU-resistant human colorectal cancer cell line.

Kazuaki Matsuoka, Takashi Kobunai, Teiji Takechi. _Translational Research Laboratory, Taiho Pharmaceutical Co., Ltd., Tokushima, Japan_.

Background: In 2015, the FDA approved TAS-102 (also named TFTD)—a 1:0.5 molar ratio of trifluridine (FTD), an antitumor agent, and tipiracil (TPI), a thymidine phosphorylase inhibitor—for the treatment of refractory metastatic colorectal cancer. In this study, we investigated whether cytotoxicity was enhanced when FTD was used simultaneously or sequentially with SN-38, which is an active metabolite of irinotecan hydrochloride (CPT-11). Method: The colorectal cancer cell line DLD-1 and 5-FU-resistant DLD-1/5-FU were treated with one of four combinations of SN-38 and FTD: (1) exposure to only 0.1-2.0 µM FTD or only 0.0005-0.02 µM SN-38 for 24 hours; (2) simultaneous exposure to 0.1-2.0 µM FTD and 0.01 µM SN-38 for 24 hours, followed by no drug exposure for 24 hours; (3) sequential exposure to 0.1-2.0 µM FTD for 24 hours followed by 0.01 µM SN-38 for 24 hours; or (4) sequential exposure to 0.01 µM SN-38 for 24 hours followed by 0.1-2.0 µM FTD for 24 hours. All treatments were evaluated by the colony formation assay. Results: The 5-FU-resistant DLD-1/5-FU demonstrated no cross-resistance to both FTD and SN-38. Survival fractions (SFs) of DLD-1 and DLD-1/5-FU after simultaneous exposure to 0.5 µM FTD and 0.01 µM SN-38 were 0.30 and 0.26, respectively, which were lower compared to those after exposure to FTD alone (0.92 and 0.94, respectively). After sequential exposure to 0.5 µM FTD followed by 0.01 µM SN-38 or vice versa, SFs of DLD-1 were 0.49 and 0.36, respectively, and those of DLD-1/5-FU were 0.47 and 0.22, respectively; in both cases, SFs were lower than those after exposure to FTD alone (0.92 and 0.94, respectively). Taken together, sequential exposure to SN-38 followed by FTD was more effective in both cell lines. Conclusion: Simultaneous and sequential exposure to FTD and SN-38 were effective against both parent and 5-FU-resistant cell lines. These results suggest that a combination of TAS-102 and CPT-11 might be useful to relapsed colorectal cancer after 5-FU-based treatment.

Table

---

DLD-1, mean ± SD, survial fractions, n = 3 (independently)

FTD

(µM) | FTD

schedule (1) | FTD + SN-38

schedule (2) | FTD ⇒ SN-38

schedule (3) | SN-38 ⇒ FTD

schedule (4)

0.0 | 1.00 ± 0.00 | 1.00 ± 0.00 | 1.00 ± 0.00 | 1.00 ± 0.00

0.1 | 1.04 ± 0.03 | 0.37 ± 0.05 | 0.64 ± 0.10 | 0.42 ± 0.18

0.5 | 0.92 ± 0.08 | 0.30 ± 0.11 | 0.49 ± 0.12 | 0.36 ± 0.16

1.0 | 0.79 ± 0.06 | 0.29 ± 0.12 | 0.27 ± 0.12 | 0.25 ± 0.09

2.0 | 0.50 ± 0.11 | 0.20 ± 0.07 | 0.05 ± 0.07 | 0.04 ± 0.02

DLD-1/5FU, mean ± SD, survial fractions, n = 3 (independently)

FTD

(µM) | FTD

schedule (1) | FTD + SN-38

schedule (2) | FTD ⇒ SN-38

schedule (3) | SN-38 ⇒ FTD

schedule (4)

0.0 | 1.00 ± 0.00 | 1.00 ± 0.00 | 1.00 ± 0.00 | 1.00 ± 0.00

0.1 | 1.01 ± 0.05 | 0.25 ± 0.11 | 0.65 ± 0.06 | 0.31 ± 0.22

0.5 | 0.94 ± 0.06 | 0.26 ± 0.12 | 0.47 ± 0.03 | 0.22 ± 0.14

1.0 | 0.92 ± 0.07 | 0.22 ± 0.12 | 0.25 ± 0.04 | 0.17 ± 0.07

2.0 | 0.61 ± 0.15 | 0.14 ± 0.07 | 0.09 ± 0.01 | 0.05 ± 0.01

#1307

ATM and ATR protein kinase activity confers cellular resistance to the toxic effects of the anthelminthic drug niclosamide.

Junaid Ansari,1 Hazem El-Osta,1 Reinhold Munker,1 Felicity N. E. Gavins,2 Jie Chen,3 Glenn M. Mills,1 Rodney E. Shackelford3. 1 _Feist-Weiller Cancer Center, LSU Health, Shreveport, LA;_ 2 _Department of Molecular and Cellular Physiology, LSU Health, Shreveport, LA;_ 3 _Department of Pathology, LSU Health, Shreveport, LA_.

Background and Purpose: Niclosamide is an anthelminthic drug which has recently exhibited anti-cancer effect in various malignancies acting via multiple intracellular growth-promoting cascades. One of the under studied aspects of niclosamide's activity is its ability to induce DNA damage. Here we examined the role of the DNA damage response (DDR) protein ataxia-telangiectasia mutated (ATM) and the ataxia-telangiectasia mutated and RAD3-related (ATR) protein kinases in cellular resistance to the toxic effects of niclosamide.

Materials and Methods: Wild-type cells and cells derived from individuals with the rare ATM protein loss disease ataxia-telangiectasia (A-T) and from individuals with hypomorphic ATR mutations (Seckel syndrome) where exposed to 0.5 µM niclosamide in colony efficiency forming assays. The effects of niclosamide on cell survival with and without ATM and ATR loss/deficiency, respectively was examined. Additionally, western blot on cell lysates was used to examine the effects of higher concentrations of niclosamide on ATM wild-type and ATR (wild-type and hypomorphic mutant) protein kinase activation. Lastly, the effect of niclosamide on ATM and ATR-dependent gene phosphorylation was examined.

Results: Niclosamide exerted preferential toxicity towards cells carrying either ATM protein loss (A-T cells) or Seckel syndrome cells carrying a hypomorphic ATR protein kinase mutation. Niclosamide also activated these proteins as measured by increased ATM serine-1981 and ATR protein serine-428 phosphorylations, markers of each kinase being activated. Interestingly, while niclosamide exposure increased activating ATR serine-428 phosphorylation, it failed to induce the same phosphorylation in the hypomorphic ATR mutation carrying cells that are otherwise syngeneic to the Seckel syndrome cells. Niclosamide exposure also induced serine-345 CHK1 protein phosphorylation in wild-type cells, but not in the syngeneic ATR hypomorphic mutant cells, indicating that niclosamide activates some DNA damage response pathways downstream of the ATR kinase. Lastly, niclosamide exposure increased serine-139 gamma-H2AX phosphorylation indicating it induces an important DNA repair event downstream of and regulated by both the ATM and ATR kinases.

Conclusion: Niclosamide activates two large protein kinases that are central to the DNA damage repair and the DDR and loss or attenuation of normal ATM or ATR protein function, respectively, results in increased cellular sensitivity to niclosamide, and loss of some of the cell-protective activities associated with normal ATM and ATR activities. For the first time, we show that niclosamide activates the DDR and the ATM and ATR proteins play a central role in cellular resistance to niclosamide toxicity. Possibly inhibition of tumor ATM and/or ATR activities might increase the effectiveness of chemotherapeutic regimes using niclosamide.

#1308

Enhanced antitumor activity of rapamycin and genipin, a UCP-2 inhibitor, in lung cancer.

Wen-Pin Su,1 Jang-Yang Chang,2 Ching-Chuan Kuo,3 Wu-Chou Su2. 1 _Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan;_ 2 _Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan;_ 3 _Institute of Biotechnology & Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli County, Taiwan_.

mTOR is constitutively activated in lung cancer. Deletion of TOR1 increases expression of OXPHOS proteins, and mitochondrial uncoupling proteins 2 (UCP-2) expression promotes chemoresistance. Therefore, we evaluated whether adding genipin, a UCP-2 inhibitor, to rapamycin has better antitumor effect in lung cancer. Combined with genipin and rapamycin have a synergistic effect with more cellular apoptosis in A549, H460 and CL1-0 cells. The similar phenomenon was found in the combination treatment with genipin and everolimus or BGT226 (a dual PI3K/mTOR inhibitor) in this three lung cancer cells. It is said that NrF2 may affect UCP-2 expression, and we found NrF2 was activated in lung cancer A549, H460 and CL1-0 cells treated with rapamycin. Everolimus and dual PI3K/mTOR inhibitor, BGT226, also induced NrF2 nuclear translocation in these lung cancer cells. It is said that under oxidant stress, p62 activates NrF2 through P62 Ser351-phosphorylation. We found that rapamycin induced ROS generation, p62 serine phosphorylation and NrF2 downstream mRNA and protein expression, including UCP-2. ROS inhibitor, NAC, and down-regulation of p62 by siRNA suppressed rapamycin-induced UCP-2 expression. Overexpression of p62 by cDNA transfection enhanced NrF2 activation and then UCP-2 expression in lung cancer cells. Moreover, rapamycin-induced increased UCP-2 expression, leading decreased mitochondrial membrane potential and mild increased oxygen consumption rate. Finally in A549 subcutaneous tumor mode in SCID mice, combined genipin and everolimus also demonstrated the greatest antitumor activity. Therefore, inhibition UCP-2 by genipin enhances anticancer effect of rapamycin in lung cancer.

#1309

CYC065, a novel CDK2/9 inhibitor, is an effective inducer of cell death and synergizes with BCL2 and BET inhibitors in B-cell lymphoma, including double-hit lymphomas.

Sheelagh M. Frame, Elizabeth Pohler, Craig MacKay, Daniella Zheleva, David Blake. _Cyclacel Ltd, Dundee, United Kingdom_.

Introduction: Double-hit lymphomas (DHL), defined by concurrent MYC and BCL2 rearrangements, have poor prognosis compared to standard-risk diffuse large B cell lymphomas (DLBCL). Current treatment regimens involve multiple chemotherapies, but do not specifically exploit these molecular features of the disease.

Several studies have established that: (i) DLBCL show frequent overexpression of Mcl-1, an anti-apoptotic member of the Bcl-2 family (30% and 50% in the ABC and GCB subtypes, respectively), (ii) MYC-driven lymphomas are highly sensitive to depletion of Mcl-1, (iii) MYC overexpression and inhibition of CDK activity are synthetically lethal and (iv) resistance to BH3 mimetics targeting Bcl-2 can be conferred by upregulation of Mcl-1.

CYC065 is a specific and potent CDK2/9 inhibitor, currently in a Phase 1 trial in patients with advanced cancer (NCT02552953). The mechanism of action of CYC065 involves rapid reduction of the levels of both Mcl-1 and MYC, suggesting a therapeutic rationale for investigating this agent in DLBCL. This preclinical study explored the single agent activity of CYC065 in B cell lymphoma, and its potential to combine with other molecularly targeted agents of interest, including venetoclax (ABT-199, a Bcl-2 inhibitor), and BET inhibitors.

Methods: Single agent activity of CYC065 was explored using short pulse treatments (6-8 h) in B cell lymphoma cell lines. Viability and total cell number were assessed 24, 48 or 72 h following treatment. Levels of MYC, Bcl-2 and Mcl-1 were determined by Western blotting at baseline and following treatment with CYC065. Cell fate was examined by flow cytometry. CYC065 was combined with venetoclax or BET inhibitors ((+)-JQ-1, GSK525762 and OTX-015), which were administered concomitantly for a 6 h pulse or up to 72 h. Combination data were analyzed by the Chou & Talalay method.

Results: The median IC50 for CYC065 in 13 B cell lymphoma cell lines was 0.43 µM. No obvious difference was observed between ABC and GCB subtypes of DLBCL, and DHL DLBCL lines had similar IC50 values to non-DHL DLBCL lines (median 0.47 µM vs 0.29 µM; p=0.95). As expected from the target inhibitory profile, CYC065 caused a rapid decrease in the phosphorylation of S2 of the CTD of RNA polymerase II followed by downregulation of Mcl-1 and MYC, and rapid induction of apoptosis in sensitive cell lines. CYC065 had no impact on Bcl-2 levels. Combining CYC065 with venetoclax was highly synergistic in DHL lines (median CI values range from 0.1-0.8), and resulted in >90% cell death. The combination of CYC065 and BET inhibitors was also highly synergistic.

Conclusions: CYC065 targets key oncogenic and survival pathways in DLBCL. CYC065 is a potent and effective inducer of cell death and combines synergistically with Bcl-2 or BET inhibitors in B cell lymphoma cell lines, including DHLs, which represent an unmet clinical need.

#1310

Targetingprolactin signaling to suppress pancreatic cancer stem cells.

Prasad Dandawate, Gaurav Kaushik, Dharmalingam Subramaniam, Prabhu Ramamoorthy, Scott J. Weir, Roy A. Jensen, Shrikant Anant. _University of Kansas medical Center, Kansas City, KS_.

Background: Pancreatic cancer (PCa) is a major cause of cancer related mortality in United States with < 6% survival rate. It is an aggressive and devastating disease, which is characterized by poor prognosis, invasiveness, rapid progression, profound resistance to drug treatment and recurrance after surgery. Presently, single agent based chemotherapy (e.g. Gemcitabine) is the major treatment for metastatic adenocarcinoma of pancreas but it has a tumor response rate of below 10%. Moreover combination therapy of gemcitabine and erlotinib only marginally improved survival rate. Hence, there is a dire need to identify novel ways to inhibit pancreatic cancer growth.

Methods: PCa cells (MiaPaCa-2 and PanC-1) were grown to 70-80% of confluency and treated with and without prolactin (PRL) and JAK2, STAT3 and ERK phosphorylation in presence and absence of antipsychotic compound were evaluated by western blot. Growth of PCa lines (MiaPaCa-2, PanC-1, BxPC-3, AsPC-1) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, respectively. Pancosphere formation was used to identify effects on stem cells.

Results: We have recently identified that the receptor for the pituitary hormone prolactin is overexpressed in pancreatic cancers, and in pancreatic cancer cell lines. When prolactin (PRL) binds its cognate receptor (PRLR), it induces various downstream events including the JAK-STAT and ERK MAPK pathways. In pancreatic cancer cell lines, we observe that PRL treatment induced dose- and time-dependent JAK2, STAT3, and ERK1/2 phosphorylation. Furthermore, there was an increase in the expression of cancer stem cell (CSC) markers DCLK1 (doublecortin calmodulin like kinase 1) and CD44. In addition, PRL-induced pancosphere formation further suggesting that PRL affects stem cells. Based on these data, we conclude that PRL signaling enhances stemness in pancreatic cancers, and therefore we decided to target it for therapeutic intervention. For this, we developed a homology model for the C-terminal intracellular region of the receptor and performed a virtual screening in silico with FDA approved drugs. One compound, a first generation antipsychotic drug diphenylbutylpiperidine, also called Penfluridol was found to interact with the region of the receptor that also binds site for JAK2. The compound has a long half-life, and is used in the treatment of chronic schizophrenia and similar psychotic disorders. We have further determined that Penfluridol inhibits PRL-induced STAT3 and ERK phosphorylation. In addition, the compound reduced proliferation, colony formation, and spheroid formation. Moreover, it induced cells to undergo autophagy by activating LC3B and increasing expression of autophagy-related genes ATG5, 7 and 12.

Conclusions: PRL signaling through its cognate PRLR receptor is critical for aggressive pancreatic cancer behavior, and therefore may be an effective therapeutic strategy.

#1311

High order drug combinations are required to effectively kill colorectal cancer cells.

Thomas Horn,1 Stephane Ferretti,2 Nicolas Ebel,2 Angela Tam,1 Samuel Ho,1 Fred Harbinski,1 Ali Farsidjani,1 Matt Zubrowski,1 William R. Sellers,1 Robert Schlegel,1 Dale Porter,1 Erick Morris,1 Jens Wuerthner,2 Sebastien Jeay,2 Joel Greshock,1 Ensar Halilovic,1 Levi A. Garraway,3 Giordano Caponigro,1 Joseph Lehar4. 1 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 2 _Novartis Institutes for BioMedical Research, Basel, Switzerland;_ 3 _DFCI, Boston, MA;_ 4 _Google Life Sciences, Mountain View, CA_.

Tumors are complex biological systems that often retain proliferative capacity even when challenged with drug treatment. Given this resiliency, drug combinations may provide greater therapeutic benefit, however, which molecules to combine and how many to include in combinations for effective responses is not clear yet. Using image-based proliferation and apoptosis assays in colorectal cancer cell lines we systematically investigated combinations that ranged in number from two to six drugs and targeted critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds, but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In many cases, where cell lines were resistant to two- and three-way drug combinations, increased expression of anti-apoptotic proteins was observed and induction of cytotoxic responses required up to fourth-order combinations. Our results demonstrate that high-order drug combinations might be needed to kill cancers and show how systematic drug combination screening together with a molecular understanding of drug responses can guide their identification.

## CANCER CHEMISTRY:

### Drug Delivery 1

#1312

PSMA antibody functionalized docetaxel-loaded magnetic nanoparticles for prostate cancer therapy.

Prashanth Kumar Bhusetty Nagesh, Nia Johnson, Vijaya K.N. Boya, Pallabita Chowdhury, Aditya Ganju, Bilal Hafeez, Sheema Khan, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. _The University of Tennessee Health Science Center, Memphis, TN_.

Objectives: Prostate cancer (PrCa) is the second most leading cause of cancer-related death in men in the United States. Chemotherapy (Docetaxel, Dox) is currently the most common first-line therapeutic option. However, adverse side effects and chemo-resistance of docetaxel limit its clinical use. Improving docetaxel targeted delivery and its activity at the tumor site using a targeted nanoparticle system could be an attractive strategy for PrCa therapy. Prostate Specific Membrane Antigen (PSMA) is highly overexpressed in PrCa cells, thus is a highly attractive molecular target for PrCa therapy. In this study, we developed and determined anti-cancer efficacy of a novel docetaxel loaded, PSMA targeted magnetic nanoparticle (PSMA-MNP-Dox) formulation for PrCa therapy.

Methods: Docetaxel loaded magnetic nanoparticle (MNP-Dox) formulation is composed of an iron oxide core coated with cyclodextrin (for drug loading) and F127 polymer (for particle stability and chemosensitization). Therapeutic efficacy of this unique nanoparticle formulation was evaluated using clinically relevant cell line models (C4-2, PC-3, and DU-145) through cell proliferation and colony formation assays. Molecular effects of this formulation on apoptosis, anti-apotosis, and drug resistance associated proteins were evaluated using immunoblotting assays. Contrast imaging property of MNP-Dox formulation was examined using Phantom Gel MR imaging model. For active targeting, PSMA antibody conjugation to this formulation was achieved through N-hydroxysuccinimide group containing PEG polymer. Active targeting potential of this formulation was evaluated in PSMA+ (C4-2) and PSMA- (PC-3) cell lines, C4-2 generated tumor xenografts.

Results: MNP-Dox formulation showed optimal particle size and zeta potential which can efficiently internalized in PrCa cells. Our formulation showed anti-cancer efficacy in prostate cancer cell lines. Additionally, it induces the expression of apoptosis associated proteins, Bax and Bad, cleaved PARP, and caspase 3, and downregulated the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Moreover, it also inhibited the expression of chemoresistance associated proteins (PSMA and MDR1). Our PSMA antibody targeted MNPs-Dox formulation exhibited a profound uptake pattern in PSMA+ cells (C4-2) compared to PSMA null (PC-3)- cells, suggesting its targeting potential. A similar targeting potential was also observed in ex-vivo studies while using C4-2 tumor xenografts, however, no intense targeting was observed in normal tissues due to lack of PSMA expression.

Conclusion: PSMA antibody functionalized MNP-Dox formulation can efficiently target PSMA + PrCa cells and deliver docetaxel into prostate tumors. This targeted drug delivery system could reduce the dose of docetaxel required to kill cancer cells, thus minimizing long-term docetaxel associated systemic toxicity and drug-resistance.

#1313

Targeting micelles eradicate acute myeloid leukemia stem cells in a patient-derived leukemia xenograft model.

Tzu-Yin Lin, Yuanpei Li, Yanjun Zhu, Hongyong Zhang, Rick Harse, Kit Lam, Brian Jonas, Chong-xian Pan. _UC Davis, Sacramento, CA_.

Patients with acute myeloid leukemia (AML) have a very poor prognosis related to a high rate of recurrence rate drug-related toxicity. The current standard "7+3" regimen, with cytarabine and an anthracycline such as daunorubicin (DNR), can achieve complete remission in up to 70-80% of patients, but the majority of patients ultimately suffer from relapse and as many as 30% of patients die from drug-related toxicity during induction therapy. The ability of leukemia stem cells (LSC) to survive chemotherapy is primarily responsible for relapse, and eliminating LSC is ultimately essential for cure. The goal of this project is to develop novel LSC-targeting nanometer-scale micelles, called disulfide-crosslinked CLL1-targeting micelles (DC-CTM), which can deliver high concentrations of DNR into both leukemia and leukemia stem cells and eradicate AML from the very root. In this project, we successfully synthesized DC-CTM-DNR. The nanoparticles were 17+/- 6 nm in size and had a payload of 2mg/ml DNR with 100% loading efficiency. DC-CTM were very stable, while an intracellular reductive agent, Glutathione, could reduce the disulfide bonds resulting in micelle dissociation after uptake. Furthermore, using an AML patient-derived xenograft (PDX) model, we showed that 1 mg/kg DC-CTM-DNR-treated mice had significantly less leukemia burden in the bone marrow at week 2 and 4 than both DNR- and PBS-treated mice. Although all mice developed end-stage leukemia by week 8, the DC-CTM-DNR-treated group had significantly smaller leukemia-infiltrated spleens than the PBS group. We further evaluated the effect of treatment on LSC in vivo by secondary transplantation of total bone marrow cells from DNR- and DC-CTM-DNR-treated AML PDX bearing mice into naïve NSG mice. At 8 weeks post secondary transplantation, 100% (7/7) and 37.5 % (3/8) of mice developed leukemia in the bone marrow from the 1 mg/kg DNR- and DC-CTM-DNR-treated groups, respectively. Spleens from secondary transplant mice in the DC-CTM-DNR group were significantly smaller than the DNR group indicating less leukemia infiltration. Collectively, DC-CTM-DNR exhibited superior anti-leukemia efficacy and could eradicate LSC more effectively than free DNR. Lastly, pharmacokinetic studies were performed with free DNR and DC-CTM-DNR in rats. Consistent with previous results, DC-CTM-DNR had a longer T1/2a, T1/2b, and accumulated under curve area, than free DNR. Toxicology studies also confirmed that DC-CTM-DNR exhibited a superior toxicity profile, with significantly less cardiotoxicity. In conclusion, we have developed a novel LSC-targeting micelle delivery system that can effectively target AML LSC and bulk leukemia cells with decreased systemic toxicity. Ultimately, DC-CTM-DNR has the potential to significantly enhance the complete remission rate and disease-free survival time and decrease the relapse rate and treatment-related mortality for patients with AML.

#1314

Evaluating payload delivery to the CNS from surface-modified nanoparticles.

Kyle T. Householder, Danielle DiPerna, Layla Ghaffari, Rachael Sirianni. _Barrow Neurological Institute, Phoenix, AZ_.

The blood-brain barrier (BBB) remains a major obstacle to treatment of intracranial tumors. Most drugs are poorly bioavailable in the brain, and peripheral toxicity can severely limit administerable dose. Drug loaded nanoparticles are capable of accumulating preferentially in the core of angiogenic tumors by a mechanisms known as the enhanced permeation and retention (EPR) effect, which can improve therapeutic efficacy of encapsulated molecules compared to free form. However, to achieve complete tumor kill, chemotherapy must target both the leaky tumor core and cells that reside behind an intact blood-brain barrier.

The goal of this work was to evaluate payload delivery to the CNS from nanoparticles modified with one of four targeting ligands: TAT, antennapedia (AP), angiopep2 (Ang2) or Tet-1. Poly(lactic acid)-co- poly(ethylene glycol) nanoparticles (NPs) encapsulating Nile Red, a small fluorescent dye, were produced by solvent-evaporation technique. Peptides were covalently attached to the nanoparticle surface by a maliemide-thiol reaction. The effect of targeting ligands on NP payload delivery to healthy brain and spinal cord compared to unmodified NPs was evaluated in mice at 2 and 6 hours post injection. Mice were perfused with saline, and the brains and spinal cord were removed, homogenized and payload delivery quantified by fluorescence.

All targeting ligands evaluated significantly increased payload delivery to the brain at both 2 and 6 hours, with the cell-penetrating peptides, TAT and AP, producing the greatest effect of a 4-fold increase over control. Additionally, the cell-penetrating peptides significantly increased payload delivery to the spinal cord at 2 hours but had no significant effect on delivery at 6 hours. Interestingly, the receptor-mediated peptides, Ang2 and Tet-1, did not significantly change spinal cord delivery at 2 hours but significantly increased payload concentration at 6 hours. These data suggest both receptor and nonspecific targeting ligands affect payload delivery and kinetics in a regionally specific manner. Future work is focused on evaluating additional ligands to better understand how the type of cell-nanoparticle interactions affects payload distribution.

#1315

Nanoparticles targeting RAF/ERK-driven cell-autonomous resistance to sorafenib for effective treatment of hepatocellular carcinoma.

Yunching Chen,1 Ya-Chi Liu,1 Ts-Ting Lin,1 Rakesh Ramjiawan,2 Dan Duda3. 1 _Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan;_ 2 _Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA;_ 3 _Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA_.

Sorafenib is the only systemic therapy approved for advanced hepatocellular carcinoma (HCC). Sorafenib's efficacy has been attributed in part to inhibition of cancer cell proliferation due to B- and CRAF targeting in HCC cells. However, cell autonomous mechanisms promote evasion from sorafenib treatment, leading to moderate survival benefit. Herein, we demonstrated that the effects of sorafenib on HCC cell viability were initially independent of RAF kinase inhibition and were mediated in part by p38MAPK inhibition. Moreover, sorafenib treatment led to RAF heterodimerization (BRAF/CRAF) and subsequent ERK activation and thus converted HCC cell dependence for survival signaling to the RAF/ERK pathway. Inhibition of ERK pathway by the MEK inhibitor AZD6244 significantly enhanced the therapeutic efficacy of sorafenib efficacy on HCC in vitro and in vivo. Furthermore, we developed HCC-targeted nanoparticles (NPs) to efficiently co-deliver both sorafenib and AZD6244 into HCC, downregulate RAF/ERK pathway, suppress angiogenesis and enhance anti-cancer effect in the orthotopic HCC model. In conclusion, sorafenib treatment leads to rapid MAPK activation due to BRAF heterodimerization as a cell-autonomous mechanism of treatment resistance. Co-delivery of sorafenib and MEK inhibitor AZD6244 using HCC targeted NPs downregulates the compensatory activated RAF/ERK pathway and overcomes resistance to sorafenib in HCC.

#1316

Targeted therapeutic approach for triple negative breast cancer using paramagnetic nanoparticle.

Meser M. Ali, Li Zhang, Hassan Bagher Ebadian, James R. Ewing. _Henry Ford Hospital, Detroit, MI_.

Breast cancer (BC) is a spectrum of diseases with distinct molecular alterations accounting for differences in treatment response and outcome. Gene expression profiling defines much of this molecular heterogeneity and has been used to stratify the disease into different molecular subtypes. Triple-negative breast cancer (TNBC) refers to a subtype of breast cancer that is negative for estrogen receptors (ER) and/or progesterone receptors (PR), and lacks HER2 overexpression. Therefore, this subtype of breast cancer lacks the benefits of specific therapies which target these receptors. TNBC has been characterized by an acidic extracellular environment. In human aggressive breast tumors, extracellular pH (pHe) has been measured by microelectrode and the values are in good agreement with the values observed in animal systems i.e. that the pHe is significantly acidic in the range from 6.2 to 7.0. However, tumor intracellular pH is either neutral or alkaline. Interestingly, a similar pH gradient is not observed in normal tissues. Therefore, this acidic extracellular pH (pHe) within tumor tissues can be exploited for targeted delivery of drugs and imaging agents.

Recently, A pH low insertion peptide (pHLIP) derived from the protein bacteriorhodopsin has been found to target tumor acidic pHe. The peptide inserts across cell membranes as an alpha-helix when extracellular pH (pHe) is acidic, but does not form the helix at normal or alkaline pH. Since aggressive TNBC has an acidic environment, the pHLIP will insert into the cancer cell membrane, but the pHLIP will not insert into the cell membranes of normal tissues, providing excellent specificity for targeting TNBC. Our initial study demonstrated the detection of breast tumor in rodent by dendrimer-based paramagnetic nanoparticles nonspecifically. To develop this nanoparticle as a platform, it should easily accommodate targeting ligands for selective localization at the acidic tumor microenvironment and for therapeutic drugs that can be released selectively into TNBC tumor cells. Here, we demonstrate that pHLIP-tagged nanoparticles bind to and are internalized by TNBC cells in in-vitro. Systemic delivery of paramagnetic nanoparticle, Gd-G5-pHLIP (NP-pHLIP) leads to accumulation of the NPs in a flank mouse model of TNBC tumor that are detected by both optical and MR imaging.

Finally, we have conjugated doxorubicin (dox) with pHLIP3-NP via hydrazone bond formation. We have successfully incorporated 20 dox molecules with pHLIP3-NP via a pH-sensitive hydrazone bond as evidenced by maldi-tof analyses. A cohort of 6 mice was treated with pHLIP-NP-dox and a second cohort of 6 mice was treated with vehicle. A solution of 0.04 mmol/kg pHLIP-NP-dox (12mg/kg equivalent dose of dox) was injected through the tail vein catheter at day 8 and 14. We generated T2 maps on day 21`to calculate tumor volume by following our previous reports. We observed a short-term tumor regression by pHLIP-NP-dox infusion.

#1317

IT-143: A nanoparticle delivery platform that encapsulates daunorubicin and widens therapeutic window.

Tara Lee Costich,1 Rick Crouse,2 Adam Carie,1 Taylor Buley,1 Tyler Ellis,1 Lauren Repp,1 J.Edward Semple,1 Tomas Vojkovsky,1 Suzanne Bakewell,1 Kevin Sill1. 1 _Intezyne Technologies, Tampa, FL;_ 2 _Yale University, New Haven, CT_.

Daunorubicin is an anthracycline family chemotherapeutic with a narrow therapeutic window. Employing our nanoparticle drug delivery platform we encapsulated daunorubicin non-covalently in the hydrophobic core of a polymer micelle (IT-143). IT-143 is a nanoparticle formulation comprised of a hydroxamic acid triblock polymer with ferric crosslinking that increases stability for improved drug circulation and results in a pH dependent release of the encapsulated daunorubicin. Formulation characterization demonstrates a 3.7% weight loading (v/v) of daunorubicin resulting in an average micellar size of 58 nm. In vitro cytotoxicity is comparable to daunorubicin free drug with an IC50 ranging between 60-100 nM, dependent on cell line, compared to 67-114 nM for daunorubicin. In vivo we compared IT-143 plasma pharmacokinetics to daunorubicin administered as free drug and the Cmax was extended from 0.18 µg/mL for daunorubicin to 27.0 µg/mL for IT-143 at the 3 mg/kg dose, and to 49.4 µg/mL at the 6 mg/kg dose. We increased the maximum tolerated dose (MTD) from 7.5 mg/kg to 17.5 mg/kg in mice and improved anti-tumor efficacy. Furthermore, IT-143 eliminated in vivo toxicity compared to equivalent daunorubicin dosed at its MTD. Efficacy studies in 3 xenograft models (HCT116, HT-1080 and HOS-MNNG) compared intravenous bolus administration of IT-143 at equivalent and equitoxic dose to daunorubicin treatment. IT-143 inhibited HCT116 colorectal tumor volume growth by 21% compared to 7% at the equivalent daunorubicin dose, and by 51% at the 17.5 mg/kg dose. In the HT-1080 fibrosarcoma model IT-143 inhibited tumor volume by 38% compared to 37% inhibition at the equivalent daunorubicin dose, and by 94% at the 17.5 mg/kg dose. In the HOS-MNNG osteosarcoma model IT-143 did not inhibit tumor volume compared to 21% inhibition at the equivalent daunorubicin dose, but inhibited tumor volume by 34% at the 17.5 mg/kg dose. The encapsulation of daunorubicin as IT-143 exhibits increased dosage of daunorubicin with decreased toxicities not possible with free daunorubicin. These studies indicate that IT-143 widens the therapeutic window of daunorubicin treatment, providing a safer way to reduce patient toxicity yet increasing antitumor efficacy.

#1318

Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel.

Jun Zhao, Chunhui Wu, Xiaoxia Wen, Junjie Li, Qian Huang, Ying Ma, Stephen Ullrich, Huamin Wang, Jason Fleming, Chun Li. _UT MD Anderson Cancer Center, Houston, TX_.

Sonic hedgehog (SHH) signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine, a potent inhibitor of the SHH pathway, has shown promising antitumor efficacy. However, cyclopamine is insoluble in water and is highly toxic, and cyclopamine monotherapy did not effectively inhibit tumor growth in a range of preclinical pancreatic cancer models. We hypothesized that polyion complex polymeric micelles that simultaneously deliver cyclopamine and paclitaxel (M-CPA/PTX) would display greater efficacy against pancreatic tumor than micelles loaded with cyclopamine alone (M-CPA) or paclitaxel alone (M-PTX). We tested this hypothesis in cell cultures and in an orthotopic human pancreatic cancer Miapaca-2 xenograft model and a genetically engineered mouse model of pancreatic ductal carcinoma (PDAC), the KPC model. Our results showed that M-CPA interrupted the SHH pathway by antagonizing the smoothened receptor, reduced expression of Gli-1 transcription factor, and inhibited proliferation of Miapaca-2 cells and stroma-producing human pancreatic stellate cells. In Miapaca-2 cells, M-CPA decreased the fraction of CD133+ cells (cancer stem cells) and decreased tumorsphere formation, whereas M-TXL increased the fraction of CD133+ cells and tumorsphere formation. M-CPA negated the stimulation of human pancreatic stellate cells by exogenous SHH ligands. In vivo, M-CPA/PTX reduced tumor cell proliferation, depleted tumor stroma, and increased tumor vasculature density, leading to effective inhibition of tumor growth in Miapaca-2 xenografts. In the KPC model, M-CPA/PTX significantly prolonged media survival of mice with PDAC compared to non-treatment control mice. In conclusion, M-CPA/PTX was an effective strategy to treat pancreatic cancer. (supported in part by the Viragh Family Foundation and the John S. Dunn Foundation)

#1319

A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models.

Ada W.Y. Leung,1 Malathi Anantha,1 Kathleen E. Prosser,2 Mohamed Wehbe,1 Charles J. Walsby,2 Marcel B. Bally1. 1 _BC Cancer Research Centre, Vancouver, British Columbia, Canada;_ 2 _Simon Fraser University, Burnaby, British Columbia, Canada_.

CX-5461 is a RNA polymerase I inhibitor currently in Phase I clinical trial in Australia for patients with advanced hematologic malignancies. In the pre-clinical setting, CX-5461 is efficacious in a wide range of hematologic and solid tumor models when given orally or intraperitoneally. Currently, the compound is given intravenously (iv) in the first-in-human clinical trial. CX-5461 is solubilized in low pH (3.5) 50 mM sodium phosphate as it is sparingly soluble in water (<1 mg/mL) and at physiological pH. The use of such low pH phosphate buffers affects infusion tolerance. While CX-5461 has nanomolar range activity in vitro, in vivo studies are conducted with either high dosages or frequent dosing. The chemical structure of CX-5461 also suggests potential drug instability under physiological conditions. We have addressed these solubility and stability issues by developing a lipid-based nanoparticulate (LNP) formulation which relies on a previously unrecognized interaction between CX-5461 and copper. Copper (Cu) coordination with CX-5461 was demonstrated using 1H NMR and UV-Vis. Cu-LNPs were prepared with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio) using extrusion methods. CX-5461 was loaded into Cu-LNPs at 60oC for 30 minutes and the external buffer was exchanged to Hepes Buffered Saline (pH 7.4). Pharmacokinetic (PK) studies were performed in CD-1 mice given a dose of 30 mg/kg (iv) and plasma concentrations of CX-5461 were determined by HPLC. Efficacy studies were performed in RAG2M mice with established subcutaneous (sc) MV-4-11 tumors and in a MV-4-11 bone marrow engraftment model (30 mg/kg given iv, Q4Dx3). Our 1H NMR data suggests that CX-5461 binds to copper at a 1:1 molar ratio. Copper complexation does not affect the anticancer activity of CX-5461 as determined in several cancer cell lines in vitro. Using copper-complexation as a driving force, CX-5461 was efficiently loaded into LNPs (~100 nm) prepared to contain copper (300 mM CuSO4). Resulting LNPs have a drug-to-lipid-ratio of 0.2. The LNP formulation has significantly improved the PK profile of CX-5461 and increased the total exposure to drug by an order of magnitude (AUC = 4156 µg-hr/mL for LNP and 319 µg-hr/mL for free CX-5461). The LNP formulation of CX-5461 is more active than the current low-pH formulation of CX-5461 when given iv in leukemia xenograft models. Specifically 37 days after sc tumor cell inoculation the tumor sizes were 720 mm3 and 240 mm3 in control and LNP-CX5461-treated animals respectively. The clinical formulation administered at the same dose exhibited tumor volumes that were comparable to the untreated controls. We have generated the first LNP formulation of CX-5461 using the copper complexation technology developed in our laboratory. LNP-CX-5461 is suitable for iv administration and is more efficacious than the current formulation in our pre-clinical studies.

#1320

Local and sustained delivery of tamoxifen for the prevention of ER+ breast cancer using a nanochannel delivery platform.

Carly S. Filgueira, Eugenia Nicolov, Andrea Ballerini, R. Lyle Hood, Priya Jain, Giacomo Bruno, Alessandro Grattoni. _Houston Methodist Research Institute, Houston, TX_.

A high incidence (~75%) of primary breast cancers are estrogen receptor positive (ER+), and a large fraction of these patients can pursue chemopreventive therapies. However, due to adverse side effects, only 5% to 20% of the women at high risk who could benefit from chemotherapeutics enroll in preventive treatment. There is a clear need for alternative preventive strategies that minimize side effects and improve enrollment and compliance. Selective estrogen receptor modulators, such as tamoxifen (TMX), have been shown to reduce ER+ breast cancer incidence by up to 50% among high-risk women. Importantly, along with raloxifene, it is one of only two FDA-approved drugs for breast cancer prevention. TMX has been in use for over 40 years and has a proven record in pre- and post-menopausal women. However, the drug is marred by side effects, the most common being symptoms of menopause. Further, women treated systemically and chronically with TMX were found to have an increased incidence of endometrial carcinoma. Although rare, this side effect, along with other serious adverse effects (such as blood clots, strokes, and cataracts), has resulted in a debate concerning TMX use in cancer prevention. As the key for breast cancer chemoprevention relies upon long-term delivery of drugs while circumventing side effects, we have developed a novel local delivery strategy for the constant and sustained administration of TMX. We maintain a long-term, local release of TMX in mammary tissues by utilizing a novel implantable nanochannel Delivery System (nDS). The nDS consists of a bioinert, implantable, and mechanically robust silicon membrane which houses an exact number of densely packed slit-nanochannels as small as 2.5 nm with tight tolerances on size, geometry, and surface properties. Providing steady levels of TMX at the mammary gland target through nDS delivery maximizes the therapeutic index while limiting the unwanted secondary effects, which will ultimately improve patient compliance. In this work we chemically induced tumorigenesis in Sprague-Dawley rats by N-methyl-N-nitrosourea (NMU) injection to promote development of estrogen-dependent tumors. We performed ovariectomies seven days after NMU injection to mimic post-menopausal biology. nDS implants loaded with either TMX or PEG400 (negative control) were inserted under the left abdominal mammary gland to determine effects of nDS-TMX on tumor growth and biomarkers. Utilizing LC/MS we were able to determine the amount of TMX released from the nDS. Rats were examined for palpable tumors to assess breast tumor incidence, latency to onset, and multiplicity. Our results show that the nDS implant enables the effective delivery of TMX in this breast tumor model. Further, this technology has the potential to rapidly provide long-term breast cancer protection with significant improvement in the quality of life of patients at high risk, thereby saving thousands of lives every year.

#1321

IT-147: A stabilized polymer micelle formulation of epothilone D for treatment of solid tumors.

Adam Carie,1 Taylor Buley,1 Bradford Sullivan,1 J.Edward Semple,1 Tyler Ellis,1 Tara Lee Costich,1 Jyothi Sethuraman,1 Rick Crouse,2 Kevin Sill,1 Suzanne Bakewell1. 1 _Intezyne Technologies, Tampa, FL;_ 2 _Yale University, New Haven, CT_.

IT-147 is a formulation of epothilone D encapsulated in a stabilized polymer micelle. Epothilones are a class of cytotoxic chemotherapeutics that induce apoptosis via microtubule stabilization. Epothilone D is a poor substrate for P-glycoprotein efflux pumps and has demonstrated equivalent activity against drug-sensitive and multidrug-resistant human cancer cell lines. This offers a distinct advantage over other microtubule stabilizing agents such as taxanes, however clinical utility is limited by a narrow therapeutic window due to dose limiting toxicities such as peripheral sensory neuropathy, GI toxicities, and cognitive abnormalities. We have optimized a lead formulation comprising 2% epothilone D (w/w) encapsulated in a polymer micelle that is crosslinked with iron to provide micelle stability upon dilution. IT-147 has an average diameter of 70 nm. The iron-mediated crosslinking provides pH-dependent release of epothilone D such that drug can be released in the tumor microenvironment as well as in the endosome/lysosome upon cellular entry. In addition, the geometrical arrangement of iron molecules around the core of the micelle imparts superparamagnetic properties that allow for imaging of intact nanoparticles by MRI. IT-147 demonstrates a low nanomolar IC50 against a panel of human cancer cell lines in vitro. In vivo, IT-147 demonstrates a 4-fold increase in maximum tolerated dose in a mouse model compared to epothilone D free drug. Pharmacokinetics (PK) in a cannulated rat model for a 20 mg/kg epothilone D dose demonstrates a 6.5-fold increase in area under the time versus concentration curve (AUC), and a 14.3-fold increase in the terminal half-life compared to free drug. Dose escalation of IT-147 to 30 and 40 mg/kg epothilone D in the rat PK model results in linear increases in AUC from 53.5 h*µg/mL at 20 mg/kg to 82.9 and 110.8 h*µg/mL at 30 and 40 mg/kg, respectively. Treatment with IT-147 at the MTD in an HCT116 tumor xenograft model led to tumor stasis during the course of treatment, and MRI imaging of mice treated with IT-147 demonstrates tumor accumulation over 24 and 48 hours post administration. Taken together, data for IT-147 demonstrates successful encapsulation of epothilone D leading to a safer, more effective formulation with the potential for MRI imaging as a predictor for response.

#1322

Folate receptor-targeted lipid coated albumin nanoparticles (F-LCAN) for therapeutic delivery of RX-0201 (Archexin®), an antisense oligonucleotide against Akt-1.

Hong Li,1 Xinwei Cheng,1 Yang Liu,1 Young Bok Lee,2 Deog Joong Kim,2 Chang-ho Ahn,2 Robert J. Lee1. 1 _The Ohio State University, Columbus, OH;_ 2 _Rexahn Pharmaceuticals, Inc., Rockville, MD_.

Akt-1 is a protein kinase that contributes to cancer progression by promoting proliferation and inhibiting apoptosis of tumor cells. RX-0201 (Archexin®), a 20-mer phosphorothioate antisense oligonucleotide that inhibits the expression of Akt-1, is currently in a Phase IIa clinical trial for metastatic renal cancer. To further improve the efficacy of RX-0201, folate receptor-targeted lipid coated albumin nanoparticles containing RX-0201 (F-LCAN-RX-0201) were synthesized and evaluated for Akt-1 inhibition and antitumor activity. These nanoparticles are composed of DOTAP/soyPC/TPGS/folate-PEG-DSPE (25:70:4:1), a cationic human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, and RX-0201. F-LCAN-RX-0201 had a particle size of 108.6±5.8 nm, zeta potential of 10.5±3.2 mV and 71.5% antisense loading efficiency. F-LCAN-RX-0201 in folate receptor-expressing KB cells inhibited cell growth at IC50 of 11.9 ± 4.0 µM while free RX-0201 did not show significant cytotoxicity at concentration up to 250 µM. Cellular uptake and Akt-1 inhibition of F-LCAN-RX-0201 was greater than the non-targeted LCAN-RX-0201 and was partially blocked by excess free folate. Pharmacokinetic studies showed that F-LCAN-RX-0201 had a longer half-life than that of free RX-0201 (30 ± 4 min vs 10 ± 2 min). In a KB xenograft tumor model, F-LCAN-RX-0201 exhibited higher tumor suppressive activity compared to the non-targeted LCAN-RX-0201 at 16 mg/kg (as a RX-0201 amount) without body weight changes. Moreover, the antitumor activity of F-LCAN-RX-0201 at a dose of 16mg/kg (as a RX-0201 amount) was similar to free RX-0201 at a dose of 90mg/kg. In conclusion, LCAN is a highly effective formulation for therapeutic delivery of antisense agents and F-LCAN-RX-0201 could be a potential therapeutic agent for tumors with increased folate-receptor expression.

#1323

Targeting tMUC1 for the diagnostics and treatment of pancreatic cancer.

Shuta Wu, Anthony J. Fowler, Craig Ogle, Pinku Mukherjee. _UNC Charlotte, Charlotte, NC_.

Background:

Pancreatic Cancer is (PC) is the 4th leading cause of cancer-related deaths in the United States with 94% of PC patients dying within 5 years of diagnosis. Treatment options for PC patients are limited as PC is diagnosed at a late stage from the lack of early symptoms and inaccessibility of the pancreatic region. Typical early detection methods involve ultrasound tests or cholangiopancreatography which can be intrusive or use radioactive materials.

Typical treatments involve ablation, chemotherapy, or a combination of chemotherapy with radiation. But these methods can have adverse side and off-target effects due to systemic delivery of drugs

There is a need to diagnose pancreatic cancer efficiently and to target the delivery of anti-cancer treatments at optimal dosages to avoid adverse side-effects in patients.

Pancreatic Ductal Adenocarcinoma (PDA) represents 90% of PCs and over 80% of PDAs over-express tumor-associated Mucin-1 (tMUC1), a membrane-tethered glycoprotein protein. Tumor-associated MUC1 exhibits changes to its glycosylation pattern that expose its protein core, thus making it identifiable to the TAB004, a specific antibody we developed and have shown to only recognizes this form of MUC1.

Thus, we hypothesize that TAB004 antibody can be utilized as a targeting agent to accurately diagnose PDA and to enable the use of highly cytotoxic anti-cancer treatments at localized concentrations, which would make the treatment more effective at lower doses overall.

Methods:

We determined the specificity and internalization of TAB004 (OncoTab Inc) in several MUC1-expressing PDA cell lines in vitro using confocal and high resolution fluorescence microscopy. NuLink kit (NuChemie) was used in TAB004-PLGA nanoparticle (NPs) conjugation. Confirmation of NPs to TAB004 conjugation (NP-TAB) was determined using flow cytometry. NP-TAB and NPs internalization was compared using confocal microscopy in vitro in several tMUC1-expressing PDA cell lines. Viability of cell lines was determined using the Dojindo CCK -8 Cell Proliferation Assay and Cytotoxicity Assay in response to NPs and NP-TAB treatments. Imaging of mice injected with TAB004 conjugated with ICG (Dojindo ICG labeling Kit-NH2), NPs with ICG, and NP-TAB with ICG was performed using and IVIS Spectrum.

Results:

TAB004 was specific for and internalized significantly more in tMUC1 expressing cell lines. NP-TAB persist longer in tMUC1 expressing cells in vitro. TAB004 was able to target tMUC1 expressing orthotopic PDA tumors. NP-TAB had significantly increased accumulation at the tumor site than NPs. Further experiments are currently being performed in vivo in an appropriate mouse model of PDA to determine treatment efficacy.

Conclusion:

TAB004 is a promising targeting agent for diagnosing and treating tMUC1 expressing PDA. Given that tMUC1 is associated with aggressive form of PDA and patients with MUC1-positive PDA have poor prognosis, these results are significant and clinically relevant.

#1324

**Enhancing therapeutic efficacy of albumin bound paclitaxel in breast cancer liver metastasis by homing to tumor associated macrophages: I** n vitro **,** in vivo **and** in silico **studies.**

Fransisca Leonard,1 Curtis T. Louis,2 Pooja Yesantharao,1 Tomonori Tanei,1 Jenolyn F. Alexander,1 Liu Xuewu,1 Ferrari Mauro,1 Kenji Yokoi,1 Hermann B. Frieboes,2 Biana Godin1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _University of Louisville, Louisville, KY_.

This study is aimed to evaluate and enhance the albumin-bound paclitaxel (nab-PTX) in multistage vector (MSV) for liver metastasis treatment using in silico modeling based on in vitro data, and verified via in vivo study. The resulting model will help for therapy optimization in the clinical setting.

Clinically, breast cancer liver metastases can be observed as hypoattenuated in MRI and CT images caused by low penetration of drugs or contrast agents into the lesions along with a high washout rate, which correlates to poor chemotherapeutic response. Nab-PTX is clinically used for treatment of advanced breast tumors, but not for liver metastasis. We designed MSV-nab-PTX, a new nab-PTX delivery system for specifically homing to tumor associated macrophages in the liver. MSV-nab-PTX consists of nab-PTX loaded in porous silicon multistage nanovectors which were previously shown to be efficient in delivering siRNA and other therapeutics. MSV-nab-PTX was evaluated in in vitro, in vivo and in silico models of liver metastasis. Co-culture of breast cancer tumor cells (3D-spheroids) and macrophages was developed and utilized to evaluate the drug efficacy and to study the mechanism of nab-PTX transport. Addition of macrophages in the 3D model significantly increased the efficacy of MSV-nab-PTX, but not nab-PTX, revealing the major role of macrophages in the drug transport into the lesion. Primary mouse and human macrophages were shown to uptake up to 10 ng of PTX/cell in MSV-nab-PTX with no significant effects on their viability, and later release the PTX over 24h. Treatment with MSV-nab-PTX increased chemokine production by tumor cells when compared to nab-PTX, increasing macrophage migration into the tumor sphere by more than 2-fold. Based on these data, we implemented a mathematical model linking drug release and retention from macrophages to project MSV-nAb-PTX efficacy based on the presence of macrophages in tumor biopsies. Simulation of repeat treatment every 3 days showed a significant reduction of tumor size, and was verified by the in vivo data. MSV-nab-PTX as compared to nab-PTX enhanced PTX concentration in the target lesions, increased apoptosis rate and reduced cancer cells proliferation rate, ultimately reducing the tumor size and prolonging the survival of treated mice.

This showed that MSV association with macrophages can increase drug efficacy compared to bolus injection, and that it is feasible to reach sustained lesion regression with the macromolecule-bound formulation. The in silico simulations also revealed that the timing interval is crucial for the treatment strategy, and the effect depends on the size and vascularization stage of the lesion. We conclude that an integrated in vitro, in vivo, and in silico framework may be of use to assess response to albumin bound paclitaxel targeted to breast cancer liver metastasis via tumor associated macrophages.

#1325

Oral delivery of drug-loaded microspheres for cancer.

Duc P. Do. _Chicago State University College of Pharmacy, Chicago, IL_.

The oral route remains the preferred method of administering drugs; however, this delivery mode faces many challenges, including low bioavailability of the administered drug, generalized toxicity and drug decomposition. Microsphere drug delivery systems have evolved to improve patient compliance, reduce toxicity, and increase efficacy. Additionally, the use of microspheres to deliver drugs has many other advantages, such as control-release of the active drug, increase bioavailability and target delivery of the drug to the desired tissue/cell. Our research explored the utility of encapsulating vorinostat in biodegradable microsphere delivery system, to be delivered orally via a capsule, to attain effective therapeutic effect. The strategy is to develop an oral, biodegradable albumin-based microsphere delivery system containing vorinostat that would elicit targeted inhibitory effect on cancer to enhance therapeutic efficacy. Vorinostat-loaded microspheres were prepared by a microencapsulation method through spray drying. The parameters for spray drying were optimized to find the best formulation for enhanced uptake and efficacy in vitro in cultured HEPG2 cells. Microspheres were loaded into capsules. Microspheres and capsule formulations were assessed for particle size, particle size distribution, surface morphology and properties and Zeta potential measurements. Drug release characteristics were quantified using dissolution studies. Chemical and thermal stability of the encapsulated drug and microsphere formulations were evaluated using FTIR and DSC, respectively. The uptake and cellular internalization of the microspheres were evaluated using co-cultures of Caco-2 cells and Raji cells. In vitro cytotoxicity was evaluated using MTT and clonogenic assays. Histone deacetylase (HDAC) activity levels in cells from treatment of free vorinostat and microparticle formulation were compared. Global histone modifications in HEPG2 cancer cells due to vorinostat-loaded microsphere treatment were also analyzed. Our data indicated that vorinostat microspheres were 1-2 microns in size and possessed spherical morphology. Zeta potential measurements show values of -30 mV, which indicated colloidal stability of the microsphere dispersion. Uptake studies indicated that more than 50% of the microspheres were internalized inside the cells within 12 hours. Additionally, dissolution studies showed that within 24 hours, 80% of the drug is sustained-release from the microsphere formulations. HDAC activity levels were shown to be decreased in cultured cells when vorinostat microspheres were used. Our in vitro studies showed that drug-loaded microsphere formulations exert cytotoxic effects on cultured cells. In summary, microspheres were shown to be a potential effective oral delivery system to administer anticancer drugs, with enhanced efficacy due to their controlled-release manner.

#1326

1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside a novel c-Myc inhibitor induces GNMT expression and apoptosis in hepatocellular carcinoma.

Rajni Kant,1 Chia Hung Yen,2 Chung Kuang Lu,3 Chien Yi Tung,1 Yi-Ming Chen2. 1 _National Yang Ming University, Taipei, Taiwan;_ 2 _Kaohsiung Medical University, Taiwan;_ 3 _National Research Institute of Chinese Medicine, Taipei, Taiwan_.

1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) possesses potent anticancer activities. We identified PGG as GNMT inducer by using GNMT based drug screening platform. In this study we aimed to find the mechanism of action of PGG in hepatocellular carcinoma (HCC). We used microarray analysis to identify the differently expressed genes induced by PGG. Microarray experiment showed that c-Myc was involved in all detected pathways in enrichment analysis. PGG suppressed c-Myc mRNA and protein expression in Huh7 and Hep G2 cells. Knocked-down of c-Myc resulted in significant induction of GNMT promoter activity and endogenous GNMT mRNA expression. Furthermore, overexpression of c-Myc significantly inhibited GNMT promoter activity and endogenous GNMT protein expression. Moreover, PGG not only inhibited c-Myc mRNA expression but also induced degradation of c-Myc protein in Huh7 cells. Finally we found that PGG induces apoptosis in Huh7 cells. Taken together, PGG is a novel c-Myc inhibitor and induces GNMT through c- Myc modulation in HCC. Our results also suggest that PGG may potentially be a good drug candidate for HCC and c-Myc overexpressing cancers.

#1327

Gold nanorods based hypoxia-targeted photothermal cancer therapy.

Huan Xie,1 Sunil Krishnan2. 1 _Texas Southern University, Houston, TX;_ 2 _UT M.D. Anderson, Houston, TX_.

PURPOSE: Tumor hypoxia is a well-recognized driver of resistance to traditional cancer therapies such as chemotherapy and radiation therapy. Gold nanorods (GNR), with unique dimension and optical properties, have shown superior capability for tumor photothermal ablation. This study aimed to develop a new nanoconstruct composed of gold nanorods (GNRs) conjugate to anti-carbonic anhydrase IX (CAIX) antibody that specifically binds to CAIX, a biomarker of hypoxia, to facilitate targeting tumor hypoxic areas for focused photothermal ablation. METHODS: GNR were synthesized according to the seed-mediated growth method with modification. Transmission electron microscopy (TEM) were used to characterize the size and shape of the particles. The GNR/anti-CAIX conjugates were prepared through a bifunctional polyethylene glycol (PEG). The conjugates were characterized by UV-Vis spectra, particle sizes and zeta potentials. The binding affinity and specificity of the GNR/anti-CAIX were evaluated with ELISA, cell adhesion assay and cell photothermal treatment. HT29 colon cancer cell line (overexpressing CAIX) and NIH3T3 cell line (no expressing CAIX) were used in our study. Near-infrared (NIR) ablation was performed on HT29 tumors in mice that treated with PBS, GNR-PEG and GNR/anti-CAIX. Biodistribution were conducted on the latter two groups. RESULTS: TEM images showed that the synthesized GNR had an aspect ratio of about 3 (8 X 24 nm). The changes of UV-vis spectra, particle sizes and zeta potentials confirmed the success of the conjugation. ELISA suggested that the number of anti-CAIX per GNR was 4.8 ± 0.3. In the cell binding assay, GNR/anti-CAIX only showed specific and high degree binding to HT29 cells but not to NIH3T3 cells. NIR laser treatment of HT29 cells resulted in significantly higher killing of GNR/anti-CAIX-targeted cells than of controls. NIR ablation of HT29 tumors in mice showed no tumor regression in the sham-treated group, regression but recurrence in the non-targeted-GNR group, and complete tumor regression in the targeted-GNR group. Biodistribution study showed significant higher accumulation in tumor in the targeted-GNR group. CONCLUSIONS: GNR/anti-CAIX nanoconstructs show promise as highly efficient targeting and photothermal ablation agents for cancer treatment.

GRANT SUPPORT: This work was performed under funding from NIH/NIGMS grant (1SC3GM102018-01) and NIH/NIMHD/RCMI grant (5G12RR003045-21).

#1328

Arenobufagin induces apoptosis, autophagy and cell cycle arrest in hepatocellular carcinoma cells.

Min-Feng Chen, Li-Juan Deng, Wen-Cai Ye, Dong-Mei Zhang. _College of Pharmacy, Guanghzou, China_.

Backgrounds: Hepatocellular carcinoma (HCC) is a deadly form of cancer without effective chemotherapy so far. Thus, there is an urgent need for novel chemotherapeutic agents for the treatment of HCC. Toad venom is frequently used in the treatment of liver cancer in traditional Chinese medicine. However, the exact component and the precise underlying mechanisms against tumor cells remain unclear. In the present research, we sought to study the antitumor mechanism of arenobufagin, a major active ingredient isolated from toad venom, in HCC cells.

Methods and Results: The in vitro and in vivo antitumor activity was measured by colonic formation and HepG2/ADM tumor xenograft, respectively. Arenobufagin-induced HepG2 and HepG2/ADM cell apoptosis was detected by Annexin V-FITC staining and transmission electron microscope. The mitochondrial membrane potential measured by JC-1 staining combined with the changes of expression of Caspase9, Caspase3, PARP, Bax and Bcl-2 in HepG2 and HepG2/ADM cells suggested arenobufagin induced apoptosis via the mitochondrial pathways. Increasing autophagosomes that identified by Cyto-ID® staining and transmission electron microscope were observed in HepG2/ADM cells after arenobufagin treatment. Autophagy-specific inhibitors (e.g. 3-methyladenine, chloroquine and bafilomycin A1), small interfering RNAs target Beclin1 and Atg5 enhance arenobufagin-induced apoptosis, indicated that arenobufagin-mediated autophagy may protects HepG2/ADM cells from undergoing apoptotic cell death. We also demonstrated that inhibition of PI3K/Akt/mTOR pathway promotes the development of both autophagy and apoptosis caused by arenobufagin treatment. In addition, arenobufagin arrested HCC cells in G2 phase was determined by flow cytometry and p-Histone H3 (Ser10) staining. Arenobufagin caused double-strand DNA breaks and triggered the DNA damage response was evaluated by comet assay andγH2AX staining, and these effects were partly via the ATM/ATR-Chk1/Chk2-Cdc25C signaling pathway. We used a synthetic biotinylated arenobufagin-conjugated chemical probe in live cells to show that arenobufagin accumulated mainly in the nucleus. The microscopic thermodynamic parameters measured using isothermal titration calorimetry (ITC) also demonstrated that arenobufagin directly bound to DNA in vitro. The spectral characteristics of DNA (e.g. UV-visible absorption spectrum, circular dichroism spectrum and fluorescence intensity of the ethidium bromide-DNA system) indicated that arenobufagin bound to DNA by intercalation. Molecular modeling suggested arenobufagin intercalated with DNA via hydrogen bonds between arenobufagin and GT base pairs.

Conclusion: This study provides novel insights into arenobufagin-induced HCC cells apoptosis, autophagy and cell cycle arrest. It is valuable for the further investigation of the use of arenobufagin in clinical anticancer chemotherapy.

#1329

Intravenous delivery of Erlotinib-loaded PLA-PEG nanoparticles for treatment of GB.

Lauren K. Hartman,1 Harshil D. Dhruv,1 Kyle T. Householder,2 Jeffrey E. Roth,3 Allison N. Schorzman,3 William Zamboni,3 Rachel Sirianni,2 Michael E. Berens1. 1 _Translational Genomics, Phoenix, AZ;_ 2 _Barrow Neurological Institute, Phoenix, AZ;_ 3 _UNC, Chapel Hill, NC_.

Amplification, overexpression, and mutation of the epidermal growth factor receptor (EGFR) gene represent common genetic signature abnormalities (>50%) in primary glioblastomas (GBs) that arise de novo. Although activated EGFR signaling represents the most common genetic abnormality in GB, targeted therapeutics against EGFR, such as Erlotinib and Gefitinib, have found very limited clinical success mainly because of restricted access to the tumor cells behind the blood brain barrier. Nanoparticles, such as those composed of poly(lactic acid)-poly(glycolic acid) (PLA-PEG), may be capable of overcoming delivery barriers to expand the armamentarium of drugs useful against GB. PLA-PEG is one of only two polymer NP formulations in phase II clinical trial for intravenous administration (NCT01812746). In this study, we tested the ability of Erlotinib-loaded PLA-PEG nanoparticles to inhibit the EGFR signaling pathway in short-term cultures of GB patient derived xenograft (PDX) models. We also tested the ability of systemically administered PLA-PEG nanoparticles to deliver Erlotinib payload to intracranial GB PDX cells by pharmacokinetic (PK) analysis. Potency of Erlotinib-loaded PLA-PEG was determined in four PDX models (GBM6, GBM12, GBM39, and GBM59) with known EGFR and PTEN status using immunoblot analysis and CellTiterGlo® assay. Our results demonstrate that Erlotinib PLA-PEG inhibits EGFR signaling in all PDX models tested with potency equal to free Erlotininb (EC50 range 4.9 - 16.8 μM). Pharmacokinetic measurements were done in plasma and brain tissue samples (tumor-bearing hemisphere and contralateral hemisphere were analyzed separately) collected from intracranial GBM12 tumor bearing mice at 5 min, 1 hr, 6hr, and 24 hr after intravenous (IV) dose of free Erlotinib at 7.5 mg/Kg and Erlotininb PLA-PEG at 13.125 mg/Kg. PLA-PEG-mediated brain penetration (AUCplasm:AUCBrain) of Erlotinib is ~7.5%. Using orthotopic PDX implants, perivascular/intratumoral penetration by PLA-PEG is being measured. Additional improvements in PLA-PEG to heighten uptake into brain, as well as tumor-specific targeting and enriched payload density will foster improved clinical utility of EGFR targeted therapies against GB. Supported by a grant from the Ben & Catherine Ivy Foundation.

#1330

A novel rosmarinic acid/blue light combination treatment inhibits cell proliferation and migration in head and neck squamous cell carcinoma.

Christi N. Waer, Zohra Tumur, Dee Dee Hui, Bonnie Le, Carlos Guerra, Jill Lewis, Bradley Henson. _Western University of Health Sciences, Pomona, CA_.

Our labs have observed decreased proliferation of several cultured head and neck squamous cell carcinoma (HNSCC) cell lines when treated with rosmarinic acid or blue light. The purpose of this study was to test the hypothesis that the combination therapy would be more effective than either treatment alone, and to further characterize the cellular mechanisms responsible for these effects.

UM-SCC-6 oral squamous carcinoma cells were cultured in 24- or 96-well plates, and cells were exposed to blue light delivered from a Quartz-tungsten-halogen dental curing lamp (500 mW/cm2) for 90 or 120 sec, followed 1 h later by the addition of 80 µg/ml rosmarinic acid. Cell number was assessed at 24h using a WST-1 cell proliferation assay and cell migration was measured with an Oris Universal Migration Assay Kit. Reactive oxygen species (ROS) was quantified with each single treatment and combination using the CM-H2DCFH-DA assay. Western blot analysis was used to monitor changes in levels of cell signaling proteins known to be affected by each single treatment including phosphoAKT (pAKT) and AKT, NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), and GAPDH (as a loading control).

As hypothesized, the combination of rosmarinic acid and blue light was more effective in reducing cell proliferation and migration than either single treatment alone. Both treatments also produced an early increase in ROS. Treatment with blue light significantly increased pAKT while rosmarinic acid decreased AKT activation. However, both treatments increased levels of Nrf2 and its downstream target, HO-1.

In order for the transcription factor NRF2 to translocate to the nucleus it needs to be stabilized and activated. AKT is an upstream kinase that is a known activator of NRF2 in response to an increase in ROS. Nuclear NRF2 binds to the antioxidant response element (ARE) within the promoters for phase II detoxifying/antioxidant genes, including that of HO-1. This signaling cascade has been shown to lead to anti-proliferative functions. Our results suggest that blue light induces this pathway by transiently increasing ROS that then significantly activates AKT. Subsequent NRF2 activation leads to increased levels of HO-1. Rosmarinic acid appears to use a different mechanism to upregulate NRF2 due to its downregulation of AKT. Importantly, both treatments activate NRF2, resulting in induction of antioxidant protein expression and ultimately decreasing ROS needed to promote tumor cell growth. This combination therapy may prove to be a useful, non-invasive treatment for HNSCC.

#1331

Janus nanostructures for magnetic resonance imaging and enhanced photothermal therapy for cancer theranostics.

Kanokwan Sansanaphongpricha, Hongwei Chen, Kai Sun, Bo Wen, Duxin Sun. _University of Michigan, Ann Arbor, MI_.

Photothermal therapy (PTT) using nanoparticles has been extensively studied to eliminate cancer cells. PTT is a local therapy with less harmful systemic effect compared to other treatments such as chemotherapy. Iron oxide nanoparticles have been used as a photothermal agent to absorb and convert near infrared (NIR) light to thermal energy resulting in temperature increase. However, to produce high temperature, high-energy laser power is necessary which causes non-specifically damage to normal tissues surrounding tumor tissue. We report herein the new type of nanostructure called "Janus", which composed of iron oxide and gold nanoparticles in asymmetrical fashion to significantly enhance the PTT effect compared to traditional iron oxide nanoparticles. The higher PTT efficiency of Janus nanostructure is resulted from the coupling effect between iron oxide and gold nanoparticles. In addition, Janus nanostructures also provide a great magnetic resonance imaging (MRI) capability and a photothermal effect, which could be used in cancer theranostics.

The Janus structure (JNS) was generated using 15 nm iron oxide (IONP) and 5 nm gold nanoparticles (AuNP) that were encapsulated in a thermo-cleavable polymer by using seed-mediated self-assembly method. JNS was determined by transmission electron microscope (TEM) and x-rays diffraction elemental mapping (XED). It clearly shows that IONPs and AuNPs rearrange in an asymmetrical structures. TheT2 relaxivity of JNS at different iron concentrations was determined. JNS yield almost 3 times higher R2 relaxation compared to Feridex, which is a commercially available iron oxide nanoparticles for MRI contrast agent. PTT efficiency of JNS was investigated in SUM 159 breast cancer cell line by irradiating the cell with 885 nm laser at 0.45 watt for 10 minutes. Interestingly, JNS generate a significantly higher temperature after 10 minutes of laser irradiation compared to IONPs alone, AuNP alone, and the mixture solution of IONP and AuNP at the same concentration of either iron or gold. We also examined the cell viability by alamar blue assay after photothermal treatment. SUM 159 cells were treated with JNS, IONP micelles alone, AuNP micelles alone, a mixture solution of IONP micelles and AuNP micelles. The cells treated with JNS have significant lower viability among other groups due to higher PTT efficiency.. We anticipate that AuNPs and IONPs could have an electron coupling effect among particles resulting in the enhanced temperature for PTT for cancer treatment. In conclusion, JNS provide a great MR imaging capability and PTT efficiency, which could be potentially applied for cancer treatment.

#1332

Dual antibody gold nanoconjugate significantly enhances cytotoxicity in metastatic breast cancer.

Ajit Zambre,1 Anandhi Upendran,1 Matthew Leevy,2 Sarah Chapman,2 Raghuraman Kannan1. 1 _University of Missouri-Columbia, Columbia, MO;_ 2 _University of Notre Dame, Notre Dame, IN_.

Background: Metastatic breast cancer is the second leading cause of cancer-related deaths of women in the United States. First line treatment for Her2-positive metastatic breast cancer patient is combination therapy involving pertuzumab, trastuzumab, and docetaxel. Both pertuzumab and trastuzumab targets Her2, but works in complementary ways in increasing the cytotoxicity. Indeed, combination of these two antibody drugs for treatment has shown excellent survival benefits. We hypothesized that by covalently attaching these two antibodies to a single nanoparticle would accelerate the cancer cell death in lower concentrations of antibodies. In this study, we covalently conjugated trastuzumab and pertuzumab to gold nanoparticle (AuNP) and investigated the cytotoxicity in metastatic breast cancer cells.

Methods: Gold nanoparticles of core size 16 nm were prepared and modified using heterofunctional end terminal carboxylic polyethylene glycol (PEG) linker. The carboxyl terminals were activated by conventional EDC/NHS procedure followed by simultaneous addition of antibodies. The dual antibody gold nanoconjugates (AuNP-Dual Ab) was purified by HPLC using size exclusion column and characterized in detail by routine analytical techniques. Detailed proteomic analysis was performed on the dual antibody gold nanoconjugate. The binding affinity of AuNP-Dual Ab was investigated on HER2+ve SkBR3 cells using dark field microscopy. In vitro cytotoxicity evaluations of AuNP-Dual Ab were performed on HER2 expressing SKBR3 and BT474 cell lines. The effect of AuNP-Dual Ab on signal transduction and phosphorylation status mediated by HER family proteins were evaluated by monitoring the HER2, HER3, AKT, ERK, MEK protein expression through western blot analysis. In vivo studies have been performed in metastatic breast tumor mice models to evaluate the therapeutic efficacy of the nanoconjugate.

Results: AuNP-Dual Ab was isolated from the free antibodies by HPLC. We confirmed both the presence and the structural identity of antibodies covalently attached to AuNP using proteomics. TEM immunohistochemistry and dark field microscopy showed AuNP-Dual-Ab possess high receptor binding affinity with Her2+ve breast cancer cells. We investigated the cytotoxicity of AuNP-Dual-Ab in SKBR-3 and BT474 human breast cancer cells. Our studies reveal that AuNP-Dual-Ab is 60 fold higher cytotoxic than non-antibody coated AuNPs, antibodies, and combination antibodies. Western blot analysis showed down regulation of AkT and phospho-AkT proteins while expression of MEK and phospho-MEK proteins was un-altered. We believe the increase in cytotoxicity is possibly due to effective transport of antibodies to cancer cells.

Conclusions: The in vitro studies demonstrate that AuNP-Dual Ab is promising and can potentially overcome the problem of resistance. In vivo studies are ongoing on metastatic breast tumor mice models for therapy evaluation.

#1333

**Bigelovin inhibits colorectal cancer by suppressing the growth and inducing apoptosis both** in vitro **and** in vivo **.**

Ming-Yue Li,1 Grace Gar-Lee Yue,2 Stephen Kwok-Wing Tsui,1 Kwok-Pui Fung,3 Ning-Hua Tan,4 Clara Bik-San Lau2. 1 _School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong;_ 2 _Institute of Chinese Medicine, The Chinese University of Hong Kong; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, Hong Kong;_ 3 _School of Biomedical Sciences, The Chinese University of Hong Kong; Institute of Chinese Medicine; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, Hong Kong;_ 4 _State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China_.

Colorectal cancer (CRC) is one of the most common cancers with high incidence and mortality worldwide. In 2014, CRC was the second commonest cancer and second leading cause of cancer deaths in Hong Kong. National Cancer Institute estimates that 132,700 people will be diagnosed with CRC and 49,700 people will die from the disease in 2015 worldwide. Despite effectiveness of the chemotherapeutics, severe side effects often affect patients' normal life. Traditional Chinese medicines can be a good source of anti-tumor agent with better safety profile. Here, we reported a sesquiterpene lactone (bigelovin) isolated from Inula helianthus- aquatica, which was shown to induce anti-angiogenesis1 and apoptosis2 in vitro. Although previous studies reported its apoptotic effects on various cell lines, there were no detailed mechanistic or animal studies on its effect on colon cancer cells. To further explore its effect on CRC, we used both in vitro and in vivo assays. The effects of bigelovin in vitro were determined by Annexin V/PI double staining, cell cycle assay and western blotting. Bigelovin was shown to have better selectivity in colon cancer cells than primary normal colon cells. Besides, CRC cells treated with bigelovin resulted in decrease in cell proliferation and increase in apoptosis in both time- and dose-dependent manner. Western blot results also showed caspase activation and protein changes of key molecules (p-H2A.X-DNA damage marker, Cyclin B1-cell cycle, etc). Furthermore, bigelovin (20 mg/kg) significantly reduced the growth of CRC cells-driven tumors in vivo without severe side effects comparing to FOLFOX (containing folinic acid, 5-fluorouracil and oxaliplatin) treatment. Taken together, these results suggested that bigelovin can be a promising therapeutic candidate for colorectal cancer.

References:

1. Yue, G.G.L., et al. Anti-angiogenesis and immunomodulatory activities of an anti-tumor sesquiterpene bigelovin isolated from Inula helianthus-aquatica. Eur J Med Chem, 2013, 59: 243-52.

2. Zhang, H.H., et al. Bigelovin inhibits STAT3 signaling by inactivating JAK2 and induces apoptosis in human cancer cells. Acta Pharmacol Sin, 2015, 36(4): 507-16.

#1334

Lipid-based nanoparticulate hydroxychloroquine (HCQ) formulations for use in combination with autophagy inducing drugs for treatment of breast cancer.

Jagbir Singh,1 Wieslawa H. Dragowska,2 Malathi Anantha,2 Ashleen S. Prasad,2 Jenna S. Rawji,2 Norman S. Chow,2 Marcel B. Bally3. 1 _Experimental Therapeutics, BC Cancer Agency; Precision NanoSystems, Vancouver, British Columbia, Canada;_ 2 _Experimental Therapeutics, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 3 _Experimental Therapeutics, BC Cancer Agency; Faculty of Pharmaceutical Sciences, Department of Pathology and Laboratory Medicine, University of British Columbia; Centre for Drug Research and Development, Vancouver, British Columbia, Canada_.

Many targeted and broad spectrum anticancer drugs used to treat breast cancer trigger survival responses exemplified by the induction of cytoprotective macroautophagy (autophagy). Previously, we and others have shown that the anti-malarial agent hydroxychloroquine (HCQ) can improve the effects of anticancer drugs by inhibiting autophagy when used in high concentrations (1-20 µM). These levels are difficult to attain in vivo, thus, we developed novel liposomal formulations of HCQ (L-HCQ) designed to maintain therapeutic concentrations in plasma and tumor sites over extended periods of time.

Liposomes (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio)) were prepared by extrusion to exhibit a mean particle size of 100 ± 20 nm. Copper HCQ complexation or ammnonium sulphate methods were used for loading HCQ into liposomes achieving >99% encapsulation efficiency (HCQ to lipid ratio: 0.22 ± 0.02 (mol:mol)). In vitro stability studies indicated that more than 80% of the liposomal associated HCQ was retained in the formulation for at least 24 h at 37 °C. In vivo pharmacokinetic studies, demonstrated that free HCQ was eliminated from the plasma compartment within 30 minutes following i.v. injection while the L-HCQ formulations maintained significantly higher plasma HCQ levels (>100 µM) over 24 h regardless of the loading method. Tolerability studies in non-tumor bearing CD1 mice showed no signs of toxicity following single and multiple doses (3 x week, i.v., 75 mg/kg). Inhibition of autophagy in vivo was examined in liver, heart and pancreas tissues of C57B1/6 mice 6 h after dosing with L-HCQ or free HCQ in combination with the autophagy inducing drug rapamycin. The results show that L-HCQ inhibited rapamycin-induced autophagy more effectively than free HCQ, as evident by a significant increase in LC3-II levels in all the examined tissue. Finally, the efficacy of L-HCQ alone (3 x week, i.v., 60 mg/kg) or in combination with the autophagy promoting drug gefitinib, an EGFR tyrosine kinase inhibitor (5 x week, oral gavage, 100 mg/kg), was tested in the JIMT-1 breast cancer xenograft model (s.c.) established in Rag2M mice. After four weeks of treatment, there were no significant differences in tumor volume between untreated and L-HCQ or gefitinib alone treated animals (p>0.05). In contrast, the gefitinib and L-HCQ combination engendered a significant inhibition of tumor growth compared to untreated controls (p<0.05). Moreover, molecular analysis confirmed inhibition of gefitinib-induced autophagy in vivo by L-HCQ, as judged by increased LC3-II and p62 protein levels in tumor tissue.

In summary, this study established that L-HCQ was able to inhibit autophagy and improved sensitivity in an in vivo model of breast cancer treated with gefitinib.

#1335

Dietary compound Diosgenin inhibits proliferation of BGC-823 gastric cancer cells through demethylation of miR-34a.

Fengqin Shi, Li Hou, Yamei Xu, Jing Wang, Yayue Zhang, Xinyi Chen. _Dongzhimen hospital, Beijing, China_.

Background: DNA methylation-mediated epigenetic silencing of microRNA is a critical event during the pathogenesis of gastric cancer. Accumulating evidence has supported that low levels of miR-34a in gastric cancer were largely attributed to its aberrant hypermethylation. Diosgenin is a steroidal sapogenin, which is extracted from the tubers of Dioscorea wild yam, such as the Kokoro. Diosgenin possesses anti-cancer activities possibly via proliferation reduction, cell cycle arrest, and apoptosis induction. In addition, its chemopreventive role in gastric cancer has been reported recently. However, the cancer prevention activities of Diosgenin and the epigenetic-based effects in gastric cancer remain largely unknown. In this study, we test the hypothesis that Diosgenin could play anti-proliferative effect on gastric cancer cells through demethylation of miR-34a.

Material and Methods: GES-1 normal epithelial cells and BGC-823 gastric cancer cells were investigated by methylation and the expression of miR-34a using methylation-specific PCR (MSP) and real time-PCR (RT-PCR). The proliferation of BGC-823 gastric cancer cells after Diosgenin treatment was measured using MTS proliferation assay. The methylation status and the expression of miR-34a were assessed using MSP and RT-PCR in BGC-823 cells after Diosgenin treatment.

Result: In comparison with GES-1 normal epithelial cells, hypermethylation of miR-34a was detected in BGC-823 gastric cancer cells. The expression of miR-34a was significantly down-regulated in BGC-823 gastric cancer cells when compared to GES-1 normal epithelial cells. Diosgenin treatment led to inhibition of proliferation in BGC-823 cells as measured by the MTS assay. Compared to untreated BGC-823 cells, the expression of miR-34a was significantly up-regulated through demethylation of miR-34a in Diosgenin treated BGC-823 cells.

Conclusion: In this study, we demonstrate that expression of miR-34a is frequently decreased in gastric cancer because of DNA hypermethylation. Diosgenin, a dietary compound, plays anti-proliferative effect on gastric cancer cells through demethylation of miR-34a. Our findings not only provide preclinical evidence to demonstrate the use of Diosgenin as a dietary supplement to inhibit gastric cancer but also shed novel light on miR-34a as an epigenetic target for gastric cancer prevention. These observations suggest that the therapeutic potential of Diosgenin, a novel, safe, and epigenetic agent, to be used in addition to chemotherapy for gastric cancer patients.

#1336

Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor.

Florent Beaufils,1 Denise Rageot,2 Anna Melone,2 Marc Lang,1 Jürgen Mestan,1 Vladimir Cmiljanovic,1 Petra Hillmann,1 Paul Hebeisen,1 Doriano Fabbro,1 Matthias P. Wymann2. 1 _PIQUR Therapeutics AG, Basel, Switzerland;_ 2 _University of Basel, Basel, Switzerland_.

Mammalian target of rapamycin (mTOR) signaling pathway plays a fundamental role in cell proliferation, differentiation, growth and survival.[1] As a consequence, various tumors and central nervous system (CNS) disorders share aberrant activation of the mTOR pathway. Drugs targeting the mTOR pathway represent therefore a valuable path to address multiple therapeutic areas.[1-2] Here, we report the lead optimization of PQR620, a novel potent and selective brain penetrant inhibitor of mTORC1/2.

The development of selective mTOR inhibitors is particularly challenging due to extensively conserved amino acid residues in the ATP binding pocket within the PI3K and PI3K-related protein kinase family. Here, we present a detailed ligand-based structure activity relationship study allowing selective targeting of mTOR kinase activity without the interference of other PI3K family members. Systematic variation of the hinge region and affinity binding motifs led to the identification of PQR620, a morpholino-triazinyl derivative, as potent and selective mTOR inhibitor. Substitution of the morpholine binding to the hinge region and introduction of a 2-aminopyridine, substituted with a difluoromethyl group, induced a >1000-fold selectivity towards mTOR over PI3Kα in enzymatic binding assays.

In A2058 melanoma cells PQR620 demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values of 0.2 µM and 0.1 µM, respectively. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR620 demonstrated its potency to prevent cancer cell growth in an NTRC 44 cancer cell line panel, resulting in a 10log(IC50) of 2.86 (nM). Further pharmacological properties and in vivo efficacy of PQR620 are presented in detail in Ref. [3].

The preparation of PQR620 was optimized towards a robust synthetic route involving only 4 steps, allowing for a rapid access to quantities required for pre-clinical testing. In conclusion, PQR620 inhibits mTOR potently and selectively, and shows anti-tumor effects in vitro and in vivo. PQR620 is currently in pre-clinical development.

[1] M. Laplante, D. Sabatini, Cell 2012, 149, 274-293.

[2] Z. Z. Chong, Y. C. Shang, L. Zhang, S. Wang, K. Maiese, Oxid. Med. Cell. Longev. 2010, 3, 374–391.

[3] F. Beaufils, D. Rageot, A. Melone, A. M. Sele, M. Lang, J. Mestan, R. A. Ettlin, P. Hillmann, V. Cmiljanovic, C. Walter, E. Singer, H. P. Nguyen, P. Hebeisen, D. Fabbro, M. P. Wymann, "Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620" presented at AACR Annual Meeting 2016, April 16-20, New Orleans, Louisiana, USA.

#1337

Exploration of 2-aryl fused thiophene analogs as selective inhibitors of MEK5.

Patrick T. Flaherty,1 Dhruv Shah,1 Mohit Gupta,1 Thomas Wright,1 Jane E. Cavanaugh,1 Matthew E. Burow2. 1 _Duquesne University, Pittsburgh, PA;_ 2 _Tulane University, New Orleans, LA_.

In several cancers intermediate enzymes in the MAPK cascades are up-regulated and may represent adaptive changes giving these cancers a competitive metabolic edge over normal cells. Additionally these alterations may induce resistance to previously useful chemotherapeutic agents. The enzyme MEK5, a component of the MEK5/ERK5 cascade, is up-regulated in several cancer types including triple negative breast cancer and prostrate cancers. Our group has previously described the synthesis and testing of anthranillic acid derived compounds and their ring constrained analogs. To explore chemical functional group tolerance and preference on the central arene ring by MEK5, a series of fused-thiophene ring variations, obtainable by the Gewald reaction were prepared and tested. These compounds were examined at 10 μM with MDA-MB-231 cells treated with EGF to induce MEK1/2 and MEK5 phosphorylation. Western blot analysis using IR-tagged antibodies against pERK1/2, pERK5, ERK1/2, or ERK5 was used to determine total and relative inhibition of MEK-mediated phosphorylation of respective pERK products. Incubation with PD0325901 resulted in full suppression of both pERK1/2 and pERK5. For the variations explored, several novel compounds were identified that resulted in no inhibition in the formation of pERK1/2 and 70 % inhibition of pERK5. This establishes a new structural class of selective Type III inhibitors of MEK5 that are more amenable to structural variation, facilitating additional structure activity relationship studies.

#1338

Ferulic acid as a natural bioactive compound enhances PARP inhibitor sensitivity in breast cancer cells.

Eunmi Park, Hyuna Lee. _Hannam University, Daejeon, Republic of Korea_.

Homologous-recombination (HR)-dependent repair defective cells are hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Combinations of defective HR pathway and PARP inhibitors have been an effective chemotherapy strategy. We previously showed that knockdown of the WD40-repeat containing protein, Uaf1, is HR repair defect in mouse embryo fibroblast cells and is sensitive to ABT-888, a chemotherapy drug commonly used for inhibiting PARP. Consistent with the HR defective mouse genetic study, here, we show that ferulic acid inhibits Rad 51 foci formation and accumulates γ-H2AX in breast cancer cells. Ferulic acid treatment reduces HR repair and causes breast cancer cells to become hypersensitive to ABT-888 treatment. Our study indicates that ferulic acid with PARP inhibitor treatment may be useful for the combination chemotherapy as a natural bioactive compound.

#1339

Effects of INCB052793, a selective JAK1 inhibitor, in combination with standard of care agents in human multiple myeloma (MM) cell lines and xenograft models.

Eric Sanchez, Mingjie Li, Cathy Wang, Puja Mehta, George Tang, Haiming Chen, James R. Berenson. _Institute for Myeloma & Bone Cancer Research, Beverly Hills, CA_.

Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow of myeloma patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken. The human MM cell lines, RPMI8226 or U266, were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents, cyclophosphamide (CY), melphalan (MEL), and bendamustine, the proteasome inhibitor, carfilzomib, the corticosteroid, dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). After 48 hours, cell viability was assessed. Combinations of INCB052793 plus the three alkylating agents or carfilzomib synergistically inhibited the viability of both cell lines in vitro. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1S MM cell line. In vivo, mice bearing the human patient derived MM tumor LAGκ-1A had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at Day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant effect on tumor growth compared to all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through Day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. Studies to further understand the mechanistic effects of these combinations on myeloma signaling and the tumor microenvironment are ongoing. In conclusion, these in vitro and in vivo studies demonstrate that the combination of INCB052793 with a broad spectrum of anti-MM agents is effective, and provide further support for the clinical evaluation of these drug combinations in MM patients.

#1340

Dextran-Catechin conjugate: An anticancer nano-modified natural compound targeting copper metabolism in neuroblastoma.

Orazio Vittorio,1 Miriam Brandl,1 Giuseppe Cirillo,2 Kathleen Kimpton,1 Elizabeth Hinde,3 Hien T. T. Duong,4 Cyrille Boyer,4 Claudia Flemming,1 Eugene M.H. Yee,5 Naresh Kumar,5 Arvind Parmar,6 Giancarlo Pascali,6 Arnaud Charil,6 Michelle Haber,1 Murray Norris,1 Maria Kavallaris1. 1 _Children's Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Australia, Randwick, Australia;_ 2 _Department of Pharmacy Health and Nutritional Science University of Calabria, Arcavacata di Rende, Italy;_ 3 _ARC Centre of Excellence in Advanced Molecular Imaging UNSW Australia, Randwick, Australia;_ 4 _School of Chemical Engineering, UNSW Australia, Randwick, Australia;_ 5 _School of Chemistry, UNSW Australia, Randwick, Australia;_ 6 _Australian Nuclear Science and Technology Organisation, Lucas Heights, Australia_.

Background: Neuroblastoma is an aggressive childhood cancer that is poorly responsive to therapy. Moreover, survivors experience long term side effects from their treatment highlighting the need for effective and less toxic therapies. Catechin is a natural polyphenol with anti-cancer properties and limited side effects. Low serum stability of Catechin limits its clinical use that we overcame by chemical functionalization with Dextran. However its mechanism of action is unknown. Here, we investigated the mechanism of action of Dextran-Catechin, and its efficacy against neuroblastoma in vitro and in vivo.

Methods: Anticancer activity was tested in 4 independent neuroblastoma cell lines using the cell viability assay Alamar Blue and apoptosis was assessed using PARP cleavage. Gene expression was determined using qRT-PCR and protein expression of CTR1 by western blotting. Intracellular copper was measured by spectrophotometric analysis. Fluorescence-lifetime imaging microscopy was used to study NADH/NAD+ ratio to determine the induction of oxidative stress. Cellular levels of the antioxidant GSH was examined using a colorimetric assay. PET imaging studies with Cu64 were performed in a xenograft neuroblastoma model to monitor copper uptake in tumors. In vivo anticancer activity of Dextran-Catechin was assessed in human xenograft and syngeneic models of neuroblastoma.

Results: The neuroblastoma cell lines SH-SY5Y, IMR-32, BE(2)C and doxorubicin-resistant BE(2)C-ADR were sensitive to Dextran-Catechin (IC50 9.7 µg/ml, 17.83 µg/ml, 16 µg/ml and 18.2 µg/ml, respectively) at concentrations that were not toxic to non-malignant MRC-5 cells. However, Cisplatin-resistant neuroblastoma cells, IMR-32-CisRes, were 2.5-fold resistant against Dextran-Catechin compared to the parental cells. Copper transporter 1 (CTR1) mediates cisplatin uptake and IMR-32-CisRes exhibited 50% lower expression of CTR1 and lower intracellular copper compared to the IMR-32 cells. In contrast, Dextran-Catechin sensitive neuroblastoma cells had elevated intracellular copper implicating copper in the activity of this conjugate. We demonstrated that Dextran-Catechin reacts with copper generating reactive oxygen species and inducing cancer cell death. Dextran-Catechin treatment caused a decrease of NADH/NAD+ ratio and GSH levels, confirming oxidative stress. PET imaging analysis of the IMR-32-neuroblastoma xenograft model revealed high accumulation of copper in the tumor mass. The high levels of copper were maintained in the tumor mass for up to 48h, while Cu64 was cleared by the other organs. Importantly, we showed that Dextran-Catechin significantly reduced tumor growth in human xenograft and syngeneic models of neuroblastoma with no evidence of side effects.

Conclusion: Dextran-Catechin targets copper, inhibits tumour growth, and has therapeutic potential for cancers dependent on copper for their growth.

#1341

PRINT: A protein bioconjugation method with exquisite N-terminal specificity.

Surojit Sur,1 Yuan Qiao,1 Anja C. Fries,1 Robert N. O'Meally,2 Robert N. Cole,2 Kenneth W. Kinzler,1 Bert Vogelstein,1 Shibin Zhou1. 1 _Ludwig Center at the Johns Hopkins Kimmel Cancer Center, Baltimore, MD;_ 2 _Mass Spectrometry and Proteomics Facility, Johns Hopkins University, Baltimore, MD_.

The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal biodistribution. Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life as well as reduced renal clearance, immunogenicity and protease mediated degradation. However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior. Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation.

We have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bioconjugation of proteins and peptides. Conceptually, PRINT can be performed on any protein that has any N-terminal tag for purification and a protease cleavage site following the tag. The recombinant protein is first treated with an excess of citraconic anhydride to reversibly block all reactive primary amine sites. Proteolytic cleavage then exposes only a single amine (the primary amine at the N-terminus) for desired bioconjugation by amine-reactive NHS ester chemistry. Lowering of reaction pH results in removal of the citraconates, leaving N-terminal specific mono PEGylated protein molecules. We used Tumor Necrosis Factor-α (TNF-α) as a model protein as it suffers from inherent instability and short biological half-life, and exhibits toxic side effects at therapeutic concentrations in both small animals and human patients.

We demonstrate that PRINT results in a single product with exquisite selectivity and specificity in contrast to conventional reaction using the same NHS reagent, which was further confirmed by mass spectrometric analyses. Subsequent de-blocking generated an N-terminal protected TNF-α molecule with enhanced serum stability, superior pharmacokinetic properties, and reduced systemic toxicity. Importantly, N-terminal protection by PRINT did not affect the bioactivity of TNF-α. Existing site-selective bioconjugation approaches are either specific to amino acid tags or involve substantial non-trivial chemical or biotechnological manipulations to synthesize a desired bioconjugate. In contrast, PRINT employs ubiquitously used recombinant DNA techniques and easily acquired commercial reagents to generate exquisite N-terminal selective protection. We show that PRINT is a robust, reproducible and mild strategy which is able to target the α -amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues. We believe that this approach is strongly orthogonal to current methods and will be applicable to many biotherapeutics and bioprobes that are currently being designed to treat cancer. 

### Drug Delivery 2

#1342

A production-scalable cabazitaxel cellulose nanoparticle exhibits significant efficacy in a bone model of docetaxel-resistant prostate cancer.

Joseph Bteich,1 Bryan Hoang,1 Elijus Undzys,1 Mohammed Mohammed,1 Ai Lin Su,1 Kevin Ou,2 David Emerson,3 Kenneth Sokoll,4 Shyh-Dar Li,5 Mohit Trikha,6 Henry Lowman,6 Mark J. Ernsting1. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Precision Nanosystems Incorporated, Vancouver, British Columbia, Canada;_ 3 _Bolder BioPATH Incorporated, Boulder, CO;_ 4 _Fight Against Cancer Innovation Trust, Toronto, Ontario, Canada;_ 5 _University of British Columbia, Vancouver, British Columbia, Canada;_ 6 _Triphase Accelerator US Corporation, San Diego, CA_.

Purpose: This study aimed to: (1) evaluate the antitumor efficacy of a controlled release cabazitaxel cellulose nanoparticle (CBZed-Nano) in an orthotopic bone model of docetaxel-resistant LNCaP C4-2B prostate cancer, and (2) confirm the efficacy of dose preparation by scalable instrumentation (NanoAssemblr), in a subcutaneous PC3 prostate xenograft model.

Methods: mPEG-OH and cabazitaxel (CBZ) were coupled to a carboxymethylcellulose acetate polymer to yield CBZed-Nano, according to established coupling and purification methods (1). Particles were prepared by nanoprecipitation of polymer into saline by either a manual vortex technique (1) or a NanoAssemblr™ (NA) microfluidic-based instrument. Particles were purified by dialysis and sterile filtration (1). Particle size was measured by a DLS Malvern Zetasizer, morphology was screened by TEM, and residual substances were measured by LC.

Male NOD-SCID mice were inoculated via intrafemoral injection with docetaxel-resistant LNCaP C42B prostate cancer. One day after inoculation, mice were treated with vortex prep CBZed-Nano (55 mg/kg) or CBZ (2 mg/kg) on a q1,5,9d schedule. Survival curves were generated (n=10 mice per group). To directly compare preparation technique, nude mice bearing s.c. PC3 prostate cancer xenografts were treated at Bolder BioPath (Boulder, US) with CBZed-Nano (NA and vortex prep, 85 to 142 mg/kg) and CBZ (15-25 mg/kg) on a q1,5,9d schedule.

Results: The CBZed-Nano polymer conjugate contained 36 wt% CBZ and 11 wt% PEG, with residual free CBZ < 0.01 wt% of CBZ content. Particles prepared by vortex and NA were 94 and 70 nm respectively (by DLS). By TEM analysis, NA particles exhibit a more uniform spherical morphology.

Mice bearing docetaxel resistant LNCaP C42B tumors exhibited a partial response to CBZ treatment, with a median survival of 61 days, compared to 34 days for vehicle controls. In contrast, mice treated with CBZed-Nano exhibited a significantly improved response, with 70% durable cure.

Efficacy was observed regardless of CBZed-Nano preparation technique. Mice treated with CBZ exhibited a 38-63% durable cure in the 60, 75, and 100% MTD groups. In comparison, mice treated with CBZed-Nano exhibited a 92-100% durable cure, confirming both the superior activity of CBZed-Nano relative to free CBZ, and confirming antitumor activity of the scalable NanoAssemblr production.

Conclusion: The CBZed-Nano preparation technique is scalable, with manual and NanoAssemblr™ produced dose being equally efficacious in a PC3 prostate cancer model. Significant efficacy (70% durable cure) in a docetaxel-resistant prostate cancer of the bone model was also observed. The data suggests that CBZed-Nano is more tolerable than CBZ, and that it may overcome taxane-resistant solid tumor indications.

(1) Bioconjugate Chem 22 2011, 2474-2486

(2) Biomaterials 33 2012, 3931-3941

#1343

P32-targeting TT1 peptide delivers nanoparticles to intracranial glioblastomas.

Pille Säälik,1 Hedi Hunt,1 Allan Tobi,1 Anne-Mari Anton Willmore,1 Kadri Toome,1 Shweta Sharma,2 Ramana Kotamraju,2 Gabriele Bergers,3 Rolf Bjerkvig,4 Erkki Ruoslahti,2 Tambet Teesalu,1 Tambet Teesalu2. 1 _Institute of Biomedicine and Translational Medicine, Tartu, Estonia;_ 2 _Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA;_ 3 _UCSF Comprehensive Cancer Center, University of California, San Francisco, CA;_ 4 _Department of Biomedicine, University of Bergen, Bergen, Norway_.

Targeted delivery of cancer therapeutics using affinity ligands can dramatically improve antitumor efficacy. Over the years a number of homing peptides that upon systemic injection accumulate in solid tumors have been identified by in vivo peptide phage display. In a quest to find homing peptides optimally suited for drug delivery to high-grade gliomas, our laboratories are using advanced mouse models of glioblastoma (GBM) to systematically audit known tumor-homing peptides and to perform new in vivo screens using peptide phage libraries.

P32 is a mitochondrial chaperone that is aberrantly expressed on the cell surface in activated malignant and stromal cells in tumors. P32 is a receptor for widely used LypP-1 peptide and for recently identified TT1 peptide. Here we show that iron oxide nanoworms (IONW) functionalized with linear TT1 peptide (CKRGARST) strongly home to intracranial GBMs grafted in immune deficient mice. IONW are paramagnetic nanoparticles that are PEGylated to extend blood half-life, and have, because of their elongated shape, more effective targeting properties than spherical nanoparticles. Five hours after intravenous injection of IONW (7.5 mg/kg), macroscopic fluorescence imaging demonstrated robust homing of TT1-IONW in GBMs of murine origin (WT GBM and VEGF KO GBM from the G. Berger lab) and in a patient-derived glioma model (P13 model from the Bjerkvig lab). Confocal microscopy confirmed the presence of TT1 but not control IONW in gliomas, with TT1-IONW signal showing partial overlap with blood- and lymphatic vessel markers (CD31 and LYVE-1) in WT GBM and P13 gliomas, whereas lower level of colocalization with these markers was detected for mouse GBM not expressing VEGF. In addition, moderate colocalization between TT1-IONW and the macrophage marker CD11b was detected in the P13 tumors. Detailed phenotyping and functional characterization of TT1-positive macrophages is ongoing.

Our data suggest that TT1 peptide has potential applications as a glioma-targeting vehicle. Currently, we are evaluating TT1-targeted IONWs as a contrast agent for glioma MRI and as carriers for cytotoxic compounds.

#1344

Novel targeting all-in-one nanoporphyrin platform against bladder cancer.

Yuanpei Li, Tzu-yin Lin, Chongxian Pan, Kit S. Lam. _UC Davis Comp. Cancer Ctr., Sacramento, CA_.

We have developed an extremely versatile and highly innovative theranostic nanoporphyrin (NP) platform for the integration of a broad range of clinical relevant imaging and therapeutic functions, including magnetic resonance imaging (MRI), positron emission tomography, fluorescence optical tomography, photodynamic therapy, photothermal therapy and targeted drug delivery. NPs exhibit architecture-dependent fluorescence- and magnetic resonance- properties, which could significantly increase the sensitivity of optical imaging and MRI for tumor detection. We recently integrated the NP platform into our previously reported bladder cancer targeted nanoparticles that were coated with a bladder cancer-specific ligand named PLZ4, resulting in a novel bladder cancer targeting and multifunctional PLZ4-nanoporphyrin (PNP) platform. PNP selectively delivered photosensitizer and chemotherapy into bladder cancer cells 30-50 times more than to adjacent normal urothelial cells, compared to only 2-3 times with reported photosensitizer, 5-aminolevulinic acid (ALA). Photodynamic therapy with PNP generated reactive oxygen species was over 100 times more potent than 5-ALA in vitro. PNP-mediated intravesical photo-therapy completely eliminated orthotopic patient-derived bladder cancer xenografts (PDXs). Image-guided photodynamic and photothermal therapies synergized with targeted chemotherapy of doxorubicin and significantly prolonged overall survival of mice carrying PDXs. This uniquely engineered targeting PNP platform has tremendous potential to improve the management of bladder cancer in clinic.

#1345

Tumor selective localization of CRLX101, an investigational nanoparticle-drug conjugate of camptothecin.

Christian G. Peters, Douglas Lazarus, Donna Brown, Ningning Zhang, Adam P. Stockmann, Roy Case, Ellen Rohde, Scott Eliasof, Lata Jayaraman. _Cerulean Pharma, Waltham, MA_.

CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin, is currently being clinically evaluated in multiple treatment-refractory solid tumors. In preclinical models, CRLX101 is believed to release camptothecin in the tumor in a slow and prolonged manner due to its long circulation half-life. CRLX101 has been shown preclinically to be a dual inhibitor of topoisomerase 1 and hypoxia-inducible factor 1α. It has demonstrated striking anti-tumor activity in several different tumor models. Camptothecin itself was identified as an active anti-tumor agent preclinically but was not developed clinically due to its poor tolerability in patients. The development of CRLX101, which has not shown significant toxicity in over 300 patients to date, offers a unique opportunity to improve cancer treatment in a meaningful way.

We hypothesized that CRLX101 utilizes the enhanced permeability and retention (EPR) effect to accumulate selectively in tumors. In this study, we sought to mechanistically dissect the process of CRLX101 entry and accumulation into tumor cells using multiple methods, both in vitro and in xenograft tumors in vivo. Using confocal microscopy, we detected camptothecin fluorescence in CRLX101-treated tumor cells in culture as well as in tumor tissue from mice treated with CRLX101. We can co-localize this camptothecin with intact nanoparticles using an anti-PEG antibody that specifically detects the PEG loops in the NDCs. More recently, we have shown that camptothecin and anti-PEG co-localize specifically in tumors of patients treated with CRLX101 but not in adjoining normal tissue. We can also demonstrate that macropinocytosis and activation of actin polymerization play a role in the process by which tumor cells take up CRLX101. Using an anti-CD31 antibody, we can visualize the distance traversed by CRLX101 from the tumor vasculature over time. We have developed novel analytical methods to precisely quantify both released and CRLX101-conjugated camptothecin over time in CRLX101 treated tumor cells in vitro, as well as in tumor tissue from mice treated with CRLX101 in vivo. Using cell viability assays, we can correlate the kinetics of camptothecin released inside tumor cells to the degree of tumor cell kill. We believe that these data are an important step forward in understanding the precise mechanism(s) underlying selective delivery of CRLX101 into tumor tissue.

#1346

Magnetoelectric particles cross blood brain barrier to deliver anti-tumor peptide to glioblastoma cells with on-demand release.

Tiffanie Stewart,1 Emmanuel Stimphil,1 Rakesh Guduru,1 Alexandra Rodzinski,1 Ping Liang,2 Carolyn Runowicz,1 Ren-Zhi Cai,3 Luis Salgueiro,3 Andrew Schally,3 Sakhrat Khizroev1. 1 _Center for Personalized Nanomedicine, Herbert Wertheim College of Medicine, Florida International University, Miami, FL;_ 2 _Cellular Nanomed, Weston, FL;_ 3 _V.A. Medical Center, and Department of Medicine, Division of Hematology Oncology, University of Miami, Miller School of Medicine, Miami, FL_.

Background: Despite significant progress in targeted drug delivery, glioblastoma is one of the least treatable tumors today. Delivering therapy across the blood-brain barrier (BBB) and overcoming resistance of tumor cells to current treatments remains a formidable task. The objective of the current in vitro and in vivo study was to show how a new class of multifunctional nanoparticles termed magnetoelectric nanoparticles (MENs) are used to achieve high-efficacy delivery of a therapeutic load across BBB, then release the load on demand, without affecting normal cells, via application of a specially tailored command sequence of D.C. and A.C. magnetic fields.

Methods: Growth hormone (GH)-releasing hormone (GHRH) antagonists, a class of anti-tumor peptides, block the release of tumorigenic insulin-like growth factors. Glioblastoma cell line U87 MG were treated with 1 μM of GHRH antagonist MIA-690 bound to GMO-MENs in MEM media supplemented with 5% FBS and 1% penstrep. In parallel, an in vitro BBB experiment, designed using transwells, applied a D.C. magnetic field gradient to determine optimal field and exposure time for MENs to penetrate the BBB. Next, experimental cells were placed on a D.C. magnetic field (100 Oe) for 12 h to induce nanoelectroporation, then an A.C. field (~50 Hz) for 2 h to release the peptide intracellularly. Control cells were treated with MIA-690 peptide only, GMO-MENs only, and MIA-690 bound to GMO-MENs at varying magnetic fields and exposure times. Cells were incubated at 37°C with 5% CO2 in a humidified atmosphere. At 24 h and 48 h post-treatment, trypan-blue vital stain assessed cell growth inhibition. Cells were then washed and lysed to measure the concentration of MENs, and MIA-690 using ICP-MS and spectrophotometry, respectively. AFM and SEM-EDS imaged MENs in cell lysate and brain tissue from mice IV injected with MENs, to find signature peaks of Ba and Ti representing the outer core of MENs, to ascertain if particles cross the BBB in vivo.

Summary of data: Glioblastoma cells treated with MIA-690 bound to GMO-MENs on a D.C. magnetic field for 12 h, then an A.C. field for 2 h exhibited the greatest penetration of MENs and inhibition of tumor growth. AFM and SEM-EDS imaging showed MENs in cell lysate treated with D.C. for 12 h, but not 2 h or with no magnetic field, confirming application of a low D.C. field for 12 h triggers MENs' penetration. On-demand MIA-690 intracellular release was highest after exposure to 12 h of D.C. field, then A.C. field at 50 Hz. Confocal microscopy confirmed a transfer of at least 50% of drug-loaded MENs across in vitro BBB, requiring 200 Oe/cm for 5 h. SEM-EDS images of brain slices taken from mice treated with MENs confirmed the presence of Ba and Ti, indicating MENs are able to cross BBB in vivo.

Conclusion: Functionalized MENs are effective in treating glioblastomas using targeted, on-demand release of anti-tumor drug with the ability to cross the BBB.

#1347

NTRK1/TRKA as a therapeutic target in castration resistant prostate cancer.

James E. Korkola, Moqing Liu, Rebecca Smith, Tiera Liby, Joe W. Gray. _Oregon Health & Science University, Portland, OR_.

Treatment options remain limited for men with castration resistant prostate cancer, with current chemotherapy offering mainly palliative benefits. Thus, there is an urgent need to identify new targets for therapeutic intervention in men who have progressed on anti-androgen therapy. We have compiled a panel of 20 prostate cancer cell lines that has been extensively characterized at the molecular level that enables us to perform high throughput drug and siRNA screens to identify novel therapeutic targets. We performed an siRNA screen targeting kinases using eight representative lines, both in the presence and absence of the anti-androgen MDV3100. Knockdown of NTRK1 (TRKA) significantly inhibited growth in 4/8 of the cell lines tested. To confirm this, we screened the entire panel of cell lines with the inhibitor CEP-701, which targets FLT3, JAK2, and TRK-A/B/C. CEP-701 showed GI50 values (dose required to inhibit growth by 50%) less than 400 nM in 8/20 cell lines. Since this drug had already been tested in clinical trials for prostate cancer and the trials were discontinued due to lack of PSA decline, we looked for other drugs that also inhibit TRKA. Both BIBF-1120 and GSK-1363089 are also known to inhibit TRKA, but neither have been tested in prostate cancer expressing TRKA. We tested GSK-136089 in a subset of 9 prostate cell lines, and found that 4/9 lines reached GI50 at 1 uM or less, including AR mutant or null lines such as 22rv1 and PC3 that are non-responsive to MDV3100. Additional tests with BIBF-1120 and GSK-1363089 are underway. These data suggest that NTRK1 may be a potential target for patients with castration resistant prostate cancer.

#1348

A novel bicistronic reporter-based high-throughput screening platform for discovery of transcription-targeted lead compounds.

Liwei Lang, Chunhong Yan. _Georgia Regents University, Augusta, GA_.

Given that aberrant gene expression is widely spread in cancer, there is an imperative need of developing reliable high-throughput drug screening (HTS) platforms in search of lead compounds that can rectify abnormal expression of cancer-driving genes for treating this deadly disease. Although it has been demonstrated that reporter gene assays are useful in the identification of transcription-targeted lead compounds, these assays are often unreliable and can yield high rates of false compounds due to the facts that reporter genes are driven by cloned promoters and localized in foreign chromatin environments. While cloned promoters lack essential cis-regulatory elements (e.g., enhancers) far removed from transcription start sites, foreign chromatin environment can alter gene expression in a way atypical to endogenous genes. To address these limitations, we employed emerging genome-editing tools to develop a novel reporter-based HTS assay whereby the reporter gene is inserted immediately downstream of the coding region of an endogenous gene, and thereby co-expressed bicistronically with the endogenous gene as a single transcript under the control of the native transcriptional regulatory machinery. As a proof-of-principle, we first employed the CRISPR-Cas9 system to knock a fused selection gene tk-ble (encoding Zeocin resistance for selection for targeting events and thymidine kinase for negative selection for the removal of the selection gene, respectively) flanked by a wild-type and a mutated FRT fragment into the eukaryotic translation initiation factor 4E (EIF4E) gene locus, and then replaced the selection gene with a firefly luciferase (FLuc), or a Renilla luciferase (RLuc), by cassette exchange mediated by the recombinase Flp. We demonstrated that the FLuc gene was expressed as single transcripts with EIF4E by Northern blotting, and responded to chemical treatments in a way analog to the endogenous EIF4E gene. Using the cells carrying bicistronic FLuc reporter, we screened a chemical library containing 4,800 compounds for small molecules inhibitory for EIF4E expression, and identified 11 hits, out of which 6 compounds were validated to decrease EIF4E expression by qRT-PCR. While the 5 false-positive compounds were likely yielded due to their direct inhibition of FLuc activity, 4 of these false positives could be readily identified by an orthogonal assay using recombinant cells carrying RLuc in the same EIF4E locus. Therefore, our results have demonstrated that screening assays based on bicistronic in-situ reporters are more reliable, and powerful in searching for transcription-targeted lead compounds with high confidence.

#1349

Systematic deconvolution of kinase inhibitor profiles identifies synthetic lethal targets in ERBB2-mutant and BRD4-NUT rearranged cancer.

Johannes Braegelmann,1 Peter Habenberger,2 Felix Dietlein,3 Johannes M. Heuckmann,4 Sascha Menninger,2 Uwe Koch,2 Axel Choidas,2 Daniel Rauh,5 Bert Klebl,2 Martin L. Sos,1 Roman K. Thomas6. 1 _Molecular Oncology & Department of Translational Genomics, Cologne, Germany; _2 _Lead Discovery Center GmbH, Dortmund, Germany;_ 3 _Department I of Internal Medicine and Center for Integrated Oncology, University Hospital of Cologne, Cologne, Germany;_ 4 _NEO New Oncology AG, Cologne, Germany;_ 5 _Department of Chemistry and Chemical Biology, Technical University of Dortmund, Dortmund, Germany;_ 6 _Department of Translational Genomics, Cologne, Germany_.

The development of targeted therapies that efficiently inhibit cancer signaling pathways is one of the main goals of modern precision cancer medicine. Consequently, genetic and biological phenotypic data of in vitro screens are increasingly utilized to develop compounds directed against distinct oncogenic alterations. However, current targeted therapies are often limited to small genetically defined patient cohorts due to the very finite number of proteins amenable to direct chemical inhibition. An alternative approach is the exploitation of synthetic lethality, i.e. inhibition of an unaltered protein required for cell viability in a certain genetic background. Systematic chemo-genomic analyses of cancer cell lines have been shown to be suitable tools for the identification of novel synthetic lethal dependencies in cancer (Chan et al. Sci Trans Med, 2011; Sos et al. PNAS, 2012; Kim et al. Cell 2013).

To systematically extend this strategy to non-small cell lung cancer (NSCLC) we characterized the efficacy of 1505 chemical compounds based on a variety of kinase inhibitor motifs in a high-throughput screen against 80 NSCLC cell lines. We extracted patterns of biological activity based on chemical and genetic information and found that potency and selectivity of compounds are strongly related to their molecular scaffold, but independent of their overall chemical complexity. We thereby discovered a sunitinib derivative that exhibited exquisite activity against ERBB2-mutant cell lines but was devoid of ERBB2 kinase activity. Instead a kinome scan and an shRNA screen suggested a mechanism of synthetic lethality by activity against NTRK family members. Moreover a CDK9 inhibitor was identified as selective and potent against a midline carcinoma cell line - a tumor entity characterized by recurrent BRD4-NUT gene fusions. Using additional cell lines we validated the upregulation of c-Fos and selective induction of apoptosis in BRD4-NUT positive midline carcinoma compared to control cell lines following CDK9 inhibition. This can augment existing therapeutic approaches, which have primarily focused on directly targeting the fusion product with bromodomain inhibitors, and offers a novel target in this entity.

In conclusion, by systematically screening a large number of compounds against a panel of genetically well characterized NSCLC cell lines and incorporating chemical information we were able to derive structure activity relationships and to identify potential synthetically lethal targets in two genetic entities in clinical need of advanced selective therapies.

#1350

New NCI experimental therapeutics program (NExT) small molecule libraries for academic investigator hts projects.

Raj N. Misra,1 Michael Eckert,2 Christian Laggner2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Evotec (US), South San Francisco, CA_.

The NCI Experimental Therapeutics Program (NExT) is making available two new smaller (3.5K) screening libraries suitable for academic investigator HTS drug discovery programs. The NExT program is a partnership between NCI's Division of Cancer Treatment and Diagnosis (DCTD) and the Center for Cancer Research (CCR). NExT consolidates NCI's anticancer drug discovery and development resources with the goal of maintaining a robust and balanced therapeutics pipeline. It encompasses tasks from new target validation through Phase III clinical trial evaluation. The Program is designed to streamline development and testing of promising new anticancer drugs and to expedite their delivery to the bedside. As part of the Program to support HTS drug discovery, NCI has previously offered academic oncology investigators access to our full 83K NExT Diversity Library in single-use, 384-well plate format. The set was designed to identify lead small molecules for drug discovery programs. We are now making available 2 smaller pre-plated subsets of this library, the NExT Diversity 3500 and NExT Diversity 3500 SAR, for when screening the full set is not appropriate. Each library contains 3,500 compounds: NExT Diversity 3500 is a diverse sampling across the entire 83K library while the NExT Diversity 3500 SAR is designed to facilitate rapid SAR follow-up by sampling only those compounds that have at least 5 close neighbors in the 83K library. The new libraries were designed by initially filtering the full NExT Diversity Library for undesirable compounds such as PAINS. Both atom-pair based 2D pharmacophore fingerprints and ECFP6 topological fingerprints from ChemAxon were then employed to generate the two non-overlapping subset libraries. The NExT screening libraries are provided as a resource to academic oncology investigators through the NExT Program (see NExT Resources: http://next.cancer.gov/). The design details and physiochemical characteristics of the two new sets, NExT Diversity 3500 and NExT 3500 SAR, and procedures and criteria for accessing this resource are presented along with several other screening sets available through the Developmental Therapeutics Program (DTP).

#1352

High-throughput matrix screening reveals synergistic chemotherapeutic combinations with blockade of CD47 to enhance cytotoxicity in breast cancer.

David D. Roberts,1 Ashley Smith,2 John M. Sipes,1 Adam Wilson,2 Lesley Mathews-Griner,3 Rajarshi Guha,3 Craig J. Thomas,3 Marc Ferrer,3 David R. Soto-Pantoja2. 1 _Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _Wake Forest School of Medicine, Winston-Salem, NC;_ 3 _National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD_.

CD47 is a widely expressed cell surface receptor that serves as a counter-receptor for SIRPα in recognition of self by the innate immune system. On the other hand CD47 also serves as a signaling receptor for the matricellular protein thrombospondin-1 (TSP-1), regulating cell growth and survival. Clinical studies consistently show that increased expression of CD47 is an independent poor prognosis factor in several types of cancer, including breast cancer. Moreover elevated expression of CD47 is associated with development of resistance to chemotherapy by inhibiting cell death pathways. Therefore, agents targeting CD47 are attractive to overcome therapeutic resistance in breast cancer. Using a high-throughput assay in a 1536-well microplate format we screened a comprehensive set of approved and late stage development oncology drugs in combination with an anti-CD47 morpholino to find combinatorial strategies that are more cytotoxic against breast cancer cells. Potency and efficacy measurements during the primary screen identified compounds that synergized with anti-CD47 to augment their cytotoxicity effect. One of the compound groups that demonstrated increased synergism with CD47 were the anthracycline family of drugs. We further validated these results in a syngeneic model of breast cancer and demonstrated that blockade of CD47 in combination with doxorubicin improves chemotherapeutic response when compared to mice administered doxorubicin alone. Furthermore the anti-tumor response with anti-CD47 combination is associated with a reduction in glycolytic flux and a selective up regulation of mitophagy. Overall this indicates that blockade of CD47 in the clinic may synergize with chemotherapeutic strategies to improve patient curative responses.

#1353

Validation and implementation of screens for partial agonists and antagonists of the ErbB4/HER4 receptor tyrosine kinase: Targeted melanona drug discovery.

Richard Cullum, Allan David, David J. Riese. _Auburn University, Auburn, AL_.

Introduction: Gain-of-function mutations in the gene encoding the ErbB4 (HER4) receptor tyrosine kinase have been detected in melanoma cell lines and these cell lines are dependent on ErbB4 expression for proliferation. Consequently, ErbB4 antagonists hold promise as targeted melanoma therapeutics and we have sought to identify small molecule ErbB4 antagonists. Our approach is based on the observation that the Q43L mutant of the ErbB4 peptide growth factor agonist Neuregulin 2beta (NRG2b) functions as a partial agonist at ErbB4; NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4 coupling to cell proliferation, antagonizes stimulation of cell proliferation by ErbB4 full agonists, and causes ErbB4 down-regulation.

Experimental procedures: We have developed a high-throughput screening (HTS) strategy that can be used to identify ErbB4 antagonists. The primary screen identifies molecules that stimulate ErbB4 tyrosine phosphorylation. The secondary screen distinguishes between molecules that stimulate and fail to stimulate ErbB4-dependent proliferation. The tertiary screen identifies molecules that antagonize agonist stimulation of ErbB4-dependent proliferation.

Results: An phospho-ErbB4 sandwich ELISA assay identifies molecules that stimulate ErbB4 tyrosine phosphorylation with high sensitivity and fidelity (Z' >0.5). MTT assays using a cell line that displays ErbB4-dependent proliferation distinguish between molecules that stimulate and fail to stimulate ErbB4-dependent proliferation (Z'>0.5) and identify molecules that antagonize agonist stimulation of ErbB4 dependent proliferation (Z'>0.5). These assays have been used to identify small molecules that stimulate ErbB4 tyrosine phosphorylation. Efforts to determine whether these hits function as ErbB4 full agonists or partial agonists (antagonists) are underway and will be reported. Structures of these small molecule ErbB4 full and partial agonists may be reported, pending submission of a provisional patent application.

Conclusions: We have validated an HTS strategy for identifying ErbB4 partial agonists that function as ErbB4 antagonists and deployment of that strategy has led to the identification of several hits. Such molecules may hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors.

#1354

Developing 384-well and 1536-well cell growth inhibition assay workflow for screening drug-drug combination in tumor cell lines.

Yvonne Li, Mike Ma, Julie Chan, Yang Tian, Nikos Pagratis, Derek Stonich, Victor Chen, Louis Zhang, Mark Kenney. _Gilead Sciences, Foster City, CA_.

Drug combination has been widely used in treating the most debilitating diseases such as cancer. The ideal drug-drug combination will broaden and/or deepen therapeutic efficacy while overcoming resistance and unwanted off-target effects. We have developed a 384-well combination compound plating method in 8x7 format (8 dilutions of Drug A and 7 dilutions of Drug B), and screened a number of compound pairs that showed synergistic effect in inhibiting tumor cell growth in either suspension or solid tumor lines. Compound vehicle DMSO was used as negative control and puromycin treatment as positive control for calculating % inhibition, and both HSA and Bliss independence synergy models were applied for calculating synergy scores. As 384-well 8x7 format contains one compound pair per plate, we aimed to increase the throughput by developing 384-well 5x5 format (5 dilutions of Drug A and 5 dilutions of Drug B) which contains 3 drug pairs per plate, and 1536-well 8x7 and 5x5 format which contains 4 and 12 drug pairs per plate, respectively. We compared EC50s and synergy scores generated from 384-well and 1536-well with both 8x7 and 5x5 formats, single agent EC50s within combination pairs were generally within 3 fold difference and synergy scores are largely consistent. Therefore, we have validated and enabled higher throughput drug-drug combination screen by using 384-well 5x5, 1536-well 8x7, or 5x5 format. The high-throughput method presented here can be readily adopted for combination studies in other disease areas.

#1355

A novel derivative of benzoxazole enhances radiosensitivity of human colon cancer cells through p53-mediated G2/M arrest and mitochondrial apoptotic pathway.

Hwani Ryu, Bitna Seo, Jooyoung Kim, In-Sung Jung, Jie-Young Song, Jiyeon Ahn. _Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, Republic of Korea_.

The tumor suppressor p53 plays a critical role in cell cycle regulation and apoptotic cell death, and its activation can sensitize cancer cells to radio- or chemotherapy. To identify small molecules that induce apoptotic cell death via increasing p53 transcriptional activity, we designed and synthesized 44 benzoxazole derivative small molecule compounds. Using a cell-based screening with p53-responsive luciferase reporter assay system of benzoxazole derivatives, we found AU14022, which significantly increased p53 transcriptional activity in a concentration dependent manner. Treatment of AU14022 increased p53 protein expression, its phosphorylation on serine 15 and its target p21 gene/protein expression and induced apoptotic cell death measured by FACS analysis using Annexin V/PI double staining in wild-type p53 HCT116 human colon cancer cells but not in p53 knockout HCT116 cells. Furthermore, treatment of HCT116 cells with AU14022 exhibited mitochondrial dysfunction including bcl-2 family modulation and cytochrome c release. Combination treatment of AU14022 and ionizing radiation (IR) synergistically induced apoptotic cell death, compared to IR or AU14022 alone. Further investigation demonstrated that cell cycles are significantly arrested at G2/M phase, and marked inductions of p21 protein expression and phosphorylation of Chk1 protein and a decrease of cyclin B1 protein expression were observed by treatment of AU14022 alone and combination treatment with AU14022 and IR. Our findings indicate that AU14022 induced apoptosis through p53-mediated cell cycle arrest with mitochondrial dysfunction, leading to enhanced radiosensitivity of colon cancer cells. These results provide a basis for the further investigations required for its assessment as a promising anticancer agent.

#1356

Cell-based screening to identify repressors of wild type and mutated telomerase reverse transcriptase gene promoter activity.

Alan Bilsland,1 Yu Liu,1 Sharon Burns,1 David Jenkinson,2 Jon Roffey,2 W. Nicol Keith1. 1 _Institute of Cancer Science, University of Glasgow, Glasgow, United Kingdom;_ 2 _Cancer Research Technology, London, United Kingdom_.

Cellular immortality is a near-universal phenotype of cancer cells with the central role played by the multicomponent telomerase complex firmly established. The reactivation of telomerase is found in approximately 85% of all cancers. Furthermore, frequent activating non-coding mutations in the hTERT promoter region have been identified in multiple tumour types. A considerable body of evidence supports the concept of targeting the signal transduction pathways that contribute to telomerase overexpression in cancer cells. We have exploited our knowledge in this area to develop cell-based luciferase reporter gene assays to measure hTERT and hTR gene promoter activity. To this end we undertook high throughput screening in the A2780 human ovarian cancer cell line against both hTR and hTERT promoters. Secondary specificity and cytotoxicity assays reduced the initial hits to a single chemical series of dual inhibitors which strongly suppress both hTR and hTERT promoter activity without primary cytotoxicity, as predicted of telomerase inhibitors. Through a process of Hit to Lead we have further developed the chemical series. Lead compounds are active across a panel of cell lines from distinct histological origins, reduce endogenous hTERT mRNA levels and decrease telomerase catalytic activity at nM concentrations. Furthermore, these compounds repress both wild type and mutant C228T and C250T hTERT promoter activity. In specificity assays no off-target activity has been identified in an expanded promoter screen against a panel of 15 off-target promoter reporters.

#1357

In silico design and biological evaluation of benzofused polyamides targeting G-quadruplex DNA structures.

Khondaker M. Rahman,1 AKM Azadur Rahman,1 Paul J. M. Jackson,2 David E. Thurston1. 1 _King's College London, London, United Kingdom;_ 2 _Femtogenix Limited, London, United Kingdom_.

Guanine-rich nucleic acids can fold into distinctive four-stranded G-quadruplex structures which are found in telomeric DNA repeats as well as in sequences in the promoter and other regulatory regions of genes, especially those involved in cellular proliferation. Small molecules that can selectively bind and stabilize the G-quadruplex structure have become of significant interest to researchers, and are gaining momentum as a possible new class of anticancer agents.

We recently reported a series of novel biaryl polyamides with significant selectively toward G-quadruplex compared to duplex DNA. Using a distamycin scaffold as a starting point, we introduced biaryl building blocks in place of pyrroles to switch preference from duplex to G-quadruplex DNA. This alteration in shape ensured that the molecules had low affinity for duplex DNA while increasing their interaction with a G-quadruplex structure. We have now used a molecular modeling approach to modify the structure of the previously reported biaryl polyamides by incorporating benzofused building blocks to improve affinity for G-quadruplexes while further reducing affinity for duplex DNA to enhance selectivity for G-quadruplex versus duplex DNA.

A small, focused benzofused-polyamide library (17 molecules) was initially synthesized and evaluated for the ability of members to stabilize G-quadruplex structures using a FRET-based DNA thermal denaturation assay and molecular dynamics (MD) simulations. However, these compounds failed to stabilize the human telomeric G-quadruplex and c-Kit quadruplexes, and MD simulations suggested that the shape of the molecules required further modification to facilitate G-quadruplex interaction. A second library of molecules (50 in total) was then designed and synthesized using a molecular modeling based approach. In this series, the shape of the polyamide fragment was changed, while retaining the original scaffold, by introducing benzofused moieties with 3,5-substitutions. Evaluation of these molecules in the same FRET assay showed a notable increase in stabilization of the F21T quadruplex for many library members. For example, compounds AR-130 and AR-168 stabilized the G-quadruplex by 15°C and 17°C, respectively (at 1 µM concentration), while showing insignificant affinity for duplex DNA. Cytotoxicity in the micromolar region is anticipated based on recent studies on related compounds, and a full growth inhibition evaluation is presently underway in MiaPaCa2, MDA-MB 231, HeLa and NCIH1975 cell lines.

Given their low molecular weight (e.g., between 422-646 Daltons), reasonable water solubility and good cellular penetration properties compared to other known G-quadruplex inhibitors which are mostly non-drug-like, molecules of this type have the potential to be developed into reagents that can probe DNA structure, and novel therapeutic agents based on G-quadruplex targeting.

#1358

Structure-based drug design of 3-site binding, high affinity inhibitors of S100B in malignant melanoma.

Michael C. Cavalier,1 Paul T. Wilder,1 Diane Luci,2 David J. Maloney,2 Lei Fang,1 Mohd. Imran Ansari,1 Alexander D. MacKerell,1 Andrew Coop,1 Ajit Jadhav,2 David J. Weber1. 1 _University of Maryland, Baltimore, Baltimore, MD;_ 2 _National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD_.

An especially predictive biomarker when used in combination with other diagnostic indicators, S100B is elevated in >90% of malignant melanoma (MM) patients and its protein level correlates directly with poor survival (<1 yr.) and relapse. For patients with low levels of S100B (5-10%), MM vaccines are uniquely effective. While this correlation is not fully understood, mechanistic studies show that S100B is capable of blocking p53 oligomerization, promoting p53 phosphorylation in its C-terminus, and contributing to p53 degradation in concert with hdm2. Correspondingly, lowering S100B levels via siRNA or via small molecule inhibitors restore p53 levels and its tumor suppressor activities including UV-activated apoptosis pathways found in normal melanocytes. Additionally, elevated S100B and overexpression of the receptor for advanced glycation end products (RAGE) confers a metastatic phenotype. Studies into how S100B affects tumor progression and metastasis are ongoing, novel S100B inhibitors (termed SBiXs) to ablate S100B-dependent effects in cancer are in development. Structure-activity relationship (SAR) studies are ongoing to improve the affinity and selectivity of SBiXs. These studies reveal that the binding surface of S100B can be divided into "hot spots" or "persistent sites" that contribute more energetically to the protein-protein interactions (PPI) than the surrounding protein surface. Described here are descriptions of SBiXs which bind these Sites (1, 2, and 3), details to the SAR studies involved in the chemical optimization of these ligands, and information into the tethering of these SBiXs into a single-molecule capable of binding all 3 sites simultaneously. Also provided is a discussion regarding how protein dynamics affect the on/off rates of SBiXs and the resulting apparent binding affinity. The results described here represent a Structure-based drug design approach to the development of PPI inhibitors nearing the threshold required for a useful therapeutic window.

#1359

Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor cells.

Aleem Gangjee,1 Manasa P. Ravindra,1 Mike R. Wilson,2 Adrianne W. Povrik,2 Lisa P. Hou,3 Carrie O'Connor,2 Zhanjun Hou,3 Larry H. Matherly4. 1 _Duquesne University, Pittsburgh, PA;_ 2 _Department of Oncology, Wayne State University School of Medicine, Detroit, MI;_ 3 _Department of Oncology, Wayne State University School of Medicine, Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI;_ 4 _Department of Oncology, Wayne State University School of Medicine, Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI_.

Folates are important single carbon transferring cofactors required for the biosynthesis of purine and pyrimidine nitrogen bases and DNA. Antifolates inhibit synthesis of these nitrogenous bases and function as antineopastic agents. The use of currently marketed antifolates such as methotrexate (MTX and pemetrexed (PMX) is limited by dose-limiting toxicity due to non-selective transport by the ubiquitously expressed reduced folate carrier (RFC). We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT expressing tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and circumvent the important limitation of currently marketed clinical antifolates that lack selective uptake into tumors. As an extension of the SAR, we now report on the role of fluorine atom(s) in improving selectivity as well potency. Fluorine was incorporated into our 6-substituted pyrrolo[2,3-d]pyrimidine antifolates to improve pharmacodynamics by improving binding affinity to target proteins via a number of well documented electrostatic and hydrophobic interactions of fluorine. In addition, fluorines were strategically placed on the side chain of our targeted antifolates for improved potency and selectivity through induced conformational restriction due to a plausible intramolecular fluorine-hydrogen bonding interaction. The fluorinated analogs had IC50s in the subnanomolar range (IC50 = 0.33 to 0.29 nM) for KB tumor cells and nanomolar (IC50 = 2.77 to 3.47 nM) for IGROV-1 cells. In addition, these analogs showed 600- to 1000-fold selectivity for FRα expressing cells over RFC. Protection studies suggest that glycinamide ribonucleotide formyl transferase (GARFTase) is the intracellular targets. In vivo activity was established toward the SKOV3 tumor xenograft model. These preclinical studies suggest that these fluorinated analogs are candidates for possible clinical trial as antitumor agents.

#1360

Genistein-AR antagonist conjugate potently induce growth arrest in both androgen dependent and independent prostate cancer cell lines.

Idris O. Raji,1 Alex George,1 Omer Kucuk,2 Adegboyega K. Oyelere1. 1 _Georgia Institute of Technology, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

The ability of prostate cancer to transition from androgen dependent- to androgen independent state (castration resistant), during androgen deprivation therapy, requires that new chemotherapeutic agents be introduced that will retain activity in the two prostate cancer types. Though described as androgen independent, castration resistant prostate cancer (CRPC) still relies heavily on androgen receptor (AR) signaling for survival. Enzalutamide, an AR antagonist, is probably that most effective drug used in androgen deprivation therapy for CRPC and AR dependent prostate cancer. Despite its clinical success, there is the fear of resistant to enzalutamide due to mutation in the ligand binding domain of AR. This poses a challenge that requires developing new agents devoid of resistance, in the event of mutation. One of the approaches being proposed is to develop new agents that can selectively induce AR degradation, an approach that was successfully used to develop the selective estrogen receptor degrader, fulvestrant.

In our study, we incorporate genistein, a soy isoflavone known to perturb multiple biochemical pathways relevant for cancer survival, into an AR binding template to give AG-1-33. AG-1-33 was evaluated for growth inhibitory activity in LNCaP (AR dependent) and DU-145 (AR independent) prostate cancer cell lines. This compound retained its AR antagonist activity and also showed low micromolar anti-proliferation activity in the two prostate cancer cell lines. We also evaluated the effect of AG-1-33 on AR expression, as well as its ability to act as a selective AR degrader in LNCaP.

#1361

Progress in light activatable prodrug for the combinational treatment of PDT and site-specific chemotherapy: paclitaxel prodrugs.

Pritam Thapa, Mengjie Li, Moses Bio, Pallavi Rajaputra, Yajing Sun, Sukyung Woo, Youngjae You. _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

We developed the photoactivatable prodrug of Paclitaxel (PTX) for the combinational treatment of chemo and photodynamic therapy (PDT). PTX causes dose-limiting side effects as other anticancer drugs when administered systemically. On the other hand, PDT suffers from incomplete ablation and subsequent reoccurrence in part due to the short half-life and poor diffusion rate of singlet oxygen. We prepared a conjugate of PTX with phthalocyanine via a singlet oxygen cleavable linker, as a unique prodrug of PTX, to overcome the problems of PDT and systemic chemotherapy. The PTX prodrug was evaluated for tubulin polymerization, the release rate of PTX from prodrug upon illumination at 690 nm, stability in complete media, and the combination effect in killing ovarian cancer cells in vitro (SKOV-3). The PTX prodrug did not enhance the tubulin polymerization unlike PTX. While it was stable in the media under dark, it rapidly released PTX upon illumination with far-red light: > 90% release in 30 min. The prodrug showed much lower dark toxicity compared to PTX. When illuminated with 690 nm at 5.6 mW/cm2, the prodrug showed very potent phototoxicity: IC50 = 3.9 nM, through the combinational effect of PDT and PTX. In conclusion, we found that the PTX prodrug have desired properties as light-activatable prodrug expressing the combinational effect of PDT and local PTX chemotherapy. Animal study is underway to evaluate the antitumor effect of the PTX prodrug.

#1362

Novel proton coupled folate transporter (PCFT) and reduced folate carrier (RFC) pharmacophore models for development of transporter-selective antifolates.

Sudhir Raghavan,1 Aleem Gangjee,1 Larry H. Matherly2. 1 _Duquesne University, Pittsburgh, PA;_ 2 _Barbara Ann Karmanos Cancer Institute, Detroit, MI_.

RFC is a ubiquitously expressed folate transporter that is present in normal tissues and tumors. RFC is utilized by reduced folates and by all the clinically used classical antifolates. In contrast, PCFT shows more limited expression in normal tissues and is highly effective in folate transport under low pH conditions associated with hypoxic solid tumors. Antifolates that are selectively transported by PCFT over RFC offer significant promise for development of targeted therapies of tumors (over)expressing PCFT. We previously reported 6-substituted pyrrolo[2,3-d]pyrimidines and thieno[2,3-d]pyrimidines as part of a continued effort to elucidate the SAR for substrate binding and cellular uptake for RFC, PCFT as well as folate receptors (FRs). While the X-ray crystal structure of FRs bound to antifolates has been resolved, the absence of X-ray crystal structures of PCFT and RFC has hindered structure-based medicinal chemistry efforts for development of antifolates selectively transported by PCFT. To address this, pharmacophore models were developed for the first time using Discovery Studio for PCFT and RFC using a series of thirty six 6-substituted bicyclic pyrrolo[2,3-d]pyrimidines and/or thieno[2,3-d]pyrimidines that included highly potent and selective compounds such as AGF94 (PCFT = 0.3 nM; RFC = 101 nM) previously reported by us. Compounds used for model development displayed a 3-log range inhibition of cancer cells overexpressing PCFT or RFC. The best models display a 4-point pharmacophore with excellent regression values (r2 PCFT = 0.96; RFC = 0.92). The models were internally validated by leave-one-out analysis (q2 PCFT = 0.752; RFC = 0.59) and externally validated using a test set of 6-substituted bicyclic analogs not used in model development. Comparison of the pharmacophore models indicates different conformational and structural requirements for compounds for binding to PCFT or RFC. We have used these models to explain the potent PCFT inhibitory activity and selectivity of compounds such as AGF94 . These models are being used in the development of selectively transported targeted antifolates that would result in decreased toxicity compared to standard antifolates such as methotrexate (MTX) or pemetrexed (PMX) which utilize all three folate transport systems.

#1363

Novel Nrf2 inhibitors and their application to overcome chemoresistance of anticancer drugs.

Vineet Kumar, Kira Foygel, Ramasamy Paulmurugan, Sanjay V. Malhotra. _Stanford University School of Medicine, Palo Alto, CA_.

A large number of malignant tumors, for example, colonic, thyroid, endometrial, lung, ovarian, breast and pancreatic cancers, exhibit an increased Nrf2 expression and constitutive activation. This enhanced Nrf2 activation originates from rare gain-of-function mutations of Nrf2 itself, and from loss-of-function mutations, promoter hypermethylation or microRNAs targeting Nrf2 inhibitory protein Keap1/INRF. As a consequence of the enhanced Nrf2 activity, tumor cells acquire protection from apoptosis and are more capable of proliferation, both conditions favoring tumorigenesis on one hand and making tumor cells more refractory to chemo- and radiotherapy on the other hand. These findings suggest that Nrf2 exhibits profound protumorigenic activity, and can be a crucial target for anticancer therapy, particularly by overcoming chemoresistance.

In the current study, we have rationally created a library of about 150 flavonoid-based compounds and screened them for Nrf2 inhibition effect using a novel intact cell based bioluminescence imaging assay which directly measures the expression of NQO1, the downstream target gene of Nrf2. HeLa cells transfected with Luciferase reporter gene expressed under the promoter of NQO1 gene were treated with the library compounds at 10 µM concentration for 16 hours in a 96-well plate format. After treatment, the cells were imaged by the addition of 25 µg/ml of D-Luciferin using IVIS bioluminescence imaging system. The NQO1-ARE-luciferase signals measuring its expression in different wells were quantified. In parallel, the drug-associated toxicity was assessed by MTT assay, the results were used to normalize the bioluminescence signal, and the inhibitory effect was assessed by comparing the signal induced by each compound with the DMSO control. In this preliminary screening, 11 compounds that inhibited the expression of NQO1 by more than 3-fold, as compared to the DMSO control, were identified. Dose-response studies showed that these compounds inhibit Nrf2 in a linear dose-dependent pattern. Moreover, 3 of the hit compounds exhibited 6-10 fold of Nrf2 inhibition (compared to DMSO) at as low as 0.16 µM concentration. We also evaluated Hela cells treated with Doxorubicin (Dox, 1 µM) and four hit compounds (10 µM conc.) for induced apoptosis after 48 hours of treatment, to evaluate synergistic effect. Quantification studies by FACS assay using PI staining revealed that our hit inhibitors enhanced the apoptosis effect of Doxorubicin.

In conclusion, we have identified new potent inhibitors of Nrf2, which also enhanced the apoptotic effect of Doxorubicin. Further studies to probe the mechanism of action of these inhibitors and medicinal chemistry efforts to study the structure activity relationship are underway.

#1364

Novel 4-(pyrimidin-2-yl)morpholines targeting the colchicine-binding site of tubulin.

Alexander M. Sele,1 Denise Rageot,1 Thomas Bohnacker,1 Florent Beaufils,1 Andrea E. Prota,2 Michel O. Steinmetz,2 Matthias P. Wymann1. 1 _University of Basel, Basel, Switzerland;_ 2 _Paul Scherrer Institute, Villigen, Switzerland_.

Microtubules are dynamic polymers and integral components of the cytoskeleton. They play important roles in the regulation of cellular signaling, motility, maintenance of cell shape, secretion, intercellular transport and spindle formation during mitosis.1 As a consequence, small molecules that interfere with the dynamics of microtubules have been recognized as powerful tools for the treatment of cancer.2, 3

A systematic structure-activity relationship (SAR) study starting from morpholino-substituted biheteroaryls with moderate microtubule disrupting activities allowed the optimizations of biological activity, metabolic stability, and drug-like properties. Interestingly, the type of core (aryl, pyridine or pyrimidine) was of importance, as well as the regioisomeric arrangement of the pyrimidine nitrogens. Pyrimidines substituted with four- or five-membered N-heterocycles proved to be superior both in terms of biological activity and metabolic stability. Finally, optimization of the heteroaryl substituent of the pyrimidine derivatives culminated in the identification of a novel series of substituted 4-(pyrimidin-2-yl)morpholines targeting microtubule polymerization in the nM range.

Selected compounds potently inhibit cellular microtubule polymerization with EC50 values of 20-90 nM. This was confirmed by phosphorylation of Histone3, nuclear DNA condensation, and cell cycle arrest in G2/M or induction of cell death across multiple cell lines. Moreover, substituted 4-(pyrimidin-2-yl)morpholines were shown to be poor substrates for P-gp multi-drug resistance pumps, and therefore caused efficiently mitotic arrest and cell death in colchicine-resistant cells.

The co-crystal structure of tubulin with selected compounds showed that 4-(pyrimidin-2-yl)morpholines bind to the colchicine pocket located between the α and β subunits of the αβ-tubulin dimer. Relevant inhibitor contact residues include Lys352, Met259, Ala316, Leu248, Val238, Tyr202 and Cys241 of β-tubulin. Moreover, two water molecules link the morpholine oxygen to the β-tubulin bound GTP. Conformational changes induced by inhibitor binding suggest that free or plus end β-tubulin is targeted by this compound series.

Pre-clinical studies characterized a lead compound selection with excellent stability in human hepatocytes, and human, mouse and rat microsomes. Overall, these compounds qualify as a novel class of microtubule destabilizing agents that target the colchicine-binding site, and which warrant further investigations currently in progress (PK studies, xenograft tumor mouse models).

1) Walczak, C. E, Curr. Opin. Cell Biol. 2000, 12, 52-56.

2) Risinger, A. L.; Giles, F. J.; Mooberry, S. L, Cancer Treat. Rev. 2009, 35, 255-261.

3) Downing, K. H.; Nogales, E., Curr. Opin. Struct. Biol. 1998, 8, 785-791.

#1365

Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC.

Aleem Gangjee,1 Weiguo Xiang,1 Aamod Dehkne,2 Mike R. Wilson,2 Adrianne Wallace-Povrik,2 Carrie O'Connor,2 Zhanjun Hou,2 Larry H. Matherly2. 1 _Duquesne University, Pittsburgh, PA;_ 2 _Wayne State University School of Medicine, Detroit, MI_.

Drugs usually bind to different targets in different conformations. As antitumor agents, antifolates first need to be selectively transported by tumor cells via the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) or folate receptors (FRs). The second step is the binding of antifolates with the intracellular targets. We are systematically developing novel folate analogs with selective membrane transport for FRs and PCFT over the ubiquitously expressed RFC. These 6-substituted pyrrolo[2,3-d]pyrimidine antifolates are principally inhibitors of de novo purine biosynthesis at the steps catalyzed by glycinamide ribonucleotide formyl transferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). We previously demonstrated that classical antifolates bind to FRα and GARFTase in different conformations. An amide in the bridge region of classical antifolates has two interchangeable lowest energy conformers, pseudo-cis and pseudo-trans. We reported that a novel antifolate (AGF238) with an amide in the bridge region binds to FRα in a pseudo-trans conformation and binds to GARFTase in a pseudo-cis conformation. As part of our continued efforts to pursue targeted antifolates as antitumor agents, we designed an expand series of amide-bridged classical antifolates with different energy barriers between the pseudo-cis and pseudo-trans conformers. Among these, AGF266 was exclusively transported by Chinese hamster ovary (CHO) cells engineered to express human FRα (IC50 = 0.42 nM) or FRβ (IC50 = 0.88 nM) over CHO expressing human RFC (IC50 > 1000 nM). AGF266 was also found to exhibit highly potent antitumor activity (IC50 = 0.79 nM in KB human tumor cells). AGF266 inhibited GARFTase as its principal cellular target, based on patterns of protection from growth inhibition by adenosine and AICA. Our in vitro findings of antitumor activity associated with FR selectivity suggest that further preclinical evaluation of AGF266 as a potential antitumor agent is warranted.

#1366

Fabrication of minocycline loaded PLGA microparticles for the treatment of intracranial tumors.

Sue Anne Chew, Marco Arriaga, Jesus R. Franco, Daniela Barbosa, Victor A. Hinojosa, Jose-Carlos Martinez, Paul Lenz. _University of Texas Rio Grande Valley, Brownsville, TX_.

Minocycline is a tetracycline derivative that was originally used clinically as an antibiotic and is currently being investigated as an anti-angiogenic factor for the treatment of different cancers including intracranial tumors such as glioblastomas. Due to its lipophilic nature, it has difficulty dissolving completely in organic or aqueous solvents and thus, presents a challenge for the fabrication of these particles via the emulsion-solvent evaporation method. The aim of this study is to investigate the effects of different methods of fabricating minocycline loaded microparticles on particle properties, such as the diameter, drug loading and release kinetics of the microparticles. Microparticles loaded with drug during the fabrication process were produced via two different methods: 1) an oil-in-water (O/W) single emulsion-solvent evaporation method where the drug was dissolved in the oil phase (denoted as "O/W particles") and 2) a water-in-oil-in-water (W/O/W) double emulsion-solvent evaporation method where the drug was dissolved in the first water phase (denoted as "W/O/W particles"). Empty microparticles were also fabricated by a W/O/W method and loaded with drug after fabrication by dripping a drug solution onto freeze dried particles which were then left overnight at 4°C to allow the drug to adsorb onto and absorb into the particles (denoted as "prefabricated particles"). Light microscopy images of the particles were obtained and used to measure the diameters of the particles with the ImageJ software. The drug loading and release kinetics were determined by measuring the absorbance of minocycline at 350 nm with a microplate reader. The prefabricated particles resulted in larger diameters compared to the O/W and W/O/W particles which were loaded during the fabrication process. This could have resulted from the use of a vortex instead of a homogenizer/sonicator for the prefabricated scaffold which produces an emulsion with a lower speed compared to a homogenizer/sonicator. The prefabricated particles had a higher amount of drug loaded compared to O/W and W/O/W particles which were loaded during the fabrication process. By loading the drug after fabricating the particles, almost all of the drug can be adsorbed onto and/or absorbed into the particles without resulting in much loss of the drug. Alternatively, by loading the particles during the fabrication process, a lot of drug is lost into the water and/or oil phase and thus reducing the loading efficiencies of the particles. However, the particles loaded during the fabrication process were able to prolong the release of the loaded drug compared to the prefabricated scaffold which lost almost all of its drug by Day 1. In conclusion, although the prefabricated particles allow for the complete loading of the drug, the particles loaded during the fabrication process are more promising as controlled release vehicles as they are able to better sustain the release of the loaded drug.

#1367

Intra-cerebral pharmacokinetic monitoring of a tyrosine kinase inhibitor (theranostic 18[F]-PET/NIRF labeled dasatinib) delivered via convection-enhanced delivery.

Melinda Wang, Zhiping Zhou, Hari Krishna Kommidi, Melanie Schweitzer, Mark Chan, Yue Linda Wu, Ranjodh Singh, Richard Ting, Mark M. Souweidane. _Weill Cornell Medical College, New York, NY_.

Introduction: Convection enhanced delivery (CED) has been recently explored as an advantageous therapeutic strategy for central nervous system (CNS) tumors. One current limitation is the inability to quantitatively monitor distribution of chemotherapeutic agents. The use of surrogate tracers probably underestimates differences in distribution and clearance owing to discordant features between contrast molecules and therapeutic agents, including bioactivity, degradation, conductivity, and diffusivity. Ideally, direct labelling of a therapeutic compound would eliminate these concerns and afford a noninvasive method to monitor critical pharmacokinetic information. This ability would pave the way for designing infusion parameters and drug schedules that are expected to be unique for CED-based therapy.

Methods: The small molecule kinase inhibitor dasatinib was modified with a dual-probe technology that utilizes a boronate trap to conjugate 18[F] with a near-infrared fluorophore into a single molecule, 1, allowing for visualization by positron emission tomography (PET) and near-infrared fluorescence (NIRF). The novel boronate trap allows for direct conjugation of 18[F] with minimal disruption of dasatinib's mechanism of action, resulting in a small, versatile probe with potential for other clinically relevant targets.

Results: The modified drug was first tested in vitro in a PDGF-B driven p53 deficient DIPG tumor line from Nestin tv-a; p53 floxed mouse. 1 was shown to enter cells on fluorescence microscopy and inhibit cell proliferation. Antagonist drug potency of 1 as evaluated on pontine glioma in ATP-dependent luminescent cell viability, cell-permeant calcein AM, and an MTS cell proliferation assays show that the IC50, of 1 is ~10nM. In addition, infusions of 1 were performed via CED and intravenous systemic delivery in a RCAS/tv-a PDGF-B driven with p53 deficiency Ntv-a mouse model of high-grade glioma. Compared to systemic delivery, CED was shown to be roughly 230 times more effective in delivery of the infusion to the tumor site. Maximal glioma delivery occurred at 115 min post a 20 min infusion, with clearance occurring with a half-life of 45 min after maximum delivery.

Conclusion: Dasatinib was modified to give 1, an agent that can be imaged non-invasively by fluorescence and positron emission tomography (PET). The modified dasatinib showed little modification in biologic activity when compared to unmodified dasatinib and exhibited cytotoxic effects in vitro in glioma cell culture. CED shows a clear benefit to intravenous delivery when the target site is in the CNS. The dual-label probe technology's versatility and minimal biologic profile lends itself to various clinical uses, including the study of different therapeutic agents and delivery routes without the need to rely on surrogate tracers.

#1368

Preclinical evaluation of NC-6201, an antibody/drug-conjugated micelle incorporating novel hemiasterlin analogue E7974.

Mitsunori Harada, Masami Tsuchiya, Ryusuke Miyazaki, Tadashi Inoue, Ryosuke Tanaka, Yuuki Yanagisawa, Masayoshi Ito, Yu Ito, Kenichiro Naito. _NanoCarrier Co., Ltd., Kashiwa, Chiba, Japan_.

Background:

Antibody-drug conjugates (ADCs) have been recognized as a promising anticancer agent. There have been a lot of registered clinical trials for ADCs. However, not all ADC compounds were successful. To overcome the difficulties and drive the next generation of ADCs, we have recently developed Antibody/Drug-Conjugated Micelle (ADCM) system. ADCM is composed of polyethylene glycol-poly (amino acid derivative) polymers, which can form a micellar nanoparticle spontaneously in aqueous media with a diameter of 20-100 nm. Antibodies are attached to the surface of the nanoparticle, while payloads are incorporated in the inner core at a payload-to-antibody molecular ratio of 100-200. In this study, anti-EGFR monoclonal antibody NCAB001 and novel hemiasterlin analogue E7974 were used as a targeting sensor and a payload of the ADCM (NC-6201), respectively. Here we report the results of in vitro evaluation and in vivo efficacy and toxicity studies.

Methods:

NC-6201 was prepared as described previously (Japan Patent No.4538666) with slight modification. The antigen affinity and the cytotoxicity of NC-6201 were evaluated using a Biacore system and Cell Counting Kit-8, respectively. NC-6201 was administered intravenously to BALB/c-nu/nu mice xenografted with various human tumor cell lines. Tumor volumes and animal body weights were monitored 2 or 3 times a week. Also, the dose- and schedule-dependency of the antitumor effect were evaluated. Single-dosed toxicological studies of NC-6201 in SD rats and cynomolgus monkeys were performed.

Results:

NC-6201 showed similar antigen affinity to the unconjugated NCAB001 and had a broad range of in vitro cytotoxicity against a panel of tumor cells. NC-6201 potently suppressed tumor growth in nude mice bearing subcutaneous human tumor xenografts expressing EGFR, such as BxPC-3 (pancreas) and MDA-MB-231 (triple-negative breast) tumor models. The efficacious NC-6201 dose schedules were achieved at one tenth or two fifth of its MTD. In an EGFR-null tumor model, NC-6201 and untargeted micelle incorporating E7974 showed comparable tumor growth inhibition. Overall, NC-6201 at efficacious doses was well tolerated without significant body weight loss, indicating that NC-6201 has an excellent therapeutic window. Relative to E7974, NC-6201 showed an unaltered toxicity profile in rats and monkeys, and the potential for reduced toxicity and an improved therapeutic window.

Conclusion:

Based on these promising results, NC-6201 is advanced in our project pipeline as a clinical candidate for cancer therapy.

#1369

**In** vitro **and in** vivo **antitumor activity of TH3424: Preclinical rationale for a highly selective AKR1C3 prodrug for treating hepatocellular carcinomas.**

Jianxin Duan,1 Zhong Wang,2 Qing Li,2 Yeyu Cao,1 Ping He,2 Fanying Meng,1 Changhua Zhou,2 Yanhong Wang,2 Gavin Qu,3 Henry Li,3 Jiang Li,4 Meng Yang,5 Hui Qi,5 Don Jung,1 Mei Song,1 Mark Matteucci1. 1 _Threshold Pharmaceuticals Inc., South San Francisco, CA;_ 2 _School of Pharmaceutical Sciences and Centre for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China; _3 _Crown Bioscience Inc., Santa Clara, CA;_ 4 _State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Department of Biotherapy, Guangzhou, China;_ 5 _AntiCancer Biotech (Beijing) Co., Ltd, Beijing, China_.

Aldo-keto reductase family 1 member C3 (AKR1C3) catalyzes the reduction of a diverse group of substrates, including prostaglandin (PG) D2 and PGH2. It has been reported that AKR1C3 is overexpressed in the majority of hepatocellular carcinomas (HCC) with 58% of HCC patient surgical tumor samples having strong expression of the enzyme1. Tumors overexpressing AKR1C3 can be resistant to radiation therapy2 and chemotherapies3. AKR1C3 is also expressed in normal tissues,but its expression is much lower than in HCC tissue1.

HCC is the sixth most common cause of cancer. It has very poor prognosis, and is the second leading cause of cancer death in all cancers. Treatment options for late stage liver cancer are very limited, with Sorafenib, a multi-tyrosine kinase inhibitor, being the only approved drug with limited efficacy. More effective therapies are urgently needed.

TH3424 is a prodrug which selectively releases a DNA alkylating agent upon exposure to activated AKR1C3. In vitro cell proliferation tests with TH3424 in several HCC cell lines showed that it is very potent (IC50 ≤ 10

nM with 2 hour exposure) in killing cancer cells with high levels of AKR1C3, but less active (IC50 ≥ 10 µM) in killing cells with low or no

AKR1C3 reductase. The activity of TH3424 correlates with the expression level of AKR1C3 as the potency of TH3424 is inhibited when used with a specific AKR1C3 inhibitor:4 (IC50: 4 nM vs 6.3 µM with SN33638) in a non-small cell lung cancer cell line (H460). In vivo orthotopic and patient derived disease (PDX) liver cancer model studies have shown promising efficacy with TH3424 being administer weekly with doses as low as 1.5mg/kg. TH3424 showed better efficacy than Sorafenib in an orthotopic HepG2 mouse model. Three of 8 mice treated with TH3424 at 2.5 mg/kg, Q7Dx3, and 8 of 8 mice treated with TH3424 at 5 mg/kg, Q7Dx3 were tumor free at day 35.

Reference:1: Guise C. P.; Abbattista M. R.; Singleton R. S.; Holford S. D.; Connolly J.; Dachs G. U.; Fox S. B.; Rollock R.; Harvey J.; Guiford P.; Daňate F.; Wilson W. R.; and Patterson A. V.; Cancer Res.70(4), 2010, 1573

2: Xiong W.; Zhao J.; Yu H.; Li X.; Sun S.; Li Y.; Xia Q.; Zhang C.; He Q.; Gao X.; Zhang L.; Zhou D.; Plos One, V. 9 (11), 2014, e111911

3: Liu C.; Lou W.; Zhu Y.; Yang J.; Nadiminty N.; Gaikwad N.; Evans C.; Gao A.; Cancer Res.; 2015, 75(7), 1413

4: Flanagan J. U.; Atwell G. J.; Heinrich D. M.; Brooke D. G.; Silva S.; Rigoreau L. J. M.; Trivier E.; Turnbull A. P.; Raynham T.; Jamieson S. M. F.; Denny W. A.; Bioorg. Med. Chem.; 22(2014); 967

## CLINICAL RESEARCH:

### Clinical Assay Development

#1370

A new RAS G12D specific rabbit polyclonal antibody for immunohistochemical FFPE application.

Chi-Kuan Chen. _Mackay Memorial Hospital, Taipei, Taiwan, New Taipei City, Taiwan_.

Background: Determining RAS mutation status is considered as prognostic factor for colorectal cancer patients in anti-epidermal growth factor receptor (EGFR) antibody therapy. Because genetic testing remains costly and time-consuming, it is necessary to develop mutation specific antibody for immunological analysis. However RAS G12D mutation antibody for immunohistochemical (IHC) staining is not available.

Results: Here we report a novel RAS G12D rabbit polyclonal antibody (GTX132407) for immunochemistry (IHC) testing, which was developed by cooperating with GeneTex In. Based on analyzing 15 clinical colorectal cancer specimens, the result of IHC staining using RAS G12D mutation specific antibody (GTX132407) was correlated with genetic testing of RAS G12D mutation using ultra-sensitive total RAS mutation screen kit [Hong Jing Biotechnology Inc. (FemtoPath)].

Conclusion: In summary, the result showed that IHC testing with RAS G12D specific mutation antibody could be a reliable tool for predicting RAS genetic mutation status.

#1371

Spatially-resolved, multiplexed digital characterization of protein distribution and abundance in FFPE tissue sections.

Alessandra Cesano,1 Joseph Beechem,1 Philippa Webster,1 Chris Merritt,1 Jaemyeong Jung,1 Dwayne Dunaway,1 Gary Geiss,1 Sarah Warren,1 Gordon Mills2. 1 _NanoString Technologies, Seattle, WA;_ 2 _MD Anderson Cancer Center, Houston, WA_.

Intratumoral heterogeneity has emerged as a critical challenge to the implementation of targeted therapeutics. Historically, immunohistochemistry (IHC) has been used to assess spatial heterogeneity of proteins; however, it has been difficult to quantify protein abundance at high multiplex and wide dynamic range. Here, we report the development and validation of a spatially-resolved, antibody-based proteomic approach with a "barcoding-potential" to quantify up to 800 targets with 5.5 logs (base 10) of dynamic range in a single formalin-fixed paraffin-embedded (FFPE) slide. By labeling antibodies with photocleavable oligos which are recognized by NanoString® nCounter® fluorescent barcodes and subsequently exposing them to focused UV light, we have developed an nCounter assay capable of quantifying protein abundance in a predefined spatial region of a tissue section.

Methods: A slide-mounted FFPE tissue section is bound with a multiplexed cocktail of primary antibody-oligo conjugates, and a microfluidic flow cell is attached to the slide. Using a simple modification of a standard microscope, regions of interest are identified by light or fluorescence microscopy and are sequentially illuminated with UV light to release the oligos. Following each illumination cycle, an eluent is collected and analyzed, resulting in digital counts that correspond to the abundance of each targeted protein in sequentially illuminated areas. We demonstrate a high degree of linearity (0.97 < R2 < 0.99) for the number of observed counts versus area of UV illumination, with current detection spatial resolution down to 100 µm x 100 µm, or approximately 100 cells.

Application: FFPE slides from resected breast cancers are bound with an antibody cocktail (10+ plex, including HER2, EGFR, PR and others) and visualized by light microscopy. Regions of interest are identified, and oligo barcodes from those regions are released by UV illumination and digitally quantified by nCounter analysis. This enables multiplexed detection and comparison of proteins of interest from discrete regions within the tumor and adjacent normal tissue, enabling systematic interrogation of the heterogeneous tumor microenvironment.

Conclusion: Application of this NanoString barcoded antibody platform to ongoing clinical studies is intended to elucidate novel responses to immunotherapy and other targeted therapies. Further development of this technology will enable the multiplexed analysis of up to 800 protein targets from a single FFPE section and facilitate detailed interrogation of spatial interactions within a tissue. The ability to measure DNA, RNA, and protein from FFPE tissue may enable the discovery of immune biomarkers in tumors and the development of companion diagnostics.

#1372

Quantification of PD-L1 and PD-1 expression on tumor cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry.

Amanda Chargin,1 Rian Morgan,1 Uma Sundram,2 Navneet Ratti,3 Keith Shults,1 Bruce Patterson1. 1 _IncellDx, Menlo Park, CA;_ 2 _Stanford, Stanford, CA;_ 3 _Tissue Diagnostics, Hayward, CA_.

Objective: The objective of this study was to develop a truly quantitative technology for PD-L1 expression in NSCLC. In addition, we also present a non-enzymatic technology that creates a tumor cell suspension from fresh tumor tissue so that either fine needle aspiration or fresh tissue can be used in this assay.

Methods: 4 mm punches were taken from each tumor. Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, CA) was performed. A French technique FNA was also taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, PD-L1, fixed and permeabilized then stained with DAPI to identify intact, single cells, and to analyze cell cycle.

Results: Both FNA and IncellPREP generated greater than 1 million cells per mL with the IncellPREP yields being 2.5 times the number of cells per preparation compared to FNA (Mann-Whitney, p=0.003). Comparing the IncellPREP homogenization and FNA, a strong correlation (r2-0.8) was demonstrated for expression of PD-L1. We compared PD-L1 expression by flow cytometry using a 1%a cut-off for positivity in the tumor cell population and a 1% cut-off of cells with at least 1+ intensity in immunohistochemically stained tissue sections as positive (Table 1). As demonstrated in the table, 10 of 12 lung tumor samples were concordant while 2 were discordant, one positive by flow and negative by IHC and one negative by flow and positive by IHC. PD-L1 expression by flow cytometry varied widely (1.2% to 89.4%) even in the positive concordant cases. In addition, PD-L1 expression in the aneuploid tumor population did not necessarily agree with the expression in the diploid tumor population.

Summary: Fine, unequivocal, quantification of PD-L1 on tumor and immune cells in NSCLC may allow for better prediction of response to therapies. The present study also offers a technology that can create a universal sample type from either FNA or fresh tissue.

Table 1

---

Diagnosis | DNA Index Tumor | Aneuploid Index | PD-L1 (1% CO) | PD-L1 % (Aneuploid) | IHC (1% CO)

Squamous cell carcinoma | 0.94 | 1.64 | 18.9% (+) | 28.8% | Pos

Adenocarcinoma | 0.97 | None | 1.2% (+) | - | Neg

Adenocarcinoma | 0.90 | 2.18 | 86.6% (+) | 99.6% | Pos

Adenocarcinoma | 0.96 | 1.40 | 85.1% (+) | 97.0% | Pos

Squamous cell carcinoma | 0.95 | 1.34 | 94.8% (+) | 97.7% | Pos

Squamous cell carcinoma | 1.01 | None | 13.7% (+) | - | Pos

Adenocarcinoma | 0.93 | None | 0.8% (-) | - | Neg

Squamous cell carcinoma | 0.94 | None | 0.5% (-) | - | Pos

Acinar Adenocarcinoma | 0.96 | 1.21 | 22.9% (+) | 88.5% | Pos

Adenocarcinoma | 0.87 | None | 0% (-) | - | Neg

Squamous cell carcinoma | 0.91 | None | 25.8% (+) | - | Pos

Adenocarcinoma | 0.96 | None | 0.1% (-) | - | Neg

#1373

IHC and RT-PCR assays for detection of cancer antigen NY-ESO-1 in human tissues.

Abdel Halim,1 Rebecca G. Bagley,2 Lisa Dauffenbach,3 Eric P. Olsen,3 Tibor Keler1. 1 _Celldex Therapeutics, Inc., Hampton, NJ;_ 2 _Celldex Therapeutics, Inc., Needham, MA;_ 3 _Mosaic Laboratories, Lake Forest, CA_.

Introduction: NY-ESO-1 is a cancer testis antigen frequently expressed in a broad range of tumors: lung, breast, ovarian, bladder, liver cancer, melanoma, sarcoma and myeloma. CDX-1401, a fully human monoclonal antibody linked to full length NY-ESO-1, binds to dendritic cell (DC) receptor DEC-205 to stimulate NY-ESO-1 specific CD4 and CD8 responses. Immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) assays were developed to determine NY-ESO-1 expression in human tumors and normal adjacent tissues (NAT). A qRT-PCR assay for the detection of LAGE-1, a cancer testis antigen with significant DNA homology to NY-ESO-1, was also developed but no commercially specific antibody was available for IHC.

Methods: Formalin fixed, paraffin embedded tumors and NAT were procured from a commercial source or obtained from Mosaic Laboratories; split anonymized samples were analyzed for NY-ESO-1 by IHC at Mosaic and for NY-ESO-1 and LAGE-1 by qRT-PCR at MolecularMD (OR) under IRB-approved protocols allowing in vitro analysis of remnant human samples. The study included: 18 each melanoma and ovarian cancers; 13 each colorectal (CRC), head & neck (H&N), and non-small cell lung cancers (NSCLC); and 38 NAT. HT1080 fibrosarcoma and SKOV3 ovarian cancer cell lines (ATCC, VA) and normal testis were used as controls. The qRT-PCR assays were validated using in vitro transcribed RNA on NY-ESO-1 and LAGE-1 kits provided by Life Technologies and the NY-ESO-1 IHC was developed using a mouse monoclonal antibody (Sigma, MO).

Results: The NY-ESO-1 and LAGE-1 qRT-PCR assays had a linear range of 100-1,000,000 copies/reaction with limit of detection between 1-10 copies. An excess of LAGE-1 copies did not impact the specificity of the NY-ESO-1 assay and vice-versa. NY-ESO-1 protein was detected by IHC and NY-ESO-1 transcripts and LAGE-1 transcripts were detected in 9 (12%), 12 (16%) and 16 (21.3%), respectively, of the 75 tumor tissues while only 2 of the 38 normal samples were positive for LAGE-1. When stromal staining was considered in addition to epithelial staining, 16 (21.3%) tumor samples were positive by IHC. NY-ESO-1 protein was more prevalent in NSCLC (38%), ovarian cancer (11%) and melanoma (11%) vs. other tumor types. Positive staining by IHC was achieved in tumor tissues shown positive for NY-ESO-1 and negative for LAGE-1 transcripts. In general, tumor tissues obtained from Mosaic were of better quality, as assessed by the pathologist, and more positive than commercially procured samples.

Conclusions: Two qRT-PCR assays were developed to specifically quantify NY-ESO-1 or LAGE-1 mRNA without cross reaction. The antibody used in IHC assay seemed specific to NY-ESO-1. Inadequate sample quality impacted target prevalence. Implementing these assays in clinical trials may aid in prospectively identifying patients with NY-ESO-1 positive malignancies who could derive benefit from investigational NY-ESO-1 vaccines designed to induce anti-tumor immunity, such as CDX-1401.

#1374

Isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH testing in breast cancer.

Amanda Gerber,1 Trisky Clarin,1 Ambica Bhandari,2 Valeria Sero,1 Chiara Bolognesi,1 Gianni Medoro,1 Nicolò Manaresi,1 Mathew Moore,2 Philip D. Cotter,2 Farideh Bischoff1. 1 _Silicon Biosystems, La Jolla, CA;_ 2 _Research Dx, Irvine, CA_.

Body: Guidelines worldwide focus on the importance of precise, reproducible, and quality assurance of Fluorescent In Situ Hybridization (FISH) methods for testing companion diagnostic markers, including Human Epidermal Growth Factor Receptor 2 (HER2) gene amplification in breast cancer. Despite these guidelines, variations in test results due to pre-analytical sampling and tissue processing are observed. In this study, we demonstrate a unique approach to isolating pure and intact tumor cells from breast cancer Formalin-Fixed, Paraffin-Embedded (FFPE) samples for precise subsequent FISH analysis.

Methods: Fifty-micron thick FFPE curls from both HER2 non-amplified breast cancer tumors (n=4; each with a reported HER2/CEP17 ratio 1.8) and positive control SKBr3 breast cancer cells were tested. Isolation of ∼250 pure and intact cytokeratin-positive/vimentin-negative/DAPI positive tumor cells from each sample was achieved using the DEPArray™ platform, an automated system enabling image-based cell sorting with single-cell resolution for pure cell population isolation and collection. Recovered cells were then cyto-spun onto poly-L coated glass slides prior to standard dual-color HER2/CEP17 FISH (Path Vysion Abbott/Vysis) analysis.

Results: Positive HER2 amplification levels for the FFPE derived control SKBr3 cells were observed (HER2/CEP17 ratio >4.4) and consistent with levels reported in the literature. Among the patient samples, ≥75% of the DEPArray™ isolated tumor cells were recovered onto slides prior to FISH. Through routine FISH scoring, an expected non-amplified result was observed for each patient sample, with observed HER2/CEP17 ratios only ranging from 1.1 to 1.4.

Conclusion: We demonstrate feasibility in performing FISH for HER2/CEP17 on pure and intact tumor cells isolated from breast cancer derived FFPE using the DEPArray™ platform. Using a 50-micron section permitted recovery of whole, intact, tumor cells based on immunostaining for cytokeratin, vimentin, and DAPI. Efficient recovery of the DEPArray™ sorted cells onto slides further permitted routine FISH analysis of only tumor cells. These preliminary results imply the possibility of more precise FISH analysis when standard FISH results are inconclusive or when insufficient tumor content prohibits downstream analysis. Evaluation of larger numbers of patient samples is underway.

#1375

An ultra-sensitive cell free DNA liquid biopsy assay for cancer treatment monitoring.

Grace Zhao,1 Li Weng,1 Paul Tang,1 Johnny Sun,1 Yi Huang,2 Lingchen Guo,2 Hongyan Wang,2 Xiaozheng Kang,3 Wei Shen,4 Kang Ying,2 Shengrong Lin1. 1 _AccuraGen, Menlo Park, CA;_ 2 _AccuraGen, Shanghai, China;_ 3 _Beijing Cancer Hospital, Beijing, China;_ 4 _Xinhua Hospital, Shanghai, China_.

In order to take advantage of the narrow time window for optimal treatment efficacy, highly sensitive disease monitoring is critical in the successful management of cancer. Currently, treatment efficacy is assessed by using a combination of protein cancer biomarkers and imaging. However, both methods present limitations with regard to specificity or sensitivity due to their dependency on tumor size. Recently, a number of studies have suggested that monitoring cell free DNA (cfDNA) may provide a more specific alternative for tracking cancer treatment with greatly improved sensitivity. Here we introduce a novel next-generation sequencing based mutation detection system aimed at improving the sensitivity, reliability, and clinical utility of cancer treatment monitoring. Our system, comprised of Nebula, a whole genome amplification technology that is capable of amplifying nanogram quantities of cfDNA >1000-fold, and Firefly, a proprietary technology combining molecular biology and computational algorithm for error-suppression, has reduced the rate of random sequencing errors to 10-6. As a result, we are able to detect 1.5 variant copies from 10ng of input cfDNA with a detection rate of 46%. We have validated the Nebula-Firefly assay on a patient cohort with either colorectal (CRC) or lung cancer. Here we report the successful detection of drug resistant mutations and various genomic alterations associated with minimal residual detection (MRD) in sample cfDNA. These initial findings have led to the exploration of Nebula-Firefly as the technological backbone for a noninvasive, scalable approach for the early detection, treatment, and monitoring of cancer.

#1376

Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients.

Yasuhiro Koh,1 Tomoya Kawaguchi,2 Masaru Watanabe,1 Shun-ichi Isa,3 Masahiko Ando,4 Akihiro Tamiya,5 Akihito Kubo,6 Hideo Saka,7 Sadanori Takeo,8 Hirofumi Adachi,9 Tsutomu Tagawa,10 Seiichi Kakegawa,11 Motohiro Yamashita,12 Kazuhiko Kataoka,13 Yukito Ichinose,14 Yukiyasu Takeuchi,15 Kazuhiro Sakamoto,16 Akihide Matsumura5. 1 _Wakayama Medical University, Wakayama, Japan;_ 2 _Osaka City University, Osaka, Japan;_ 3 _Kinki-chuo Chest Medical Center, Osaka, Japan;_ 4 _Nagoya University Hospital, Nagoya, Japan;_ 5 _Kinki-chuo Chest Medical Center, Sakai, Japan;_ 6 _Aichi Medical University School of Medicine, Aichi, Japan;_ 7 _Nagoya Medical Center, Nagoya, Japan;_ 8 _Kyushu Medical Center, Fukuoka, Japan;_ 9 _Hokkaido Cancer Center, Wakayama, Japan;_ 10 _Nagasaki Medical Center, Nagasaki, Japan;_ 11 _Nishigunma National Hospital, Gunma, Japan;_ 12 _Shikoku Cancer Center, Matsuyama, Japan;_ 13 _Iwakuni Clinical Center, Yamaguchi, Japan;_ 14 _Kyushu Cancer Center, Fukuoka, Japan;_ 15 _Toneyama National Hospital, Osaka, Japan;_ 16 _Yokohama Medical Center, Yokohama, Japan_.

BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction.

METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated.

RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency (<0.1%) was also detected in the same reaction. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for exon19 deletion, and 0.9959 for T790M, respectively.

CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation.

#1377

Impact of biospecimen pre-analytical factors on molecular analysis.

Rachana Agarwal,1 Ping Guan,2 Mary Barcus,1 Jasmin Bavarva,1 Robin Burges,1 Philip Branton,2 Latarsha Carithers,2 Corinne Camalier,3 Biswajit Das,3 Jason Lih,3 Hana Odeh,2 Nancy Roche,1 Dan Rohrer,4 Michael Sachs,2 Leslie Sobin,1 Jewell Scott,4 Anna Smith,1 Conrado Soria,1 Kimberly Valentino,1 Dana Valley,4 Mickey Williams,3 Helen Moore2. 1 _Biospecimen Research Group, Clinical Research Directorate, Leidos Biomedical Research Inc., Rockville, MD;_ 2 _Biorepositories and Biospecimen Research Branch, Cancer Diagnosis Program, National Cancer Institute, Bethesda, MD;_ 3 _Leidos Biomedical Research Inc., Rockville, MD;_ 4 _Van Andel Institute, Grand Rapids, MI_.

The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens.

Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected.

The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification.

The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF).

Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens.

This project is funded by NCI Contract No. HHSN261200800001E.

#1378

Generating quality control reference standards to evaluate ultrasensitive variant detection.

Marija Debeljak, Lisa Haley, Derek A. Anderson, Emily M. Adams, Masaya Suenaga, Katie Beierl, Ming-Tseh Lin, Michael Goggins, Christopher D. Gocke, James R. Eshleman. _Johns Hopkins University, Baltimore, MD_.

Introduction: Ultrasensitive point mutation detection can facilitate minimal residual disease detection, early detection of cancer, and therapeutic monitoring of cancer patients. However, nearly identical mutant and wildtype molecules exhibit crosstalk with most technologies, thus producing high background levels. To evaluate technologies against each other for accurate ultrasensitive point mutation detection it is crucial to supply laboratories with reference samples of various dilutions.

Methods: We tested a series of sample mixes with digital-droplet PCR and next generation sequencing. Using linearity and accuracy as the gold standard, we empirically introduced two corrections to generate improved quality control reagents. Baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation of variant, while haplotype counting was used to correct technical errors (cell counting and pipetting).

Results: Correlation between the level of dilution and the measured VAF was relatively good (R2=0.80) when measured using ddPCR. However, correcting for the parental VAFs substantially improved the correlation (R2=0.97). In contrast, correlation of the cell count dilution and the measured VAF, as determined by next generation sequencing (AmpliSeq), was initially relatively poor (R2=0.63). However, substantial improvement in correlation was achieved by correcting for the parental VAFs (R2=0.94). In addition, correcting by haplotype counting alone only slightly improved accuracy in both technologies (ddPCR, from R2=0.80 to R2=0.81; AmpliSeq, from R2=0.63 to R2=0.64). Furthermore, correcting for both factors resulted in the best correlation for ddPCR-generated data (R2=0.99); however, introducing both corrections for AmpliSeq data did not prove more beneficial than correcting for parental VAFs alone (both corrections, R2=0.94 compared to parental VAF correction only, R2=0.94).

Conclusions: Validation of ultrasensitive variant detection requires standardized reference samples. These will also permit assessing various ultrasensitive detection technologies against one another.

#1379

Identification of the functional significance of mutations using the novel precision cancer analysis system.

Gabi Tarcic,1 Nir Peled,2 Zohar Barbash,1 Naama Barabash-Katzir,1 Shlomo Yaakobi,1 Naama Barabash-Katzir,1 Hani Nevo,1 Michael Vidne,1 Mariusz Adamek,3 Mordechai R. Kramer,4 Nikolai Goncharenko,5 Yakov Fellig,6 Karen Meir,6 Keith Mostov,7 Yoram Altschuler1. 1 _NovellusDx, Jerusalem, Israel;_ 2 _Tel Aviv University Sackler School of Medicine, Tel Aviv, Israel;_ 3 _Medical University of Silesia, Zabraze, Poland;_ 4 _Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel;_ 5 _IBBL (Integrated BioBank of Luxembourg), Luxembourg, Luxembourg;_ 6 _Hadassah-Hebrew-University-Medical-Center, Jerusalem, Israel;_ 7 _UC San Francisco School of Medicine, San Francisco, CA_.

Mounting evidence indicates that growth of pathologically identical cancers in each individual patient is fueled by different sets of driving mutations. The need to identify these drivers stems from the recognized necessity for tailoring therapy and scheduling future surveillance. This personalized medical approach has been shown to result in better treatment outcomes. We present a novel Precision Cancer Analysis system (PCAS) capable of identifying activated signaling pathways by means of a transfected cell-based fluorescent reporter assay yielding a quantitative output of particular pathway activation levels. Being a functional platform PCAS reveals activated pathways regardless of the type of mutation behind it, i.e. whether it is already a known mutation or a variant of unknown significance (VUS) mutation. 20 cancer patients were sequenced for a panel of 37 genes and analyzed by the PCAS. This system quantifies oncogenic activity in the majority of the oncogenic signaling pathways altered by the patients' mutations through a functional assay and does not rely on prior knowledge of the mutations. The system produces a quantitative output enabling grading the different mutants of the same patient, providing prioritization for better drug selection. In 3 tested genes, 16 different mutations were identified- 4 in EGFR, 4 in PIK3CA and 8 in KRAS. Of these 10 were classified as known mutations for which functional annotation exists, and 6 were VUS. In addition to correctly annotating all known mutations, the PCAS further quantified oncogenic activity in all the VUS tested. Measuring the functional mechanism behind known mutations and VUS provides another layer of critical information to the physician. These results clearly demonstrate the value of a functional assay in accurately identifying the optimal course of treatment, particularly by its ability to add actionable information to VUS. The study produced a comprehensive delineation of the oncogenic activity of each patient's individual mutations demonstrating the ability of the PCAS to 1) accurately deliver comparable actionable information as found by NGS, 2) functionally characterize mutations annotated as VUS, and 3) monitor oncogenic activity of signaling pathways induced by different mutations and mutation-combinations enabling informed treatment decisions.

#1380

Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring.

Christina M. Wood,1 Billy Lau,2 Laura Miotke,1 Hanlee P. Ji,2 Stephanie Greer2. 1 _Division of Oncology, Stanford, CA;_ 2 _Stanford Genome Technology Center, Stanford University, CA_.

We have developed a new molecular assay that utilizes digital PCR to detect and quantify cancer mutations within poor quality and limited quantity samples such as archival tissue DNA and circulating tumor DNA (ctDNA). This digital PCR assay is highly sensitive and is capable of detecting the targeted mutant fraction at an individual DNA molecule resolution. Thus, this assay is ideal for applications where DNA is in low abundance. An example is where DNA is shed from tumors that can be extracted from the plasma fraction of routine blood draws. The resulting circulating DNA (ctDNA) is extremely low in concentration, which makes it a prime candidate for our highly sensitive molecular assay.

Our assay incorporates small amplicon PCR primers and can be configured for nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. Mutation quantitation relies on two DNA primer sets that are identical with the exception of the mutant or wild type specific base at the 3' end of the "detecting" primer sets, to amplify the genomic region of interest. Through the addition of artificial 5' non-complementary tails to our mutant and wild type specific "detection" primers, we are able to consistently differentiate between droplets that contain the mutant or wild type alleles based upon their differential amplicon lengths. The synthetic amplicon extension tails minimize bias within the PCR reaction by allowing our primers to target an identical region of genomic DNA, with the exception of the single nucleotide variant specific base at the 3' end of the detecting primer. The dPCR technology allows the standard PCR reaction to be partitioned into 20,000 independent wells; we can then assay each individual droplet to assess whether the individual DNA molecule partitioned into the droplet is "wild-type" or the target "mutant". The resulting data provides an absolute count of mutant and wild-type templates in a given patient sample.

We have optimized this new molecular assay technology for quantitatively measuring clinically actionable mutations including BRAF V600E, KRAS G12D among others. We have successfully validated the sensitivity, specificity and reproducibility of our assays through controlled cell line DNA mixed dilution samples ranging from 66% mutant to 0.1% mutant in each of our optimized primer sets. Our assay improves the limit of detection to seven DNA molecules containing a mutation among a total number of 7,000 genome equivalents. Because our assay is sensitive down to the single DNA molecule resolution, we have also been able to reduce the amount of clinical sample DNA required to determine the presence of clinically actionable mutations such as those occurring in KRAS or BRAF. We are testing this extremely low cost, highly sensitive diagnostic technology for detecting nearly any cancer mutation from longitudinal samples of ctDNA.

#1381

NGS-based CNV detection sensitivity is dependent upon nucleic acid input quality.

Josh Haimes, James Covino, Namitha Namoj, Elina Baravik, Laura Johnson, Joshua Stahl, Brady P. Culver, Brian Kudlow. _ArcherDX, Boulder, CO_.

Copy number variations (CNV) impact more of the cancer genome than all other mutation types combined. Recent advances in next-generation sequencing (NGS) have enabled simultaneous detection of CNVs and other somatic mutations from FFPE-derived samples, but NGS-based detection of low level CNVs (ie 2-3x) remains challenging. Nucleic acid from FFPE is a common starting material for NGS-based cancer genotyping; however, this material is often of low complexity due to a variety of factors including limited mass amount, excessive fragmentation, or chemical crosslinking. Current practices often measure input mass, or the nanograms of DNA that are added to a reaction, yet it is input complexity, or the amount of nucleic acid available for NGS library generation, that truly dictates the amount of information that can be recovered from a given sample.

Archer™ VariantPlex™ assays are targeted NGS panels that permit simultaneous detection of SNVs, in/dels, and CNVs using Anchored Multiplex PCR (AMP™). Molecular barcoded adapters are ligated to each input molecule prior to any amplification. This permits the unique identification of individual input molecules thus facilitating precise copy number measurements. In addition, AMP enables amplification of highly fragmented FFPE inputs as short fragments are captured between the ligated adapter and the enrichment probe. To determine the effect of input quality on sensitivity of CNV calling we characterized over 150 tumor sample input qualities and their resulting library metrics. In addition we modeled the effect of low tumor cellularity on CNV sensitivity by carrying out dilution experiments of CNV-positive samples into samples of normal copy number.

Using Archer VariantPlex assays in conjunction with Archer Analysis, we have successfully detected CNVs as small as 2X in both FFPE and cell line DNA. We found that input nucleic acid quality, as measured by a qPCR-based assay called Archer PreSeq™ DNA QC, strongly impacted the sensitivity of CNV calling. Assessment of input complexity using the PreSeq DNA QC Assay is predictive of limit of detection for CNVs and identifies an input quantity that will result in high quality NGS libraries. Our dilution experiments confirmed the expected relationship between actual and measured copy number in our population-averaging assay.

Nucleic acid damage typical of FFPE samples reduces CNV calling sensitivity; however, this loss of sensitivity can be partially mitigated by increasing the input quantity. This corroborates the notion that input complexity is the major driver of information-capture from NGS based assays. Finally, tumor cellularity displays a predictable effect on the measured CNV value.

#1382

A complete workflow for high-throughput isolation and analysis of cell-free DNA from urine.

Alex J. Rai,1 Robert A. Setterquist,2 Xingwang Fang,2 Hannah E. Saunders,2 Matthew Carter,2 Charmaine San Jose Hinahon,2 Sarah E. Larocca,2 Susan M. Magdaleno2. 1 _Columbia University Medical Center and New York Presbyterian Hospital, New York, NY;_ 2 _Thermo Fisher Scientific, Austin, TX_.

Circulating cell-free DNA (cfDNA) shed from tumors has gained considerable attention as a source of nucleic acid for testing cancer biomarkers. Critical to finding and implementing a diagnostic biomarker for cfDNA is the consistent and efficient isolation of the nucleic acid from blood. Sample preparation technologies are now commercially available for cfDNA isolation from plasma and serum, making these sample types used for most applications. However, plasma and serum require blood drawn by trained phlebotomist and only limited amounts can be obtained; additionally, individuals with advanced disease usually require routine monitoring and may not be able to spare additional blood drawn for cfDNA testing. Recently, it has been appreciated that urine may also serve as a valuable source for cfDNA. DNA from tissues and organs of the genitourinary system may be shed directly into urine and cfDNA circulating in blood can filter through the glomeruli in the kidneys to end up in urine. Compared to plasma and serum, urine is much easier to obtain, does not require a needle stick, and it can be collected in larger volumes making longitudinal studies more accessible. Urine presents a number of new challenges for the preparation of cfDNA that need to be overcome before this sample source can truly be utilized.

The objective of this project was to develop reagents and workflows optimized for analysis of cfDNA from urine. Through our studies, we found that the slightly shorter cfDNA in urine requires optimized chemistry to maximize yield, a larger volume of urine may be necessary to isolate sufficient cfDNA compared to plasma/serum and urine must be treated with stabilization agents to minimize further degradation of cfDNA after collection. Using the MagMAX™ cell-free DNA isolation kit, we have developed a magnetic bead-based sample preparation protocol specific for isolating cfDNA from urine. Workflows for preparing cfDNA from urine through manual processing or by automated high throughput sample processing on the KingFisher™ instruments were developed. A small cohort of healthy donors was used to demonstrate compatibility of the cfDNA with qPCR, dPCR and next generation sequencing platforms. The effectiveness of this fast and easy workflow will be further tested on cfDNA from urine samples from donors with and without metastatic disease. We will analyze cfDNA from paired urine and plasma to understand the applicability to different tumor types.

#1383

NGS-based measurement of gene expression of 2560 oncology-related biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissues.

Monica M. Reinholz, Debrah M. Thompson, Ihab Botros, Matt Rounseville, Patrick C. Roche. _HTG Molecular Diagnostics, Inc, Tucson, AZ_.

Background: HTG Molecular (HTG) developed a targeted Next Generation Sequencing (NGS)-based gene expression assay that measures mRNA levels of 2,560 genes (2,532 oncology-related biomarker genes). The HTG EdgeSeq Oncology Biomarker Panel Assay (OBP) is based on a novel derivative of our quantitative nuclease protection chemistry (qNPA) that enables extraction-free quantitation (detection) of mRNA from variety of sample types including formalin-fixed, paraffin embedded (FFPE) tissue.

Purpose: To determine (1) linearity across a range of sample inputs, (2) recommended sample input amounts for FFPE, cells, and extracted RNA, and (3) reproducibility of the HTG EdgeSeq Oncology Biomarker Panel Assay in measuring the mRNA expression of 2,560 genes.

Methods: Lysates of 5 micron sections of FFPE tissues (lung, breast, prostate, and colon carcinoma; melanoma; 25 mm2 to 0.78 mm2 per reaction), THP-1 and HCC78 cell lines (7500 cells to 234 cells per reaction), and Universal RNA (URNA; 25 ng to 1.56 ng per reaction) were used for the linearity and sample input studies. URNA (25 ng per reaction) was used to demonstrate reproducibility of the assay across multiple days and processors (one processor across three days and three processors on one day). Sequencing libraries were generated from the qNPA reactions and run on an Illumina MiSeq sequencing platform. The HTG EdgeSeq Parser was used for post-sequencing data processing. Linear regression (R2) and Pearson correlation coefficients (r) were used to assess linearity and reproducibility of the assay.

Results: The R2 for linearity across four concentration points for lung FFPE tissue (6.25-0.78 mm2), cell lines (1875-234 cells), and URNA (12.5-1.56 ng) were >0.97, 0.99, and 0.99, respectively. The (r) between low (1.56 mm2) and high (12.5 mm2) sample inputs for each FFPE tissue type was > 0.98. The (r) for intra-run, inter-day, and inter-run reproducibility were > 0.95, > 0.98, and > 0.98 respectively. In addition, differential expression of tissue-specific genes was identified in the respective FFPE tissues, including NKX2 and MUC1 in lung, ERBB2 in breast, NKX3, KLK2, and KLK3 in prostate, and SPP1 and PRAME in melanoma.

Conclusions: The HTG EdgeSeq Oncology Biomarker Panel Assay for a 2,560-gene panel of oncology-related biomarkers is linear over a wide range of sample inputs, can comprehensively analyze very small, clinically relevant tissues, and is highly reproducible. The demonstrated performance of the assay in breast, lung, colon, and prostate cancer and melanoma FFPE samples enables multiplex oncology biomarker profiling of these and other malignant neoplasms.

#1384

A comprehensive gene panel for precise diagnosis and treatment of childhood cancer.

Timothy J. Triche, Jackie Biegel, Alex Judkins, Tracy Busse, Alan Wayne, Deepa Bhojwani, Shahab Asgharzadeh, Matthew Oberley, David Parham. _USC/Children's Hospital Los Angeles, Los Angeles, CA_.

We have created and are validating a targeted gene panel that utilizes 20ng of FFPE DNA and 10 ng of FFPE RNA in a simple, 3-tube Ion AmpliSeq assay suitable for sequencing on the Ion Torrent platform with rapid turn around time (<3 days in lab) and automated report generation that is designed specifically to improve pediatric cancer diagnosis, identify prognostic features, and detect genomic alterations (including mutations, insertions, deletions, gene amplification, and RNA expression levels) that match current targeted therapeutic targets. The core content is identical to the Oncomine Comprehensive Assay as employed by the NCI MATCH program. In addition, a total of 128 genes known to be genomically altered in pediatric cancer are interrogated (either hotspots or coding exons), 21 genes frequently amplified in pediatric cancer, and 157 gene fusions (including over 1,500 variants) unique to these tumors are included. Unique to this panel are 22 pharmacogenomic gene polymorphisms that influence cytotoxic drug metabolism, to guide patient-specific dosing. Content was chosen to cover all common pediatric malignancies, including pediatric leukemia (ALL, AML, and multiple variants within each), lymphoma, brain tumors, neuroblastoma, and sarcomas, by a working group composed of pediatric oncologists and pathologists. Specific genomic alterations were identified from a comprehensive search of the pediatric cancer literature by the panel and categorized by level of evidence (FDA approved drug, open clinical trial, or peer-reviewed literature identifying a feature in a pediatric cancer). This panel includes 45 of the 51 gene targets identified by the COG TAP committee for pediatric cancer specific gene target-therapeutic agent matching. In contrast to the NCI MATCH program, which currently employs the Ion Torrent Personal Genome Machine and Ion 318 Chip, this panel is intended for use on the newly available Ion S5 platform, due to its significantly enlarged content. Verification of individual feature identification by orthogonal assays (Sanger sequencing, PCR, FISH, SNP arrays) is in process. The assay will be released initially as an LDP, with intended updated content as new features and therapeutics are identified. Unlike current panels for specific features like single gene mutations or known gene translocations, this panel is intended to detect all currently known genomic alterations with level I, II, or III evidence in pediatric cancer, as well as the ability to detect new gene mutations and fusion variants (subject to secondary assay validation). Also unique is the ability to measure gene amplification and RNA expression levels in one comprehensive panel starting with 30 ng or less of nucleic acid extracted from routine clinical FFPE specimens. This is the only comprehensive gene panel for pediatric cancer that encompasses diagnostic, prognostic, pharmacogenomic, and therapeutic targets in one assay.

#1385

Patient-derived xenografts as validation material for NGS assays: Analysis of genetic homogeneity.

Yves Konigshofer,1 Bharathi Anekella,1 Russell Garlick2. 1 _SeraCare Life Sciences, Inc., Gaithersburg, MD;_ 2 _SeraCare Life Sciences, Inc., Milford, MA_.

Full process controls and validation materials for next generation sequencing (NGS)-based molecular diagnostics in Oncology that mimic a biopsy sample and are renewable and scalable have been elusive. Patient-derived Xenografts (PDXs), which are similar in morphology and somatic mutations to original tumors, may present a solution to this pressing need.

In order to evaluate the abilities of PDXs to serve as full process controls and validation materials, we had PDXs that contained established driver mutations KRAS G12C analyzed by clinical laboratories using a variety of different NGS-based assays. The resulting data was analyzed for genetic homogeneity as well as for the presence of mouse-derived variants.

PDXs were found to be generally compatible with NGS-based assays and similar variant frequencies of driver mutations were reported. High concordance was also observed in variant frequencies of PDXs at passage 5 that were derived from the same tumor but grown in different mice. For example, the observed differences in over 90 % of variants and all driver mutations were not statistically significant.

In amplicon-based assays, which represent the majority of current assays that look at mutational hotspots, mouse-derived variants were rare. Typically, variant data was usable without any further processing. In hybrid/capture-based assays, mouse-derived variants were common because of the lower binding stringency used by these assays. Without further processing, this manifested in slightly lowered variant frequencies for driver mutations - where wild-type "reference" sequences are often conserved between species and thus inflate the number of reference sequences - and greatly elevated numbers of total variants. However, by processing the data at the FASTQ stage using in-house developed software, it was possible to effectively remove mouse-derived variants in a manner that would be compatible with patient testing as well.

In conclusion, we show that PDXs are compatible with NGS-based assays and can be used to control the full process from the point where the Pathologist first fixes the tissue. Those containing driver mutations that have been previously characterized in cancer also enable laboratories to control and validate their assays by showing that they can consistently detect these mutations.

#1386

Clinical research results for a NGS-based kit for targeted detection of clinically relevant gene rearrangements in lung tumor samples.

Jeoffrey J. Schageman,1 José Luis Costa,2 Orla Sheils,3 John E. Glassco,4 David Chi,4 Jon Sherlock,5 John Bishop,6 Rosella P. Petraroli,7 Kelli S. Bramlett1. 1 _Thermo Fisher Scientific, Austin, TX;_ 2 _ipatimup, Porto, Portugal;_ 3 _Trinity College Dublin, Austin, Ireland;_ 4 _Life Technologies Clinical Services Lab, West Sacramento, CA;_ 5 _Thermo Fisher Scientific, London, United Kingdom;_ 6 _Thermo Fisher Scientific, Carlsbad, CA;_ 7 _Thermo Fisher Scientific, Milan, Italy_.

In recent years, advances in next-generation sequencing (NGS) technologies have enabled faster and cheaper methods for uncovering the genetic basis of disease. For cancer, NGS based screening for known tumor subtypes may inform diagnosis and allow the clinician to tailor a specific therapeutic approach in the future. Here, we present the testing results of one such NGS based kit used to detect specific chromosomal translocations in retrospective non-small cell lung cancer (NSCLC) samples by targeting specific breakpoints in known fusion transcripts.

The included panel tested consists of a single primer pool containing amplicon designs to simultaneously screen for over 75 specific rearrangements involving the receptor tyrosine kinase (RTK) genes ALK, RET and ROS1 as well as NTRK1. The panel was compatible with formalin-fixed paraffin-embedded (FFPE) lung tumor research samples and achieved high-sensitivity down to 10 ng of RNA input. In addition, amplicon assays designed at the 5' and 3' ends the RTK genes provide non-specific evidence that a translocation exists in a sample by comparing expression imbalance between the two ends.

Testing was carried out at three external clinical research laboratories. In addition to positive and negative control samples, each site contributed FFPE lung tumor research samples for which ALK fusion status was known prior to NGS library preparation carried out using the Ion AmpliSeq™ workflow. For site-specific samples (n=144, 16 samples per sequencing run), high concordance, sensitivity and specificity were measured at 97.2%, 90.5% and 98.4%, respectively.

#1387

Application of target enrichment combined with unique molecular identifiers to determine allelic frequencies of human cancers.

Daniel C. Kraushaar,1 Sarah K. Bowman,1 Theodore B. Davis,2 Amy B. Emerman,1 Noa Henig,1 Kruti M. Patel,1 Salvatore Russello,2 Cynthia L. Hendrickson1. 1 _Directed Genomics, Ipswich, MA;_ 2 _New England Biolabs, Ipswich, MA_.

Existing target enrichment and library preparation for next-generation sequencing typically utilize PCR amplification steps that introduce substantial bias and sequencing of duplicate reads, which in turn affect the quantification accuracy of somatic allele frequencies. Unique molecular identifiers (UMIs) are random molecular tags that uniquely label each molecule prior to library amplification and aid in the identification of true PCR duplicates and thereby improve the accuracy in estimating the relative molecular copy number of the original sample. Here, we applied a target enrichment method for hybridization-based capture of regions of interest, combined with unique molecule labeling and next-generation sequencing to determine allelic frequencies of select cancer targets. Target enrichment and library preparation can be performed in just under seven hours, and our UMI method is compatible with sample indexing using separate barcodes. To demonstrate the robustness and accuracy of our method, we present the detection and assessment of allelic frequencies of multiple cancer genes across multiple reference standards. The result is a fast and cost-efficient method that enables the determination of even low allelic frequencies with high confidence.

#1388

Multi-template analysis in metastatic breast cancer blood samples.

William M. Strauss,1 Chris Carter,1 Erich Klem,1 Jill Simmons,1 Keerthi Gogineni,2 Ruth O'Regan,3 Laura Austin,4 Paul W. Dempsey,1 Massimo Cristofanilli5. 1 _Cynvenio Biosystems, Inc., Westlake Village, CA;_ 2 _Emory University School of Medicine, Atlanta, GA;_ 3 _University of Wisconsin, Madison, WI;_ 4 _Thomas Jefferson University, Philadelphia, PA;_ 5 _Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL_.

Introduction

The presence of Circulating Tumor Cells (CTC) has been observed in advanced cancer patients and studies have indicated that these cells contribute to the process of metastasis. Historically CTCs with metastatic potential have been characterized as cells that are EpCAM positive, Cytokeratin positive (CK+), and CD45 negative. We report the results of a prospective study challenging these historical definitions of circulating tumor cells.

Description

In a prospective study, patients with metastatic breast cancer there were about to start a new systemic therapy were enrolled The samples and representative tissue were collected at baseline. The patients were predominantly female (97%) and all had stage IV disease. From these whole blood samples a LiquidBiopsy® was performed using the "MultiTemplate" format which directly compares CTC, circulating cell free (CCF), and WBC patient-matched DNA in a NGS based resequencing test. Of 22 patients, 18 biopsy samples were successfully evaluated; the balance had insufficient tissue or DNA for analysis.

Summary

The LiquidBiopsy compared two phenotypic definitions for CTC capture. Tumor cell populations were enriched using epithelial targeted capture and compared to a novel cocktail of antibodies. The cocktail of reagents gave 77% average recovery of engineered samples when a target receptor was present on cell lines representing the spectrum of breast cancer subtypes. This enrichment protocol was applied to serial patient samples. Epithelial based enrichment served as a control and showed an average recovered purity of 10.7% CK+ cells. By comparison, cocktail selection enriched populations with on average 8.8% CK+ cell populations but a larger range of cells than epithelial selection. Importantly, the median number of CK+ cells recovered from cocktail capture was almost twice that of epithelial capture alone. (median of 35.8 vs 22.1 for cocktail and epithelial respectively). The median CD45+ non-target cells background was 80 and 155 cells respectively. This background supports sequencing detection of mutations present at >1%. ccfDNA sample was purified from the same tube of blood. The average concentration of patient matched ccfDNA recovered was 7.3 ng/mL. The combined results of ctcDNA and ccfDNA template sequencing gave productive samples showed similar detection frequency (55% vs 58%), were temporally flexible, and were complementary both to each other and the "gold standard" (FFPE).

Conclusions

The data from this prospective clinical study reveals subsets of CTC, including an important cohort that is not detected using the standard definition for epithelial CTCs. Furthermore, we present evidence for the successful use of a "Multi-Template" approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and justifies the redefinition of CTC phenotype based upon cell surface biomarkers and mutation content.

#1389

A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer.

Gary J. Latham,1 Julie Krosting,1 Michael Dodge,1 Robert Zeigler,1 Liangjing Chen,1 Jason Plyler,1 Shobha Gokul,1 Junya Fujimoto,2 Vassiliki Papadimitrakopoulou,2 Ignacio Wistuba,2 Richard Blidner,1 Brian Haynes1. 1 _Asuragen, Austin, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction: The promise of precision medicine relies on the identification of DNA and RNA markers that can individualize patient management. Methods such as next-generation sequencing (NGS) can deduce DNA or RNA sequences, but both types of nucleic acid have not been efficiently and effectively combined into a single NGS workflow. We describe a comprehensive methodology for targeted clinical NGS that reports DNA and RNA variants, provides a streamlined workflow, and accommodates low-input total nucleic acid (TNA) from challenging clinical specimens.

Methods: Sample QC was performed using a novel qPCR assay that quantifies discrete populations of amplifiable DNA and RNA from TNA material. PCR-based target enrichment was performed using QuantideX® NGS reagents (Asuragen) and sequenced on the MiSeq® System (Illumina). Bioinformatic analyses were conducted using QuantideX® Reporter (Asuragen), a software suite that directly incorporates pre-analytical QC information into the variant calling.

Results: Targeted DNA- and RNA-seq panels were developed that query 54 lung cancer DNA targets and 135 RNA targets, including >100 gene fusions and mRNA expression markers associated with clinical actionability. Gene-specific primers were formulated as multiplex PCR or RT-PCR reactions to independently interrogate DNA or RNA variants, respectively, from TNA or combined into a 189-plex RT-PCR to report multi-categorical nucleic acid variants. Integration of the bioinformatics pipeline and variant caller with wet-lab QC results enhanced mutation call sensitivity at <10% abundance, improved PPV for low-input specimens, and achieved absolute quantification of RNA.

The NGS panels were assessed with cell-line and synthetic controls and a residual clinical cohort of 97 NSCLC FFPE specimens. Mutations were accurately detected from as few as 5-10 DNA templates and down to <5% abundance, and RNA translocations could be observed in sub-nanogram quantities of fusion-positive FFPE TNA. The unified DNA/RNA panel identified clinically-relevant DNA and RNA variants and maintained similar coverage uniformity to the separate DNA and RNA panels. Analysis of 61 lung adenocarcinoma and 36 squamous cell carcinoma specimens revealed mutation distributions in driver genes and RNA fusions consistent with the known spectrum of variants from each tumor type as previously reported by TCGA and other groups.

Conclusions: QuantideX targeted NGS chemistries can unify the analysis of DNA and RNA markers associated with lung cancer and report SNVs, indels, fusions, and aberrantly expressed mRNA transcripts from a single NGS run. This novel technology offers reliable, accurate and comprehensive molecular characterizations of challenging tumor specimens by integrating wet-bench and dry-bench methods and improving the efficacy of routine laboratory testing.

#1390

**High sensitivity detection of** EGFR **exon 20 T790M and C797S mutations using ICE COLD-PCR.**

Grant Wu, Sheena Jensen, Karissa Scott, Erin Montagne, Jason Stoddard, Phil Krzycki, Courtney Schweikart, Benjamin L. Legendre, Philip Eastlake, Katherine A. Richardson. _Transgenomic, Inc., Omaha, NE_.

Introduction: The EGFR Exon 20 T790M mutation confers resistance to the first generation tyrosine kinase inhibitors (TKI), such as Tarceva®. A new generation of treatments, including AZD9291, targets the T790M mutation thus allowing a new approach to cancer therapy for NSCLC patients. Recently EGFR Exon 20 C797S mutations (c.2389T>A and c.2390G>C) have been identified as resistance mutations to the third generation TKI AZD9291. High sensitivity detection of these mutations, as well as the T790M mutation, in patients with acquired resistance to third generation TKI is an important requirement for best practice NSCLC treatment.

Materials and Methods: ICE COLD-PCR (Improved & Complete Enrichment CO-amplification at Lower Denaturation temperature PCR) is a technology that preferentially enriches mutant DNA sequences in an excess of wild-type DNA through selective amplification of the mutant DNA population using an oligonucleotide (RS-oligo) complementary to the wild-type sequence. Due to the inherent sequence characteristics of the region between the codons of interest (790 and 797), a RS-oligo focusing on the C797S was developed and is independent of the RS-oligo for T790M, and thus tiling across EGFR Exon 20 can be performed. The RS-oligo in this assay was designed to prevent PCR amplification of wild-type sequences while allowing exponential amplification of any mutations including C797S within the RS-oligo binding region. Serial dilutions of spiked gBlocks® DNA containing C797S mutations mixed with K562 DNA and samples extracted from plasma were amplified with ICE COLD-PCR followed by Sanger sequencing analysis.

Result: For mutations c.2389T>A and c.2390G>C, limits of detection as low as 0.05% were achieved. Forty DNA samples extracted from plasma were also tested. These indicated that the assay is suitable for fragmented samples with low quantities of input DNA such as liquid biopsies.

Conclusion: The pre-amplification PCR product covers both codons 790 and 797. This allows ICE COLD-PCR to be used for the analysis of both T790M and C797S using the same liquid biopsy sample aliquot. This new ICE COLD-PCR method to detect both C797S mutations c.2389T>A and c.2390G>C may provide a highly sensitive, simple and cost effective assay for monitoring acquired resistance to AZD9291 in the liquid biopsies obtained from patients and clinical trials. Coupled with our existing highly sensitive assay for T790M, ICE COLD-PCR offers a highly sensitive method to determine the emergence of resistance mutations, thus potentially contributing to therapeutic optimization.

#1391

Gene expression profiling for cancer classification in circulating tumor cells.

Harris S. Soifer,1 Ranelle C. Salunga,1 Tristan G. Harris,1 Jose Ramirez,1 Priyadarshini Gogoi,2 Yixin Wang,2 Saedeh Sepehri,2 Kalyan Handique,2 Catherine A. Schnabel1. 1 _bioTheranostics, San Diego, CA;_ 2 _Celsee Diagnostics, Plymouth, MI_.

Background: Enumeration and molecular characterization of circulating tumor cells (CTCs) offer a non-invasive method for tumor analysis, particularly in cases where the biopsy is difficult to obtain or has been exhausted by conventional diagnostic methods. The 92-gene assay (CancerTYPE ID®, bioTheranostics, Inc.) is a clinically-validated cancer classifier based on the collective expression of 87 informative genes, many of which are involved in lineage commitment and signal transduction. The objective of this study was to determine the feasibility of a blood-based application using differential gene expression.

Methods: Technical feasibility studies for cancer cell enrichment and RNA viability were conducted by purifying cancer cell lines that were spiked into donor blood using a microfluidic chip (Celsee PREP™100 system, Celsee Diagnostics) followed by immunodepletion of leukocytes. Quantitative RT-PCR and immunoflourescence were performed on cells harvested at different steps of the purification workflow. Quantitative RT-PCR on individual genes specific to each lineage compartment was used to estimate cell types and cell numbers that were co-purified by the workflow. Gene expression was evaluated by the 92-gene assay.

Results: An integrated workflow was established that allows for the molecular characterization of cancer cells purified from blood. Cell enrichment on the microfluidic chip followed by purification using immunodepletion resulted in efficient recovery (> 80% of input cells) and viable RNA from cancer cells spiked into whole blood. A key result of this study was the demonstration that the level of cancer-specific gene expression in purified samples was similar to the level of expression from an equivalent number of unspiked cancer cells suggests that significant cell loss does not occur during the purification workflow. A comparison of gene expression profiles from purified and mixed cell populations allowed for the identification of subsets of genes that are differentially expressed in cancer cells compared with leukocytes.

Conclusion: A functional workflow with viable RNA and efficient recovery of cancer cells that are within the technical specifications of the 92-gene assay was established. Findings from this feasibility study are fundamental to the potential for CTC-based cancer classification.

#1392

Development of highly multiplexed, whole process reference materials for monitoring oncology RNA fusions.

Catherine E. Huang, Yves Konigshofer, Lequan Nguyen, Bharathi Anekella. _SeraCare Life Sciences, Gaithersburg, MD_.

Introduction: Genomic structural alterations are increasingly actionable for targeted therapeutics and personalized medicine. Next-generation sequencing methods are increasingly used to detect these fusion RNAs in FFPE material. However, quality control materials for these assays are lacking, and validation materials for rare fusions are frequently not available. We developed a novel FFPE Fusion RNA Reference Material to fill this unmet need.

Methods: Highly multiplexed RNAs were designed which contain multiple fusion targets observed predominantly in solid tumors including ALK, RET, ROS1 FGFR3, and NTRK1 fusions as well as PAX-PPARG fusion and ETV6-NTRK3 fusion. These RNAs were transcribed in vitro and contained a 5' guanosine cap and a poly-adenosine tail to improve intracellular stability. The RNA was introduced into GM24385 reference cell line (The 1000 Genomes Project, Coriell). After recovery from transfection, the cells were collected and fixed in formalin. Digital PCR with TaqMan chemistry was used to determine the average number of synthetic RNA transcripts per cell. These transfected cells were then mixed with non-transfected GM24385 cells to achieve a consistent "low positive", formulation. The cell mixture was embedded in a paraffin block and 10 micron sections were produced. Quality control was performed by extracting RNA and testing using the ArcherDx FusionPlex™ Lung Thyroid Panel and the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel.

Results: All twelve (12) fusions present in the prototype were detected as high confidence calls on the ArcherDx Lung Thyroid panel. Whereas embedded cell lines (with genomic mutations) give extremely high positive results (typical results are thousands of reads across the fusion junction), Seraseq™ FFPE Fusion RNA Reference material gave low positive results, similar to patient samples. The reads spanning the fusion junction ranged from 38 to 396. The Ion AmpliSeq™ RNA Fusion Lung Cancer panel assays only 6 of the fusions in the Seraseq reference material due to their assay design; however, all assayed fusions were appropriately detected.

Conclusions: Highly multiplexed reference materials in FFPE format are needed for quality control of NGS-based detection of oncology RNA fusions. Seraseq FFPE RNA Fusion Reference Material allows simultaneous evaluation of detection for twelve fusions observed in a variety of solid tumors, both common and rare. It generates low positive results on two leading assays, and this is important to truly challenge the assay system and verify performance at levels expected for patient samples.

#1393

Developing a next-generation noninvasive clinical test for non-small cell lung cancer.

Christopher Kasbek, Yang Song, Danielle Quintanilha, Si Chen, QingXuan Song, Tianjiao Wang, Jun T. Huang. _Admera Health LLC, South Plainfield, NJ_.

Significance: Non-Small Cell Lung Cancer (NSCLC) is responsible for the largest number of cancer-related fatalities, accounting for 22.8% of all cancer deaths in the United States in 2015. Targeted therapy precisely treats mutated cancer pathways and has significantly higher response rates compared to chemotherapy. However, tumor heterogeneity is prevalent in NSCLC patients, and the common practice of detecting mutations using tissue biopsy from a fraction of a single tumor can be less than satisfactory. Failure to detect molecular resistance emerging from heterogenic tumor cell population is a major reason that, even though highly effective, targeted therapy has not contributed greatly to prolong patient survival. We aim to develop a cost-effective, highly sensitive and specific liquid biopsy for advanced stage NSCLC patients that is able to capture a comprehensive tumor profile and monitor tumor response to therapy. Innovation: BEST (Blocker-based Enrichment System for Tumor DNA) blocks primer extension of over 90% of wild type DNA and allows preferential amplification of the mutant allele. BEST NSCLC liquid biopsy is highly sensitive (limit of detection of >=0.005%), comprehensive (detects 56 mutations in the EGFR, KRAS, BRAF, ALK, and ROS1 oncogenes), and rapid (estimated 4-day turnaround time). BEST is comprised of a multiplex of circulating tumor DNA enrichment reaction and a multiplex plasma cDNA enrichment reaction. Our BEST NSCLC liquid biopsy panel enriches >100 fold for the above driver mutations. The ability to detect a myriad of mutations in heterogenic tumor cell populations allows dynamic and personalized treatment during the course of the disease. Summary: Our primary focus is to offer a test to detect actionable mutations and subsequently monitor tumor dynamics and the rise of molecular resistance. Our liquid biopsy will be also ideal for patients in follow-up visits in conjunction with the typical X-ray or CT image scans. Our test is also suitable for vulnerable patients from whom a solid biopsy cannot be obtained due to illness or inaccessibility.

#1394

RT-PCR and NGS comparison: detection of known EGFR mutations in non-small-cell lung cancer clinical samples with routine laboratory testing.

John F. Palma,1 Yu Chuan Tai,1 Wei-min Liu,1 Steve Anderson,2 Anup Madan,2 John W. Longshore,3 Arundhati Rao4. 1 _Roche, Pleasanton, CA;_ 2 _LabCorp, Seattle, WA;_ 3 _Carolinas Healthcare, Charlotte, NC;_ 4 _Baylor Scott & White, Temple, TX_.

Next-generation sequencing (NGS) promises to deliver clinical mutation analysis for multiple actionable biomarkers at once. However, most targeted therapies are developed with companion diagnostic assays after rigorous analytical and clinical validation. In order to compare the consistency of biomarker results under standard laboratory workflow, we measured the detection of known EGFR mutations in 17 clinical and 2 reference samples using the cobas® EGFR Mutation Test version 1(cobas test), the TruSeq and TruSight panels using the Illumina MiSeq and AmpliSeq panel using the Thermo Fisher PGM instruments. Samples were tested in triplicate at 2 sites for each platform with 2 sites performing cobas testing and sequencing. Archived clinical samples were NSCLC adenocarcinoma with average tumor cell content at 60.3% ± 21.0% and mutant allele frequency of 36.0% ± 17.0% based on pyrosequencing during initial clinical testing. Three samples had scar tissue present and the remainder had little to no necrosis. The average age was 67.6 ± 7.0 years and samples came from 10 women, 6 men and 1 gender not specified. Data were analyzed to assess the inter- and intra- platform discordant rates, the mean error rates relative to the known mutation status, platform invalid rates, average turnaround time and repeat testing rates. All platforms had low discordant and mean error rates when using the 2.5% mutation frequency cutoff and a read depth (RD) limit = 100. One PGM site had a sample invalid rate between 20%-51% for different EGFR amplicons at RD= 100; the upper limit of the invalid rate for PGM sites was >70% with RD = 500. The cobas z 480 platform had a 0-0.32% intra-platform discordant rate and a 0.65-0.93% mean error rate. The MiSeq platform had a 0-0.69% intra-platform discordant rate and a 0-0.69% mean error rate. The PGM platform had a 0.99-3.15% intra-platform discordant rate and a 1.5-4.02% mean error rate. The mean numbers of all non-silent COSMIC mutations detected by PGM and MiSeq at the 2.5% cutoff were 4.36 and 2.32, respectively. However, the mean number of EGFR mutations detected relative to the expected number was 100% for MiSeq and 92.4% and 100% for PGM at 2.5% and 10% frequency cutoffs, respectively. The 3 platforms were assessed for samples that required repeat testing, re-extraction of DNA and the turnaround time (TAT) from DNA isolation to result. In all three cases, the cobas test required the least TAT, fewer repeats and re-extractions relative to PGM and MiSeq. One MiSeq site required the longest turnaround time (14 days) but did not require repeat testing for invalid runs. Re-extraction from backup sections was common for both PGM and MiSeq but not cobas.

The cobas test provides accurate, precise, and fast actionable results for NSCLC patients. NGS approaches can result in accurate and precise results when adequate RD is achieved but TAT remains a challenge relative to the cobas test.

#1395

Development and validation of NGS-based clinical cancer panels for precision medicine in Asian and Caucasian adult cancers.

Winston Patrick Kuo, Amy Wang, Tak Cheng, Pan Du, Shidong Jia. _Predicine, Inc, Palo Alto, CA_.

In the new era of precision medicine, comprehensive cancer panels are needed to identify actionable cancer genes and match patients to personalized therapies. Here, we report a Pan-Cancer NGS Panel designed by leaders in the field that comprises of all exon coding and selected hotspots of critical cancer genes in the most common cancer types for both Asian and Caucasian cohorts, including but not limited to lung, colon, breast and prostate cancer. The panel allows detection of known and novel variants in indication-specific signaling pathways, disease biology, DNA repair, and in particular drug resistance, to advance research into personalized cancer treatment and for use in clinical trials to help the development of new targeted therapies. Technical validation was performed to characterize point mutations, indels, copy number variations and fusions across clinically relevant, actionable cancer genes in both liquid biopsies and formalin-fixed paraffin-embedded (FFPE) clinical specimens. Assay sensitivity, specificity and accuracy were tested using reference samples with known genetic profiling and further validated on a variety of orthogonal platforms such as digital PCR, allele-specific PCR and Sanger sequencing. To the best of our knowledge, this is the first ethnic-specific NGS diagnostic test designed specifically for Asian and Caucasian adult cancer patients.

#1396

Validation of a MEK/MET-specific NGS panel for early phase trial interrogation.

Perry Maxwell,1 Jurgen Del Favero,2 Marc-Aurel Fuchs,1 Josep Tabernero,3 Tim Maughan,4 Mark Middleton,4 Richard Addams,5 Christian Rolfo,6 Brian Hennessey,7 Pierre Laurent-Puig,8 Alberto Bardelli,9 Thierry Andre,10 Vlad Popovici,11 Patrick Johnstone,1 Richard Wilson,1 Mark Lawler,1 Sandra Van Schaeybroeck,1 Manuel Salto Tellez1. 1 _Queen's University Belfast, Belfast, United Kingdom;_ 2 _Multiplicom N.V., Niel, Belgium;_ 3 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 4 _University of Oxford Department of Oncology, Oxford, United Kingdom;_ 5 _Velindre Cancer Centre, Cardiff, United Kingdom;_ 6 _University Institution Antwerp, Antwerp, Belgium;_ 7 _Royal College of Surgeons , Ireland, Dublin, Ireland;_ 8 _Paris Descartes University Medical School, Paris, France;_ 9 _Institute for Cancer Research and Treatment, Turino, Italy;_ 10 _Saint-Antoine Hospital, Paris, France;_ 11 _Masaryk University, Masaryk, Czech Republic_.

Introduction - MErCuRIC is a Phase Ib/II clinical trial study using a combined MEK1/2 - cMET inhibition in RAS MT and RAS WT (with aberrant c‐MET) colorectal cancer patients. As part of the discovery efforts on the cases enrolled in Phase I, we aimed to analyze the mutation status of 10 genes that could potentially be associated to the doublet MEK/MET inhibition. This study compared the MEK/MET-specific panel with the Ion Torrent 50 gene panel, aiming to compare: long-established, commercially available panels against this newly developed panel; the Ion Torrent PGM2 platform against Illumina MiSeq v.3 600 bp chemistry; hot-spot-based against full exomes-designed; and to compare the use of different bioinformatics reporter systems. The overlapping genes between the panels were: EGFR (n=3); BRAF (n=4); KRAS (n=8); NRAS (n=1); MET (n=8); PIK3CA (n=6); and ERBB2 (n=5).

Summary of Method

NGS Design - The Multiplicom - MErCuRIC specific MASTR assay includes PTEN, MAP2K1 (MEK), EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET and PIK3R1, involving 257 amplicons in 4 plexes covering all coding exons of the 10 genes. Of the 257, 21 are control amplicons to allow for gene deletions/amplifications. The average length of the amplicons is 198 bp ranging from 124 bp to 255 bp.

Validation - From a pool of 120 routine tumour samples characterised with a 50 gene mutation panel (Ion Torrent PGM2) and confirmed by Sanger sequencing and/or COBAS (Roche) QPCR, 24 FFPE cases were selected representing colorectal cancer and 4 other solid tumour types; all included a variety of DNA quality, and DNA concentration was standardized prior to library preparation. Pre-analytical handling was in accordance with established protocols in a laboratory, clinically-accredited in the UK for tissue-based, anatomical pathology testing.

Results

The evaluation of this MEK/MET-specific panel (Illumina MiSeq platform) resulted in 100% coverage of all targets, a higher than 97% on target reads and higher than 99% of all amplicons within 20% of mean coverage. The design minimized the areas of low coverage. A small part of exon 9 of ERBB2 was covered suboptimally: the low covered region is 30 bp in size located at the 5' end of exon 9. It is unlikely that this low coverage led to false negative results since no mutations are reported in the COSMIC database for this DNA fragment. The results were concordant in relation to mutations involved in the genes stated above. Importantly, the percentages of allele frequency between both methods were similar, with variations ranging from 0.2% to 11.5% with an average variation of 5.2%. Insertion/deletion (Indel) detection however, required alternative bioinformatics pathways.

Conclusion

After combining well-established quality metrics to cover pre-analytical aspects with suitable technologies such as the MiSeq platform (Illumina) and appropriate bioinformatics, we recognize that this MEK/MET-specific NGS panel is fit for purpose.

#1397

Oncomine focus assay: Simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in 52 oncogenes relevant to solid tumors.

Peng Fang,1 Zhenyu Yan,1 Paul Labrousse,1 Weihua Liu,1 Jennifer Biroschak,1 Jennifer Wright,2 Selby Weeks,2 Cindy Spittle,2 Chad Galderisi,2 Jin Li1. 1 _MolecularMD Corp., Cambridge, MA;_ 2 _MolecularMD Corp., Portland, OR_.

Introduction: The Oncomine™ Focus Assay (OFA) is aimed to simultaneously detect and report hotspot mutations, Copy Number Variants (CNVs) and gene fusions in 52 oncogenes clinically relevant to solid tumors. With minimal DNA/RNA input, OFA employs AmpliSeq™ library preparation chemistry to enrich target regions for Ion Torrent™ Next Generation Sequencing. Variants are annotated for actionability by Oncomine® Knowledgebase. Here we report an analytical validation of OFA.

Methods: DNA and RNA, extracted from the FFPE processed GM12878, was used as the negative control to evaluate the specificity of OFA. A RNA sample containing multiple oncogenic gene fusions and a DNA sample containing multiple hotspot SNV and indels were used as the positive controls. Fresh DNA from cancer cell lines (HCC1143 or NCI-H2122) along with DNA from matched normal cell lines, HorizonDx NGS standards TruQ-1, TruQ-2, several engineered FFPE samples with gene fusions or copy number changes, and 43 clinical FFPE samples of a variety of solid tumor types (non-small cell lung cancer, colorectal cancer, gastrointestinal stromal tumor, ovarian cancer, pancreatic cancer, and melanoma) were used to evaluate the OFA performance. The analysis of the sequencing data was primarily performed with the OFA pipeline integrated with the Oncomine® Knowledgebase. Only the genetic alterations with clinical utility were selected as the final output from the data pipeline. The SBS and indels detected by OFA were confirmed by Sanger sequencing. Any CNVs detected were confirmed by FISH, if possible, and detected fusions were confirmed by RT-PCR or FISH.

Results: No clinically relevant genetic alterations were detected from the negative control FFPE-GM12878, indicating the high specificity of the OFA. The LOD for SBS and short indel detection was 5%, as assessed by TruQ-1 and TruQ-2, each containing 9-10 variants. The assay can detect

gene fusion RNA present as 0.1%-1% of the total RNA, and gene amplifications are detected with a minimum of 50% tumor cell content. A known 12X CCND1 amplifications in HCC1143 and a 13X MYC amplification in NCI-H2122 were detected. In addition, a 13X EGFR amplification and 11X CDK4 amplification were detected in a lung cancer FFPE sample and confirmed by FISH. The analytical specificity for detection of SBS and indels was 100% (25/25). The analytical specificity for detection of CNV gain and gene fusions, and the assay sensitivity are currently under assessment.

Conclusions: These results demonstrate the high sensitivity and specificity of OFA. With as little as 10 ng RNA and 10 ng DNA, OFA provides biomarker analysis of patient FFPE tumor specimens focusing on the detection of genetic alterations that have been targeted by oncology drugs or supported by published clinical evidence.

#1398

RNA DIRECT: A novel strategy for targeted RNA enrichment of transcripts associated with solid tumors.

Kruti M. Patel,1 Sarah K. Bowman,1 Theodore B. Davis,2 Salvatore Russello,2 Daniel C. Kraushaar,1 Amy B. Emerman,1 Noa Henig,1 Cynthia L. Hendrickson1. 1 _Directed Genomics, Ipswich, MA;_ 2 _New England Biolabs, Ipswich, MA_.

RNA sequencing (RNA-seq) has become a powerful platform for profiling transcriptomes that enables gene expression analysis and identification of nucleotide or structural variations. RNA-seq, however, can be cost prohibitive due to the size and complexity of most transcriptomes, which require deep sequencing coverage to achieve the necessary sensitivity for a reliable analysis. By combining targeted enrichment strategies with next-generation sequencing (NGS), a subset of transcript regions or sequences can be enriched for and analyzed with the resolution required for both research and clinical applications.

The RNA DIRECT targeted enrichment method utilizes probe-based hybridization to capture only relevant cDNA sequences. Targeted sequences are then ligated to NGS platform specific adaptors and amplified by PCR. The RNA DIRECT strategy provides a robust and cost effective method for the enrichment of transcript sequences for the purpose of identification and detection of known or novel variants and gene fusions. To demonstrate the high specificity, sensitivity and uniformity of this method, we have applied it to the capture of targets commonly associated with solid tumors. 

### Immune Response Monitoring: Clinical

#1400

Development of a quantitative, multiplexed imaging approach for comprehensive immune profiling of the tumor microenvironment using amplification-free, photocleavable oligo-tagged primary antibodies.

Phillip J. Sanchez,1 Mariam Vanetsyan,1 Nooriel Banayan,1 Joseph M. Beechem,2 Philippa J. Webster,2 Sarah E. Warren,2 Chris R. Merritt,2 Jung J. Jaemyeong,2 Dwayne L. Dunaway,2 Paul C. Tumeh1. 1 _UCLA Medical Center, Los Angeles, CA;_ 2 _Nanostring Technologies, Seattle, WA_.

BACKGROUND: Quantitative, multiplexed immunohistochemistry has emerged as an area of great interest within oncology since it has the capability of identifying discrete microenvironments with unique cell-cell interactions that drive or inhibit response to checkpoint blockade. The purpose of this study was to establish a practical and feasible approach that could (1) quantify protein expression levels without signal amplification, (2) achieve higher-order target antigen multiplexing, and (3) be applied to formalin-fixed paraffin embedded (FFPE) tissue sections. METHODS: The strategy involves a one-step, amplification-free staining method where an oligo conjugated primary antibody binds to the target antigen within a single FFPE tissue section. Illumination with ultraviolet (UV) light cleaves a photolabile linker that frees the oligo from the antibody. The free oligo is captured and digitally counted using nCounter technology to quantify antigen abundance. RESULTS: We investigated a variety of conjugation methods and established a reducing cysteine bioconjugation method that is stable, site-specific to the hinge-region heavy-chain, and controllable in terms of oligo:antibody stoichiometry. We next performed linear regression analysis to determine the relationship between UV illumination area and measured digital protein counts. A high degree of linearity was found between illumination area and digital counts of protein (0.97 < R2 < 0.99), confirming the mechanism underlying this amplification-free, protein counting method in FFPE tissue sections. To determine the impact of oligo-labeling on antibody-antigen interaction, we compared the performance of oligo-conjugated antibody to the unmodified antibody under identical conditions in FFPE tissue sections according to oligo:antibody ratio (1-2 oligos/antibody vs. 3-4 oligos/antibody), sensitivity, specificity, and signal intensity. The selected antibodies targeted antigens that were localized to the nucleus, cytoplasm and membrane and included Histone H3, P-S6, PD-L1, PD-1, CD3, CD4 and CD45RO. A conjugation ratio of 1-2 oligos per antibody showed sensitivity, specificity and signal intensities that were independent of subcellular location and comparable to the unmodified antibody. CONCLUSIONS: These results provide proof of concept data for the use of photocleavable, oligo-labeled antibodies in FFPE tumor samples. Future development of this technology will be focused on establishing a quantitative, antibody-based proteomic approach that is practical and accessible to investigators.

#1401

A systems immunology analysis to detect prognostic biomarkers in patients with squamous cell carcinomas of the head and neck.

Cariad Chester,1 Ajay Fernandez,1 Atsushi Yonezawa,2 Xing Zhao,3 Naren Rajasekaran,1 Holbrook Kohrt1. 1 _Stanford University, Palo Alto, CA;_ 2 _Kyoto University, Kyoto, Japan;_ 3 _Guizhou Medical University, China_.

In 2015, an estimated 59,340 people will develop head and neck cancer and an estimated 12,290 deaths will occur. Recent evidence has demonstrated that the immune system plays a key role in the development, establishment, and progression of head and neck squamous cell carcinoma (HNSCC). Here, we conduct comprehensive immune profiling to better understand the immunophenotype of HNSCC patients, assess the applicability of immune-modulatory drugs, and identify prognostic biomarkers of response to cetuximab treatment.

Our systems immunology approach is composed of three complimentary, high-dimensional technologies: time-of-flight mass cytometry (CyTOF), luminex, and the HIMChip microarray platform. Mass cytometry quantifies protein expression on a single-cell basis by utilizing transition element, isotope-tagged antibodies. The Luminex immunoassay measures plasma cytokine levels. The "HIMChip" microarray is a custom Agilent SurePrint HD 8x15k format array containing over 7,000 unique probes for over 4,274 human immune-related genes. We employed these technologies to study the global immune status of thirty HNSCC patients receiving treatment with cetuximab, an IgG1 monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR). Patients were consented to participate on the Phase 0 biomarker-focused clinical trial (NCT01114256) and peripheral blood was obtained before and after cetuximab treatment. Peripheral blood mononuclear cells and plasma were isolated from each time point and utilized in downstream analysis.

Via manual gating, our CyTOF-based phenotyping measured 24 distinct populations and the expression of 5 activation markers and 5 checkpoint receptors. The results of manual gating were confirmed with an automated, unsupervised algorithmic analysis combining stochastic neighbor embedding and k-means clustering. The resulting 2D representation of our single-cell data preserved global geometries and facilitated exploration of unique subsets within the natural killer (NK) compartment. To assess the prognostic value of these NK cell subsets, we applied a lasso-regularized logistic regression model to stratify patients based on their clinical response to cetuximab therapy. We identified an NK cell subset that is associated with clinical benefit to therapy. Luminex analysis revealed a cetuximab-induced cytokine signature composed of VCAM1, IP-10, and VEGFD that may play a role in increasing NK cell trafficking to tumor (T-statistics of 1.77, 2.17, and 1.55 respectively). The HIMChip microarray results support our proteomic findings.

These results provide the first known comprehensive immune profiling of HNSCC patients as they undergo treatment with cetuximab. Validation in a large cohort of patients is needed and future mechanistic studies will investigate the efficacy of our novel NK cell population in disease control.

#1402

Exploratory biomarkers that predict for clinical outcomes in a Phase II trial with ipilimumab plus finite androgen deprivation therapy for metastatic non-castrate prostate cancer.

Sumit K. Subudhi,1 Ana Aparicio,1 Jianjun Gao,1 Amado Zurita-Saavedra,1 John C. Araujo,1 Christopher J. Logothetis,1 Korivi R. Brinda,1 Jim P. Allison,1 Luis Vence,1 Ryan O. Emerson,2 Erik Yusko,2 Marissa Vignali,2 Harlan S. Robins,2 Jingjing Sun,1 Padmanee Sharma1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Adapative Biotechnologies Corporation, TX_.

Background: Androgen deprivation therapy (ADT) is a standard treatment that, although not curative, often induces dramatic responses in patients with advanced prostate cancer by inducing tumor cell death, which may provoke antigen release to initiate T cell responses. Ipilimumab blocks the inhibitory immune checkpoint, CTLA-4, which enhances T cell responses and induces both clinical benefit and unique toxicities in a subset of patients with advanced cancer. We designed a clinical study combining ipilimumab with finite ADT in men with metastatic prostate cancer to estimate therapeutic efficacy, determine safety, and identify predictive biomarkers for clinical outcomes.

Methods: We screened 30 patients and enrolled 27 patients between July, 2011 and June, 2013 for an open-labelled, single-center, Phase II study of ipilimumab plus finite ADT as first-line therapy for metastatic non-castrate prostate cancer. Eligible patients had histological confirmation of prostate carcinoma and radiographic documentation of metastatic disease. ADT was given for a finite duration of eight months with concurrent ipilimumab (10 mg/kg) for up to four doses, each given four-weeks apart. The primary endpoint was the proportion of patients with undetectable PSA (≤0.2 ng/mL) at seven months post-treatment initiation with ADT. In addition, we performed comprehensive immune monitoring of the peripheral blood using multi-parametric flow cytometry and next-generation sequencing of T cell receptors.

Findings: All 27 of the enrolled patients were evaluable for safety and toxicity, and 24 of these patients were evaluable for therapeutic effectiveness. Ten of the 24 (42%) patients achieved the primary endpoint of undetectable PSA levels at seven months post-treatment initiation. The median time to PSA progression was 10.0 (IQR 5.9-13.3) months from treatment initiation, and one (4%) of the 24 patients continues to have a PSA response that is ongoing for 40.7 months. The trial was closed as prespecified due to grade 3 toxicities that occurred in 12 (44%) of 27 patients. The most common grade 3 adverse events were transaminitis, colitis/diarrhea, and hypophysitis. Immunological biomarkers were identified that potentially predict for clinical benefit and grade 3 toxicities in this small study. No grade 4 or 5 toxicities were observed.

Interpretation: Although the combination of 10 mg/kg ipilimumab plus finite ADT led to toxicities that met a prespecified endpoint for closure of the trial, our current knowledge and experience about combination therapies and dose of ipilimumab leads us to postulate that additional clinical trials evaluating ipilimumab at 3 mg/kg plus ADT is warranted. In addition, we identified candidate immunological biomarkers that need to be further tested as predictive markers of clinical benefit and grade 3 toxicities.

#1403

Correlations between overall survival and patient regulatory T-cell levels at initiation of Folfiri therapy in colorectal cancer.

Caroline Jochems,1 Vittore Cereda,2 Romaine I. Fernando,1 Jacob Richards,1 Italia Grenga,1 Patrizia Ferroni,2 Fiorella Guadagni,2 Jeffrey Schlom,1 Mario Roselli2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Medical Oncology, Tor Vergata Clinical Center, University of Rome, Rome, Italy_.

Twenty-three patients with metastatic colon or rectal carcinoma were enrolled in a first-line study of Folfiri therapy consisting of irinotecan (180 mg/m2 day 1), levo-leucovorin (200 mg/m2 day 1), 5-FU (400 mg/m2 bolus day 1 and 2400 mg/m2 continuous infusion over 48h), and bevacizumab (5 mg/kg day 1). This treatment schedule was repeated every 2 weeks. Peripheral blood samples were collected prior to the start of cycle I, and after 30 days, and assayed by 4-color flow cytometry and regulatory T-cell (Treg) suppression assay.

Cytofluorometric analysis of PBMCs of the entire group of patients revealed no statistical differences between the PBMCs collected at baseline and post 30 days of therapy in terms of PBMC amount, percent of CD4+ T cells, CD8+ T cells, or Tregs, or the ratios of CD4+ T cells or CD8+ T cells vs. Tregs.

Lower levels of Tregs (< 2.5% of PBMC) at baseline, i.e., prior to chemotherapy, were associated with a better PFS by log-rank analysis. The median PFS for patients with < 2.5% Tregs at baseline was 23.3 weeks, and for patients with >2.5% Tregs at baseline PFS was 10.7 weeks (p = 0.037, n = 23).

Furthermore, a forward step-wise regression analysis model demonstrated that lower Tregs at baseline (regression coefficient = 0.467, p = 0.004) and the decrease in Tregs (as % of PBMC) at 30 days of chemotherapy (regression coefficient = 0.454, p = 0.006) were both independent predictors of better OS, independent of age, gender, ALP, LDH, mucinous vs. non-mucinous phenotype, serum CEA, serum 19.9, and number of metastatic sites.

Of the 23 patients on study, 14 demonstrated clinical responses by RECIST criteria and nine did not. Eight of the 14 (57%) responders by RECIST criteria displayed Treg levels < 2.5% of PBMC at baseline, whereas only two of the seven (22%) non-responders demonstrated < 2.5% Tregs at baseline. In addition, nine of the responders by RECIST criteria (64%) showed a trend in the decrease of Tregs as % of PBMC at 30 days of therapy, whereas only 22% of the non-responders had a decrease in Tregs (p = 0.049, Chi2 = 3.9 with Pearson test).

In conclusion, we found lower frequencies of Tregs at baseline in responders than non-responders, which was associated with longer PFS. In addition, lower Tregs at baseline and a decrease in Treg frequency after 1 month of Folfiri therapy were both independent predictors of longer OS.

#1404

Pemetrexed decreases circulating S100A9-myeloid derived suppressor cells in patients with non-small cell lung cancer.

Po-Hao Feng, Kang-Yun Lee. _Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan_.

Background: We previously reported that S100A9-myeloid derived suppressor cells (MDSCs) are negatively associated with chemotherapy effect in non-small cell lung cancer (NSCLC) patients. Pemetrexed-cisplatin chemotherapy is a standard treatment for advanced lung adenocarcinoma. The effect of chemotherapy on MDSCs is still not clear.

Methods: We prospectively enrolled nineteen stage 4 lung adenocarcinoma patients without known driving mutation before chemotherapy. Fourteen patients received pemetrexed-cisplatin and five with docetaxel-cisplatin as first line chemotherapy. Peripheral blood MDSCs percentage was evaluate by flow cytometry before first cycle and second cycle of chemotherapy.

In vitro migration assay with transwell MDSC/A549 cell co-culture system was used to test the effect of chemotherapy on chemotactic ability of A549 cell. Chemotherapy pretreated-A549 cells were cultured in the bottom chamber, and MDSCs, isolated from PBMC with CD14+ magnetic bead, were cultured in the upper chamber.

Results: MDSCs percentage decreased after patients received first cycle pemetrexed-cisplatin treatment (mean ±SD MDSC% in PBMC: 25.9±13.9% vs 16.3±11.3%, p<0.05, n=5). In the subgroup patients that had partial response to pemetrexed and cisplatin, the MDSCs percentage decreased most significantly. (25.0±9.1 to 7.1±5.2, p<0.05, n=5) In vitro study confirmed that pemetrexed reduced numbers of CD14+ MDSC (pemetrexed vs control: 15.0±6.6 vs 107.8± 56.8, p0.05) or cisplatin (96.7±98.2 vs 107.8 ±56.8, p>0.05)

Conclusion: Pemetrexed-cisplatin, but not docetaxel-cisplatin, decreased S100A9-MDSC in patients with lung adenocarcinoma, an effect might be due to a reduction in chemotactic ability of tumors by pemetrexed. In addition to cytotoxic effect, clinical benefit of pemetrexed might be also due to the effect on microenvironment.

#1405

Vesigenurtacel-L stimulates tumor infiltration of unique polyclonal T cell clones in non-muscle invasive bladder cancer patients.

Taylor H. Schreiber,1 George Fromm,1 Xin Xu,1 Eric Yusko,2 Brandon Early,1 Melissa Price1. 1 _Heat Biologics, Inc., Durham, NC;_ 2 _Adaptive Biotechnologies, Seattle, WA_.

Non-muscle invasive bladder cancer affects approximately 50,000 Americans each year, requires chronic medical management with a combination of surgical resection and intravesical BCG instillations for treatment. While BCG improves recurrence-free survival in this population, there remains a significant unmet medical need for therapeutics in patients who have a particularly high risk of recurrence and/or progression to muscle-invasive disease. Vesigenurtacel-L is an allogeneic, cell based, vaccine engineered to secrete the heat shock protein, gp96-Ig, which facilitates cross-presentation of cell-derived antigens on MHC I to activate CD8+ T cell responses across MHC barriers. We recently reported that in patients treated with Vesigenurtacel-L in a Phase I clinical trial, a minimum of 15 antigens were shared between the vaccine and individual patient tumors. Although no patient received a complete course of maintenance BCG, 70% of patients remained recurrence free beyond 12 months. Baseline biopsies were collected from all patients, and serial biopsies for several patients when clinically indicated. Analysis of these tissues by RNA Sequencing revealed that recurring patient tumors expressed higher levels of B7-H4, GITR and KIR3 at baseline, while non-recurring patient tumors expressed an immune profile consistent with an interferon defense signature. In addition, patients with progressive elimination of residual disease in repeat biopsies showed increased levels of CD3, CD8, IL-23A and OX40. Deep sequencing of peripheral blood T cells and total tumor DNA for T cell receptors was performed at baseline and at several post-treatment time points. These data demonstrated polyclonal expansion of TCR clones that were either absent, or present at low frequencies at baseline in the peripheral blood. Remarkably, approximately 40% of these clones could also be found expanded within the tumor. These data correlate positively with the absolute increase in tumor infiltrating lymphocyte percentages observed by immunohistochemistry. ELISPOT assays were performed to demonstrate the specificity of expanded T cell clones toward shared tumor antigens, since the antigens in both the patients tumors and Vesigenurtacel-L were known. These data provide exciting clinical evidence of patient benefit derived from an allogeneic immunotherapy vaccine and compelling evidence for a vaccine therapy converting 'cold' tumor to (predominantly CD8+) TIL-rich tumors, with a corresponding analysis of the antigenic determinants of the response.

#1406

Analysis of immune responses as a consequence of androgen deprivation therapy in patients with biochemical progression of prostate cancer.

Caroline Jochems, Ravi A. Madan, Romaine I. Fernando, James L. Gulley, Kwong Y. Tsang, Jeffrey Schlom. _National Cancer Institute, Bethesda, MD_.

We evaluated the immune responses in 28 patients enrolled in a phase II trial at the National Cancer Institute (NCI). The aim was to investigate the effects of 3 months of androgen deprivation therapy (ADT) with goserelin on the immune responses in patients with biochemical progression of prostate cancer following local definitive therapy. PBMC and sera were collected before treatment and after 3 months. We performed flow cytometry to evaluate the frequencies of subsets of CD4+ and CD8+ T-cells, regulatory T-cells (CD4+ CD25hi CD127- FoxP3+), MDSCs and CD4 and CD8 recent thymic emigrants. CD4 T-cell proliferation and NK-cell lytic activity were evaluated. Serum samples were analyzed for cytokines, sCD27 and sCD40L.

The baseline characteristics were: median age 64 years, testosterone 361 ng/dl, PSA 2.2 ng/ml, PSA-doubling time 7.0 months, and Gleason score 7. After 3 months of ADT we found no significant changes in NK-cell function or frequencies of NK subsets. CD4+ T-cell proliferation decreased slightly. The number of CD4+ effector memory T-cells decreased, but other CD4 subsets were unchanged. The frequencies of CD4 and CD8 recent thymic emigrants did not change. Regulatory T-cells (including the CTLA4+ subset), and the effector:Treg ratio did not change. In contrast, the frequency of granulocytic MDSC decreased (P<0.01). There was an increase in the sCD27:sCD40L ratio (P=0.012), due to decreased sCD40L. There was a decrease in the serum levels of IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, GM-CSF and IFNγ. There were no changes in the serum levels of TNFα or CXCL2 after therapy.

In conclusion, three months of ADT with goserelin did not alter the immune response in a way that would decrease the likelihood of successful immunotherapy after this treatment. The decreased MDSC and the increased sCD27:sCD40L ratio could indicate a more favorable immune environment.

#1407

FPA144, a therapeutic monoclonal antibody targeting the FGFR2b receptor, promotes antibody dependent cell-mediated cytotoxicity and stimulates sensitivity to PD-1 in the 4T1 syngeneic tumor model.

Janine Powers, Servando Palencia, Susan Foy, Barbara Sennino, Toni Rose Hidalgo, Abigael Gemo, Thomas Brennan, Kisten Pierce. _Five Prime Therapeutics, South San Francisco, CA_.

Five Prime Therapeutics, Inc. has developed an FGFR2b-specific humanized monoclonal antibody, FPA144, to treat patients with cancer bearing overexpression of the FGFR2b receptor, and is currently in clinical trials as a single agent for gastric cancer (NCT02318329). FGFR2 gene amplification and FGFR2b overexpression occur in approximately 5% of gastric cancers and are associated with a poor prognosis in gastric cancer patients. In addition to blocking ligand binding and inducing FGFR2b internalization, FPA144 is glycoengineered for enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). We have shown previously that FPA144 can produce complete and durable tumor growth inhibition in FGFR2b-over-expressing and FGFR2-amplified gastric cancer xenografts in immune-compromised mice (Gemo et al., Poster 5446, AACR 2014). In order to understand the contribution of the immune system to the mechanisms of action of FPA144, we evaluated the anti-tumor effects and immune cell recruitment of FPA144 in the 4T1 model of cancer in immune-competent mice. Although this model expresses FGFR2b, it is not FGFR2 amplified. Therapeutic treatment with FPA144 in the orthotopic 4T1 model resulted in a reduction in tumor burden (33%, P<0.001) and concomitant recruitment of NK cells to the site of tumor implantation, while a modified antibody lacking Fc effector function neither inhibited tumor growth nor lead to the recruitment of NK cells. Together these data support a potential role for ADCC as a mechanism of FPA144 tumor growth inhibition. In addition, treatment with FPA144 increased PD-L1 expressing cells within the tumor microenvironment, providing a strong rationale that FPA144 may combine effectively with PD-1 blockade for additional tumor growth inhibition. PD-1 blockade by the RPM1-14 antibody did not inhibit tumor growth as a single agent in the 4T1 model. Treatment with RPM1-14 in combination with FPA144, however, inhibited tumor growth by 49% (P<0.001), demonstrating an additive benefit of combination therapy. Overall, these data suggest that the enhanced ADCC activity of FPA144 may be critical for anti-tumor efficacy in tumors that have modest expression of FGFR2b. In addition, FPA144 may reprogram the tumor micro-environment in a way that primes the tumor for additional anti-tumor activity when combined with PD-1 blockade.

#1408

Tumor-targeting Salmonella typhimurium A1-R promotes CD8+ T cell infiltration in pancreatic cancer in an orthotopic syngeneic mouse model.

Takashi Murakami,1 Yukihiko Hiroshima,2 Yong Zhang,3 Ming Zhao,3 Tasuku Kiyuna,3 Ryusei Matsuyama,2 Takashi Chishima,2 Kuniya Tanaka,2 Michael Bouvet,4 Itaru Endo,2 Robert M. Hoffman3. 1 _Department of Surgery, San Diego, CA;_ 2 _Department of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, Japan;_ 3 _AntiCancer, Inc., San Diego, CA;_ 4 _Department of Surgery, University of California San Diego, San Diego, CA_.

The effect of tumor-targeting Salmonella typhimurium A1-R on CD8+ cell infiltration was determined on Pan02 murine pancreatic adenocarcinoma cells implanted orthotopically on the pancreatic tail of C57BL/6 mice. Three weeks after orthotopic implantation, mice were randomized into G1: untreated control group (n=8); and G2: Salmonella typhimurium A1-R treatment group (n=8, 1 × 107 CFU/body, iv, weekly, 3 weeks). On the 22nd day from initial treatment, all mice were sacrificed and tumors were harvested. The tumor volume ratio was defined as ratio of tumor volume at the 1st day to the 22nd day. H&E staining was performed to evaluate tumor destruction which was classified into 4 categories according to the Evans grading system: Grade I; 0-9% destruction, Grade IIA; 10-50% destruction, Grade IIB; 51-90% destruction, Grade III; few (<10%) viable cancer cells. Immunohistochemical staining using anti-mouse CD8+ antibody was performed to count tumor infiltrating CD8+ cells. The tumor volume ratio was significantly lower in G2 (3.0 ± 2.8) than G1 (39.9 ± 30.7, p < 0.01). Six mice in G1 had peritoneal dissemination whereas no mice showed peritoneal dissemination in G2 (p < 0.01). Significant tumor destruction was observed in G2 (Grade I: 0, Grade IIA: 1, Grade IIB: 6, Grade III: 1) compared to G1 (Grade I: 5, Grade IIA: 3, Grade IIB: 0, Grade III: 0, p < 0.01). Tumor infiltrating CD8+ cells per high-magnification field were significantly higher in G2 (133.5 ± 32.2) than G1 (45.1 ± 19.4, p < 0.001). Strong tumor destruction correlated with higher numbers of tumor-infiltrating CD8+ cells per high-magnification field (Grade I: 35.5 ± 18.3, Grade IIA: 77.3 ± 32.8 Grade IIB and III: 134.7 ± 34.8). The present study demonstrates that Salmonella typhimurium A1-R promotes CD8+ T cell infiltration in a syngeneic pancreatic cancer mouse model.

#1409

Is it what's on the inside that counts? Melanoma mutation profiling and outcomes with immunotherapy.

Morganna L. Freeman, Howard McLeod. _H Lee Moffitt Cancer Center, Tampa, FL_.

Extraordinary advances have been made in the treatment of melanoma over the last few years; immune checkpoint blockade has provided durable responses and meaningful survival outcomes for patients with metastatic disease, yet predicting such responses remains a nascent science. More importantly, the toxicity and financial burden of receiving such therapy warrants a personalized, or precision medicine, approach to appropriate selection of treatment, thus molecular predictors of response may become as important as biomarkers such as PDL1 expression. Recently the high mutational burden of malignant melanoma was shown to be related to the degree of response to the anti-CTLA4 antibody ipilimumab, but mutational burden alone was not sufficient to predict benefit1. Another study has shown NRAS-mutant had a trend to superior outcomes compared with other cohorts in terms of response to therapy with anti-PD1 antibodies and progression-free survival2. Moreover, insight into the molecular evolution of melanoma was recently established3, wherein somatic mutations in oncogenes such as BRAF, NRAS, GNAQ, or GNA11 occur early during progression and additional mutations of TERT, CDKN2A, TP53, and PTEN are seen in late-stage disease. To better evaluate the relationship of these mutations to immunotherapy response, our institution has profiled 95 patients with melanoma with next-generation sequencing (NGS). Of the 95 patients profiled, the most common mutations identified were BRAF (38%), NRAS (21%), TP53 (18%), PTEN (14%), TERT (9%), and CDKN2A (8%). We are presently conducting a retrospective analysis of these selected oncogenes as they relate to clinical responses, and intend to report disease outcomes (duration of response, progression free survival, and overall survival) as they relate to checkpoint inhibition.

#1410

PI3K inhibitors reduce breast cancer tumor growth and enhance anti-tumor leukocyte activity.

Nicole Lavender, Jinming Yang, Jiqing Sai, Sergey Novitskiy, Linda Horton, Andrew Johnson, Vandana Abramson, Ingrid Mayer, Ingrid Meszoely, Mark Kelley, Ann Richmond. _Vanderbilt University Medical Center, Nashville, TN_.

Background: PI3K inhibitors have shown anti-tumor activity in breast cancer and are currently being investigated in multiple clinical trials. Despite the number of trials studying treatment efficacy, tolerability, and survival, little attention has been given to how these agents affect the anti-tumor capacity of leukocytes. However, one potential reason for diminished success of these therapies could be that systemic inhibition of this kinase results in a loss of 'entrainment' of leukocytes such that they fail to appropriately kill tumor cells. Therefore, the goal of this research was to evaluate the effects of PI3K inhibitors on leukocyte function in breast cancer.

Methods: Blood samples were obtained from patients enrolled in the VICCBRE1320 clinical trial aimed to evaluate GDC0032 and docetaxel. Entrainment properties of circulating leukocyte cells were compared from Day 0 to on treatment samples for multiple cycles of therapy. Blood samples were drawn before administration of the treatment. Peripheral blood leukocytes were isolated and analyzed by FACS. Neutrophils as well as CD4+/8+ cells were also isolated from these blood samples to measure their ability to kill breast cancer tumor cells in vitro. We also examined the effects of PI3K inhibitors on human breast cancer tumors and leukocytes using humanized mouse models. For these models, tumors were implanted into the mammary fat pad of NSG mice engrafted with the patient's CD34+ hematopoietic stem cells and infused with the patient's in vitro expanded CD4+/8+ T cells.

Results: In two of the clinical trial patients studied we observed a decrease CD4+ T cells and DCs over the first two cycles of therapy (p<0.007). We also saw a drop in B, NK, Treg cells in one patient (p=0.004). No changes in circulating neutrophils or macrophages have been detected. We found that neutrophils as well as CD4+/8+ cells isolated from the blood of these patients are able to kill ER+ MCF7 or Triple-Negative MDA-MB-231 tumor cells in vitro (p<0.038). Throughout the treatment period, leukocytes from patients receiving PI3K inhibitor are able to kill tumor cells regardless of the breast cancer subtype or PI3K mutation status. When we treated humanized mice bearing patient derived xenografts of triple negative breast tumors with BKM120, we observed a 20% decrease in CD4+ cells and an increase in Th1 phenotype among tumor infiltrating T cells. BKM120 also resulted in a decrease in tumor growth compared to vehicle (p<0.01).

Conclusions & Future Directions: Our findings suggest that pan-Class I PI3K inhibitors possess anti-tumor activity, but are also capable of enhancing anti-tumor immune cell function. The latter could improve treatment outcomes as well as prevent metastasis. Hence, our preliminary data demonstrate that PI3K inhibitors can serve as effective therapies in breast cancer regimens to reduce tumor growth and progression. As part of a combination, these agents may also effectively treat metastatic patients.

#1411

Plasma and cerebrospinal fluid pharmacokinetics of tocilizumab in a nonhuman primate model.

Anandani Nellan,1 Nalini Jayaprakash,2 Cindy McCully,2 Brigitte C. Widemann,2 Daniel W. Lee,2 Katherine E. Warren2. 1 _National Cancer Institute/Johns Hopkins University, Bethesda, MD;_ 2 _National Cancer Institute, Bethesda, MD_.

Background: Cytokine release syndrome (CRS) is a life-threatening complication of effective immunotherapies, particularly chimeric antigen receptor (CAR) T-cells. CRS is characterized by fever, hypotension, cardiac dysfunction, and at times end organ dysfunction and is mediated by elevated cytokines including IL-6, IFN-γ, and TNFα. A mainstay treatment for severe CRS is intravenous (IV) tocilizumab, a humanized, monoclonal antibody against membrane-associated and soluble IL-6 receptor. Most CRS symptoms resolve within hours of a single dose of IV tocilizumab in contrast to neurotoxicities, which are exacerbated or develop de novo after tocilizumab by a mechanism that is not well understood. IL-6 is elevated in the cerebrospinal fluid (CSF) of patients with CRS and neurotoxicity. The CSF penetration of tocilizumab is unknown. The objective of this study is to determine the CSF penetration of tocilizumab after IV administration in a nonhuman primate model.

Methods: Four adult male rhesus monkeys (Macaca mulatta) each received 10 mg/kg tocilizumab IV (human equivalent dose of 8 mg/kg). The macaques have an implanted CNS ventricular reservoir for CSF collection and two central venous lines; one for drug administration and a second for sample collection. Serial, paired blood and CSF samples were collected 0-8 hours, 1-3 days, 5 days, & 10 days post-infusion. A fluorescence bridging ELISA was used to determine serum and CSF levels of free tocilizumab (AbD Serotec).

Results: Three of the four animals were evaluable. Mean serum Cmax = 1.55 μM (1.20-2.13) and mean CSF Cmax = 0.00065 μM (0.00037-0.00083). Mean serum and CSF Tmax were 10 hours and 48 hours, respectively. Mean serum and CSF half-life were 127 and 339 hours, respectively. Serum AUC (mean) = 63 μM/hour (25-130) while CSF AUC (mean) = 0.099 μM/hour (0.07-0.125). CSF penetration was low with mean CSF:plasma AUC ratio of 0.26%. All samples and standards were plated in triplicate and had a coefficient of variation less than 20%. In one animal, tocilizumab concentrations were markedly lower with no detectable tocilizumab in the serum after 4 hours; results from this study were considered unevaluable.

Conclusion: Studies in this nonhuman primate model reveal low CSF penetration of tocilizumab and delayed time to maximum concentration in the CSF, potentially explaining the persistent neurologic toxicity observed in patients with CRS treated with tocilizumab. However, tocilizumab has a long half-life in the CSF, suggesting alternate means of delivery may be beneficial. Free tocilizumab concentrations were extremely low in one out of four animals and may have been due to preexisting inhibitors. Intranasal delivery of tocilizumab may result in significantly higher CSF exposure and provides a convenient route in patients that are otherwise critically ill. Studies of intranasal tocilizumab are ongoing and will be presented.

#1412

Prediction of the treatment response to eribulin chemotherapy in breast cancer using tumor-infiltrating lymphocytes (TILs).

Kashiwagi Shinichiro, Yuka Asano, Wataru Goto, Kento Kurata, Tamami Morisaki, Satoru Noda, Tsutomu Takashima, Naoyoshi Onoda, Sayaka Tanaka, Masahiko Ohsawa, Masaichi Ohira, Kosei Hirakawa. _Osaka City Univ., Osaka, Japan_.

Background: Eribulin mesylate (eribulin) is currently indicated for treatment of locally advanced or metastatic breast cancer (MBC). It is a cytotoxic agent with unique mechanisms that suppress epithelial-mesenchymal transition (EMT) of cancer cells. On the other hand, Tumor-infiltrating lymphocytes (TILs), which are considered indicators of immune response monitoring, have been reported as prognostic factors and predictors of therapeutic efficacy. We thought that eribulin, which has an EMT-inhibiting mechanism, may produce an antitumor effect by improving the immune microenvironment, and in this study investigated the effects of breast cancer eribulin chemotherapy on the immune microenvironment with TILs as a marker.

Materials and Methods: TILs was evaluated in 52 patients with MBC who underwent chemotherapy with eribulin. The correlation between TILs evaluated according to the standard method, and prognosis, including the efficacy of eribulin chemotherapy, was investigated retrospectively. TILs were defined as the infiltrating lymphocytes within tumor stroma and were expressed in proportion to the field investigated. The area of in situ carcinoma and crush artifacts were not included. Proportional scores were defined as 3, 2, 1, and 0 if the area of stroma with lymphoplasmacytic infiltration around invasive tumor cell nests was > 50%, > 10 - 50%, ≤ 10%, and absent, respectively. TILs were considered positive when scores were ≥ 2, and negative when scores were 1 and 0.

Results: Of the 52 MBC patients, 29 (55.8%) were in the high TILs group and 23 (44.2%) were in the low TILs group. The high TILs group included significantly more triple-negative breast cancer (TNBC) (p = 0.008) than the low TILs group. In an analysis of outcomes, TNBC patients in the high TILs group had significantly longer disease-free survival than TNBC patients in the low TILs group (p = 0.033, log-rank), but no significant differences were seen in all breast cancer patients (p = 0.489, log-rank) or in non-TNBC patients (p=0.878, log-rank). In a univariate analysis of recurrence in TNBC patients, being in the high TILs group was a factor for a good outcome (p = 0.047, HR = 0.260). In a multivariate analysis, being in the high TILs group was again an independent factor for a good outcome (p=0.031, HR=0.063).

Conclusions: The results of this study suggest that TILs may be useful as a predictive marker of the therapeutic effect of eribulin chemotherapy in TNBC.

#1413

Treatment of mouse liver and brain colon cancer metastases with Toca 511 and 5-fluorocytosine for intratumoral production of 5-fluorouracil leads to increased survival, induction of antitumor immune responses, and reduction of MDSC.

Maria Rodriguez-Aguirre, Kader Yagiz, Fernando Lopez Espinoza, Daniel Mendoza, Tiffany T. Huang, Carlos Ibanez, Harry E. Gruber, Douglas J. Jolly, Joan Robbins. _Tocagen, San Diego, CA_.

Approximately 50% of patients with colorectal cancer (CRC) develop metastases (mCRC) during the course of disease, with liver the most frequent site. Standard treatment for mCRC is 5-fluorouracil (5-FU) based combination therapy, with median survival now >20 months. As a result of prolonged survival with metastatic disease, incidence of brain metastases from colorectal cancer is increasing. Approved immunotherapeutic agents have had limited effect in mCRC except in the subset of subjects (3-6%) with deficient mismatch repair. Recent studies suggest myeloid-derived suppressor cells (MDSC) contribute to cancer immune evasion by suppressing T cell anti-tumor functions and modulating innate immune responses.

We are pursuing a unique approach to treat cancer via in situ production of 5-FU. Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector, selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) protein. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an oral anti-fungal drug) to 5-FU. Direct tumor cytotoxicity and extended immunotherapeutic effects have been reported using this approach.

Toca 511 and Toca FC (extended release 5-FC) is under investigation in subjects with primary brain tumors, delivered intratumorally (NCT01156584), by injection into the resection bed (NCT01470794, NCT02414165), or intravenously (NCT01985256). We tested the intravenous (IV) approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci in the liver. IV delivery of Toca 511 followed by courses of 5-FC resulted in tumor response, improved survival, and immune mediated inhibition of rechallenge with tumor. IV delivery resulted in expression of vector encoded transgene in tumor foci but not in adjacent normal liver. Similar published results have been obtained in a number of brain tumor models with Toca 511+5-FC including a CT26 colon carcinoma brain metastasis model. We further investigated the effect of Toca 511+5-FC treatment on CRC tumor-induced MDSC in the CT-26 brain metastasis model. Intracranial cell implantation and intratumoral Toca 511 vector injections were followed by one course of 5-FC. Expansion of MDSC occurred in brain tumors and spleens of tumor bearing animals. A significant decrease in MDSC in spleens and tumors was observed with Toca 511+5-FC treatment compared to control (p=0.03, p<0.0001; respectively).

The results reported here support the development of Toca 511 and Toca FC as a novel immunotherapeutic approach for patients with mCRC. A phase 1 study of IV Toca 511 and Toca FC in solid tumors, including mCRC, is planned (NCT02576665).

#1414

Treatment outcome under cetuximab-based therapy and individual ADCC capability in head and neck cancer.

Cristiana Lo Nigro,1 Laura Lattanzio,1 Daniela Vivenza,1 Martino Monteverde,1 Federica Tonissi,1 Giuliana Strola,1 Marco Merlano,1 Gerard Milano2. 1 _S. Croce & Carle Teaching Hospital, Cuneo, Italy; _2 _Centre Antoine Lacassagne, Nice, France_.

Introduction

Cetuximab is a IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) used for head and neck squamous cell carcinoma (HNSCC) treatment. In addition to EGFR targeting, cetuximab is able to develop an antibody-dependent cell-mediated cytotoxicity (ADCC), triggered by Fc receptors (Fcγ-R) on Natural Killer cells, macrophages and polymorphonucleated leukocytes.

This prospective study examined the possible prognostic value of cetuximab-mediated ADCC response in a cohort of HNSCC patients.

Patients and Methods

There were 63 patients treated with curative intent with chemo-radiotherapy (CT-RT), associated (N=39) or not (N=24) with cetuximab. Peripheral blood samples were collected at start of therapy. Intrinsic ADCC was evaluated from ex vivo NK-dependent activity measuring LDH release through the Cytotox 96® non radioactive cytotoxicity assay, previously standardized in our laboratory (Crit Rev Oncol Hematol, 2015). EGFR tumoral expression was analyzed by immunohistochemistry.

Results

Median ADCC response at treatment start for all patients was 56.1% (range 5-99%). Analysing the whole population, patients with ADCC above the median value (high) showed a better OS as compared to patients with ADCC below the median (low), HR=0.3827 (95% CI, 0.1464 to 1.000), p=0.05 (Long-rank Mantel-Cox Test). A sub-group analysis confirmed a possible link between high ADCC and a better OS (CT-RT group, p=0.223 and CT-RT+cetuximab group, p=0.244).

Importantly, considering CT-RT+cetuximab treatment and EGFR tumoral expression (N=21), patients with EGFR 3+ status and high ADCC showed an improved OS as compared to patients with ADCC below median value, HR=0.09031 (95% CI, 0.009983 to 0.8170), p=0.032 (Long-rank Mantel-Cox Test). Median OS values for patients with high EGFR expression were 34.8 months (range 9.1-53.7) and 13.1 months (range 2.1-39.5) for patients with respectively high ADCC and low ADCC.

Conclusion

ADCC is shown to be a strong driver of cetuximab-based therapy outcome in HNSCC. High tumoral EGFR expression and high ADCC confer a particularly long overall survival. These data suggest an ADCC-based and EGFR expression-based precision therapy under cetuximab in HNSCC and open perspectives for NK boosting.

#1415

Targeting the TGFβ pathway with galunisertib, a small molecule inhibitor of TGFβRI, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint inhibition.

David Schaer, Kyla Driscoll. _Eli Lilly and Company, New York, NY_.

TGFβ signaling is known to play a central role in tumor biology, via inducing and/or enhancing tumor cell growth and differentiation, modulating the extracellular matrix in the stroma, inducing epithelial to mesenchymal transition, modulating angiogenesis, and inhibiting immune surveillance and anti-tumor immunity. Galunisertib is a pharmacological inhibitor of the TGFβ pathway which acts by inhibiting signaling though TGFβRI. Galunisertib is currently being evaluated clinically in several Phase I and II studies; as a monotherapy, galunisertib has shown antitumor activity against a variety of tumors, including durable and long-term responses in patients with glioma.

To explore the impact of Galunisertib monotherapy on anti-tumor T cell immunity, we utilized murine tumor models. Treatment of mice with 4T1-LP (poorly immunogenic 4T1 breast tumor engineered to express luciferase) implanted in the mammary fat pad resulted in strong dose-dependent anti-tumor activity with nearly 100% inhibition of tumor growth across doses during the dosing period, with complete tumor rejection upon cessation of treatment in ~50% of animals at the highest dose tested; depletion studies demonstrated that rejection of 4T1-LP was dependent on the presence of CD8+ T cells. Rechallenge of treated, tumor free mice resulted in complete rejection of 4T1-LP tumor cells but no rejection of EMT6-LM2 tumor cells, demonstrating the establishment of a durable response and immunological memory. Treatment of mice bearing parental 4T1 tumors in the mammary fat pad resulted in no significant inhibition of tumor growth, indicating that the presence of a foreign antigen (i.e. LP), potentially enhanced the ability to reject the 4T1-LP derivative. Animals that rejected the immunogenic 4T1-LP cells were able to also reject 4T1 parental cells upon rechallenge, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. In the CT26 murine colon carcinoma model, treatment with galunisertib or anti-PD-L1 as monotherapies resulted in tumor growth inhibition compared to control of 75% and 86%, respectively (T/C values of 25% and 14%); complete responders were observed in about 20% of treated animals in both monotherapy groups. Combination of galunisertib with anti-PD-L1 resulted in an enhanced tumor growth inhibition of 98% (T/C value of ~2%), and a complete response rate of ~50%, suggesting at least additive activity with potential for synergy when targeting the TGFβ and PD-1 pathways. Taken together, these data demonstrate the potential for galunisertib treatment to enhance the development of anti- tumor T cell immunity, which can be enhanced by combinations with immune check point inhibitors.

#1416

Peritumoral IL-15 potentiates radiation-induced anti-tumor immunity.

Karsten Pilones, Silvia Formenti, Sandra Demaria. _Weill Cornell Medicine, New York, NY_.

Radiotherapy (RT) can induce T cell-mediated anti-tumor immune responses by multiple mechanisms but is often unable to overcome immunosuppression in the tumor microenvironment. The common gamma-chain cytokines interleukin (IL)-2 and IL-15 promote the proliferation of activated T cells and, therefore, are prime agents for immunotherapy strategies aimed at sustaining anti-tumor T cell responses. The benefits of high dose IL-2, however, are undermined by serious toxicity and by regulatory T cell (Treg) stimulation. In contrast, IL-15 is well-tolerated and lacks Treg stimulatory activity, making it an attractive candidate for testing in combination with RT. Here we tested the hypothesis that IL-15 strengthens the pro-immunogenic effect of local RT to potentiate a durable anti-tumor immune response.

The poorly immunogenic mouse TSA breast cancer cells were implanted s.c. in syngeneic BALB/c mice and randomly assigned to one of 4 treatment groups when tumors reached 5mm average diameters: control, RT, IL-15 or RT+IL-15. RT was delivered locally in 8 Gy fractions on days 13, 14 and 15. IL-15 (2 μg/mouse) was administered s.c. peritumorally daily for 10 days starting on day 12. Mice were followed for tumor growth. A parallel experiment was done to characterize tumor-infiltrating lymphocytes (TILs) at the end of treatment (day 22).

Low dose IL-15 given peritumorally as a monotherapy induced marginal tumor growth control and had no effect on survival (median survival = 45 days compared to 76 days for control). Local RT significantly delayed tumor growth (p < 0.05 compared to control) and improved survival (median= 76 days, p<0.05). However, highest survival advantage was seen in mice given IL-15+RT (median= 102 days, p<0.05 compared to all groups) with 1 of 6 mice showing complete tumor rejection and development of anamnestic response against tumor re-challenge. Analysis of TILs showed marked infiltration of CD8+ T cells expressing the activation marker CD137 (35.3% in RT+IL-15 versus 5.90% In control, p < 0.05) while the increase was modest with each monotherapy (18.8% in RT, 20.7% in IL-15, p < 0.05 compared to control). In addition, we found a significant increase in the ratio of effector CD4+ T-cells to Tregs (2.5 in RT+IL-15 versus 0.78 in control, p < 0.05) whereas monotherapy had no effect (1.14 in RT, 0.96 in IL-15). Additionally, IL-15 treatment, regardless of RT, led to significant intratumoral enrichment of NKp46+ NK cells.

Overall these results support the rational combination of low dose intratumoral IL-15 with local RT. We are currently elucidating the mechanisms involved in pre-clinical models in preparation for future testing in patients.

#1417

Combination cancer immunotherapy using checkpoint blockade and intratumoral virus-like particles containing CpG ODN.

Caitlin Lemke,1 Aliasger Salem,1 Arthur Krieg,2 George Weiner1. 1 _University of Iowa, Iowa City, IA;_ 2 _Checkmate Pharmaceuticals, Cambridge, MA_.

Checkpoint blockade of immune inhibitory pathways is an exciting new approach to inducing durable and clinically relevant anti-tumor immune responses. Nevertheless, there remains considerable room for improvement despite significant improvements in patient survival following therapy of a variety of malignancies with monoclonal antibodies that target CTLA4 or the PD-1/PD-L1 pathway.

Our goal is to improve the efficacy of this exciting new approach to cancer therapy by increasing intratumoral release and presentation of tumor associated antigens. More specifically, we evaluated the combination of anti-PD-1 antibody with intra-tumoral injection of a novel CpG ODN-containing virus-like particle (VLP) formulation in murine models of melanoma and lymphoma. The CpG VLP (designated CMP001) was designed to enhance antigen presentation and development of the T cell response. CMP001 was previously tested in >700 human volunteers and patients with asthma and found to be well tolerated and to stimulate Th1 immune responses. A modified version of CMP-001 in which a tumor antigen was conjugated to the VLP surface was also found to be well tolerated and to induce CD4 and CD8 T cell responses as a cancer vaccine adjuvant for melanoma.

Mice were given a bilateral subcutaneous challenge of either A20 B cell lymphoma or B16F0 melanoma. Anti-PD-1, or saline control, was delivered intraperitoneally (i.p.) starting 3-7 days after tumor challenge and continued twice weekly. CMP001, or saline control, was delivered intratumorally (i.t.) on one side starting 3-7 days after tumor challenge for a total of 3 doses. This allowed us to assess the local (treated tumor) and systemic (untreated tumor) effect of CMP001 therapy. Body weights, tumor volumes (treated and untreated), and overall survival were measured.

CMP001 enhanced survival, and reduced tumor growth of both the treated and untreated tumors in the A20 model. Each of these effects was enhanced when anti-PD1 was added. In the B16 model, CMP001 treatment reduced growth of the local tumor - an effect that was modestly enhanced by anti-PD1 therapy. There was no detectable synergy between CMP001 anti-PD1 on the untreated tumor, likely because of the rapid outgrowth of the untreated tumor before there was time for development of a robust anti-tumor immune response.

Results to date suggest that intratumoral CMP001, both alone and in combination with checkpoint blockade, can stimulate anti-tumor responses. These responses can result in regression of systemic tumor growth if they are generated before systemic disease becomes too advanced. Studies are ongoing to elucidate the protective role of immune cell populations that develop following this novel combination immunotherapy.

#1418

Marine ω-3 polyunsaturated fatty acid intake and risk of colorectal cancer according to tumor-infiltrating T cells.

Mingyang Song, Reiko Nishihara, Yin Cao, Eunyoung Chun, Zhi Rong Qian, Kosuke Mima, Kentaro Inamura, Yohei Masugi, Jonathan Nowak, Katsuhiko Nosho, Kana Wu, Molin Wang, Edward Giovannucci, Wendy S. Garrett, Charles S. Fuchs, Shuji Ogino, Andrew T. Chan. _Harvard University, Boston, MA_.

Importance: Marine ω-3 polyunsaturated fatty acids possess potent immunomodulatory activity and may protect against cancer development. However, evidence relating marine ω-3 polyunsaturated fatty acids to colorectal cancer risk remains inconclusive.

Objective: To test the hypothesis that marine ω-3 polyunsaturated fatty acid intake may be associated with lower risk of colorectal cancer subsets characterized by immune infiltrate.

Design: Prospective cohort study

Setting: Nurses' Health Study and Health Professionals Follow-up Study

Participants: In the two cohorts, 125,172 participants provided data about marine ω-3 polyunsaturated fatty acid intake every 4 years through a validated food frequency questionnaire and followed up for incident colorectal cancer over 24-26 years. We documented 614 colorectal cancer cases from which we could assess T-cell infiltration in the tumor microenvironment. Cause-specific Cox proportional hazards regression was used to estimate hazard ratios and 95% confidence intervals for risks of colorectal cancer subtypes.

Exposure: Intake of marine ω-3 polyunsaturated fatty acids

Main Outcome and Measures: Incidence of colorectal cancer according to tumor-infiltrating T cells

Results: The inverse association of marine ω-3 polyunsaturated fatty acids with colorectal cancer risk differed according to FOXP3+ T-cell infiltration (P for heterogeneity = 0.006). Compared to intake of <0.15 g/day of marine ω-3 PUFAs, intake of ≥0.35 g/day was associated with a multivariable hazard ratio of 0.57 (95% confidence interval, 0.40 to 0.81) (P for trend < 0.001) for FOXP3+ T-cell-high tumors. In contrast, the corresponding hazard ratio was 1.14 (95% confidence interval, 0.81 to 1.60) (P for trend = 0.77) for FOXP3+ T-cell-low tumors. No statistically significant differential association was found according to tumor-infiltrating CD3+, CD8+, or CD45RO+ T cells (P for heterogeneity ≥ 0.34). In functional assays, the suppressive activity of regulatory T cells was approximately two-fold lower when pre-incubated with marine ω-3 polyunsaturated fatty acids at 50, 100 and 200 μM than without marine ω-3 polyunsaturated fatty acids (P<0.0001).

Conclusion and Relevance: High marine ω-3 polyunsaturated fatty acid intake was associated with lower risk of colorectal cancer with high-level, but not low-level, FOXP3+ T-cell density, suggesting a potential role of ω-3 polyunsaturated fatty acids in cancer immunoprevention through modulation of regulatory T cells.

#1419

Characterization of the evolution of immune response in lung squamous carcinogenesis.

Celine Mascaux,1 Mihaela Angelova,2 Jennifer Beane,3 Kahkeshan Hijazi,3 Geraldine Antoine,4 Karen Willard Gallo,4 Vincent Ninane,4 Arsène Burny,4 Jean-Paul Sculier,4 Avrum Spira,3 Jérôme Galon2. 1 _Aix Marseille University, Marseille, France;_ 2 _Université Paris Descartes, Paris, France;_ 3 _Boston University, Boston, MA;_ 4 _Université Libre de Bruxelles, Bruxelles, Belgium_.

Molecular profiling of from pre-invasive bronchial lesions, and particularly the evolution of immune response, may identify promising new biomarkers for early detection and targets for chemoprevention/treatment of lung cancer.

mRNA expression was analyzed (Agilent microarrays) from fresh frozen human bronchial biopsies (N=122, 77 patients) at successive morphological stages of lung squamous carcinogenesis. Modules of co-expressed genes were identified among the stage associated genes, selected using a linear mixed-effects model adjusting for smoking status, gender, and history of cancer as fixed effects and patient as a random effect. Cell type specific signatures were applied to the genes co-expressed in these modules in order to identify the immune response signal through the stages of lung carcinogenesis.

Genes expression alterations were grouped into 4 successive molecular steps. Based on their behavior across the 4 steps, 8 modules of genes with different patterns were observed: gene modules primarily up-regulated among the early or late steps, up-regulated in a linearly across the 4 steps, as well as down- then up-regulated (biphasic). Genes signing the presence of activating CD8 and CD4 T cells, as well as some memory B cells were progressively and linearly up-regulated from metaplasia through all stages of carcinogenesis. At the later stages, the transition to high-grade dysplasia, the most significant gene expression changes included immune/inflammatory response genes. At this stage a very strong adaptive immune response is noted involving T cells - mainly Th1 and T regulators - and dendritic cells. In addition, immunosuppressive signals, corresponding to negative co-inhibitory molecules, also significantly appears at the high-grade dysplasia stage.

The adaptive CD4/CD8 related immune response starts from the earlier stages (metaplasia) of lung carcinogenesis. The significant modification of inflammatory/immune response genes in association with high-grade lesions suggests, at this critical stage of carcinogenesis, a major role of the surrounding microenvironment with an adaptive immune response, driven by Th1 and Treg cells, and but also, before tumor invasion across the basal membrane, a tumor versus host immunosuppressive response.

#1420

IL17F-rs9463772 independently predicts long-term outcome in locally advanced rectal cancer.

Cecchin Erika,1 Eva Dreussi,1 Sara Gagno,1 Chiara Zanusso,1 Marcella Montico,1 Claudio Belluco,1 Antonino De Paoli,1 Salvatore Pucciarelli,2 Luca Quartuccio,3 Salvatore De Vita,3 Vincenzo Canzonieri,1 Angela Buonadonna,1 Maria Luisa Friso,4 Sara Lonardi,4 Giuseppe Toffoli1. 1 _CRO-National Cancer Institute, Aviano, Italy;_ 2 _Padova University, Padova, Italy;_ 3 _Santa Maria Degli Angeli General Hospital, Udine, Italy;_ 4 _Istituto Oncologico Veneto, Padova, Italy_.

Host genetic variation in the immune factors has the potential to highlight individual characteristics of tumor risk, prognosis, and response to treatment. In locally advanced rectal cancer (LARC) no predictive and prognostic factors are available to select patients for multimodality treatment comprising neoadjuvant chemoradiation followed by radical surgery, and possibly adjuvant chemotherapy. This work aimed at identifying immunogenetic markers of prognostic significance in LARC patients treated by multimodality treatment.

A panel of 192 tagging polymorphisms (SNPs) in 34 immuno-system related genes was selected and analyzed in a Veracode (Illumina) platform on 250 LARC patients (training set) treated with neo-adjuvant therapy, and followed up for at least 24 months after radical surgery. The primary clinical end-point of the study was 2-years recurrence status which has been reported to be the most reliable long-term prognostic factor in these patients. Significant SNPs were then tested on 10-years overall survival (OS). An independent validation set comprising of 63 rectal cancer patients was used to replicate the results.

Training set analysis identified 4 markers significantly associated with 2-years recurrence status by multivariate logistic regression which were still significant after bootstrap validation, according to a dominant genetic model: three of them were risk factors, IL17F-rs641701 (P=0.002), IL17F-rs9463772 (P=0.009), TGF-beta- rs9867701 (P=0.020); one of them was a protective factor, STAT3-rs8069645 (P=0.025). Three of them were also associated to 10-years OS by multivariate COX regression: IL17F-rs641701 (P=0.003); IL17F-rs9463772 (P=0.002) were risk factors for death; STAT3-rs8069645 (P=0.003) was a protective factor. The prognostic effect of IL17F-rs9463772 on 10-years OS was replicated using the validation set (P=0.045).

IL17F is a cytokine with onco-suppressing and anti-angiogenic effects, previously reported to be down-regulated in colorectal tumor as compared to healthy tissue. Although no functional data are available for the intronic rs9463772, it might be involved in the regulation of IL17F expression level affecting tumor prognosis.

In conclusion IL17F-rs9463772 represents a validated prognostic marker of long term survival in LARC patients, further studies are warranted to provide functional evidence to support these findings.

#1422

Dietary 2-deoxy-D-glucose (2-DG) reduces TLR-4 induced acute inflammation and prevents chronic inflammation driven carcinogenesis.

Sanjay Pandey,1 Saurabh Singh,1 Vandana Anang,2 Dhananjay K. Shah,1 Saurabh Seth,1 Anant N. Bhatt,1 Kailash Manda,1 Bal G. Roy,1 K Natarajan,2 Bilikere S. Dwarakanath3. 1 _Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, Delhi, India;_ 2 _Ambedkar center for Biomedical Research (ACBR), University of Delhi, Delhi, India;_ 3 _Sri Ramachandra University, Porur, Chennai, India_.

Inflammation is a critical component and contributor of carcinogenesis. Toll like receptors (TLRs) are key regulators of innate immunity and inflammatory signaling. Besides various microbe-associated molecular patterns, TLRs can also bind to endogenously generated damage associated molecular patterns (DAMPS) ligands such as heat shock proteins, surfactant protein A18 and homobility group box-1(HMGB1). While HMGB1, a TLR-4 ligand is over-expressed in several human neoplasms, preclinical studies in mouse tumor models have strengthened the role of TLR-4-HMGB1 axis in promoting carcinogenesis. TLR-4 dependent inflammation and stimulation is accompanied with a switch from respiratory to glycolysis dominated metabolism, which is critical for the differentiation and maturation of myeloid cells thus affecting the outcome of an inflammatory disease. In the present studies we investigated the potential of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) as an energy restriction mimetic agent for countering the acute and chronic inflammation which can be employed as a cancer preventive strategy. Oral administration of 2-DG in drinking water (0.4% w/v) for 7 days reduced the diethyl nitrosamine (DEN) induced acute inflammation reflected by reduced the serum levels of TNF(~4 fold) and IL-6( ~7 fold). when administered chronically, 2-DG reduced the incidence (~50%) and the number of papillomas (~84%) in DMBA-TPA skin carcinogenesis model, which correlated with the reduced levels of TNF. In an acute inflammation model established using LPS (a TLR-4 ligand), we observed that 2-DG reduced the systemic and macrophage specific inflammatory response in LPS injected mice. 2-DG (1mM) significantly reduced the levels of co-stimulatory surface markers (CD80 and CD86) and associated ROS and NOS effecter functions in mouse macrophagic cell line (RAW 264.7). These studies suggest that dietary administration of 2-DG can be used as a strategy for preventing acute inflammation and associated carcinogenesis.

#1423

The addition of bevacizumab to chemoimmunotherapy prolongs progression-free survival in patients with chronic lymphocytic leukemia (CLL) through modulation of the microenvironment.

PAOLO STRATI, TAIT D. SHANAFELT, BETSY LAPLANT, ADAM PETTINGER, CONNIE LESNICK, DANIEL NIKCEVICH, TIMOTHY CALL, WEI DING, CURTIS HANSON, NEIL KAY. _MAYO CLINIC, ROCHESTER, MN_.

Introduction. First-line chemoimmunotherapy (CIT) has significantly increased the complete remission (CR) rate for patients with CLL; however, relapse is a common event and strategies aimed at eradicating disease more effectively are needed. Vascular Epithelial Growth Factor (VEGF) plays a crucial role in the cross talk between CLL B cells and their microenvironment. In addition low baseline VEGF levels predict a better response to CIT. Testing the combination of an anti-VEGF agent with CIT was therefore assessed by us in upfront CLL patients.

Methods. Here we report the results of a phase II open-label randomized trial comparing the combination of bevacizumab, an anti-VEGF monoclonal antibody, with pentostatin, cyclophosphamide and rituximab (PCR-B) to PCR alone as front-line therapy in previously untreated patients with CLL. VEGF, b-fibroblast growth factor (FGF), thrombospondin (TSP)-1, chemokine ligand (CCL)-3 and CCL-4 plasma levels were measured at baseline and at time of response assessment.

Results. Between 01/2009 and 01/2013, 62 patients were accrued in the study, 32 in the arm A (PCR-B) and 30 in arm B (PCR alone). A higher rate of grade 3-4 cardiovascular toxicity was observed with the use of PCR-B (34% vs 0%, p<0.001): this included 7 cases of hypertension, 2 cases of congestive heart failure, 1 myocarditis, and 1 case of torsade de point. A higher CR rate (50% vs 33%, p=0.21) and a significantly longer median progression-free (p=0.04) and treatment-free (p=0.05) survival were achieved with the use of PCR-B. No difference in chemokine kinetics was observed between the 2 arms, except for VEGF, which significantly increased after PCR-B. In the PCR-B arm, chemokine kinetics for CCL-3 (median drop 5.0 vs. median drop 31.8, respectively; p=0.02) and CCL-4 (median drop 23.8 vs. median drop 120.9, respectively; p=0.01) were significantly associated with achievement of CR. No significant correlations were found between cytokine plasma levels and responses for patients treated in the PCR only arm.

Conclusions. The addition of bevacizumab to CIT is safe and effective, although associated with higher cardiovascular toxicity, in particular hypertension. The addition of bevacizumab produces higher CR rates, translating into improved progression-free survival, and is associated with decreased plasma levels of CCL-3 and CCL-4.

#1424

Predicting response to immune checkpoint therapy using an mutation burden threshold.

Anshuman Panda,1 Anil Betigeri,2 Kalyanasundaram Subramanian,2 Kim Hirshfield,3 Lorna Rodriguez,3 Shridar Ganesan,3 Gyan Bhanot3. 1 _Department of Physics, Rutgers University, Piscataway, NJ;_ 2 _Strand Life Sciences, Bangalore, India;_ 3 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Treatment with antibodies to PD-1 or CTLA-4 leads to prolonged response in some cancer patients. Patients whose tumors have a high mutational burden have a better response to anti-CTLA-4 treatment with ipilimumab in melanoma5, and to anti-PD-1 treatment with pembrolizumab in non-small cell lung6 and colorectal cancer7. These results suggest that a sufficiently high non-synonymous somatic mutation burden in the tumor may lead to protein alterations that serve as neo-antigens to stimulate CD8 T-cell immune response. This immune response may be blocked by the tumor by engaging the immune checkpoint pathway, making such tumors especially vulnerable to immune checkpoint disruption. However, the specific criteria to identify patients likely to respond to immune checkpoint therapy have remained elusive so far.

Using somatic mutation and RNA-Seq data from TCGA, we propose that there exists a mutational threshold, which we call the "Activated Immune Mutational" (AIM) Threshold, which can identify patients likely to respond to immune checkpoint therapy. Compared to AIM- patients (potential non-responders), AIM+ patients (potential responders) have tumors that meet four criteria: (1) a high non-synonymous mutation burden; (2) a high level of immune infiltration in the tumor; (3) a high CD8 T cell fraction in the leukocyte component of the immune infiltrate plus high expression of the T cell marker CD8A; and (4) a high expression of immune checkpoint genes PD-1, PD-L1, PD-L2, CTLA-4.

We find that in melanoma, endometrial, colon, cervical and breast cancer, a clear AIM threshold satisfying all four criteria exists. The distribution of mutations in AIM- versus AIM+ tumors for these cancers is very different. In the former, there are high frequency somatic mutations in only a few genes, while in the latter, high frequency somatic mutations are found in many genes distributed over the whole genome.

Pathological analysis of high-resolution images from 150 TCGA tumors (30 from each of the five tumor types) validated the presence of significantly higher lymphocytic infiltration in the AIM+ tumors compared to AIM- tumors. We show that AIM+ tumors can be identified using sequencing assays which are currently in clinical use. Finally, survival analysis in melanoma patients from TCGA treated with immunotherapy will be presented.

### Special Populations, Supportive Care, and Survivorship Research

#1425

Prospective evaluation of serum unbound fraction of docetaxel as a key determinant of its pharmacokinetics and neutropenia in the elderly.

Hirotsugu Kenmotsu,1 Chiyo K. Imamura,2 Akira Ono,1 Shota Omori,1 Kazuhisa Nakashima,1 Kazushige Wakuda,1 Tetsuhiko Taira,1 Tateaki Naito,1 Haruyasu Murakami,1 Toshiaki Takahashi,1 Yusuke Tanigawara2. 1 _Shizuoka Cancer Center, Shizuoka, Japan;_ 2 _Keio University School of Medicine, Tokyo, Japan_.

Background: Although elderly patients are at increased risk for treatment-related toxicities of docetaxel (DOC), total body clearance (CL) is unaltered by advanced age. Because the elderly patients show large interindividual variability in pharmacokinetics and toxicities, the profiles of DOC in older patients remain unclear. The present prospective study was conducted to elucidate a key determinant of pharmacokinetics and hematological toxicities of DOC in the elderly focusing on the unbound fraction in serum.

Patients and Methods: DOC was administered at a dose of 60 mg/m2 once every 3 weeks to patients with non-small cell lung cancer. Pharmacokinetics and toxicity were assessed during the first cycle of therapy. Blood samples were taken immediately before and at the end of the DOC infusion, and at 3 points until 24 h. Unbound DOC was obtained by equilibrium dialysis, and DOC concentrations were determined by the UPLC-MS/MS method. Population pharmacokinetic (PPK) analysis was performed using NONMEM version 7.3.

Results: Of 51 patients treated, median age was 66 years (range 34-84 years), alpha1-acid glycoprotein (AAG) level was 1.21 g/L in median (range 0.5-2.64 g/L), and albumin (ALB) level was 37 g/L in median (range 22-48 g/L). The estimated CL, area under the concentration-time curve (AUC), volume of distribution of the central component (V1), volume of distribution at steady state (Vss) and serum unbound fraction were 23.91 ± 5.78 L/hr, 4.22 ± 1.24 mg/hr/L, 9.10 ± 0.94 L, 128.44 ± 9.09 L, and 0.08 ± 0.03, respectively (mean ± SD). In the PPK analysis, levels of serum proteins were significant covariates affecting pharmacokinetics of DOC but age was not statistically significant. AAG and ALB were associated with CL and V1 (both p<0.01), respectively. In addition, significant correlation was observed between unbound fraction and AAG (p<0.01) which is the major binding protein of DOC, whereas ALB showed marginal correlation (p=0.055). In 49 patients evaluable of adverse events, decrease in neutrophil counts was well correlated with AUC of unbound DOC (p<0.05), but not with total drug AUC.

Conclusion: Pharmacokinetics of DOC, which is bound to serum proteins extensively (>90 %), was influenced by not only AAG but also ALB levels in serum. The key determinant of DOC pharmacokinetics was the unbound fraction caused by individual variations in AAG and ALB levels regardless of patients' age. The present results suggest that the severe hematologic toxicity induced by higher unbound exposure can be predicted by serum protein levels in patients treated with DOC and it is useful especially for the elderly.

#1426

Racial variation in annexin A2(AnxA2) gene expression and poor outcome in triple-negative breast cancer.

Lee D. Gibbs, Jamboor K. Vishwanatha. _University of North Texas Health Science Center, Fort Worth, TX_.

Background: Triple-negative breast cancer (TNBC) is identified by the absence of three major receptors that drive most breast cancers. The incidence of TNBC is also associated with health disparity due to its disproportionate occurrence in African American (AA) women. Our previous studies and others have shown that AnxA2 is overexpressed in TNBC, but its association with racial variation and outcomes is unknown.

Methods: AnxA2 gene expression was evaluated in breast cancer subtypes from The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) mRNA database that include 1,094 patients. Associations between clinical outcomes and AnxA2 gene expression were tested in a genome wide association study of combined publicly available datasets that include 4,147 patients.

Results: AnxA2 gene expression was significantly increased in TNBC in comparison to other breast cancer subtypes. Furthermore, AnxA2 gene expression was significantly elevated in AA women in comparison with Caucasian (CA) women and was associated with the disproportionate occurrence of TNBC in AA women. High expression levels of AnxA2 was associated with reduced overall survival (hazard of death = 2.66; 95% confidence interval {CI] = 1.14 - 6.25, P = 0.0192), reduced relapse free survival (hazard of relapse = 1.45; 95% confidence interval {CI] = 1.12 – 1.89, P = 0.0051), and reduced distant metastasis free survival (hazard of metastasis = 1.7; 95% confidence interval {CI] = 1.00 – 2.91, P = 0.048). AnxA2 gene expression was not associated with poor outcome in other intrinsic breast cancer subtypes, such as Luminal A, Luminal B, and Her-2, indicating the specific association of AnxA2 with the aggressive behavior of TNBC.

Conclusion: AnxA2 overexpression is associated with racial variation and is a potential prognostic candidate for TNBC.

Impact: AnxA2 has potential prognostic value, implicating a role for AnxA2 in the

aggressive biology of TNBC in AA women.

#1428

The program for cancer detection, diagnosis, and treatment technologies for global health: Translating affordable, minimally invasive point-of-care technologies to less-resourced settings.

Michael Gwede,1 Paul Pearlman,1 Pushpa Tandon,1 Miguel Ossandon,1 Lokesh Agrawal,1 Houston Baker,1 Vinay Pai,2 Tiffani Lash2. 1 _National Cancer Institute, Rockville, MD;_ 2 _National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD_.

Cancer kills more people worldwide than HIV/AIDS, tuberculosis and malaria combined, and low-and-middle income countries (LMICs) bear the majority of this burden. Success in detection, diagnosis and treatment has been reported in LMICs through the use of low-cost point-of-care (POC) technologies, and the program presented offers a unique pathway to this POC market by funding multidisciplinary investigative teams to adapt and clinically validate existing technologies for cancer detection, diagnosis and treatment in low-resource settings.

Each project consists of an adaptation phase (2 years: $500k total costs/year) and validation phase (3 years: $1M total costs/year). Projects are selected through NIH peer review process by a carefully-selected special emphasis panel briefed on the goals of the program. Projects are competitively vetted for Phase II funding based on completion of Phase I milestones.

The program currently supports seven technologies for cancer detection, diagnosis and treatment, each of which is progressing towards experimental and clinical validation. The first project is an LED-based photodynamic therapy device for oral cancer, that has similar efficacy in vivo and ex vivo as existing laser phototherapy. Another supported project is an automated high resolution microendoscope for cervical cancer detection, with an impressive histological concordance in detecting CIN2/3 (90%+ for CIN3). Two cervical cancer cryotherapy projects are funded: a cryopen, that can achieve an approximately 4.0 mm depth of necrosis (>90% of disease) for cervical cancer treatment, and an efficient cryopop device that consumes less than 10% of CO2, compared to commercially-available devices and exhibits comparable therapeutic efficacy in ballistic gel studies. The program is also supporting two POC tests, a HPV test and a Hepatitis C viral antigen level and viral load detection. Additionally, a breast cancer triaging device/algorithm, with 95% sensitivity and capabilities to reduce false positive detection rate by 40%, is also being supported. Each project has its own detailed outline for Phase I and Phase II studies, which will be highlighting in our presentation.

The program is in the process of adding another six projects, and it is anticipated that by year seven of the program, at least nine projects will have progressed through optimization, clinical validation, and business planning for commercialization and field/clinic dissemination. Through these process, we will uniquely accelerate these technologies for success in clinical translation.

#1429

The effect of payer status on early stage laryngeal cancer survival.

Samip R. Master, Peter Zhang, Glenn Mills, Runhua Shi. _Louisiana State University Health Sciences Center, Shreveport, LA_.

Background

It is estimated that there will be 13,560 new cases of larynx cancer and an estimated 3,640 people will die of this disease, in 2015. There are many risk factors affecting survival outcomes in larynx cancer. Previous studies on laryngeal cancer found several discrepancies for mortality in patients with various racial and socioeconomic backgrounds. However, further studies indicate that reduced survival outcomes for African-American or uninsured patients are not affected by tumor biology hinting that some disparities can be overcome through healthcare equity. In the wake of the Affordable Care Act and its impact on insurance coverage, evaluating the effect of insurance status on health outcomes is urgently necessary. This study characterizes the relationship between payer status and overall survival for larynx cancer patients by analyzing data from the large National Cancer Data Base (NCDB).

Methods

We analyzed data from the National Cancer Data Base of 22,177 patients with laryngeal cancer of stage I and II who received laser surgery or radiation treatment. The outcome variable was overall survival and the primary predictor variable was payer status. Additional variables addressed and adjusted for included sex, age, race, Charlson Comorbidity index, education, income, distance traveled facility type, diagnosing/treating facility, treatment delay, stage, grade and type of treatment received.

Results

Among these 22,177 patients, approximately 90% were over age 50 years, and 80% of the patients were male. 48.5%, 39.4%, 5.4%, 3.8% and 2.7% of patients had Medicare, private, Medicaid, uninsured, and unknown status, respectively. There was 83 % of patients received radiation treatment only whereas 5.2 % received laser surgery and 3.2 % received both laser surgery and radiation.

In multivariate analysis, after adjusting for secondary predictor variables, payer status was a statistically significant predictor of overall survival.

Relative to privately insured patients, patients with Medicaid had a 76% increased risk, no insurance had a 29% increased risk, Medicare had a 38% increased risk and unknown insurance had a 25% increased risk of mortality. Compared to patients without treatment (no radiation, no laser surgery), patient with laser surgery, patients with laser and radiation therapy were 77% and 76% less likely to die while patients receiving radiation therapy was not significant different from no treatment.

Conclusion

We observed that payer status has a statistically significant relationship with overall survival from laryngeal cancer. This remained true after adjusting for other predictive factors. Medicaid Medicare and uninsured patients had the highest mortality while privately insured patients had the lowest mortality. Further research is necessary how the disparities associated with different types of insurance result in inferior treatment outcomes and the ways to address them.

#1430

Do race and gender independently predict risk factors associated with the Human papillomavirus.

Rebecca L. Rohde,1 Nosayaba Osazuwa-Peters,2 Eric Adjei Boakye,3 Rajan Ganesh,1 Ammar Moiyadi,1 Adnan S. Hussaini,1 Mark A. Varvares4. 1 _Saint Louis University School of Medicine, Saint Louis, MO;_ 2 _Saint Louis University, Department of Otolaryngology - Head and Neck Surgery, Saint Louis, MO;_ 3 _Saint Louis University, Center for Outcomes Research (SLUCOR), Saint Louis, MO;_ 4 _Harvard Medical School, Department of Otolaryngology, Boston, MA_.

Purpose

To assess racial and gender differences, among a drag racing population, as predictors of sexual risk factors associated with HPV infection, including number of oral sexual partners, age of first vaginal sex, and age of first oral sex.

Methods

A cross-sectional study was conducted at a drag racing event on September 12-13, 2015 in Madison, Illinois. Independent variables were gender and race. Outcome variables were number of oral sexual partners (high vs. low), age of first vaginal sex, and age at first oral sex. Multivariable logistic regression was used to examine the association between race and gender, and number of sexual partners. Multivariable linear regression analysis assessed the association between race and gender, and age of first vaginal sex, and age at first oral sex.

Results

Three hundred and one individuals took our survey. Of these respondents, 63% were male, and 65% identified as African Americans. Mean age for this population was 48 ± 13 years. Approximately 45% of participants reported they have had four or more oral sexual partners. Mean age for first vaginal sex was 17 ± 3 years, 20 ± 6 years for first oral sex. Multivariate logistic regression revealed that gender was a significant predictor of number of oral sexual partners, but race was not significant. Men compared to women were more likely to have a high number of oral sexual partners (OR = 2.08; 95% CI: 1.23, 3.57). In multivariable linear regression, gender and race were significant predictors of age of first vaginal sex, and age at first oral sex. Men were more likely to have earlier vaginal sexual debut as compared to women (β = -1.13; 95% CI: -1.82, -0.43) as were African Americans compared to Caucasians (β = -1.14; 95% CI: -1.82, -0.46). Men were also more likely to have earlier oral sexual debut as compared to women (β = -2.51; 95% CI: -3.98, -1.04). Caucasians were more likely to have earlier oral sexual debut as compared to African Americans (β = 2.44; 95% CI: 1.02, 3.87).

Conclusions

Race and gender were independent predictors of HPV risk factors. Males were more likely to initiate earlier and engage in both oral and vaginal sexual behaviors that are high risk for developing HPV. Caucasians were more likely to initiate earlier, and engage in oral sexual behaviors that are high risk for developing HPV. These findings provide impetus for the development of targeted educational interventions aimed at improving knowledge of these sexual risk factors among Caucasians and males, in the community.

#1431

Incidence and natural history of cardiovascular toxicity associated with biologic and targeted therapies used in hematologic malignancies.

Jan S. Moreb, Haneen Mohammad, Lindsay McCullough, Anita Szady, Leslie Pettiford, Alexandra Lucas. _University of Florida, Gainesville, FL_.

INTRODUCTION: Unanticipated cardiac toxicity has been reported with anti cancer targeted and biological treatments. In this retrospective study, we aim at identifying the number of cardiac toxicity cases among patients with hematologic malignancies (HM) who were treated with targeted therapies over 10-year period with emphasis on natural history and outcomes.

METHODS: With the help of the Faculty Practice Decision Support (FPDS) team, we used 114 diagnosis codes for HM and 17 codes for cardiac diseases in order to mine our electronic medical records (EPIC) and identify patients with HM and specific cardiac problems, then sorted them according to whether they received known biologic agents such as thymidine kinase inhibitors (TKIs), proteasome inhibitors, monoclonal antibodies, hypomethylating agents, and immunomodulatory drugs.

RESULTS: FPDS team provided us with a file that contained medical records of 820 patients that had both HM and cardiac abnormalities. We pulled out 53 patients who received the drugs of interest. We were able to confirm cardiac toxicity defined mainly by left ventricular ejection fraction (LVEF) of < 50%, arrhythmias, or ischemic cardiovascular event in 44 patients. Ten patients were excluded because of pre-existing cardiac disease. Thus the final analysis was performed on the remaining 34 patients. The median follow-up was 9 mo (range, 1-78). The median age of this group was 66 years (range, 27-81), male/female 19/15, 26 Caucasians, and 15 were still alive. The median time from exposure to development of cardiac events was 120 days (range, <1-300). The diagnosis distribution was as follows: multiple myeloma 16, B-cell non-Hodgkin's lymphoma (NHL) 10, follicular NHL 4, Philadelphia positive acute lymphoblastic leukemia 3, MDS 1. Number of patients (n) who received suspected drugs contributing to the cardiac toxicity: bortezomib (8), carfilzomib (4), imatinib (2), dasatinib (4), Rituximab (13), and lenalidomide (3). Twenty one patients had no prior cardiac disease, while 11 had known coronary artery disease and 2 had known arrhythmias, while only 9 had no chronic co-morbidities. Fourteen patients did not receive anthracyclines including 2 who received rituximab. Nine patients had elevation in troponin and were diagnosed with NSTE myocardial infarction at the time of cardiac toxicity. Among the 19 dead patients, only 6 died of cardiac causes, while the rest died of their cancers. Repeat echocardiograms showed worsening of LVEF in 5 patients and improvement in 15 patients.

CONCLUSIONS: About 4.1% of patients with HM experience unanticipated cardiac toxicity with related mortality of 17.6%. Most patients do well with stable compensated cardiac function with objective improvement in LVEF. Whether Rituximab can cause cardiac toxicity is controversial and we will present specific arguments.

#1432

One stop screening for multiple cancers: 10 year experience of an integrated cancer prevention center.

Ari Leshno, Shiran Shapira, Eliezer Liberman, Eyal Gur, Hanoch Elran, Sarah Kraus, Miri Sror, Amira Harlap-Gat, Lior Galazan, Maayan Jean, Galit Aviram, Arie Blachar, Ada Kessler, Orit Golan, Shlomi Benjamin, Menachem Moshkowitz, Nadir Arber. _Tel Aviv Sourasky Medical Center, Tel Aviv, Israel_.

BACKGROUND: Cancer is the leading cause of mortality worldwide. Prevention and early detection is the key strategy for reducing cancer morbidity and mortality

METHODS: Between 2006 and 2015 consecutive, asymptomatic, apparently healthy adults, aged 20-80 year, were screened on the same day for early detection and prevention of 11 common cancers (Skin, oral cavity, thyroid, breast, lung, ovarian, cervix, uterus, prostate, testicles, colon, lymph nodes) that account for 70-80% of cancer mortality. Extensive clinical and epidemiological data was obtained from all patients. Peripheral blood DNA was extracted from all patients.The APC I1307K and APC E1317Q polymorphisms were evaluated on all patients

RESULTS: A total of 4,984 (49.1%) men and 5,157 (50.9%) women, aged 47.8±11.5 and 46.6±11.6 year respectively were screened. Malignant lesions were found in 333 (3.3%) of the general screenees. The most common cancers were skin (153, 1.5%), breast (40, 0.4%), gastrointestinal tract (38, 0.3%), gynecological (30, 0.3%) and prostae (26, 0.3%).Forty one patients (0.4%) had more than one cancer. Fifty (0.5%) patients died at a mean age of 69.3±13.9. 1,854 participants returned for at least one yearly follow-up. First degree family history of cancer, positive I1307K, alcohol, female gender, smoking and advanced ageassociated with increased cancer risk (Table 2). First degree family history of cancer and advanced age were associated with increased risk for multiple malignancies (OR 2.4, P< 0.01 and 1.1, P <0.001 respectively).

CONCLUSIONS: One stop shop screening for 11 common cancers in the setting of a multidisciplinary outpatient clinic is feasible and can prevent and detect cancer at an early stage  | |  | |

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

Table 1. Multivariate logistic regression of risk factor associated with malignancy

Risk factor | Sig. | OR | 95% C.I.for OR

|  | |

Lower | Upper

Family history of cancer | <0.001 | 3.039 | 2.386 | 3.871

APC I1307K | 0.001 | 1.941 | 1.321 | 2.852

Age | <0.001 | 1.056 | 1.047 | 1.066

Smoking | 0.001 | 1.524 | 1.198 | 1.940

Female gender | <0.001 | 1.609 | 1.277 | 2.029

Alcohol | <0.001 | 1.674 | 1.330 | 2.108

APCE1317Q | 0.461 | .681 | .245 | 1.891

#1433

A mobile app for health related quality of life and symptom assessment in patients with primary brain tumors in an outpatient oncology clinic.

Lindsay Rowe,1 Tuo Dong,2 Terri Armstrong,3 Megan Mackey,1 Mark Gilbert,1 Andra Krauze,1 Kevin Camphausen1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _Howard University, Washington, DC;_ 3 _The University of Texas Health Science Center at Houston, School of Nursing, Houston, TX_.

Purpose: Primary brain tumors, and their treatments, can have a significant impact on patient quality of life due to altered mentation, mood changes, memory loss, and neurologic deficits. It has therefore been suggested that patient related outcomes need to be included as endpoints for treatment efficacy. The objective of this project was to develop and evaluate a mobile application (GlioNCI), in reporting health-related quality of life measures and symptom scoring in patients with primary brain tumors treated and followed in an outpatient oncology clinic.

Experimental Procedures and Results: Apple's Xcode Integrated Development Environment (IDE) was used to develop GlioNCI using the Swift programming language. The parameters and scores chosen had previously been validated in primary CNS tumors. Medication and steroid use is captured at each encounter, as are seizure characteristics, frequency and management. Health related quality of life questionnaires included portions of the EORTC QLQ-C30, and EORTC QLQ-BN20, and MD Anderson Symptom Inventory Brain Tumor Module, which have been validated in patients with cancer and primary CNS tumors respectively. Patients' symptoms and effects on function are collected with the Activities of Daily Living scale, and the Instrumental Activities of Daily Living Scale. Mood and mental state were addressed using the Hospital Anxiety and Depression Scale. Separate questionnaires for different encounters and time points in outpatient treatment were included to assess care provider, as well as patient related measures, allowing for flexibility of the app to the needs of both. A mobile app was developed to collect, and archive in a database, input and feedback from both patients and care providers in oncology clinics.

Conclusion: The creation of a patient centered quality of life and symptom assessment mobile app, following patients with primary brain tumors over their course of treatment and in follow-up is feasible. Pilot testing and patient evaluation of the application in an oncology clinic will be used to validate GlioNCI as a tool in outpatient clinics.

#1434

Diabetic metastatic castration-resistant prostate cancer patients administered metformin during docetaxel chemotherapy have improved prostate cancer-specific and overall survival.

Michelle Mayer, Laurence Klotz, Vasundara Venkateswaran. _Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada_.

Introduction and Objective: Docetaxel (DTX) chemotherapy is currently one of the only treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC). Although DTX treatment has been shown to provide survival benefits, it is not curative. Thus, there is a continued need to improve the therapeutic options available for mCRPC patients. One approach is combining DTX with other agents that enhance its effectiveness (i.e. chemosensitizers).

Metformin, a commonly prescribed and well-tolerated oral biguanide used to treat type II diabetes, has been shown to exert anti-neoplastic effects in several types of solid tumors, including prostate cancer. More specifically, metformin has been shown to improve the efficacy of chemotherapy in breast cancer and colon cancer models. Niraula et al (2013) evaluated the effect of metformin combined with DTX in prostate cancer patients who participated in the TAX 327 trial. Metformin did not have a statistically significant additive or synergistic effect with docetaxel, but only 38 patients were included in the analysis.

There is very limited data examining the combined effect of metformin and DTX in prostate cancer patients. Therefore, the objective of this study is to determine if diabetic mCRPC patients administered metformin during DTX treatment have improved prostate cancer-specific (PCa-specific) and overall survival (OS) when compared to nondiabetic mCRPC patients receiving DTX therapy.

Methods: The primary objective of this retrospective study is to analyze patient data from the Ontario Cancer Registry (OCR), Ontario Diabetes Database (ODD), Canadian Institute for Health Information Discharge Abstract Database (CIHI DAD), National Ambulatory Care Reporting System (NACRS), Ontario Health Insurance Plan (OHIP), and the Registered Persons Database (RPDB) evaluate whether there is a difference in PCa-specific and OS between diabetic mCRPC patients administered metformin during DTX treatment and non-diabetic mCRPC patients receiving DTX treatment. The databases described previously are all accessible through the Institute for Clinical Evaluative Sciences (ICES) in Ontario, Canada.

Data has been collected and linked at ICES and a research-ready, anonymized dataset has been provided to our research group. Since receiving access to the dataset from ICES, statistical analysis (including multivariate Cox proportional hazards regression, logrank test, and Kaplan Meier survival curves) is in progress using SAS software.

Results and Conclusions: Data analysis is currently in progress. If metformin is shown to have a chemosensitizing effect in diabetic mCRPC patients, this would indicate a novel therapeutic approach that is needed for this patient population.

#1435

Psychoimmunological effect of depression on cervical carcinoma: Genome-wide long noncoding RNA implications.

Wanjun Dai, Yinhua Yu, Yu Kang. _Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China_.

Objective: Cervical cancer is the second most commonly diagnosed cancer and the third leading cause of cancer death among females in developing countries. Depression is a vital psychological factor for tumor progression. Long noncoding RNAs(LncRNAs) are a novel group of gene regulators and may contribute to the disease development. Herein, the study aims to discover the mechanisms of psychoimmunological effect of depression on cervical carcinoma from the perspective of long noncoding ribonucleic acid.

Methods: Six tumor tissues were obtained from Ib-IIa invasive squamous cell cervical carcinoma patients during laproscopic surgeries. They were divided into high-depressed group and low-depressed group according to the scoring system. LncRNA microarray experiments were performed. Expression levels of six lncRNA-mRNA pairs were confirmed by quantitative real-time PCR in other 20 patients' materials.

Results: We found that 1621 lncRNA transcripts are differently expressed between high-depressed and paired low-depressed samples, with 510 up-regulated and 1111 down-regulated (fold-change>2.0). Most of lncRNAs were found trans-regulated by three TFs with most credentiality: BCL11A,MEF2A and TBP. In the KEGG analysis, the most down-regulated mRNA involved was systemic lupus erythematosus (Maximun transcriptional domain coverage: 25.5%). In the LncRNA and mRNA Coexpression, the function of lncRNAs with most differently expressions was predicted in relationship with interleukin-1 receptor activity (Number of lncRNAs annotated: 15).

Conclusions: Our study identified that genome-wide LncRNA profiles are significantly altered in cervical carcinoma patients with worse depressed profiles, and the psychoimmunological effect of tumor progression needs further investigation.

Keywords: Depression, Cervical Cancer, Immunology, Long-noncoding RNA

#1436

The possible role of embryonic polarity axes for the normalization of tissue function induced by the interaction between human bilateral parts.

Ming Cheh Ou,1 Dennis Ou,2 Chung Chu Pang3. 1 _Taipei City Hospital, Taipei City, Taiwan;_ 2 _Department of Mechanical Engineering and Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA;_ 3 _Su Woman Hospital, Taipei City, Taiwan_.

Objective Ou MC decrescendo phenomenon (OuDP) is produced by placing the contralateral hand over a diseased location (Ou MC handing remedy [Ou HR]) to produce a zone under the hand with decreased pain or inflammation (Am J Emerg Med, 2012). Our objective is to report a series of patients with various diseases that resolved with application of the Ou HR, and review other studies of the OuDP. Methods During 2015, 18 patients with various diseases were treated at our clinic: 3 patients had infectious processes; 4 had joint and/or soft tissue pain; 3 had dysmenorrhea; 1 had preterm labor; 2 had abdominal pain/fullness after surgery; 1 had diabetes mellitus and difficulty walking; 2 had suspected malignancies and 1 had a malignancy. All patients were treated with the Ou HR, and their clinical outcomes were presented. Other studies examining the Ou HR and OuDP were reviewed (Proc Physiol Soc, 2014, 2015, Mascc/Isoo, 2015). Results Of the 18 patients, 17 self-administered the HR and 1 had the Ou HR administered by a therapist. In all 18 patients the symptoms or disease process was improved or cured. Prior published studies including 121 patients indicated that the Ou HR was effective in 92.3 to 100% of patients. Including prior studies, there were 3 patients with pathology-identified cancer: (1) uterine endometrioid carcinoma, stage IIIB, (2) uterine endometrial carcinoma, stage IA, (3) uterine leiomyosarcoma stage IB, and 5 patients with suspected cancer: (1) suspected pancreatic cancer in stage IA with isodense pancreatic nodule, elevated CA199 and dilated tortuous main pancreatic duct, (2) Ovarian tumor with solid and cystic part and omentum metastasis by CT scan, (3) menopausal ovarian solid tumor, (4) suspected inguinal lymph node metastasis after operation for cervical cancer IB, (5) suspected gluteal metastatic lesion of chronic myelogenous leukemia. All these 8 patients showed suppression or cure of the tumor with Ou HR treatment. Conclusions The OuDP shows to accelerate the recovery of a number of disease states, and the effect mainly occurs with the use of the contralateral hand, which suggests that the axes of embryonic polarity may impart the OuDP leading to the normalization of tissue function. The normalization of the tumor cells and microenvironment function may make tumor cells conform to the regulations with apoptosis, metastasis suppression, preventing uninhibited proliferation, minimizing angiogenesis and supervision by host immunological systems, which suppresses or cures the cancer. These findings warrant further investigation.

#1437

Relationship of G-CSF related bone pain (BP) with Bv8/PK2 expression in breast cancer (BC) patients (pts) treated with FEC100 adjuvant chemotherapy (CT).

Luigi Rossi, Anselmo Papa, Federica Tomao, Lucia Negri, Silverio Tomao. _University of Rome Sapienza, Rome, Italy_.

The Bv8/Prokineticins (PKs) are a new family of peptides identified in frog, fish, reptiles and mammals that signal through two highly homologous G-protein coupled receptors, PKR1 and PKR2. Over the past few years, several biological functions of Bv8/PK proteins have been elucidated. The high expression level of human Bv8/PK2 in bone marrow, lymphoid organs and leukocytes suggested an involvement of these peptides in hematopoiesis and in inflammatory and immunomodulatory processes. Neutrophils are the main endogenous source of Bv8/PK2 , while G-CSF is the the only cytokine able to activate PK2 expression in neutrophiles. Moreover G-CSF derived pain seems to be stronlgly related to blood levels of Bv8/PK. According to these results , we started a clinical experimental trial in order to explore role and expression of Bv8/PK proteins in patients treated with chemotherapy for early breast cancer and receiving G-CSF (filgrastim vs peg-filgrastim), investigating also the relationship with G-CSF related pain.

Methods: 20 BC pts were submitted to adjuvant FEC100. G-CSF was given as primary prophylaxis. 12 pts received Lenograstim (L: 5 doses, beginning 96 hours after CT) while 8 pts received Pegfilgrastim (P: 1 dose 24 hours after CT). Real-Time PCR and Elisa test were used to analyze Bv8/PK2 expression both in pts receiving L and P. Blood samples for Bv8/PK2 analysis were taken in different steps and days, in order to evaluate Bv8/PK2 expression according to CT time and G-CSF administration. Incidence of neutropenia (N), febrile-N and BP (NRS) were evaluated in all CT cycles. 6 healthy women were recruited as controls to evaluate their Bv8/PK2 basal level. Results: An impressive increase of Bv8/PK2 expression was observed both in L and P group. N occurred in 41.6% of pts treated with L (G3: 8.3%; G4: 16.7%) and in 75% of pts with P (G3: 12.5%; G4: 62.5%; 1 FN). In pts receiving L an incidence of BP occurred in 59.3% (NRS 7-8: 41%) with a mean duration of 6 days; in P group incidence was 37.5% (NRS 7-8: 37.5%), BP lasting 4 days. In all pts BP started within 24-48 hours after G-CSF administration, concurrently with Bv8/PK2 overexpression. Conclusions: Our preliminary results suggest an emerging role of Bv8/prokineticin system in occurrence of G-CSF related BP in primary prevention of CT induced N. Therefore, inhibition of Bv8/PK2 activity should constitute a promising therapeutic strategy to prevent inflammatory pain and perhaps other cancer characteristics.

#1438

Preserving genital nerve function in prostate cancer: imaging with a molecular imaging agent.

Lucia Le Roux, Daniela Ramos, Leo Flores, Dawid Schellingerhout. _MD Anderson Cancer Center, Houston, TX_.

Purpose: To establish proof-of-concept for the use of a molecular nerve imaging agent in demonstrating nerve tissue at risk during prostate surgery, and thus preventing iatrogenic sexual dysfunction. Erectile Dysfunction (ED) is a common complication of all forms of prostate cancer treatment, with incidence varying from 13-97% depending on the series quoted. There is broad agreement that treatment related damage to the neurovascular bundles is a root cause of the condition, with strong indications from many studies that preservation of the neurovascular bundles avoids post-treatment ED. The goal of this study is to use a novel neural imaging agent based on the non-toxic Tetanus Toxin C-fragment (TTc) to allow the visualization of the anatomy of the genital nerves, thus facilitating nerve-sparing therapies.

Materials and methods: C57 Black male mice were anaesthetized (1-2% isofluorane/oxygen) and injected with 40 µg/10 µl of fluorescently labeled TTc790 in the tunica albuginea of the left corpus cavernosum. Animals were imaged live over a period of 3 hours post injection with a Xenogen IVIS 200 small animal imager. Animals were then euthanized, and the complete male reproductive system was surgically exposed and imaged ex-vivo with a Zeiss AxioZoom16 dissection microscope. The cavernosal nerves, prostatic tissue with intact nerve plexi were dissected and prepared for cryo-sectioning followed by fluorescent immuno-histology using standard methods.

Results: En bloc excision of the prostate with the penis allowed imaging of the genital nerves using the retrograde transported neural marker, TTc790. We noted fluorescent TTc-related signal uptake along the expected course of the nerve tract ipsilateral to the injection site, and were able to clearly demonstrate the pelvic ganglion within the capsule of the prostate. Immuno-histological assessment with post cryo-sectioning confirmed our surgical microscopy findings, including our demonstration of the pelvic ganglion.

Conclusions: We demonstrate the use of TTc790 as a nerve imaging agent for showing the anatomy of the cavernosal nerves and pelvic ganglion complex by intra-operative fluorescent imaging. This suggests that, with further work, image guidance could be provided to physicians and surgeons to spare these nerves during prostate carcinoma treatments, allowing the preservation of genital nerve function and improving quality of life in prostate cancer survivors.

#1439

Pathological complete response after neoadjuvant chemotherapy predicts improved survival in all major subtypes of breast cancer: systematic review and meta-analyses of over 18,000 patients.

Laura Spring,1 Rachel Greenup,2 Kerry Reynolds,1 Barbara L. Smith,1 Beverly Moy,1 Aditya Bardia1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Duke, Durham, NC_.

Introduction:

Several randomized clinical trials have shown that neoadjuvant chemotherapy has similar long-term survival outcomes as compared to adjuvant chemotherapy. However, the prognostic significance of pathological complete response (pCR) after neoadjuvant chemotherapy remains unclear, particularly for HR+ (Hormone Receptor positive) and HER2+ (Human Epidermal growth factor Receptor-2 positive) breast cancer where adjuvant therapy after surgery could also have an impact on survival. The primary objective of this study was to conduct a systematic review of published neoadjuvant chemotherapy studies to comprehensively evaluate the association between pCR with subsequent breast cancer recurrence (BCR) and mortality.

Methods:

Based on PRISMA guidelines, a librarian-led search of PubMed from inception until November 2015 was performed to identify potentially eligible studies. Inclusion criteria were clinical trials or retrospective studies with at least one arm featuring neoadjuvant chemotherapy that reported pCR results as well as recurrence and/or survival stratified by the presence or absence of pCR. A total of 1,171 citations with associated abstracts were reviewed. Pooled odds ratios (ORs), 95% confidence intervals (CI), and p values were estimated for endpoints using the fixed and random effects statistical model.

Results:

18,772 patients from 49 studies met inclusion criteria. The majority of studies featured patients with stage II-III breast cancer and defined pCR as no residual invasive cancer in breast or nodes with residual noninvasive breast cancer allowed (ypT0/is ypN0). The overall pCR rate was 21.5% (range: 5.7-62%), with the highest pCR rates seen in HER2+ (range: 16.7-76%) and triple negative tumors (range: 14.3-67%). Achievement of pCR, as compared to absence of pCR, was associated with significantly reduced BCR (OR 0.33, CI: 0.28-0.39, p = < .001) and mortality (OR 0.28, CI: 0.21-0.36, p = < .001) overall. Similar association of pCR with reduced mortality was seen in all major breast cancer subtypes. The highest survival differential between pCR and no pCR was seen among patients who did not receive subsequent adjuvant chemotherapy.

Conclusions:

Achieving pCR following neoadjuvant chemotherapy is associated with significantly improved disease recurrence and survival across the various breast cancer subtypes, including HR+ and HER2+ breast cancer. These results provide further support for using pCR as a surrogate marker for survival outcomes as newer targeted therapies are evaluated in the neoadjuvant setting and might provide an efficient model for drug development in early breast cancer, but impact of adjuvant therapy needs to be carefully weighed in the analytical interpretation.

## IMMUNOLOGY:

### Immune Microenvironment and Antitumor Immunity

#1441

Systems-level interrogation of resistance mechanisms to immunotherapy through pooled shRNA screens.

Zhe Wang, Shruti Malu, Weiyi Peng, Jodi McKenzie, Rina Mbofung, Leila Williams, Sahil Seth, Tim Heffernan, Patrick Hwu. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Despite the impressive clinical efficacy of immunotherapy in some patients, many still do not respond or progress following an initial response. The molecular mechanisms underlying the tumor resistance in those non-responders remain largely undefined. To address this issue, we set out to perform high-throughput unbiased pooled shRNA screens to identify critical genes that confer immune resistance. Patient-derived melanoma cells were transduced with barcoded pooled lentiviral shRNA libraries that targeted the human kinome, followed by exposure to cytotoxicity mediated by autologous tumor infiltrating lymphocytes. Tumor cells were then subject to genomic deep sequencing and integrated analysis to identify depleted barcodes and corresponding genes. One identified candidate of particular interest is Aurora Kinase A (AURKA), a cell cycle regulator that has previously been shown to contribute to tumorigenesis and correlate with poor prognosis for cancer patients. Importantly, our independent screening with a bioactive compound library also implicated Aurora Kinase as an immune resistance candidate against T cell immunotherapy. Further studies showed that suppression of Aurora kinase activity with a pan inhibitor - AMG900, exhibited a synergistic cytotoxic effect with tumor infiltrating lymphocyte-mediated killing on autologous tumor cells from patients. Furthermore, our Nanostring analysis of tumor biopsies from patients that received adoptive T cell therapy revealed significantly increased expression of AURKA in tumors from non-responding patient when compared with responder counterparts. These results have substantiated the validity of our pooled shRNA screening platform. Further investigations are ongoing to elucidate the underpinnings of Aurora kinase-mediated tumor resistance to immunotherapy and to functionally and physiologically validate other putative targets we have identified.

#1442

Comprehensive TIL profiling by simultaneous DNA barcoding of proteins, RNA and natively paired immune receptors from millions of single cells.

Katherine L. Connor, Adrian W. Briggs, Stephen J. Goldfless, Sonia Timberlake, Brian J. Belmont, Christopher R. Clouser, David Koppstein, Francois Vigneault. _Juno Therapeutics Inc., Seattle, WA_.

We present high resolution single cell profiling of tumor infiltrating lymphocytes (TILs) for immune research and cancer target discovery. TILs hold the key to understanding anti-cancer immune responses but remain challenging to study due to their vast range of phenotypes, functions and abundance. Current approaches to TIL analysis such as immune receptor sequencing, flow cytometry, and RNA expression profiling are limited, since each approach gives only partial insights into a TIL repertoire and their results cannot be connected at the single cell level. We therefore built a technology for simultaneous determination of immune receptor sequences, surface protein phenotypes and RNA expression profiles from single TILs at unprecedented scale. Able to process millions of cells per experiment, the throughput of our emulsion-based DNA barcoding method allows deep and unbiased TIL profiling directly from tumor tissue without the need for cell sorting, stimulation or culture.

Briefly, single-cell dissociated biopsy tissue samples are stained with DNA-labeled surface marker antibodies and isolated into individual ~65-pL microdroplets by a microfluidic emulsion chip. Within the droplets, cellular RNAs are reverse transcribed into cDNA, and a barcoding step attaches droplet-specific DNA barcodes to all of the cDNAs as well as to the DNA labels carried by the marker antibodies. At the end of the reaction, all products associated with a single input cell carry the same DNA barcode sequence, allowing subsequent high throughput sequencing to assign the products to their cell of origin. During analysis the full-length immune receptor pairs for each B- and T-cell are reconstructed and the marker antibody tags are identified and quantified along with dozens of additional mRNA markers, resulting in a high-dimensional dataset containing integrated immune receptor clone, protein marker and RNA expression profiles of thousands to hundreds of thousands of individual lymphocytes in the input sample.

We have successfully applied our method to a variety of healthy blood and tissue samples including human cancers such as pancreatic, ovarian and lung cancer, profiling millions of B-cells and T-cells present in the samples. We identify likely tumor-reactive TILs by finding BCR or TCR clonal lineages associated with particular protein and RNA signatures, including expression of immunosuppression markers such as PD-1 and CTLA-4. Following candidate selection, full-length BCR and TCR pairs are synthesized and expressed for functional screening and target antigen identification. By providing an unprecedented view into TIL diversity across a wide range of disease and sample types, our platform also has widespread potential for basic and applied research into the patient immune response to cancer and the dynamics of immunotherapy.

#1443

IL-18 is associated with the onset and progression of gastric cancer.

Paul Nguyen,1 Rita Busuttil,2 Lisa Mielke,1 Gabrielle Belz,1 Alex Boussioutas,2 Matthias Ernst,3 Tracy Putoczki1. 1 _Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 3 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia_.

INTRODUCTION: Gastric cancer (GC) is the fourth most prevalent, and the third most common cause of cancer-related death worldwide. The disease is generally asymptomatic, and consequently is often diagnosed at an advanced stage when metastasis is present, and limited treatment options are available. Chronic inflammation is recognized as an integral component in the development and progression of GC, and is associated with increased infiltration of immune cells into the tumor microenvironment. The production of pro-inflammatory cytokines by these cells may contribute to tumor progression through activation of pathways promoting tumor cell survival and proliferation. Previous work from our lab has shown that therapeutic inhibition of the inflammatory cytokine interleukin (IL)-11 is effective in ameliorating disease progression in gastrointestinal cancer, however, it is unclear what role other pro-inflammatory cytokines, such as IL-18, might have in disease progression.

METHODS: To characterize the role of different pro-inflammatory cytokines in GC, we first analyzed microarray and qRT-PCR data of from human GC specimens and adjacent non-tumor tissue. Following the generation of a candidate list of cytokines deregulated in human GC, the role of individual cytokines in disease progression was examined using a validated mouse model of intestinal-type GC, referred to as gp130Y757F, by crossing into cytokine knock-out strains and monitoring tumor burden and the expression of genes and proteins classically associated with tumorigenesis.

RESULTS: We found that the expression pro-inflammatory cytokines including IL-1β and IL-18 were significantly elevated in the tumors of human GC patients compared to non-tumor tissue. In gp130Y757F mice, genetic ablation of IL-18, but not of IL-1β significantly reduced gastric tumor burden, which was associated with a reduction in the number of intratumoral macrophages, but not lymphocytes. This observation correlated with decreased expression of pro-inflammatory (Ifng, Tgfb1), antimicrobial (Reg3b, Reg3g), and tissue remodeling (Mmp9) genes, and a reduction in apoptosis in the gastric lesions in these mice.

CONCLUSION: Our results demonstrate that IL-18 has an important role in GC disease progression, and may serve as a potential therapeutic target.

#1445

STING activation in the tumor microenvironment with a synthetic human STING-activating cyclic dinucleotide leads to potent anti-tumor immunity.

Laura Hix Glickman,1 David B. Kanne,1 Shailaja Kasibhatla,2 Jie Li,2 AnneMarie Culazzo Pferdekamper,2 Kelsey Sivick Gauthier,1 Weiwen Deng,1 Anthony L. Desbien,1 George E. Katibah,1 Justin J. Leong,1 Leonard Sung,1 Ken Metchette,1 Chudi Ndubaku,1 Lianxing Zheng,3 Charles Cho,2 Yan Feng,3 Jeffrey M. McKenna,3 John A. Tallarico,3 Steven L. Bender,2 Thomas W. Dubensky,1 Sarah M. McWhirter1. 1 _Aduro Biotech, Inc., Berkeley, CA;_ 2 _Genomics Institute of the Novartis Research Foundation, San Diego, CA;_ 3 _Novartis Institutes for BioMedical Research, Cambridge, MA_.

Stimulator of Interferon Genes (STING) is a critical signaling sensor of the innate immune system. STING binds cyclic dinucleotides (CDN) produced by an intracellular enzyme in response to presence of intracellular DNA, including tumor-derived DNA. STING-mediated production of host type I interferon within the tumor microenvironment (TME) leads to the priming and activation of systemic tumor antigen-specific CD8+ T-cell immunity and tumor regression. A novel synthetic CDN derivative (ADU-S100), with superior STING-activating and anti-tumor properties, was developed for clinical translation. ADU-S100 has enhanced cellular uptake properties and stability, as compared to bacterial- and mammalian-derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING alleles, including a homozygous haplotype for the most refractory human allele (R232H), indicate that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through intratumoral (IT) administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provides long-lived immunologic memory. Mechanistic studies demonstrate that STING-mediated anti-tumor immunity is due in part to an acute pro-inflammatory cytokine response as well as a tumor-specific CD8+ T cell response. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, for example anti-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors.

#1446

Discovery of a distinct human lung tissue microbiome and its epidemiologic and clinical features.

Guoqin Yu. _National Cancer Institute, Bethesda, MD_.

The human lung tissue microbiota remains largely uncharacterized although a number of studies based on airway samples suggest the existence of a viable human lung microbiota. Here we characterized the microbiota taxonomic and derived functional profiles in 165 non-malignant lung tissue samples from cancer patients. We show that the lung microbiota is unique, differing from the microbiota of oral, nasal, stool, skin, and vagina samples. Microbiota diversity increases with environmental exposures, such as air particulates, residence in low to high population density areas, and pack-years of tobacco smoking. Moreover, the non-malignant lung tissue microbiota is altered in subjects with chronic bronchitis or advanced stage of lung cancer, and differs significantly from the microbiota in lung tumor samples. These findings provide insights on the human lung microbiota composition and function, and their link to human life style and clinical outcomes.

#1447

Influence of IL-17-secreting immune cells in pancreatic cancer stemness.

Yu Zhang,1 Ismet Sahin,2 Sonal Gupta,1 Anirban Maitra,1 Jennifer Bailey,3 Steven Leach,4 Florencia McAllister1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Texas A &M University-Kingsville, Kingsville, TX; _3 _The University of Texas Medical School at Houston, Houston, TX;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Little is known about how the immune system influences pancreatic cancer stemness. Using a murine model of pancreatic intraepithelial neoplasia (PanIN), we have previously reported that KrasG12D induces expression of IL-17 receptors (IL-17RA) on emerging PanINs, as well as pancreatic infiltration by IL-17-producing CD4+ and gamma-delta-T cells. Forced IL-17 over-expression dramatically accelerates PanIN initiation and progression, while inhibition of IL-17 signaling using either genetic or pharmacologic techniques effectively prevents PanIN formation. When IL-17 signaling was blocked using monoclonal antibodies on transgenic mice harboring PanINs, transcriptional analysis of Kras-mutated epithelial cells revealed significant down-regulation of multiple stemness-related genes: doublecortin-like kinase 1 (Dclk1) [2], Trefoil factor 1 (Tff1)[3], Sonic Hedgehog (SHH) [4], and others. Dclk1 has been recently described as a marker of tumor initiating cells and its functional role in cancer initiation and development is currently being studied. We found that adenovirus mediated-IL-17 overexpression in the murine pancreas harvesting Kras mutation results in PanINs with significantly higher number of Dclk1+ cells that PanINs generated in the presence of a luciferase control virus. Reversely, the genetic ablation of IL-17 in the hematopoietic compartment of the same type of mice resulted in PanINs with significantly lower number of Dclk1+ cells when compared with PanINs from mice transplanted with wild type (WT) bone marrow. We have found that in vitro stimulation of KPC cells (obtained from KrasG12D/+; LSL- Trp53R172H/+; Pdx1−Cre mice) with IL-17A results in direct induction of Dclk1, Aldh1 and SHH protein expression in a time-dependent and dose-dependent fashion, validating the results of our gene microarray in a different system and at the protein level. Furthermore, we have found that one of the mechanisms by which IL-17 induces Dclk-1 and ALDH1a1 is through activation of NF-KB canonical pathway. We found that IL-17 had no effect in KPC cells proliferation in 2D culture. However, stimulation of matrigel embedded- KPC cells with IL-17A resulted in significant increase in tumorspheres formation in vitro. But even more important, KPC cells in vitro incubated with IL-17A for 7 days have accelerated capacity for in vivo- tumor initiation when injected subcutaneously into syngeneic immune-competent mice, confirming the functional in vivo role that IL-17-immune secreting cells have in inducing pancreatic cancer stemness. Better understanding of the regulation that IL-17-secreting immune cells can impose on pancreatic cancer- tumor initiating cells would be of high importance given the potential translational value of blocking IL-17 signaling for pancreatic cancer prevention and treatment with the currently available IL-17 neutralizing monoclonal antibodies.

#1448

MDSC depletion delays primary tumor growth in syngeneic models of oral cavity cancer.

Paul E. Clavijo, Ruth Davis, Zhong Chen, Carter Van Waes, Clint T. Allen. _NIH, Bethesda, MD_.

Carcinogen-associated oral cavity cancers are a heterogeneous group of aggressive cancers with a high recurrence rate after definitive treatment and a poor 5-year survival. The genetic alteration rate of these cancers tends to be high, and many oral cancers express immune checkpoint molecules in the tumor microenvironment with potential to respond to checkpoint blocking immunotherapy. Local immunosuppression mediated by both the tumor cells and other infiltrating immune cells are likely a major mechanism of resistance to immunotherapy. Here, we investigated the role of myeloid derived suppressor cells (MDSCs) in a highly aggressive but poorly immunogenic syngeneic model of carcinogen-induced oral cavity cancer (MOC2). Performing a time course analysis of immune infiltrates, MOC2 tumors demonstrated robust recruitment of CXCR2+CSFR1+CCR2- MDSCs that peaked at 15 days following tumor transplantation. This intratumoral accumulation of MDSCs preceded draining lymph node and splenic MDSC accumulation by 6 and 9 days respectively. The accumulation of MDSCs corresponded to a sharp decrease in the presence of tumor infiltrating CD8+ T-lymphocytes (TIL), CD4+ TIL, FoxP3+CD4+ Tregs and CD3-NK1.1+ NK cells. As CD8+ TIL numbers decreased, cell surface expression of the lymphocyte activation markers CD69, CD44 and ICOS decreased with the checkpoint molecule CTLA-4 increased. Phenotypically, the great majority of tumor-infiltrating MDSCs were granulocytic MDSCs (Ly6GhiLy6Cint), while few were monocytic MDSCs (Ly6GloLy6Chi). In a CFSE-based functional T-lymphocyte suppression assay, sorted peripheral and tumor-infiltrating MDSCs strongly suppressed T-lymphocyte proliferation at MDSC:T-lymphocyte ratios as low as 1:32. Hypothesizing that decreased tumor MDSCs would impact primary tumor growth, MOC2 tumors were allowed to engraft to 100mm3 and depletion of granulocytic MDSCs was performed with a rat anti-mouse Ly6G depleting antibody (clone RB6-8C5). This depletion resulted in a statistically significant delay in MOC2 primary tumor growth. Flow cytometric analysis of tumor tissues revealed that MDSC depletion was transient with rapid rebound of MDSC tumor infiltration within days of depleting antibody administration. This data highlights the critical role that MDSCs play in local immunosuppression and suggest that the syngeneic MOC model represents a powerful tool to study MDSC pathobiology. Functional inhibition or elimination of MDSCs from the tumor microenvironment represents an exciting adjuvant therapy that may enhance the response to checkpoint inhibition in patients with oral cavity cancer.

#1449

In vivo **targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity.**

Serena Zilio,1 Jennifer Vella,1 Adriana De La Fuente,1 Vincenzo Bronte,2 Emilia Mazza,3 Silvio Bicciato,3 Paolo Serafini1. 1 _University of Miami, Miami, FL;_ 2 _University of Verona, Verona, Italy;_ 3 _University of Modena and Reggio Emilia, Modena, Italy_.

Introduction: Depending on their polarization, myeloid cells can either promote or restrain tumor progression. By secreting myelopoietic factors tumours promote myeloid cells polarization toward a phenotype that provides immune protection and that stimulate neoplastic cells growth, invasiveness, and metastasis. Chemokines and their cognate receptors are thought to play a key role in myeloid cell trafficking, however, their study in vivo is hindered by their signal redundancy and integration.

Methods: By taking advantage of a newly developed nanoplatform that allows the in vivo preferential transfection of tumor infiltrating myeloid cells (TIMC, aka Myeloid derived suppressor cells, MDSC, and tumor associated macrophages, TAM), we evaluated the effect of CCR-1, CCR-2, CCR-4, CCR-5 and/or CCR-7 silencing on TAMC trafficking, phenotype, and function.

Results: While CCR-4 and -7 targeted silencing did not affect tumor progression, simultaneous silencing of CCR-1 and CCR-5 significantly restrained tumor growth and greatly modifies tumor micro-environment. Mechanistic in vivo and in vitro analysis surprisingly indicate that myeloid cell trafficking was not altered whereas myeloid cell polarization was. In particular, CCR-1 and CCR-5 blockade restrained MDSC differentiation and promote the accumulation of retinoblastoma positive, neutrophil-like cells with a strong tumoricidal activity.

Discussion: Our data indicate that CCR-1 and CCR-5 engagement (most likely via CCL-3 and CCL-4) is necessary for the pro-tumoral polarization of myeloid cells in cancer. Our findings not only improve our understanding of myeloid cells differentiation in cancer but, also, strongly suggest that targeted inhibition of CCR1 and CCR5 on TIMC can be a new and effective anti-tumor immune-therapeutic strategy that convert pro-tumoral MDSCs into tumoricidal neutrophils.

#1450

Tumor-associated glycans interact with macrophages through class-A scavenger receptor.

Behjatolah Monzavi Karbassi, Thomas Kieber-Emmons, Steven R. Post. _University of Arkansas for Medical Sciences, Little Rock, AR_.

Introduction. Tumor initiation and growth are associated with modifications to the glycan structure of glycoproteins, glycolipids and proteoglycans present on the cell surface. Suppression of the anti-tumor immune response is a putative mechanism linking tumor-associated-glycans to tumor progression. Within a tumor microenvironment, the conversion of macrophages from an activated, immunological type (referred to as M1) to an alternatively activated, IL-10 secreting type (referred to as M2) suppresses the immune response and potentiates tumor progression. We previously reported that the class A scavenger receptor (SR-A)-mediated macrophage activation enhances production of IL-10, indicating promotion of an immunosuppressive M2 phenotype. Therefore, tumor glycans may suppress immune responses by interacting with macrophages and promoting an M2 phenotype. Such interactions are most likely mediated by pattern recognition receptors such as SR-A. In the current study, we investigated whether tumor glycans interact with macrophages via binding to SRA.

Methods. Binding of recombinant soluble SR-A to breast cancer cells and relevant glycan structures was assayed by flow cytometry and ELISA assays. N-glycosylation was inhibited by incubating cells with tunicamycin. Peritoneal macrophages were harvested from wild type (SR-A+/+) and knock-out (SR-A-/-) mice and used for ligand competition and functional assays. A peptide mimic of GlcNAc/GalNAc-containing tumor-associated carbohydrates was used for binding and competition assays.

Results. To determine whether carbohydrates present on tumor cells are SR-A ligands, we examined the binding of a soluble SR-A protein to tumor cells. We found that soluble SR-A bound to breast cancer cells but not to normal epithelial cells, and that this tumor-specific binding of soluble SR-A was abolished by treating cells with tunicamycin. These results demonstrate that cell surface tumor-associated glycans are ligands for SR-A. Using purified glycans, we found that soluble SR-A bound to multiple breast cancer-associated glycans. To assess the potential consequence of glycan binding to SR-A on macrophage function, we adhered primary macrophages to plates coated with a carbohydrate-mimetic peptide and found that SR-A+/+ macrophages bound to CMP-coated plates showed increased IL-10 production relative to SR-A-/- macrophages. The CMP also competed for acetylated LDL binding to macrophages, indicating that it is a ligand for SR-A. These results suggest that SR-A binding to carbohydrate promotes macrophages to adopt an M2 phenotype.

Conclusions. Together, our results indicate that tumor-associated glycans interact with macrophages via SR-A, and that this interaction leads to production of IL-10, which is a key characteristic of immunosuppressive (M2-like) macrophages.

#1451

Analysis of expression MHC class I chain-related gene A and B (MICA/B) in normal and tumor tissue.

Hormas Ghadially,1 Lee Brown,1 Arthur Lewis,1 Meggan Czapiga,2 Viia Valge-Archer,1 Robert W. Wilkinson1. 1 _MedImmune Ltd., Cambridge, United Kingdom;_ 2 _MedImmune LLC, Gaithersburg, MD_.

MHC class I chain-related gene A and B (MICA and MICB) are highly polymorphic proteins that are induced upon stress, damage or transformation of cells which act as a "kill me" signal through the NKG2D receptor expressed on Natural Killer, CD8+ and γδ T cells.

Experimentally, the MIC/NKG2D axis has been shown to be important for the recognition of tumour cells by cytotoxic cells of the immune system and many tumours have evolved strategies to evade the detection by NKG2D expressing cells, e.g. by shedding MIC from the cell surface.

Expression of MIC has been reported for most tumour types and in normal gastrointestinal tract epithelium but the published data is often difficult to interpret. Additionally, it is not clear how much of the protein is expressed on the cell surface as MIC cell surface expression is known to be regulated tightly on multiple levels.

A validated MICA/B IHC assay was developed using an in-house tool antibody to profile multiple frozen human normal and tumour tissue microarrays (TMA's) by both standard and confocal microscopy techniques.

Using a stringently characterised novel antibody that detects MICA as well as MICB this study describes the expression patterns in a wide range of tumours and normal tissues. With this method we generated data with unprecedented resolution, which enabled us to analyse the expression of MICA and MICB not only on the cellular but also on the sub-cellular level.

#1452

IL-15 overexpression protects cerulein-induced fibrosis in the mouse model of chronic pancreatitis.

MURLI MANOHAR, SATHISHA UPPARAHALLI VENKATESHAIAH, CHANDRASHEKARA PUTANAPURA MAHADEVAPPA, ALOK KUMAR VERMA, ANIL MISHRA. _DEPARTMENT OF MEDICINE, PULMONARY DISEASES, TULANE EOSINOPHILIC DISORDER CENTER, TULANE UNIVERSITY SCHOOL OF MEDICINE, New Orleans, LA_.

Introduction: Earlier, interleukin (IL)-6, IL-8, IL-1β and IL-10 are implicated in the pathogenesis of pancreatitis. Recently, we observed reduced level of IL-15 in cerulein- induced acute and chronic mouse model of pancreatitis. IL-15 is a pleiotropic cytokine that has an important role in innate immunity. Therefore, we tested the hypothesis that IL-15 overexpression improves the pathogenesis of cerulein-induced pancreatitis.

Experimental procedure: Accordingly, we tested our hypothesis by inducing chronic pancreatitis in rIL-15 pretreated Balb/c mice. Cerulein was given by repetitive intraperitoneal (i.p.) injections as reported earlier (50 μg/kg, 6 hourly injections/day, 3 days/week) along with the rIL-15 (5 μg in 100μl saline/ two-times/week/mice) for up to 4 weeks; the control mice received 100μl saline or saline with 5 μg IL-15. Mice were sacrificed 3 days after the last cerulein injection. Histopathological evaluation of H&E stained tissue sections was performed including tissue remodeling and the accumulation of collagen by Masson's trichrome stain in the tissue sections. Additionally, pro-inflammatory and pro-fibrotic gene i.e. TGF-β1, IL-6, IL-8, Collagen 1 and α-SMA levels were measured by ELISA, immunofluorescence, qPCR and immunoblot analysis.

Summary: We report acinar cells atrophy, induced inflammatiory cells and cytokines in the pancreas of cerulein treated mice along with perivascular collagen deposition compare to saline treated mice. Interestingly, rIL-15 treated and cerulein given mice showed significantly improved morphology of acinar cells and reduced perivascular collagen. Further, we also report that protein levels of TGF-β1 and α-SMA is also reduced in the pancreas of cerulein injected and IL-15 treated mice compare to cerulein treated mice by performing western blot and immunohistochemistry.

Conclusion: Our data show that IL-15 overexpression protects mice from cerulein-induced pancreatic pathogenesis including fibrosis. Taken together, rIL-15 therapy may be a novel strategy to treat pancreatic fibrosis, and may also helpful to treat pancreatic cancer patients by down-regulating tissue fibrogenesis.

Funding: NIH R01 DK067255 (AM), NIH R01 AI080581 (AM),

#1453

CXCR4 and CXCR7 homodimers and heterodimers play differential roles in breast cancer.

Irene del Molino del Barrio, Simi Ali, John Kirby, Annette Meeson. _Newcastle University, Newcastle upon Tyne, United Kingdom_.

The majority of breast cancer deaths are due to the metastasis of tumor cells to secondary organs. One of the most studied metastatic processes is the migration of chemokine receptor-expressing cancer cells towards their ligands in other organs, a mechanism where the receptor CXCR4 and the chemokine CXCL12 play key roles. This process, however, may be affected by the expression of the atypical receptor CXCR7, which has recently been linked to breast cancer progression. Indeed, staining of CXCR7 in patient samples is strongly positive in both lobular and ductal breast cancers. Given CXCR7's ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differs from their lone expression in terms of their signaling and internalization pathways.

In this study we created stable single CXCR4 or CXCR7 CHO transfectants or double CXCR4-CXCR7 transient transfectants to examine ligand-dependent receptor internalization. The presence of heterodimers was evaluated using fluorescence resonance energy transfer (FRET). We found that in CHO-CXCR4 the receptor was degraded after a 30 minute stimulation with 10 nM CXCL12, whilst in CHO-CXCR7 the receptor returned to the surface in under 2 hours, reaching higher CXCR7 levels than before treatment. RT-PCR data suggested that this increase was partially due to induction of further CXCR7 expression. When the receptors were coexpressed, a similar pattern could be seen, although the CXCR7 recycling levels were not as high. CXCR4 degradation could be partially inhibited by pretreating cells with 1 μM lactacystin, a proteasome inhibitor. When cells were stimulated for 1 hour with 1 nM VUF11207, a CXCR7 agonist, no significant effect was seen on CHO-CXCR4 whilst CHO-CXCR7 cells showed internalization and recycling. Supporting RT-PCR data showed that the agonist did not trigger CXCR7 expression like seen with CXCL12. Interestingly, when coexpressed, both CXCR4 and CXCR7 showed degradation after internalization. Similar results were observed with MDA-MB-231 cells transfected with CXCR4 or CXCR7.

The effects of the binding were also assessed by measuring ERK phosphorylation by Western blot and cell-based ELISA. An early but transient (5-15 minutes) ERK activation was seen in CHO-CXCR4 cells, whilst CHO-CXCR7 cells showed a sustained activation of up to 2 hours. On the other hand, CHO-CXCR4-CXCR7 cells showed a very strong phosphorylation at 5 minutes that quickly diminished. The role of the receptors in invasion and migration in the presence of CXCL12 was also assessed.

In summary, this study highlights how CXCR4/CXCR7 heterodimerization can create a distinct signaling entity with unique properties and alter receptor pharmacology. This could be used to create therapeutic compounds to reduce breast cancer cell migration.

#1454

IL-4 modulate the tumor microenvironment and response to cancer therapies.

Shuku-ei A. Ito, Hidekazu Shirota, Chikashi Ishioka. _Tohoku University, IDAC, Sendai, Japan_.

recent findings show that immune cells constitute a large fraction of the tumor microenvironment and modulate tumor progression. Clinical data indicate that chronic inflammation is present at tumor sites and that IL-4 in particular is up-regulated. Thus, we tested whether depletion of IL-4 affected in tumor immunity. Current results demonstrate that administration of neutralizing antibody against IL-4 enhances anti-tumor immunity and improve the response to Taxol-based chemotherapy and CpG ODN immunotherapy. Depletion of IL-4 also alter the tumor microenvironment, reducing the generation of immunosuppressive M2 macrophages and myeloid derived suppressor cells and enhancing tumor specific CTLs. These findings suggest that IL-4 impact anti-tumor immunity and constitute an attractive therapeutic target to reduce immune suppression in the tumor microenvironment and thus enhance the efficacy of cancer therapy.

#1455

IL-4 derived from T follicular helper cells in tumor draining lymph nodes regulate myeloid cell properties and anti-tumor immunity.

Hidekazu Shirota,1 Dennis M. Klinman,2 Shuku-ei A. Ito,1 Chikashi Ishioka1. 1 _Tohoku University, Sendai, Japan;_ 2 _National Cancer Institute, Frederick, MD_.

Recent findings show that immune cells constitute a large fraction of the tumor microenvironment and modulate tumor progression. Clinical data indicate that chronic inflammation is present at tumor sites and that IL-4 in particular is up-regulated. Current results demonstrate that T follicular helper (Tfh) cells arise in tumor draining lymph nodes where they produce an abundance of IL-4. Deletion of IL-4 expressing Tfh cells improves anti-tumor immunity, delays tumor growth and reduces the immunosuppressive activity of myeloid-derived suppressor cells (MDSC) and M2 macrophages. These findings suggest that IL-4 from Tfh cells impact anti-tumor immunity and constitute an attractive therapeutic target to reduce immune suppression in the tumor microenvironment and thus enhance the efficacy of cancer immunotherapy.

#1456

**Dexamethasone suppresses cytokine-induced dual oxidase 2 (Duox2) and VEGF-A expression in human pancreatic cancer cells** in vitro **and pancreatic cancer growth in xenografts.**

Yongzhong Wu, Smitha Antony, Jennifer L. Meitzler, Jiamo Lu, Agnes Juhasz, Guojian Jiang, Krishnendu Roy, James H. Doroshow. _NCI-DCTD, Bethesda, MD_.

Chronic pancreatic inflammation is strongly associated with pancreatic cancer. We previously demonstrated that inflammatory cytokines interact to produce a Duox2 (one of 7 members of the NADPH Oxidase family)-dependent, reactive oxygen species (ROS)-related, pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation, enhancing malignant transformation. Furthermore, we have shown that Duox2 expression is upregulated in patients with chronic pancreatitis as well as pancreatic ductal adenocarcinoma (PDAC). Dexamethasone (Dex) has been reported to inhibit PDAC invasiveness, the formation of pancreatic intraepithelial neoplasia in genetically-engineered mouse models of pancreatic cancer, as well as epithelial to mesenchymal transition, and local tumor recurrence and metastasis in vivo. Using cultured human pancreatic cancer cell lines, we found that in a dose- and time-dependent fashion Dex inhibited cytokine (IFN-γ/LPS, IL-4, and IL-17)-mediated up-regulation of Duox2 and VEGF-A expression in several human pancreatic cancer cell lines, including BxPC-3, ASPC-1, and CFPAC-1. The effects of Dex were abolished by pre-treatment with the Dex antagonist RU-486. As expected, we did not observe anti-proliferative effects of Dex on PDAC cells in vitro. However, Dex strongly repressed Duox2 mRNA and protein expression as well as the growth of xenografts initiated from BxPC-3 cells; in contrast, for the MIA-Paca line that is unresponsive to cytokines in culture, Dex produced no effect on Duox2 expression and tumor growth when these cells were grown as xenografts. Examination of the human Duox2 promotor in silico revealed a putative negative glucocorticoid receptor (GR) binding element. Western analysis, using nuclear extracts from pancreatic cancer cells treated with Dex, revealed that both activated glucocorticoid receptor and certain co-repressors, such as NCOR-1/2 and histone deacetylases (HDAC1, 2, and 3) exist in human pancreatic cancer cell nuclei. Our ongoing experiments are focused on understanding the molecular mechanism of Dex-mediated Duox2 repression both in vitro and in vivo. In summary, these studies suggest that cytokine-related oxidant stress, generated by Duox2, could play a role in the progression of pancreatic cancer.

#1457

Regulatory T cells and its impact on prostate cancer development and clearance.

Sanjay Kumar,1 James Stokes III,1 Karyn Scissum Gunn,1 Udai Singh,2 Selvarangan Ponnazhagan,3 Manoj K. Mishra1. 1 _Alabama State University, Montgomery, AL;_ 2 _University of South Carolina, Columbia, AL;_ 3 _University of alabama, Birmingham, AL_.

Prostate Cancer (PCa) is the most common non-skin malignancy and the most commonly diagnosed cancer in men in United States. The immunotherapeutic role of immune cells in regulation of PCa have been studied but still the specific roles of regulatory T cells needs further investigation. In this study, the role of immune cells, particularly the role of regulatory T cells, in tumor development and progression was analyzed using TRAMP (transgenic adenocarcinoma of mouse prostate) model system. TRAMP cells (TRAMP C1, C2 and C3) were used to induce tumor in C57/B6 mouse. Interestingly, the TRAMP-C3 is non-tumorigenic whileTRAMP-C1 and TRAMP-C2 are tumorigenic. Briefly, tumor induction studies were performed on different groups of mice. Mice were inoculated with these three cell lines, tumors were analyzed at different time points, and the percentage and absolute number of different immune cells (CD4, CD8, NK, NKT, Macrophages, regulatory T cells, and Dendritic cells) were analyzed. Our data demonstrated that the capacity of TRAMP-C1 and TRAMP-C2 cells to form tumors and the inability of TRAMP-C3 cells to induce tumors is mediated by number of regulatory T cells in the tumor microenvironment. Therefore, the data suggest an understanding of function and effect of regulatory T cells during tumor progression and clearance is needed to successfully develop a targeted therapy to modulate the number of immune cells in the tumor microenvironment.

#1458

Toll-like receptors 1, 2, 4 and 6 expression levels in diffusely infiltrative astrocytomas.

Isabele F. Moretti, Daiane Gil Franco, Roseli Silva, Thais F. Galatro, Sueli M. Oba-Shinjo, Suely K.N. Marie. _University of Sao Paulo, Sao Paulo, Brazil_.

Toll-like receptors (TLRs) are the first receptors of the immune system that identify disturbances in homeostasis, capable of recognizing molecules associated to cell and tissue damage. TLRs signaling pathway activates inflammatory transcription factors, mainly Nuclear Factor-kappa B (NF-kB) and Interferon Regulatory Factors (IRF3/7). Due to their importance in inflammatory process, TLRs are research hot targets for therapy in several diseases, including cancer. This study aims to analyze TLRs (1, 2, 4 and 6) expressions in human diffusely infiltrative astrocytomas, WHO grades II to IV of malignancy (AGII-AGIV), and correlate their expressions among them and with the clinical outcome of AGIV (glioblastoma, GBM) patients. TLRs mRNA levels were evaluated by qRT-PCR in 143 astrocytoma samples and 22 non-neoplastic (NN) brain specimens from epilepsy surgery. Upregulated mRNA levels of all studied TLRs were observed in astrocytomas compared to NN cases (p<0.05), except for TLR2 in AGII. TLR1 and TLR2 median expression values increased according to astrocytoma malignancy. TLRs mRNA expressions presented positive correlation with significance (p<0.05) in all grades of astrocytoma, except for TLR2 and TLR4 in AGIII. Strong associations (r<0.7) were detected with TLR1-TLR2 mRNA expressions among AGII, AGIII and AGIV, TLR1-TLR4 in AGIII, and TLR6-TLR1 in AGII. In GBM patients, TLR2 upregulation was associated to better outcome with longer overall survival (p<0.007). The same was verified for concomitant upregulation of TLR1, 2 and 4 (p<0.05). Aiming to analyze the distribution of these receptors in different cells present in the tumor, we performed immunofluorescence staining of GBM cell lines (A172 and U87MG). All TLRs were detected in both cell lines, indicating that their expression were not limited to immune cells. The current literature has described controversial roles for the TLRs in cancer, either activating an anti-inflammatory condition leading to tumor progression or a pro-inflammatory state leading to tumor abrogation. TLRs distribution among different cells in the tumor environment might be relevant for the inflammatory process. Our findings of TLRs on tumor cells may suggest a tendency towards an anti-inflammatory state. The comprehension of TLRs role in each cell type, that compose the tumor, may help to determine the signaling pathways involved in diffusely infiltrative astrocytoma progression, and potential new therapeutic strategies.

#1459

Revealing the underlying causes of the gender disparity in melanoma: Role of testosterone.

Janet Markman, Daiko Wakita, Timothy Crother, Moshe Arditi. _Cedars Sinai Medical Center, Los Angeles, CA_.

Introduction: At advanced age, men have increasingly higher incidence of melanoma compared with women, as well as continuously decreasing levels of testosterone. With the low rate of survival for distant metastatic patients with melanoma, especially in men, studies aimed at elucidating the immunological and hormonal mechanisms underlying the gender disparity are warranted.

Procedures: To investigate the gender disparity observed between men and women affected by melanoma, we used an experimental murine model where B16 melanoma cells were injected i.v. into C57BL/6 mice and lung colonization was assessed at day 14. The roles of neutrophils and NK cells were then investigated by specific cell depletion using anti-Ly6G(1A8) and anti-NK1.1 monoclonal antibodies, respectively. Depleting antibodies or control IgG were injected every 3 days, starting one day before melanoma cell injection. To elucidate the role of hormones, ovariectomy or castration surgeries were performed 4 weeks before B16 injection and 60 day slow release pellets were inserted at the time of surgery. Findings were reproduced with a second murine melanoma cell line (YUMM1.7) derived from a genetically engineered mouse harboring human mutations in BrafV600, as well as loss of Cdkn2a and PTEN.

Data: Compared with males, naïve female mice injected with melanoma (B16 or YUMM1.7) cells have higher lung tumor burden, reduced neutrophil infiltration, and decreased NK cell activation. Ovariectomy of female mice did not affect lung tumor burden, indicating that estrogen and progesterone are not protective. Interestingly, castrated males revealed increased lung metastasis and worse survival rates compared with sham male mice, suggesting a protective role for testosterone. Further, testosterone replacement in castrated mice significantly reduced the elevated lung tumor burden to equal levels observed in sham males. Depletion of NK cells greatly increased tumor burden in both male and female mice in a gender independent manner. In contrast, neutrophil depletion increased lung tumor burden only in male mice and had no significant affect in female mice, indicating a potential gender difference in neutrophils. Moreover, neutrophil depletion in male mice also reduced NK activation and IFN-γ production, suggesting a stimulatory relationship between neutrophils and NK cells.

Conclusion: Our data indicates that both neutrophils and NK cells are important for the initial response against melanoma colonization of the lung. Neutrophil depletion, as well as our castration data, indicate that there is a gender specific differential response, which may contribute to the gender disparity seen in human melanoma incidence and survival. Further, testosterone replacement at physiological levels may benefit a subset of melanoma patients.

#1460

**Bone marrow-derived CD11b** + **/Podoplanin** + **cells are lymphatic progenitors directly responsible for breast cancer lymphatic formation.**

Caitlin Griggs, Kelly Hall, Lisa Volk-Draper, Kathy Robinson, Sophia Ran. _Southern Illinois University School of Medicine, Springfield, IL_.

Introduction: Lymphatic metastasis, a key factor for poor outcome of breast cancer (BC), strongly depends on lymphatic vessel (LV) formation. Recent studies suggest that lymphangiogenesis is strongly promoted by bone marrow (BM)-derived CD11b\+ monocytes that differentiate into Lymphatic Endothelial Cell Progenitors dubbed here M-LECP. While BC are known to recruit BM monocytes, a specific BM subset responsible for tumor lymphatic formation has not been identified. We recently discovered that podoplanin (Pdpn) is the highest upregulated lymphatic marker that signifies transdifferentiation of either human or mouse immature myeloid cells into M-LECP. We hypothesized that BM-derived M-LECP are represented by a subset expressing podoplanin in CD11b\+ myeloid cells that are capable of inducing tumor lymphatics. Methods: Tumor lymphatic vessels expressing myeloid markers were detected immunohistochemically by triple-staining in clinical specimens and experimental breast tumors. Expression of Vegfr-3, Lyve-1, Sca-1, and other markers in BM-derived CD11b+/Pdpn+ and Pdpn-negative subsets was determined by FACS. Expression of lymphatic-specific markers in CD11b+/Pdpn\+ and Pdpn- myeloid cells isolated from MDA-MB-231 tumors was determined by RT-qPCR. CD11b+/Pdpn\+ and Pdpn-negative subsets from BM of GFP-expressing mice were adoptively transferred to mice with orthotopic breast tumors followed by quantifying mobilized GFP+/CD11b+/Pdpn+ cells and the density of tumor LV. Results: M-LECP were absent in normal human breast tissues but highly present in tumors. Analysis of clinical BC specimens showed significant correlations between tumor-mobilized M-LECP, density of LV and metastasis to regional nodes. All examined BC models exhibited very high densities (60-90%) of double-positive macrophages expressing lymphatic markers, and lymphatic vessels expressing myeloid proteins. At least a half of CD11b\+ cells isolated from tumors were positive for podoplanin and nearly 90% of CD11b+/Pdpn+ subset expressed markers of progenitor cells. Only CD11b+/Pdpn\+ subset (but not other myeloid cells) expressed a broad panel of proteins specific to the lymphatic endothelial lineage. Adoptive transfer of BM-derived CD11b+/Pdpn\+ or Pdpn-negative fractions into tumor-bearing mice showed that only Pdpn+ BM myeloid cells integrated into preexisting LV and caused a 10-fold increase in peritumoral and intratumoral LV density. Conclusion: We show for the first time in clinical BC samples that tumor-recruited M-LECP significantly correlate with LN metastasis. We also identified a phenotypically distinct BM fraction of CD11b+/Pdpn+ cells that directly promote generation of new tumor lymphatic vessels. Our data suggest that tumors induce myeloid BM cells to undergo differentiation into M-LECP that subsequently promote cancer lymphatics thereby promoting tumor spread.

#1461

MHCII-expressing breast cancer cells can induce anti-tumor immune response.

Mei Li, Tyler R. McCaw, Selene Meza-Perez, Troy D. Randall, Amy Weinmann, Donald J. Buchsbaum, Andres Forero, Albert F. LoBuglio. _University of Alabama at Birmingham, Birmingham, AL_.

We recently reported that the mRNA expression of genes in the Major Histocompatibility Complex class II (MHCII) antigen processing and presentation pathway in breast tumor tissues was strongly prognostic of good clinical outcome in triple negative breast cancer (TNBC) patients (J Clin Oncol 33, 2015 suppl; abstr 1066). Although MHCII expression is most often observed on antigen-presenting cells, such as dendritic cells, macrophages and B cells, we observed MHCII protein expression on TNBC tumor cells. These observations suggested that TNBC cells expressing MHCII may have the ability to directly stimulate CD4 T cells and promote anti-tumor immune responses. To test this hypothesis, we transfected murine TS/A tumor cells (syngeneic, non-immunogenic, and metastatic breast cancer model) with a vector encoding the human MHCII Transcriptional Activator (hCIITA) under the control of a doxycycline (Dox)-inducible promoter. We found that TS/A-hCIITA cells did not express MHCII or CD74 until exposed to Dox, whereupon they expressed MHCII and CD74 mRNA and protein in a dose-dependent fashion. We also found that TS/A-hCIITA cells injected into the mammary fat pad expressed MHCII genes and cell surface MHCII proteins on tumor cells if mice received Dox in their drinking water. Importantly, tumor-bearing, Dox-exposed mice exhibited significantly impaired tumor growth compared to mice that were not exposed to Dox. We also found that tumor-bearing, Dox-exposed mice had a significantly enhanced tumor-specific CD8 T cell response in both the tumor-draining lymph node and tumor bed. Thus, breast cancer tumor cells expressing MHCII proteins promote anti-tumor immune responses, which likely contribute to the control of tumor growth in vivo.

#1462

Non-inflammatory role of ASC-dependent inflammasomes in promoting gastric tumourigenesis via IL-18.

Brendan Jenkins,1 Virginie Deswaerte,1 Alison West,1 Paul Nguyen,2 Tracy Putoczki2. 1 _Hudson Institute of Medical Research, Clayton, Australia;_ 2 _The Walter and Eliza Hall Institute of Medical Research, Clayton, Australia_.

Introduction

Gastric cancer is the third most lethal cancer worldwide and represents a growing number of cancers linked with inflammation. However, the identity of innate immune regulators with oncogenic potential in the host gastric mucosal epithelium remains obscure. We aim to identify the molecular basis by which specific pattern recognition receptors belonging to the inflammasome family promotes gastric tumorigenesis.

Experimental procedures

We have employed the gp130F/F gastric cancer mouse model coupled with mice lacking specific inflammasome-related genes. In addition, human gastric cancer tumor (and matched non-tumor) biopsies and cell lines were used. Immunohistochemistry on gastric tissue sections was used to assess the extent of cellular proliferation, apoptosis, inflammation and angiogenesis, and quantitative real-time PCR was used to measure the expression of genes of interest. Immunoblotting and ELISA were used to measure the activation and expression levels of inflammasome-related proteins.

Results

A causal role for ASC-dependent inflammasomes in gastric tumorigenesis was confirmed upon the genetic ablation of ASC in gp130F/F:Asc-/- mice, which resulted in a ~50% reduction in tumor burden compared to parental gp130F/F mice. The suppressed gastric tumorigenesis in gp130F/F:Asc-/- mice was associated with increased gastric epithelial (tumor) cell apoptosis, as determined by elevated numbers of TUNEL and caspase-8 positive cells. Surprisingly however, no changes in the number and activation status of inflammatory cells were observed. The suppressed gastric tumorigenesis in gp130F/F:Asc-/- mice was also characterized by a gene signature comprising apoptotic-related genes. In gastric tumor tissue, IL-18, but not IL-1β, protein levels were augmented compared to matched non-tumour tissue, and genetic ablation of IL-18 in gp130F/F:IL-18-/- mice suppressed gastric tumorigenesis comparable to that observed upon ASC deficiency. The treatment of human gastric cancer cells with IL-18 also potentiated their growth compared to IL-1β.

Conclusions

Collectively, these data reveal that ASC-dependent inflammasomes and their downstream mediator IL-18 represent a novel oncogenic mechanism in gastric cancer.

#1463

Ionic immune suppression within the tumor microenvironment limits T cell effector function.

Robert L. Eil, Rahul Roychoudhuri, Madhu Sukumar, David Clever, Jenny H. Pan, Shashank Patel, Douglas C. Palmer, Nicholas P. Restifo. _NCI/NIH, Bethesda, MD_.

Tumors progress in immunocompetent hosts despite the local infiltration of tumor-specific effector T cells. Recent advances have identified ion transport as important for T cell activation and function. We report that the concentration of potassium is markedly elevated within murine and human tumors in comparison to other tissues while other ions are minimally perturbed. Additionally, ex vivo stimulation of effector T cells in hyperkalemic conditions led to profound suppression of T cell activation, cytokine production, and glycolytic metabolism without affecting viability - suggesting a role for potassium in tumor immune evasion. Characterization of this phenomenon with whole-transcriptome RNA-Sequencing revealed that elevated potassium specifically repressed TCR induced transcripts with the effector cytokine interferon gamma (IFNγ) being among the most depressed. Moreover, hyperkalemia limited TCR induced glucose uptake and consumption, metabolic changes required for full effector function.

Prior investigations into the role of potassium transport in cellular physiology have centered on the presumption that the ion's sole function is to maintain cellular membrane potential and calcium influx. However, we find that elevated potassium had no effect on TCR induced calcium signaling. Although hyperkalemia increased the cytoplasmic membrane potential of effector T cells as expected, other methods to similarly depolarize the cell without the addition of extracellular potassium did not produce the inhibitory phenomenon. Therefore, we conclude that the immunosuppressive effect induced by hyperkalemia is independent of potassium's previously understood role in the regulation of cytoplasmic membrane potential and calcium signaling. Causatively, elevated potassium led to blunted serine/threonine phosphorylation within the Akt/mTOR signaling cascade following TCR ligation, while pharmacologic or genetic means to restore Akt signaling rescued effector function.

Mechanistically, the suppressive effect of elevated extracellular potassium was directly related to an increase in intracellular levels, and depletion of intracellular potassium restored full T cell activation and cytokine production. T cells genetically reprogrammed to express the potassium efflux channel Kcna3 were resistant to potassium mediated suppression - displaying lower intracellular potassium levels and increased cytokine production. Finally, tumor-specific Kcna3 expressing cells exhibited augmented in vivo intratumoral IFNγ production, Akt/mTOR signaling, and enhanced anti-tumor activity against established melanoma in tumor bearing mice. These results uncover a novel axis of tumor-induced immune suppression via an 'ionic' checkpoint. The manipulation of cellular ion transport represents an entirely new approach for cancer immunotherapy.

#1464

The tumor immune microenvironment is similar in anal squamous cell carcinomas (SCCs) from HIV-infected and uninfected patients.

Elizabeth L. Yanik,1 Suzanne L. Topalian,2 Genevieve Kaunitz,3 Jessica Esandrio,3 Tricia Cottrell,3 Janis M. Taube1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Johns Hopkins University School of Medicine and Kimmel Cancer Center, Baltimore, MD;_ 3 _Johns Hopkins School of Medicine and Kimmel Cancer Center, Baltimore, MD_.

Tumors may evade immune attack by constitutive (oncogene-driven) and/or adaptive (IFN-g inducible) expression of PD-L1. PD-1/PD-L1 blockade can mediate tumor regression in immunocompetent patients. In HIV-infected patients, developing tumors may face little immune selection pressure and therefore may not evolve to evade immune attack. Expression of PD-L1 has not been systematically assessed in cancers from HIV-infected people. In the current study, immunohistochemistry for PD-L1, and CD3 and CD68 (immune cells, ICs) was performed on biopsies from 46 anal SCCs, including 27 from HIV-infected and 19 from uninfected patients. The proportion of cases with tumor cell PD-L1 expression was similar in patients with and without HIV (52% vs. 47%, respectively, p=0.76), as was the presence of moderate/severely dense ICs (33% vs. 37%, p=0.81) (Table). Among HIV-infected patients, 19% of anal SCC tumors (5/27) both expressed PD-L1 and had moderate/severe IC infiltration. Tumors from HIV-infected and uninfected patients had similar densities of CD68+ macrophages (mean 517 vs. 404 cells/mm2, p=0.33) and CD3+ T- lymphocytes (mean 501 vs. 428 cells/mm2, p=0.57). A component of adaptive PD-L1 expression (juxtaposed to tumor infiltrating ICs) was also observed in both groups (56% vs. 47%, p=0.58), consistent with comparable T-cell functionality. Further studies will explore the expression of other immune checkpoint proteins and lymphocyte subsets. Despite expectations that cancers from HIV-infected patients would show reduced inflammation, our preliminary findings demonstrate an immune-reactive microenvironment in both HIV+ and HIV- anal SCCs and suggest that anti-PD-1/PD-L1 therapies should be evaluated in anal SCC patients.

Tumor infiltrating IC | |

Total | HIV- a

(n=19) | HIV+ a

(n=27) | P-value c

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

None-mild | Total | 30 | 12 (40%) | 18 (60%)

|

PD-L1(+) b | 14 | 5 (36%) | 9 (64%) | 0.66

PD-L1(-) | 16 | 7 (44%) | 9 (56%)

Moderate-severe | Total | 16 | 7 (44%) | 9 (56%)

|

PD-L1(+) b | 9 | 4 (44%) | 5 (56%) | 0.95

PD-L1(-) | 7 | 3 (43%) | 4 (57%)

a Anal SCC tumors in HIV-infected patients include 19 tumors identified through the AIDS-Cancer Specimen Resource and 8 tumors identified at Johns Hopkins Hospital. Anal SCC tumors in HIV-uninfected patients include 1 tumor identified through the AIDS-Cancer Specimen Resource and 18 tumors identified at Johns Hopkins Hospital. | b Defined as ≥5% of tumor cells expressing cell surface PD-L1 by immunohistochemistry with the 5H1 monoclonal antibody. | c P-values are for the comparison of PD-L1 expression between HIV+ and HIV- anal SCCs within strata of tumor infiltrating IC | IC=immune cells

#1465

Analysis of tumor-infiltrating lymphocytes (TILs) reveals biologically different subgroups of breast ductal carcinoma in situ.

Marie Beguinot-Cornillon,1 Marie-Melanie Dauplat,2 Fabrice Kwiatkowski,3 Guillaume Lebouedec,4 Lucie Tixier,5 Christophe Pomel,6 Frederique Penault-Llorca,2 Nina Radosevic-Robin2. 1 _Surgical Oncology, Pathology, Jean Perrin Cancer Center (JPCC); ERTICa Research Team, University of Auvergne EA4677, Clermont-Ferrand; Master Program in Biology and Health Sciences, University Paris-East Val-de-Marne (UPEC), Creteil, France;_ 2 _Pathology, JPCC; ERTICa Research Team, University of Auvergne EA4677, Clermont-Ferrand, France;_ 3 _Clinical Research, JPCC; ERTICa Research Team, University of Auvergne EA4677, Clermont-Ferrand, France;_ 4 _Surgical Oncology, JPCC, Clermont-Ferrand, France;_ 5 _Pathology, JPCC, Clermont-Ferrand, France;_ 6 _Surgical Oncology, JPCC; ERTICa Research Team, University of Auvergne EA4677, Clermont-Ferrand, France_.

Background: Treatment of breast ductal carcinoma in situ (DCIS) aims to prevent invasive recurrence. Recent studies have shown an important impact of TILs on the outcome of invasive breast cancer, however their influence on breast DCIS prognosis has not been fully explored. In this study we investigated whether the amount and/or phenotype of TILs can help recognizing DCIS subgroups of different biology and recurrence risk.

Methods: The study included 134 patients, diagnosed and treated for a DCIS from 2001 to 2005 in our institution. Formalin-fixed and paraffin-embedded (FFPE) cancer tissue samples, taken before any treatment, were retrospectively collected. H&E whole tissue sections served for assessment of the cancer size, grade, histotype, architecture, mitotic index and amount of stromal lymphocytic (Ly) infiltrate. The latter was semi-quantitatively graded into 4 grades (0 - absent, 1 - mild, 2 - moderate, 3 - intense). Ly-phenotype was assessed by immunohistochemistry (IHC) on tissue microarrays (TMAs), constructed by sampling each case at the area of the densest Ly-infiltration (3 cores of 0.6 mm diameter per case). The number/mm2 of the CD8+, CD4+, FOXP3+, CD20+ and CD38+ mononuclear cells was determined. TMAs served also for estrogen receptor (ER), progesterone receptor (PR), HER2 and Ki67 IHC staining, as well as for HER2 amplification status.

Results: There were 97 DCIS and 37 microinvasive DCIS (micDCIS). The micDCIS displayed significantly more diffuse architecture, frequent HER2 amplification (HER2amp), higher grade, lower ER expression (0.029<p<0.044 for each) and more peritumoral Ly-infiltrate (grades 2+3, micDCIS vs DCIS, 51.5% vs. 39.0%, p=0.036). All but CD20+ cells were more numerous in micDCIS than in DCIS (0.0016<p<0.05). Within the entire cohort, the cases having the (CD8+/CD4+):(CD20+/CD38+) ratio higher than 1 had a significantly greater risk of containing a microinvasive component (OR 3.47 (1.26-9.57), p=0.029). Interestingly, that ratio was significantly higher (p=0.012) in micDCIS than in the DCIS with grade 2 or 3 Ly-infiltrate (Ly-DCIS, n=38). On the other side, there was no difference between micDCIS and Ly-DCIS in architecture, histograde, HER2amp rate and ER expression. Cluster analysis further confirmed significant similarities between micDCIS and Ly-DCIS, putting them both apart from non-Ly-DCIS (p=0.0034).

The overall 10-year recurrence rate was 13% (18/134 pts). No parameter significantly correlated with recurrence risk, however the micDCIS have received significantly more treatment than DCIS (axillary lymphadenectomy (p<10-7), chemotherapy (p=0.031) or hormonal therapy (p=5.4x10-6)).

Conclusion: These results indicate that Ly-DCIS might be biologically and immunologically similar to micDCIS. TILs in DCIS are worth investigating in larger studies, as they could be a marker of microinvasion and help tailoring the initial treatment of the disease.

#1466

Interferon-induced microRNA turnover leading to epithelial-to-mesenchymal transition (EMT) in cancer.

U-Ging Lo, Rey-Chen Pong, Jer-Tsong Hsieh, Leah Gandee. _UT Southwestern Medical Center, Dallas, TX_.

Introduction: Interferon-γ (IFN-γ) is a cytokine that has been shown to be associated with antitumor mechanisms during tumor immune surveillance. However, a steady flow of reports suggests that it has pro-tumorigenic effects. In this study, we have investigated the role of IFN-γ in cancer progression and its mechanism of action in regulating microRNA turnover.

Methods: Three cell lines from different cancer types (prostate PC-3, renal 786-0 and hepatoma, HepG2) were employed. Luciferase reporter gene assay and real-time RT-PCR were used for examining the IFN-γ-induced gene transcription. Site-directed mutagenesis, in vitro transcription, RNA pull down, and in vitro RNA degradation assays were used for determining miRNA biogenesis. Western blot assay was used to determine EMT marker expression. Transwell assay was used for determining cell migration and invasion.

Results: In the presence of IFN-γ, both the migration and invasion of PC-3 increased, which was accompanied with elevated EMT marker (such as Vimentin) as well as factors (such as Slug and ZEB1). IFN-γ is known to induce transcriptional activation of IFN-stimulated genes (ISGs) via JAK-STAT signaling pathway. Subsequently, we identified that IFN-γ could induce interferon-induced tetratricopeptide repeat 5 (IFIT 5) mRNA and protein levels in these cell lines. IFIT5 is first identified as viral RNA binding protein and we recently demonstrate its new function as a post-transcriptional machinery for miRNAs turnover. We therefore screened the potential IFIT5-regulated miRNAs based on microRNA microarray screening and identified several candidates such as miR-101, miR-335, miR-203, miR-128, miR-363, miR-153, miR-146a, miR-125b and miR-200a. We further examine the functional roles of these miRNAs contributing to cancer metastasis. We demonstrated that miR-363 could target Slug to suppress EMT as well as cell migration and invasion. Also, the seed regions of miR-101, miR-335, miR-203 and miR-128 have sequence-matched target sites at the 3'UTR of ZEB1, and Slug mRNA, and we further showed that they could indeed suppress the expression of these EMT factors. Clinically, IFIT5 mRNA level is elevated in higher-grade prostate cancer (PCa), and positively correlated with ZEB1, ZEB2 and Slug in PCa. By knocking down IFIT5, we observed suppression of both ZEB1 and Slug, along with decreased migration motility in these cells. Taken together, our data demonstrate the pro-progression role of IFN-γ in different cancer types.

Conclusion: IFN-γ can potentiate cancer progression by inducing EMT, which is mediated through IFIT5-mediated miRNA turnover. IFIT5 complex represents unique machinery for the turnover of a specific population of tumor suppressor miRNAs.

#1467

Multiplexed immunofluorescence quantitation and validation of multiple immune cell types in colon cancer epithelium and stroma.

Christopher Sevinsky,1 Alberto Santamaria-Pang,1 Anup Sood,1 Yunxia Sui,1 Qing Li,1 Nicole LaPlante,2 Raghav Padmanabhan,2 Fiona Ginty1. 1 _GE Global Research Center, Niskayuna, NY;_ 2 _GE Healthcare Clarient, Aliso Viejo, CA_.

Immune response to tumor growth and development is highly complex and varies with stromal and tumor cell composition and secreted factors. Different immune cell types can have opposing functions (tumor suppressive or immune suppressive) and their density, location and activation state can have profound effects on tumor outcome. Routine immune-profiling methods based on sample homogenization or limited in situ detection capabilities may be insufficient to measure this diversity. To fully decipher tumor immune response and contribution of different cell types, there is urgent need to develop new imaging tools for in situ immune cell-typing and quantification. MultiOmyx® technology allows repeated staining, imaging and cell-level analysis of multiple biomarkers (at least 60) on the same FFPE tissue section. Together with epithelial and stromal cell analysis, the relative location and quantity of immune cells can be established, enabling regional assessment of immune infiltration and activation status.

Using this workflow, 747 stage I-III colorectal cancer patient samples in TMA format were multiplexed for a total of 61 proteins, including CD3 (all T-cells), CD8 (cytotoxic T-cells), CD20 (B-cells), CD68 (macrophages) and pan-cytokeratin (epithelial tumor cells). All images were registered, auto-fluorescence subtracted and illumination corrected. Individual cells were segmented using automated image analysis workflow, consisting of nuclei segmentation, epithelium segmentation and stroma-epithelial cell nuclei classification. We applied a probabilistic multi-class, multi-label classification algorithms based on multi-parametric models to build statistical models of CD protein expression and classify immune cells. Using selected images, expert annotations of the following immune cells were made [CD20+ (n=4282 (cells)), CD3+ (n=5600), CD8+ (n=5346), and CD68+ (n=4261), and defects (n=1739)]. Support Vector Machines (SVM) were used to derive a statistical model for cell classification. To objectively evaluate the cell classification accuracy, we performed 10-fold cross validation.

The methods described performed well in classifying cell-types, yielding ≥97% accuracy, relative to expert user annotations, for all immune cell types. Classification accuracy was slightly higher for lymphocytes vs. macrophages, likely due to more diffuse localization of CD68. In line with previous reports, higher T-cell infiltration was significantly correlated with recurrence-free survival in the entire cohort. In summary, we have developed a highly accurate quantitation method for in situ analysis of immune cells in tumor and stroma. Using the same workflow, additional cell lineage and functional markers can be added for deeper phenotyping and identification of innate and adaptive immune cell lineages, generating new insights into role of immune response in tumor progression.

#1468

Intratumoral tertiary lymphoid structures are associated with favorable prognosis of patients with gastric cancer.

Chie Sakimura, Hiroaki Tanaka, Tatsuro Tamura, Go Ohira, Masatsune Shibutani, Sadaaki Yamazoe, Katsunobu Sakurai, Hisashi Nagahara, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Kazuya Muguruma, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. _Department of Surgical Oncology Osaka City University Graduate School of Medicine, Osaka, Japan_.

Background: The host immune system is one of the key players of anti-tumor functions. High numbers of tumor infiltrating lymphocytes has been showed to be a predictor of favorable clinical outcome in several solid cancers. The role of B cells in tumor microenvironment is still unclear. Recent studies have reported a correlation between the densities of B cells and the favorable clinical outcome of cancer patients. Moreover, some studies considered the organization of tumor infiltrating B cells as criteria in addition to the cell density. Tertiary lymphoid structures (TLSs) are defined as transient ectopic lymphoid organizations that can develop in non-lymphoid tissues at the site of chronic inflammation, and are anatomically and functionally similar to secondary lymphoid organs. In the present study, we investigated the association between tumor infiltrating B cells and clinicopathological features in gastric cancer.

Materials and Methods: Tumor blocks were obtained from 226 patients with stage Ib to stage IV gastric cancer who had undergone initial surgical resection. The density of CD20+ B cells within the tumor and invasive margin area was assessed by immunohistochemistry. We also evaluated CD3+ T cells, CD21+ follicular dendritic cells (FDCs), Bcl6 germinal center B cells, and PNAd+ high endothelial venules (HEVs) to show the presence of TLSs in gastric cancer.

Results: Tumor infiltrating B cells mostly organized clusters surrounded by CD3+ T cells. B cell area contained FDCs and some clusters had Bcl6+ B cells. There were HEVs around follicles. We observed most of TLSs at the tumor invasive margin. Kaplan-Meier survival analysis showed that high number of CD20+ B cells was associated with significantly better survival (P<0.0001). Univariate analysis demonstrated a correlation between CD20 high and longer OS, and multivariate analysis also showed that CD20 high was one of the independent predictors of OS.

Conclusion: We demonstrated that B cells mostly infiltrated as TLSs and were associated with better prognosis in patients with gastric cancer. Our results suggested that B cells, perhaps as the structure of TLSs, might play an important role for immune response against gastric cancer.

#1469

The inflammatory pre-metastatic niche in reliably metastatic models of triple negative breast cancer.

Kassondra Balestrieri, H Keith Pittman, Matthew Britt, Mohamed Ramez, Nasreen Vohra, Kathryn Verbanac. _East Carolina University/ Brody School of Medicine, Greenville, NC_.

The purpose of this study was to evaluate Triple Negative Breast Cancer (TNBC) murine models to use for investigations of the pre-metastatic niche. We selected two murine TNBC based on published gene expression studies which demonstrated molecular profiles that mirrored human breast tumor subtypes. The 2225L murine tumor has similar gene expression patterns to human tumors of the basal-like phenotype. When 2225L was implanted in the mammary fat pad or sc flank of syngeneic naïve Balb/c female mice, no metastases were observed. Tumors were subsequently resected at approximately 600-800 mm³ to promote the growth of seeded metastases. To select for a consistently metastatic 2225L variant, primary tumors or lung metastases from the most consistently metastatic 2225L tumors were serially passaged sc. The resulting variant (2225LM) is highly metastatic, with lung metastases in 90% (43/48) of mice from the most recent 4 cohorts compared to 27% (47/176) of mice in 66 previous passages. Metastases were not observed at any other site. We also studied the T11 murine tumor, which is representative of the claudin-low phenotype. However, when tested for mouse pathogens prior to implantation, T11 was positive for lactate dehydrogenase elevating virus (LDEV). To eliminate LDEV, T11 tumors were serially passaged into athymic nude rats. The resulting LDEV-free T11 tumor was used for in vivo studies. After sc implantation of T11 in Balb/c mice, consistent lung metastases were observed (89%; 24/27 mice). Tumor-infiltrating immune cell subsets were assessed using flow cytometry. CD11b+ Ly6G- and CD11b+ Ly6G+ cells comprised the majority of tumor-infiltrating immune cells and both subsets were more prevalent in 2225LM than T11. Infiltrating lymphoid (CD3+) cells were a minor component of both tumors. In preliminary studies to quantify lung-infiltrating immune cells, levels of CD11b+ Ly6G+ cells were 1.5 fold higher in 2225LM and T11 tumor-bearing mice compared to controls. Prior to the development of frank metastases (12-30 days post-2225L implant), lung analysis by ELISA showed a steady increase in the neutrophil chemoattractant KC and the pro-tumor M2/N2 chemokine marker MCP-1. Analysis of lungs with visible 2225LM metastases detected increased levels of KC (6-27 fold) and MCP-1 (11-23 fold), when compared to naïve mice. These chemokines were not elevated at non-metastatic sites (i.e. kidney). To conclude, our lab has selected, developed and characterized two TNBC mouse models. Both tumor cell lines are reliable metastatic models of TNBC and will be used to further characterize the inflammatory microenvironment of the pre-metastatic niche, including the continuum of gene expression patterns and changing cellular components.

### Immune Modulating Agents and Therapeutic Antibodies

#1470

Cancer treatment by near infrared photoimmunotherapy targeting intratumoral regulatory T cells.

Kazuhide Sato, Noriko Sato, Biying Xu, Yuko Nakamura, Tadanobu Nagaya, Peter L. Choyke, Hisataka Kobayashi. _NCI/NIH, Bethesda, MD_.

Introduction and Purpose:

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to suppress immune responses against cancers. Treg-mediated immunosuppression is a key mechanism for tumor immune-evasion, which could lead cancer immunotherapies to failures. Systemic depletion of Tregs have been tried by using antibodies against them; however, intravenously delivering these antibodies might not sufficiently deplete Tregs in the tumor microenvironment or could substantially deplete effector cells, in case anti-CD25 antibody was used. In addition, development of auto-immune responses could cause potential side effects. To overcome these problems, we exploited near infrared photoimmunotherapy (NIR-PIT) at the tumor to quickly deplete only intratumoral Tregs, aiming to induce anti-tumor immune activation.

Experimental Design:

F(ab')2 fragments of an anti-mouse CD25 antibody, PC61.3, were generated and conjugated with a phthalocyanine dye, IRDye-700DX (αnti-CD25-F(ab')2-IR700). Using the anti-CD25-F(ab')2-IR700, in vitro NIR-PIT effect was examined against CD25-expressing mouse T lymphocytes, HT-2 clone A5E cells. In vivo CD25-target-NIR-PIT of the tumor was performed after an intravenous injection of the conjugate to mice bearing subcutaneous, luciferase transfected, LL/2 (Lewis lung carcinoma, LL/2-luc) or MC38 (mouse colon cancer, MC38-luc) cancers. Tumor volume, bioluminescence signals (BLI), and Immune responses following a local CD25-target-NIR-PIT were examined.

Results:

In vitro NIR-PIT-induced cytotoxicity was light dose dependent. CD25-target-NIR-PIT at the tumor quickly and selectively depleted Tregs in the cancers, yet, Tregs in any other organs were not affected. The local CD25-target-NIR-PIT induced a rapid activation and cytotoxic action of CD8 T cells and NK cells within LL/2-luc or MC38-luc tumors. This led to significant reductions of tumor volume (p < 0.0001) and BLI (p < 0.05) in both LL/2-luc and MC38-luc models and prolonged the survival of the mice (p < 0.0001) compared to the non-treated controls. Intriguingly, this local CD25-target-NIR-PIT induced a transient systemic cytokine storm and antitumor-effects on distant non-irradiated specific tumors. Effects of local CD25-target-NIR-PIT were significantly (p < 0.0001) inhibited by a CD8-, NK-, or INFγ−depletion, suggesting the anti-tumor roles of CD8 T cells and NK cells.

Conclusions:

Quick depletion of intratumoral Tregs by a local CD25-target-NIR-PIT rapidly induced CD8 T- and NK-cell activation, thereby restoring local anti-tumor immunity. Consequently, activated immunity led to regression of not only NIR-PIT-treated tumors but also non-NIR light exposed tumors in separate parts of the body. These observations suggest that using local CD25-target-NIR-PIT may be a promising new strategy for cancer immunotherapy.

#1471

Immunotherapy revised: Ipilimumab potentiates the vascular response to stereotactic radiosurgery in patients with brain metastases.

Ingrid Digernes,1 Cathrine Saxhaug,2 Oliver Geier,1 Edmund Reitan,2 Einar Waldeland,3 Kunt Håkon Hole,2 Kari Dolven Jacobsen,4 Åslaug Helland,4 Kyrre Eeg Emblem1. 1 _The Intervention Centre, Oslo University Hospital, Oslo, Norway;_ 2 _Dept. of Radiology and Nuclear Medicine, Oslo University Hospital, Oslo, Norway;_ 3 _Dept. of Medical Physics, Oslo University Hospital, Oslo, Norway;_ 4 _Dept. of Oncology, Oslo University Hospital, Oslo, Norway_.

Introduction: While immunotherapy is a promising treatment option for advanced metastatic disease, the survival benefit of adding ipilimumab to stereotactic radiosurgery (SRS) in unselected patients with metastases to the brain seems limited [1, 2]. Moreover, because conventional diagnostic methods for assessing treatment response were not designed for these therapies, the mechanisms of action in vivo are poorly understood. To reveal potential synergistic effects of adding ipilimumab to SRS, we here present preliminary data on the vascular response to SRS and ipilimumab in patients with brain metastases from malignant melanomas and non-small cell lung cancer.

Methods: Voxel-wise estimations of blood volume and vessel calibers were acquired by perfusion MRI and Vessel Architectural Imaging [3], respectively, in 13 patients with brain metastases from lung cancer receiving SRS only (N=6; 7 lesions) and malignant melanomas receiving SRS only (N=4; 5 lesions) or SRS and ipilimumab (N=3; 5 lesions). Regions of enhancing tumor were identified on contrast-enhanced MRIs, whereas peri-tumoral tissue regions included a region containing a 2mm wide dilation of the enhancing tumor and outside the SRS target area. MRIs were performed at baseline (pre SRS) and repeated every three months. Ipilimumab (3mg/kg) were administered intravenously over 90 min every third week for four cycles. SRS was delivered to the tumor (visual metastasis + 2 mm margin) with a peripheral dose of 18 Gy or 25 Gy, depending on tumor size.

Results: For patients treated with SRS only, the vessel calibers decreased with a median value of 15% in the peri-tumoral area at 3 months following SRS (P<0.05, Wilcoxon signed rank test) and more in metastases from lung cancer than in melanomas (75% vs 95%, P<0.05, Mann-Whitney U test). In contrast, for patients treated with SRS and ipilimumab, there was almost a two-fold increase in the average vessel caliber size in the peri-tumoral area at 3 months (P<0.05 vs SRS only, Mann-Whitney U test). Non-significant trends towards lower blood volume values and tumor volumes were observed in all groups at 3 months.

Conclusion: The enlarged average vessel calibers and the subsequent lack of an increase in blood volume suggest ipilimumab helps clean up the vascular bed by removing small caliber vessels and effectively reducing the capillary vessel density. Our advanced MRI data provide valuable and novel insights into the biological mechanisms of response to ipilimumab and at study end may help identify patients with metastatic disease that benefit from this therapy by prolonged survival.

References:

1. Mathew et al, Melanoma Res 2013. 23(3):191-5

2. Patel et al, Am J Clin Oncol 2015. May 16:[Epub]

3. Emblem et al, Nat Med 2013. 19(9):1178-83

#1472

Enzymatic depletion of adenosine by pegylated, engineered adenosine deaminase 2 (PEG-ADA2): A novel immunotherapeutic approach to treat solid tumors.

Lin Wang, Jessica Cowell, Sanna Rosengren, Lei Huang, Xiaoming Li, Qiping Zhao, Jennifer Souratha, Mathieu Marella, Barbara Blouw, Keri Cannon, Chunmei Zhao, Kim Phan, Curtis Thompson, Michael Shepard, Christopher Thanos. _Halozyme Therapeutics, San Diego, CA_.

Adenosine is an endogenous immunosuppressant that binds to adenosine receptor checkpoints and protects tissue from immune-mediated rejection. Abnormally high adenosine levels (up to 100-fold greater than other tissues) contribute to a highly immunosuppressive tumor microenvironment (TME). We hypothesized that adenosine deaminase 2 (ADA2), a human enzyme that catalyzes the deamination of adenosine, could be administered at therapeutic levels to deplete high levels of TME adenosine and stimulate anti-tumor immune activity. Recombinant wild-type ADA2 (wtADA2) was cleared extremely rapidly from circulation (t1/2 = 69 min, 7.5 mg/kg iv, n=9 mice), rendering it unsuitable for therapeutic testing. Therefore, a series of variants was designed to attenuate the heparin binding properties of ADA2 to improve bio-distribution and conjugated with 20K PEG to improve pharmacokinetics (PK). The variant PEG-ADA2-K374D displayed 94% less binding to heparin compared to wtADA2, enzymatic activity comparable to wtADA2, and 33-fold improved PK (t1/2 = 2,256 min); and consistently induced at least 60% (p<0.0001) tumor growth inhibition (TGI) of established subcutaneous syngeneic CT26 tumors (b.i.w., 0.3mg/kg, n=8). Treatment with PEG-ADA2-K374D resulted in a 5-fold increase in tumor infiltrating CD3+ T-cells (p<0.0001, 6 hours post dose), as assessed by histology. We hypothesized that CD73, which catalyzes the turnover of AMP to adenosine, could be used as a biomarker to identify tumors with elevated adenosine levels. Gene expression studies against a panel of syngeneic tumors revealed that lung KLN205 and pancreatic MH194/PSC4 tumors had high CD73 levels. PEG-ADA2-K374D inhibited the growth of established MH194/PSC4 and KLN205 tumors, with TGI reaching 47% (p<0.0001) and 78% (p<0.0001) after 2 weeks of treatment (b.i.w, 0.3 mg/kg, n=8). A second series of variants was generated based on structure-based design to have significantly improved kcat/km for adenosine deaminase activity. ADA2-R222Q/S265N had the highest improvement, with a 15-fold greater kcat/km than wtADA2. After pegylation, the circulating half-life of PEG-ADA2-R222Q-S265N in mice was extended from 69 min to 2,790 min (>40-fold increase). This variant induced a maximum TGI of 69% (p<0.05) in the MH194/PSC4 model at 0.003 mg/kg, a 100-fold lower dose than PEG-ADA2-K374D (n=8). A third variant, PEG-ADA2-E182T lacked detectable enzymatic activity and displayed no tumor growth inhibition, suggesting that ADA2 enzyme activity is required for efficacy. These data

suggest that engineered PEG-ADA2 variants induce significant tumor growth inhibition activity in several syngeneic solid tumor models, validating enzymatic depletion of high TME adenosine levels as novel immunotherapeutic approach to treat solid tumors.

#1473

CXCL12 inhibition with NOX-A12 (olaptesed pegol) increases T-cell infiltration in tumor-stroma spheroids and synergizes with PD-1 immune checkpoint blockade.

Dirk Zboralski, Lisa Bauer, Dirk Eulberg, Axel Vater. _NOXXON Pharma, Berlin, Germany_.

Immune checkpoint inhibition promotes T cell-mediated killing of cancer cells and can induce striking responses, but objective control of tumor growth is observed in only 10-30% of patients with cancer types that generally respond to this treatment (Fearon 2014, Cancer Immunol Res 2:187). A possible cause for this limitation of checkpoint inhibition may be an immune-privileged tumor microenvironment (TME) which excludes the cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 has recently been described as an important T cell exclusion factor in the TME-driven immune suppression. In this study we aimed to investigate whether CXCL12 inhibition by the clinical stage L-aptamer (Spiegelmer®) NOX-A12 (olaptesed pegol) is able to enhance T cell infiltration in 3D tumor-stroma spheroids, thereby facilitating effective immunotherapy.

We established 3D multicellular microtissues that mimic a solid tumor with a CXCL12-abundant TME. For this purpose, CXCL12-expressing murine stromal MS-5 cells were co-cultured with solid human cancer cell lines in ultra-low attachment plates for three days. Primary human T cells isolated from healthy donors were added to the spheroids in the presence of various concentrations of NOX-A12. The next day, spheroids were washed and dissociated for T cell quantification by flow cytometry. T cell localization in the 3D microtissues was assessed by immunohistochemistry (IHC). In order to examine T cell activation in the spheroids, a bioluminescent reporter-based PD-1/PD-L1 blockade bioassay (Promega) was adapted to the 3D format: Jurkat-PD-1/Luc T cells were incubated with anti-PD-1 and added to NOX-A12-treated spheroids (CHO-PD-L1 + MS-5).

We found that NOX-A12 increases the amount of T cells in tumor-stroma spheroids in a dose-dependent manner; flow cytometry analyses revealed a 2-3 fold increase in spheroid T cell infiltration at 10 nM NOX-A12 in all examined 3D co-culture types. Enhanced T cell infiltration in the presence of NOX-A12 was corroborated by IHC. In line with this, we found increased infiltration and activation of Jurkat-PD-1/luc T cells in the MS-5/CHO-PD-L1 spheroids treated with NOX-A12. Importantly, NOX-A12 synergized with anti-PD1-induced T cell activation.

Taken together, in heterotypic 3D models that mimic the complexity of the TME, the CXCL12 antagonist NOX-A12 improved T cell-based tumor immunotherapy by increasing T cell infiltration. By modulating the CXCL12 gradients within the complex 3D structure, NOX-A12 appears to break the immune-privilege of the TME, thereby paving the way for T cell migration into the tumor. These data provide a rationale for the combination of NOX-A12 with checkpoint inhibitors as well as other T cell-based therapies in patients with solid cancer.

#1474

A novel oncolytic adenovirus expressing tumor microenvironment modulators that activates myeloid cells, lymphocytes and endothelial cells.

Angelica Loskog,1 Emma Eriksson,1 Ioanna Milenova,1 Rafael Moreno,2 Ramon Alemany2. 1 _Uppsala University, Uppsala, Sweden;_ 2 _IDIBELL-Institut Català d'Oncologia, L'Hospitalet de Llobregat, Barcelona, Spain_.

Immunotherapy is becoming a cornerstone in cancer treatment of many indications. So far, pancreatic cancer has shown little response to so called checkpoint blockade antibodies. However, animal data suggests that activating immunotherapies releases the effect of checkpoint blockade also in pancreatic cancer. The tumor microenvironment (TEM) supports the growth of the tumor cells and consists of stroma cells, fibroblasts, blood vessels and immune cells. In some tumor lesions, such as those in pancreatic cancer, the TEM is dense and comprises most of the lesion. The TEM regulates immune activity via its high content of M2 macrophages, myeloid derived suppressor cells and T regulatory cells. Further, the dysfunctional blood vessels in lesions are not optimal for recruiting lymphocytes. With these aspects in mind, LOAd703 was developed. LOAd703 is an oncolytic adenovirus carrying TEM modulators.

LOAd703 was constructed from the ICOVIR system of oncolytic adenoviruses in which replication depends on a dysfunctional, hyperphosphorylated retinoblastoma pathway. The genome was further altered by removing E3-6.7K and gp19K, changing the serotype 5 fiber to a serotype 35 fiber to target CD46 expressed by most tumors, as well as by adding a CMV-driven transgene cassette with the human transgenes for TMZ-CD40L and 4-1BBL. Hence, the transgenes will be expressed in both tumor and stroma while oncolysis is initiated in the tumor cells. We demonstrate herein that LOAd703 infection of a panel of pancreatic cancer cell lines efficiently induced tumor cell death within 48-72 hrs post infection while LOAd703 infection of dendritic cells demonstrated an increased maturation of myeloid cells including dendritic cells (DCs). These DCs could in turn potently activate and promote expansion of both T- and NK cells. Further, LOAd703 infection of endothelial cells (HUVEC) induced upregulation of molecules involved in lymphocyte attachment, rolling and transmigration. In conclusion, LOAd703 is a novel oncolytic virus that targets both the tumor and its TME and a clinical trial is underway to elucidate its effect in pancreatic cancer.

#1475

Local immune activation resulting in tumor growth inhibition with MEDI9197 - an intratumorally administered TLR7/8 agonist.

Stefanie Mullins,1 Iwen Grigsby,2 Lester I. Harrison,2 Song Ren,3 Serguei Soukharev,3 Lesley Young,1 James M. Elvecrog,2 Robert W. Wilkinson,1 Mark A. Tomai,2 Ronald Herbst,3 John P. Vasilakos,2 Andrew J. Leishman1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _3M, St. Paul, MN;_ 3 _MedImmune, Gaithersburg, MD_.

MEDI9197 (formerly 3M-052) is a sustained-release imidazoquinoline toll like receptor (TLR) TLR7/8 agonist designed with a lipid tail that, when injected, is retained within the tumor. This has been shown in pharmacokinetic studies in mice and rats injected with MEDI9197 via the subcutaneous (SC) or intratumoral (IT, mouse only) routes. Stimulation of TLR7 and TLR8 in primary human dendritic cells induces the release of interferon-alpha (IFN-a) from plasmacytoid dendritic cells (pDCs) and interleukin 12 (IL-12) from myeloid dendritic cells (mDCs). Intratumoral administration of MEDI9197 induces a local immune response characterized by upregulation of genes involved in activation of innate and adaptive immunity both from the injected tumor and tumor draining lymph node. Furthermore, flow cytometric analysis of tumor infiltrating lymphocytes (TILs) show increased expression of activation markers, such as CD69, on natural killer (NK) cells and CD8 cytotoxic T cells. This local stimulation of immune cells with MEDI9197 results in tumor growth inhibition as shown in the B16F10 luc syngeneic mouse tumor model using in vivo imaging system (IVIS) equipment. Additionally, combination of MEDI9197 with immune checkpoint inhibitors enhances the efficacy observed in syngeneic mouse tumor models. The data presented shows intratumoral administration of MEDI9197 induces local immune activation leading to tumor growth inhibition in preclinical models of cancer. MEDI9197 is currently being evaluated as a monotherapy for safety and efficacy in human clinical trials.

#1476

A therapeutic antibody that inhibits CD73 activity by dual mechanisms.

Bryan C. Barnhart,1 Emanuela Sega,1 Aaron Yamniuk,2 Sandra Hatcher,2 Ming Lei,2 Haben Ghermazien,1 Anne Lewin,2 Xi-Tao Wang,2 Haichun Huang,1 Pingping Zhang,1 Alan Korman1. 1 _Bristol Myers Squibb, Redwood City, CA;_ 2 _Bristol Myers Squibb, Princeton, NJ_.

CD73 has a central role in dictating the adenosine concentration within the tumor as it is the final step in converting extracellular ATP to adenosine. Thus, substantial reduction of CD73 enzymatic activity has the potential to reduce immunosuppression of effector immune cells within the tumor. We present data describing an anti-human CD73 antibody that suppresses CD73 by two mechanisms: 1. direct inhibition of enzymatic activity upon binding to CD73 and 2. rapid, near-complete internalization of the enzyme. Durable reduction of cell-surface CD73 was observed in multiple tumor cell lines both in vitro and in vivo. The unique properties of this antibody are a result of the use of a human IgG2-IgG1 hybrid antibody with effector function eliminated by specific mutations of the Fc. The IgG2 sequence of this antibody drives superior internalization of CD73 and enhanced CD73 inhibition. Syngeneic tumor models demonstrate that CD73 contributes to resistance to anti-tumor therapy. Combination therapy with PD-1 blockade and a surrogate anti-mouse-CD73 antibody resulted in a better anti-tumor efficacy than either treatment alone. Finally, we demonstrate a novel technique for assessing CD73 enzymatic activity in situ that has potential for clinical application. These data support antibody-based anti-CD73 therapy in cancer and highlight a novel mechanism for inhibition of CD73 enzymatic activity.

#1477

Targeting immunosuppressive myeloid cells in oral cavity cancer with the PI3Kδ/γ isoform inhibitor duvelisib.

Ruth Davis,1 Ellen Moore,1 Paul E. Clavijo,1 Chris Silvin,2 Carter Van Waes,1 Zhong Chen,1 Clint Allen1. 1 _Tumor Biology Section, Head and Neck Surgery Branch, National Institutes of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD;_ 2 _National Institutes of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD_.

BACKGROUND: IPI-145 (duvelisib) is an oral inhibitor of the p110δ/γ isoforms of phosphinositide 3-kinase (PI3K) that are differentially expressed in leukocytes. IPI-145 is currently under study in phase II/III trials for the treatment of hematologic malignancies, but has not previously been studied in solid tumors. In this study we sought to determine if p110δ/γ inhibition could suppress solid tumor growth through functional inhibition of myeloid derived suppressor cells (MDSCs) within the tumor microenvironment of syngeneic murine oral cavity (MOC) tumor models of head and neck cancer.

METHODS: In pilot experiments, highly immunogenic MOC1 and poorly immunogenic MOC2 tumor-bearing mice were treated with daily oral doses of 15 mg/kg IPI-145 for 14 days and monitored for tumor progression. Flow cytometry was used to study cellular immune correlates in both the periphery and tumor microenvironment. MDSCs were sorted using FACS and functionally evaluated in a carboxyfluorescein succinimidyl ester (CFSE)-based T-cell proliferation assay.

RESULTS: Selective inhibition of p110δ/γ with low-dose IPI-145 resulted in a trend toward growth suppression of established MOC1 tumors, and had no effect on the growth of poorly immunogenic MOC2. IPI-145 increased infiltration and activation of CD8 T-lymphocytes and NK cells along with increased tumor cell expression of MHC class I and PD-L1 in MOC1 tumor bearing mice. MDSCs sorted from IPI-145 treated MOC1 tissues demonstrated a reduction in their capacity to suppress CD3/28 stimulated T-cell proliferation. MOC2 tumor bearing mice demonstrated no immune correlative changes.

CONCLUSION: Daily oral treatment with low-dose IPI-145 resulted in a trend toward inhibition of primary tumor growth and enhanced immune activation in the tumor microenvironment in an immunogenic murine model of head and neck cancer. This data shows promise for future studies of IPI-145 as a monotherapy at higher doses or as a sensitizer for immunotherapies such as checkpoint inhibitors, and these studies are underway.

This work was supported by the NIDCD Intramural Program ZIA-DC-000087-01

#1478

Anti-EMP2 IgG1 combined with anti-PD1/PDL1 antibodies synergistically reduce tumor load in animal models of breast cancer.

Negin Ashki, Jessica Tsui, Madhuri Wadehra. _UCLA, Los Angeles, CA_.

Purpose: Despite significant advances in detection and medicine, the incidence and mortality due to breast cancer world-wide is still unacceptably high. Our recent work reveals that tetraspan protein epithelial membrane protein-2 (EMP2) is up-regulated in up to 70% of patients with invasive breast cancer and its expression is further augmented in metastatic lesions. New data suggests that EMP2 expression correlate with an increase in tumor associated PDL1 expression. We thus hypothesized that EMP2 expression regulated tumor immunogenicity through either direct control of PDL1 expression or MHC Class I processing.

Recently, to determine the therapeutic potential of EMP2, our group created a panel of anti-EMP2 antibodies and new data shows that tumors treated with anti-EMP2 IgG1 show an increase in immune infiltrates. We thus proposed that EMP2 positive tumors will be amendable to anti-PD1/PDL1 therapy.

Methods:A panel of breast cancer cell lines, both TNBC as well as hormone positive cell lines, were transfected with a vector to overexpress EMP2 or to reduce its levels. To determine if EMP2 expression influenced the expression of immune associated genes, RNAseq was performed. In addition, PDL1 protein expression was measured using standard immunohistochemistry or western blot analysis. Immune cell populations in various xenografts models were quantitated using standard immunohistochemistry. To determine the efficacy of combination anti-EMP2 and anti-PD1 therapy, syngeneic 4T1/firefly luciferase mouse models were created in BALB/c mice. Briefly, cells were injected into the mammary fat pad of female BALB/c mice, and once they approached 100 mm3, were injected IP with a)10 mg/kg dose twice a week of anti-EMP2 antibody, b) 5mg/kg twice a week with anti-PD-1 antibody (BioXCell), c) 10mg/kg ant-EMP2 and 5mg/kg of anti-PD-1 antibody twice a week, or d) saline control.

Results: In multiple breast cell lines, EMP2 levels correlated with an increase in PDL1 expression, and treatment with anti-EMP2 IgG1 increased the number of infiltrating leukocytes. Using syngeneic mouse models, tumors treated with a combination of anti-EMP2 and anti-PD-1 antibodies grew slowly and had the smallest total volume compared to all other treatment groups. Anti-EMP2 IgG1 treatment was superior in reducing overall tumor volume compared to anti-PD-1 antibody treatment alone.

Conclusions: Our preliminary data strongly supports the synergistic efficacy of anti-EMP2 IgG1 and anti-PD-1 antibody in reducing tumor load, suggesting that using anti-EMP2 treatment with an anti-PD1/PDL1 antibody may improve survival without inducing systemic toxicity as seen with chemotherapy. We predict that this is the direct result of EMP2 expression altering the immunogenicity of the tumor and in support of this, RNAseq data and western blot data suggest that EMP2 levels regulate PDL1 and MHC associated gene expression.

#1479

Preclinical model of human melanoma for evaluation of targeted drug treatment and for immunotherapy validation.

Rajaa El Meskini,1 Michelle Gumprecht,1 Alan Kulaga,1 Anthony Iacovelli,1 Terry Van Dyke,2 Chi-Ping Day,3 Glenn Merlino,3 Zoe Weaver Ohler1. 1 _Leidos Biomedical Research, Inc., Frederick, MD;_ 2 _Basic Research Laboratory, National Cancer Institute, Frederick, MD;_ 3 _Laboratory of Cancer Biology and Genetics, National Cancer Institute, Frederick, MD_.

Malignant melanoma accounts for less than 5% of skin cancer cases, yet it represents 75% of deaths from skin cancer. The high mortality rate is due to the malignant, metastatic nature of the disease and resistance to chemotherapeutic treatments. Most mouse melanoma models have not fully recapitulated the histopathology of the disease and its metastatic nature. At NCI's Center for Advanced Preclinical Research (CAPR), we have adapted the HGF/SF; CDK4R24C transgenic mouse model to an optimized allograft transplant model for preclinical therapeutic studies in primary and metastatic melanoma. This genetically engineered mouse-derived Allograft (GDA) model recapitulates the features of the original GEM, including the epithelioid histopathology and key marker expression of human melanoma. It is an efficient and tractable tool for monitoring of both tumor growth and therapeutic responses in primary and metastatic melanoma in the context of a normal immune system. Additionally, aberrant expression of c-Met and upregulation of the downstream signaling pathway in HGF-GDA tumors is relevant for targeted therapeutics in melanoma. Thus, the model is a useful platform for evaluating therapies that target tumor cells and/or immunomodulatory pathways in intervention or adjuvant settings. Although drugs such as the c-Met inhibitor crizotinib and the MEK inhibitor trametinib were potent in cell culture, PD analyses of short-term (4-6 hour) treatment with small molecule therapies indicated that treatment incompletely suppresses the pathway in vivo compared to the corresponding primary cell line, and does not inhibit tumor growth. Therefore the HGF-GDA can be exploited to examine combination treatments that either prevent feedback activation of downstream pathway nodes in vivo, or modify alternate pathways, such as immunomodulatory targets. Hence, we are currently exploring rational combinations of oncogene-targeted therapy with immune-targeted therapy, for example, combined trametinib and anti-CTLA4 antibody treatment. In the HGF-GDA, complete response was observed in a subgroup of mice treated with anti-CTLA-4, i.e. established tumors fully regressed, yet the durable response and increased survival time (based on tumor volume) was not enhanced by concurrent treatment with trametinib. Future treatment studies will involve alternative regimens. Additionally, since metastasis, not the primary tumor, leads to progression of melanoma in patients, we have characterized a primary tumor resection model in which only metastatic disease is treated. Lung metastases develop after resection of the HGF-GDA primary tumor, which

may be treated in an intervention or adjuvant setting. Therefore we evaluated the effect on survival of the same combination therapy (trametinib and anti-CTLA-4) we previously used to treat the primary tumor, but as adjuvant treatment for the metastatic melanoma model.

#1480

Systemic immunotherapeutic efficacy of an immunocytokine, NHS-muIL12, in a superficial murine orthotopic bladder cancer model.

Amanda J. Vandeveer, Jeffrey Schlom, John W. Greiner. _National Institutes of Health, Bethesda, MD_.

Interleukin-12 (IL-12) is one of the most powerful proinflammatory cytokines capable of supporting T and NK cell function, inducing interferon-gamma while driving a TH1 adaptive immune response. Yet, to date, its success as an antitumor agent in many preclinical models has yet to be realized in a clinical setting due to systemic toxicity. Investigators have developed IL-12 delivery systems to maximize deposition of the cytokine directly in the tumor microenvironment that may be the preferred site for IL-12 while mitigating the dose-limiting systemic effects. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of human (hu) or murine (mu) IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). NHS76 recognizes exposed chromatin-DNA often found in human/murine tumors that have outpaced their blood vessels and the inadequate perfusion quickly results in tumor necrosis. Indeed, previous studies have shown selective tumor uptake of NHS-IL12 in necrotic subcutaneous murine tumors. In the present study, we evaluated the use of NHS-muIL12 in a murine orthotopic bladder cancer model (MB49 Luc). MB49 luciferase positive cells, instilled into the bladder form superficial, multifocal tumors which can be monitored in real time with a luciferase-based intravital imaging system. Urothelial bladder cancer is known to respond favorably to immunotherapeutic agents due to the presence of multiple somatic mutations, a high number of TILs, and a response to the live bacterium Bacillus Calmette-Guerin (BCG). NHS-muIL12 was found to be a very potent anti-tumor agent in both subcutaneous and intravesical MB49 tumor models, reducing tumor volume in a dose-dependent manner. For example, in the intravesical bladder model, antitumor effects were initially seen at 2.5 ug/kg administrated as three separate systemic injections. Mice were completely cured of their bladder tumors when treated at 20 ug/kg x 3 NHS-muIL12 injections with durable tumor-free long-term survival. Immune analyses revealed potent p15E-specific CTLs and IFN-γ responses, indicating the development of a specific anti-tumor immune response in mice treated with NHS-muIL12. Underlying the durable tumor-free long-term survival was an immune memory response that protected mice following re-challenge with either subcutaneous or intravesical MB49 tumor cells. Anti-tumor efficacy required the presence of NK or CD8+ T cells as depletion of either abrogated the anti-tumor effects of this agent. NHS-huIL12, is currently being evaluated against solid tumors, in a Phase 1 clinical trial (NCT01417546).

We acknowledge the kind contribution of NHS-muIL12 from EMD Serono, Billerica, MA.

#1481

Preclinical validation of a novel CD33/CD3 bispecific T-cell engager (BiTE) antibody to target patient-derived glioblastoma cells.

Parvez Vora,1 Chitra Venugopal,1 Jarrett Adams,2 James Pan,2 Chirayu Chokshi,1 Maleeha Qazi,1 Minomi Subapanditha,1 Mohini Singh,1 David Bakhshinyan,1 Ksenia Bezverbnaya,1 Nicole McFarlane,1 Jonathan Bramson,1 Sachdev Sidhu,2 Jason Moffat,2 Sheila Singh1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Glioblastoma (GBM), an aggressive primary brain tumor in adults, is feared for its near uniformly fatal prognosis and is characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. We developed patient-derived brain tumor initiating cell (BTIC) early passage lines that describe the extent of intertumoral heterogeneity, presenting a powerful preclinical model of GBM. Numerous studies have implicated CD133+ BTICs as drivers of chemo- and radio-resistance in GBM. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of a recombinant RW03xCD3 bispecific T-cell engager (BiTE) antibody that redirects human polyclonal T cells to CD133+ GBM cells, inducing very potent anti-tumor response.

Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and high-throughput DNA sequencing technology, we developed the CD133-specific monoclonal antibody 'RW03'. We constructed CD133-specific BiTEs or RW03xCD3 that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The BiTEs were constructed in four different conformations and dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. In order to test the ability of BiTEs to functionally elicit CD133-specific cytotoxic responses in vitro, we performed Presto blue-based killing assays. We observed CD133-specific BiTEs redirect T cells to kill CD133-expressing GBM cells in a coculture of T cells and GBM cells. The killing was more efficient in CD133high GBMs compared to CD133low GBMs, validating its specificity to target CD133+ BTICs. Incubating T cells with BiTEs and GBMs resulted in increased surface expression of T-cell activation markers CD69 and CD25 in both, CD4+ and CD8+ T cells populations. Treatment with BiTEs yielded extended survival in mice and significant reductions in brain tumor burden.

This rigorously obtained data offers compelling evidence that BiTE-mediated cytotoxicity against treatment-resistant and evasive CD133+ GBM BTICs could provide a very potent, specific and novel therapeutic strategy for GBM patients.

#1482

**Anti-GPC3 TRAB, a first-in-class T cell-redirecting bispecific antibody targeting glypican-3 with potent** in vitro **and** in vivo **antitumor efficacy against solid tumors.**

Yasuko Kinoshita,1 Takahiro Ishiguro,1 Yuji Sano,1 Yumiko Azuma,1 Toshiaki Tsunenari,1 Natsuki Ono,1 Yoko Kayukawa,1 Otoya Ueda,1 Naoko A. Wada,1 Hiroshi Hino,1 Koichi Jishage,1 Hirotake Shiraiwa,1 Mika Kamata-Sakurai,1 Junichi Nezu,2 Mika Endo1. 1 _Chugai Pharmaceutical Co., Ltd., Japan;_ 2 _Chugai Pharmabody Research Pte. Ltd., Singapore_.

We present efficacy data for the T cell-redirecting antibody (TRAB) with highly potent anti-tumor efficacy. Anti-Glypican-3 (GPC3) TRAB is a humanized IgG4 bispecific antibody that simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface. Anti-GPC3 TRAB utilizes T cells as effectors to induce strong TRAB dependent cellular cytotoxicity (TDCC) in the presence of GPC3-expressing cells. Treatment with anti-GPC3 TRAB first activates T cells by increasing the expression of CD25 and CD69 and also upregulating cytokines IL-2, IL-4, IL-6, IL 10, IFNγ, and TNF, and then it enhances the proliferation of T cells. Anti-GPC3 TRAB showed antitumor activity against xenograft tumors derived from various cancer types — MKN-74 (human gastric adenocarcinoma), PC-10 (human lung squamous cell carcinoma), TOV-21G (human ovarian clear cell carcinoma), and KYSE70 (human esophageal squamous cell carcinoma) — in a NOD-SCID mouse model injected with human T cells. Although recent immunotherapy, as represented by immune check point inhibitors PD-1, PD-L1, and CTLA-4 antibodies, showed promising efficacy in human, not every patient can benefit from this immunotherapy, because the significant efficacy shown in patients by a blockade of immune checkpoints is closely related to the tumor microenvironment. The immune check point inhibitors show high efficacy against inflamed tumors, because these have been sufficiently infiltrated by cytotoxic T cells that recognize cancer-specific antigens. However, they do not have efficacy against non inflamed tumors. In an immunocompetent mouse model using human CD3 transgenic mice, neither the inhibitors that block immune checkpoints (such as PD-1, PD-L1 and CTLA-4) nor a conventional ADCC antibody recognizing GPC3 could show significant efficacy against a poorly immunogenic LLC1/hGPC3 tumor. However, anti-GPC3 TRAB showed efficacy against this poorly immunogenic tumor by utilizing any kind of T cell as effectors irrespective of TCR specificity, including not only CD8-positive but also CD4-positive T cells.

The studies we present show that anti-GPC3 TRAB is a promising drug with high efficacy utilizing all kinds of T cells as effectors. The compound is expected to have efficacy even in patients with poorly immunogenic tumors, in which an immune checkpoint blockade fails to show efficacy.

#1483

MT-3724, an engineered toxin body targeting CD20 for non-Hodgkin's lymphoma.

Garrett L. Robinson, Sangeetha Rajagopalan, Brigitte Brieschke, Jennifer Erdman, Jane Neill, Rodney E. Flores-Lefranc, Julia Foree, William Null, Jensing Liu, Jack P. Higgins, Erin K. Willert. _Molecular Templates Inc., Georgetown, TX_.

Molecular Templates has developed engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit to specifically destroy target expressing cells. The use of other immunotoxins and antibody-drug conjugates is limited to targets with tumor-specific cell surface expression that efficiently internalize. The ETB scaffold has been designed to overcome this limitation and promote forced internalization of ETBs, a property that expands the potential targets to receptors with poor internalization kinetics. An additional benefit to ETBs is the mechanism of action (MOA) which is unique to that of other oncology treatments.

CD20, a receptor that does not undergo rapid internalization, is a well characterized target for agents used to treat non-Hodgkin's lymphoma (NHL). Thus, successful CD20-targeted clinical strategies include monoclonal antibodies (mAbs) and radiolabeled mAbs that act on the cell surface. Molecular Templates' lead compound, MT-3724, is a CD20-targeted ETB that has been engineered to force the rapid internalization of the immunotoxin after binding to CD20. MT-3724 has potent direct cell kill activity on CD20 positive lymphoma cells and is the first immunotoxin to CD20 to enter the clinic. The on-going phase I study being conducted at Memorial Sloan-Kettering Cancer Center, MD Anderson Cancer Center, and New York University Langone Medical Center has shown promising safety and efficacy in highly refractory NHL patients.

Once MT-3724 is delivered to appropriate cells, the Shiga toxin A subunit inhibits protein synthesis and promotes apoptosis of tumor cells. The difference in MOA allows for activity in the refractory setting, where resistance to other treatments has emerged, and may also allow for combination therapy. Pre-clinical studies with immunomodulatory drugs (IMiDs), PI3K and Bcl-2 inhibitors that are used in treatment of NHL have shown promising results when combined with MT-3724. MT-3724 is a promising novel agent in the treatment of NHL and other CD20 positive hematological malignancies. Data around the internalization kinetics, enzymatic activity, and combination with other agents will be presented.

#1484

Combined treatment with anti-LAG-3 and anti-PD-1 fully human monoclonal antibodies inhibits tumor growth in immunocompetent double-humanized LAG-3/PD-1 mice.

Elena Burova, Omaira Allbritton, Chandrika Taduriyasas, Venus Lai, William Poueymirou, Nicholas Papadopoulos, Douglas MacDonald, William Olson, Markus Mohrs, Ella Ioffe, Gavin Thurston. _Regeneron Pharmaceuticals, Inc., Tarrytown, NY_.

Lymphocyte-activation gene 3 (LAG-3) receptor is expressed on activated CD4 and CD8 T cells, γδ T cells, Treg, NK, NKT, B and plasmacytoid dendritic cells. LAG-3 binds to major histocompatibility complex class II (MHC II) and delivers inhibitory signals that regulate T cell proliferation and cytokine production. LAG-3 blockade augments T cell proliferation and activation. Programmed cell death 1 (PD-1) receptor upon binding to its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273) also delivers an inhibitory checkpoint signal that is critical for the establishment and maintenance of peripheral T cell tolerance. PD-1 signaling plays a critical role in the tumor microenvironment by allowing tumors to escape immune surveillance.

We tested in vivo activity of anti-mouse PD-1 and anti-mouse LAG-3 antibodies in several preclinical syngeneic tumor models and showed that combination treatment with both antibodies resulted in an additive anti-tumor effect compared to either single antibody treatment. TaqMan analysis in the MC38 tumor model demonstrated CD8+ T cell expansion in both the draining lymph nodes and spleens of mice in the combination treatment group.

Anti-human LAG-3 antibody is a fully human monoclonal antibody developed for cancer immunotherapy. It binds with high affinity to human LAG-3 and blocks LAG-3/MHC II interaction. Fully human monoclonal antibody REGN2810 binds with high affinity to human PD-1 and blocks PD-1 interaction with PD-L1 and PD-L2.

Double humanized LAG-3/PD-1 mice were engineered using VelociGene® technology to replace the extracellular domains of mouse Pdcd1 and Lag3 genes with the corresponding regions of human PD-1 and human LAG-3 genes. To validate humanized protein expression, we examined PD-1 and LAG-3 protein expression on T cells after anti-CD3/anti-CD28 antibody stimulation. We confirmed binding of human LAG-3 and PD-1 to the corresponding mouse ligands by cell adhesion assay for human LAG-3 and mouse MHC II interactions and by SPR-Biacore for human PD-1 and mouse PD-L1 interactions, respectively.

Combination of REGN2810 and anti-hLAG-3 antibodies in MC38.ova tumor model in double humanized LAG-3/PD-1 mice, which allows testing of clinical antibodies that do not cross to mouse receptors, demonstrated improved efficacy, including reduced tumor growth and improved survival, compared to REGN2810 and anti-hLAG-3 monotherapies.

Robust anti-tumor efficacy of REGN2810 and anti-hLAG-3 combination in preclinical setting supports their clinical development as a combination cancer immunotherapy.

#1485

Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment.

Luuk van Hooren, Maria Georganaki, Hua Huang, Sara Mangsbo, Anna Dimberg. _Uppsala University, Uppsala, Sweden_.

CD40-activating immunotherapy has potent anti-tumor effects due to its ability to activate dendritic cells and induce cytotoxic T-cell responses. However, its efficacy is limited by accumulation of immunosuppressive cells in the tumor and by endothelial anergy induced by pro-angiogenic growth factors. Here, we show that combining agonistic CD40 monoclonal antibody (mAb) therapy with vascular targeting using the tyrosine kinase inhibitor sunitinib significantly decrease tumor growth and improve survival in B16.F10 melanoma and T241 fibrosarcoma. Treatment of tumor-bearing mice with anti-CD40 mAb lead to increased activation of CD11c+ dendritic cells in the tumor draining lymph node, as evidenced by enhanced CD86 expression, while sunitinib treatment reduced vessel density. CD11b+Gr1+ myeloid derived suppressor cells accumulated in the tumor draining lymph nodes after anti-CD40 mAb treatment. This effect was reversed by co-treatment with sunitinib. Tumor endothelial expression of ICAM1 and VCAM1 adhesion molecules were increased only when anti-CD40 mAb treatment was combined with sunitinib. This was associated with enhanced tumoral infiltration of CD8+ T-lymphocytes. Our results show that combining CD40-stimulating immunotherapy with sunitinib treatment exert potent complementary antitumor effects mediated by dendritic cell activation, reduction in myeloid derived suppressor cells and increased endothelial activation, resulting in enhanced recruitment of cytotoxic T cells.

#1486

Pharmacodynamic response to HER2/CD3 bispecific antibody (HER2-TDB): evidence of T-cell recruitment and further rationale for combination treatment with anti-PD-L1.

Ji Li, Maria Hristopoulos, Robyn Clark, Jennifer Johnston, Dion Slaga, Bu-Er Wang, Rafael Cubas, Klara Totpal, Melissa R. Junttila, Teemu T. Junttila. _Genentech, South San Francisco, CA_.

Based on the recent clinical success of tumor immunotherapies that block immune suppressive mechanisms to restore T cell function, there is a profound interest in the clinical development of T cell targeted therapies. We have produced a trastuzumab-based HER2 T cell dependent bispecific antibody (HER2/CD3; HER2-TDB; Junttila et al Cancer Res 2014, 74:5561) that conditionally activates T cells resulting in lysis of HER2 expressing cancer cells at low picomolar concentrations. In vivo, HER2/CD3 can inhibit growth of established mammary tumors in mice at doses as low as 0,01 mg/kg. Due to its unique mechanism of action, which is unrelated to HER2 signaling or sensitivity to chemotherapeutic agents, HER2/CD3 can eliminate cells refractory to currently approved HER2 therapies and inhibit growth of Kadcyla insensitive mammary tumor models. Treatment with HER2-TDBs also results in tumor regression as a second line treatment for Kadcyla relapsed mammary tumors in mice.

Spontaneous mammary tumors arising in MMTV-huHER2 transgenic animals are poorly immunogenic or "immunological deserts" due to their low mutational load and low T cell infiltration. Despite these attributes, HER2/CD3 shows remarkable activity in this model characterized by a robust increase in the number of infiltrating lymphocytes and CD8+ T cells as well as a significant enhancement of tumor CD8+ cytotoxic effector function as measured by increased IFNg production. In addition to inducing T cell proliferation, we further show that HER2/CD3 strongly recruits new T cells from the periphery and this leads to significantly increased T cell numbers in the mammary tumor tissue. Another notable feature of the response to HER2/CD3 treatment is a significant upregulation of PD-1 expression on tumor infiltrating CD8+ T cells. This observation adds a strong rationale for combining HER2/CD3 with anti-PDL1 treatment. Our in vitro experiments further demonstrate that PD-1/PD-L1 signaling can inhibit killing mediated by CD3 bispecific antibodies and that combining two immune therapies - direct polyclonal recruitment of T cell activity together with inhibiting the T cell suppressive PD-1/PD-L1 axis results in enhanced and durable long term responses in vivo.

#1487

Gene expression and linkage analysis implicates CBLB as a mediator of rituximab resistance.

John Jack,1 George Small,2 Tammy Havener,2 Chad Brown,1 Howard McLeod,3 Alison Motsinger,1 Kristy L. Richards4. 1 _North Carolina State, Raleigh, NC;_ 2 _University of North Carolina - Chapel Hill, Chapel Hill, NC;_ 3 _Moffitt Cancer Center, Tampa, FL;_ 4 _Cornell University, Ithaca, NY_.

Drug resistance remains one of the largest challenges in the curative treatment of cancer. We investigated the problem of monoclonal antibody resistance in vitro in a high throughput, complement-dependent cytotoxic (CDC) assay using immortalized as well as cancerous cell lines. First, the heritability of rituximab and ofatumumab sensitivity was explored using Epstein Barr Virus-immortalized human lymphoblastoid cell lines from two independent sources: a collection of trios (95 samples, 13 trios) from the Centre d'Etude du Polymorphisme Humain and an unrelated collection (486 samples) from the Children's Hospital Oakland Research Initiative. Cell viability in the presence or absence of 10ug/ml rituximab or 10ug/ml ofatumumab treatment was quantitated using AlamarBlue. Heritability was estimated using variance component analysis using MERLIN: H2=33.11% (p<0.001) and H2=31.11% (p<0.001) for rituximab and ofatumumab, resp. Next, public information on genotype and gene expression was used to analyze associations between loci/genes and drug response with three methods: genome-wide association, linkage, and gene expression analysis. GWAS were performed on the 486 unrelated samples: (a) rituximab responses, (b) ofatumumab responses, and (c) the vector of both responses modeled jointly. There was one suggestive (-log10(P)>6) result across all three analyses; this result for rituximab response occurred within the gene, SMOC2. Next, linkage analysis on the 95 related samples revealed one significant linkage peak on chromosome 12 for rituximab and two significant peaks for ofatumumab, on chromosome 12 and 3. Also, we identified genes whose mRNA expression level was correlated with the degree of either rituximab or ofatumumab sensitivity. Quantitative Significance Analysis of Microarrays revealed 13 genes whose expression was correlated with rituximab sensitivity and 25 genes whose expression was correlated with ofatumumab sensitivity (false discovery rate cutoff<0.001%). CBLB, present on both gene lists, was the only gene that was located in either the chromosome 3 or chromosome 12 linkage peak. As such, CBLB was chosen for further functional validation. We used RNA silencing in several cell line and cancerous models to functionally validate the role of CBLB expression on rituximab resistance. Knockdown of CBLB in cell lines, both LCLs and lymphoma, led to increased resistant to both rituximab and ofatumumab. Additionally, we performed immunofluorescence in CBLB knockdown cells. CD20 localization was altered in cells with CBLB knockdown. Our current work has uncovered new mechanistic insight into the relationship between anti-CD20 antibody susceptibility, CBLB, and CD20, illustrating the power of comprehensive genetic analyses to discover a previously unknown mediator of rituxumab response. Additional studies are ongoing to further elucidate the mechanisms of CBLB-mediated resistance as well as its clinical relevance.

#1488

Development of anti-nucleolin antibodies with broad spectrum anticancer activity and negligible toxicity to normal cells.

Daniel J. Fernandes,1 Baby G. Tholanikunnel2. 1 _CharlestonPharma, Charleston, SC;_ 2 _Medical University of South Carolina, Charleston, SC_.

Nucleolin has multiple, unique functions in cancer cells, including the shuttling of ligands from the cell surface to the cytoplasm and the stabilization of oncogene and cytokine mRNAs that have an AU-rich nucleolin binding element in their 3'-UTRs. We developed a panel of anti-nucleolin antibodies that exploit the temperature-dependent shuttling function of nucleolin to gain access to the cytoplasm of human tumor cells and induce oncogene mRNA destabilization. Several lines of evidence indicate that our fully human monoclonal IgG1 antibody, CP101.2C8, targets nucleolin and penetrates tumor cells. CP101.2C8 bound tightly to human recombinant nucleolin (Kd =26 ± 7 nM, SEM.) and to plasma membrane nucleolin of human tumor cells. Confocal microscopy of Panc-1 and DU-145 tumor cells incubated at 37 0C with CP101.2C8 revealed punctate localization of the antibody in the plasma membranes of these cells and internalization of the antibody into the cytoplasm. The localization of the antibody within foci in the plasma membrane suggested that the antibody was bound to nucleolin that was incorporated into lipid rafts within the plasma membrane. MCF-7 human breast cancer cells were made more than 100-fold resistant to CP101.2C8 by growing the cells in increasing concentrations of the antibody. The resistant cells regained contact inhibition and had a 10-fold lower level of cytoplasmic nucleolin compared to the parental MCF-7 cells. CP101.2C8 is a potent inhibitor of tumor cell viability in vitro. IC50 values of less than 1 µg/ml were obtained for CP101.2C8 versus MV4-11 AML cells, colon, prostate, and lung cancer cells as well as CD33+-CD24- stem cells from MDA-MD-231 breast cancer cells. In contrast, the IC50 concentrations of CP101.2C8 versus normal human B and myeloid cells, breast epithelial cells and lung fibroblasts were greater than 10 µg/ml. Unlike the tumor cells, these normal cells did not express detectable levels of nucleolin in either the plasma membrane or cytoplasm. A fully human recombinant CP101.2C8 is also potent in reducing the viability of MV4-11 cells (IC50 = 0.4 µg/ml). Groups of 10 nu/nu mice bearing SC MV4-11 tumor xenografts were injected IV (every 3 days x 6) with either 10 mg/kg CP101.2C8 or 10 mg/kg of a human IgG1 isotype control antibody. Tumor progression to the 2,000 mm3 endpoint was observed between 24-41 days in all mice treated with the control antibody, while 3/10 of the CP101.2C8-treated mice were long-term survivors that did not reach the endpoint by day 76. The hazard ratio calculated from the Kaplan-Meier plot was 0.22. CP101.2C8 was well tolerated by all the mice; the only adverse event observed was a transient loss in mean body weight of 16%. The widespread and aberrant expression of the multi-functional protein nucleolin in human tumor cells, in contrast to the corresponding normal cells, explains both the broad-spectrum anticancer activity and tumor selectivity of antibody CP101.2C8.

#1489

Development of a humanized ROR1 x CD3 bispecific DART® molecule for the treatment of solid and liquid tumors.

Bhaswati Barat,1 Gurunadh Chichili,1 Valentina Ciccarone,1 James Tamura,1 Sergey Gorlatov,1 Michael Spliedt,1 Francine Chen,2 Scott Koenig,1 Paul Moore,1 Ezio Bonvini,1 Ralph Alderson,1 Syd Johnson1. 1 _MacroGenics, Inc., Rockville, MD;_ 2 _MacroGenics, Inc., San Francisco, CA_.

Introduction: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in chronic lymphocytic leukemia and a subset of solid tumors, including lung, breast, ovarian, colon, and pancreatic cancers, as well as sarcoma. Limited adult tissue expression and its absence in normal leukocytes makes ROR1 a promising cancer therapeutic target. We have developed a Dual-Affinity Re-Targeting (DART®) protein for redirecting T lymphocytes to lyse tumor cells via monovalent recognition of ROR1 on tumors and CD3 on T cells. ROR1 x CD3 DART protein was engineered for improved half-life with the incorporation of a modified Fc domain, lacking effector function.

Methods: The ROR1 x CD3 DART protein was stably expressed in CHO cells and purified to homogeneity by a standard antibody platform. Bispecific binding was evaluated by ELISA and SPR analysis. In vitro functional studies were performed with lymphoma and solid tumor cell lines in the presence of primary human T cells. Tumor growth inhibition was evaluated in NOD/SCID/IL-2 gamma chain KO (NOG) mice coimplanted with human T cells and either mantle cell lymphoma (MCL) or lung cancer cell lines (5:1 effector : target cell ratio) followed by treatment with ROR1 x CD3 DART protein by intravenous (IV) administration. In vivo activity was also evaluated in human PBMC-reconstituted NOG/B2m deficient mice bearing established intradermal tumor xenografts following IV treatment with ROR1 x CD3 DART molecule. Pharmacokinetic analysis of the DART molecule was performed in human neonatal Fc receptor (hFcRn) transgenic mice.

Results: The ROR1 x CD3 DART protein displayed the expected bispecific binding for ROR1 and CD3 antigens and retained the affinity and specificity of the parent mAbs. The DART molecule mediated dose-dependent lysis of ROR1-positive MCL and solid tumor (breast, lung, and osteosarcoma) cell lines through recruitment of human T cells. DART molecule-mediated killing of ROR1-expressing target cells was accompanied by target-dependent T-cell activation and cytokine release; however, no activity was observed in the absence of target cells and no cytokine release was observed with human PBMCs alone. The ROR1 x CD3 DART protein displayed extended circulating half-life after administration to hFcRn-transgenic mice. In mouse efficacy studies, the growth of HBL-2 (MCL), HOP-92 (lung cancer), or NIH-1975 (a lung cancer line resistant to erlotinib) cells co-implanted with human T cells in NOG mice was inhibited by treatment with the ROR1 x CD3 DART protein at doses in the mcg/kg range. The ROR1 x CD3 DART molecule also demonstrated antitumor activity with high complete response rates in human PBMC-reconstituted mice bearing established HBL-2 cell xenografts.

Conclusion: The promising in vitro and in vivo activity of the Fc-bearing ROR1 x CD3 DART molecule supports further investigation as a potential candidate for the cancer treatment.

#1490

Potentiating immunotherapy by targeting complement deposited on cancer cell surfaces.

Elizabeth J. Carstens,1 Martin Skarzynski,1 Vicent Butera,1 Margaret Lindorfer,2 Berengere Vire,1 Mohammed Farooqui,1 Christoph Rader,3 Ronald Taylor,2 Adrian Wiestner1. 1 _National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health, Bethesda, MD;_ 2 _University of Virginia School of Medicine, Charlottesville, VA;_ 3 _Scripps Research Institute, Jupiter, FL_.

Treatment of lymphoid malignancies with anti-CD20 antibodies (mAbs) can be frustrated by the loss of cell surface CD20 through trogocytosis, creating "escape variants" that are no longer sensitive to the anti-CD20 mAb. In patients with chronic lymphocytic leukemia (CLL) treated with the anti-CD20 mAb ofatumumab, we observed that these CD20 escape variants carried covalently bound C3d complement fragments and that these C3d opsonized CLL cells persisted for weeks in circulation. Therefore, we hypothesized that C3d is a neoantigen that could be exploited to re-target cells that have escaped from anti-CD20 mAb therapy.

To target complement opsonized cells we generated a human IgG1 mouse chimera mAb specific to C3d that is not competed by full length C3 in serum. To test whether targeting C3d can eliminate escape variants after anti-CD20 therapy, we collected blood samples from CLL patients before (day 1) and 24 hours after administration of ofatumumab (day 2). As expected, CLL cells on day 2 had lost CD20 expression and could neither bind, nor be killed by ofatumumab. In contrast, the anti-C3d mAb did not bind CLL cells obtained pre-treatment but bound cells obtained on day 2 with high affinity (kD=6.7nM) and were effectively killed through CDC, NK cell mediated ADCC, and phagocytosis. Importantly, non B lymphocytes were neither bound nor killed by the anti-C3d mAb, consistent with the highly targeted and selective deposition of C3d on CD20+ cells by ofatumumab. Interestingly, when C3d opsonized CLL was exposed repetitively to anti-C3d mAb ex vivo, the amount of cell bound C3d and the fraction of cells killed increased with successive rounds of treatment consistent with an auto-amplification of C3d targeting.

We tested the efficacy of a chimerized anti-C3d mAb in two mouse models. First, we transferred PBMCs obtained from CLL patients on day 2 of ofatumumab treatment (containing the C3d opsonized CD20 escape variants) into NSG mice and three days later injected either isotype control mAb (trastuzumab) or anti-C3d mAb. One injection of anti-C3d mAb effectively reduced tumor burden in both peripheral blood (from 42.5 to 0.59 CLL cells/ul of blood; p<0.01) and spleen (from 574 to 2.19 CLL cells/100,000 splenocytes; p<0.01). Second, we subcutaneously xenografted HBL2 cells, a CD20+ mantle cell lymphoma line, into SCID mice. Mice were treated three days after cell injection with ofatumumab alone, ofatumumab and anti-C3d mAb or isotype control (trastuzumab). Caliper measurements of the tumor dimensions and survival were recorded. The combination of the anti-C3d mAb with ofatumumab extended time to tumor development and prolonged overall survival compared to ofatumumab alone (median survival 88 days vs 22 days, respectively, p<0.02).

We conclude that targeting C3d deposited on cancer cells can eliminate antigen escape variants and potentiate complement fixing antibodies.

#1491

IPH4301, an antibody targeting MICA and MICB exhibits potent cytotoxic activity and immunomodulatory properties for the treatment of cancer.

Ariane Morel,1 Nicolas Viaud,1 Cécile Bonnafous,1 Sylvia Trichard,1 Alix Joulin-Giet,1 Samia Mizari,1 Gwendoline Grondin,1 Nadia Anceriz,1 J. Zhang,2 J. Jarzen,2 J. Wu,2 Gwendoline Grondin,1 Laetitia Cohen-Tannoudji,1 Yannis Morel,1 Benjamin Rossi,1 Carine Paturel,1 Renaud Buffet,1 Laurent Gauthier,1 Nicolai Wagtmann,1 Mathieu Blery1. 1 _Innate Pharma, Marseille, France;_ 2 _Medical University of South Carolina, Charleston, SC_.

MICA and MICB, and ULPB1-6, are ligands for NKG2D, an activating receptor expressed on NK cells and subsets of T cells. Expression of MICA and MICB is induced by cellular stress in transformed tumor cells, upon infections or at sites of chronic inflammation. Their expression is tightly regulated by complex mechanisms both at the mRNA and protein levels. As markers of cellular stress and tumorigenesis, MICA and MICB proteins are attractive candidates for targeting by a cytotoxic antibody. Moreover, ionizing radiation and various chemotherapies that cause cellular stress have been shown to induce expression of NKG2D ligands, opening interesting options for combination therapies. MICA and MICB are also compelling targets for immunomodulation. MICA and -B cause internalization of NKG2D, leading to reduced cell surface NKG2D levels and desensitization of cytotoxic effector cells in cancer patients. It was recently reported that blockade of the interactions between NKG2D and its ligands could lead to significant anti-tumor responses in mouse models. Moreover, NKG2D ligand expression was induced on immunosuppressive macrophages in cancer patients and in mouse tumor models, raising the possibility that anti-MICA/B antibodies may be used to counter local immunosuppression by targeting myeloid derived suppressor cells.

We have selected the IPH4301 antibody for its ability to bind to all allotypes of MICA and MICB, and for its dual action as an immunomodulatory agent, as well as direct cytotoxicity towards MICA/B-expressing tumor cells. First, IPH4301 induces killing of MICA/B expressing tumor cells through antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) measured towards MICA expressing cells in vitro. In vivo ADCC/ADCP efficacy was demonstrated in several preventive and curative settings using MICA expressing cell lines or endogenous tumors.

Second, IPH4301 blocks the binding of MICA/B to NKG2D. In a tumor context of chronic triggering, NKG2D downmodulation has been described in several studies of cancer patients. This modulation is mainly induced by expression of MICA/B, and less by the ULBPs. By blocking the MICA/NKG2D interaction, IPH4301 effectively restored NKG2D expression and function in vitro on primary NK and T cells.

Third, we show that IPH4301 can override immunosuppression induced by suppressive myeloid cells. In vitro differentiated M2 macrophages, but not M1 macrophages, have the capacity to impair cytotoxic functions of autologous NK cells towards MICA expressing tumor cell lines. This suppression could be overcome by IPH4301, which triggered ADCC by these otherwise impaired NK cells.

Altogether, IPH4301 is a novel, first-in-class anti-MICA/B mAb with both cytotoxic and immunomodulatory properties. Ongoing work aims to perform regulatory toxicology studies and manufacture a clinical grade product for testing in a clinical trial.

#1492

Development of a CD123xCD3 bispecific antibody to treat acute myeloid leukemia (AML).

Francois Gaudet, Jennifer F. Nemeth, Ronan McDaid, Yingzhe Li, Benjamin Harman, Hillary Millar, Alexey Teplyakov, John Wheeler, Jinquan Luo, Susan Tam, Sheng-Jiun Wu, Emily Chen, Alexander Babich, Yusri Elsayed, Ricardo Attar. _Janssen R &D, Spring House, PA_.

AML is a heterogeneous disease characterized by uncontrolled clonal expansion of leukemic stem cells (LSCs). Current therapies for AML are not curative, in part due to their inability to eradicate LSCs from the bone marrow. T cell redirection has been shown to be effective in heme malignancies and represents a promising approach to treat AML by targeting markers differentially expressed on the cell surface of cancer cells. One marker, CD123 (α-chain of the interleukin-3 receptor) is often present on AML LSCs and blasts. We developed a human bispecific antibody (CD123xCD3; Ab-178) capable of binding to the extracellular domain of CD123 and the ε chain of CD3 on T cells to induce T cell-mediated tumor cell killing. This bispecific IgG4 antibody can recruit T cells to CD123+ AML cells (MOLM-13, KG-1 and OCI-AML5) and induce T cell activation as evidenced by CD69 and CD25 up-regulation on T cells. Ab-178 potently killed these CD123+ AML cell lines in vitro (EC50 = 0.51-0.91 nM) but not a CD123- cell line (JIM3). Ab-178 was also able to induce tumor inhibition (MOLM-13 cells) and regression (KG-1 cells) in murine xenograft CD123+ AML models in the presence of human PBMCs. Furthermore, this antibody was able to kill AML blasts in primary AML blood samples ex vivo in the absence of exogenous T cells (autologous setting; EC50 = 0.83 nM). Related bispecific antibodies directed against a viral epitope (nullxCD3 or CD123xnull) did not activate T cells or cause tumor cell killing in the various assays tested. Ab-178 had no impact on T cell activation when incubated with T cells alone. These results indicate that Ab-178 can potently and specifically activate T cells in the presence of CD123+ AML cells and induce their killing. Furthermore, because of the antibody format, this molecule is expected to have a longer half-life compared to smaller bispecific biologic scaffolds. Ab-178 is currently being evaluated pre-clinically for its potential to treat patients with AML.

#1493

Improvement of the bispecific antibody ADCC platform by genetic insertion of IL-15 as a cross-linker to create NK cell reactive TriKEs.

Daniel A. Vallera,1 Joerg U. Schmohl,2 Martin Felices,1 Jeffrey S. Miller1. 1 _University of Minnesota Masonic Cancer Center, Minneapolis, MN;_ 2 _University Hospital of Tuebingen, Tuebingen, Germany_.

Natural killer (NK)-cell related anti-tumor surveillance is limited by the ability of the tumor to escape killing. Previously, we constructed a bispecific NK-cell engager (BiKE) consisting of two scFV against CD16 (FcγRIII) on NK cells and EpCAM on tumor cells (EpCAM16). Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein with prevalent expression on carcinomas making it a valuable marker for cancer targeting. This BiKE facilitated ADCC and tumor elimination, but did not account for the cellular expansion required for the success of T CARs. To improve this, we incorporated a modified interleukin (IL)-15 crosslinker to create a trispecific construct (TriKE) to enhance activation, proliferation, and to prolong survival of NK-cells. IL-15 was chosen since it is an established immunostimulatory cytokine with known effects on NK cells and is recognized as a promising cancer cure drug in NIH guided review. TriKE was assembled, expressed in E.coli, extracted, refolded, and purified to >90% with a molecular weight of 68,860 daltons. To determine the functional activity of 1615EpCAM, its killing ability was measured in standard Cr-51 release assays with EpCAM+ HT-29 colorectal cancer cells. The 1615EpCAM TriKE induced the highest level of killing compared to BiKE and other controls. To determine if the effect of IL-15 in the drug correlated with NK-cell levels, donors were selected with different naturally occurring NK cell levels. Freshly isolated PBMCs were added to HT-29 cells at E:T ratios of 20:1, 6.6:1, and 2:1. Donors showed increasingly higher levels of Cr-51 kill with TriKE, but not with BiKE indicating that greater the presence of NK-cells, the greater the TriKE effect. In order to study lytic degranulation as a function of NK-cell activity, CD107a expression was measured. Cells treated with TriKE showed significantly elevated degranulation when co-cultivated with targets (p<0.001) compared to BiKE. IL-15 alone did not enhance lytic degranulation. When PBMC were exposed to TriKE, only NK-cells, but not T-cells showed a proliferation specific pattern. Direct comparison of the BiKE and the TriKE showed that the TriKE had the ability to induce proliferation and expansion, the BiKE did not. Only with TriKE or IL-15 exposure was there a significantly enhanced expansion index. IFN-γ production from the same CD56+/CD3- NK-cell population was enhanced by 1615EpCAM, but levels did not approach levels seen with the IL12/IL18 combination that is known to stimulate cytokine production at supraphysiologic levels. We believe that this is a platform technology since anti-CD133 also can be substituted for anti-EpCAM to create functional anti-cancer stem cell TriKEs. These results indicate that we have successfully developed an immune engager that simultaneously mediates ADCC and also provides a self-sustaining costimulatory signal inducing NK effector cell expansion.

#1494

Combination of CEA TCB, a novel T-cell bispecific antibody for the treatment of solid tumors, with PD-L1 checkpoint blockade.

Marina Bacac, Tanja Fauti, Sara Colombetti, Linda Fahrni, Valeria Nicolini, Christian Gerdes, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. _Roche Innovation Center Zurich, Schlieren, Switzerland_.

Recent results from clinical trials have shown that immune therapies, particularly immune checkpoint inhibitors, can extend the overall survival of cancer patients and lead to durable responses. Despite these promising results, current immune-based therapies are only effective in a proportion of patients and combination strategies are needed to improve therapeutic benefit. Programmed death-ligand 1(PD-L1) is found on the surface of immune and tumor cells and its expression is induced by interferon gamma (IFNg). It prevents the immune system from destroying cancer cells by interacting with the inhibitory programmed death-1 (PD-1) and B7.1 receptors on activated T cells, which results in a T-cell inhibitory signal. CEA TCB (RG7802, RO6958688) is a novel T cell bispecific antibody targeting the carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent in a Phase I study in patients with advanced and/or metastatic CEA-expressing tumors.

CEA TCB-mediated killing of tumor cells led to T cell activation, IFNg secretion and subsequent up-regulation of the PD-1/PD-L1 immune suppressive pathway in vitro and in vivo, similarly to what happens during tumor adaptive immune resistance mechanisms where upon recognition of tumor antigens, TILs produce IFNg, which drives PD-L1 expression in the tumor microenvironment and delivers a suppressive signal to T cells upon binding to PD-1. Incubation of the high-CEA expressing gastric carcinoma cell line (MKN45) with human PBMCs (E:T 10:1) and increasing concentrations of CEA TCB, led to a dose-dependent up-regulation of PD-1 receptor on both CD4+ or CD8+ T cell subsets as well as of PD-L1 on surviving tumor cells as early as 24 h following treatment. PD-1 expression was specific as it did not occur in the absence of CEA-expressing tumor cells or upon treatment with un-targeted control TCB. In vivo studies performed using MKN45 tumor xenografts (MKN45) in fully humanized NOG mice demonstrated that CEA TCB treatment led to increased frequency of intra-tumoral T cells expressing PD-1 and to a strong induction of PD-L1 expression in tumors. Combination of CEA TCB with a PD-L1 blocking antibody showed significant increase of anti-tumor activity as compared to the respective single agents.

These preclinical data indicate that CEA TCB treatment leads to up-regulation of the PD-1/PD-L1 immune suppressive pathway and that the combination of CEA TCB with PD-L1 blocking agents results in enhanced anti-tumor activity. Phase Ib clinical trials investigating the combination of CEA TCB and atezolizumab are currently ongoing.

#1495

Neutralization of CD47 in cancer cells with bispecific antibodies harnesses the phagocytic potential of tumor-infiltrating macrophages.

Krzysztof Masternak, Valéry Moine, Lucile Broyer, Xavier Chauchet, Vanessa Buatois, Elie Dheilly, Stefano Majocchi, Giovanni Magistrelli, Yves Poitevin, Ulla Ravn, Eric Hatterer, Susana Salgado Pires, Limin Shang, Zoë Johnson, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer. _Novimmune SA, Geneva, Switzerland_.

The inhibitory "don't eat me" signal of phagocytosis, CD47, is commonly overexpressed in cancer cells, a feature generally associated with poor prognosis. CD47 overexpression in cancer is believed to promote immune evasion by allowing tumor cells to "hide" from innate immune phagocytes like macrophages or dendritic cells. CD47 is therefore a new type of immune checkpoint and an attractive target for cancer immunotherapy. However, as CD47 is also universally expressed on healthy cells, clinical development of anti-CD47 monoclonal antibodies is inevitably limited by toxicity and/or pharmacokinetic issues. To overcome these liabilities, we engineered dual-targeting bispecific antibodies (biAbs) for selective blockade of CD47 in malignant cells. By tethering the biAbs strongly to cells expressing a tumor-associated antigen (TAA), such as CD19 or mesothelin, CD47 is blocked selectively on the target cell. In contrast, as these biAbs will lose the avidity effect with TAA-negative cells, they will bind with very low affinity to healthy cells which express CD47. In this manner, dual-targeting should help to sidestep safety and pharmacokinetic "sink" problems resulting from ubiquitous CD47 expression. Studies in non-human primates performed with the CD47/CD19 therapeutic candidate NI-1701 confirmed this prediction, demonstrating normal IgG1 pharmacokinetics and absence of toxicity, even at high antibody doses (100 mg/kg per week).

Hence, the mechanism of action of CD47/TAA dual-targeting antibodies is heavily contingent upon target co-engagement. In vitro, CD19-positive or mesothelin-positive cancer cells are efficiently killed through antibody dependent cellular phagocytosis (ADCP) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of effector cells, such as macrophages or natural killer cells, and the corresponding dual-targeting CD47/TAA antibodies. Their enhanced ability to induce tumor cell phagocytosis was also demonstrated in vivo, in xenograft models: Mice implanted with subcutaneous human B cell lymphoma xenografts controlled tumor growth following therapy with NI-1701, contrary to mice treated with an anti-CD19 mAb. Importantly, tumor microenvironment (TME) studies revealed that mouse macrophages infiltrating human tumors engulfed tumor cells more frequently—and at a significantly higher rate—in animals treated with NI-1701 as compared to controls. Moreover, the observed superior phagocytic activity of tumor-infiltrating macrophages was associated with a reduction of granulocytic myeloid-derived suppressor cell infiltrates, suggesting that NI-1701 may favor the establishment of a tumor-hostile, immunostimulatory TME. We conclude that dual-targeting CD47/TAA bispecific antibodies may open the way to the safe and efficacious therapeutic neutralization of CD47, the universal 'don't eat me' signal hijacked by cancer cells.

#1496

Identification of target and cytotoxicity of novel monoclonal antibody NEO-201 in ovarian and uterine cancer subtypes.

Monica K. Neuman,1 Lidia Hernandez,1 Xue-Ping Wang,2 Olga Saric,2 Alex Dubeykovskiy,2 Philip Arlen,2 Christina M. Annunziata1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Precision Biologics, Rockville, MD_.

Objectives: Given the heterogeneity of ovarian cancer, it is imperative to identify subtype-directed treatments. The novel antibody NEO-201 targets a specific tumor-associated antigen (TAA) expressed on some ovarian and uterine malignancies, providing a tumor-directed approach. Here, we aimed to identify the cancer subtypes which express the NEO-201 target and demonstrate its cytotoxic effects in vitro and in mouse models of ovarian cancer.

Methods: NEO-201 is a genetically humanized novel monoclonal antibody developed through vaccines with TAAs. This antibody targets malignant tissues that express tumor-specific epitopes in membrane-anchored protein CEACAM-6. We performed immunohistochemistry (IHC) on tissue microarrays from formalin-fixed paraffin-embedded subtyped ovarian and uterine cancers to estimate the incidence of cancers expressing the NEO-201 target. Ovarian and uterine cell line pellet arrays were stained by IHC to identify in vitro models. We examined the cytotoxicity of NEO-201 in two high- and three low-expressing cell lines using Calcein AM cell viability assays alone and with purified natural killer (NK) cells with and without IL-2 stimulation. Studies of NEO-201 in ovarian cancer mouse models are ongoing.

Results: IHC of NEO-201 in tissue microarrays demonstrated 51% and 12% reactivity in uterine and ovarian samples, respectively. Similar expression patterns were identified in representative cell lines by IHC and Western blot. NEO-201 killed cell lines expressing its target in the range of 0.5-20μg/mL. Over 3 weeks, NEO-201 treatment of tumor-bearing mice demonstrated control of tumor growth with 3 doses of 250μg NEO-201 and tumor regression with 100μg in combination with IL2-stimulated PBMC. Studies are ongoing to further investigate NEO-201 cytotoxicity in ovarian cancer mouse models as well as identify the mechanism of tumor cell death and the target epitope of NEO-201.

Conclusions: NEO-201, a novel monoclonal antibody, specifically targets epithelial malignancies including ovarian and uterine cancer. This potentially therapeutic antibody demonstrates tumor-specific cytotoxicity in cancer cell lines expressing its target in vitro and in xenografts with IL2-stimulated PBMC, suggesting both antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity mechanisms are involved in tumor inhibition and regression. These studies lay the groundwork for future examination of TAA-directed therapy for ovarian or uterine malignancies.

#1497

Development of a novel TCR-like bi-specific T-cell engager targeting endogenous PR1/HLA-A2 leukemia antigen.

Amanda Cernosek Herrmann, Jin S. Im, Sijie Lu, Anna Sergueeva, Jeffrey Molldrem. _UT MD Anderson Cancer Center, Houston, TX_.

PR1 (VLQELNVTV) is a HLA-A2 restricted peptide derived from endogenous tumor associated antigens proteinase-3 and neutrophil elastase that are aberrantly expressed in myeloid leukemia blasts. Previously, we have developed a high affinity T-cell receptor-like monoclonal antibody (h8F4) specific for PR1/HLA-A2, and demonstrated the effective killing of myeloid leukemia blasts via antibody dependent cellular cytotoxicity (ADCC). To improve the therapeutic efficacy of h8F4, we adopted a novel antibody engineering strategy, bi-specific T-cell engaging (BiTE) antibody that can bring T cells in close proximity to leukemia blasts through the binding of both PR1/HLA-A2 and CD3, resulting in T-cell mediated cytolysis of leukemia blasts. Here we successfully generated h8F4-BiTE proteins using a eukaryotic expression system and investigated binding characteristics, target specificities, and subsequent T-cell activation with the h8F4-BiTE. The h8F4 BiTE showed binding affinities in the nM range for PR1/HLA-A2 and CD3 moiety of T cells. Within 18 hours of h8F4-BiTE engagement with PR1 pulsed T2 cells, polyclonal T cells from healthy donors up-regulated surface expression of CD69 and down-regulated CD3 in a PR1/HLA-A2 specific and concentration dependent manner, suggesting successful T-cell activation. In conclusion, we developed a novel h8F4 BiTE antibody that recognizes both the CD3 on T cells and endogenous PR1/HLA-A2 complexes on myeloid leukemia, and demonstrated high affinity bindings and subsequent T cell activation. We envision our novel TCR-like BiTE antibody will provide a safer and more potent treatment option for patients with aggressive myeloid leukemias that will improve upon currently available highly toxic standard therapies.

#1498

Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors.

Jill Rillema,1 Monica Licea,1 Shereen Saini-Lal,1 Doug Smith,1 Francine Chen,1 Annie Lam,1 Yinhua Yang,2 Liqin Liu,2 James Tamura,2 Ralph Alderson,2 Ezio Bonvini,2 Syd Johnson,2 Paul Moore2. 1 _MacroGenics, Inc., San Francisco, CA;_ 2 _MacroGenics, Inc., Rockville, MD_.

Introduction. IL13Rα2 is a membrane-bound protein expressed on a number of malignant tumors, but not at significant levels in most normal tissues. Clinically tested therapies to treat cancer by targeting IL13Rα2 to date have included a ligand toxin conjugate (IL13-PE) and IL13Rα2 directed CAR-T cells. Both strategies appear to be well tolerated, although with mixed results in tumor eradication. An alternative strategy for targeting IL13Rα2 is an IL13Rα2 x CD3 Dual-Affinity Re-Targeting (DART®) bispecific molecule designed to co-engage IL13Rα2 on tumor cells and cytotoxic T cells through CD3, resulting in killing of tumor cells.

Methods. Mouse monoclonal antibodies (mAbs) were generated using standard immunization protocols followed by binding and epitope binning analyses. Cell binding was performed by flow cytometry and frozen normal and tumor tissues analyses by immunohistochemistry (IHC). In vitro studies were performed with cancer cell lines and primary human T cells or peripheral blood mononuclear cells (PBMCs). In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human PBMCs.

Results. A panel of mAbs that bind human and cynomolgus monkey IL13Rα2 proteins were selected, representing a range of binding characteristics and epitope diversity. IHC showed favorable normal vs tumor tissue reactivity, with high levels of IL13Rα2 expression in glioblastoma and melanoma. The mAb panel was converted to DART molecules with an anti-CD3 arm, assessed for the ability to mediate cytotoxic T lymphocyte (CTL) activity and cytokine release and a lead selected for humanization and engineering into an IL13Rα2 x CD3 DART molecule incorporating an Fc domain to prolong circulating half-life. The IL13Rα2 x CD3 DART protein bound human and cynomolgus IL13Rα2 (KD = 0.64 nM and 0.5 nM, respectively) and mediated CTL activity against LOX-IMVI (melanoma) and SK-MES-1 (lung adenocarcinoma) target cells. Cytokines (IFN-γ, IL-10, and TNF-α) were released in the presence of IL13Rα2-expressing target cells and human PBMC, but not with PBMCs alone. Administration of the IL13Rα2 x CD3 DART molecule (≤50 µg/kg IV for 4 consecutive days) prevented tumor growth when tumor cells (A375 and LOX-IMVI melanoma, U87 glioblastoma, or DAOY medulloblastoma cell lines) were co-mixed with activated human T cells and implanted subcutaneously in NOD/SCID/IL2gamma-chain null mice. Tumor eradication was also observed in NOD/SCID/IL2gamma-chain/MHCI KO mice reconstituted with human PBMCs and bearing established LOX-IMVI melanoma tumors following 2 weekly IV doses of 50 µg/kg IL13Rα2 x CD3 DART molecule.

Conclusions: Robust in vitro and in vivo antitumor activity for an IL13Rα2 x CD3 DART protein was demonstrated. Additional studies are underway to further characterize the molecule as a development candidate for treatment of IL13Rα2-positive cancers.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Applications of Bioinformatic Tools to Analyze Cancer Data

#1499

Genomic instability alters the activity of transcription factors through competition for their binding.

Pavel Sumazin, hua-sheng Chiu. _Baylor College of Medicine, Houston, TX_.

Somatic copy-number alterations are frequently observed in cancer and are known to promote tumorigenesis. They affect a larger proportion of the cancer genome than any other genetic alteration, and they are routinely used in the clinic as predictors of therapeutic efficacy in multiple cancer types. Alarmingly, nearly 3/4 of recurrent focally-altered regions contain no known cancer genes, and the functional relevance of majority of whole-arm duplications and deletions remain unknown. We set out to evaluate whether copy number alterations can regulate cancer genes in trans by modulating the activity of transcription factors. We produced a pan-cancer catalog of copy number alterations that are predicted to regulate cancer genes in trans by titrating transcription factors and altering their regulatory functions in tumors. Focusing on recurrent amplifications in breast cancer we biochemically showed that, when acting in concert, recurrent amplifications can alter the expression of known cancer genes including ESR1 and AR.

#1500

How K-Ras4B attaches to the membrane and forms a dimer: A new paradigm.

Hyunbum Jang, Ruth Nussinov. _National Cancer Institute at Frederick, Frederick, MD_.

Ras is a small GTPase, controlling signal transduction pathways and promoting cell proliferation and survival. KRAS is frequently mutated in cancer. Ras consists of highly homologous catalytic domains (G-domains) and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Recent nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations discovered that the HVR of K-Ras4B in the GDP-bound state extensively interacts with the catalytic domain. However, it weakly interacts with the catalytic domain in the GTP-bound state. Here, using MD simulations we modeled K-Ras4B membrane interaction and dimerization. Membrane binding of K-Ras4B through the anchoring of the positively charged HVR is thought to be critical to its function as an oncogene and initiates signaling events. At the membrane, the catalytic domain takes on multiple orientations, including perpendicular and parallel alignments of the allosteric helices with respect to the membrane normal. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with the farnesyl anchored into the membrane and the HVR unrestrained, the catalytic domain fluctuates reinlessly, exposing its effector binding site. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states that display GDP-bound-like orientations of the helices. We thus propose that GDP/GTP nucleotide exchange may not be sufficient for K-Ras4B activation; composite mechanisms including HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain on the membrane can determine the functional state of K-Ras4B. Remarkably, K-Ras4B-GTP, but not GDP-bound, is able to form stable homodimers with different dimer interfaces, suggesting that the nucleotide-dependent dimerization with various dimer interfaces can resolve nanoclustering and cluster reorganization accomplishment with Raf's activation. Ras was believed to function as a monomer; however, since Raf dimerizes, it has been suspected that Ras can also dimerize. Dimerization and clustering could rein the fluctuations producing more productive pre-organized conformations. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

#1501

Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer.

Jora M.j. Lin,1 Jiang-Liang Chou,2 David E. Frankhouser,3 Yu-Ming Chuang,1 Alex Liang-Yu Chang,1 Li-Han Zeng,1 Szu-Shan Chen,1 Ru-Inn Lin,4 Cheng-Shyong Wu,2 Kuo-Liang Wei,2 Enders K.W. Ng,5 Pearlly S. Yan,6 Alfred S.L. Cheng,7 Chin Li,1 Michael W. Y. Chan1. 1 _Department of Life Science and Advanced Institute of Manufacturing with High-tech Innovations, National Chung Cheng University, Chia Yi, Taiwan;_ 2 _Division of Gastroenterology, Chang Gung Memorial Hospital, Chia Yi, Taiwan;_ 3 _College of Medicine, Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH;_ 4 _Departments of Radiation Oncology, Buddhist Dalin Tzu Chi Hospital, Chia Yi, Taiwan;_ 5 _Department of Surgery, The Chinese University of Hong Kong, Hong Kong, China;_ 6 _Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH;_ 7 _School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China_.

Gastric cancer is the fourth leading cause of cancer-related death worldwide. Previous studies demonstrated that activation of the JAK/STAT3 signaling pathway is frequently observed in H. pylori infected gastric cancer. However, the role of aberrant JAK/STAT signaling in the global epigenetic changes remains unclear. In this regard, we compared the global methylomic changes in AGS gastric cancer cells showing constitutive activation of STA3 vs its STAT3 knock-down subclone by MBDcap-Seq followed by PrEMeR-CG analysis. Together with RNA-Seq, we identified 97 targets showing concomitant hypomethylation and over-expression while 76 targets showing concomitant hypermethylation and down-regulation after STAT3 knock down. Genes showing hypomethylaton/over-expression were subjected to transcription factor binding site analysis by MEME CentriMo. Interestingly, the transcriptional repressors binding site for ETS1 (p=2.90E-06) and EHF (p=3.50E-06) were overrepresented in those identifed STAT3 targets suggesting the cooperative binding with STAT3 in the epigenetic silencing of the targets. Further gene ontology analysis by DAVID showed that genes involved in cell cycle and apoptosis were significantly enriched in the hypomethylated/over-expressed targets while genes involved in protein degradation and ubiquitination were found among the hypermethylation/down-regulated targets. To experimentally confirm our result, we analyzed the functional role of one of the hypomethylated targets, miR-193a in gastric cancer. Concomitant with MBDcap-Seq, bisulphite pyrosequencing confirmed that promoter region of miR-193a was hypomethylated in AGS cells depleted with STAT3 but hypermethylated in MKN28 gastric cancer cells overexpressed with constitutive activated STAT3. Cell lines studies also found that promoter region of miR-193a was hypermethylated in gastric cancer cells which did not express miR-193a. Over-expression of miR-193a in AGS cells inhibited cell proliferation (p<0.001) and migration (p <0.01) by colony formation assay and wound-healing assay respectively. Clinically, significantly higher promoter methylation of miR-193a was observed in gastric cancer patient samples (Hong Kong, n=70; Taiwan, n=38) as compared to gastritis (n=9, p<0.05). Interestingly, gastritis with H. pylori infection (p <0.05) had higher methylation of miR-193a than that without H. pylori infection. Patients with higher methylation of miR-193a tended to have shorter overall survival. Importantly, overexpression of miR-193a suppressed the expression of a predicted miR-193a target, YWHAZ (14-3-3ζ). As YWHAZ has been previously found to be a positive regulator in TGF-β-mediated EMT in human cancer, the role of JAK/STAT signaling in promoting TGF-β-mediated EMT program deserves further investigation.

#1502

Three intrinsic subtypes of prostate cancer with distinct pathway activation profiles differ in prognosis and treatment response.

Sungyong You,1 Beatrice Knudsen,1 Nicholas Erho,2 Mohammed Alshalalfa,2 Mandeep Takhar,2 Hussam Al-deen Ashab,2 Elai Davicioni,2 Robert Jeffrey Karnes,3 Eric A. Klein,4 Robert B. Den,5 Ashley E. Ross,6 Edward M. Schaeffer,6 Isla P. Garraway,7 Jayoung Kim,1 Michael R. Freeman1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _GenomeDx Biosciences Inc., Vancouver, British Columbia, Canada;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _Cleveland Clinic, Cleveland, OH;_ 5 _Thomas Jefferson University, Philadelphia, PA;_ 6 _Johns Hopkins Hospital, Baltimore, MD;_ 7 _UCLA, Los Angeles, CA_.

In the era of precision medicine, genomic classifications of prostate cancer (PC) have not broadly impacted patient care. Here we present an integrative transcriptome analysis of 14 disease-related pathways in over 4,600 clinical specimens and 25 PC preclinical models. This approach provides a novel classification scheme, inclusive of 3 PC subtypes (PCS1-3), which predict disease progression and drug resistance. The PCS1 and PCS2 categories possess characteristics of luminal cells, while PCS3 exhibits basal features. Castration-resistant and metastatic cancers are over-represented in PCS1 and PCS3 tumors. All 3 subtypes are represented in commonly used PC cell lines; however, none of the mouse models we analyzed resembles PCS3. PCS classification using RNA expression data appears to be stable across primary and metastatic human tumors, cell lines, xenografts and mouse models. This new subtyping method provides novel opportunities for patient stratification that reflect specific features of tumor biology and may influence therapeutic decisions.

#1503

Discovery of new breast cancer relevant genes using the cancer genomics public database.

Ritika R. Samant,1 Rajeev S. Samant2. 1 _Vestavia Hills High School, Vestavia Hills, AL;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

We used the cBio portal to query the importance of key players in breast cancer. Telomerase (TERT) is an important protein that ensures successful immortalization of cancer cells. Immortalization is a key step to establish uncontrolled cell division. MYC is an oncogene that is very relevant in breast cancer. Our searches showed that both genes (TERT and MYC) are amplified in a high percent of patients. We wanted to explore if there are genetic events that co-occur with telomerase amplification. We found that NONO gene mutation had a high tendency of co-occurrence with telomerase amplification. We wanted to understand if this subset of tumors with mutation of NONO reveals any additional correlations. Our query revealed that PDE4DIP (Phosphodiesterase 4D interacting protein) is amplified concurrent with the NONO mutation. There is no notable cancer related data on PDE4DIP in publicly available peer-reviewed publication records (PubMed). However, when we queried PDE4DIP, we discovered that it is amplified in a large majority of the breast cancer cases. Thus, it is surprisingly more common than TERT or MYC amplification. We propose that it is very important to understand the relevance of PDE4DIP and NONO to breast cancer.

#1504

Identification of novel kinase fusion transcripts in endometrial cancer.

Ryo Tamura, Kosuke Yoshihara, Kazuaki Suda, Kaoru Yamawaki, Tatsuya Ishiguro, Sosuke Adachi, Takayuki Enomoto. _Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan_.

The prognosis of patients with advanced stage or recurrent endometrial cancer remains poor and there is at present no effective therapeutic molecular target for this disease. Our aim is to identify therapeutic targetable targets in endometrial cancer. We focused on fusion transcripts whose inhibitors show impressive activity in some tumors and have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts. We have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications but have not detected recurrent fusion transcripts. We performed Sanger sequencing of RT-PCR amplification products and validated 27 (93.1%) of the 29 fusion transcripts that were randomly selected. As targetable fusion candidates, we extracted three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. Western blotting did not show the detectable protein level of the CPQ-PRKDC similar to CAPZA2-MET, and VGLL4-PRKG1. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. In conclusion, we have described the landscape of fusion transcripts in endometrial cancer by using RNA-sequencing data. We could not identify therapeutically targetable kinase fusions in a small set of 25 endometrial cancer cell lines. Our findings suggest that the transcript allele fraction is a good predictor for determining bona fide targetable fusion transcripts when applying fusion detection methods that use RNA-sequencing data.

#1505

Radiogenomics defines key genomic network driving GBM invasion.

Rivka R. Colen,1 Markus Luedi,1 Sanjay K. Singh,1 Islam Hassan,1 Joy Gumin,1 Erik P. Sulman,1 Frederick F. Lang,1 Pascal O. Zinn2. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

PURPOSE

Clinical care and outcome in Glioblastoma (GBM) remains challenging due to the tumor's invasive grwoth. To establish personalized treatment options in GBM, discovery of genetic mechanisms essential for the tumor's invasion is needed. We have previously described radiogenomic approaches to diagnose gene networks non invasively by analyzing genomic data from TCGA. The purpose of the current reseach is to identify a genetic network that drives GBM invasion and can be targeted specifically.

METHOD AND MATERIALS

Using Kaplan-Meier statistics, the data of the two independent databases TCGA and REMBRANDT were used to validate the genetic netowork's impact on clinical outcome. The genes' staus was assessed in a panel of human glioma stem cells (GSCs) and conventional proneural, classical and mesenchymal GBM cell lines using RT-PCR. Differentiation potential (Tuj1+ve, S100A+ve, and GFAP+ve), self-renewal (limiting dilution assays), invasion (Boyden chamber) and proliferation (BrdU) were assessed. Gain (lentiviral vectors) and loss (SMARTchoice Inducible shRNA) of function experiments were performed. Orthotopic xenograft models (nude mice) were used to characterize the genes impact in vivo. Potential FDA approved therapeutics were identified using connectivity map.

RESULTS

Texture analysis based on radiogenomics significantly predicted the genes responsible for invasion of GBM in a non-invasive manner. Invasion in both, in vitro and in vivo was significantly decreased upun downregulation of this gene network. Transcriptome micro-array analysis showed that an upregulation of the described genes results in class switching from proneural to mesenchymal sub-types. Cmap derived therapeutics could significantly inhibit the gene network's activitiy and hence invasion.

CONCLUSION

The describend genes could be essential drivers of molecular subtypes and invasion in GBM. The therapeutics defined with cmap offer a targetted therapy to adress these key features of GBM pathogenesis. Noninvasive radiogenomics-based identification of tumor subgroups and potential treatment approaches can significantly contribute to personalized therapy.

CLINICAL RELEVANCE/APPLICATION

The described gene network seems to be key for GBM pathogenesis. Noninvasive, radiogenomics-based subgroup identification and specific novel treatment approaches can significanty contribution to personalized GBM therapy.

#1506

Identification of genes with age-dependent expressions in breast cancer patients.

Xiang Gu, LuZhe Sun. _University of Texas Health Science Center San Antonio, San Antonio, TX_.

Age is the number one risk factor for breast cancer development. The breast cancer incidence rate increases with age, following beta distribution, which is approximately linear in range from 30 to 70 years old [1]. Transcriptome alterations have been shown to promote tumorigenesis for many types of cancers. Therefore, we hypothesize that the genes with altered expression during aging may promote breast cancer development. Using TCGA data, we extracted whole transcriptome profiling data of matched normal tissues from 82 female patients with age at diagnosis and menopausal status available. The RSEM estimated raw reads counts data were normalized by library sizes, log2 transformed and further normalized by quantile normalization. Removal of 25% of genes with lowest mean expressions resulted in subsequent analyses on 15,398 genes. We applied simple linear regression to study the association between gene expression level and age at diagnosis on all the 82 patients, and on the 54 post-menopausal patients to reduce the leverage effect of pre-menopausal patients with limited age range. The genes with significant correlation are filtered by the criteria: 1) R2 value is among the highest 5%, 2) absolute value of slope for age is among the highest 5% and 3) adjusted p value for the slope is among the lowest 5%. We identified 210 upregulated and 98 downregulated genes in all patients, as well as 103 upregulated and 164 downregulated genes in post-menopausal patients only. The unions of these genes (258 upregulated and 240 downregulated) are considered as genes affected by age. A combination of methods using moderate hierarchical t test (R/limma) and moderate fitting based on negative binomial distribution (R/DESeq2), with and without Surrogate Variable Analysis for potential confounders (R/SVA), results in identification of 498 upregulated and 256 downregulated genes altered by menopause by comparing post-menopausal to pre-menopausal patients (FDR < 0.05). Exclusion of these menopause affected genes from those genes affected by age (258 upregulated and 240 downregulated) results in 148 upregulated and 189 downregulated genes during aging. Ingenuity Pathway Analysis shows that these genes are significantly enriched for functions associated with tumorigenesis and functions in cancer. In summary, the transcriptome profiling alterations during aging are associated with development of cancer. Functional analyses of some of the age-associated genes are currently underway.

Funding: CPRIT Research Training Award (RP140105)

Reference: 1. Francesco Pompei and Richard Wilson (2001). Age Distribution of Cancer: The incidence Turnover at Old Age. Human and Ecological Risk Assessment: Vol. 7, No. 6, pp. 1619-1650.

#1507

Investigation of estrogen receptor-modulated association between immune activities and patient survival in breast cancer.

Yi-Hsuan Chang,1 Yu-Chiao Chiu,1 Tzu-Pin Lu,1 Liang-Chuan Lai,1 Mong-Hsun Tsai,1 Tzu-Hung Hsiao,2 Eric Y Chuang1. 1 _National Taiwan University, Taipei, Taiwan;_ 2 _Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan_.

In breast cancer, estrogen receptor (ER) is a crucial biomarker for subtyping and predicting patients' prognosis. Nonetheless, its modulation of immune-related genes and survival was rarely discussed. Addressing this, in the present study we devised a comprehensive analysis to compare the survival associations of immune genes between ER+ and ER- breast tumors. Specifically, we modeled immune activities by a gene set enrichment analysis of 31 immunologic gene sets defined by a recent study. Whole-genome expression profiles and ER status of 279 breast cancer patients (245 ER+ and 34 ER-) were downloaded from the Gene Expression Omnibus (GSE4922); another dataset was incorporated for validation (GSE2034).

Nine immunologic gene sets were significantly predictive of relapse-free survival (RFS) in a ER+ specific manner (Cox P-values<0.05 in ER+ and >0.05 in ER-). In order to investigate the modulation of ER in gene set interactions, we calculated the Pearson correlation coefficients between gene sets in ER+ and ER- cohorts, respectively. The gene sets formed two coexpression clusters in ER+ patients (all correlation coefficients>0.7); one cluster was composed of protective gene sets (with hazard ratios <1) of dendritic cells, T cell regulatory, eosinophil, and mast cell, and the other included risk gene sets of CD4, CD8, and effector memory CD4. Interestingly, no significant intra-cluster correlation appeared in the ER- condition. We also identified inter-cluster inverse correlation between mast cell and CD8 or effector memory CD4 in ER+ patients (correlation coefficients<-0.6). We reason that one of the clusters accounts for naïve immune system and the other represents adaptive immune system, and crosstalk between the two systems is facilitated by inter-cluster interactions. Using an independent dataset of breast tumors (GSE2034), we corroborated all the identified intra- and inter-cluster correlated pairs (correlation coefficients>0.6 and <-0.45 for intra- and inter-cluster pairs, respectively). To further dissect the functional relevance of these ER-modulated immune activities, we performed Gene Set Enrichment Analysis (GSEA) to identify differentially activated biological functions between patients with high and low enrichment scores of an immunologic gene set. GSEA revealed significant associations with breast cancer grades, subtypes, and metastasis.

In conclusion, using a gene set framework we comprehensively investigated the involvement of ER in modulating between immune activities and prognosis. Further biological investigations into our findings are warranted.

#1508

Integration of transcriptomic and metabolomic data reveals a central role for EGR1 in regulating survival and cellular metabolism in endocrine-resistant breast cancer.

Ayesha N. Shajahan-Haq, Amrita Cheema, Lu Jin, Simina Boca, Yuriy Gusev, Krithika Bhuvaneshwar, Diane Demas, Kristopher Raghavan, Subha Madhavan, Robert Clarke. _Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC_.

Breast cancer is the most commonly diagnosed cancer in women. About 70% of all breast cancers are estrogen receptor alpha positive (ER+) and are treated with antiestrogens. However, endocrine resistance is prevalent in the clinic and the underlying mechanisms remain unclear. MYC is overexpressed in endocrine resistant ER+ breast cancers, which suggests that the metabolomic profile of endocrine resistant breast cancers may contain features that are distinct from sensitive cells. We integrated data from transcriptomics and metabolomics analysis of ER+ MCF7-derived breast cancer cells that are antiestrogen sensitive (LCC1) or resistant (LCC9; resistant to ICI182,780). To validate our model, we tested a gene-metabolite network associated with EGR1 (early growth response 1), which was significantly higher in LCC1 cells compared with LCC9 cells. In ER+ human breast tumors treated with endocrine therapy, higher EGR1 expression was associated with a more favorable prognosis. In both LCC1 and LCC9 cell lines, compared to respective controls, knockdown of EGR1 decreased cell proliferation, ERα and MYC protein levels while overexpression of EGR1 did not change these. Comparison of metabolite profiles in LCC9 cells following knockdown or overexpression of EGR1 showed interruption of several biochemical pathways such as glycolysis, lipid metabolism, glutathione, and polyamine metabolism. Treatment with tolfenamic acid (TOL), a nonsteroidal anti-inflammatory drug (NSAID) known to target EGR1, decreased EGR1 protein levels and had an additive effect with ICI182,780 in inhibiting cell proliferation in LCC9 cells. Collectively, these findings indicate that down-regulated EGR1 is an important regulator of the aberrant cellular metabolic pathways specific to endocrine resistance, and targeting EGR1 may be an effective therapeutic approach in inhibiting growth of these tumors. Furthermore, high levels of EGR1 may serve as a favorable prognostic marker in some endocrine resistant tumors.

#1509

In silico **prediction of the clinical response to the mTOR inhibitor everolimus using a Boolean model: validation from a cohort of the SHIVA trial.**

Loic Verlingue, Laurence Calzone, Maud Kamal, Nicolas Servant, Lisa Belin, Emmanuel Barillot, Christophe Le Tourneau. _Institut Curie, Paris, France_.

Introduction:

Around half of currently marketed molecularly targeted agents (MTAs) in oncology have a companion diagnostic predictive biomarker that is usually a single molecular alteration (e.g. V600E BRAF mutation for the BRAF inhibitor vemurafenib), whereas around half of MTAs lack a predictive biomarker of efficacy (e.g. mTOR inhibitors). We aimed at modeling mathematically the response to the mTOR inhibitor everolimus taking into account coexisting molecular alterations and validating the model on clinical data from the everolimus cohort of the SHIVA trial (Lancet Oncol. 2015).

Material and methods:

We constructed the molecular signaling network corresponding to the most frequent altered genes found in different cancer types. We translated this network in a Boolean model and simulated it stochastically. We also developed a method to simulate the pharmacodynamics of everolimus. A function comprising the probabilities of the phenotypes of the model (cell cycling, apoptosis, senescence and quiescence) has been used to predict the tumor growth kinetics from the molecular profiles. We evaluated whether the in silico model could be validated on clinical data from 45 patients with various tumor types treated with everolimus in the SHIVA trial following the identification of a molecular alteration involving the PI3K/AKT/mTOR pathway. The correlation between the reported progression-free survival (PFS) in the SHIVA trial and the PFS predicted by the model was evaluated.

Results:

There were 34 unique associations of alterations detected by target sequencing, comparative genomic hybridization array and immunohistochemistry in the cohort of 45 patients. We found a non-linear relation between the predicted PFS from the model and the reported PFS from the trial (Pearson correlation= 0.91, p value=1e-17). The two patients with the best PFS, one harboring a metastatic germinal tumor and the other a metastatic breast cancer, are precisely predicted by the model. Similarly, the model was able to predict efficiently the resistant tumors to everolimus.

Conclusion:

We were able to predict the response to everolimus and confirmed these predictions in data reported in the SHIVA trial using an in silico model of the molecular determinant of tumor growth. By this mean, we provide a novel approach to predict treatment response from the association of molecular alterations.

#1510

Subtype-specific cancer driver gene detection improves sensitivity to detect drivers.

Yao Fu, Aparna Chhibber, Narges Bani-Asadi, Hugo YK Lam. _Bina Technologies, Roche Sequencing, Belmont, CA_.

Identifying cancer driver mutations is a crucial step toward understanding the underlying mechanisms of oncogenesis. However, driver gene detection is complicated, considering the inherent complexity and heterogeneity of cancer.The past decade has seen the large-scale application of next-generation sequencing technologies (NGS) in cancer genomics, and many methods have been published utilizing NGS to detect novel oncogenic drivers. However, the low concordance across methods raises the concern about false positives and negatives in those findings. In particular, proper modeling of the background mutation profile (known to be affected by multiple factors) is critical to improving precision and recall in driver detection. Though intra-sample heterogeneity in the background mutation profile is considered to some extent in multiple methods, most computational driver detection methods assume a homogeneous mutational landscape across cancer samples. With more and more subtypes being discovered in various cancers, the assumption is largely untenable.

In this study, we present a driver gene detection framework taking into account the heterogeneous mutational context in a cancer cohort. Our approach improves sensitivity to detect drivers by first pre-selecting a more uniform sample subset to apply driver detection algorithms. We combine this method with enhancements to an existing ensemble approach that combines methods with different assumptions about the characteristics of driver mutations (recurrence across samples, functional impact bias and positional clustering). We apply this combined approach to ~750 breast invasive carcinoma samples from The Cancer Genome Atlas. In this dataset, genes identified by our approach show higher enrichment in known cancer driver genes compared to other methods, and the subtype-driven method identifies additional potential drivers that are missing in overall analysis.

#1511

Mutational signatures from RNA-seq data distinguish HPV(+) and HPV(-) HNSCC.

Pelle Hall, Shama Virani, Charles Warden, Yanxiao Zhang, Lada Koneva, Katie M. Rentschler, Alisha Virani, Laura S. Rozek, Maureen A. Sartor, The UM Head and Neck SPORE Investigators. _University of Michigan, Ann Arbor, MI_.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide, and an increasing percentage of HNSCC cases are associated with human papillomavirus (HPV) which have improved prognosis compared to HPV(-) HNSCC. Identifying distinctions between HPV(+) and HPV(-) HNSCC may lead to improved prognosis or new treatments, such as determining mutagen(s) that are driving accumulation of genetic variants in each tumor. A recent approach uses the fact that different mutagens cause different types of mutations, referred to as a mutagen's signature. By comparing the mutations in a tumor sample with the mutagen signatures, the mutagens responsible for most of the mutations in the tumor can be identified. We hypothesized that mutational signatures could distinguish HPV(+) from HPV(-) HNSCC tumors.

Materials and Methods

We performed RNA-sequencing on 18 HPV(+) and 18 HPV(-) tumor samples collected from HNSCC patients by the University of Michigan Head and Neck SPORE. Variant calling was performed on each sample using a GATK best practices for RNA-seq pipeline. These variants were then filtered to include only variants that were both rare (<5%) and predicted to have a functional effect. The bases 5' and 3' of each variant were extracted to identify its sequence context. The observed mutations in each sample were then approximated by a non-negative linear combination of previously reported mutagen signatures. Certain signatures are associated with dysregulation of particular pathways; this dysregulation was validated with expression and mutation analysis of the RNA data.

Results

In the studied HNSCC tumors, signatures related to aging and impaired DNA repair were responsible for the largest proportion of the mutations found. The signatures that were significantly different between HPV(+) and HPV(-) tumors were associated with APOBEC editing and defective DNA mismatch repair. Expression and mutational analysis validated that APOBEC is more active in HPV(+) than HPV(-) tumors. Interestingly, signatures associated with smoking were not among the most relevant.

Conclusion

The evaluation of mutational signatures in HNSCC tumors identified the main sources of mutations for HNSCC. The differences in mutational signatures between HPV(+) and HPV(-) identify distinct methods of carcinogenesis for these two subtypes of HNSCC.

#1512

Functional validation of microRNA activity inferred by ActMiR in bladder cancer.

Mireia Castillo-Martin, Ana Collazo Lorduy, EunJee Li, Jun Zhu. _Icahn School of Medicine at Mt. Sinai, New York, NY_.

Background: MicroRNAs (miRs) play a key role in cancer, both in tumorigenesis and tumor progression. In the past years, miR expression signatures have been reported as prognostic biomarkers in different tumor types including bladder cancer (BC). However, miR's expression does not always correlate with activity. We recently developed a novel computational method, named ActMiR, for explicitly inferring the activity of miRs based on the changes in expression levels of target genes. The main objective of this study was to validate the inferred miRs' activity using BC as a model.

Design: We applied ActMiR in 405 BC cases from The Cancer Genome Atlas (TCGA) database for which information from both mRNA and miR expression was available. Different BC cell lines (5637 and HT1376 for basal BC, SW780 and HT1197 for p53-like BC, and RT4 and RT112 for luminal BC) as well as the immortalized urothelial cell line [Human Urothelium cell (HUC)] were used to perform the in vitro functional validation. RNA was extracted from these cells basally and after inhibition of miR106-5p with specific anti-miR inhibitor (Taqman, Life Technologies); miR and target genes' expression was assessed by qRT-PCR.

Results: ActMir analyses revealed that a subset of 306 out of 1044 miRNAs were differentially expressed between tumor and normal samples at p-value<10-4. More importantly, at p-value<10-4, 155 out of 556 miRNAs were functionally active. From these, only four (miR106b, miR532, mir556, and mir134) were significantly differentially expressed, functionally active and showed prognostic significance. We chose to further analyze mir106b because it showed the biggest and most significant difference in activity between normal and cancer cells (p-value<2*10-11). As inferred by ActMir, mir106-5p was significantly overexpressed in all cancer cells when compared to HUC. Basal cells showed the highest fold increase (mean of 39; range: 31-47), followed by luminal (mean of 19; range: 5-32) and p53-like cells (mean of 8; range: 3-13). Inhibition of mir106b-5p was performed to assess whether the predicted target genes were consequently upregulated.

Conclusion: Our results underscore the value of ActMir for inferring miR activity in BC from a systems biology perspective. It endorses ActMir as a promising tool for studying casual effects of miR activity on target genes, and ultimately for determining survival outcomes of BC patients.

#1513

Pan-cancer analysis of alternative splicing patterns and association to genomic aberrations.

Sergei Häyrynen, Matti Nykter. _University of Tampere, Tampere, Finland_.

Alternative splicing of pre-messenger RNA is responsible for the diversity of transcriptome and proteome, with the majority of multi exon genes producing multiple transcripts. Aberrations in regulative factors of alternative splicing are known to produce transcript isoforms associated with cancer progression and prognosis. Both mutations in the proximity of spliced regions and mutations in and regulation of previously identified splicing factor genes are known to alter splicing patterns.

In this study, we utilized publicly available TCGA data to study association patterns between alternative splicing and its regulatory elements across multiple cancer tissue types. We collected evidence of splicing patterns of annotated isoforms in addition to novel patterns identified using our previously developed tools using RNA-seq data. We mined for associations between somatic mutations and changes of splicing patterns as well as genome and transcriptome level changes in splicing factors. Selective transcriptome assembly was done in loci with differentially expressed patterns and novel patterns with sufficient evidence to identify transcript isoforms and quantify isoform specific expression.

In our study we compiled a catalogue of splicing patterns and their changes across multiple cancer types. Integration of this comprehensive set of quantified splicing patterns with data on proximal mutations and aberrations in splicing factors resulted in statistically relevant associations with clinical phenotypes. Our analysis enables identification of mechanisms behind splicing aberrations common to all cancers and cancer specific splicing patterns.

#1514

Significance assessment of mutations in 944 MDS patients using publicly available variant databases and mutation impact prediction software.

Niroshan Nadarajah, Manja Meggendorfer, Wolfgang Kern, Claudia Haferlach, Torsten Haferlach. _MLL Munich Leukemia Laboratory, Munich, Germany_.

Introduction: In 2015 the FDA issued a call to the public to receive feedback on FDA's regulatory approaches to diagnostic tests using next generation sequencing technology. For the clinical performance of such tests one of the proposals was to use community-derived databases to classify variants, especially ClinVar, conceived as a clinically grade database. To test this we here applied ultra-deep sequencing and subsequent mutation profiling in patients with myelodysplastic syndomes (MDS).

Aim: Investigate the performance of public databases and mutation impact prediction software for the interpretation and distinct classification of variants of a well-characterized MDS mutation dataset (Haferlach et al, Leukemia 2014).

Patients and Methods: A total of 944 patients with various MDS subtypes were screened for gene mutations in 104 known/putative genes relevant to MDS using targeted deep-sequencing (Illumina, San Diego, CA). For this assessment the following databases were used: ClinVar (release 2015-11), COSMIC (v73) and dbSNP (v142). Additionally, mutations were computationally tested for their severity of impact on protein level using PolyPhen-2 and SIFT.

Results: In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). A total of 2764 variants were called, among them 1,608 being distinct in 96 genes. Assessment was conducted by submitting positional information of each mutation to the database/prediction software. ClinVar yielded information for 141 (9%) of the mutations, with TP53 being the best-characterized gene out of 34, comprising of 33 entries (23%). Querying COSMIC yielded information for 671 (42%) mutations in 61 genes, with a subset of them being particularly well characterized (TET2, TP53, DNMT3A, and ASXL1). 255/1608 variants were listed in dbSNP. In the majority of instances, no global minor allele frequency (MAF, frequency of occurrence in the population of a variant base) is given and the validation status lists only a single submitter, indicating poorer reliability.

Additionally, we analyzed the mutations with two tools (PolyPhen-2, SIFT) to predict the possible impact of an amino acid substitution on the structure and function of human proteins. Results were available for 1137/1608 mutations. In 72% (820/1137) results were concordant, but surprisingly in 28% (317/1137) instances, results were contradicting, leaving them non-interpretable based on the combined use of both tools.

Conclusion: 1) Assessment demonstrates that current methods for variant interpretation using publicly available databases have to be improved for the characterization of mutations in patients with myeloid neoplasms. 2) So far COSMIC seems to outperform ClinVar. 3) Tools for novel mutations (no record in any databases) seem to perform well in quite a few instances, but a consensus of multiple tools is needed due to contradicting results.

#1515

Identification and characterization of HPV-host fusion transcripts in HNSCCs.

Lada A. Koneva, Yanxiao Zhang, Pelle Hall, Shama Virani, Alisha Virani, Thomas E. Carey, Laura S. Rozek, Maureen A. Sartor. _University of Michigan, Ann Arbor, MI_.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most incident cancer worldwide. Human papillomavirus (HPV) is implicated in at least 70% of oropharyngeal squamous cell carcinomas in the US. HPV genomic integration events are a likely critical step in progression to cancer and occur as a consequence of HPV oncogene-induced chromosomal instability. The majority of viral integration events in the human genome appear to occur within or near genic regions. Identified HPV integration events in HNSCC were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and inter-/intrachromosomal rearrangements.

Materials and methods

We analyzed 84 HPV-positive HNSCC samples collected at the University of Michigan (18 tumors) and from The Cancer Genome Atlas (66 tumors). We used VirusSeq software for detection of integration events. Integrations were evidenced by host-virus fusion transcripts in RNA-seq, considering only breakpoints supported by at least four discordant read pairs and at least one junction spanning read.

Results

We identified 50 integration-positive tumors, which consisted of 41 with HPV16, one tumor with HPV18, 5 tumors with HPV33 and 3 with HPV35. We found an overall 271 breakpoints of integration within or near 83 human genes. Fusion virus-host transcripts with breakpoints within E6 and E7 HPV oncogenes were more common (59.3%) compared with breakpoints into other viral genes: E1 and E2 (19%), E4 and E5 (16.8%), L1 and L2 (4.7%).

The detected viral integration events were widespread across the human genome with a few hotspots of recurrent integrations in genic regions at 1p36, 9p24, and 13p22. After accounting for regions covered by the data, we did not find an association of integration sites with aphidicolin-induced common fragile sites (CFSs), as observed for HPV integration sites in cervical cancer.

Analysis of the protein interaction network constructed from the 83 host genes harboring viral integrants showed that 56 of them were linked into a highly connected sub-network with direct interactions. ETS2, TP63, FOXA1, CTGF and KLF5 were hubs in this network. The genes were statistically enriched for cancer genes known as important in "Head and Neck Neoplasms"(p= 1.74E-11).

Conclusion

HPV integration events in HNSCC are largely different from those common to cervical cancers. While TP63 appears to be a recurrent target in both cervical cancer and HNSCC, HNSCCs have a surprisingly high percent of integration events in known HNSCC-related genes. Overall, our results suggest that there is strong selective pressure for integration events that occur in HNSCC relevant genes.

#1516

Biomedical informatics applied to the analysis of colon cancer genome: looking for a mutational pattern associated to infectious-origin cancer.

Claudia Machicado,1 Enrique Machicado2. 1 _Universidad Peruana Cayetano Heredia, Lima, Peru;_ 2 _Hospital Nacional Arzobispo Loayza, Lima, Peru_.

Colon carcinoma, one of the most common malignancies globally, is the fourth cause of death due to cancer in Peru. Number of diagnosed cases have increased notably in the latest 5 years, which can be partially explained due to eating habits and environmental factors. In the present work we investigated the presence, in colon cancer genome, of genetic mutations reported in infectious-related cancer. Since most of gastric cancer is associated with chronic infection by Helicobacter pylori and Epstein-Barr virus (EBV), we investigated if the mutational pattern of gastric cancer caused by such infectious agents was similar to the colon carcinoma. Biomedical informatics tools were used to analyze the genomes of both gastric and colon carcinoma. Taken the 20 most frequently mutated genes both in gastric and colon cancer, a total of 8 genes in common resulted broadly mutated in both malignancies including p53, ATM, PIK3CA, ARID1A, CREBBP, KMT2D, KMT2C, and TRRAP. From such list, PIK3CA gene has been associated with gastric cancer caused by H. pylori and the mutational profile matched with that found in colon carcinoma. The point mutations of the oncoprotein PIK3CA identified equally in gastric and colon carcinoma were K111E, M1043I and E545G, all of them associated with gastric cancer caused by infection. Further screening of the gastric cancer genome allowed us to identify a point mutation of the oncoprotein β-catenin reported in infective-origin gastric cancer (D32N). Gene amplifications reported in infection-caused gastric cancer were also identified in the colon carcinoma genome in JAK2, CD274 and PDCD1LG2 genes. Finally downregulation of the tumor supresor gene FOXD3 associated to gastric cancer caused by pathogens was also found in colon carcinoma. Given that the origin of such genetic mutations have not been determined in colon cancer, our study brings evidence that colon carcinoma contain genetic mutations associated to infectious-related cancer. Further experimental studies will be required to verify the existence of mutations induced by parasites in colon carcinoma.

#1517

Impact of poly-A and ribo-depletion RNA-seq library construction protocols on transcriptomic analysis of samples from patients with haematological malignancies.

Ashwini Kumar, Matti Kankainen, Alun Parsons, Olli Kallioniemi, Pirkko Mattila, Caroline Heckman. _Institute for Molecular Medicine Finland, Helsinki, Finland_.

RNA sequencing (RNA-seq) is a versatile tool for characterization and quantification of transcriptomes. It has become the main approach to elucidate the transcriptomic landscape in leukemia. However, the knowledge about the advantages and disadvantages of RNA-seq in clinical decision-making and the suitability of mainstream RNA-seq library preparation methods for leukemia research are still in their infancy. Here, we have generated and sequenced polyadenylation selection (PA) and ribo-depletion (RD) RNA-seq libraries from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples and compared their fusion-gene, differential expression and related pathways and variant discovery abilities.

Our analysis demonstrated that the RD protocol provided, on average, 5% more uniquely mapped reads than the PA protocol (81.5% of reads). Overall, 60%, 35%, and 3% of the reads in the RD libraries mapped to exonic, intronic, and intergenic regions, respectively. The corresponding numbers in the PA libraries were 76%, 21%, 3%, suggesting that PA captures more mature RNAs than the RD preparation and provides a higher read coverage for exon regions. The RD preparation was also marginally better in removing ribosomal RNAs (rRNAs) than the PA protocol. Regarding transcript quantification, both RD and PA produced fairly equivalent measures of RNA abundance. The patient-matched PA and RD libraries showed a high correlation (r=0.96) that was slightly less than that seen between technical replicates (r=0.98) but slightly more than that seen between library-matched sample from patients with the same disease (r=0.93), indicating that inter-patient variance is the main source of variance in RNA-seq studies. Interestingly, based on a receiver operating characteristic (ROC) analysis using known leukemia gene signatures or gene targets of 17 clinically used drugs in AML and ALL treatment, the PA is more optimal than the RD method for disease classification (area under curve 0.69 vs. 0.65) and personalized cancer therapy guidance (AUC of 0.73 vs, 0.71). Finally, both methods equally detected known fusion genes, which were supported with high number of reads on fusion

junctions. However, in the case of lowly expressed fusion transcripts, only the RD method was able to detect fusions.

In conclusion, both protocols produced consistence measures and were of similar usability. However, RD preparation captured more transcriptomic features and was more suitable for fusion-gene discovery, which may be of clinical relevance for sub-typing leukemia patients. The PA method on the other hand had higher overall sensitivity and specificity in gene expression analyses and was, based on our results, more suitable than RD for clinical classification and drug treatment guidance.

#1518

Lung adenocarcinoma differential expression analysis using the Maverix RNA-Seq pipeline.

Michael S. Fitzsimons, Mei-Chong Wendy Lee, Byung-In Lee, Lenin Subramanian. _Maverix Biomics, San Mateo, CA_.

Background

RNA-Seq is a powerful means of identifying changes in gene expression in cancerous tissue. Widespread adoption of RNA-Seq is hampered by lack of familiarity with the appropriate analysis tools and excessive turn around time. Maverix Biomics, Inc offers a scalable, reliable, validated, and easily accessible differential expression pipeline for translational and clinical researchers to analyze and explore RNA-Seq data. Here we describe the pipeline, demonstrate the available tools and visualizations, and provide benchmarks for completion time from lung adenocarcinoma.

Results

Lung adenocarcinoma samples appear very different compared to their normal counterparts in terms of overall gene expression and exhibit many differentially expressed genes. For analysis completion time, parallelization is far superior than the theoretical curve without parallelization. It took 7.9 hours to analyze two samples, but took only 13.1 hours to analyze 25 samples. We found 2246 significant genes common to three different differential expression tools: DESeq, edgeR, and cuffdiff.

Conclusions

The Maverix RNA-Seq pipeline is demonstrated here to be highly scalable, easy to use, and includes a range of tools and visualizations for understanding differential expressions results. This pipeline is a great analytic option to accelerate translational and clinical research in the field of oncology.

#1519

Myb-regulated gene networks and signaling pathways in pancreatic cancer.

Shafquat Azim, Sanjeev K. Srivastava, Arun Bhardwaj, Haseeb Zubair, Mohammad Aslam Khan, Seema Singh, Ajay P. Singh. _USA Mitchell Cancer Institute, Mobile, AL_.

The MYB proto-oncogene (a cellular progenitor of v-Myb oncogene) encodes an oncogenic transcription factor that regulates the expression of a wide array of genes responsible for different cellular functions. We have recently identified MYB as a key driver of pancreatic cancer (PC) pathogenesis. Our findings demonstrated that MYB was overexpressed in majority of PC tissues and cell lines, while remained undetectable in normal pancreatic cells. In addition, MYB overexpression was shown to be associated with pancreatic tumor growth and metastasis. To gain further insight into the molecular mechanisms involved in MYB-potentiated effects in PC cells, we conducted Next-Generation Sequencing of transcriptome of control and MYB-silenced MiaPaCa cells on an Illumina HiSeq 2500 platform. By keeping a cut-off of fold change > 1.5 and p ≤ 0.05, we identified a total of 774 genes to be differentially expressed in MYB-altered MiaPaCa cells. Out of these, 485 genes were downregulated, while 289 genes exhibited upregulated expression. The differentially-regulated genes were subsequently analyzed for functions and cellular pathways alterations using Ingenuity Pathway Analysis (IPA) software. The top three networks to be affected in MYB knockdown PC cells included i) RNA post-transcriptional modifications, molecular transport, RNA trafficking, ii) cellular assembly and organization, cancer, and, iii) cell cycle, DNA replication, recombination and repair. These networks are centered on splicing factors, EGFR and NF-κB complex, respectively. IPA predicted pancreatic adenocarcinoma signaling as one of the most significantly affected canonical pathway upon MYB-silencing. Altogether, our findings identify novel MYB-regulated gene networks and signaling pathways in PC and thus suggest potential molecular mechanisms involved in mediating MYB action in pancreatic tumorigenesis.

#1520

Identifying differential dependency networks accounting for response to NEDD8-inhibitor in large-scale cancer cell line data.

Gil Speyer,1 Harshil Dhruv,1 Jeff Kiefer,1 Stuart Schreiber,2 Paul Clemons,2 Michael E. Berens,1 Seungchan Kim1. 1 _TGen, Phoenix, AZ;_ 2 _Broad Institute of Harvard and MIT, Cambridge, MA_.

The Cancer Cell Line Encyclopedia (CCLE) houses molecular profiles of ~800 human long term cell lines spanning several different histological types of cancer. In addition, Cancer Therapeutics Response Portal (CTRP) provides drug response measurements for 481 small molecules. Integration of these data enables investigation of the molecular correlates of drug response (sensitivity and resistance). In this current effort, we studied the NEDDylation small molecule inhibitor, MLN4924, in the context of genomic data to uncover novel mechanistic correlates of drug response across the panel of cell lines. We recently reported (Jung and Kim 2016 NAR) development of a robust computational method that shows promise to identify novel insights when applied to multi-dimensional data sets as outlined above. The Evaluation of Differential Dependency (EDDY) employs Bayesian networks to represent statistically distinct differences in relationships between genes within a specific biological pathway as queried between two conditions, in this instance, cell lines that are sensitive and those that are non-sensitive to MLN4924. While EDDY has been successfully employed in the analysis of specific diseases such as TCGA adrenocortical carcinoma, its statistical rigor incurred a prohibitive computational load to assess conditional differences across larger datasets. Recent computational enhancements to EDDY enable processing of larger datasets in reasonable time while maintaining sensitivity. The capability of analyzing broader pan-cancer datasets such as CCLE has enabled EDDY to become more capable in identifying general trends across disease subtypes. Specifically, we demonstrate the enhanced EDDY in analysis of MLN4924 response across the CCLE data set combined with CTRP data set.

Initial outcomes from EDDY point to both anticipated and unanticipated biological determinants of response. For example, it is noted that specific oncogenic pathways, such as those centered on PIK3CA, appear to show differential dependencies in the sensitive and non-sensitive cell lines. We also observe genes and candidate pathways related to apoptotic mechanisms that may reveal mechanistic insights to predicting drug response. Specifically, genes and pathways associated with certain apoptotic mechanisms around mitochondrial proteins and glutathione peroxidase may serve as unique determinants of drug response. Multidimensional data analyzed by EDDY uncovers candidate mechanisms of vulnerability to specific small molecule inhibitors, which may guide development of predictive models for treatment planning when using agents with highly context-dependent efficacies. Supported by NIH U01CA168397

#1521

Transcriptional networks of the normal human kidney.

David Lindgren,1 Jennifer Hansson,1 Helen Nilsson,2 Krzysztof Krawczyk,2 Martin Johansson,2 Håkan Axelson1. 1 _Laboratory Medicine, Lund University, Sweden, Lund, Sweden;_ 2 _Department of Translational Medicine, SUS Malmö, Malmö, Sweden_.

The kidney is a complex but highly structured organ composed of more than 20 different cell types. Disturbances among these cell types leads to a plethora of diseases of acute or chronic type, some of which are life threatening. In addition, kidney cancer represents about 3 percent of all malignancies. Considerable efforts have been made to profile the different cell types of the kidney. However, these are mainly based on studies in rodents. In the current study we present a method, called composition based cellular stratification that define gene signatures specific to different cell types and regions of the kidney. The method uses correlation-based analyses of gene expression microarrays of needle core biopsies obtained from histologically normal kidney tissues. For example, a HNF-driven gene expression signatures of proximal tubuli cells could be defined. This signature was validated in publically available data at the gene expression as well as at the protein level. We employ these gene expression signatures as a framework to study expressional changes observed in renal cell carcinoma, thus increasing the possibility to pin-point cell specific alterations associated with the transformation of renal epithelial cells.

#1522

Predicting drug sensitivity based on gene array data for cytotoxic chemotherapeutic agents.

Joshua Mannheimer, Jared S. Fowles, Katherine Shaumberg, Dawn L. Duval, Ashok Prasad, Daniel L. Gustafson. _Colorado State University, Fort Collins, CO_.

The increasing availability of genetic data has led to a demand for robust methods to extract information and an increased need for complex computational analysis. The DREAM project consisted of 44 teams assessing various computational methods to predict drug sensitivity in 53 breast cancer cell lines. (Nat Biotechnol 32:1202). Results from this and other studies suggest that gene expression data from microarrays are one of the more valuable and robust data sets for predicting drug sensitivity. Whole exome sequencing is a valuable tool for predicting sensitivity to targeted agents, however the mutational spectrum is not as predictive for cytotoxic agents (Cancer Res 73:4372). Thus, a systematic analysis of the predictive ability of gene expression on cytotoxic chemotherapy drug sensitivity (IC50 values) using multiple cell line panels is an important component to understanding the versatility and applicability of gene expression for predicting cytotoxic drug IC50 values in tumor cells. Using GDSC and NCI60 cell panels, linear regression models were constructed to predict IC50 values from array data for 13 cytotoxic agents. To address the limited amount of data compared to a possible feature space > 22,000 genes, the methods of Principle Least Squares Regression (PLSR) and Principle Component Regression (PCR) were employed. Models were generated using four different training and validating scenarios to evaluate performance. Mean Absolute Differences (MAD) achieved between expected and predicted IC50 values as low as 11% and correlations as large as 0.7 (P<0.05) were achieved for some drugs. However, several obstacles have presented themselves. Mainly, among 35 cell lines shared by the GDSC and NCI60 direct correlation across common cell lines averaged only 0.4 highlighting the challenge to obtain, evaluate, and address consistent data suitable for robust models.  | |

|

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

Model Performance Metrics showing high, average, and low values across all thirteen drugs.

Model | Measurement | PCR

Low : Avg. : High | PLSR

Low : Avg. : High

GDSC: Train

GDSC: Valid | Correlation | 0.16 : 0.36 : 0.7 | 0.12 : 0.36 : 0.66

P-Value | 0a : 0.008 : 0.009 | 0 : 0.02 : 0.16

MAD % Range | 11 : 13 : 16 | 11 : 13 : 16

Classification Accuracy | 0.39 : 0.66 : 0.94

|

NCI60: Train

NCI60: Valid | Correlation | -0.11 : 0.36 : 0.67 | -0.21 : 0.4 : 0.79

P-Value | 0.007 : 0.31 : 0.755 | 0.0007 : 0.24 : 0.87

MAD % Range | 11 : 17 : 27 | 11 : 17 : 29

Classification Accuracy | 0.33 : 0.62 : 0.75 | N/A

GDSC: Train

NCI60: Valid | Correlation | 0.2 : 0.36 : 0.55 | 0.16 : 0.34 : 0.56

P-Value | 0 : 0.03 : 0.13 | 0 : 0.06 : 0.23

MAD % Range | 11 : 23 : 37 | 11 : 23 : 39

Classification Accuracy | 0.32 : 0.59 : 0.82 | N/A

NCI60: Train

GDSC: Valid | Correlation | 0.023 : 0.19 : 0.48 | -0.03 : 0.18 : 0.43

P-Value | 0 : 0.08 : 0.75 | 0 : 0.09 : 0.72

MAD % Range | 12 : 19 : 36 | 12 : 19 : 35

Classification Accuracy | 0.37 : 0.53 : 0.69 | NA

a Low p values of 0 correspond to p values < 10-5

In summary, PCR and PLSR based gene expression models predict tumor cell sensitivity to some cytotoxic drugs with varying degrees of accuracy.

#1523

Cross-disciplinary methods for personalizing screening modalities for early gastric cancer intervention.

Rachel Walker,1 Jaime Mejia,2 Heiko Enderling,1 Jose M. Pimiento,1 Domenico Coppola1. 1 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _Instituto de Patologia Mejia Jimenez, Cali, Colombia_.

Background.

Up to 80% of patients in the early stages of gastric cancer are asymptomatic, making early diagnosis and hence effective treatment challenging. The poor prognosis of patients with late diagnoses clearly necessitates the identification of markers for early detection and the development of streamlined screening protocols. This is particularly crucial in high-risk populations, for example regions in which Helicobacter Pylori infection and associated chronic inflammation, responsible for over 70% of gastric cancers worldwide, is prevalent. At present, an efficient and personalized program for early detection does not exist.

Methods.

Specific biomarkers for the progression of gastric cancer have been identified in preliminary studies, including CD44, Lgr5 and CD133. In retrospective tissue samples incremental increases in expression of these markers was observed during the progression from normal gastric tissue to precancerous histologic lesions such as intestinal metaplasia and ultimately dysplasia and carcinoma. We developed a mechanistic mathematical model capable of simulating these marker-positive population dynamics, calibrated using the existing clinical data. The predictive capability of the model is validated by comparison of simulated to actual marker expression patterns in an independent test cohort of endoscopic gastric biopsies taken at sequential points during individual patients' disease progression.

Results.

Based on clinically obtainable, patient-specific tissue conditions, the computational model can simulate the dynamics of marker-positive cells and, when calibrated and validated with clinical data from specific patient populations, may forecast disease progression and suggest optimal screening schedules for individual patients.

Conclusions.

The tools of mathematical oncology have the potential to directly inform clinical screening protocols. Verified prognostic factors for disease progression can be incorporated into mathematical models to improve diagnostic accuracy and allow clinically-actionable screening optimization. This can guide personalized screening schedules according to current marker expression on a case-by-case basis to achieve more efficient prognosis and reduced healthcare costs. 

## TUMOR BIOLOGY:

### Biomarkers and Profiling of Metastasis

#1524

Mutations and gene copy number variations landscape of metastases of various cancer types from patients enrolled in the SHIVA trial.

Maud Kamal, Nicolas Servant, Gaelle Pierron, Celine Callens, David Gentien, Alban Lermine, Georges Lucotte, Virginie Bernard, Anne Vincent-Salomon, Ivan Bièche, Christophe Le Tourneau. _Institut Curie, Paris, France_.

Background

The molecular landscape of primary tumors of several cancer types is available and tissue-independent classification of tumors on the basis of genetic and epigenetic alterations is emerging1. The molecular profile of metastatic cancers remains to be unraveled. Here we present mutations and gene copy number variations (CNV) of metastases across tumor types from patients enrolled in the SHIVA trial2.

Patients and methods

The SHIVA trial is a multicentric randomized proof-of-concept phase II trial comparing molecularly targeted therapy based on tumor molecular profiling versus conventional therapy in patients with any type of refractory cancer. Mutations using targeted NGS (AmpliSeq cancer panel on Ion Torrent / PGM (Life)), and CNV using Cytoscan HD (Affymetrix) were assessed on a mandatory biopsy from a metastatic site with ≥30% of tumor cell content. Sequencing data were processed in order to detect actionable somatic mutations. Detected variants were filtered according to their frequency (≥4% for SNVs and 5% for indels), strand ratio (≥0.2), and reads coverage (≥30X for SNVs and 100X for indels). Non polymorphic deleterious mutations were reported. Copy number and allele difference profiles were processed taking into account % of tumor cells and ploidy of the sample. Homozygous and heterozygous deletions and focal amplifications were reported for tumor suppressor genes and oncogenes. Between October 2012 and July 2014, 741 patients with any type of solid cancer were enrolled in the SHIVA trial. Among these, 507 and 496 patients had high quality NGS and CNV profiling, respectively.

Results

Major cancer types for which molecular analyses were possible on a metastatic biopsy were: breast (14%), colorectal (11%), lung (11%), ovarian (11%), pancreatic (8%), sarcoma (6%), head and neck (4%), cervical (4%), urothelial (4%), endometrial (3%), oesogastric (3%) cancers, adenoid cystic carcinoma (3%) and others (18%). TP53 mutations were present in 44% of metastatic patients. Major mutated oncogenes were KRAS (20%), PIK3CA (12%), MET (6%), FGFR3 (5%), EGFR (2%) and BRAF (2%). Tumor suppressor genes PTEN, FBXW7 and CDKN2A were mutated in 3% of patients. Alternatively, homozygous and heterozygous deletions of PTEN, FBXW7 and CDKN2A were observed in around half of the patients analyzed (40%, 40% and 54%, respectively).

Conclusions

Mutations and CNV analyses on metastatic tumors from patients enrolled in the SHIVA trial showed similar results in terms of frequency of previously reported genetic alterations1,3. The frequency of KRAS mutations (20%) was however higher than the frequency (7%) reported in the pan cancer cohort of the tumors portal3. This result needs to be interpreted in accordance with tumor types before suggesting potential resistance mechanisms in heavily pretreated patients. Clustering of these alterations in specific signaling pathways is currently ongoing.

#1525

Vortex technology for label-free enrichment of CTC from mouse xenograft models.

Kyra Heirich,1 Melanie M. Triboulet,1 Corinne M. Renier,2 Vishnu C. Ramani,1 Elodie Sollier,2 Stefanie S. Jeffrey1. 1 _Stanford University, Stanford, CA;_ 2 _Vortex Biosciences Inc., Menlo Park, CA_.

Background

Non-invasive liquid biopsies, such as CTCs, have been of growing interest due to their potential use in cancer detection, prognosis, and monitoring of therapeutic resistance [1]. Beyond enumeration, characterization of CTCs could help guide treatment selection and the development of targeted cancer therapy. Here, we show that Vortex technology can be used successfully for the size-based capture of CTCs in a preclinical mouse model of breast cancer.

Method

To establish that human epithelial cancer cells can be reliably detected in small volumes of mice blood, 50-100 MDA-MB231 and MCF7 breast cancer cells were spiked in 500 μL mice blood, diluted 20 fold, and processed through Vortex chip [2]. Recovered cells were immunostained (CK, CD45, DAPI), and enumerated. For breast cancer xenograft model, 8x106 MDA-MB-231-fLuc/GFP cells were implanted orthotopically into the mammary fat pad of NOD-SCID Gamma mice (n=35). Tumors were measured in 2 dimensions 3 times/week and tumor volume calculated. Blood from cardiac puncture (500 μl) and lateral saphenous vein (100 μl) was collected starting 1 week post implantation, diluted 40X and processed. Mice were euthanized, organs harvested, formalin fixed, paraffin embedded and H&E stained.

Results

Spiking of MCF7 cells in mouse blood resulted in a capture efficiency of 56.9±13% and a purity of 80.1± 9% (n=3), with the flow-through from each cycle being re-processed for a total of 4 cycles. Recycling the flow-through led to capture efficiency of 36.7%, 23.4%, 5.2% and 7.9% during cycles 1, 2, 3 and 4 respectively. Similar results were obtained for MDA-MB-231, with a capture efficiency of 54.4% and a purity of 41.3%. In an orthotopic tumor xenograft model of breast cancer, CTCs were successfully recovered from cardiac puncture as early as day 7. CTC counts, ranging from 0.4-3649 CTCs/100 μl, increased over time and correlated with tumor burden. CTC clusters were also captured from day 7 (6 clusters/100 μl; frequency 1/3), with number and frequency increasing over time up to 147-485 clusters/100 μl by day 42. No CTC were recovered from lateral saphenous vein blood until day 28 post implantation, and their number remained low (mean 2.15±0.65). Microscopic metastases were evident in lung of all mice starting at day 28 and in liver of all mice starting at day 35.

Conclusion

CTCs were isolated in a label-free manner from mice blood with both high capture efficiency and purity. In a preclinical model of metastatic breast cancer, CTC counts correlated well with primary tumor volume and metastases occurrence. Thus the Vortex chip appears to be well suited for the enrichment of CTCs from murine xenograft models. Future works will focus on mice implanted with patient derived xenograft (PDX) and their therapeutic response. Information gathered from these studies should facilitate discovery of new therapeutic targets and the development of personalized medicine.

[1] Ignatiadis et al. Clin Cancer Res 2015.

[2] Sollier et al. Lab Chip 2014.[K.H. and M.T. contributed equally to this work.]

#1526

KPNA4 promotes prostate cancer metastasis through TNFα/β mediated cytokine crosstalk in tumor microenvironment.

Jian Yang, Cuijie Lu, Juncheng Wei, Wendi Liu, Yuqi Guo, Xin Li. _New York University College of Dentistry, New York, NY_.

Prostate cancer (PCa) is one of the most common types of cancer for man in worldwide. Distant metastasis to multiple organs is the key lethal factor for PCa patients, still, little is known about the molecular mechanism. Here, we reveal that Karyophorin α4 (KPNA4), an importin family member, mediated cytokine crosstalk plays a critical role in PCa skeletal metastasis. Both IHC staining of KNNA4 on human PCa tissue microarray slides and KPNA4 expression in a TCGA (The Cancer Genome Atlas) dataset demonstrates a positive association between KPNA4 and the pathological stages as well as the Gleason scorse of human prostate cancer. Accordingly, transient knockdown of KPNA4 in multiple PCa cell lines reduces cell migration in vitro. Furthermore, stable knockdown of KPNA4 in PC3 cell line (PC3-shKPNA4) significantly attenuates the primary tumor invasion and bone metastasis in our experimental orthotopic and metastatic mouse models. Cytokines array assays reveal that KPNA4 knockdown reduces to the production of primary cytokines TNF-α and β in PCa cells. Since TNF α and β are the central inflammatory cytokines in the complicated cytokines network as well as immune response, TNF α and β could mediate the effects of KPNA4 on PCa metastasis through priming tumor microenvironment. As expected, TNF-α/β can rescue the migration capacity of PC3-shKPNA4 as well as the M2 polarization of tumor-associated macrophages. Furthermore, PC3-shKPNA4 derived conditioned medium suppresses the inflammatory response of RAW264.7 cells, supporting the critical role of KPNA4 in regulating the tumor-microenvironment. It is known that the formation of metastatic lesion in bone environment is enhanced by activated bone resorption. In addition to modulating the tumor microenvironment adjacent to the primary tumors, TNF-α/β also favor PCa metastasis by restoring the osteoclast formation and activity suppressed by PC3-shKPNA4. Interestingly, only the protein but not the RNA level of KPNA4 is dramatically increased in PCa cells. Therefore, the dysregulation of KPNA4 in PCa cells could be mediated by a post-transcriptional process. Indeed, through bioinformatical screening and transient infection, KPNA4 is identified as a target of miR-708, a tumor suppressive miRNA reported in multiple types of cancer. Supporting the increased expression of KPNA4, miR-708 level is radically inhibited in PCa cells. Collectively, our data reveal a novel mechanism mediated by KPNA4 that links the loss function of miR-708 and the activation of cytokines in facilitating the metastasis of prostate cancer. The KPNA4/TNF axis stimulates PCa metastasis by promoting cancer cell mobility, priming tumor micro-environment nearby and distantly. These findings shed light on the molecular mechanism of prostate cancer metastasis in terms of the regulation of cytokines crosstalk which provides a potential new diagnosis biomarker and therapeutic target for prostate cancer treatment.

#1527

Urinary miRNAs as predictors of tumor metastasis in localized patients with clear cell renal cell carcinoma: a pilot study.

Xiang Shu,1 Michelle AT Hildebrandt,1 Rodolfo Borges dos Reis,2 Yuanqing Ye,1 Jian Gu,1 Christopher G. Wood,2 Xifeng Wu1. 1 _Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction: 25-50% of localized renal cell carcinoma (RCC) patients will eventually develop metastasis which has a dramatic adverse effect on prognosis following treatment. Unfortunately, reliable biomarkers predicting RCC metastasis are still lacking. MicroRNAs (miRNAs) are important regulators for vital biological processes in humans. Urine miRNAs(UmiRNAs) present promising potential as biomarkers for diagnosis of urological cancers. However, no study has assessed the predictive value of UmiRNAs for RCC metastasis.

Materials and Methods: We prospectively collected pre- and post-surgery urine samples from 117 patients with localized clear cell RCC (ccRCC,T1-3 N0/x M0). UmiRNAs were extracted and purified from 250 μl urine supernatants and measured using RT-PCR. Relative quantifications (RQ) of 137 selected miRNAs were calculated by 2-∆∆ct method. The ratio of post/pre RQs were tested for the association with distant metastasis using multivariable Cox regression models.

Results: More than half of the patients were diagnosed at pT3 (53%) or grade III-IV (69%). Twenty-two patients (18.8%) had metastasis during a mean follow-up of 73 months. The levels of 34 UmiRNAs were significantly changed after nephrectomy (p<0.05, 29 of 34 significantly reduced). The 50% or more reduced post-nephrectomy levels of five UmiRNAs (miR-191-5p, miR-324-3p, miR-186-5p, miR-93-5p, and miR-30b-5p) independently conferred a 2- to 4-fold increased risk of metastasis with miR-191-5p being the most significant one (hazard ratio [HR]=4.16, 95% confidence interval [CI]=1.38-12.58, p=0.011). Stratified analyses by stage and Fuhrman grade revealed that the associations were most profound in pT3 patients for miR-191-5p, miR-324-3p, and miR-186-5p. Ingenuity pathway analysis showed that the predicted target genes of these five UmiRNAs were enriched in cell survival and proliferation networks.

Conclusion: Our study suggests that urinary miR-191-5p, miR-324-3p, and miR-186-5p are potential biomarkers for the prediction of metastasis in localized, particularly pT3 ccRCC patients after nephrectomy. These findings may help improve individualized patient management in the clinic.

#1528

Selective gene-expression profiling of metastasizing breast tumor cell subpopulations complements the predictive power of Mammaprint Dx and Oncotype Dx.

George S. Karagiannis, Sumanta Goswami, Joan G. Jones, Maja H. Oktay, John S. Condeelis. _Albert Einstein College of Medicine, Bronx, NY_.

Gene-expression profiling has yielded prognostic tests, such as MammaPrint and Oncotype Dx, which facilitate clinical decision-making in breast cancer patients. These tests are based on signatures constructed from whole tumor tissue, including tumor as well as stromal cells. Since metastatic tumor cells are not uniquely identified in these signatures, these tests may have reduced clinical specificity and sensitivity in predicting risk of metastasis. In contrast, we captured breast cancer cells in the act of migration/dissemination using an in vivo invasion assay and expression profiled these cells to create a distinct signature named the human invasion signature (HIS). A comparison between HIS and MammaPrint in the NKI295 cohort demonstrated that both signatures perform comparably in selecting a group of patients with significantly poorer outcomes. However, when compared to MammaPrint, the initial slope of the high-risk patients identified by the HIS is significantly steeper, suggesting that the HIS may identify patients at higher risk of early distant metastatic recurrence. Therefore HIS carries prognostic information beyond that captured by MammaPrint. Additionally, a more thorough examination of candidate genes present in HIS yielded two predictive tissue-based biomarkers: TMEM score which measures the density of cancer cell intravasation sites, and MenaCalc which measures the expression of invasive isoforms of the actin-regulatory protein Mena (MenaCalc=PanMena-Mena11a), thereby identifying tumor cells that disseminate. Both, TMEM density and MenaCalc independently predict the development of distant metastases in breast cancer patients. Here, we show that in a small patient cohort (n=58), there is no correlation between TMEM density and the Oncotype recurrence score (RS). In particular, a subgroup of patients had tumors with high-Oncotype DX and low TMEM score while another subgroup had low Oncotype DX and high TMEM score. These findings indicate that TMEM and Oncotype Dx address different tumor biology and could be used in a complementary fashion for more accurate patient stratification, as well as better selection of systemic therapies to avoid over- and under-treatment.

#1529

RASAL2 promotes tumor progression and metastasis in colorectal cancer.

Yi PAN, Joanna Hung Man TONG, Raymond Wai Ming LUNG, Samantha Wei Man LUN, Kwok Wai LO, Anthony Wing Hung CHAN, Ka Fai TO. _Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Background:

Metastatic colorectal cancer (mCRC) is a heterogeneous disease with a poor prognosis. By using high resolution array comparative genomic hybridization (aCGH) and mRNA gene expression microarray of mCRC samples, we found up-regulation of RASAL2, a RAS GAP gene that is located on chromosome 1q, in colorectal tumors. Oncogenic RAS represents one of the most common molecular changes in human colorectal adenocarcinoma. However, the role of RASAL2 in colorectal adenocarcinoma metastasis and the related mechanisms are still unclear. We analyzed its aberrant expression, clinical significance and biological effects in colorectal cancer.

Methods:

Expression of RASAL2 was examined by QRT-PCR, western blot and immunohistochemistry in CRC cell lines, primary and metastatic CRC and the corresponding normal colonic mucosa. The biological effects of RASAL2 on proliferation, apoptosis, cell cycle, and cell motility and invasiveness were evaluated by siRNA knock down and RASAL2 reconstitution in CRC cell lines.

Results:

Up-regulation of RASAL2 mRNA was observed in CRC cell lines (n=9) than normal colon mucosal tissues. Interestingly, significantly higher RASAL2 mRNA was found in cell lines derived from advanced stage tumors (n=4, Dukes' C and D) than those from early stage tumors (n=5, Dukes' A and B) (P=0.0317). Up-regulation of RASAL2 mRNA was also found in primary CRCs (n=77) compared with normal colon mucosa (n=18, P<0.0001). In 15 cases that paired primary and metastatic tumors were available, 12 (80%) demonstrated higher RASAL2 in metastatic tumors than their primary counterparts. RASAL2 protein was detected in 37% (81 of 219) of CRC by immunohistochemistry. Whereas in the paired normal colonic mucosa, the positive rate is 14% (20 of 142, P<0.0001).

We knocked down RASAL2 by siRNA in 2 CRC cell lines (DLD1 and HCT116) with high endogenous RASALs level. Successful knockdown was demonstrated by western blot analysis. SiRASAL2 significantly inhibited cell proliferation (P<0.05), reduced colony formation (P<0.05) and repressed cell invasion and migration ability (P<0.05) in both cell lines. Flow cytometry analysis revealed G1 arrest in cells treated with siRASAL2. Ectopic expression of RASAL2 was performed in 2 CRC cell lines with low endogenous RASAL2 (SW480 and LoVo). Over expression of RASAL2 did not change the cell proliferation and anchorage-dependent growth of these cell lines but effectively enhanced cell invasiveness by transwell invasion assay.

Conclusions:

Up-regulation of RASAL2 was frequently found in CRC cell lines and primary and metastatic tumors. Our experimental findings suggested that RASAL2 might play an oncogenetic role in CRC and promotes tumor invasion and metastasis. A better understanding of the molecular mechanism of RASAL2 in promoting CRC cell metastasis may lead to a more effective management of mCRC patients.

#1530

Dissecting breast cancer dormant CTC phenotypes.

Monika Vishnoi,1 Sirisha Peddibhotla,2 Wei Yin,1 Zhong Xue,1 Antonio T. Scamardo,3 Goldy C. George,3 David S. Hong,3 Dario Marchetti1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Tumor relapse is a clinically relevant problem in breast cancer where patients are asymptomatic because disseminated cells appear to become dormant for periods longer than 20 years and are undetectable by current clinical tools. Uncovering phenotypes of circulating tumor cells (CTCs) - the "seeds" of intractable metastasis-offers the promise to dissect CTC heterogeneity in relation to metastatic competence, to predict biomarker assessment, and to significantly improve monitoring and treatment of cancer. However, little is known about CTC biology and how CTCs differ in their capacity to circulate while maintaining a metastatic potential. We hypothesized that EpCAM-negative breast cancer CTC subsets exist, and avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties. We collected peripheral blood of clinically diagnosed breast cancer patients with or without brain metastasis, and performed multiparametric flow cytometry to isolate EpCAM-negative CTC subsets with stem-cell properties (CD44+/CD24-), along with combinatorial expression of two neoplastic markers: urokinase plasminogen activator receptor (uPAR) and integrin beta1 (int β1). EpCAM-negative CTCs were further interrogated at a single-cell level employing DEPArray platform. Second, we were able to culture FACS-sorted CTC subsets, selected for six cell-surface expression markers (CD45-/EpCAM-negative/CD44+/CD24-/uPAR+/-/int β1+/-), as long-term in-vitro 3D CTC tumorspheres. Third, CTC subsets were interrogated for biomarker profiling and biological characteristics. We identified adhesive, proliferative and invasive properties of 3D CTC tumorspheres which were distinct per uPAR/int β1 combinatorial expression. Lastly, we performed next-generation whole-genome sequencing and mutation analyses to discover unique genomic signatures of uPAR/int β1 CTC subsets and verified as putative CTCs originally disseminated from primary breast tumor. Additional investigations are being pursued assessing the molecular and genomic characterization of uPAR/int β1 CTC subsets comprehensively. Clinical relevance of this research includes that this may enhance abilities to prospectively identify patients who may be at high-risk of developing breast cancer brain metastasis.

#1531

Isolation of both EpCAM+ and EpCAM- tumor cells in microfluidic devices.

Z. Hugh Fan, Jinling Zhang. _University of Florida, Gainesville, FL_.

Isolation and enumeration of circulating tumor cells (CTCs) in peripheral blood have been used for cancer diagnosis, prognosis, and theragnosis. The majority of the CTC isolation methods, including the FDA-approved CellSearch®, employ antibodies against epithelial cell adhesion molecule (EpCAM). However, it is known that some CTCs express little or no EpCAM, partially due to epithelial-to-mesenchymal transition (EMT). As a result, an ideal method should be able to isolate all types of tumor cells including epithelial tumor cells (EpCAM positive), mesenchymal tumor cells (EpCAM negative), and those cells in the transition.

We have investigated in using fibrin-immobilized microfluidic devices to isolate both EpCAM+ and EpCAM- cells, since it has been found that fibrin deposits on the surfaces of carcinoma and sarcoma cells where normal cells and tissue showed no deposits of fibrin during tumor progression/metastasis [1]. The microfluidic devices we used [2] are in the size of a microscope slide and each of them consists of an inlet, an outlet, and eight parallel channels connected via consecutive bifurcation between the inlet and outlet. There are micromixers inside the channels for enhanced interactions. The channels were first exposed with thrombin and then fibrinogen, followed by a buffer to stop the thrombin-activated fibrinogen's polymerization. At the end, a thin layer of fibrin polymer was formed on the surface of microchannels and the device was then used to isolate tumor cells.

We tested the device for isolation of Pan-1 (EpCAM+ pancreatic cancer cells), CCRF-CEM (EpCAM- leukemia cells), and MIAPaCa-2 cells (EpCAM- pancreatic cancer cells) in PBS (phosphate buffered saline). All of them showed capture efficiency of 80% to 90% at a flow rate of 1.0 μL/s. The capture efficiency, calculated by dividing the number of cells captured by the number of cells introduced into the device, decreased as the flow rate increased from 0.5 μL/s to 3 μL/s, because there were less interaction time between cells and fibrin at a higher flow rate. Capture efficiency of 86+4% was obtained when CCRF-CEM cells were spiked in human whole blood at a concentration of 1 thousand to 1 million cells/mL. We also studied the effects of combining aptamers with fibrin on the channel surfaces and found an enhanced interaction between aptamer-specific tumor cells and the aptamer-fibrin surface.

In summary, microfluidic devices with fibrin-immobilized channel surfaces were able to capture both EpCAM+ and EpCAM- cells, suggesting a possibility to isolate both epithelial and mesenchymal tumor cells. Moreover, the fibrin surface could be enhanced by tumor-cell-specific capture agents such as aptamers or antibodies.

References:

[1] F. W. Schardt, B. Schmausser and E. Bachmann, Histol Histopathol, 2013, 28, 993-998.

[2] W. A. Sheng, O. O. Ogunwobi, T. Chen, J. L. Zhang, T. J. George, C. Liu and Z. H. Fan, Lab Chip, 2014, 14, 89-98.

#1532

The isolation of CTC from diagnostic leukapheresis.

Kiki C. Andree,1 Anouk Mentink,1 Martin Scholz,2 Roland Kirchner,2 Rui P. Neves,3 Christiane Driemel,3 Rita Lampignano,3 Hans Neubauer,3 Dieter Niederacher,3 Tanja Fehm,3 Wolfram T. Knoefel,3 Johannes C. Fischer,3 Nikolas H. Stoecklein,3 Leon WMM Terstappen1. 1 _University of Twente, Enschede, Netherlands;_ 2 _Leukocare AG, Munich, Germany;_ 3 _University Hospital of the Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany_.

Introduction

At present, the CellSearch system is the only validated method for the detection of circulating tumor cells (CTC) that has been cleared by the U.S. Food and Drug Administration. This system, designed for the enumeration of CTC in 7.5 mL of blood, detects CTC based on their expression of EpCAM and cytokeratins and negativity for CD45. However, the number of CTC that are detected in patients with metastatic carcinomas is in most cases too small to reliably determine tumor heterogeneity and to be representative as a 'liquid biopsy'. Our aim is to identify and isolate a sufficient number of circulating tumor cells in virtually all metastatic cancer patients to enable their characterization and to represent a real-time liquid biopsy. For this purpose we used Diagnostic LeukApheresis (DLA) to increase the blood volume to be analyzed. We developed several techniques to isolate CTC from DLA to enable a multicenter comparison of CTC detection in DLA products.

Methods

DLAs were performed for ~1 hour to obtain 40 mL of product containing ~4 x10^9 mononuclear cells representing ~1 liter of blood. Using CellSearch a maximum of 2 mL of DLA could be processed for EpCAM+ CTC (Fisher et al. doi: 10.1073/pnas.1313594110) and EpCAM- CTC (de Wit et al doi: 10.1038/srep12270). Using filtration through microsieves with 5 μm pores a maximum of 1.0 mL of DLA could be processed. To process 18 mL DLA product protocols were developed for leukocyte depletion using RosetteSep™ (StemCell Technologies, USA) and for EpCAM selection using an anti-EpCAM coated column (Leukocare AG). All enriched cell fractions were stained using CD45 PerCP, Cytokeratins PE and the nuclear dye DAPI, followed by fluorescence microscopy scanning and analysis.

Results

Leukocyte depletion using the RosetteSep™ CTC Enrichment cocktail was first optimized using small sample volumes (1 mL) spiked with cells from cancer cell lines. Depletion of leukocytes ranged from 3.1 to 3.9 logs with an average recovery of spiked cancer cells of 50-60%. Isolation of CTC expressing EpCAM was pursued using anti-EpCAM coated columns and optimized for selection and release of EpCAM expressing cells by passage of cells from cancer cell lines through the column resulting in 34-100% recovery. Both procedures were scaled up to enable processing of 18 mL of DLA. Leukocytes were depleted using RosetteSepTM by 3.1 - 3.9 logs whereas with anti-EpCAM columns only 1.7 - 1.8 logs depletion were reached. Using RosetteSepTM 21% and with the anti-EpCAM coated columns 2% of the tumor cells spiked into 18ml DLA were recovered.

Conclusion

Standard operating procedures were developed to isolate CTC in DLA's from breast, prostate cancer and lung cancer patients for evaluation and comparison in the EU sponsored consortiums CTCTrap (www.utwente.nl/tnw/ctctrap/) and CANCER-ID (www.CANCER-ID.eu). Isolation of EpCAM expressing CTC using the anti-EpCAM coated columns will need further optimization before it can proceed to multicenter comparison.

#1533

Circulating melanoma cells (CMCs) in metastatic disease: a cut-off validation study.

Elisabetta Rossi,1 Antonella Facchinetti,1 Maria Chiara Scaini,2 Mariangela Manicone,2 Stefania De Faveri,2 Sara Valpione,2 Jacopo Pigozzo,2 Vanna Chiarion-Sileni,2 Paola Zanovello,1 Rita Zamarchi2. 1 _University of Padova, Padova, Italy;_ 2 _IOV-IRCCS, Padova, Italy_.

Background

Melanoma is characterized by both genomic instability and continuously arising mutations. The current model suggests that it metastasizes hematogenously from primary sites via Circulating Melanoma Cells (CMCs); mutations can arise in the primary tumor, metastases, or both. As an intermediate step toward metastasis, CMC analysis could be a valid tool for monitoring melanoma evolution. As well as in solid tumors, a validated assay for the enumeration and characterization of CMCs is expected to facilitate the development of more effective therapies for metastatic melanoma (MM) patients.

Patients and Methods

From June-2011 until November 2015, we enrolled 174 MM patients, 105 male and 69 female, median age 61 years (range from 27 to 87 years).

The CellSearch technology for the enumeration of circulating tumor cells (CTCs) in peripheral blood was designed as prognostic and predictive tool for breast, prostate, colorectal, and lung cancer, all of them express EpCAM marker. A similar technology allows the detection of CMCs, defined as CD146+, HMW-MAA+, CD45-, and CD34- cells.

Fluorescent anti gamma-H2AX mAb (specific for the phosphorylated form of H2AX) was integrated in the standard CMC assay to assess DNA damage and to measure cell viability and apoptosis in CMCs.

We enumerated CMCs at the diagnosis of MM, before the start any therapy (prognostic study); we also quantified CMCs at any clinical/radiological reevaluation, at any therapeutic change and/or at progression (predictive study). Overall, we performed from 1 up to 17 CMC tests per patient throughout their follow-up.

Results/Conclusions

We included in this analysis informative CMC results from 169 out of 174 enrolled pts; 3 pts were excluded because the enumeration was not ultimate in time, meanwhile in 2 cases we cannot perform the test due to preparatory failure.

At baseline, 88 patients out of 169 (52%) were CMC-positive (range 1 to 94 cells); 63 out of 88 CMC-positive patients had at least 1 apoptotic CMC (range 0 to 18 cells), the median percentage of apoptotic CMCs was 50.

Furthermore, we could obtain survival data in 139 patients to compare the overall survival probability (OS) between CMC-positive and CMC-negative groups of patients.

We demonstrated that the CMCs were associated with worst prognosis for a CMC number > 1 (p=0.028, according to Kaplan Meier, log rank test), > 2 (p=0.003), and > 3 cells (p<0.001).

Our data show that CMCs were ever associated with poor prognosis, and suggest the idea that this parameter should be included in patients staging.

#1534

Amplification of PITPNC1 affects breast cancer progression.

Peiqi Wang,1 Ranju Nair,2 Nisha Kanwar,2 Grace Cheung,2 Susan J. Done2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _The Campbell Family Institute for Breast Cancer Research, Toronto, Ontario, Canada_.

Introduction

Identification of driver mutations at single gene level and determination of their respective contribution to the enhanced invasive potential of cancer cells, via expression profiling and functional assays, are indispensable steps toward elucidating breast cancer pathobiology. In our previous array comparative genomic hybridization studies, gain in a region of chromosome 17, 17q23.3-24.3, was frequently observed in invasive duct carcinoma (IDC) patients with lymph node metastasis. Particularly, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1), found in 17q24.2, was associated with higher histological grade, larger tumor size, positive HER2 staining, and poorer prognosis from the analysis of a NKI dataset. PITPNC1 is involved in signal transduction and intracellular lipid transport, and may be an essential part of the epidermal growth factor signaling pathway. Additionally, amplification of PITPNC1 via the loss of microRNA-126 suppression has been implicated in metastatic angiogenesis and colonization. In this study, we aim to assess the role of PITPNC1 in the development of aggressive breast cancer by evaluating its effects on breast cancer progression and invasion.

Method

A total of 21 samples - 13 pure duct carcinoma in situ (DCIS), 8 IDC with or without lymph node metastasis - were used for the preliminary round of this study. Formalin-fixed and paraffin-embedded tissue blocks were microdissected and whole genome amplified using ligation-mediated PCR. Amplified DNA was subjected to quantitative real-time PCR. Copy number alterations of PITPNC1 were calculated with Livak method, using B2M as the reference gene. Lipofectamine transfection overexpressing PITPNC1 in non-invasive MCF-10A and invasive MCF7, MD-MB-231 cell lines was prepared for subsequent in vitro assays. Proliferation and migration capabilities associated with PITPNC1 overexpression is being evaluated using MTT assay and transwell migration assay.

Result

From preliminary qPCR data, PITPNC1 was shown to be amplified in DCIS samples (p=0.0025) and IDC samples (p=0.0093). The 95% confidence intervals of fold difference against normal breast tissue control are [7.1, 23.4] and [5.7, 24.4] respectively. PITPNC1 overexpressing MCF-10A cells exhibit a higher proliferation rate in culture compared to controls.

Conclusion

Consistent amplification of PITPNC1 is seen in both DCIS and IDC cases. However, it is still unclear if PITPNC1 amplification is a result of a single mutational event occurring early in cancer progression or a cumulative effect occurring over time. Additional qPCR on paired samples against a pooled control would be necessary to determine its contribution to invasion. The regularity of copy number gain in the PITPNC1 locus and its association with HER2 expression highlight its predictive potential as a novel biomarker for tumorigenesis or invasion.

#1535

Next generation sequencing of brain metastasis in non-small cell lung cancer.

Bryan Thibodeau, Paola Yumpo Cardenas, Samreen Ahmed, Marc Dunn, Matthew Johnson, George Wilson. _Beaumont Hospital, Royal Oak, MI_.

Introduction: Molecular analysis of non-small cell lung cancer (NSCLC) has provided a detailed classification of NSCLC. Activating mutations in the EGFR gene are both prognostic and predictive in that they are associated with improved survival irrespective of therapy and are associated with a significant response to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib. Patients with EGFR mutations show a significant improvement in progression free survival compared to standard chemotherapy. In addition, a recent study showed that ALK gene rearrangement was significantly associated with pericardial spread and pleural disease and those patients with either ALK gene rearrangements or EGFR mutations were predisposed to liver metastasis. These results support the hypothesis that the dominant molecular oncogenes in NSCLC are associated with different biological behaviors which manifest as distinct patterns of metastatic spread at the time of diagnosis. To date, no pattern of molecular variation has been linked to brain metastasis. Here we use next generation sequencing to identify a pattern of genomic variation associated with the development of brain metastases in NSCLC. Methods: Three patient groups were analyzed: samples from NSCLC patients who did not develop metastasis (n=8), samples from NSCLC patients who developed metastasis not associated with the central nervous system (n=7), and brain metastasis samples of NSCLC patients (n=16). DNA was isolated, and libraries prepared using Qiagen's Comprehensive Cancer Panel which focuses on 160 cancer-related genes. Libraries were sequenced on the Illumina NextSeq 500. Alignment was done using SoftGenetic's NextGENe with subsequent biological interpretation with Ingenuity Variant Analysis. Results: At the variant level, a missense mutation in CDK12 (chr1:37686934) was found in 7 of the 16 cases with brain metastasis while only 1 of 8 cases with no metastasis and 0 of 7 cases with non-CNS metastasis had the variant. However, no other variants were found in ≥6 cases of brain metastasis and 1 or less of the control cases. After filtering variants that occurred in more than 1 control (no metastasis or non-CNS metastasis), the TP53 gene was altered in 11 of the 16 cases with brain metastasis. This included 14 different variants with none of these variants present in the controls. Additionally, BRCA1 and CDK12 had variants in 9 cases with brain metastasis, but the variant was also present in 1 of the controls. Taken together, all 16 cases contained alterations in at least 1 gene involved in PI3K/AKT signaling including variants in JAK1, MTOR, TP53, TSC1, and TSC2. Conclusion: This pilot study has delivered valuable results to better characterize the pattern of genomic variants in NSCLC. While no single variant was associated with brain metastasis, this study implicates PI3K/AKT signaling and, in particular, variants of TP53 as crucial for determining the potential development of NSCLC brain metastasis.

#1536

In silico discovery of target genes and pathways associated with breast cancer brain and bone metastases.

Su-Ni Tang, Peixin Jiang, Xinli Liu. _Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX_.

Metastasis is the primary cause of death from breast cancer. Bone and brain are the most frequent sites for breast cancer metastasis. The aim of this study was to explore candidate genes associated with breast cancer bone and brain metastases. We compared the gene expression profiles of bone and brain metastases with primary tumors by compiling 55 microarray data sets from Gene Expression Omnibus (GEO: GSE12763, primary tumor n=30; GSE14017, bone metastasis n=10, brain metastasis n=15). A total of 160 differentially expressed genes were screened with a two-fold change, among which 109 genes were up-regulated and 51 genes were down-regulated in bone metastasis. Pathway analysis showed these differentially expressed genes were significantly related to bone development and cell adhesion (p<0.01). Similarly, a total of 150 differentially expressed genes were identified in brain metastasis, 36 genes were up-regulated and 114 genes were down-regulated, which were enriched in immune response (p<0.01). Moreover, protein-protein interaction analysis revealed highly connected hub genes that are expected to play an important role in brain and bone metastases. These hub genes were FOS, EGR1, CTNNB1, DCUN1D1, HIST1H4E, VIM, UBD, EGFR in bone metastasis, and HIST1H4E, DCUN1D1, CDC42, ESR1 in brain metastasis. Interestingly, two genes, DCUN1D1 and HIST1H4E were both up-regulated in bone and brain metastases. It is known that DCUN1D1 could induce extracellular matrix invasion while HIST1H4E played a critical role in regulating chromosomal accessibility and stability. These findings reveal potential biomarkers and therapeutic targets of breast cancer bone and brain metastases, which provide a molecular basis for our future in vitro and in vivo studies.

#1537

The biological role of BAT-1 in prostate cancer.

Aileen M. Garcia-Vargas,1 Maylein C. Juan-Rivera,1 Milaris M. Sánchez-Cordero,2 Maria Sánchez-Vázquez,3 Krizia Rohena,1 Magaly Martínez-Ferrer1. 1 _University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico;_ 2 _University of Puerto Rico Rio Piedras Campus, Rio Piedras, Puerto Rico;_ 3 _University of Puerto Rico Comprehensive Cancer Center, San Juan, Puerto Rico_.

Prostate cancer is the most common cancer in men in the United States and is the second cause of cancer death in men. In Puerto Rico, prostate cancer is the most common type of cancer and the leading cause of cancer death in men. The current available biomarkers are unable to predict malignant outcomes such as recurrence. Thus, there is a critical demand for the development of innovative diagnostic and prognostic tools for the management of prostate cancer. In the current study, we evaluated the biological role of BAT-1 in prostate cancer. Preliminary data from patients who had prostate cancer recurrence identified that HLA-B associated transcript 1 (BAT-1) was down-regulated in patients with prostate cancer recurrence when compared with non-recurrent patients. We down-regulated BAT-1 in PC3 and 22RV1 prostate cancer cell lines using small interfering RNA (siRNA). In vitro assays were performed to measure de-differentiation, proliferation, apoptosis, migration and invasion. Flow cytometry analysis using the cell Viability/Annexin V cell staining kit showed that down-regulation of BAT-1 significantly increased the proliferation of PC3 cells (P<0.05) but decreased apoptosis. Down-regulation of BAT-1 showed no significant change in the expression of vimentin, desmin, and alpha-smooth muscle actin (α-SMA). Wound healing and Boyden assays showed that cells treated with BAT-1 siRNA significantly increased migration and motility at 12h and 24h of PC3 cells when compared to control (P<0.05). Our results showed that BAT-1 down-regulation increased migration and cell motility. Cells also tend to proliferate more, but have reduced apoptosis. These results were expected due to its down-regulation in recurrent prostate cancer patient samples, suggesting that BAT-1 down-regulation promotes aggressiveness in prostate cancer recurrence. A possible mechanism of action for BAT1 is the modulation of cell proliferation and migration.

#1538

Vimentin expression as a potential immunomarker of predicting aggressive disease in clear cell renal cell carcinoma.

Mahmoud Mohamed, Lulin Hu, Haiyan Liu, Fan Lin, Heinric Williams. _Geisinger Health System, Danville, PA_.

Vimentin is a type III intermediate filament protein that is expressed in mesenchymal cells. Overexpression of vimentin has been detected in prostate cancer, lung cancer, gastrointestinal cancer, and melanoma. Although vimentin has been used for diagnosing and classifying renal neoplasms, little is known regarding its prognostic value. This study examines the prognostic significance of vimentin expression in clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma (chRCC) and oncocytoma. Our patient group (n=88) consisted of 63 men and 25 women with a mean age of 59 years at the time of diagnosis. Paraffin embedded specimens from these patients' renal tumors were selected for this study which included ccRCC (n=66), chRCC (n=11) and oncocytoma (n=11). Vimentin expression was determined by a tissue microarray technique (TMA) performed by a single pathologist (HL). Tumor staining results were classified as negative (<5%), weak positive (5-25%), intermediate positive (25-50%), moderate positive (50-75%), or strong positive (>75%). Clinicopathologic data was also included tumor histology, tumor size, T stage, grade, presence of sarcomatoid differentiation, presence of metastasis, and survival outcome following diagnosis were determined for each patient. Chi square statistical test analysis was performed in order to test the association of clinical parameters with vimentin immunoreactivity. Our results show that positive (>5%) vimentin expression was present in 100% of ccRCC cases, 9% of oncocytoma, and 0% of chRCC cases. In addition, vimentin expression in ccRCC was associated with metastasis (p<0.047) but was not significantly associated with pathologic T stage (P<0.185), Fuhrman grade (P<0.636), or the presence of sarcomatoid differentiation (P<0.855). With a median follow up of 7.2 years, there were 26 deaths in the ccRCC and none in the chRCC or oncocytoma groups. Positive vimentin staining was not associated with overall survival in the ccRCC group. Our results suggest that vimentin may be valuable predictor of metastatic disease in ccRCC patients.

#1539

Live circulating tumor cells as a predictive biomarker of response to neoadjuvant chemoradiotherapy for resectable pancreatic cancer.

Masahiro Tanemura,1 Kenta Furukawa,1 Masaki Wakasugi,1 Kentaro Kishi,1 Yasuo Urata,2 Kiyomi Taniyama,3 Hiroki Akamatsu1. 1 _Department of Surgery, Osaka Police Hospital, Osaka, Japan;_ 2 _Oncolys BioPharma Inc., Okayama, Japan;_ 3 _Department of Pathology, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Hiroshima, Japan_.

The detection of increase in the number of circulating tumor cells (CTCs) during a patient's clinical course may be a harbinger of forthcoming overt metastasis. To detect live CTCs (l-CTCs) in blood samples of cancer patients (pts), we employed a new genetically modified telomerase-specific replication-selective adenovirous, expressing GFP (TelomeScanF35), containing both type 35 fiber that induce broad infectivity and miR-142-3p target sequence that can prevent a false positive toward blood cells. Recently, we indicated that preoperative chemoradiation therapy comprising gemcitabine and S-1 administration concurrent with full-dose radiation (NACRT) lead to encouraging survival rate in pts with resectable pancreatic cancer. But, NACRT may have disadvantages. NACRT requires several months, potentially delaying surgical resection, and can be waste of time and may result in the pts missing the chance for surgery. We assessed the l-CTCs burden in pts treated with NACRT. This study was approved by the Osaka Police Hospital IRB. Pts with resectable cytologically or histologically proven ductal adenocarcinoma of the pancreas were enrolled. Treatment consisted of an intervenous infusion of Gem 800 mg/m2 on day 1, 8, 22, and 29; and S-1 80 mg/m2 orally on day 1-5, 8-12, 22-26, and 29-33 given concurrently with IMRT to 60 Gy. Surgical exploration was scheduled 4-7 weeks after the final radiation fraction. 7.5 ml of blood samples were obtained from the pts included in this clinical study before NACRT, after 1 month of NACRT, and after 2 months of surgical resection. To distinguish between leucocyte and cells with epithelial origin, cells were stained with anti-CD45 and anti-Cytokeratin Abs. To distinguish cells with mesenchymal origin, cells were labeled with anti-Vimentin Ab. GFP-positive and CD45-negative cells were counted as l-CTC. 16 pts aged 44-78 years (5 males and 11 females) were enrolled. No treatment-related deaths occurred. CA19-9 was reduced to <50 % of baseline values in 10 of 12 measureable pts. 13 of 16 enrolled pts successfully underwent surgical resection. 5 of 16 enrolled pts had l-CTCs (1~4CTCs,Vimentin[+]) detected before NACRT. An increase in CTCs was seen in 4 of 5 pts after NACRT, but no detectable l-CTCs were observed in only 1 pt after NACRT. These 4 CTC-positive pts continuously had CTCs detected after surgery. 2 of 4 CTC-positive pts early developed liver metastasis and died, despite R0 resection. On the contrary, small increase (1~2CTCs) of CTCs was seen after NACRT in 4 of 11pts, who had no CTCs detected before NACRT. But, no l-CTCs were detected in these 11 pts after surgery and they survived without recurrence. CTC-positive pts before NACRT presented a higher rate of liver metastasis after surgery than CTC-negative pts (50% vs. o%). We may consider surgery first for the CTC-positive pts before NACRT. The capture of l-CTCs may be useful biomarker for prognosis assessment or stratification.

#1540

Midkine-raised oral cancer growth, migration and invasion is required for RBPJk modulation.

Tai-Jan Chiu, Yi-Ching Chen, Chang-Han Chen. _Chang Gung Memorial Hospital, Kaohsiung, Kaohsiung, Taiwan_.

Oral squamous cell carcinoma (OSCC) represents 95% of all forms of head and neck cancer, and its incidence has increased by 50% during the past decade. Despite the nowadays available therapeutic managements, such as surgery, radiotherapy, chemotherapy or in combination, the five-year survival rate is still improved. The current view is to present that the accumulations of genetic and epigenetic alterations in OSCC lead to the progression of cancer. Our previous study indicated that midkine, heparin-binding growth factor, was overexpressed in tumor tissues of head and neck cancer patients and its expression also correlated with poor 5-year survival. In this study, we tried to explore the biological functions and mechanisms of midkine in vitro and in vivo. Established gain-of-function of midkine in two oral cancer cell lines not only promoted cell growth by MTT, soft agar and BrdU assays, but also induced tumor growth in xenograft model. Midkine-overexpressing cells induced cell migration and invasion by wound healing and Transwell assays. Conversely, cell growth, migration and invasion were also inhibited; while endogenous midkine was abolished by midkine-mediated siRNA in oral cancer cells. By using bioinformatics, Q-RT-PCR, Western blotting, promoter assay and ChIP assay, we identified that RBP-Jk, a transcriptional factor, could bind to the promoter regions of midkine and to induce midkine expression in oral cancer cells. In the specimens of oral cancer patients, the expressions of midkine and RBP-Jk had a positive correlation by immunohistochemistry. Taken together, these results demonstrated that RBP-Jk regulates midkine expression and involves in midkine-elicited tumor formation in oral cancer.

#1541

Heterogeneous response to platinum in metastatic ovarian cancer detectable by biodynamic imaging.

Daniel Merrill,1 Hao Sun,1 Daniela Matei,2 Bakhtiyor Yakubov,2 John Turek,1 David Nolte1. 1 _Purdue University, Lafayette, IN;_ 2 _Indiana University School of Medicine, Indianapolis, IN_.

The mechanisms by which tumors develop multi-drug resistance are not well understood. Biodynamic imaging (BDI) is a label-free assay that detects phenotypic response of ex vivo tissue to chemotherapeutic drugs. In the current study, we used BDI to measure the response of primary and metastatic ovarian tumors to cisplatin. Xenografts from two human ovarian cancer cell lines, A2780 and SKOV3, were grown orthotopically in the ovaries of nude mice and allowed to metastasize in the peritoneal space. Primary and metastatic tumors were harvested and tested for response to 25 µM cisplatin by using BDI. Sensitivity was assessed through selected response biomarkers. Metastatic tumors of the platinum-sensitive A2780 cell line showed 20% decrease in sensitivity to cisplatin compared with the response of the primary tumors, while the less sensitive SKOV3 tumors exhibited a 6% decrease in response of metastatic vs. primary tumors. In parallel, RNA sequencing profiled primary ovarian and metastatic implants. Differential gene expression was detected in primary tumors vs. metastatic sites. Genes upregulated in metastases include TIMP3, WNT2, OSR1, elastin (SKOV3 tumor model) and MMP1, MMP3, MMP10, MET, EGFR, PDGF-C (A2780 tumor model). The study demonstrates the ability of BDI to detect differences in drug sensitivity between primary and metastatic tumors and provides support for the association of metastasis and chemoresistance in ovarian cancer. Supported by NIH R01 EB016582.

#1542

Identification and utilization of molecular pathways correlated with osteosarcoma metastasis to predict targeted therapies.

Laird Klippenstein,1 Jared S. Fowles,1 Sierra K. Lear,2 Daniel L. Gustafson1. 1 _Colorado State University, Fort Collins, CO;_ 2 _University of Colorado Cancer Center, Denver, CO_.

Osteosarcoma (OSA) is the most prevalent primary bone tumor and accounts for approximately 2% of childhood cancers. Approximately 40% of all OSA patients succumb to metastatic disease within 5 years. Canine OSA occurs about 10 times more frequently than human OSA and has been shown to be very similar to pediatric OSA biologically, histologically, molecularly, and in treatment. This suggests that canine OSA is an excellent pre-clinical model to investigate better treatment options for metastatic OSA. We compared in-vitro markers of metastatic potential with gene expression data in eight canine OSA cell lines to identify molecular pathways that will be potential chemotherapeutic targets to treat and prevent metastatic OSA.

The metastatic potential of 8 canine OSA cell lines was characterized utilizing Incucyte Zoom based migration and invasion scratch wound assays. Gene expression analysis was performed using Affymetrix GeneChip Canine Genome 2.0 Arrays. Genes that were statistically correlated with migratory and invasive phenotypes were used to enrich molecular pathways described in several databases utilizing Enrichr software. Altered pathways and associated targeted agents that would inhibit the pathways enriched by the correlated gene sets were identified.

Chemotherapeutics targeting significantly enriched pathways were screened for their ability to inhibit metastatic potential. Dasatinib and Stattic were the only inhibitors screened to date that inhibit migration of the osteosarcoma cell lines at clinically relevant doses. The remaining significantly correlated pathways will be targeted as well as rational combinations of pathways. An orthotopic murine model is currently being tested to further explore the utility of efficacious chemotherapeutics in-vivo.

Pathway Inhibition and Anti-Migratory Effects

---

Pathway | P-Value | Targeted Agent | IC50 vs Migration

Map Kinase | 0.0037 | Selumetinib | >25 uM *

Focal Adhesion | 0.0059 | Dasatinib | 18 nM - 214 nM

Erbb2/3 | 0.0214 | Lapatinib | >8 uM *

Tubulin | Control | Paclitaxel  | 7 nM - 54 nM

mTOR | 0.0054 | Rapamycin | >20 nM *

STAT3 | 0.0004 | Stattic | 2.4 uM - 4.6 uM

BMPR2 | <0.0005 | Tacrolimus | >12.5 nM *

ALK | <0.0005 | Crizotinib | >900 nM *

VEGFR | 0.0226 | Vandetanib | TBD

ITK | <0.0005 | Ibrutinib | TBD

PDPK1 | 0.0011 | Celecoxib | TBD

* We were not able to reach an IC50 at clinically relevant doses

#1543

Clinical significance of ZNF185 intracellular localization in pancreatic ductal carcinoma.

Daisuke Furukawa,1 Tsuyoshi Chijiwa,2 Masahiro Matsuyama,1 Masaya Mukai,3 Ei-ichi Matsuo,4 Osamu Nishimura,5 Kenji Kawai,2 Hiroshi Suemizu,2 Toshio Nakagohri,1 Seiei Yasuda,1 Kazuaki Shimada,6 Nobuyoshi Hiroka,7 Masato Nakamura8. 1 _Department of Surgery, Tokai University School of Medicine, Tokyo, Japan;_ 2 _Central Institute for Experimental Animals, Kawasaki, Japan;_ 3 _Department of Surgery, Tokai University Hachioji Hospital, Tokyo, Japan;_ 4 _Global Application Development Center, Shimadzu Corporation, Japan;_ 5 _Institute for Protein Research, Osaka University, Osaka, Japan;_ 6 _Hepatobiliary and Pancreatic Surgery Division, National Cancer Center Hospital, Tokyo, Japan;_ 7 _Division of Pathology and Clinical Laboratories, National Cancer Center Hospital, Tokyo, Japan;_ 8 _Department of Regenerative Medicine, Tokai University School of Medicine, Tokyo, Japan_.

Pancreatic cancer is one of the most aggressive human tumors because of the challenges associated with detecting it at an early stage and its high potential for dissemination and distant metastasis. Therefore, clarifying the biological mechanisms underlying distant metastasis from pancreatic cancer and accelerating the development of new treatment strategies are needed. We previously isolated ZNF185, a metastasis-related protein, using a global quantitative proteome analysis. ZNF185 contains two zinc-finger motifs in the C-terminus that fit the consensus pattern of the LIM domains inducing tumor formation and cytoskeleton organization. In the present study, we investigated the expression of ZNF185 in human pancreatic cancer in vivo and in clinical cases, and also evaluated its prognostic significance. Immunohistochemical staining of resected specimens and a xenograft model of liver metastasis was used to examine the relationship between distant metastasis in and the prognosis of pancreatic cancer and the expression of ZNF185. Cells expressing ZNF185 in the plasma membrane were detected in 71 (39.0%) out of 182 patients with resected pancreatic cancer. Patients not expressing ZNF185 in the plasma membrane had a median survival of 30.2 months, whereas those expressing ZNF185 in the plasma membrane had a median survival of 21.3 months. A multivariate analysis indicated that the expression of ZNF185 in the plasma membrane was an independent poor prognostic factor (p=0.016). A multivariate analysis indicated that patients with cells expressing ZNF185 in the plasma membrane were more likely to develop hematogenous metastasis (p=0.081). NOD/Shi-scid/IL-2Rγnull(NOG) mice were used to compare the expression of ZNF185 in the subcutaneous tumors of the parent cell line BxPC-3 and liver metastatic tumors of the highly liver-metastatic subline LM-BxPC-3 established in our laboratory. Cells expressing ZNF185 in the plasma membrane were more prevalent in metastatic disease in the liver than in subcutaneous tumors, and these cells were often located at the tumor margins rather than in the center of the tumor. In conclusion, ZNF185 is a protein related to hematogenous metastasis, the expression of which in the plasma membrane was identified as an independent prognostic factor for pancreatic cancer.

#1544

Overexpression of Rab11 and E-cadherin promotes collective cell migration and indicates a poor prognosis in colorectal carcinoma.

Yuan-Chiang Chung,1 Wan-Chen Wei,2 Chia-Nung Hung,3 Chih-Ping Hsu,4 Hsiang-Ling Chiu,3 King-Jen Chang,1 Wei-Ting Chao3. 1 _Department of Surgery, Cheng-Ching General Hospital, Taichung, Taiwan;_ 2 _Cheng-Ching General Hospital, Taichung, Taiwan;_ 3 _Department of Life Science, Tunghai University, Taichung, Taiwan;_ 4 _Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsin Chu, Taiwan_.

Collective cell migration, whereby the cell-cell contacts such as E-cadherin are maintained during migration, has recently emerged, and the detailed mechanisms of this process are still unclear. The present study identified the role of Rab11 which functions in recycling endosome and the relation to E-cadherin in colorectal carcinoma and clarified the mechanism of Rab11 in the collective cell migration of cancer cells. The relationships between the overexpression of Rab11 and E-cadherin and survival were evaluated from colorectal carcinoma patients. A total of 107 patients with surgically resected colorectal carcinoma were enrolled in immunohistochemical study. The relationships between the overexpression of Rab11 and E-cadherin and survival were evaluated. GFP tagged Rab11 or Rab11 shRNA was introduced into HT-29 colon cancer cells for overexpression or knockdown. The interaction between E-cadherin and Rab11 was determined by immunoprecipitation. Rac1 and matrix metalloproteinase (MMP) activities were evaluated as functional effectors of collective cell migration. In the results, the expression of Rab11 and E-cadherin was not correlated with the stages of cancer or lymph node metastasis. However, the overall survival was poor in the group of 60 patients with duo-positive Rab11 and E-cadherin expression compared to the group (47 patients) without duo-positive expression (p=0.038). In the cell biology assay, Rab11 was demonstrated through its physical interaction with E-cadherin, and overexpression of Rab11 was found to promote collective cell migration through the increased distribution of E-cadherin, which enhanced cell-cell connections. In addition, Rac1 activation and MMP2 expression were up-regulated upon Rab11 expression. This study demonstrated that Rab11and E-cadherin expression are poor indicators of survival time in colorectal carcinoma, but that Rab11 overexpression may contribute to increased collective cell invasion in colorectal carcinoma.

#1545

**Metastasis blood test by** in vivo **tumorsphere detection.**

Viktoria Denes,1 Laszlo Graf,2 Gabor Barna,2 Balint Tegze,2 Pal Jakso,1 Eva Pallinger,2 Peter Geck3. 1 _University of Pecs, Pecs, Hungary;_ 2 _Semmelweis University, Budapest, Hungary;_ 3 _Tufts University School of Medicine, Boston, MA_.

INTRODUCTION Metastasis is the turning point in the progression of most cancers. The prognosis of systemic, disseminated cancer is usually negative, but methods for early metastasis detection are not available in the clinical practice. We present a novel approach that may have the potential to detect metastatic predisposition early from patient blood.

BACKGROUND It is almost universally accepted that metastases are disseminated by cancer stem cells. It is also known that hypoxia in a cancer is a major factor in therapy-resistance and dissemination. The findings are consistent with other observations that stem cells rapidly adapt to hypoxia and prefer hypoxic metabolism. We studied cancer stem cell differentiation and found that most stem cells (cancer and normal) readily form spheroids in suspension culture in vitro. Further studies showed that these spheroids develop hypoxic centers and are positive for stem cell markers.

HYPOTHESIS These observations suggest that in vitro spheroid formation may reflect a fundamental biological feature of cancer stem cells and they may find a perfect niche in the hypoxic microenvironments in spheroids. The fact that spheroid formation is universally found in vitro raised the possibility that stem cells may also form spheroids in vivo, and by providing a "portable" hypoxic milieu, circulating tumorspheres may be critical in metastatic dissemination. We investigated, therefore, whether cancer spheroids can enter circulation and worked out the methodology to detect them in blood.

EXPERIMENTAL PROCEDURES Blood samples were collected from 106 patients, 62 metastatic and 44 controls. The samples were analyzed by flow cytometry using forward scatter and fluorescent labels. For spheroid capture we used simple nylon mesh filters (10 um and 20 um pore size). From the collected spheroids total RNA was isolated and gene expression studies were performed for stem cell, hypoxia and other markers. Both the collected spheroids and in vitro spheroid models were analyzed by immunohistochemistry.

RESULTS We found that spheroids show complex metabolic architecture with hypoxia, stem and other markers. We tested several flow cytometers and found that the wide scatter of spheroids was not optimal in some instruments, but performed well in Partec and Beckman-Coulter systems. In our pool almost half of the metastatic samples were spheroid positive, while all healthy samples were negative. Follow up clinical studies will investigate the prospective significance of the results.

CONCLUSIONS Our data demonstrate significant correlation between cancer spheroid positivity in blood and metastasis. The results suggest that circulating in vivo spheroids may be involved in the dissemination of metastatic cancer stem cells and can be used as a new prognostic/diagnostic approach.

#1546

Differential gene expression analysis by NGS of primary tumor xenograft cells, circulating tumor cells, and metastasis cells purified from the same mouse hosts to identify drivers of cancer dissemination.

Tong Xu, Gareth Morrison, Yucheng Xu, Timothy Triche, Jonathan Buckley, Amir Goldkorn. _University of Southern California, Los Angeles, CA_.

Introduction: Cancer dissemination involves ongoing dynamic transit of tumor cells between primary and metastatic sites via a hematogenous or "liquid phase". To identify molecular mechanisms related to the liquid phase of cancer dissemination, we developed a tractable mouse xenograft model wherein GFP-labeled pure primary tumor (PT) cells, circulating tumor cells (CTCs) and lung metastasis (LM) cells are isolated and characterized from the same host.

Methods: GFP+ MDA-MB-231 human breast cancer cells were inoculated subcutaneously into NOD/SCID/IL2r-γnull (NSG) mice. When a 1cm diameter primary tumor xenograft formed, 500 µl blood were collected by intracardiac puncture, and CTCs were enriched by wbc/rbc depletion (StemCell Tech). PT and LM samples were fluorescence microdissected from the same mouse hosts and disaggregated into single cell suspensions. Using a micromanipulator pipette, matched PT, CTC, and LM samples (100 pure GFP+ cells each) were isolated, lysed for RNA extraction (Ambion), and analyzed using the Ion AmpliSeq Transcriptome Human Gene Expression workflow for library preparation and pooled sequencing (Ion Proton). Sequence data from biologic triplicates was aligned and curated using the Genetrix bioinformatics suite, and candidate genes were further analyzed using Ingenuity Pathway Analysis (IPA).

Results: The sequence panel provides gene-level expression information from over 20,000 genes covering > 95% of the RefSeq gene database. Initially, we focused on genes that were up- or down- regulated in CTCs compared with both PT and LM (i.e. differentially expressed in the liquid phase: 402 genes, 184 up-regulated in CTCs, 218 down-regulated in CTCs, fold change range 1.2x-22x). After further filtering for false discovery, 4 up-regulated and 39 down-regulated genes were selected for IPA analysis, which revealed associations with cellular movement pathways (16 genes) and cancer pathways (24 genes). Of these, the top-ranked 12 differentially expressed genes in CTCs relative to PT and LM (9 down-regulated and 3 up-regulated) were selected for validation by qPCR and further functional characterization in the mouse xenograft model.

Conclusions: Here we leveraged new techniques for pure CTC capture, AmpliSeq transcriptome amplification and sequencing to identify and validate new candidate drivers of cancer dissemination. This model holds the potential to uncover key mechanisms engaged by cells that leave the primary and metastatic tumor niche and enter the cancer's liquid phase. Further validation and functional analysis of these candidates in vitro, in vivo and in patient samples may identify new therapeutic targets that specifically disrupt cancer dissemination.

#1547

Circulating tumor cells from a syngeneic mouse model of hepatocellular carcinoma demonstrate epithelial-mesenchymal transition, decreased MHC I expression, and increased CCR7 expression.

Jeannette A. Huaman,1 Ubayed Muhith,2 Dibash K. Das,1 Michelle Naidoo,2 Olorunseun O. Ogunwobi1. 1 _CUNY Graduate Center/CUNY Hunter College, New York City, NY;_ 2 _CUNY Hunter College, New York City, NY_.

Metastasis is the leading cause of death in cancer patients, worldwide. A better understanding of how cancer cells detach from the primary tumor, escape from immunosurveillance while in circulation, and successfully colonize distant parts of the body, is needed. Circulating tumor cells (CTCs) are excellent tools to study this process. Using a syngeneic hepatocellular carcinoma mouse model, we were able to isolate cancer cells from the primary tumor and CTCs from the blood and establish novel primary tumor-derived cell lines and novel CTC lines. Wound healing assays revealed that CTCs have greater migratory capacity than the cancer cells from the primary tumors. However, MTT assays revealed that CTCs had less proliferative capacity than the cancer cells from the primary tumors. Furthermore, molecular characterization of the cancer cells from primary tumors and CTCs using Western blotting showed a decreased expression of E-cadherin and an increased expression of fibronectin by CTCs, suggesting that epithelial-to-mesenchymal transition (EMT) takes place in CTCs in comparison to cancer cells from primary tumors. To address the question of how CTCs may evade immunosurveillance, we investigated the profile of expression of two cell surface proteins involved in immune system function: the major histocompatibility complex class I (MHCI)- required for cytotoxic T cell activation and subsequent cancer cell killing, and chemokine receptor 7 (CCR7)- exploited by cancer cells for immune evasion. Our analysis revealed that there is decreased cell surface expression of MHCI and increased expression of CCR7 in the CTCs in comparison to the cancer cells from primary tumors. These findings indicate that although CTCs are not more proliferative, they have acquired significantly increased migratory capacity than cancer cells in primary tumors. This may be explained by their demonstration of EMT. Furthermore, their loss of MHC I expression and their gain of CCR7 expression may help with evasion of killing by cytotoxic T cells and result in enhanced metastasis.

#1548

Re-isolated metastatic pancreatic cancer cells show distinct modulation of gene expression during liver colonization.

Khamael M K Al-Taee, Martin R. Berger, Hassan Adwan. _German Cancer Research Ctr., Heidelberg, Germany_.

Gastrointestinal cancers typically metastasize into the liver. By re-isolation of pancreatic ductal adenocarcinoma (PDAC) cells from the liver of rats harboring these cells in their liver we assessed gene expression during various stages and investigated whether extracellular matrix (ECM) genes play a role in metastatic pancreatic cancer cells. We used ASML rat pancreatic cancer cells, which were inoculated into the portal vein of isogenic BDX rats. These cells had been marked by eGFP, which allowed their re-isolation following liver perfusion by FACS sorting after early (1,3 days), intermediate (9 days), advanced (16 days), and terminal (21 days) stages of liver metastasis. Re-isolated ASML cells were used for total RNA isolation and subsequently their gene expression was investigated by Illumina chip array for mRNA and miRNA species, followed by Ingenuity pathway analysis (IPA).

Pending on the time span following re-isolation, 7-15% of all known genes and 10% of miRNA species were modulated significantly in expression. By IPA analysis, significant alterations in diseases, disorders and canonical pathways were found. Interestingly, cancer was ranked second within the five top diseases and disorders at early and terminal stages, and third at intermediate and advanced stages. Within the cancer category, the gene groups related to metastasis and advanced malignant tumor were most significantly altered, but found decreased at early and advanced stages, and increased at the terminal stage only. In addition, ECM genes were modulated significantly. These genes included gene families as chemokines, transforming growth factor -beta (TGF-β), laminins, A Disintegrin and Metalloproteinase (ADAM) peptidases, matrix metallopeptidases (MMPs), collagens, and others. From all 59 chemokines investigated, 18 and 11 mRNAs were significantly up- and down regulated. Similarly, from 21 MMPs, 6 and 2 mRNAs were significantly up and down modulated. Finally, from 39 collagens, 16 and 7 were significantly up and down regulated in expression. Also, the respective miRNAs species were modulated: In case of collagens, the miR-29b-3p species was significantly down modulated (2-fold) in accord with the upregulation of respective target collagens. Also, the miRNA species regulating chemokines were altered as shown e.g. for CCR5, which was more than 10 fold increased at mRNA level with the corresponding miR-125b-5p being ca. 2 fold decreased.

In conclusion, among the top five modulated diseases and disorders,

cancer ranked only at intermediate positions. In addition, the expression profiles of ECM genes were altered significantly in metastatic pancreatic cancer cells and thus hypothetically contributed to successful colonization of the liver by these cells.

#1549

Molecular profiling of circulating tumor cells as a surrogate for distant metastasis in stage IV breast cancer.

Alexander Ring,1 Victoria Forte,1 Dany Barrak,1 Tania Porras,1 Vasu Punj,1 Steven Carrasco,1 Min Yu,1 Debu Tripathy,2 Julie Lang1. 1 _University of Southern California, Los Angeles, CA;_ 2 _The University of Texas MD Anderson Cancer Center, Texas, TX_.

Background: Metastasis is responsible for virtually all breast cancer related deaths. While circulating tumor cells (CTCs) have been shown to be prognostic in metastatic breast cancer (MBC), their use as a biomarker to date has been limited. While initial treatment recommendations are based on primary tumor biology, ASCO guidelines call for biopsy of a metastatic site to guide decision making for systemic therapy. The ANGLE Parsortix system is a microfluidics device that separates CTCs based on size and deformability, without the need for cell-surface marker selection. We hypothesize that the ANGLE Parsortix system permits isolation and gene expression profiling of pure CTCs and allows comparison of the biopsied metastatic site, thereby acting as a surrogate for macrometastases.

Methods: We are currently enrolling metastatic breast cancer patients to a prospective, observational clinical study in which CTCs are enumerated and captured from 10-20 mL peripheral blood (PB) via the ANGLE Parsortix system. CTCs, peripheral blood (PB), and metastatic sites were profiled with RNA Seq via the Illumina HiSeq (primary predictor). We received fresh frozen tissue biopsies from the following metastatic sites: skin, brain, pleural effusion, pericardial effusion, breast, cerebrospinal fluid and bone tissue. Bioinformatics analysis was then performed. NanoString PAM50 and real-time polymerase chain reaction will be used as validation studies.

Results: To date we have successfully isolated and molecularly profiled CTCs and metastatic tissue (MT) from 9/9 stage IV breast cancer patients. Final bioinformatics analysis on the last five patients is underway. Principal component analysis demonstrated clustering of MT and CTCs, with clear separation from PB. These results demonstrated high purity of CTCs, eliminating the need for subtraction of PB background signal, and demonstrated common gene expression between CTCs and MT. Differential gene expression analysis (FDR p<0.05) identified a 214 gene-expression signature that statistically significantly correlated (R>0.98) between CTCs and MT. Gene Set Enrichment Analysis (GSEA) analysis of 214-gene signature identified biological pathways related to metastasis in CTCs and MT. Individual intra-patient analysis confirmed gene-expression correlation between CTCs and MT, but not PB.

Conclusion and outlook: Cell surface marker independent CTC isolation is feasible for RNA Seq analysis without background subtraction. Furthermore, RNA Seq of CTCs can identify gene expression signatures that correlate with distant macrometastatic sites. The gene-expression patterns revealed biological relevant information that could be used as biomarkers or identify potential therapeutic targets.

#1550

Multi-biomarker subtyping of circulating tumor cells using sequential fluorescence quenching.

Daniel L. Adams,1 Susan Tsai,2 Cha-Mei Tang,3 Steingrimur Stefansson4. 1 _Creatv MicroTech, Inc., Monmouth Junction, NJ;_ 2 _The Medical college of Wisconsin Milwaukee, Milwaukee, WI;_ 3 _Creatv MicroTech, Inc., Rockville, MD;_ 4 _HeMemics Biotechnologies, Inc., Rockville, MD_.

Background: Circulating tumor cells (CTCs) are rare but clinically valuable indicators of cancer status. However their clinical utility is limited to 2-3 positive fluorescent markers and 1 negative fluorescent marker. By contrast, tissue biopsies allow for numerous subtyping markers, yielding information about the tumor's biology and predicted treatment response. If CTC analysis is to be more useful, it must move beyond 2-3 identification markers. We describe a simple and inexpensive method to capture and identify CTCs using traditional fluorescence biomarkers with repeated restating of 9 unrelated fluorescent antibodies. In this study, we sought to subtype CTCs with the epithelial to mesenchymal-like phenotype (EMTCTCs) from pancreatic cancer samples first identified using a conventional CTC marker panel: Cytokeratin (CK), EpCAM, and CD45. We further subtyped these EMTCTCs with an immunosuppression therapy panel (PD-L1, CXCR4, PD-1) and then with a mesenchymal marker panel (CD14, CD34, Vimentin) to better interrogate the EMT phenotype. Alternative predictive and prognostic biomarkers may also be utilized. The order of staining does not affect the result. Our data demonstrate the ability to sequentially analyze, subtype and track 9 distinct cancer markers on each individual cell.

Methods: We developed a method of fluorescence quenching using cell lines: A2058, MB231, MCF7, HUVEC, and LnCAP. The technique consists of the steps: quench, underivatize, amine strip and restain ("QUASR"). Cells isolated on CellSieveTM microfilters were stained using the CTC marker panel, after which the QUASR protocol was applied and the cells were stained with a second mesenchymal marker panel. After imaging, QUASR was repeated for a third immunotherapy marker panel, and cells imaged a third time. QUASR was then used on 12 pancreatic cancer patient blood samples previously identified with EMTCTCs provided by Medical College of Wisconsin.

Results: No degradation was observed in cell surface, or intracellular markers for the 3 rounds of QUASR. There were 764 EMTCTCs identified as CK+/CD45- in the cancer patient samples, while EpCAM was positive in only 2% of these EMTCTCs. Post quenching, most EMTCTCs had additional mesenchymal phenotypes, i.e. 97% of cells were vimentin+ and 11% were CD34+. Expression of the immunotherapy markers were highly heterogeneous between patients, ranging from 0% to 100% positivity for PD-L1 and from 0% to 90% positivity for CXCR4. None of the CTCs were PD-1+ nor CD14+.

Conclusions: Our data demonstrates that sequential multi-panel restaining of clinically applicable cancer biomarkers can provide a greater amount and broader variety of information from patient blood samples. The ability to analyze CTCs beyond simple enumeration will greatly enhance the clinical utility of blood based biopsies, as patient samples can now be screened for multiple prognostic and predictive biomarkers.

#1551

High-resolution parallel analysis of genome and transcriptome of single disseminated prostate cancer cells.

Stefan Kirsch,1 Urs Lahrmann,1 Miodrag Guzvic,2 Zbigniew T. Czyz,1 Giancarlo Feliciello,1 Bernhard Polzer,1 Christoph A. Klein3. 1 _Project Group Personalized Tumor Therapy, Fraunhofer ITEM, Regensburg, Germany;_ 2 _Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany;_ 3 _Project Group Personalized Tumor Therapy, Fraunhofer ITEM, and Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany_.

Introduction: Manifestation of bone metastases is the most frequent cause of death among prostate cancer patients. Since the progression from non-metastatic (M0) to metastatic (M1) is highly unpredictable, molecular analysis of disseminated cancer cells (DCCs) detected before overt metastasis may provide an opportunity to dissect the early stages of systemic cancer and enable detection of critical therapy targets. However, we previously showed that early stage prostate DCCs acquire a bone marrow-associated transcriptomic phenotype (Guzvic et al. Cancer Res. 2014, 15;74(24):7383-94) impeding the definite identification of DCCs. To address this problem we developed a workflow of combined single-cell RNA-Seq and single-cell aCGH allowing high-resolution analysis of genomes and transcriptomes of individual cells. Here, we present a proof-of-concept study using DCCs isolated from bone marrow of prostate cancer patients.

Materials and methods: We isolated 24 EPCAM-positive DCCs from bone marrow of prostate cancer patients. In addition, we analyzed two VCaP prostate cancer cells and two peripheral blood lymphocytes of healthy individuals. All isolated cells were subjected to combined whole genome and whole transcriptome amplification using Ampli1TM WGA Kit and a WTA approach (Klein CA et al., Nat Biotechnol. 2002, 20(4):387-92), respectively. The suitability of WTA and WGA products for downstream analyses were assessed using PCR-based QC-assays. Subsequently, WGA products were hybridized on high-resolution SurePrint aCGH arrays and analyzed with Agilent Genomic Workbench Software. WTA products were sequenced using Roche 454 GS FLX+ or Illumina HiSeq 2000 systems. Single-cell RNA-Seq data were evaluated using a bioinformatic pipeline adjusted to the needs of the single cell WTA method.

Results: We provide two experimental protocols for single-cell RNA-Seq: (i) allowing detection of low-abundant transcripts and (ii) enabling comprehensive gene-expression analysis. The protocols were validated using VCaP cells, in which we could successfully detect low expression levels of TMPRSS2-ERG fusion transcripts. The established analysis allowed also detection of novel fusion transcripts in a M1-stage prostate cancer patient. Comprehensive gene expression analysis revealed presence of 4.000 to 13.000 transcripts expressed in bone marrow-derived cells. The comparison of RNA-Seq and aCGH data allowed to simultaneously detecting copy number alterations and corresponding changes in gene expression dosage. Thereby, the combined genome and transcriptome analysis of single cells enables to uncover the impact of genomic gains and losses on cellular transcriptomes.

Conclusions: Combined genome and transcriptome analysis of patient-derived single DCCs is feasible and enables unambiguous identification and genotypic and phenotypic characterization of DCCs.

#1552

Nucleotide excision repair gene expression in childhood acute lymphocytic leukemia is a predictor of early relapse.

Omar Ibrahim, Homood As Sobeai, Stephen G. Grant, Jean J. Latimer. _Nova Southeastern University College of Pharmacy, Fort Lauderdale, FL_.

Double strand break repair and its relationship with genomic instability have been well studied in Acute Lymphocytic Leukemia (ALL). In contrast, base repair mechanisms, such as Nucleotide Excision Repair (NER) have not been well characterized in ALL. The NER pathway addresses base damage resulting in helix distortion and replication stalling, making it important for all types of cancer. Since increased NER is a known mechanism of chemotherapeutic drug resistance, we hypothesize that there will be an increase in the expression of the NER genes at the time of relapse in ALL patients. We have studied the role of NER in ALL by secondary analysis of 2 independent gene expression microarray studies performed by Staal et al. (Set A, GSE18497) and Hogan et al. (Set B, GSE28460). Both studies analyzed matched samples of ALL patients at diagnosis and relapse (41 and 49 pairs, respectively). In Set A we found that 17/20 NER genes have higher expression at the time of relapse, than at the time of diagnosis. 4 of these genes were individually significant. However, in a subset analysis of these data sets, based on the time to relapse, patients who have early (<3 years) vs. late (>3 years) relapse gave distinct results. In Set A, 18/20 NER genes were upregulated at the time of diagnosis in the early relapse group vs. the late relapse group. 4 genes were individually significantly increased. In set B, NER gene expression at the time of diagnosis of patients in the early relapse group was increased in all 20 NER genes when compared to the late relapse group. 7 genes showed individually significant increases in expression. In addition patients in the late relapse group showed an increase in expression of many of the NER genes at time of relapse when compared to time of diagnosis. In conclusion, there are two types of ALL. Early relapsing ALL manifests high NER gene expression at diagnosis with little change at relapse. Late relapsing ALL manifests low NER gene expression at diagnosis with substantial increase at the time of relapse. NER gene expression at the time of diagnosis may allow us to predict which patients are at greatest risk for earlier relapse. This will allow pharmacogenomic targeting of each type of ALL.

#1553

Evaluation of a tubulin, detyrosinated tubulin and vimentin expression in CTCs; identification of the interaction between CTCs and blood cells through cytoskeletal elements.

Galaktea Kallergi,1 Stuart S. Martin,2 Panagiotis Katsarlinos,1 Vassilis Georgoulias3. 1 _Laboratory of Τumor Cell Biology, School of Medicine, University of Crete, Heraklion, Greece;_ 2 _Marlene and Stewart Greenebaum Cancer Center, University of Maryland, School of Medicine, Department of Physiology, Baltimore, MD;_ 3 _Hellenic Oncology Research Group, Athens, Greece_.

Background: Circulating tumor cells are the major player in metastatic procedure (CTCs). A potential mechanism of cell migration and invasion is the formation of microtentacles in tumor cells. These structures are supported by alpha-Tubulin, detyrosinated, alpha Tubulin (GLU) and Vimentin.

The goal of the current study is to identify the expression of the above cytoskeletal proteins on CTCs.

Methods: We used three different representative breast cancer cell lines MCF7, SKBR3 and MDA-MB 231 spiked in of normal donor's blood as controls. Furthermore 15 metastatic breast cancer patients were enrolled in this study. Tumor cell were isolated using the ISET platform. Spots were then stained with two different combinations of antibodies; pancytokeratin/Vimentin/a-Tubulin and pancytokeratin/Vimentin (VIM)/GLU. Cells were then analyzed with both confocal laser scanning microscopy and the ARIOL system.

Results: We observed that Vimentin was highly expressed in MDA-MB231 cells whereas lower expression was observed in SKBR3 and MCF7 cells. Fluorescence intensity quantification, using the ARIOL system, revealed that the ratio CK/VIM was 41.11, 29.03 and 4.58 in MCF7, SKBR3 and MDA-MB 231 respectively. CK/Tubulin ratio was also higher in MCF7 compared to other cell lines 6.24, 2.29 and 4.18 respectively. In accordance with this, CK/GLU was 20.13 in MCF7 cells, 11.87 in SKBR3 and 11.68 in MDA-MB 231. Interestingly, in SKBR3 and MDA-MB 231 cell lines, alpha-tubulin and vimentin filaments were observed to mediate tumor to blood cell interaction through filamentous bridges between cells.

Consequently, we analyzed 15 breast cancer patients. Eight (53%) of them harvested CTCs in their blood. One of these patients had clusters of CTCs in her blood. In this patient as well as in other CK(+) patients, CTC-to-CTC communication through cystoskeletal bridges could be revealed. These cell connections show localization of alpha-tubulin, Vimentin and GLU; moreover, the same proteins form cytoskeletal structures which allow connections between CTCs and blood cells.

In all breast cancer patients the CK/Vimentin and CK/Tubulin ratio was lower than that observed in MCF7 cells. Conversely, there was a heterogeneity of the CK/GLU ratio since in four patients was lower than that of MCF7 cells and in three others higher.

Conclusions: The importance of understanding the mechanisms that underlie CTC aggregation have been highlighted by recent reports showing that clusters of CTCs have up to 50-fold higher metastatic potential. Our study shows that CTCs can aggregate with each other and with blood cells through cytoskeletal structures supported by. Vimentin, a-Tubulin and Glu-tubulin. Quantification of these cytoskeletal elements revealed that CTCs present a phenotype similar to the most aggressive cell lines.

### Cellular and Molecular Mediators of Metastasis

#1554

Myeloid derived suppressor cells-mediated inflammation in metastasis and cancer cachexia.

Maria Ouzounova,1 EunMi Lee,1 Raziye Piranlioglu,1 Ena Novakovic,1 Mehmet Demirci,1 Sumeyye Korkaya,2 Alicia Hudson,1 Hasan Korkaya1. 1 _Georgia Regents University Cancer Center, Augusta, GA;_ 2 _University of Michigan, Ann Arbor, MI_.

Despite recent advances and better diagnostics, the major challenge is that metastatic breast cancer is still incurable and remains leading cause of cancer related death. Cachexia is considered to be a chronic inflammatory syndrome which is defined by loss of skeletal muscle mass (with or without loss of adipose mass), negative energy and metabolic balance, and systemic inflammation. Cancer patients who develop cachexia are more susceptible to infections and sepsis. Clinical studies suggest that cachexia in cancer patients cannot be fully reversed by conventional nutritional supports, which distinguishes this condition from anorexia. Due to its complexity and lack of clinical biomarkers, currently there is no standard treatment for these patients. Therefore cachexia remains a largely underestimated and untreated condition. Nearly 60-80% of the advanced/ metastatic cancer patients experience cachexia, a condition that accounts for 20% of cancer-related deaths.

Chronic inflammation has been recognized as a risk factor contributing to the etiology of many human malignancies. Accumulating evidence suggest that tumor infiltrating immune cells (mainly myeloid origin) differentiate into cells that promote tumor growth and metastasis via inducing a systemic inflammation. Our preliminary studies suggest that systemic induction and infiltration (tumor, bone marrow, spleen, liver, and lung) of myeloid derived suppressor cells (MDSC) generate a pro-inflammatory micro-environment and are a major source of inflammatory cytokines, many of which are implicated in cancer cachexia. Using a murine breast cancer in a syngeneic (immunocompetent) mouse model we show that metastatic (4T1) murine tumor produce significantly higher level of inflammatory cytokines and is able to induce systemic expansion and infiltration of MDSC compared to non-metastatic murine tumor (EMT6). Furthermore, injection of condition media from metastatic 4T1 tumor cells is also able to induce MDSC expansion in vivo suggesting that tumor-produced factors play role in this process. Moreover, we demonstrate the involvement of the inflammatory cytokines in muscle wasting as shown by co-culture experiments with C2C12 myoepithelial cells and analysed the expression of cachexia markers such as E3 ubiquitin ligases Trim63 and Fbxo3, Myh1(myosin heavy chain), Stat3 and NFkB pathway activation, and elevation of pro-cachexia cytokines.

Our preliminary studies demonstrated that the monocytic MDSC induce EMT phenotype and contribute to the dissemination of tumor cells while the granulocytic MDSC promote the metastatic outgrowth, and present higher infiltration in the lungs. We therefore propose that tumor-induced inflammatory cytokines play role in induction of MDSC and further elevation of inflammatory markers leading to metastasis and cancer cachexia.

#1555

MDSC mediated spatiotemporal tumor plasticity in breast cancer metastasis.

Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El-Andaloussi, Ena Novakovic, Alicia Hudson, Sumeyye Korkaya, Mhmet F. Demirci, Gang Zhou. _Georgia Regents University, Augusta, GA_.

Metastatic breast cancer is the second leading cause of cancer-related death among women. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5.

MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice.

We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naïve animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice.

#1556

Tumor-associated osteoblasts are major mediators in facilitating bone metastatic breast cancer cell quiescence.

Karen M. Bussard,1 Frank C. Marini2. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Wake Forest University, Winston-Salem, NC_.

Breast cancer (BC) has a predilection for bone metastasis, where the five-year survival rate is bleak (<10%). Deadly disseminated BC cells invade the bone and can remain undetectable and untreatable for ≤25 years during a period of proliferative quiescence. We have evidence that disseminated BC cells create a proliferatively quiescent niche by co-opting osteoblasts (OBs) to alter their production of microRNAs that regulate cell cycle and cellular dormancy.

MC3T3-E1 murine OBs were grown to various stages of maturity: 4 (growth), 10 (early differentiation), and 20 days (late differentiation) and incubated with conditioned medium (CM) from human BC MDA-MB-231 cell variants (parental MDA-231 or metastasis-suppressed MDA-231BRMS) to produce tumor-associated osteoblasts (TAOs). TAO-derived exosomes were assayed for microRNAs (miRs) using species-specific Qiagen miScript miRNA PCR arrays. Also, tumors from mice inoculated via flank injection with TAO cells plus MDA-231-GFP BC cells were assayed ex-vivo for RUNX2 production.

Metastasis-suppressed BC cells formed Connexin 43 gap junctions and exchanged cellular material with TAOs as determined by a parachute assay. Additionally, TAO cells produced substantial amounts of exosomes, with the largest induction seen in TAOs generated from metastasis-suppressed BC CM, that were taken up by the BC cells. TAO-derived exosomes were found to contain increased amounts of microRNAs that decrease cellular proliferation and induce G0 phase of the cell cycle (miR 320a), and repress cellular proliferation and regulate cyclin D1 expression (miR 193b). Knock-down of miRs 320a and 193b in BC cells resulted in a reduced number of cells in G0 and increased numbers of cells in G1/S/G2/M phases of the cell cycle as indicated by acridine orange and propidium iodide staining.

This same phenomenon was found in-vivo in mice inoculated with TAO cells plus proliferatively quiescent BC cells. Tumors composed of an admix of TAO cells plus BC cells grew slower and were not as large as tumors composed of OBs plus BC cells, or BC cells alone. Furthermore, after over 60 days in-vivo, tumors composed of TAO cells plus BC cells exhibited the presence of OB differentiation marker RUNX2, indicating that TAOs were still present in the tumor population, contributing to the tumor microenvironment through culmination of the experiment.

Combined, these data suggest that TAOs produce exosomal miRs that contribute to the induction of BC cell proliferative quiescence in the bone. The nature of this study also suggests the importance of OBs and their crosstalk in orchestrating the proliferative quiescence of disseminated BC cells in the bone. Thus, these findings clearly implicate the bone microenvironment and cancer cell manipulation thereof in facilitating disseminated tumor cell colonization and survival.

Supported by NIH 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; (NRSA) T32 CA079448, NIH 2 K99 CA178177 for KMB.

#1557

Metastasis suppressors regulate the tumor microenvironment by blocking recruitment of prometastatic TAMs.

Daniel C. Rabe,1 Casey Frankenberger,1 Russell Bainer,1 Devipriya Sankarasharma,2 Kiran Chada,2 Thomas Krausz,1 Yoav Gilad,1 Lev Becker,1 Marsha R. Rosner1. 1 _University of Chicago, Chicago, IL;_ 2 _Rutgers University, Piscataway, NJ_.

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. While physiological inhibitors of metastasis (metastasis suppressors) play key roles in regulating tumor growth, invasion and metastasis, their role in regulating the tumor microenvironment and immune system is unknown. We hypothesized that the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) regulates stromal cells, which then affect tumor invasiveness.

Using species-specific RNAseq we determined that expression of RKIP in tumors markedly reduces the number and metastatic potential of infiltrating TAMs. While TAMs isolated from TNBC xenografts drive in vitro invasion, RKIP+ derived TAMs did not drive invasion and had decreased secretion of pro-metastatic factors including SLPI, OPN, MMP-12, Galectin-3, VEGF-A, VEGF-D, TNFR2, and PGRN. We determined that RKIP regulates TAM recruitment by blocking HMGA2, which activates CCL5 expression. CCL5 rescued pro-metastatic TAM infiltration as well as tumor intravasation. We additionally showed that factors decreased in RKIP-derived TAMs were restored in CCL5-derived TAMs. CCL5 derived TAMs were also able to promote metastasis when co-injected with MDA-MB-231 tumors. These tumor cells demonstrated permanent increases in both growth and invasive potential after co-injection with highly pro-metastatic CCL5 derived TAMs.

To determine the clinical utility of these TAM genes we combined their expression with RKIP signaling in the tumor to create a signature that strikingly separates TNBC patients based on outcome. Our results demonstrate for the first time that metastasis suppressors can regulate the microenvironment, regulating invasion through TAMs. Our results also suggest aggressive triple negative breast cancers could be controlled by attacking CCL5 derived TAMs crucial for promoting metastasis.

Funded by: GM087630, CA184494, and CA192780

#1558

The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis.

Xin Zhang,1 Ryan Sullivan,1 Nathan Hite,2 Grace Maresh,1 Linh Hellmers,1 Zhen Lin,3 Erik Flemington,3 Carlos Salomon,4 Heather Green,2 David Margolin,5 Li Li6. 1 _Lab of Translational Cancer Research, Ochsner Health System, New Orleans, LA;_ 2 _Department of Colon and Rectal Surgery, Ochsner Health System, New Orleans, LA;_ 3 _Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, LA, New Orleans, LA, New Orleans, LA;_ 4 _Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland, Brisbane, Australia;_ 5 _Department of Colon and Rectal Surgery, University of Queensland Ochsner Clinical School, Ochsner Health System, New Orleans, LA;_ 6 _Lab of Translational Cancer Research, University of Queensland Ochsner Clinical School, Ochsner Health System, New Orleans, LA_.

Introduction: Metastatic disease is responsible for 90% of colorectal cancer (CRC) deaths. Studies suggest that metastasis is closely associated with the presence of CRC tumor-initiating cells (Co-TIC) and their interaction with the lymph node (LN) stromal microenvironment. Prior to developing extra-nodal metastasis, these cells acquire a chemotherapy-resistant phenotype developing genetic alterations making them resistant to conventional treatments. In addition to cell-cell contact and secreted molecules, a recently discovered means of intercellular signaling is the exchange of extra-cellular vesicles. These microvesicles (MVs) carry complex biological information, including mRNA, miRNA, as well as soluble and transmembrane proteins that can affect the behavior of target cells. MVs have been detected in patient specimens with diverse malignancies and may play a role in communication between the LN stromal microenvironment and Co-TIC. We hypothesize that MVs are involved in intracellular trafficking between LN stromal cells and CRC cells promoting tumor formation and distant organ metastasis.

Methods: MVs released by human mesenteric LN stromal cells (LNSC) derived from surgical specimens and the established LN stromal cell line (HK cell) were isolated using differential centrifugation and gradient purification. The MVs were visualized using GFP-HK cell and RFP-HT-29 cell (CRC cell line) and florescence microscopy. The functional properties of LN stromal MVs and their effect on CRC proliferation and metastasis was analyzed using established in vitro co-culture models and a humanized orthotopic intra-rectal (IR) injection mouse model, tracked by bioluminescent imaging (BLI).

Results: A 100,000 g pellet containing MVs derived from LNSC and HK cells have a similar size profile when analyzed by NanoSight. Budding CD63-RFP tagged MVs were released by LNSC and HK cells and uptake by GFP tagged CRC cells was confirmed through time-lapse experiments using deconvoluting microscopy. When HK cell or LN stromal cell-derived MVs were co-cultured with HT-29 cells in vitro, they supported HT-29 cell growth at a similar level as that of HK cell or LN stromal cell conditioned media, respectively. By adding LNSC- or HK-derived MVs to HT-29-Luc cells or patient derived CRC cells (CRC-Pt-Luc cells) in our IR model, we demonstrated that MVs enhanced CRC tumor growth as well as distant organ metastasis in vivo.

Conclusion: MVs isolated from LNSCs traffic between the stromal cells and CRC cells. These MVs promote tumor formation and distant organ metastasis in vivo suggesting that they play a crucial role in the communication between the LN stromal microenvironment and CRC cells. Further analyzing the functional properties of effector MV RNAs may help identify novel targetable candidates for therapeutic strategies that target CRC metastasis using our unique patient derived orthotopic mouse model.

#1559

**CCR5-CCRL2 regulates CCL5-induced organ specific metastasis: the** in vitro **and** in vivo **studies.**

Chia-Ling Hsieh,1 Che-Ming Liu,2 Shian-Ying Sung1. 1 _Taipei Medical University, Taipei, Taiwan;_ 2 _China Medical University, Taichung, Taiwan_.

Previous studies of soluble factors in the tumor associated stromal cells demonstrated the elevation of CCL5 expression in prostate cancer patient sera that correlated wit malignant cancer progression. To evaluate the expression level of chemokine receptors in response to chemokines released by reactive stroma, quantitative PCR to evaluate the mRNA level of chemokine receptors was performed. We noticed the significant increased of CCRL2 (86-fold) and CXCR4 (45-fold) in androgen independent prostate cancer (AIPC) cells. This increased of CCRL2 expression also confirmed in malignant lung, breast and rental cells. Knockdown of CCRL2 declined 75% of migration activities induced by CCL5, suggests CCRL2 involve in CCL5 induce cancer migratory activities (t-test, p<0.001). IHC analysis of CCRL2 in paired prostate cancer patients was performed and revealed increasing of CCRL2 expression in malignant prostate cancer locus, whereas no CCRL2 can be detected in the benign region of same patient. Proximity ligation assays (PLAs) of benign, high grade tumor and tissues collected from patients less than 5-year survive demonstrated the PLA signals only in the lethal progression on patients. In vivo xenograft mice model studies demonstrated the organ specific cancer metastasis can be enhanced after overexpressed CCRL2 in cancer cells. Molecular analysis of CCRL2 demonstrated that CCRL2 regulated EZH2 expression in prostate cancer cells. In summary, out studies indicated the increased expression of CCRL2 play a central role in cancer cell migration induced by chemokine CCL5. The released of CCL5, secreted by bone stromal cells after interaction with dormancy cancer cell, may induce directional migration and invasion of circulating cancer cells. Clinical analyses demonstrated increased of CCRL2 in malignant prostate cancer. Inhibition of these homing mechanisms might significantly decreased cancer cell metastatic activities of AIPC patients.

#1560

Correlation of FAP(fibroblast activation protein)-expressing cancer associated fibroblasts (CAFs) and tumor metastasis in esophageal carcinoma.

Hajime Kashima, Kazuhiro Noma, Yuki Katsura, Takuya Kato, Ryoichi Katsube, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. _Okayama University, Okayama, Japan_.

Background: The tumor and its microenvironment have dynamic interactions and signaling that strongly affect tumor progression. Cancer associated fibroblasts (CAFs) are activated fibroblasts and thought to be an important player in the tumor microenvironment. Although there are several indicators of CAF, fibroblast activation protein (FAP) is unique in its selectivity for CAFs in comparison with other markers. The aim of this study is to investigate CAFs expressing FAP and its relationship to cancer metastasis in esophageal cancer.

Methods: Sections of paraffin-embedded resected primary human esophageal cancer specimens from 2008 through 2010 in Okayama University hospital were stained with antibody directed against FAP. Overall percentage of stromal FAP staining of the primary tumor was assessed semi-quantitatively (0, 1, 2, 3, 4, 5) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models.

Results: 49 patients with resected specimens were available for study with 26 (53.1%) stage I, 8 (16.3%) stage II, 14 (28.6%) stage III, and 1 (2.0%) stage IV patients. All patients were divided to 2 groups, FAP high group and FAP low group, which are 25 patients (51%) and 24 (49%) respectively. The cutoff for subgroups was defined at the median value. FAP (immune) expression at tumoral stroma was a significant predictive factor for lymph node metastasis (p<0.01) and vessel invasion (p<0.01). In survival analysis of DFS (disease free survival) and OS (overall survival), FAP high stroma was associated with shorter period to recurrence (p<0.05) and death (p<0.05) than those of FAP low stroma.

Conclusions: Our clinicopathological analysis indicates that patients whose esophageal carcinoma have high levels of stromal FAP expression are more likely to have an aggressive disease progression and a potential development of metastases and recurrence. Although therapeutic strategies targeting the tumor cells have been generally inadequate in esophageal carcinomas yet, here we announce that a stroma-targeted therapy should be considered. Now we are ongoing to evaluate metastatic potential of CAFs in vitro and vivo, in order to elucidate the function of FAP expressing CAFs and, in future to establish a novel therapeutic strategy.

#1561

Characterization of circulating monocyte infiltration in brain metastasis.

Vasiliki Economopoulos, Manuel Sarmiento Soto, James R. Larkin, Danny Allen, Nicola R. Sibson. _University of Oxford, Oxford, United Kingdom_.

Brain metastases are a devastating condition with extremely poor prognosis. To improve treatment outcomes for patients with brain metastases, the role of the host's immune system in their development needs to be better understood. The contribution of circulating monocytes, in particular, to the progression of brain metastases is not well understood. Histologically and molecularly, it is difficult to differentiate between resident brain macrophages (microglia) and infiltrating circulating monocytes from the systemic circulation. However, advances in genetic engineering have allowed these populations to be distinguished. We hypothesise that infiltration of circulating monocytes into brain metastases increases over time, and correlates with BBB breakdown and increased cell adhesion molecule (CAM) expression.

In this study, we have used the Lys-GFP-ki mouse strain in which bone marrow derived monocytes express GFP, but CNS microglia do not. 500 EO771 cells (mouse mammary carcinoma) were microinjected into the left striatum of Lys-GFP-ki mice (n=28) using stereotaxic guidance and a finely-drawn glass microcapillary. Some animals underwent MRI at 7T on days 7 (n = 7), 14 (n = 4) or 21 (n = 7), and were perfusion-fixed immediately after imaging. T1-weighted 3D data sets were acquired both pre- and post- intravenous injection of 30µL gadodiamide. The remaining animals were perfusion-fixed on days 7 (n = 2), 14 (n = 5) or 21 (n = 3). Immunofluorescent staining was performed on all brains and assessed the expression of F4/80 (macrophage marker), as well as the adhesion molecules VCAM-1, VLA-4, ICAM-1, LFA-1, E- and P-Selectin, PSGL-1, ALCAM and CD34.

The percentage of GFP-positive area (indicating recruitment of systemic monocytes) within the tumour increased significantly from day 7 to 14 (p < 0.001) and maintained significance at day 21. In contrast, blood vessel area and number, as determined from CD34 staining, only increased significantly at day 21 (area: p < 0.05, number: p < 0.001). With regards to MRI data, the area of signal enhancement post-gadodiamide injection increased significantly over time and correlated with GFP positive cell infiltration (p < 0.05; y = 0.5913x - 0.2513; r2 = 0.3706). Additionally, the degree of co-localisation increased significantly over time between GFP-positive macrophages and ICAM-1 (p < 0.05), ALCAM (p < 0.05), E- and P-Selectin (p < 0.001, p < 0.05), whilst ALCAM expression also increased significantly on CD34 positive vessels (p < 0.05).

In conclusion, it appears that tumour progression in brain metastasis is associated with increasing infiltration of circulating monocytes, which correlates with increasing BBB breakdown. Interestingly, circulating monocyte infiltration appears to occur prior to significant changes in vascular area and density. Analysis of CAM expression, suggests that CAMs, and in particular ICAM-1, may play a role in recruitment of circulating monocytes and in their interactions with cancer cells.

#1562

miRNAs in exosomes from prostate cancer cells modify normal fibroblasts and osteoblasts favoring tumor spread and metastasis.

Eliana Andahur,1 Enrique A. Castellon,2 Chistian Ramos,1 Juan A. Fulla,1 Catherine A. Sanchez1. 1 _Clinica Las Condes, Santiago, Chile;_ 2 _Universidad de Chile, Santiago, Chile_.

Introduction: Exosomes from bulk and cancer stem cells (CSCs) from prostate cancer primary cultures present a differential pattern of miRNAs. The most abundant miRNA in all exosomes is miR-100, while miR-21 and miR-139 were the most abundant differentially expressed miRNAs in bulk and CSCs exosomes, respectively. Bioinformatics analysis suggested that these miRNAs were related with tumor progression. The functional effect of these miRNAs on targets related with changes at primary and pre-metastatic microenvironment was evaluated in prostatic stromal cells and osteoblasts.

Methods: Normal human undifferentiated osteoblasts (hFOB1.19) and prostate fibroblasts (WPMY-1) cell lines were transfected, by separated, with 25 nM of the following miRNas: miR-100-5p, miR-21-5p, miR-139-5p. let7c was included as transfection control. After 48 hours, changes in the expression of metalloproteinases (MMP)-2, -9 and-13, RANKL, OPG and IL-8 at mRNAs and protein levels were evaluated by qPCR and western blot, respectively. As transfection control, expression of specific targets previously described for each miRNA was evaluated. Besides, the effect of miRNAs in fibroblast migration was evaluated by transwell assay.

Results: In fibroblasts, transfection with miR-21, -100 and -139 increased significantly expression of RANKL, IL8 and all MMPs, with a higher effect in MMP-9. Besides, miR-21 increased fibroblast migration. In osteoblasts, transfection with miRNAs changed the balance that triggers vicious circle at the bone, increasing RANKL and diminishing OPG expression. miRNAs also increased MMPs expression. The greater effect was observed with miR-21 transfection.

Conclusions: Through exosomes, prostate cancer cells recruit normal cells to favor their growth and spread. miRNAs in exosomes increase expression of proteins (in particular, MMPs and RANKL) and cell migration of fibroblasts that create a favorable microenvironment for tumor progression at the primary niche. Besides, through bloodstream, tumoral exosomes can reach the bone, preparing the premetastatic niche. miRNAs from these exosomes change the balance RANKL/OPG that triggers osteoclast recruitment facilitating metastases by activation of the bone vicious circle. miR-21, overexpressed in bulk cells, the main cellular component of tumor, has the higher effect in modify microenvironment, being a potential therapeutic target. (FONDECYT 11121525 and 1140417).

#1563

Interplay between perlecan/HSPG2 and matrilysin/MMP-7 in the prostate cancer tumor microenvironment directs metastatic programming through focal adhesion kinase and FoxM1.

Brian J. Grindel,1 Jerahme Martinez,1 Hamim Zafar,1 Luay K. Nakhleh,1 Leland W.K. Chung,2 Mary C. Farach-Carson1. 1 _Rice University, Houston, TX;_ 2 _Cedars-Sinai, Los Angeles, CA_.

Prostate cancer (PCa) cells interact with the stroma and extracellular matrix (ECM) during tumor expansion and invasion during metastasis. Dissecting the complex matrix-cancer interplay is essential to halting tumor progression. A vital component of the ECM is perlecan/heparan sulfate proteoglycan 2 (HSPG2). As a large extracellular proteoglycan, perlecan helps orchestrate tumor angiogenesis, proliferation, differentiation and invasion. Actively metastasizing cancer cells must proteolytically degrade several tissue borders which perlecan patrols, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. We have demonstrated that perlecan, even fully decorated with glycosaminoglycan chains and in the context of the basement membrane, is cleaved efficiently by the protease matrilysin/matrix metalloproteinase 7 (MMP-7). MMP-7 levels increase, especially in relation to its endogenous inhibitor, during metastatic programming in several cancers including PCa. Recent analysis of a tissue microarray of 157 prostatectomy patients showed perlecan and MMP-7 are increased in tandem, with perlecan disappearing at the sites where MMP-7 is high, indicative of a protease-substrate relationship in tissue. This idea is supported by the finding that perlecan fragments are present in serum of PCa patients, with a majority from domain IV. Additionally, higher perlecan staining in cancer related stroma positively correlated with higher grade cancer. Investigating the effects of perlecan on metastatic PCa cells showed full length perlecan and specifically domain IV of perlecan triggers clustering of PCa cells. Perlecan proteolysis by MMP-7 reverses this, favoring cell dispersion and tumor dyscohesion. To discover the molecular signaling events associated with perlecan binding to PCa cells, a reverse phase protein array was analyzed for protein interactions that were plotted in Cytoscape. Network analysis revealed, and western blotting confirmed, perlecan and MMP-7 mediated proteolysis altered focal adhesion kinase (FAK) signaling, with intact perlecan and domain IV suppressing FAK activity during clustering, and MMP-7 cleaved perlecan activating FAK, consistent with the induction of dispersion. Further work showed that cleavage of perlecan increased the pro-cancer transcription factor forkhead box protein M1 (FOXM1). This work reveals that degradation of perlecan by MMP-7 in the tumor microenvironment can unlock stable epithelial contacts and reprogram PCa cells for invasion and metastasis through processes involving FAK and FoxM1.

#1564

Reactive endosteum in prostate cancer bone metastases: role of tenascin-C in regulating cancer cell adhesion and proliferation.

Rebeca San Martin,1 Kenneth Pienta,2 David R. Rowley1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Johns Hopkins School of Medicine, Baltimore, MD_.

The objective of this study is to assess the regulatory roles of reactive endosteum associated with foci of bone metastatic prostate cancer cells. Previous studies evaluated tissue arrays of human prostate cancer bone metastases and we identified a "reactive endosteum" spatially associated with foci of prostate cancer cells on trabecular bone. Reactive endosteum was characterized by elevated expression of tenascin-C, a glycoprotein deposited in the extracellular matrix of reactive stroma at sites of wound repair and in the primary tumor microenvironment in adult tissues. In general, tenascin-C is stromal derived in adult tissues. Of interest, tenascin-C is also deposited at sites of fracture repair and at sites of Brodie abscess osteomyelitis-associated inflammation in human bone. Tenascin-C has many diverse functions: it regulates cell adhesion, migration and proliferation in different pathological states while playing an important role in neuropatterning and osteogenesis during development. Moreover, tenascin-C has been shown to regulate several signal transduction pathways.

To evaluate prostate cancer-bone interactions, we developed an in vitro, 3D, osteogenic organoid model composed of human mesenchymal stem cells induced to osteogenesis that were combined with human prostate VCaP cells in organ culture. In this model, foci of VCaP cells associated preferentially to regions of high tenascin-C deposition. VCaP cells also preferentially bound to purified human tenascin-C deposited on culture plates in a dose-dependent manner. VCaP cells also exhibited an elevated growth rate on osteo-mimetic plates coated with human tenascin-C as compared to control. Moreover, VCaP cells preferentially bound to human tenascin-C coated bovine trabecular bone cubes in vitro and initiated colony formation. Evaluation of potential mediators identified integrin alpha 9 beta 1 as the key mediator of attachment to human tenascin-C. Neutralization of this integrin inhibited adhesion. Evaluation of potential signaling pathways have implicated activation of EGF receptor (EGFR), Wnk1, and STAT6 pathways. Human tenascin-C exhibits EGF-like repeats and fibronectin type III domains that may mediate activation of these pathways. Of interest, adhesion and growth of VCaP cells on tenascin-C also induced elevated tenascin-C expression in these cells, a novel finding. Additional 3D organoid and in vivo xenograft studies with other cell types support the finding of induced expression of tenascin-C in epithelial cells associated with reactive stroma or matrix.

In summary, our studies characterize elevated tenascin-C at sites of a reactive endosteum associated with metastatic prostate cancer foci. Data suggests that tenascin-C mediates adhesion and proliferation of cancer cells via activation of several pathways. These data may aid in developing novel therapeutic approaches to treat metastatic disease.

#1565

Cthrc1 mediated by myeloid-specific TGF-β signaling stimulates NSCLC bone metastasis.

Paul G. Daft, Xiangqi Meng, Alexandra Vander Ark, Austin Meadows, Xiaohong Li. _Van Andel Research Institute, Grand Rapids, MI_.

One third of non-small-cell lung cancer (NSCLC) patients develop bone metastasis and die within a year. Tumor growth in the bone microenvironment results in excessive bone resorption−osteolytic bone lesions, which releases many cytokines and growth factors stored in the bone matrix, such as transforming growth factor-β (TGF-β), which further stimulates metastatic tumor growth in the bone. It is not known how TGF-β signaling in resident cells of the bone microenvironment effect NSCLC-induced bone lesions.

We found that blocking TGF-β signaling in cells of the myeloid lineage, LysMCre/Tgfbr2 knockout (KO), but not in cells of the mesenchymal lineage, Col1αcre/Tgfbr2 KO, decreases osteolytic bone lesion development of H1993 or H1975 NSCLC cells in a tibial-injected mouse model. In determining the mechanism by which LysMCre/Tgfbr2 KO decreases NSCLC bone lesion development, we demonstrated that basic-fibroblast growth factor (bFGF) of the mouse origin was significantly decreased in NSCLC injected tibiae of LysMCre/Tgfbr2 KO mice relative to controls. Exogenous bFGF partially rescues the reduced NSCLC bone lesions in these LysMCre/Tgfbr2 KO mice. bFGF is expressed by cells of the mesenchymal lineage, such as osteoblasts. However, bFGF has no effect on H1993 NSCLC cell proliferation. These data suggest that the decreases in NSCLC-induced bone lesions by loss of TGF-β signaling in myeloid lineage cells are dependent on the effects of bFGF on osteoblasts and osteoclasts. We therefore hypothesize that the regulation of bFGF expression in osteoblasts is indirect through a secreted myeloid-specific TGF-β signaling downstream target, collagen triple-helix repeat-containing 1 (Cthrc1).

Cthrc1 is a TGF-β target gene and clastokine, which are proteins secreted by osteoclasts that couple osteoblasts. Cthrc1 is also a known biomarker for NSCLC progression and promotes NSCLC cell proliferation and invasion. We found Cthrc1 was significantly decreased in H1993-injected tibiae of LysMCre/Tgfbr2 KO mice relative to controls, and stimulates H1993 proliferation in a dose-dependent manner in vitro. Additionally, Cthrc1 is known to stimulate bone formation. However, the effect of Cthrc1 on osteoblasts is not known in the context of NSCLC bone metastasis. From our in vitro studies, we found that Cthrc1 has no significant effect on osteoblast differentiation, but significantly increases bFGF gene expression in MC3T3 cells. bFGF inhibits osteoblast differentiation, but stimulates osteoclast differentiation. We therefore conclude that myeloid-specific TGF-β signaling stimulates osteoclast Cthrc1 secretion, which promotes NSCLC tumor cell proliferation. Meanwhile, Cthrc1 increases bFGF expression from osteoblasts to inhibit osteoblast, but stimulate osteoclast differentiation. Taken together, these data suggests Cthrc1 is a potential novel target for effectively inhibiting NSCLC bone metastasis.

#1566

A cancer-stroma-cancer interacting loop promotes peritoneal metastasis of ovarian cancer through TNFα-TGFα-EGFR.

Tat-San Lau, Loucia Kit-Ying Chan, Chi-Hang Wong, Chi-Wai Man, Tak-Hong Cheung, So-Fan Yim, Jacqueline Ho-Sze Lee, Tony Kwok-Hung Chung, Joseph Kwong. _The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong_.

Peritoneum is the commonest site for ovarian cancer metastasis. In peritoneum, stromal fibroblasts are the second most common cell types. In this study, we aimed to investigate the role of tumor-stromal interaction in peritoneal metastasis of ovarian cancer. Using three-dimensional (3D) organoid co-culture model, we found that the interaction between metastatic human ovarian cancer cells and normal human stromal fibroblasts promotes cancer colony formation. Using cytokine antibody array, we identified a group of cytokines are de novo produced in the 3D co-culture. Among them, transforming growth factor alpha (TGF-α) was produced anew from normal stromal fibroblasts in the 3D co-culture. In the co-culture, transcription of TGF-α in normal stromal fibroblasts is activated by tumor necrosis factor alpha (TNF-α) that derived by ovarian cancer cells. By epigenetic studies, we discovered that over-expression of TNF-α in human ovarian cancer cells is due to DNA hypomethylation and chromatin remodeling in TNF-α promoter. TGF-α is a ligand of epidermal growth factor receptor (EGFR), and we confirmed EGFR is over-expressed in a significant portion of human ovarian cancer. When applying EGFR-specific inhibitors (AG1478, Gefitini, and Erlotinib) in our 3D organoid models (3D mono-cultures and 3D co-cultures), we further found that the fibroblast-derived TGF-α promotes colony formation of metastatic ovarian cancer cells in vitro through EGFR activation. In fact, TGF-α activates Hippo/YAP signaling pathway in human ovarian cancer cells via EGFR. In addition, our in vivo experiments showed that normal stromal fibroblasts promote peritoneal metastasis of ovarian cancer through EGFR activation in a xenograft model. Finally, the TNFα-TGFα-EGFR interacting loop was detected in cancer-stroma-cancer compartments of omental metastases of ovarian cancer patients by immunohistochemistry. Based on our experimental results, we proposed a cancer-stroma-cancer interacting loop that promotes peritoneal metastasis of ovarian cancer through TNFα-TGFα-EGFR. The proposed mechanism involves: 1) TNF-α is over-expressed in human ovarian cancer cells by promoter DNA hypomethylation and chromatin remodeling; 2) the ovarian cancer cell-derived TNF-α induces de novo TGF-α production in normal human omental stromal fibroblasts; and 3) the fibroblast-derived TGF-α, in turn, promotes colony formation of metastatic ovarian cancer cells through EGFR-mediated Hippo/YAP signaling pathway. Our results thereby suggest that peritoneal metastasis in the ovarian cancer patients after cytoreductive surgery can be prevented by EGFR-targeted therapy.

#1567

Prostate cancer-derived exosomes alter the metabolic microenvironment of bone marrow pre-metastatic niche through PKM2 to promote bone metastasis.

Jinlu Dai,1 Yi Lu,2 Jian Zhang,2 Evan Keller1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Guangxi Medical University, Nanning, China_.

Tumor-derived exosomes are emerging mediators of tumor progression. We explored the role of prostate cancer (PCa)-derived exosomes (Ex) in PCa bone metastasis. Systemic pre-treatment of mice with PCa-Ex increased PCa tumor growth in bone, this was associated with increased early PCa cell seeding into the bone marrow. PCa-Ex had no direct impact on PCa growth or invasive ability in vitro; whereas, conditioned-media (CM) from PCa-Ex-treated stromal cells increased PCa cell growth, invasion and epithelial mesenchymal transition (EMT). PCa-Ex preferentially targeted bone marrow versus lung or liver stroma when injected IV. Proteomic analysis of PCa-Ex-treated cells revealed that PCa-Ex increased pyruvate kinase M2 (PKM2) protein expression in both ST2 stromal cells and primary bone marrow stromal cells, although PKM2 mRNA expression was not altered. PKM2 protein expression correlated with aggressiveness of PCa cell lines and was highest in serum of PCa patients with metastases compared to those with primary tumors. PCa-Ex induced both autophagy and β-hydroxybutarate (BHB) production from stromal cells. Knockdown of PKM2 in PCa cells resulted in production of PCa-Ex with low PKM2. The low PKM2 PCa-Ex had diminished ability to induce PCa cell growth, autophagy and BHB production. IV PCa-Ex and orthotopic tumors increased bone marrow BHB levels. PCa-Ex treatment of stromal cells induced the CM to (1) increase in oxygen consumption rate (OCR) by 80%; (2) extracellular acidification rate (ECAR) by 25%; and (3) increase ATP production in PCa cells. BHB induced OCR, but not ECAR in a dose responsive fashion. BHB administration induced a dose-responsive increase in intratibial tumor growth in mice. Our experiments indicate that primary tumor PCa-Ex can target the bone marrow microenvironment and educate stromal cells to promote PCa progression through increasing PKM2 expression in stromal cells resulting in ketone production that supports metastatic growth. Overall this demonstrates that primary tumor exosomes modulate the metabolic status of the distant pre-metastatic niche to promote metastasis.

#1568

The role of CCL2 CCL17 CCL22-CCR4 axis in prostate cancer metastasis.

AERKEN MAOLAKE. _Kanazawa University, Kanazawa, Japan_.

BACKGROUND: Multiple steps and factors are involved in prostate carcinogenesis and tumor progression. The early studies have found that tumor-associated macrophages (TAMs) have great effects on tumor developments. TAMs release a variety of cytokines, and chemokines enabling cancer cells to proliferate, migrate, invade, and metastasize. Some chemokines and their receptors were reported to play a key role during these processes of prostate cancer. Prostate cancer cells themselves also have been shown to express various chemokines and their receptors, such as CCL2, CCR2, CXCR4. Recently, CCR4 has been reported to be expressed in breast cancer cells and is associated with lung metastasis. However, the possible role of CCR4 in prostate cancer has not been well elucidated, and little is known about the relationship between TAMs and CCL2/CCL17/CCL22-CCR4 axis in tumor invasion of prostate cancer.

AIM: The aim of our study was to investigate whether TAMs and the chemokine CCL2 (a low-affinity ligand for CCR4 and a high-affinity ligand for CCR2), CCL17 and CCL22 (high-affinity ligands for CCR4) -CCR4 axis promotes prostate cancer progression.

METHODS: Human prostate cancer cell lines and monocyte cell lines were used in this study. To evaluate effects of chemokines on prostate cancer cells, we performed transwell migration and invasion assay co-cultured with or without macrophages. PCR, western blot, immunocytochemistry, immunohistochemistry, human chemokine array, ELISA were done to elucidate the mechanisms of prostate cancer progression caused by macrophages.

RESULTS: Chemokine receptor CCR2 and CCR4 were expressed in human prostate cancer cell lines both in mRNA and protein level. Prostate cancer tissue also expressed both CCR2 and CCR4. In vitro co-culture system of prostate cancer cell lines and macrophages showed that the level of chemokine CCL2 from both prostate cancer cell lines and macrophages was increased and chemokine receptor CCR2 and CCR4 expressions were increased in prostate cancer cell lines. They promoted the migration and invasion of prostate cancer cell lines via their receptors and also enhanced phosphorylation of protein kinase Akt and extracellular signal-regulated kinase (ERK). Our results suggested that CCL2/CCL17/CCL22-CCR4 axis might play an important role in prostate cancer progression.

CONCLUSION: We demonstrate that chemokine receptor CCR2 and CCR4, for the first time, were expressed by prostate cancer cell lines. CCL2/CCL17/CCL22-CCR4 axis, which is activated by TAMs, may be a potential candidate for molecular targeted therapy in the future.

#1569

IL6 mediated inflammatory loop reprograms normal to highly metastatic cancer stem cells at pre-HCC liver in β2SP+/- mice.

Abhisek Mitra,1 Xueqing Xia,1 Lopa Mishra,2 Shulin Li1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _George Washington University, Washington, DC_.

Hepatocellular carcinoma (HCC), the second-leading cause of cancer-related death world-wide, displays overexpression of the pro-inflammatory cytokine interleukin6 (IL6) and inhibition of transforming growth factor β (TGF-β) in its early phase. We aimed to identify the dynamics of liver immune cells under inflammatory conditions and to characterize early-phase tumor-initiating cells to comprehend aberrant molecular pathway(s) in HCC progression in a TGF-β disrupted murine model (β2SP+/-).

Methods: Chronic inflammation was induced in wild-type (WT) and β2SP+/- mice by hydrodynamic injection of IL6 (pIL6) encoding Sleeping Beauty transposons twice per month for 3 months. Liver stem cells were isolated by using CD133 antibody at the end of third month and cultured with suitable supplements.

Results: Culturing of stem cells enriched from WT and β2SP+/- mice treated with pIL6 showed that the stem cells derived from β2SP+/- mice were highly tumorigenic and metastatic in immune-deficient mice. Staining of these cells with EMT markers Twist and Slug were found in the nucleus. Immunophenotyping suggested that pIL6 treatment induced significant expansion and pro-inflammatory phenotypes of liver NKT and suppressed MDSCs in the β2SP+/- mice compared with WT to generate tumorigenic stem cells. By using numerous pharmacological inhibitors to identify aberrant molecular signaling event(s), we discovered constitutively activated nuclear factor κB (NF-κB) in these cancer stem cells (CSCs). Upon stable knockdown of NF-κB in the CSCs, both EMT phenotype and metastatic ability decreased significantly both in vitro and in vivo. Furthermore, screening of putative kinase(s) that could phosphorylate NF-κB identified that TGF-β activated kinase-1 (TAK-1) directly phosphorylated NF-κB. Staining of HCC tissue specimens showed that CD133+ liver stem cells expressed constitutively activated NF-κB.

Conclusions: Collectively, our data suggest that IL6, highly expressed in 40% HCC patients, can be considered an initiation driver to develop metastatic CSCs by constitutively activated TAK1-NFκB pathway at pre-HCC stage.

#1570

Defective collagen production by mammary epithelia leads to increased breast cancer metastasis: a novel role of vacuolar ATPase.

Gajendra K. Katara, Arpita Kulshrestha, Sahithi Pamarthy, Liqun Mao, Kenneth D. Beaman. _Rosalind Franklin University of Med. Science, North Chicago, IL_.

In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma as well as vesicular membranes and critically influences metastatic behavior. Recently, we have shown that by inhibiting a2 isoform of V-ATPase 'a' subunit (a2V) in cancer cells, in vivo tumor growth can be delayed through decreasing essential macrophage population in breast cancer. Here, we investigated whether the same tumor growth inhibition can be attain by deleting a2V in host tissue instead of cancer cell. For this purpose, we deleted a2V gene in the mouse mammary gland using MMTV-Cre lox system. Breast tumors were generated by inoculating E0771 mammary carcinoma in mammary fat pad of a2V knockout (KO) mice. We observed faster tumor growth in KO mice compared to wild type (WT). KO mice tumors showed more infiltration and metastasis of cancer cell as evident by the number of lung and liver lesions. KO mice did not display the characteristic solid tumor phenotype of breast cancer. Based on these observations, we evaluated expression and production of collagen protein which is an important component of the extra cellular matrix, in KO breast tissue. Trichrome staining showed less collagen protein in tumorous as well as in non-tumorous breast tissue of KO mice. Total collagen was measured by the hydroxyl proline assay and analysis showed that KO mice have significantly (P<0.05) less amount of hydroxyl proline compared to WT mice. (16.77 ± 3.74 vs 43.67 ± 6.89). To validate our observation, we knock down a2V in HMEpC mammary epithelial cell line to measure the effect on collagen production. After a2V inhibition, the production of collagen in culture media was reduced. The data show that for an efficient tumor growth, a2V is required in both cancer cell and host tissue.

#1571

PTEN is an important mediator of tumor and glia cell crosstalk in breast cancer brain metastasis.

Harriet Wikman,1 Ina Hohensee,1 Han-Ning Chuang,2 Astrid Grottke,1 Stefan Werner,1 Alexander Schulte,1 Jakob Matschke,1 Markus Glatzel,1 Stefan Horn,1 Manfred Jücker,1 Tobias Pukrop,2 Klaus Pantel1. 1 _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany;_ 2 _University Medical Center Göttingen, Göttingen, Germany_.

With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Loss of Phosphatase and tensin homolog (PTEN) is correlated with occurrence of breast cancer brain metastases, but its functional role in metastases formation remains unclear. Here, we first assessed the clinical role of PTEN expression in brain metastases, by performing immunohistochemical analyses on a TMA consisting of 132 breast cancer brain metastases samples. Loss of PTEN protein expression was found in 48.6% of brain metastases, and significantly associated with the triple-negative breast cancer subtype (67.5%, p=0.01). Loss of PTEN was furthermore significantly associated with shorter overall survival (p=0.048). We then investigated the biological role of PTEN in breast cancer brain metastasis formation by overexpressing PTEN in the triple-negative brain metastatic breast cancer cell cline MDA-MB-231 BR. Overexpression of PTEN was shown to reduce the AKT pathway activation especially by reducing the Akt1 kinase activity. Coculture experiments with glia cells (astrocytes and microglia) showed that astrocytes can inhibit cell proliferation of the tumor cells in a PTEN dependent manner. Both astrocytes and microglia promote migration of MDA-MB-231 BR cells in a PTEN-dependent manner in vitro. Using an organotypic brain slice model as a representation of the brain tumor microenvironment, the PTEN-dependent invasion of tumor cells could be confirmed. Furthermore, PTEN overexpression reduced the astrocyte activation. Coculture experiments with glia cells showed PTEN-dependent, differential cytokine expression. We could identify an endocrine and paracrine regulation between tumor cells and astrocytes, where GM-CSF, CSF2RA, EGF and PTEN play an important role. In conclusion, loss of PTEN is associated with poor prognosis and is an important mediator of a "vicious circle" mediated by the cross talk between tumor cells and glia cells.

#1572

Overexpression of Axl delays metastatic outgrowth of prostate cancer bone marrow disseminated tumor cells.

Haley D. Axelrod, Kenneth C. Valkenburg, Kenneth J. Pienta. _Johns Hopkins School of Medicine, Baltimore, MD_.

The presence of detectable metastases in the bone marrow of prostate cancer patients represents lethal, incurable disease. These metastases that may emerge years or decades after prostatectomy are derived from small colonies of dormant tumor cells that disseminated from the primary tumor. Our group has previously shown that Gas6/Axl signaling is associated with disseminated tumor cell (DTC) dormancy in mouse models of prostate cancer metastasis. In this study we explored the functional significance of this association and tested the hypothesis that overexpression of Axl in prostate cancer cells would prolong their dormant phase in the bone marrow. Axl overexpression in the AR-positive Axl-negative human prostate cancer cell line C42B slowed proliferation in vitro. Select cells were completely dormant as detected by the lack of EdU incorporation over 40 hours, and this fraction was increased when co-cultured with bone-forming osteoblasts. To assess the functional effects of Axl on prostate cancer progression in vivo, we utilized a mouse model of prostate cancer metastasis in which subcutaneous tumors are established and surgically removed upon reaching 200mm3. At 10 weeks post-tumor removal, mice bearing C42B-Axl tumors did not have overt osteoblastic or osteolytic lesions in their femurs or tibiae, whereas mice bearing C42B-WT tumors had visible lesions by x-ray. Immunohistochemistry staining of mouse femurs and tibiae corroborated these findings. To confirm that the absence of metastases in mice with C42B-Axl tumors was not due to the inability of the cells to disseminate, we used immunofluorescence to detect human cells in the bone marrow of flushed femurs and tibiae. At 12 weeks post-tumor removal, fewer prostate cancer cells were detected in the bone marrow of mice with C42B-Axl tumors compared to those with C42B-WT tumors. Notably however, both groups had detectable prostate cancer cells in the bone marrow, indicating that the absence of C42B-Axl metastasis was not due to failure of primary tumor cells to disseminate. The presence of disseminated C42B-Axl cells in the bone marrow coupled with their failure to develop into clinical metastases at the time of C42B-WT metastatic outgrowth establishes a functional role for the association of Axl expression with DTC dormancy. Axl may be sufficient to induce and maintain dormancy, and ongoing experiments will address the requirement of Axl in this setting.

#1573

The progesterone analogue, Norgestrel, impairs tumor immunity and promotes metastatic breast cancer progression.

Tomas Dalotto Moreno, Gabriel Adrian Rabinovich, Mariana Salatino. _Institute of Biology and Experimental Medicine, CONICET, Buenos Aires, Argentina_.

The immune system is responsible for recognition and elimination of cancer cells. Tumors, however, can elicit different evasive mechanisms that contribute to immune escape Thus, understanding the immunosuppressive pathways that hamper tumor immunity is critical for the identification of novel therapeutic targets. Progestin-containing hormonal supplementation (as hormone replacement therapy or contraceptive) has been associated with an increase in the risk of breast cancer incidence and recurrence. Moreover, progesterone, as a natural hormone, generally impairs cellular immune responses responsible of preventingfetal rejection during pregnancy. We postulated that progestins and in particular the widely used contraceptive Norgestrel may impair tumor immunity and foster immunosuppressive tumor microenvironments. We injected Balb/c mice with the triple-negative 4T1breast tumor, , and treated them with a Norgestrel releasing depot for 30 days. Norgestrel-treated mice displayed a higher frequency of suppressive regulatory T cells (Tregs) in draining lymph nodes and tumors which displayed a CD4+CD25+Foxp3+CD44+ phenotype . Accordingly, norgestrel-treated mice showed greater number of exhausted PD1+ Tim-3+ CD8 T cells in the tumor microenvironment. Furthermore, in vitro exposure to Norgestrel also promoted differentiation and expansion of Tregs which activated the mTOR pathway assessed by phosphorylation of S6 ribonucleoprotein. These effects could not be prevented by the progesterone nuclear receptor antagonist, Zk 230211, suggesting that Norgestrel acts preferentially through membrane progesterone receptors. Although no significant difference regarding tumor growth could be observed, Norgestrel-treated mice showed greater number of lung metastasis. Mechanistically, Norgestrel-driven Treg cells showed increased expression of RANKL, a protein involved in breast cancer development and progression. Our results identify a novel progestin-mediated tumor-promoting mechanism and indicate caution onthe use of Norgestrel-supplementation therapies. Our findings suggest that Norgestrel may impair tumor immunity, thereby promoting metastatic dissemination of breast cancer cells.

#1574

Neuropilin 2 regulates lung disseminated tumor cell escape from dormancy and progression into metastasis.

Paloma Bragado Domingo,1 Raul Alonso,2 Gemma Fuster,1 Mario Mancino,1 Patricia Fernandez-Nogueira,1 Julio Aguirre-Ghiso,3 Pere Gascon1. 1 _IDIBAPS, Barcelona, Spain;_ 2 _Fundacio clinic per la recerca biomedica, Barcelona, Spain;_ 3 _Mount Sinai School of Medicine, New York, NY_.

Despite considerable advances in the treatment of cancer, about 50% of patients will still eventually develop metastatic disease which remains the main cause of cancer-related death. Disseminated tumor cells (DTCs) are the precursors of metastasis and understanding their biology is one of the most important challenges for the future of cancer research. Our previous studies have shown that TGFβ2 through binding to TGFβR3 regulates DTCs quiescence through the activation of p38αMAPK and induction of the dormant genes DEC2 and p27. However, these DTCs dormancy may be reversed when micro-environmental conditions shift to support DTCs expansion. Nerve fibers and neural mediators are present in organs that serve as key targets for breast and head and neck cancer metastasis, including lungs and bone. In addition, neuropeptides and neurotransmitters receptors are expressed by both, nerve and cancer cells, which suggests that neurotrophic factors can act as messengers between the nervous system and DTCs and influence metastasis progression. Therefore, we hypothesized that in situations of chronic stress or inflammation, neural mediators might influence DTCs fate at secondary organs. Using bioinformatics tools we have found that the semaphorins receptor Neuropilin 2 (NRP2), a co-receptor for TGFβ ligands, is overexpressed in basal and HER2+ breast cancer patient samples and its expression in primary tumors correlate with worst prognosis. In basal breast cancer cell lines, NRP2 expression correlates with lower levels of DEC2 and p27 dormancy genes. Furthermore, we have found that NRP2 is downregulated in dormant cells and overexpressed in proliferative lung DTCs derived cell lines that express low levels of P-p38, DEC2 and p27 dormancy markers. Our results show that in lung DTCs derived cell lines, NRP2 inhibits TGFβR3 expression favoring TGFβ1 signaling and promoting DTCs growth. In agreement with this, breast cancer lung metastasis express higher levels of NRP2 than quiescence lung DTCs in vivo, suggesting that NRP2 might play a role in lung DTCs proliferation. Therefore, we conclude that NRP2 can regulate breast cancer lung DTCs progression from a dormant state to a proliferative state and promote metastasis formation.

#1575

Intratumoral injection of interleukin-17 inhibits distant metastasis of radiation-induced recurrent tumor.

Yi-Nan Li,1 Fang-Hsin Chen,2 Ji-Hong Hong,3 Chi-Shiun Chiang1. 1 _National Tsing Hua University, Hsinchu City, Taiwan;_ 2 _Chang Gung University, Taoyuan, Taiwan;_ 3 _Chang Gung Memorial Hospital, Linkou, Taiwan_.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that leads to the production of GM-CSF, G-CSF, IL-1, IL-6, TNF-α. It can be secreted by various cells, including T cells, NKT cells, fibroblasts, and even tumor cells. In addition, reports have shown that IL-17 could also promote tumor growth by recruiting and expanding granulocytic myeloid suppressor cells (G-MDSCs). In our preliminary study, we found that Tramp-C1, a murine prostate cancer cell line, had remarkable IL-17 level when it grew in pre-irradiated (PreIR) stroma. In contrast, B16F0, a murine melanoma cell line, had much less IL-17 and stronger tumor bed effect, in terms of tumor growth delay, but lung metastasis were formed gradually as B16F0 developed. When the recombinant IL-17 was directly injected into the B16F0 tumors growing in PreIR stroma, we found that the probability of lung metastasis was decreased despite a slight increase of tumor growth at primary site. This indicates that IL-17 may have different effects on irradiated tumor bed and distant metastatic site. The influence of IL-17 on tumor microenvironments of distant metastatic sites and irradiated tumor bed is currently under investigation. In conclusion, we propose a new therapeutic approach of reducing distant metastasis following radiation therapy.. This work was kindly supported by MOST-104-2314-B-182 -024 grant to Fang-Hsin Chen.

#1576

Insertional mutagenesis to identify molecular mechanisms of breast cancer dormancy and metastasis.

Laura Vera Ramirez,1 Sven Bilke,1 Robert Walker,1 Paul Meltzer,1 Tinghu Qiu,1 Roland Rad,2 Jeffrey E. Green1. 1 _National Cancer Institute, US National Institutes of Health, Bethesda, MD;_ 2 _Klinikum Rechts der Isar, Technische Universität München, Munich, Germany_.

The PiggyBac transposon insertional mutagenesis system (PBTS) is a powerful genomic screen previously demonstrated to identify cancer-causing genes and which we have adapted to identify genes regulating breast cancer cell dormancy and metastases. Transposition takes places through a cut and paste mechanism catalyzed by the PiggyBac transposase, which excises and re-integrates the transposon into TTAA chromosomal sites. We have adapted the PBTS system using the well characterized D2.0R in vitro and in vivo models of breast cancer cell dormancy. D2.0R cells grow as primary tumors in the mammary fat pad, disseminate to the lungs, but remain dormant. The PBTS system induces random genome-wide gene activations or inactivations that we expect will induce dormant cells to proliferate and form metastases in vivo. Identification of these genes is performed using high throughput sequencing and existing computational algorithms. Candidate dormancy and metastasis genes will be functionally validated for their roles in dormancy and metastases. We have validated the performance of the PBTS in our model system. D2.0R cells were transfected with 20 copies of the PiggyBac transposon ATP-1. Single clones were obtained from the pooled population and expanded under selection. Pooled D2.0R cells and D2.0R single clones were subsequently transduced with lenti-hyPBase transposase and double-selected for the stable integration of the ATP-1 transposon and the lenti-hyPBase transposase. gDNA was extracted from these cells, sheared and amplified by splinkerette-PCR and sequenced using an Illumina platform. Our data show that there is a significantly higher variability in the integration sites of the ATP-1 transposon in D2.0R cells stably expressing hy-PBase as compared with non-hy-PBase expressing D2.0R cells carrying the ATP-1 transposons. This demonstrates that cells from each clone acquire new insertions sites upon ATP-1 re-mobilization due to hyPBase transposase activity. Likewise, we found a significantly higher variability in the ATP-1 insertions in cells from the pooled cultures than in the cells from individual cellular clones. We also observed that reads from both the 5' and 3' ends of the ATP-1 insertion sites of the transposon cluster together, showing a good correlation between reads from both ends. A complimentary approach will be performed by introducing the PBTS system into the poorly metastatic MMTV-Myc transgenic mammary cancer model to identify genes and pathways that induce metastases. Since Myc is overexpressed in ~30% of human breast cancers, this is a relevant model to employ. These results demonstrate that our experimental protocols are able to induce transposon mobilization which should identify genes that regulate tumor dormancy and metastases. Identifying genes that promote dormancy and metastases will lead to novel strategies that could prevent disease recurrence or treat metastatic breast cancer.

#1577

Thrombus formation inside liver metastasis of breast cancer by pegylated liposomal doxorubicin.

Megumi Kai, Yan Ting Liu, Mauro Ferrari, Kenji Yokoi. _Houston Methodist Research Institute, Houston, TX_.

A relationship between cancer and thrombosis has been long recognized. In the patients with advanced breast cancer, chemotherapy increases risk of deep thrombosis from 2-10% to around 18%. In 1856, Virchow proposed three pathophysiological risk factors for cancer patients who will likely develop deep thrombosis, such as stasis of circulation, blood components and vessel damages. Virchow's triad explains risk only for deep thrombosis, but whether the risk for thrombus formation inside tumor can also be increased is not determined yet. In preclinical studies, it has been reported that doxorubicin can induce endothelial cell damage and induce thrombus formation. Based on these evidences, we hypothesized that thromboembolic events inside tumor can be increased by doxorubicin, therefore these events may affect transport of therapeutics, oxygen and nutrients to tumor cells to show anti-tumor effect.

In current study, we evaluated thrombus formation by IF staining of 4T1 murine breast tumors metastasized to mouse liver using antibody to fibrinogen before or after therapy with pegylated liposomal doxorubicin (PLD). There was a significant increase in the amount of fibrinogen accumulated inside tumor after PLD therapy. We also quantified the amount of blood vessels using antibody to endothelial cells. There was a significant reduction in the amount of vessels only inside, but not outside metastatic liver tumors after PLD. Next, we imaged transport of systemically injected fluorescently labeled albumin inside and outside liver metastases using intravital microscopy. The amount of circulation inside metastatic tumor was much limited as compared to those in outside tumors. We also imaged diffusion of labeled albumin into tumor. After PLD therapy, the amount of albumin inside tumor was reduced as compared to that in untreated liver metastases. Then, we evaluated relative level of hypoxia inside tumor between untreated and PLD treated tumors using antibody to CA9. There was a significant increase in the level of hypoxia inside liver metastases after the therapy.

Previously we reported accumulation of PLD in 4T1 primary breast tumors by imaging fluorescence of doxorubicin. PLD accumulated and extravasated predominantly inside tumor, suggesting direct anti-tumor effect by PLD in the primary tumors. In contrast, we imaged PLD accumulated in macrophages located in surrounding area of metastatic tumors in liver. Doxorubicin may be released, diffused close distance and affect adjacent endothelial cells, therefore inducing thrombus in vessels surrounding tumor. These changes may reduce circulation/diffusion, induce hypoxia inside tumor and kill tumor associated endothelial cells. Thus, PLD may show indirect anti-tumor cell effect in liver metastases. To determine whether thrombus formation inside tumors is key mechanism for (indirect) antitumor effect in liver metastases, combination therapy using PLD with anticoagulant will be performed.

#1578

Exploring the role of tumor-conditioned macrophage metabolism on extravasation of pancreatic ductal adenocarcinoma cells.

Giulia Adriani,1 Hweixian L. Penny,2 Je Lin Sieow,2 Siew Cheng Wong,2 Roger D. Kamm3. 1 _Singapore-MIT Alliance for Research and Technology, Singapore, Singapore;_ 2 _Singapore Immunology Network, Singapore, Singapore;_ 3 _MIT, Boston, MA_.

A persistently low survival rate and more than 80% of patients experiencing relapse after surgery combine to make pancreatic ductal adenocarcinoma (PDAC) the 4th leading cause of death from cancer. Recent studies showed that the presence of tumor-associated macrophages (TAMs) may support cancer cells in each step of the metastatic cascade: growth at the primary site followed by epithelial-mesenchymal transition (EMT), intravasation into lymph and blood vessels, circulation to a distant site, stoppage due to adhesion or size restriction, extravasation into tissue, and colonization in this secondary organ. However, the role of TAMs and their metabolic profile in tumor progression need further investigation. The objective of our studies was to examine the potential for macrophages to switch from oxidative phosphorylation to aerobic glycolysis (the Warburg effect), similar to what is observed in cancer cells, and how this metabolic conversion may influence their pro-metastatic activity. To address this question we differentiated human peripheral blood monocytes with non tumor- and tumor-conditioned media to obtain macrophages and TAMs, respectively. First, we showed in a live glycolytic stress test that TAMs had a greater glycolytic capacity compared to non tumor-conditioned macrophages. Next, a 3D in-vitro microfluidic extravasation assay consisting of an endothelial monolayer was used to assess the effect of macrophages on PDAC cell extravasation across the endothelial wall into an extracellular matrix-like hydrogel. We observed that the extravasation propensity of PDAC cells was increased by the presence of TAMs but was not significantly affected by non tumor-conditioned macrophages. This suggests that the tumor-conditioned media fostered monocyte differentiation toward pro-metastatic macrophages. To assess if the macrophage metabolic switch toward glycolysis was the key factor promoting the increment in cancer cell extravasation, we treated the TAMs with 2-Deoxyglucose (2-DG), a competitive inhibitor of glycolysis at the level of hexokinase. Indeed, we observed a significant reduction in the rate of PDAC cell extravasation when TAMs were treated with 2-DG compared to untreated TAMs. Our results confirmed that the glycolytic metabolism was involved in modulating pro-tumoural macrophage function. Taken together, our data suggest that the macrophage metabolic pathway is a potential therapeutic target for controlling immune cell contributions to tumor progression and that patient responses to current treatments might be improved by inhibition of macrophage glycolysis.

#1579

Exosomes promote ovarian cancer invasion through CD-44 transfer to mesothelial cells.

Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. _Osaka University, Suita, Osaka, Japan_.

Purpose:

The peritoneum and organs in the peritoneal cavity are covered by a single layer of mesothelial cells. Therefore, ovarian cancer cells, which metastasize within the peritoneal cavity, directly encounter mesothelial cells as the initial step of metastasis. This contact has been found to involve cell-cell communication that affects cancer progression. Possible actors in this cell-cell communication are exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, and microRNAs. Here, we aim to identify the functional roles of ovarian cancer-derived exosomes in this metastatic process.

Methods:

Exosomes were isolated from two ovarian cancer cell lines (HeyA8 and TYK-NU) and immortalized normal ovarian epithelial cell line (IOSE) using differential centrifugation. Human peritoneal mesothelial cells (HPMCs) were isolated from normal omentum of patients undergoing gynecologic surgery. The isolation of exosomes was confirmed by electron microscope, nanoparticle tracking analysis, Western blotting and electrophoresis of RNA. The transfer of exosomes into HPMCs was confirmed by fluorescent-labeled exosomes. The effects of exosome transfer from ovarian cancer cells to mesothelial cells in cancer invasion were analyzed in vitro 3D culture model, morphological assessment, Western blotting and gelatin zymography. CD-44 was enriched in cancer derived exosome. Thus, gain or loss of function of CD-44 was analyzed. CD-44 expression in ovarian cancer omental metastasis and surrounding organs was assessed by immunohistochemistry using clinical samples.

Results:

Fluorescent-labeled exosomes were evidently transferred into HPMCs. Exosome-treated HPMCs changed in cellular morphology to spindle phenotype. Ovarian cancer invasion was significantly promoted in the presence of exosome-treated HPMCs. In exosome-treated HPMCs, MMP-9 secretion was up-regulated and E-cadherin expression down-regulated. The clearance of mesothelial barrier was increased in exosome-treated HPMC monolayer. CD-44 was enriched in cancer-derived exosomes and exosome-treated HPMCs display high-level of CD-44. When CD-44 expression was knocked down by siRNA in ovarian cancer cells, these effects to HPMCs were significantly attenuated. In contrast, the enforced expression of CD-44 into HPMCs promoted cancer invasion. In human omentum with microscopic metastasis of ovarian cancer, positive CD-44 expression was confirmed in a mesothelial cell layer when cancer cells are attaching onto it, while CD-44 expression was generally negative in normal mesothelial cells.

Conclusion:

Ovarian cancer-derived exosomes transfer CD-44 to HPMCs, which can facilitate ovarian cancer invasion by up-regulating of MMP-9 secretion and increasing mesothelial clearance.

#1580

Cancer-associated fibroblasts potentiates tumor relapse after radiotherapy.

Jun Mi, Yongbin Wang, Guifang Gan, Jiangmin Zhao. _Shanghai Jiao Tong Univ., Shanghai, China_.

Cancer-associated fibroblasts (CAFs) are a major component of cancer stromal cells, about 40~50% of total cell population in cancer. Our data show that CAFs are distributed into larger area than tumor image revealed by MRI or CT since MRI- or CT- images only represent the core region containing cancer cells. Although the image-guided stereotactic body radiation therapy (SBRT) can precisely deliver radiation does to cancers and decreases radiation side effects, compared to the conventional external beam radiotherapy (EBRT), the data from our retrospective study showed that cancer recurrence including local and remote in patients with conventional EBRT was more frequent than patients with SBRT, and the survival time of patients with EBRT was also longer than patients with SBRT. We also demonstrated that CAFs could promote cancer cells recovery from radiation-induced damage through secreting cytokines/chemokines and exporting intermediate metabolites, which induce cancer cell dormancy and enhance DNA damage repair capacity.

#1581

Crosstalk of androgen sensitive prostate cancer cells and insensitive prostate cancer cells.

Yuta Takezawa, Atsushi Mizokami, Kazuaki Machioka, Kouji Izumi, Hiroaki Iwamoto, Maolake Aerken, Natsagdorg Ariunbold, Mikio Namiki. _Kanazawa University, Ishikawa, Japan_.

[Background] The mechanisms that prostate cancer (PCa) progresses to castration-resistant PCa (CRPC) are adaptation and clonal selection. We hypothesized that PCa proliferated while each mechanism cooperated when PCa recurred.

[Materials and Methods] After LNCaP cells were transfected with luciferase reporter driven by PSA promoter, LNCaP were cocultured with androgen-insensitive DU145 or PC-3. Then DHEA or DHT were added to the medium and luciferase assay was performed. Also, we added DHEA or DHT in the medium and measured various androgens level by LC-MS/MS. As for proliferation, LNCaP were cocultured with DU145 or PC -3 in 2-layer chamber and number of LNCaP were counted. DU145 and PC-3 were also cocultured with LNCaP cells for 4 days. We also checked migration by using 2-layer chamber and migrated cells were counted.

[Results] Whereas DHEA was converted into DHT in DU145 and induced PSA promoter activity in LNCaP, the effect was not found in PC-3. Moreover, DU145 elevated DHT-induced PSA promoter activity. DU145 promoted LNCaP proliferation stimulated by DHT and DHEA, but PC-3 did not. LNCaP also promoted proliferation of DU145 and PC-3. LNCaP promoted migration of PC-3, but not DU145.

[Conclusion] Cross-talk between androgen-sensitive PCa cells and androgen-insensitive PCa cells might regulate progression of CRPC.

#1582

Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering EMT induction.

Valentina Mele,1 Valeria Governa,1 Manuele G. Muraro,1 Jesus F. Glaus Garzon,2 Lubor Borsig,2 Silvio Daester,3 Raoul Droeser,3 Daniel Oertli,3 Markus Zuber,4 Raffaele Rosso,5 Giulio C. Spagnoli,1 Giandomenica Iezzi1. 1 _Department of Biomedicine and University Hospital Basel, Basel, Switzerland;_ 2 _University of Zurich, Institute of Physiology, Zurich, Switzerland;_ 3 _University Hospital Basel, Department of Surgery, Basel, Switzerland;_ 4 _Cantonal Hospital Olten, Department of Surgery, Olten, Switzerland;_ 5 _Regional Hospital Lugano, Department of Surgery, Lugano, Switzerland_.

In the last decades it has been demonstrated that the stroma surrounding tumor not only provides mechanical support, but profoundly influences the outcome of the primary tumor and its possible relapse, impacting on proliferation, metastatic progression and resistance to therapies. Major components of the stroma in most types of human carcinomas are the Tumor-Associated Stromal Cells (TASC). Different sources have been proposed for this population, ranging from the resident fibroblasts to bone marrow-derived mesenchymal stromal cells (BM-MSC) recruited to the trans-differentiated epithelial and endothelial cells, but the scenario is still unclear. Moreover, TASC have an intrinsic heterogeneity, combined also with the presence of multiple subpopulations, and they share several markers with other cell subsets of the stroma, thus making the identification of this population, based on the expression of specific markers, an arduous task.

In this intricate context, we aim to address phenotypic and functional characterization of TASC isolated from colorectal cancer specimens, and to analyze TASC-mediated effects on CRC development and progression in vivo.

TASC were characterized for phenotype and differentiation capacity. Human CRC cells were cultured in the presence or absence of TASC for five days. After co-culture tumor cells were sorted by flow cytometry and evaluated for the expression of EMT-related genes by Real Time PCR and for in vitro invasiveness by chemoinvasion assay. Furthermore, their tumorigenicity and the metastatic potential were assessed upon subcutaneous and intracoecally injection in NOD/SCID mice. Developed tumors, metastatic foci and circulating tumor cells were assessed by histology, flow cytometry and Imaging Flow Cytometry.

Our results indicate that TASC resemble BM-MSC in morphology and phenotype. They also comprise a multipotent subpopulation that is able to differentiate into adipogenic and osteogenic lineage. After co-culture with CRC cells, TASC express membrane-bound TGF-β, through which they are capable to trigger epithelial-to-mesenchymal transition (EMT). Upon subcutaneous injection in NOD/SCID mice, tumor cells co-cultured or admixed with TASC before injection, show a faster growth kinetic and develop larger tumor masses as compared to tumor cells alone. Furthermore developed tumor masses are characterized by a higher vessel density. Notably, upon intracoecal injection TASC presence or conditioning leads to a higher metastatic formation in lungs and livers and an increased number of circulating tumor cells in the peripheral blood, most importantly comprising cells undergoing EMT.

Our data clearly show that the stromal component in the human colorectal cancer microenvironment increases the malignancy of tumor cells, by providing pro-mitogenic factors and by triggering EMT initiation in vitro and in vivo, thus promoting tumorigenicity and metastasis formation in vivo.

#1583

SRI31215, a novel inhibitor of oncogenic HGF/MET signaling.

Benjamin Y. Owusu, Phanindra K. Venukadasula, Robert A. Galemmo, Lidija Klampfer. _Southern Research, Birmingham, AL_.

Constitutive activation of MET signaling, frequently observed in cancer, triggers signaling by AKT, ERK 1/2 and STAT3, and promotes survival, proliferation, migration and invasion of cancer cells. Accordingly, MET amplification or overexpression of its ligand, hepatocyte growth factor (HGF), are associated with tumor aggressiveness, resistance to therapy and poor prognosis in many cancer patients. Cancer cells can secrete HGF which triggers autocrine HGF/MET signaling, however, fibroblasts are the predominant source of HGF and activate MET expressed on tumor cells in a paracrine manner. Cancer cells and fibroblasts secrete HGF as an inactive precursor, pro-HGF. A crucial step in the regulation of HGF/MET signaling is the proteolytic processing of the inactive precursor, pro-HGF, to its active form by one of the three serine proteases, matriptase, hepsin or HGF activator (HGFA). This is the rate-limiting step in the HGF/MET signaling pathway, controlled by the inhibitors of HGF activation, hepatocyte growth factor activator inhibitors 1 and 2 (HAI-1/2).

We synthesized a novel small molecule, SRI31215, a triplex inhibitor of matriptase, hepsin and HGFA, mimicking the biological activity of HA1-1/2. We confirmed that SRI31215 inhibits the proteolytic activation of pro-HGF and blocks HGF-induced MET activation and signaling by AKT, ERK 1/2 and STAT3. SRI31215 blocked fibroblast-induced epithelial-to-mesenchymal transition (EMT) and prevented scattering and migration of cancer cells. Finally, we demonstrated that SRI31215 sensitized HGF-producing colon cancer cells to epidermal growth factor receptor inhibitors (EGFRi) and inhibited fibroblast-mediated, HGF-dependent, resistance to EGFRi in colon cancer. Thus, SRI31215 blocks signaling between cancer cells and fibroblasts and inhibits the tumor-promoting activity of cancer associated fibroblasts.

Taken together, our data demonstrate that inhibitors of HGF activation, such as SRI31215, represent a novel approach to curb the growth and metastatic spread of tumor cells that are addicted to HGF/MET signaling and to overcome primary and acquired resistance to EGFR-targeted therapy. 

### Epithelial-Mesenchymal Transition in Metastasis

#1584

Musashi-2 (MSI2) drives TGFBR1/SMAD3 dependent partial EMT and supports VEGFR2 expression and metastasis of human and mouse NSCLC cells.

Alexander Kudinov,1 Alexander Deneka,2 Anna Nikonova,2 Ilya Serebriiskii,2 Tim N. Beck,2 Qi Cai,2 Brian L. Egleston,2 Emmanuelle Nicolas,2 Hossein Borghaei,2 Don Gibbons,3 Jonathan Kurie,3 Erica A. Golemis,2 Yanis Boumber1. 1 _University of New Mexico Cancer Center, Albuquerque, NM;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _The University of Texas: MD Anderson Cancer Center, Houston, TX_.

About 221,200 new patients will be diagnosed with lung cancer and ∼158,040 will succumb to this disease in the United States in 2015. Non-small cell lung cancer (NSCLC) has a 17.4% overall 5-year survival, with metastasis contributing to the vast majority of deaths. Analyzing NSCLC tumors spontaneously arising in KrasLA1/+; P53R172HG/+ (KP) mice, we identified Musashi-2 (MSI2) protein, a stem cell-associated factor that is regulates mRNA translation, as upregulated in the metastasis-competent mouse cell lines. Importantly, MSI2 shRNA depletion in either mouse or human NSCLC cells decreased invasion in Matrigel in vitro and decreased metastasis upon orthotopic injection 129Sv immunocompetent mice in vivo. Mechanistically, by both overexpressing Msi2 cDNA in 393p murine NSCLC and shRNA depleting MSI2 in four independent mouse and human NSCLC cell lines, we defined MSI2 as a driver of a partial epithelial-mesenchymal transition (EMT) program in NSCLC cells. In support of EMT, MSI2 increases the expression of the Snail and Slug pro-EMT transcription factors, as well as the mesenchymal protein vimentin (VMN). MSI2 also downregulates expression of the extracellular matrix component fibronectin (FN1) and tight junction proteins Claudin-3, 5 and 7. However, MSI2 also inhibits protein expression of Zeb-1 and Zeb-2, while sustaining expression of E-cadherin (E-Cad), associated with epithelial identity. Moreover, MSI2 represses NOTCH-1 and upregulates VEGFR2 at the mRNA and protein levels. This is of interest, as NOTCH-1 has been shown to regulate VEGFR2 in angiogenic signaling. We found that knockdown of NOTCH-1 in MSI2-depleted mouse and human NSCLC cells rescued the loss of VEGFR2 expression, suggesting MSI2 increases VEGFR2 expression in a NOTCH-1 dependent manner. Additionally, siRNA of VEGFR2 in the highly metastatic NSCLC cell line 344sq significantly decreased Matrigel invasion, but had only a limited effect in 344sq derivative lines with stable depletion of MSI2, and human NSCLC experiments are ongoing. Together, these results indicate a possible role of MSI2/NOTCH-1/VEGFR2 axis in NSCLC, and suggest that MSI2 connects cancer cell stemness, EMT, and angiogenesis in lung cancer metastasis.

#1585

Molecular subtypes of breast cancers and normal mammary epithelial cells show hierarchical heterogeneity and complexity at single-cell resolution.

Ebrahim Azizi, Justin Colacino, Shamileh Fouladdel, Max S. Wicha. _University of Michigan, Ann Arbor, MI_.

The analysis of average gene expression in cell populations may obscure the heterogeneity of rare cells such as cancer stem cells (CSCs). The use of CSC markers to enrich for these cell populations has proven valuable in their molecular characterization. However, conventional gene expression analysis of these CSC enriched populations is still not conducive to elucidating their heterogeneity. We exploited single-cell resolution methods using C1 and BioMark HD platform and TaqMan assays in the analysis of CSCs and non-CSCs/differentiated tumor cells. The expression of 96 target genes was studied at single cell level in a multiplex RT-qPCR setting. Breast cancer cell lines including MCF7 (luminal), BT474 (HER2+), SUM149 (Basal), and MDA-MB-231 (Basal-Claudin low) that represent major different subtypes of breast cancers and normal mammary epithelial cells (NM) were studied at single cell resolution. Despite similarities, expression profiles of 96 target genes were distinctly different between subtypes of breast cancers and also with that of NM cells. Interestingly, results of single cell studies revealed significant levels of hierarchical heterogeneities within and between each analyzed population of cells. The observed heterogeneous signatures were due to both different expression levels as well as presence/absence of certain genes. We could identify CSCs with EMT (i.e. CD44+/CD24-/ALDH-/CDH2+/Vimentin+), MET (i.e. ALDH+/CD44-/CD24+/CDH1+/Vimentin-), and dual EMT-MET (CD44+/CD24-/ALDH+/CDH1+ or CDH2+/Vimentin+) characteristics. This methodology enabled us to also identify a range of several different non-CSCs/differentiated single cells with heterogeneous expression patterns for CD44, CD24, ALDH, ESR, PGR, HER2, Vimentin, CDH1, CDH2, EpCAM, and Cytokeratins that were similar to luminal, HER2 overexpressed, basal, and basal-Claudin low breast cancer cells. Additionally, NM single cells also showed significant heterogeneity that was similar to the wide spectrum patterns of some single breast cancer cells described above. This study highlights the use of single cell technologies to redefine tumor cell characterization, revealing heterogeneity of single tumor cells with respect to stem cell properties, metastatic and drug resistance characteristics. This will further improve the identification of new markers/pathways that can be used to develop new target therapies for better therapeutic responses in cancer patients.

#1586

Breast cancer cells overexpressing EMT-inducing transcription factors mediate metastasis of neighboring tumor cells via secretion of molecules that upregulate Hedgehog signaling.

Hengbo Zhou, Deepika Neelakantan, Heide L. Ford. _University of Colorado, Anschutz Medical Campus, Aurora, CO_.

Breast cancers are known to be heterogeneous, and thus it is important to understand how different tumor subpopulations influence each other to mediate metastasis. Epithelial-mesenchymal transition (EMT) is known to facilitate metastasis in breast cancer, via enabling tumor cells to be more motile and invasive (amongst other properties), yet only a small population of cells within a tumor is thought to undergo EMT at any one time. Transcription factors (TFs) such as Twist1, Snail1, and Six1 induce EMT, and are all known to increase metastasis of numerous tumor types, but are not thought to be expressed uniformly throughout tumors. While the cell-autonomous function of these EMT regulators has been extensively studied, how cells expressing these TFs influence neighboring tumor cells remains unknown. We have found that co-culture of HMLER-WT and HMLER-Snail1/Twist1 cells leads to a significant increase in migration of HMLER-WT cells. Importantly, transfer of conditioned media (CM) from HMLER-Twist1 or HMLER-Snail1 cells to HMLER-WT cells leads to increased migration and invasion, and Six1 is required downstream of Twist1 and Snail1 to mediate these non-cell autonomous phenotypes. Similarly, CM from metastatic MCF7-Six1 cells, when placed on MCF7-control (Ctrl) cells, leads to an EMT in the control cells, as observed by downregulation of cytokeratin 18 and membranous E-cadherin and upregulation of fibronectin. Importantly, while MCF7-Ctrl cells are non-metastatic when injected orthotopically into mice, co-injection of metastatic MCF7-Six1 and MCF7-Ctrl cells into nude mice results in increased metastasis of the control cells. These data demonstrate that in a heterogenous tumor where a population of cells express EMT-inducing TFs, these cells can influence their neighbors to also undergo EMT, thus promoting their metastasis. More interestingly, we have found that the non-cell autonomous effects of EMT-inducing TFs Snail1 and Six1 are mediated by Hedgehog signaling, and that the secreted factors that lead to activation of this pathway can be different in different contexts, but all impinge on Gli activation as a critical means by which neighboring cells develop metastatic characteristics. Therefore, our data suggest that treatment of heterogenous tumors with downstream, rather than upstream, inhibitors of the Hedgehog signaling pathway will be more efficacious in treating metastatic progression in breast cancer, as the pathway can be activated by means not dependent on the ligands SHH, IHH, or DHH, nor on the canonical receptors.

#1587

GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer.

Toshi Menju,1 Shigeto Nishikawa,1 Koji Takahashi,1 Shinya Neri,1 Takao Nakanishi,1 Hiroyuki Cho,1 Kei Shikuma,1 Terumasa Sowa,1 Makoto Sonobe,1 Stephan M. Feller,2 Hisataka Sabe,3 Hiroshi Date1. 1 _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan;_ 2 _Martin-Luther-University Halle-Wittenberg, Halle, Germany;_ 3 _Hokkaido University, Dept of Molecular Biology, Sapporo, Japan_.

BACKGROUND

Invasive and metastatic activities are the most challenging hallmark of cancer in clinical settings. Our previous reports have shown that GEP100 activates Arf6 by its binding to activated ErbB family receptors, such as EGFR. They also have revealed the phosphorylation at specific tyrosines in the C-terminal EGFR is necessary for the binding to the plekstrin homology (PH) domain of GEP100, which tyrosines are well known to be Grb2-binding sites as well. Additionally, this GEP100-Arf6 signal pathway is pivotal for epithelio-mesenchymal transition (EMT) leading to invasion and metastasis in various types of tumors. Here we have examined the augmentation effect of Grb2 on the binding of GEP100 with EGFR and the Arf6 activation. Furthermore, the significance of co-expression of Grb2 and GEP100 in clinical settings was analyzed

MATERIALS AND METHODS

GST-tagged proteins including PH domain of GEP100 or SH2/SH3 domain of Grb2 inserted into pGEX vector were expressed in bacteria with glutathione-beads purification. They were used for the mutual binding assays. A549 cells which HA-Grb2 or HA alone vector was transfected, were starved for 18hours and stimulated with EGF. And, their lysates were applied for the immunoprecipitation assay against GEP100. Then, Arf6 activities of these cells were examined using the pulldown assay with GST-tagged GGA protein. Furthermore the in vitro invasive activities of those cells were measured by Matrigel invasion assays. The expression levels of Grb2 and GEP100 of the tumor cells by immunohistochemical staining methods were examined in resected human lung adenocarcinoma specimens. The expression data of the two molecules integrated with their clinicopathological factors and EMT status information previously published were analyzed with regard to their invasive and metastatic activities.

RESULTS

Our results revealed that endogenous Grb2 was immunoprecipitaed with GEP100. These two molecules were physically associated through the PH domain of GEP100 and both the SH2 and N-terminal SH3 domain of Grb2, not its C-terminal SH3 domain. Exogenously aberrant expression of Grb2 in A549 lung cancer cells enhanced the association between activated EGFR and GEP100, consequently, Arf6 activation, and in vitro invasive activity, according to the expression level of Grb2. Among 239 lung adenocarcinoma specimens on tissue microarrays, 131 (55%) and 65 (27%) cases of patients were positive for Grb2 and GEP100, respectively. Tumors with double-positive for Grb2 and GEP100 (45 cases) showed significantly more aggressive EMT status (p=0.0116) and higher node-metastatic potential (p=0.0082, node-positive/negative; 12/33 to 7/77) than the double-negative one (84 cases).

CONCLUSION

Grb2 augments the binding of GEP100 to EGFR leading to Arf6 activation, and promotes lung cancer invasion and metastasis via GEP100-Arf6 pathway.

#1588

A self-enforcing CD44s/ZEB1 feedback loop maintains EMT and stemness properties in cancer cells.

Bogdan-Tiberius Preca, Jochen Maurer. _Uniklinik Freiburg, Freiburg, Germany_.

Invasion and metastasis of carcinomas are often activated by induction of aberrant epithelial-mesenchymal transition (EMT). This is mainly driven by the transcription factor ZEB1, promoting tumor-initiating capacity correlated with increased expression of the putative stem cell marker CD44. However, the direct link between ZEB1, CD44 and tumourigenesis is still enigmatic. Remarkably, EMT-induced repression of ESRP1 controls alternative splicing of CD44, causing a shift in the expression

from the variant CD44v to the standard CD44s isoform. We analyzed whether CD44 and ZEB1 regulate each other and show

that ZEB1 controls CD44s splicing by repression of ESRP1 in breast and pancreatic cancer. Intriguingly, CD44s itself activates the expression of ZEB1, resulting in a self-sustaining ZEB1 and CD44s expression. Activation of this novel CD44s-ZEB1 regulatory loop has functional impact on tumor cells, as evident by increased tumor-sphere initiation capacity, drug-resistance and tumor recurrence. In summary, we identified a self-enforcing feedback loop that employs CD44s to activate ZEB1 expression. This renders tumor cell stemness independent of external stimuli, as ZEB1 downregulates ESRP1, further promoting CD44s isoform synthesis.

#1589

Identification of EMT-selective cytotoxic compounds.

Marion Vanneste,1 Qin Huang,1 Meng Wu,2 Michael D. Henry1. 1 _University of Iowa College of Medicine, Iowa City, IA;_ 2 _University of Iowa High Throughput Screening Facility, Iowa City, IA_.

Background: Epithelial to mesenchymal transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therapeutic targeting of EMT-like phenotypes in cancer cells, thus may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance.

Methods: We have developed a cell-based high-throughput screening protocol using the high content Operetta imaging platform to identify EMT cytotoxic compounds. This screen was performed on PC3E and TEM 4-18 cell lines which were originally isolated form a parental PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer. A library of 2,640 compounds (Microsource) was screened on a co-culture of epithelial cells (PC3E GFP cells) and EMT-like cells (TEM 4-18 mCherry cells). After 72h exposure to compounds, relative numbers of GFP- and mCherry-positive cells were quantitated using the Operetta system. Dose-response curves were established for each compound exhibiting a greater toxicity against EMT-like cells. The results were validated on PC3E and TEM 4-18 cell lines cultured separately with the CellTiter Blue viability assay.

Results: Among the 2,640 compounds tested, monensin and salinomycin, both monovalent cation ionophores, exhibited a relatively greater toxicity against EMT-like cells. Nigericin, compound closely related to monensin but absent from the Microsource library, was also evaluated. The highest potency and selectivity for the EMT-like cells was obtained with nigericin (IC50 357nM for PC3E vs 7nM for TEM 4-18), followed by monensin (IC50 457nM for PC3E vs 11nM for TEM 4-18) and salinomycin (IC50 1687nM for PC3E vs 278nM for TEM 4-18). Monensin and nigericin induced apoptosis, a cell cycle arrest, and an increase in ROS production in TEM 4-18 without apparent mitochondrial disruption. In addition, monensin rapidly induced swelling of golgi apparatus likely resulting in a blockage of intracellular protein trafficking and consequently cell death. To extend our findings, we evaluated the toxicity of these three ionophores in 15 cancer cell lines from different origins and classified them as resistant or sensitive. We analyzed publically available gene expression data for these cell lines and performed a Gene Set Enrichment Analysis to identify the gene sets differentially represented in the 2 groups. Supporting our hypothesis, the gene set involving EMT was the top gene sets enriched in the sensitive group.

Conclusion: In this study we identified three chemically-related compounds (monensin, salinomycin and nigericin) exhibiting relatively greater toxicity against EMT-like cells. These compounds may serve as chemical probes for exploring the biology of EMT-like cells and point to strategies for selectively targeting these cells to disrupt metastasis and/or therapeutic resistance.

#1590

N-cadherin is a key marker for epithelial-to-mesenchymal transition in clinical prostate cancer.

Kimberley Kolijn, Esther I. Verhoef, Geert J.L.H. van Leenders. _Erasmus MC, Rotterdam, Netherlands_.

Purpose of the study: Epithelial-to-mesenchymal transition (EMT) is characterized by E cadherin downregulation and simultaneous upregulation of mesenchymal markers such as vimentin, fibronectin and N cadherin. Studies on EMT are generally performed in cell lines and mouse models, while the histopathological and phenotypical properties in clinical prostate cancer (PCa) are still unclear. Our objective was to analyze the expression of various EMT markers in clinical PCa samples.

Experimental procedures: We performed immunofluorescent double stainings with E cadherin and the mesenchymal markers N cadherin, vimentin or fibronectin, Zeb1, Twist1, and β catenin on fresh frozen radical prostatectomies.

Summary: Immunofluorescent double stainings with E cadherin and the mesenchymal markers N cadherin, vimentin or fibronectin demonstrate that E cadherin was consistently downregulated in N cadherin positive PCa cells, but not vimentin or fibronectin positive PCa cells. Moreover, co expression of mesenchymal markers was uncommon, as PCa cells did not co express N-cadherin with fibronectin and only rarely (<1%) cells with vimentin. Membranous expression of β-catenin was unaltered in N cadherin positive PCa cells, while nuclear staining could not detected. Zeb1 was expressed in the nuclei of surrounding fibroblasts, but not in PCa cells. Twist1 was localized to the nucleus of some N-cadherin negative PCa cells, but not in N-cadherin positive cells.

Conclusions: We demonstrated that N cadherin was the most reliable marker for EMT in clinical PCa as compared to vimentin and fibronectin, and that these mesenchymal markers were generally not expressed within the same cell population. The expression of N-cadherin was not associated with Twist1, Zeb1 or β-catenin expression. Despite extensive knowledge of EMT in PCa cell lines, the molecular mechanisms involved in EMT in clinical PCa is still unknown.

#1591

Identification and characterization of surface major vault protein on circulating tumor cells in patients with hepatocellular carcinoma.

Hyun Min Lee,1 Jae Won Joh,2 Hong Seo Choi,1 Won Tae Kim,1 Se Ri Seo,1 Min Kyu Kim,1 Hee Jin Chang,3 Dae Shick Kim,2 Chun Jeih Ryu1. 1 _Sejong University, Seoul, Republic of Korea;_ 2 _Sungkyunkwan University, Seoul, Republic of Korea;_ 3 _Research Institute and Hospital of National Cancer Center, Goyang, Republic of Korea_.

The prognosis of patients with hepatocellular carcinoma (HCC) is poor because of frequent metastasis and resistance to chemotherapy. Circulating tumor cells (CTCs) in blood have attracted attention as potential seeds for metastasis and an important indicator of treatment outcome. However, the biological properties of CTCs are largely unknown due to rarity and lack of CTC-specific surface markers. Previously, we produced monoclonal antibodies (MAbs) against surface molecules on human embryonic stem cells. We found that some of them bound to surface antigens on HCC cell lines but not to normal primary cells. MAb 63-B6, one of the MAbs, recognized CD45-negative CTCs in 6 out of 8 patients with HCCs, suggesting that 63-B6-reactive antigen may be a novel surface marker on CTCs. In a subsequent study, we found that 63-B6 recognized major vault protein (MVP) on the surface of cancer cells, although MVP has been known as a cytoplasmic and nuclear protein. To investigate the role of surface MVP on HCC cell lines, MVP expression was knocked down in Huh7 cells by small interfering RNA. MVP depletion decreased cell growth and increased apoptotic cell death. When Huh7 cells were treated with a polyclonal anti-MVP antibody recognizing surface MVP, Huh7 cell proliferation was decreased without apoptotic cell death, suggesting that surface MVP regulates cell proliferation. Huh7 cells treated with 63-B6 or the anti-MVP antibodies also inhibited Huh7 cell invasion and migration in vitro. Thus, surface MVP is positively associated with HCC cell proliferation, invasion and migration. To further dissect the functional role of surface MVP during HCC metastasis, blood samples from 60 HCC patients and 6 normal adults were stained with 63-B6, HSA, and/or EMT-related antibodies after the depletion of red blood cells and CD45-positive cells. CTCs were detected with 63-B6 in approximately 92% (≥2 CTCs) of patients, and the cell count measured in 7.5 ml of blood ranged between 2 and 1032. Double staining showed that 80% of HSA-positive CTCs was 63-B6-positive. Seventy-six % of EpCAM-positive CTCs was 63-B6-positive while 95% of vimentin-positive CTCs was 63-B6-positive, suggesting that 63-B6-reactive-surface MVP is more expressed on mesenchymal-phenotypic CTCs. Triple staining further showed that 36% of 63-B6-positive CTCs was EpCAM(-)Vimentin(+) and 19% of 63-B6-positive CTCs was EpCAM(+)Vimentin(+) while 3% of 63-B6-positive CTCs was EpCAM(+)Vimentin(-). Interestingly, 42% of 63-B6-positive CTCs was EpCAM(-)Vimentin(-). These results suggest that 63-B6-reactive surface MVP is a novel surface marker on CTCs in patients with HCCs, where it is expressed predominantly on mesenchymal phenotypic and EpCAM(-)Vimentin(-) CTCs.

#1592

Identifying novel drivers of the epithelial-to-mesenchymal transition across multiple cancer types: from bioinformatics to the bench.

Sarah R. Amend, Princy Parsana, James Hernandez, Alexis Battle, Kenneth J. Pienta. _Johns Hopkins, Baltimore, MD_.

To emigrate from a primary tumor to a distant site, a proliferating cancer cell must acquire the phenotypic traits necessary for migration and invasion. This shift from "growing" to "going," termed epithelial-to-mesenchymal transition (EMT), may be a genetic or epigenetic phenomenon, ultimately characterized by altered cellular function that promotes metastasis. In addition to its role in malignant progression, EMT is integral in development and for healthy wound healing. EMT has been described in all carcinomas, and, because it of its established physiologic role, we hypothesize that there are a set of central EMT regulators that are universal to all carcinoma types. To identify global regulators of EMT, we integrated data from 15 published gene expression microarray studies that include a total of 49 epithelial and 46 mesenchymal cell line samples across 6 malignant tissue types (breast, prostate, colon, esophageal, liver, retinal pigment) and with various in vitro EMT induction strategies. While accounting for batch effects and other technical variability inherent in gene expression data, we performed differential expression analysis to identify genes that vary significantly between epithelial and mesenchymal states. Importantly, we found differential expression of established EMT markers (e.g. CDH1, ZEB1) validating our approach. We also identified genes that had not previously been implicated in cancer progression, representing novel candidate drivers of EMT, for example SARG (C1orf116). We found that SARG expression is decreased in high-grade cancer and metastatic disease, and is negatively associated with chemotherapy resistance across multiple cancer types (Oncomine). In an in vitro model of prostate cancer EMT, we found that SARG had >5-fold increased RNA and protein expression in PC3-epithelial cells. Knockdown of SARG expression in PC3-epithelial cells resulted in decreased gene expression of the epithelial-marker CDH1 and elevated expression of the mesenchymal marker CDH2, suggesting a role as a driver of the epithelial phenotype. In parallel with functional in vitro experiments, we have applied a Bayesian network learning approach to identify differentially expressed genes that are likely to play regulatory roles in EMT with causal influence on other genes. Importantly, this analysis provides valuable information regarding novel downstream effectors of known key regulators of EMT such as CDH1 and ELF3. Moreover, we also identified new EMT regulators such as CEP170 that have not been previously described. Experiments are underway to further explore the functional implications of these gene networks. This integrative approach of global gene expression analysis, network learning, and functional validation may identify causal genes and regulatory interactions in EMT, both expanding upon current knowledge and identifying novel drivers of this key metastatic process.

#1593

Stat3 signaling in erbB3-mediated epithelial-mesenchymal transition in erbB2-positive breast cancer cells.

Hui Lyu, Ying Wu, Yan Zhou, Bolin Liu. _University of Colorado Denver/AMC, Aurora, CO_.

Introduction: Understanding the mechanism of metastatic program is essential to reducing the mortality of cancer patients. It is well known that epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Recently, numerous studies have documented the role of erbB3-mediated oncogenic signaling in breast cancer progression. In this study, we have focused on investigating the effect of Stat3 activation on EMT-induced by erbB3 receptor in erbB2-positive (erbB2+) breast cancer cells.

Methods: Immunofluorescence analyses were performed to study the localization and expression of EMT markers in erbB2+ breast cancer cells. Lentiviral vector containing shRNA was used to specifically knockdown erbB3. Western blot analyses were performed to assess the expression and activation of proteins. Cell growth (MTS) assays were used to determine cell viability. Wound healing and transwell assays were used to examine the motility and migration of cells.

Results: We discovered that ectopic expression of erbB3 facilitated EMT, as evidenced by change of cell morphology, induction of mesenchymal markers Vimentin, Snail, Slug, ZEB1 and repression of the epithelial marker E-cadherin in erbB2+ breast cancer cells. Wound healing and transwell assays indicated that the capability of cell motility and migration was enhanced. In contrast, specific knockdown of erbB3 abrogated the observations described above. Moreover, elevated expression of erbB3 potently stimulated activation of Stat3, which has been validated to act as a promoter of tumor invasion and metastasis in many type cancers. Suppression of Stat3 signaling via its inhibitor, S3I-201, significantly alleviated EMT and cell motility/migration that were triggered by activation of erbB3. To further assess the clinical relevance of erbB3 signaling in cancer metastasis, we took advantage of a publicly accessible portal that allows analysis of the effect of genes on survival using 4,142 breast cancer samples (http://kmplot.com/analysis/). Kaplan-Meier survival curves showed that the erbB2+ breast cancer patients with erbB3-high expression tended to have a shorter Distance Metastasis-free Survival (DMFS).

Conclusion: Activation of Stat3 signaling by erbB3 promotes EMT and shifts the cellular phenotype toward a mesenchymal state that is advantageous for cancer metastasis. Our data shed a new sight on erbB3's role in erbB2+ breast cancer metastasis and may facilitate the development of novel strategy for treatment of tumor progression.

Keywords: ErbB3, Stat3, EMT, Metastasis, Breast Cancer

#1594

Contribution of Stromal Lymphocytes to Lung Cancer Metastasis: Role in Epithelial Mesenchymal Transition.

Ylia Salazar, Magdalena Huber, Anja Schmall, Werner Seeger, SoniSavai Pullamsetti, Rajkumar Savai, Magdalena Huber, Anja Schmall, Werner Seeger, SoniSavai Pullamsetti. _Max Planck Institut, Bad Nauheim, Germany_.

The tumor microenvironment and its immune components play a critical role in cancer development, progression, and control. In this study we aim to investigate the role of tumor infiltrating lymphocyte subpopulations in lung cancer progression.

Conditioned media (CM) from co-cultures of human lymphocytes with adenocarcinoma cells (A549) induced the loss of the epithelial markers (E-Cadherin and ZO2 and an increase of mesenchymal markers (vimentin, N-cadherin on mRNA and protein level. In addition, the cells demonstrated a spindled shape-like morphological changes and an increased migratory property. In order to explore the molecular mechanism that led to lymphocyte-induced EMT and migration, we performed a cytokine array from the co-culture CM and found elevated levels of IL-8, IL-16, CCL2 and G-CSF. Furthermore, we observed that lymphocyte-induced EMT was mediated via a TGFβ-independent pathway that involves phosphorylation of ERK1/2.

Notably, this EMT phenotype induction by lymphocytes was independent of the pre-activation of lymphocytes with PMA. Furthermore, in order to identify the specific CD4+ T cell subpopulations (Th0, Th1, Th9 and Th17) responsible for the EMT effect, we generated specific subpopulations from mouse spleen T cells. Interestingly, we observed that Th9 and Th17 subpopulation CM led to EMT and increased migratory phenotype in mouse lung cancer cell lines. Additionally, we identified the T lymphocyte secretory cytokine, IL9, as responsible for the Th9 induced EMT and migratory phenotype of lung cancer cells.

This study reveals that specific T lymphocyte subpopulations, i.e. Th9 and Th17 induce EMT in lung cancer cells, suggesting T cell regulated mechanisms of metastasis.

#1595

HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin.

Ohuod A. hawsawi, Basil Smith, Jodi Dougan, Liza J. Burton, Valerie A. Odero-Marah. _Clark Atlanta University, Atlanta, GA_.

Prostate cancer is the most diagnosed cancer in men and the second leading cause of death in the United States. African American men have the highest incidence and mortality rates of prostate cancer compared to any other race. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Mesenchymal cells are migratory, invasive, and more resistant to apoptosis. Reactive Oxygen Species (ROS) has been shown to promote EMT. High mobility group A (HMGA2) is a non-histone protein that is highly expressed during the embryogenesis, whereas the gene expression is very low or absent during adulthood. Recent studies have been reported an overexpression of HMGA2 protein in malignant cancers. Loss of Let-7 miRNA (repressor of HMGA2) has been found to induce EMT via upregulation of HMGA2 in prostate cancer. There has been no link between ROS and HMGA2. We have reported that camalexin, a 3-thizol-2-yl-indole, may be a candidate treatment for aggressive prostate cancer cells by ROS-mediated apoptosis. The study demonstrated that treating the prostate cancer cell with camalexin increased ROS levels which contributed to decreased cell proliferation and increased apoptosis. We hypothesize that HMGA2 may regulate EMT in part by inducing ROS and that camalexin may antagonize HMGA2 signaling. We analysed HMGA2 and EMT marker expression in a panel of prostate cancer cell lines by western blot analysis. We also transiently and stably overexpressed wild-type HMGA2 and mutant HMGA2 (missing Let-7 binding site) in LNCaP cells. We measured ROS levels using DCFDA dye that detects hydrogen peroxide. We treated ARCaP-M (mesenchymal) cells with different concentrations of camalexin to analyse HMGA2 expression. Our results showed that HMGA2 is highly expressed in aggressive prostate cancer cell lines (C4-2, ARCaP, E006AA) as compared to RWPE1 and LNCaP cells. The transient overexpression of HMGA2 in LNCaP cells decreased E-cadherin more markedly than with mutant HMGA2 but did not show any changes in ROS. We will repeat this experiment with stable clones from HMGA2 overexpression. ARCaP-M cells treated with camalexin induced ROS and decreased HMGA2 expression. We are currently performing in vivo studies using ARCaP-M cells injected subcutaneously into nude mice followed by treatment with camalexin after the tumor grows to 50 mm³. In conclusion, HMGA2 promotes EMT, even more markedly if its Let-7 suppressor binding site is eliminated, and camalexin may target HMGA2 to decrease prostate cancer progression.

GRANT SUPPORT: 1P20MD002285; 8G12MD007590

#1596

Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer.

Van T. Hoang,1 Steven Elliott,1 Elizabeth C. Martin,1 Lyndsay V. Rhodes,1 Hope E. Burks,1 Margarite Matossian,1 Suravi Chakrabarty,2 Darlene Monlish,2 Theresa B. Phamduy,1 Lowry Curley,1 Muralidharan Anbalagan,1 Brian G. Rowan,1 Doug Chrisey,1 Jane E. Cavanaugh,2 Patrick T. Flaherty,2 Bridgette M. Collins-Burow,1 Matthew E. Burow1. 1 _Tulane University, New Orleans, LA;_ 2 _Duquesne University, PA_.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the MAPK/extracellular signal-regulated kinases (MEK) pathways has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). Here we proposed to investigate dual inhibition of MEK1/2 and MEK5 as a more efficacious method for intervention to target mesenchymal and highly metastatic breast cancer cells than MEK1/2 or MEK5 alone through the use of a novel pan-MEK inhibitor SC-151. Interestingly, TNBC cells demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET) and exhibited a significant decrease in migration potential following pan-MEK inhibition. Additionally, immuno-compromised mice inoculated with MDA-MB-231 cells and treated with SC-151 demonstrated decreased tumor volumes compared to vehicle-treated animals. To parse the roles of MEK1/2 and MEK5 in EMT and tumorigenesis, we used the CRISPR/Cas9 approach to knock out ERK5 expression in the TNBC cell line MDA-MB-231. Similar to biological changes induced by pan-MEK inhibition, loss of ERK5 promoted epithelial characteristics in TNBC cells at the morphological and molecular level and impaired tumor formation in vivo. Treatment of ERK5-ko cells with SC-151 further enhanced these effects in vitro, suggesting that MEK1/2 and MEK5 play distinct roles in maintaining the mesenchymal phenotype. Further analysis revealed that constitutive activation of MEK5 abrogated the effects of SC-151 on the reversal of EMT, highlighting the requirement for MEK5 inhibition in MET induction. Taken together, these findings show that while the MEK5-ERK5 pathway may be sufficient in EMT regulation, MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Thus, dual MEK inhibition exerts optimal effects in the reversal of EMT. These data present a novel compound and viable therapeutic strategy to target both MEK1/2 and MEK5 in phenotypically mesenchymal and clinically aggressive breast cancer cells, warranting further investigation into mechanisms by which MEK1/2 and MEK5 individually modulate the EMT axis. Additionally, as MEK inhibition has been shown to sensitize resistant cancer cells to targeted therapies, synergistic and sensitizing effects of SC-151 combined with inhibitors of alternative signaling pathways as well as kinases upstream of MEK will be examined.

#1597

Claudin-1 expression promote TNF-a-induced epithelial-mesenchymal transition and growth in colorectal adenocarcinoma cells.

Ajaz Ahmad Bhat,1 Rizwan Ahmad,2 Amar B. Singh,2 Punita Dhawan2. 1 _Hamad Medical Corporation, Doha, United Arab Emirates;_ 2 _UNMC, Omaha, NE_.

Epithelial-mesenchymal transition (EMT) is an important mechanism in colorectal cancer progression and malignancy. Importantly, inflammatory mediators are critical constituents of the local tumor environment and an intimate link between colon cancer progression and inflammation in now validated. Notably, we and others have previously reported key role of the deregulated claudin-1, a tight junction protein, in colon carcinogenesis including colitis-associated colon cancer (CAC). Of interest, the increase in claudin-1 expression in CAC was found to be associated with the inflammatory epithelium. We have further demonstrated that inflammation induced by DSS (Dextran Sodium Sulfate) administration promotes colon cancer in APCmin/Claudin-1 transgenic mice. However, what role claudin-1 plays in inflammation-induced colon cancer remains unclear. In current study, we tested the causal significance of claudin-1 expression in inflammation-associated colon cancer using colon cancer cells subjected to TNF-αα, a pro-inflammatory cytokine, treatment. HT29 colon adenocarcinoma cells cultured in the presence of TNF-α (10ng/ml), demonstrated a sharp decrease in E-cadherin expression and an increase in Vimentin expression versus control cells suggesting TNF-αα treatment induced EMT. Also, significant increase in cell proliferation and wound healing was observed in TNF-α treated cells at 24 and 48 hours of TNF-αα treatment (versus control cells). Interestingly, TNF-αα treatment also upregulated (and delocalized) claudin-1 expression in a time-dependent manner. An increase in the phosphorylation of Erk1/2 and Src, signaling associated with colon cancer survival and transformation, in TNF-αα treated cells further correlated with our published observations that claudin-1 expression promotes these signaling pathways in colon cancer cells. In support, shRNA-mediated inhibition of claudin-1 expression in HT-29 cells largely abrogated the effects of TNF-αα treatment including proliferation, p-erk and p-Src expression. Also, TNF-αα induced EMT was largely suppressed in the absence of claudin-1 expression. Taken together, our data confirm the previously described key role of claudin-1 in regulating tumorigenic abilities of colon cancer cells and further highlights a key role of claudin-1 in inflammation-induced colorectal cancer growth and progression. We predict claudin-1 expression can serve as a biomarker to predict CAC progression and malignancy.

#1598

LncRNA AK001796 as a therapeutic target in aggressive breast cancers.

Maneesh Kumar,1 Rebecca Sinnott DeVaux,1 Julia J. Shen,1 Steven P. Davis,1 Marcel E. Dinger,2 John S. Mattick,2 Charles M. Perou,3 Jeffrey M. Rosen,4 Sendurai A. Mani,5 Jason I. Herschkowitz1. 1 _University at Albany, Rensselaer, NY;_ 2 _Garvan Institute of Medical Research, Sydney, Australia;_ 3 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 4 _Baylor College of Medicine, Houston, TX;_ 5 _MD Anderson Cancer Center, Houston, TX_.

Breast cancer is a heterogeneous disease that can be classified into several distinct molecular subtypes based on gene expression. Like mRNAs and miRNAs, long noncoding RNAs (lncRNAs) differ dramatically in expression across subtypes and can be used for classification. While there has been considerable emphasis on miRNAs, our knowledge is still lacking about the role of lncRNAs that comprise the majority of the mammalian transcriptome. Recently, the importance of lncRNAs in cancer has been highlighted by several studies. We have examined the expression profiles of >17,000 lncRNAs in a large set of breast tumors and have identified a lncRNA, AK001796, that is overexpressed in aggressive breast cancers. In particular, AK001796 is enriched in the aggressive claudin-low, HER-enriched, and luminal B subtypes. Furthermore, in four different models, we find that AK001796 is significantly upregulated in cell lines induced to undergo EMT and in putative mesenchymal-like cancer stem cells within cell lines suggesting this lncRNA as an inducer/facilitator of EMT. Similar results were obtained when a lung cancer cell line was induced to EMT through TGF beta treatment. Using cell fractionation, we have discovered that AK001796 is maintained predominantly in the nucleus. By RACE we have identified two isoforms of AK001796 in breast cancer cells that differ by the presence or absence of a 94 nucleotide intron. The short form (with intron spliced out) appears to be the variant enriched following EMT and may serve as a marker of aggressiveness. Interestingly, knockdown of AK001796 using antisense oligonucleotides lead to significantly increased apoptosis in EMT positive cell lines whereas in EMT negative cells knockdown had little effect. Preliminary studies for finding out the protein interacting partners identified some mesenchymal phenotype-associated proteins in pull-down studies using biotinylated oligos. To further investigate the molecular mechanisms regulated by AK001796 and its utility as a therapeutic target, we will determine the pathways induced by AK001796 by mapping its protein interaction network and downstream signaling pathways. These results nominate AK001796 as a promising therapeutic target in aggressive breast cancers.

#1599

Epithelial mesenchymal transition and its role in TKI resistant NSCLC cell lines.

Ichwaku Rastogi,1 Tsatsral Iderzorig,1 Gagan Chhabra,1 Gregory M. Botting,1 Andrew Webb,1 Brad Foster,2 Brian Webb,3 Marie Nlend,3 Neelu Puri1. 1 _University of Illinois College of Medicine at Rockford, Rockford, IL;_ 2 _Auburn High School, Rockford, IL;_ 3 _Thermo Fisher Scientific, Rockford, IL_.

NSCLC cells acquire resistance to EGFR and c-Met TKIs after prolonged use. Our studies indicate that resistance maybe due to upregulation of alternative signaling pathways such as Wnt and mTOR. We have found that activation of Wnt/β-Catenin is also associated with EMT which results in loss of cell adhesion properties and gain of motility and invasiveness. To understand the mechanism of TKI resistance in NSCLC cells with wild type EGFR, we have developed and used H2170-P (parental) cells and the TKI resistant H2170-ER (erlotinib resistant) and H2170-SR (SU11274 resistant) cells. We aim to study EMT and determine if inhibition of β-Catenin, a key regulator of transcription by siRNA or inhibition of ZEB-1 a transcriptional repressor of cell adhesion proteins by inducing mir-200a will help overcome the TKI resistance in NSCLC cells.

Using immunoblotting, we observed modulations in key EMT-related proteins in H2170-ER cells which showed upregulation of ZEB-1, N-cadherin, active beta-catenin, Vimentin, PRMT1 and ZO1 by 1.8, 2.4, 2, 1.8, 2.6 and 2 fold, respectively, and downregulation of E-cadherin (1.8 fold) as compared to H2170-P cells. Similar results were observed for H2170-SR cells. These results were verified using qPCR where we show that β-catenin (3.4-3.2 fold) and N-cadherin (2-1.9 fold) have increased gene expression while E-cadherin (1.7-2.4 fold) has a decreased gene expression in TKI resistant H2170-ER and SR cells when compared to H2170-P cells. miR-200a induction in H2170-ER cells showed significant downregulation of ZEB-1 (3 fold) at 72 hr and an upregulation of E-cadherin (2 fold) when compared to the mock transfected cells. Morphological changes indicative of EMT were detected using immunofluorescence with Vimentin and E-Cadherin antibodies, which displayed upregulation of Vimentin filaments (2 fold) and downregulation of E-Cadherin (3 fold) in H2170-ER and H2170-SR cells when compared to H2170-P cells. We then conducted experiments where we suppressed ZEB-1 by inducing miR-200a in TKI resistant cells. The immunoblotting results suggested recovery of E-Cadherin, and downregulation of ZEB-1 and N-Cadherin in TKI resistant cells. We also observed increased sensitivity towards erlotinib and SU11274 by 20-25%. Additionally, we observed decrease in levels of β-Catenin and upon siRNA knockdown of β-Catenin, suppression of levels of ZEB-1. This indicates a direct correlation between nuclear accumulation of β-Catenin and occurrence of EMT in TKI resistant cells by increase in expression of ZEB-1.

Our results indicate that increased activation of Wnt/β-Catenin pathway in the TKI resistant NSCLC cells is due to the EMT. In NSCLC patients, L858R and T790M mutations are associated with TKI resistance which could be responsible for inducing EMT. We are further studying cell lines with EGFR mutations to determine their role in induction of EMT this may provide clinicians with novel targets to overcome TKI resistance in NSCLC patients.

#1600

Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling.

Jianguo Wu, Andrei I. Ivanov, Zheng Fu. _Virginia Commonwealth University, Richmond, VA_.

Polo-like kinase 1 (PLK1) is a key cell cycle regulator that has been implicated in the development of various cancers, including prostate cancer. However, the functions of PLK1 beyond cell cycle regulation remain poorly characterized. Here, we report that PLK1 overexpression in prostate epithelial cells triggers oncogenic transformation. It also results in dramatic transcriptional reprogramming of the cells, leading to epithelial-to-mesenchymal transition (EMT) and stimulation of cell migration and invasion. Consistently, PLK1 downregulation in metastatic prostate cancer cells enhances epithelial characteristics and inhibits cell motility. The signaling mechanisms underlying the observed cellular effects of PLK1 involve direct PLK1-dependent regulation of CRAF with subsequent stimulation of the MEK1/2-ERK1/2-Fra1-ZEB1/2 signaling pathway. Our findings highlight novel non-canonical functions of PLK1 as a key regulator of EMT and cell motility in normal prostate epithelium and prostate cancer. This study also uncovers a previously unanticipated role of PLK1 as a potent activator of MAPK signaling.

#1601

Intensive NTS/NTR1 interaction enhances epithelial-mesenchymal transition and promotes tumor metastasis via activating canonical Wnt/β-catenin signaling pathway in hepatocellular carcinoma.

Jinpu Yu, Yingnan Ye, Xinxin Long, Jieying Chen, Pengpeng Liu, Hui Li, Feng Wei, Xiubao Ren. _Tianjin Cancer Institute & Hospital, Tianjin, China_.

Neurotensin (NTS) is an endogenous 13 amino-acid neuropeptide distributed throughout the central nervous system and in parts of the digestive system. Since its first detection in breast carcinoma over two decades ago, NTS and its high-affinity receptor, neurotensin receptor 1 (NTR1), have been found to affect various pathological processes, including growth, anti-apoptosis, migration, and invasion, during the development and progression of breast, colon, prostate, pancreas, and lung carcinoma. However, limited studies have investigated the intracellular events mediated by NTS/NTR1 in HCC. In our previous study, we found NTS overexpression in certain hepatocellular carcinoma (HCC) samples that displayed stronger inflammatory response in a microenvironment, more severe epithelial-mesenchymal transition (EMT) in cancer, and worse prognosis than NTS− HCC patients. In the present study, the significant correlation between NTS upregulation and tumor metastasis was confirmed in 100 cases of HCC tissue samples, in which high NTS expression was associated with incomplete envelope and portal vein invasion, as well as early relapse and short survival after surgery. Furthermore, distinct EMT features were identified in NTS+ HCC samples via Immunohistochemistry, which expressed decreased E-cadherin, increased β-catenin translocating to the cytoplasm, and increased N-cadherin. To elucidate the molecular mechanisms involved in NTS-mediated EMT and HCC metastasis, two HCC cell lines (i.e., Hep3B and HepG2) were genetically modified by NTR1 gene transfection and NTR1-specific siRNA interference to establish different NTS-responsible HCC models in vitro. These models demonstrated that exogenous NTS stimulation and/or upregulated NTR1 expression exerted an acceleratory effect on cell migration and invasion rather than proliferation and apoptosis in both HCC cell lines. Real-time polymerase chain reaction (RT-PCR) and Western blot indicated that intensive NTS/NTR1 interaction repressed E-cadherin expression but enhanced N-caherin and β-catenin expression in HCC cell lines, accompanied by dramatic increases in the expression of Wnt1, Wnt3, Wnt5, Axin, and p-GSK3β. SR48692 and TSW119, which are specific inhibitors targeting NTR1 and GSK3β phosphorylation, significantly impaired NTS/NTR1 interaction and Wnt/β-catenin pathway activation, thereby blocking NTS-induced EMT and inhibited tumor invasion in vitro. Furthermore, SR48692 inhibited the lung metastases of HCC cells line in vivo. Thus, we proposed that the ectopic expression of NTS in HCC enhanced cell EMT and promoted tumor invasion via activating the canonical Wnt/β-catenin pathway, thereby resulting in poor prognosis after conventional surgery.

#1602

Snail transcription factor regulates nuclear cathepsin L activity.

Liza J. Burton, Valerie Odero-Marah. _Clark Atlanta University, Atlanta, GA_.

Triple negative breast cancer (TNBC) are defined as tumors lacking the expression of estrogen receptor-alpha, progesterone receptor and human epidermal growth factor receptor-2, which accounts for approximately 15% of total breast cancer patients, and is more prevalent and lethal among young African, African-American and Latino women patients. The primary cause of breast cancer death is metastasis, which is regulated by factors such as epithelial mesenchymal transition (EMT), a dynamic process that promotes cell motility and decreases cell-cell adhesion. Snail transcription factor promotes EMT by repressing epithelial genes such as E-cadherin, while increasing mesenchymal genes such as vimentin. Cathepsin L (Cat L) cysteine protease promotes cell invasion and nuclear Cat L has recently been associated with poor prognosis in colon and breast cancer. However, the mechanism by which Cat L localizes to the nucleus is unknown. CDP/CUX transcription factor is a substrate for Cat L which when proteolytically cleaved by Cat L from the p200 to the p110 isoform can transcriptionally activate Snail and repress E-cadherin. We have recently published that Snail overexpression increases Cat L activity in prostate cancer cells which can be antagonized by muscadine grape skin extract (MSKE), a natural product rich in anthocyanin. We hypothesize that a feedback loop exists whereby Snail Transcription factor may promote nuclear Cat L expression and activity in TNBC which subsequently cleaves CDP/CUX and further increases Snail transcription, leading to increased EMT; this can be abrogated by Cat L inhibitor or MSKE. We utilized various breast cancer cell lines as well as MCF-7 cells stably overexpressing Snail (MCF-7 Snail) or control (MCF-7 Neo) to evaluate the expression of Snail, Cat L, CDP/CUX and EMT marker expression (E-cadherin, vimentin) by western blot and immunofluorescence. Migration and invasion assays were performed including treatments with Cat L specific inhibitor (Z-FY-CHO) or MSKE. We observed more Snail, Cat L and CDP/CUX cleavage products (p110 and p90 isoforms) expression in MCF-7 Snail cells and TNBC cells (MDA-MB-231, MDA-MB-468) as compared to MCF-7 control cells. Interestingly, immunofluorescent and cell fractionation analyses revealed that Snail overexpression promoted nuclear Cat L and CDP/CUX p110 isoform expression, and EMT (low E-cadherin, high vimentin, migration and invasion) which could be abrogated by Z-FY-CHO or MSKE. We are currently staining for Snail and Cat L in breast cancer tissue of varying races/grades including TNBC, as well as confirming by chromatin immunoprecipitation that the CDP/CUX cleavage products seen in TNBC can bind Snail and/or E-cadherin promoters. In conclusion, our study shows that Snail promotes nuclear localization of Cat L which may promote EMT via CDP/CUX, and that inhibition with Cat L inhibitors or natural products such as MSKE may be a good therapeutic target for TNBC.

#1603

Highly expressed integrin-ɑ8 in multiple myeloma early relapse induces epithelial to mesenchymal transition-like features.

Jiyeon Ryu,1 Youngil Koh,2 Hyun Jung Lee,3 Hyun Sub Chung,4 Sung-Soo Yoon1. 1 _Cancer Reseach Institute, Seoul National University, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea;_ 3 _Department of Internal Medicine, Dongguk University Ilsan Medical Center, Koyang, Republic of Korea;_ 4 _Department of Genetic Epidemiology, SNP Genetics Inc., Seoul, Republic of Korea_.

Multiple myeloma (MM) is characterized by clonal proliferation of malignant plasma cells in the bone marrow niches. Despite the remarkable advances of treatment for MM,still many patients of MM struggle early relapses and its mechanism is not well understood. Patients with early relapse after autologous bone marrow transplantation need to be biologically characterized to better understand and reach complete remission. To define altered gene expression in MM relapse, we performed microarray with whole bone marrow aspirates from 16 relapsed MM patients who underwent auto-BMT. Gene expression profile was compared (P < 0.01) and divided into two groups on the basis of their progression-free survival (PFS) at the point of 12 months. To discern the significance of the result, only four patients with shortest and longest duration of PFS was also nexamined, and interestingly both of the analysis showed common up-regulated genes including PDGFB, ITGA7, and ITGA8. Their associated pathways were focal adhesion, ECM-receptor interaction, and regulation of actin cytoskeleton (P < 0.05). By performing qRT-PCR, we validated three genes and only ITGA8 was corresponded with microarray data that early relapsed patients showed higher expression of ITGA8. Several integrin family members are well documented to involve in MM progression that it mediates MM cell adhesion, migration and homing through BM microenvironment. However, integrin-α8 is largely unknown in MM. Due to ITGA8 was highly expressed in MM relapse, we functionally overexpressed integrin-α8 in MM cell lines. Surprisingly, it activated genes showing stem-like features including HIF1a, VEGF, OCT4, and Nanog and EMT-related genes, such as N-cadherin, Slug, Snail and CXCR4. These, consequently enhanced invasion and migration abilities and reduced adhesion activity, indicating the mechanism of MM progression, metastasis as well as relapse. Our speculated specific gene, integrin-α8, up-regulated in MM early relapse, regulates EMT feature in MM that suggesting a potential marker for relapse and attractive clinical target to achieve complete remission.

#1604

TMPRSS4 induces invasion and proliferation of prostate cancer cells through induction of Slug and cyclin D1.

Yunhee Lee, Semi Kim. _Korea Research Institute of Bioscience and Biotechnology, Daejon, Republic of Korea_.

TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates tumor cell invasion, migration, and metastasis. We also found that TMPRSS4 activates the transcription factor activating protein-1 (AP-1) to induce cancer cell invasion. Here, we explored TMPRSS4-mediated cellular functions and the underlying mechanisms. TMPRSS4 induced Slug, an epithelial-mesenchymal transition (EMT)-inducing transcription factor, and cyclin D1 through activation of AP-1, composed of c-Jun and activating transcription factor (ATF)-2, which resulted in enhanced invasion and proliferation of PC3 prostate cancer cells. In PC3 cells, not only c-Jun but also Slug was required for TMPRSS4-mediated proliferation and invasion. Interestingly, Slug induced phosphorylation of c-Jun and ATF-2 to activate AP-1, establishing a positive feedback loop between Slug and AP-1, and thus induced cyclin D1, leading to enhanced proliferation. Using data from The Cancer Genome Atlas, we found that Slug expression positively correlated with that of c-Jun and cyclin D1 in human prostate cancers. This study demonstrates that TMPRSS4 modulates both invasion and proliferation via Slug and cyclin D1, which is a previously unrecognized pathway that may regulate metastasis and cancer progression.

#1605

Cetuximab inhibits migration, invasion, metastasis and epithelial-mesenchymal transition but not proliferation via GEP100-Arf6-AMAP1 pathway in head and neck squamous cell carcinoma.

Yoshifumi Matsumoto,1 Hiroyuki Sakurai,1 Yasunao Kogashiwa,2 Koichiro Saito1. 1 _Kyorin University School of Medicine, Tokyo, Japan;_ 2 _Saitama Medical University International Medical Center, Saitama, Japan_.

Background: Recently, irradiation combined with Cetuximab (Cmab) is being one of the standard therapeutic options against head and neck squamous cell carcinoma (HNSCC), especially in advanced/recurrent cases. While this concurrent chemo-radiotherapy is widely accepted and tolerated, Cmab monotherapy has been reported to be unsatisfactory to treat HNSCC, and detailed mechanism of Cmab to show such clinical efficacy is unknown.

Methods: To assess the impact of Cmab on proliferation, migration, and invasion of HNSCC, WST assay, wound healing assay, and matrigel invasion assay were incorporated in this study. Effects of Cmab on the grade of epithelial-mesenchymal transition (EMT), expression level of membrane type 1-matrix-metalloproteinase (MT1-MMP), and activity of EGFR-GEP100-Arf6-AMAP1 pathway, were evaluated using the immunocytochemistry, western blot analysis and Arf6 activation assay, respectively. Furthermore, In vivo efficacy of Cmab was evaluated in highly aggressive and metastatic HSC-3-M3 cells using orthotopic xenograft model in nude mice nu/nu.

Results: Inhibition of migration and invasion were observed in 4 HNSCC cell lines by Cmab application. However, proliferation potential of HNSCC were not affected by this chemotherapeutic agent. Increased expression of E-cadherin, with decreased expression of N-cadherin and MT1-MMP, were observed after Cmab treatment in vitro to suggest that Cmab may inhibit EMT in HNSCC. Furthermore, inhibition of Tyr 1086 phosphorylation in EGFR, as well as the inactivation of Arf6 coupled with suppression of both GEP100 and AMAP1 expressions were observed by Cmab application. These results suggested that Cmab might have the potential to suppress the activity of EGFR-GEP100-Arf6-AMAP1 pathway. Furthermore, less metastasis of the cancer was observed in the Cmab treatment group compared with control group in vivo.

Conclusions: Our study is the first to provide that Cmab could affect HNSCC through inhibition of migration and invasion of cancer cells, regardless of its anti-proliferative action. EMT inhibition after Cmab application was also clarified in this study. Furthermore, our study suggested that EGFR-GEP100-Arf6-AMAP1 pathway may have some role in the invasion and metastasis of HNSCC. Future studies to further assess the precise mechanism of Cmab against HNSCC are warranted.

#1607

Nutritional stress promotes epithelial-to-mesenchymal transition and chemoresistance.

Justyna E. Kanska,1 Barbie Taylor-Harding,1 Wolf R. Wiedemeyer,1 Paul-Joseph Aspuria,1 Navjot Kaur Gill,2 Beth Y. Karlan,1 Amy Rowat2. 1 _Cedars Sinai Medical Center, Los Angeles, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

The mesenchymal subtype of high-grade serous ovarian cancer (HGSC) is associated with the poorest overall survival. Chemotherapy and hypoxia were shown to directly alter the epithelial state of some solid cancers. Consistent with this phenomenon, drug-resistant HGSC frequently exhibit a mesenchymal subtype upon recurrence. This suggests that environmental challenges that compromise cancer cells' survival may contribute to epithelial-to-mesenchymal transition (EMT).

We have developed a novel model system to study nutritional stress-induced EMT. Solid tumors have lower glucose levels than normal cells of the same tissue sites, possibly as a result of rapid cellular turnover, higher metabolic demands, and insufficient nutrient supply. These are likely sequelae of anti-angiogenic therapy or poorly developed vasculature of a rapidly expanding tumor. In order to study the adaptive changes to prolonged glucose deprivation, we have cultured several serous ovarian cancer cell lines (OVCAR3, OVCAR4, OAW28) in glucose-free medium for over one year. We then analyzed surviving clones for changes in DNA copy number, gene expression, and response to chemotherapy. Our data showed that glucose-independent cells acquired molecular and functional characteristics of EMT. This was evident by the loss of cell-cell interactions (E-cadherin, claudins, plakophilins) and elevated expression of mesenchymal genes (Vimentin, N-cadherin, SPARC, MMP2 and ZEB1). RNAseq followed by gene ontology analysis additionally revealed activation of the TGFβ signaling pathway and induction of angiogenic effectors. Functionally, we performed mechanotyping studies to show that glucose-independent clones become more deformable than parental cells. Moreover, these transformed clones demonstrated increased migration efficiency and anchorage-independent growth. Interestingly, they have also shown increased resistance to paclitaxel.

We next sought to identify critical mediators of EMT in this system. The metabolic enzyme Nicotinamide N-methyltransferase (NNMT) showed the highest level of induction (>140-fold) by RNAseq in OVCAR3 cells. NNMT catalyzes methylation reactions that may directly alter methylation status of histones and DNA, as well as activity of xenobiotic compounds. Our data show that NNMT expression in HGSC is restricted to the mesenchymal subtype and correlates with an aggressive, highly invasive phenotype in vitro, as well as poor survival in patients. CRISPR/Cas9 and inducible shRNA targeting NNMT significantly impaired proliferation and colony formation efficiency of glucose-independent OVCAR3 clones and decreased Vimentin and N-cadherin levels.

In summary, our data suggest that nutritional stress may promote a more aggressive phenotype of HGSC by inducing EMT in the process of metabolic reprogramming. We further identify NNMT as a potential biomarker and therapeutic target for the mesenchymal subtype of HGSC.

#1608

Resistin promotes cancer progression through induction of mesenchymal phenotypes and stem cell-like properties in breast cancer.

Shyng-Shiou F. Yuan, Chie-Hong Wang. _Kaohsiung Medical University Hospital, Kaohsiung, Taiwan_.

Growing evidence indicates that resistin plays a role in insulin resistance, obesity, inflammation, coronary artery disease, atherosclerosis and malignancies. In this study, we have explored the effects and mechanisms of resistin in promoting breast cancer progression. High serum resistin levels were positively correlated with tumor stage, tumor size and lymph node metastasis in breast cancer patients. Resistin treatment modulated the expression of epithelial-mesenchymal transition (EMT) regulators and enhanced the migratory, invasive and anti-anoikis activities in breast cancer cells. Additionally, resistin treatment promoted the formation of mammospheres and the expression of pluripotency factors (sox2, nanog and oct4). STAT3 specific inhibitor, stattic, was able to abolish the resistin mediated enhancements of cell motilities and stem cell-like properties. Using in vivo model, we found that resistin markedly enhance metastasis and tumor growth. Taken together, our data clearly demonstrated that resistin promotes cancer progression through induction of EMT and stemness by activating TLR4/STAT3 signaling in breast cancer.

#1609

The epithelial to mesenchymal transition regulates the expression of E-selectin ligands on breast cancer cell lines.

Grady Earl Carlson, Venktesh Shirure, Alexander O. Ostermann, Emily A. Blaha, Louis F. Delgadillo, David F.J. Tees, Fabian Benencia, Monica Burdick. _Ohio University, Athens, OH_.

Breast cancer cells garner invasive and stem-like characteristics through the epithelial to mesenchymal transition (EMT). Consequently, the EMT has been implicated in cancer metastasis. While many of the effects of the EMT have been elucidated, it is currently unknown how the EMT modifies the expression of E-selectin ligands on breast cancer cells. These ligands are often sialofucosylated glycans that mediate cell adhesion with E-selectin molecules presented by endothelial cells lining the blood vessel walls at the metastatic site, which is commonly bone marrow. Although E-selectin is widely recognized for its role in promoting adhesion during tumor cell extravasation, it also maintains the quiescence of hematopoietic stem cells in the vascular niche of the bone marrow. Thus in this investigation, we studied the effects of the EMT on the E-selectin ligand activities of breast cancer cell lines and their behavior in an E-selectin rich environment. The EMT was induced in breast cancer cell lines by ectopic expression of either the Snail or Twist transcription factors. Through the EMT breast cancer cells upregulated the expression of vimentin and N-cadherin, yet reduced the expression of CD24, E-cadherin, and E-selectin ligands. Breast cancer cell lines were assayed for E-selectin ligand activity using laminar flow assays, flow cytometry, qPCR, and were cultured on E-selectin-coated tissue culture plates. In laminar flow assays breast cancer cells were perfused over E-selectin substrates using physiological flow conditions. Breast cancer cells expressing Snail or Twist adhered to E-selectin from the free fluid stream in significantly fewer numbers and demonstrated fewer E-selectin ligands in flow cytometry than the vector controls. Additionally, qPCR revealed that in comparison to vector controls breast cancer cells expressing Snail or Twist downregulated the expression of α-(1,3) fucosyltransferase and α-(1,4) fucosyltransferase, which catalyze terminal fucosylations necessary for E-selectin ligand function. Interestingly, on E-selectin coated plates breast cancer cells expressing Snail or Twist avoided prolonged exposure to E-selectin by forming mammospheres, yet vector controls did not. Collectively, these data demonstrate that the EMT regulates the expression of E-selectin ligands on breast cancer cells, and causes the cells to become sensitive to culture in an E-selectin rich environment.

#1610

Inhibiting HuR, a RNA-binding protein, for inhibition of pancreatic cancer EMT and CSCs.

Ruochen Dong, Kishore Polireddy, Ying Zhang, Qi Chen. _University of Kansas Medical Center, Kansas City, KS_.

Purpose

Epithelial- mesenchymal transition (EMT) contributes importantly to cancer cell metastasis, and formation of cancer stem cells (CSCs). An RNA-binding protein, HuR, plays an important role in many solid tumors for promoting cell proliferation, metastasis, anti-apoptosis and drug resistance. However, the role of HuR in cancer cell EMT and CSC has not been elucidated. Here we aim to investigate the role of HuR in pancreatic cancer EMT and CSC, and developing a new HuR inhibitor ST-3 as an inhibitor for pancreatic cancer EMT and CSCs.

Methods

Fluorescence Polarization assay and surface plasmon resonance assay was utilized for St-3, HuR and target mRNA binding. Scratching assay and matrigel invasion assay were used for cell migration and invasion, whereas MTT assay for viability. Tumor spheroid formation assay was used as an indication of CSCs. Western blot and RT-qPCR was used for protein and mRNA expression.

Results

Knockdown of HuR with siRNA inhibited migration of pancreatic cancer cells MIAPaCa-2, and suppressed the expression of Vimentin, Snail, and increased E-cadherin expression, showing inhibition in EMT. The suppression of Snail is due to accelerated mRNA decay. Overexpression of HuR protected mRNA of Snail from degradation, suggesting that Snail is a direct target of HuR. Knockdown of HuR also decreased pancreatic cancer spheroids formation.

St-3 inhibited the proliferation of pancreatic cancer cell lines more potently in HuR-high cells MIAPaCa-2 (IC50 ~2 µM), than in HuR-low cells BxPc-3 and PANC-1 (IC50 6-20 µM). St-3 directly bound to HuR, and inhibited binding of HuR with its target mRNAs. St-3 treatment mimicked the HuR knockdown effects in inhibiting cell migration, EMT, and spheroids formation.

Conclusion

HuR is an important regulator in pancreatic cancer EMT and CSCs. ST-3 as a novel HuR inhibitor inhibited pancreatic cancer EMT and CSCs. Further investigation is under way.

#1611

Epithelial-mesenchymal-epithelial transition induced by long term exposure to TGFB1 creates cellular heterogeneity.

Patricia Oliveira,1 Joana Carvalho,1 Sara Rocha,1 Mafalda Azevedo,1 Andre F. Vieira,1 Daniel Ferreira,1 Nuno Mendes,1 Ines Reis,1 Joao Vinagre,1 Alireza Heravi-Moussavi,2 Joana B. Nunes,1 Jorge Lima,1 Valdemar Máximo,1 Angela Burleigh,2 Calvin Roskelley,2 Joana Paredes,1 Fatima Carneiro,1 David Huntsman,2 Carla Oliveira1. 1 _IPATIMUP/I3S, Porto, Portugal;_ 2 _BCCA, Vancouver, British Columbia, Canada_.

Reversible and dynamic transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression and dissemination. Whereas EMT facilitates initial steps of tumour cell detachment, MET is likely required for colonization at distant sites. Although MET is generally perceived as mirroring EMT, we hypothesize that MET entails its own set of novel and/or differentially active molecular circuitries, generating cells with features distinct from the original epithelial state.

Using an in vitro TGFβ1-induced EMT/MET model, we demonstrated that MET generates co-existing heterogeneous cell populations (Reverted-Epithelial or RE-cells) with novel phenotypic and functional properties, such as increased self-renewal, in vivo increased tumourigenicity and distinct chemoresistance properties.

Overall, our results indicate that MET is a permissive process, driving cellular plasticity towards heterogeneity and with it, creating novel biological signatures of relevance for cancer growth.

#1612

Overexpression or inducible expression of Snail, Zeb1 or TGFβ recapitulates epithelial-mesenchymal transition (EMT) in colorectal cancer cell lines in vitro.

Shripad V. Bhagwat, Nicole E. Hamm, Yu-Chih Hsu, Yuewei Qian, Robert Wild, Jonathan A. Lee, Sheng-Bin Peng. _Eli Lilly and Company, Indianapolis, IN_.

Epithelial-mesenchymal transition (EMT) is one of the hallmarks of cancer and a critical process providing tumor cells with the ability to metastasize to distant sites. Several previous studies have revealed that EMT plays a crucial role in the progression and aggressiveness of colorectal cancer (CRC). Snail, Zeb1 and TGFβ are considered as master EMT genes and their overexpression has been linked to metastatic disease. Our study was conducted to establish an EMT platform in-house to determine the role of Snail, Zeb1 and TGF-β1 in mediating EMT in CRC cell lines in vitro and using them for studying drug sensitivity and target validation. Screening of six CRC cell lines for expression of epithelial (E) and mesenchymal (M) markers by western blot analysis suggested that DLD-1 and HT-29 cells are epithelial in nature and were used to generate stable overexpression and tet-inducible models. Overexpression of Snail or Zeb1 or TGF-β1 induced EMT in both DLD-1 and HT-29 colorectal cancer cells as indicated by downregulation of E-cadherin and upregulation of vimentin by western blot analysis and immunofluorescence staining. These cells which have undergone EMT also showed significant growth advantage in 3D growth on matrigel or soft agar. Stable 'E' and 'M' cells were also used to test the sensitivity of various inhibitors including erlotinib, salinomycin and chemotherapeutic agents such as paclitaxel, gemcitabine, cisplatin, oxaloplatin and 5-FU. Consistent with previous findings, erlotinib showed sensitivity only in 'E' cells and not 'M' cells. Chemotherapy agents have also shown higher sensitivity in 'E' cells than 'M' cells. In contrast, Salinomycin showed equipotent sensitivity in 'E' and 'M' cells. We have also generated tet-inducible models of Snail, Zeb1 and TGF-β1 and they behaved very similar to overexpression models. These inducible models were useful to determine whether any inhibitor treatment or knock down of specific gene (target) can block EMT. In our studies, testing of a Focal Adhesion Kinase (FAK) inhibitor or Dasatinib (SFK inhibitor) as examples showed blockade of EMT in these models. Overall, establishing several EMT models in-house enabled us to validate new targets involved in EMT, test new inhibitors whether it can block EMT or study drug sensitivity in 'E' vs. 'M' cells.

#1613

A novel live imaging system for inflammation-induced epithelial-mesenchymal transition in colorectal cancers.

Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. _Okayama University, Okayama, Japan_.

Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire the mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. However, as EMT is a reversible process and depends on tumor microenvironment, the precise role of EMT in cancer progression remains unclear. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancers. To evaluate the implication of EMT in the inflammation-mediated colorectal cancer progression, a live imaging system for EMT is needed on the in vitro and in vivo experiments. In this study, we generated the EMT-detectable colorectal cancer cells by introducing mesenchymal cell marker promoter-driven fluorescence protein expression vector, and investigated whether inflammation-induced EMT is detectable in vitro.

Methods: To generate the EMT-detectable colorectal cancer cells HCT116-VIM635, human colorectal cancer cell line HCT116 was stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. EMT was induced in HCT116-VIM635 cells by treatment with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or by co-culture with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis.

Results: Administration of IL-1β or TNF-α induced the TurboFP635 expression in consistent with morphological change like mesenchymal phenotype in HCT116-VIM635 cells. Removal of these inflammatory cytokines attenuated the TurboFP635 expression and morphological change in HCT116-VIM635 cells, suggesting the detection of reverse EMT process in these cells. Inflammatory cytokines also induced the migration and invasion properties and the expression of EMT-related markers in HCT116-VIM635 cells. Moreover, co-culture with RAW264.7 cells stimulated with LPS also induced the TurboFP635 expression and morphological change as well as inflammatory cytokines in HCT116-VIM635 cells.

Conclusions: Our results suggest that this unique live imaging system for EMT has a great potential to detect reversible EMT process during inflammation-induced cancer progression. This system would be useful strategy to assess the role of EMT during inflammation-mediated cancer progression in vivo.

### Metastasis-Promoting and -Suppressing Genes

#1614

Identification of FBXL4 as a bone metastasis-associated gene in prostate cancer.

Elzbieta Stankiewicz,1 Xueying Mao,1 D Chas Mangham,2 Lei Xu,1 Gabrielle Fisher,3 Bernard North,3 Henrik Moller,4 Peter Scardino,1 Jack Cuzick,3 Dan Berney,1 Yong-Jie Lu1. 1 _Barts Cancer Institute, Queen Mary University of London, London, United Kingdom;_ 2 _The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, United Kingdom;_ 3 _Cancer Research UK Centre for Epidemiology, Mathematics and Statistics, London, United Kingdom;_ 4 _King's College London, Cancer Epidemiology and Population Health, London, United Kingdom_.

Prostate cancer (PCa) is the most common cancer among men in Western developed countries. While the majority of PCa diagnosed by PSA screening are indolent, advanced and metastatic disease has a significant mortality and morbidity. Bone metastases are extremely common in PCa and identification of bone metastasis associated genes may provide insights into PCa progression and assist in finding new drug targets. However, the genetic study of bone metastases is very limited due to the difficulty of sampling. By analyzing PCa bone metastases using high density microarrays, we found a common genomic copy number loss at 6q16.1-16.2, containing the FBXL4 gene, which was confirmed in a separate and larger series of bone metastatic samples by fluorescence in situ hybridization (FISH). Loss of FBXL4 was also detected in primary PCa, although at a significantly lower frequency than in bone metastases, and it was highly associated with prognostic factors including high Gleason score, clinical stage, PSA and extent of disease, as well as poor patient survival in conservatively-managed localized PCa, suggesting that loss of FBXL4 contributes to PCa progression. We also demonstrated that FBXL4 deletion is detectable in circulating tumor cells, making it a potential disease prognostic or progression monitoring biomarker by 'liquid biopsy'. Consistent with loss of FBXL4 being associated with aggressive tumors, we demonstrated in vitro that FBXL4 plays a role in regulating the migration and invasion of PCa cells. Therefore, FBXL4 is a potential novel PCa suppressor gene, which may prevent cancer progression and bone metastasis through controlling cell invasion.

#1615

Metastasis suppression by KP54 and non-KP54 kisspeptins.

Kelsey R. Hampton, Keke M. Pounds, Andrew P. Trembath, Danny R. Welch. _University of Kansas Medical Center, Kansas City, KS_.

The KISS1 metastasis suppressor blocks metastasis by inhibiting colonization at secondary sites. In order to suppress metastasis, KISS1 must be secreted and cleaved by furin into kisspeptins. Kisspeptin-54 (KP54, K⁶⁷ to F¹²¹) binds a G-protein coupled receptor, KISS1R, and the mechanism of metastasis suppression has been assumed to be through KP54 binding with KISS1R. However, since KISS1 is cleaved extracellularly, and since none of the cells tested express KISS1R, an alternate hypothesis is required - another kisspeptin is responsible for suppressing metastasis. To test this hypothesis, we engineered all ten of the possible kisspeptins based upon predicted dibasic cleavage sites (M¹-Q¹⁴⁵; M1-R⁵⁶; M¹-R⁶⁷; M¹-R¹²⁴; R⁵⁶-R⁶⁶; R⁶⁷-F¹²¹; R⁵⁶-F¹²¹; R⁵⁶-Q¹⁴⁵; R⁶⁷-Q¹⁴⁵; R¹²⁴-Q¹⁴⁵). The (KISS1 Manufactured Peptides, or KMP) each contained the same secretion signal as nascent KISS1 and were designated KMP1 to KMP10 respectively. Each KMP was expressed individually in B16-F10 melanoma cells. Following intravenous injection, experimental lung metastases were quantified. While KP54 expression suppressed metastasis, other KMP lacking the KISS1R binding motif also suppressed metastasis as or more effectively (p<0.05). In vitro, cell lines expressing suppressor KMP inhibited motility, increased mitochondrial biomass, and elevated levels of oxidative phosphorylation (phenotypes previously correlated with KISS1 function). In particular, KMP2 (M¹ - R⁵⁶) completely suppressed metastasis. Collectively, these data demonstrate that the mechanism of action for metastasis suppression by KISS1 does not necessarily require activation of KISS1R, and also suggests the existence of additional receptors.

Support: CA134981, Natl Fndn Cancer Res, Komen SAC11037, KS Bioscience Auth, P30-CA168524, Steiner Family Fund for Metastasis Research.

#1616

Galectin-4 interferes with the integrin β4/Src/FAK cascade and attenuates metastasis in urothelial carcinoma.

Li-Fang Lin,1 Meei-Maan Wu,2 Te-Chang Lee1. 1 _Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan;_ 2 _School of Public Health, Taipei Medical University, Taipei, Taiwan_.

Galectin-4 (Gal-4), a multifunctional lectin, is presented at both intracellular and extracellular sites. Recent studies have shown that Gal-4 plays certain role in attenuating cancer progression and metastasis. We have also observed that patients with urothelial carcinoma (UC) with the lower Gal-4 expression were at higher grade and stage. In addition, the Gal-4 expression is inversely associated with lymph node invasion and prognosis. Since how Gal-4 attenuates the cancer metastasis in UC has not been well investigated, the goal of the present study is to understand the role of Gal-4 in UC progression. We ectopically expressed Gal-4 in UC cells and found that increased Gal-4 significantly inhibits cell proliferation, colony formation, migration, and invasion. We also demonstrated that Gal-4 overexpression leads to a significant decrease the adhesion of UC cells to the basement membrane substrate laminin. Mechanistically, we found that Gal-4 overexpression significantly reduces the expression of the laminin receptor integrin α6β4. Besides to downregulation of integrin β4, Gal-4 overexpression also reduces phosphorylation of Src and focal adhesion kinase (FAK) and promotes anoikis. Together, the present results revealed that Gal-4 reduces the metastasis activity of UC cells by interfering integrin β4/Src/FAK cascade, which are of essential to prevent the suspended cells from anoikis. These results suggest that Gal-4 could be considered as a pivotal factor in the migration and invasion of UC cells.

Key words: Galectin-4, integrin β4, migration, invasion, metastasis, anoikis, urothelial cancer

#1617

RUNX3 inhibits metastasis and angiogenesis in colorectal cancer.

Bo Ram Kim,1 Jung Lim Kim,2 Yoo Jin Na,1 Seong Hye Park,1 Sun Il Lee,3 Sanghee Kang,3 Sung Yup Joung,3 Suk Young Lee,2 Hong-Jun Kim,2 Dae-Hee Lee,4 Byung-Wook Min,3 Sang Cheul Oh2. 1 _Korea University, Seoul, Republic of Korea;_ 2 _Division of Oncology/Hematology, Korea University Guro Hospital, Seoul, Republic of Korea;_ 3 _Department of Surgery, Korea University Guro Hospital, Seoul, Republic of Korea;_ 4 _BK21 Plus program for Biomedical Science, Korea University, Seoul, Republic of Korea_.

Recent studies proved that an inactivation of RUNX3 (runt-related transcription factor-3) expression is highly associated with lymph node metastasis and poor prognosis in various cancer types. However, the mechanism of RUNX3-mediated suppression of tumor metastasis remains unclear. Herein, we aimed to clarify the effect of RUNX3 on metastasis and angiogenesis in colorectal cancer (CRC). First, we found that the reduction of expression of RUNX3 in CRC tissues when compared with tumor adjacent normal colon tissues, as indicated by reduced RUNX3 staining, was significantly correlated with TNM stage. Second, we demonstrated that RUNX3 overexpression inhibited CRC cell migration and invasion resulting from the elevated upregulation of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) expression. In contrast, the knockdown of RUNX3 reduced the inhibition of migration and invasion of CRC cells. Last, Vascular endothelial growth factor(VEGF) is an inducer of angiogenesis and lymphangiogenesis. We found that restoration of RUNX3 decreased vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation in CRC cells. Taken together, our data demonstrated that RUNX3 may provide insights into the development of RUNX3 for CRC metastasis diagnostics and therapeutics.

#1618

Thromboxane A2 receptor controls migration and metastasis of triple-negative breast cancer cells.

Yuanqin Zhang, Xuejing Zhang, Daotai Nie. _Simmons Cancer Institute at SIU, Springfield, IL_.

Background: Triple negative breast cancers (TNBCs) have poor prognosis and there are no effective targeted therapies available. Thromboxane A2 receptor (TBXA2R) is a G protein coupled receptor whose ligand is a prostanoid produced by cyclooxygenase and thromboxane A2 synthase. There are two isoforms of TBXA2R as results of alternative splicing. TBXA2Rα and β differ in the cytoplasmic tail distal at amino acid residue 328. High expression of TBXA2R has been linked with poor progression free survival of breast cancer cells, but it is unknown how TBXA2R contributes to the progression of breast cancers and which isoform(s) is involved.

Methods: TBXA2R expression in breast cancer was determined by RT-PCR, Western blot and immunohistochemistry using isoform specific primer sets or antibodies. Small hairpin RNAs and CRISPR-CAS9 were used to knock down or knock out TBXA2R expression. Tumor cell migration was evaluated by wound healing and Boyden chamber assays. Cell proliferation and survival was evaluated by MTS and trypan blue based cell counting. Xenograft models were used to assess breast tumor growth, progression, and metastasis.

Results: While TBXA2Rα is ubiquitously expressed in breast cancer cells, TBXA2Rβ is selectively expressed in triple negative breast cancer. Activation of TBXA2R had no discernible effects on proliferation or survival of MDA-MB-231 cells. However, TBXA2R activation led to rapid activation of RhoA and cytoskeletal reorganizations of TNBC cells. TBXA2R activation also led to rapid activation of protein kinase N1 (PKN1), a kinase frequently amplified in TNBCs. Inhibition or depletion of TBXA2R led to reduced tumor cell migration in vitro and prevented breast cancer metastasis in vivo. Further analyses found that inhibition or depletion of TBXA2R compromised the ability of TNBC cells to extravasate in an animal model.

Conclusions: Our data suggest that TBXA2R can contribute to TNBC progression and metastasis by controlling tumor cell cytoskeleton reorganization and motility.

#1619

MicroRNA-466 regulates bone metastasis by targeting RUNX2 in prostate cancer.

Shahana Majid,1 Hanna Nip,1 Altaf A. Dar,2 Sharanjot Saini,1 Varahram Shahryari,1 Soichiro Yamamura,1 Yozo Mitsui,1 Nathan Bucay,1 Guoren Deng,1 Rajvir Dahiya,1 Yuichiro Tanaka1. 1 _UCSF VA Medical Center, San Francisco, CA;_ 2 _CPMC, San Francisco, CA_.

MicroRNAs are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation. The main objective of this study was to investigate the role of microRNA-466 (miR-466) in prostate carcinogenesis (PC). The expression of miR-466 was significantly suppressed in PC tissue samples and cell lines (PC3, Du145, LNCaP, MDaPCa2b and LAPC4) when compared with normal tissues and a non-malignant cell line (RWPE1). Receiver operating curve analysis showed that miR-466 expression is an independent diagnostic predictor to discriminate between normal and tumor cases. Functionally ectopic expression of miR-466 caused a robust decrease in cell migration, invasion and proliferation of prostate cancer cells. We found that miR-466 exerted these functional effects by directly targeting the oncogenic RUNX2, a key transcription factor in PC. Over-expression of miR-466 in prostate cancer cells suppressed luciferase activity of reporter plasmids containing wild type 3'UTR sequences complementary to RUNX2, which was abolished by 3'UTR mutations. miR-466 reduced RUNX2 regulated downstream molecules such as ANGPT1, ANGPT4, MMP11, Osteopontin (SPP1), and Osteocalcin (OC). MiR-466 also inhibited genes involved in cell proliferation, migration and invasion such as pAKT, FYN and FAK. To determine the biological significance of miR-466 in prostate cancer, we performed parallel experiments in the non-malignant cell line RWPE1 followed by functional assays. Attenuation of miR-466 induced oncogenic characteristics such as increased proliferation, migration and invasion in RWPE1 cells. To examine the in vivo antitumor effects of miR-466, we performed primary and metastatic mouse model experiments. Intra-prostatic implantation of stable cells expressing miR-466 significantly attenuated primary tumor growth. Whereas, bone metastatic dissemination of PC cells was significantly suppressed by intra-cardiac inoculation of stable miR-466 expressing cells compared to control cells in nude mice. We also monitored the overall survival of mice in the metastatic model. Kaplan-Meier survival analysis indicated higher overall survival in the miR-466 group compared to controls.

This is the first study demonstrating that: i) miRNA-466 is downregulated, acts as metastasis suppressor and has diagnostic implications in PC; ii) miR-466 has biological significance in PC as its attenuation induced oncogenic characteristics in normal cells; iii) miR-466 directly targeted RUNX2 and inhibited its downstream genes and iv) stable expression of miR-466 inhibited primary tumor growth and bone metastasis in in vivo models. The use of RNAi is currently being implemented as a gene-specific approach for molecular medicine. By the same principle, it is possible that restoration of silenced miR-466 may lead to down-regulation of target oncogenes thereby contributing to novel therapeutic approaches in the treatment of prostate cancer metastasis.

#1620

Elucidating metastatic signaling networks in TNBC by investigating RKIP-regulated kinome.

Ali E. Yesilkanal,1 Casey Frankenberger,1 Daniel C. Rabe,1 Gary L. Johnson,2 Marsha Rosner1. 1 _The University of Chicago, Chicago, IL;_ 2 _The University of North Carolina, Chapel Hill, NC_.

Metastatic progression of tumors is the major cause of death in patients with triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are down-regulated by RKIP in human TNBC xenograft tumors. Our results identified novel targets of RKIP whose kinase activity is either down-regulated or up-regulated by RKIP. We used bioinformatics analysis to build RKIP-regulated signaling networks based upon RKIP-induced changes in kinase activity and related gene expression. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell migration and invasion, and can potentially identify novel anti-metastatic target combinations in TNBCs.

#1621

SRGAP2 expression levels may induce a biphasic metastatic phenotype in osteosarcoma cell lines.

Tracy Marko, Ghaidan Shamsan, David Largaespada. _University of Minnesota Twin Cities, Minneapolis, MN_.

The purpose of this study is to characterize the role of Slit-Robo GTPase-Activating Protein 2 (SRGAP2) in osteosarcoma (OS) metastasis. OS is the most common primary bone tumor and the eighth leading pediatric cancer. Despite combined therapies, treatment failure is experienced within 5 years of diagnosis by over 40% of patients, generally due to metastatic disease. Using the conditional Sleeping Beauty transposon mutagenesis system, our laboratory recently identified candidate tumor promoting genes in transgenic mice that develop OS, including the potential tumor suppressor SRGAP2 involved in regulating actin dynamics. We hypothesized that loss of SRGAP2 would increase the metastatic potential of OS cell lines in vitro and in vivo, whereas gain would decrease metastatic potential. Gene expression was amplified with a stably integrated, tetracycline-inducible over expression vector containing SRGAP2 cDNA and gene silencing was accomplished with a clustered regularly interspaced short palindromic repeat (CRISPR) targeting the gene. Studies were conducted in the well-characterized murine osteosarcoma cell lines K12 and its more aggressive derivative K7M2 and human osteosarcoma cell lines HOS and its more aggressive derivative 143B. Compared to luciferase-controls, overexpression and knockout of SRGAP2 had no effect on cell proliferation in K12, K7M2, HOS, and 143B (n=4). A reduction of soft agar colony formation was observed in knockout cell lines but not in cell lines with SRGAP2 overexpression for K12 and 143B (n=3); HOS cell lines did not form colonies in soft agar. Interestingly, overexpression of SRGAP2 decreased wound-healing closure in 143B and K12 lines, but increased closure time in HOS (n=4). Tail vein injections in NRG mice are currently being investigated. Overexpression of SRGAP2 in K12 decreased development of macrometastases in lungs, whereas knockout of SRGAP2 eliminated gross detection of lung masses (n=3). Future studies include imaging fixed cells to determine if SRGAP2 knockout and overexpression affects cell morphology and completing studies with K7M2 lines. These results suggest that SRGAP2 has a biphasic phenotype in osteosarcoma cell lines, with an optima in expression for efficient metastasis. It's dual role of deforming cell membranes independent of its ability to activate Rac GTPases and alter actin dynamics may be responsible for the observed results.

#1622

Assessment of candidate host "progression genes" in the development of advanced colorectal cancer.

Stacey A. Cohen,1 Conghui Qu,2 Tabitha A. Harrison,2 Li Hsu,2 Polly A. Newcomb,2 Stephen B. Gruber,3 Stephane Bezieau,4 Graham Casey,3 Andrew T. Chan,5 Jenny Chang-Claude,6 Martha L. Slattery,7 Emily White,2 Ashley J. Vargas,8 Rami Nassir,9 Ulrike Peters,2 William M. Grady2. 1 _University of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of South California, Los Angeles, CA;_ 4 _CHU Nantes, Nantes, France;_ 5 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 6 _German Cancer Research Center, Heidelberg, Germany;_ 7 _University of Utah Health Sciences Center, Salt Lake City, UT;_ 8 _National Cancer Institute, Bethesda, MD;_ 9 _University of California, Davis, Davis, CA_.

BACKGROUND: The genetic events affecting progression from early-stage to advanced/metastatic colorectal cancer remain poorly understood. There is preclinical evidence that germline, rather than tumor somatic, variants in key mechanistic areas (host immunity, paracrine signaling, extracellular matrix, and lymphangiogenesis) can affect the ability of an early lesion to metastasize. This led us to assess these variants in pooled data from 13 epidemiologic studies within the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO).

METHODS: Genetic variants were compared in early-stage (AJCC stage 1-2; n=4,978) and advanced (AJCC stage 3-4; n=3,263) colorectal cancer cases in GECCO. Both common (minor allele frequency [MAF] >5%) and rare (MAF <5%) single nucleotide polymorphisms (SNPs) were evaluated in 88 candidate genes (range: 5kb upstream to 500bp downstream) relevant to the mechanisms of interest. A log-additive genetic model adjusting for age, sex, study, and 3 principal components was used to assess the association for each common variant. To improve power for rare variants association, these were aggregated by gene and gene-level associations were evaluated using the Mixed effects Score Test (MiST). The Bonferroni correction was used for multiple testing, resulting in two-sided p-value significance thresholds of p<3.2 x 10-5 (=0.05/1582 SNPs) for common variants and p<5.7 x 10-4 (=0.05/88 aggregates) for rare variants.

RESULTS: 1,582 common variants were analyzed after linkage disequilibrium-based SNP pruning, with top 5 results in MMP10 (p=1.24 x 10-3), EGFR (1.41 x 10-3), MMP7 (1.45 x 10-3), ANGPT2 (2.54 x 10-3), and VEGFR2 (3.16 x 10-3). None reached a priori statistical significance. The most promising aggregated rare variant candidates were CD247 (p=3.01 x 10-3), HGF (4.44 x 10-3), CD8A (1.57 x 10-2), CXCL2 (1.86 x 10-2), FGFR2 (2.07 x 10-2), and VEGFB (3.52 x 10-2), though again none reached statistical significance.

CONCLUSIONS: Despite the preclinical evidence for these candidate "progression genes," large-scale genomic analysis did not identify statistically significant germline genetic variation in advanced vs. early-stage colorectal cancer. Follow-up evaluation will include additional survival and environmental data.

#1623

CITED2 modulates metastatic ability through effects on IKK-alpha in breast cancer cells.

Swaathi Jayaraman, Michele Doucet, Wen M. Lau, Scott L. Kominsky. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators. Previously, we identified CITED2 as a novel facilitator of bone metastasis in murine mammary cancer. Extending these initial studies to human breast cancer, CITED2 mRNA expression was significantly elevated in patient samples of metastatic breast cancer relative to primary tumors, with highest levels in bone metastasis. To explore the functional impact of CITED2 in breast cancer metastasis, we stably reduced CITED2 expression in the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 cells, which are metastatic in animal models. While CITED2 knockdown had no effect on cell proliferation, it significantly reduced tumor migration and invasion in vitro and metastasis following intra-cardiac administration in athymic nude mice in vivo. To explore the mechanism responsible for these effects, we examined the impact of CITED2 knockdown on gene expression in MDA-MB-231 cells by microarray analysis. As confirmed at the mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-κB regulator IKKα was significantly reduced along with several downstream targets of the NF-κB pathway (OPN, MMP9, uPA, SPARC, IL11 and IL-1β). In line with the reduced expression of IKKα, both canonical and non-canonical NF-κB signaling was inhibited following CITED2 knockdown. By ChIP assay, CITED2 was recruited to the promoters of IKKα, as well as the NF-κB targets OPN, MMP9, and SPARC. Finally, restoration of IKKα expression in MDA-MB-231 and MDA-MB-468 cells following CITED2 knockdown rescued their invasive ability. Collectively, these results suggest that CITED2 exerts its pro-metastatic effects in breast cancer cells via regulating IKKα.

#1624

Silencing of ERK2 reverses EMT and suppresses the CSC phenotype, inhibiting lung metastasis in triple-negative breast cancer.

Mary Kathryn Pitner,1 Hitomi Saso,1 Richard Larson,1 Rachel M. Sammons,2 Huiqin Chen,1 Caimiao Wei,1 Gaurav Chauhan,1 Kimie Kondo,1 Naoto T. Ueno,1 Kevin Dalby,2 Bisrat G. Debeb,1 Chandra Bartholomeusz1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _The University of Texas at Austin, Austin, TX_.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen receptor, progesterone receptor, and HER2 overexpression. Patients with TNBC have a generally poor prognosis due to metastasis, high rates of recurrence, and lack of FDA-approved targeted therapies. We previously showed using functional proteomics that patients with high-ERK2-expressing TNBC tumors had a higher risk of death than those with low-ERK2-expressing tumors. Moreover, ERK2 but not ERK1 plays an important role in epithelial-mesenchymal transition (EMT) and is required for acquisition of stem cell-like characteristics. Compared to other breast cancer subtypes, TNBC has a higher proportion of cancer stem cells (CSCs) and is linked to EMT, two critical features associated with breast cancer progression, metastasis, and recurrence in patients. The MAPK signaling pathway is activated in TNBC, but the roles of ERK isoforms in tumor progression and metastasis are not well defined. We hypothesized that ERK2 but not ERK1 promotes EMT, the CSC phenotype, and metastasis in TNBC.

Methods and Results: Knockdown of ERK2 in SUM149 and BT549 TNBC cells significantly inhibited anchorage-independent colony formation (p<0.0001), inhibited formation of mammospheres (p=0.003), and reduced the CSC population (CD44+/CD24-) (p=0.002) in vitro. This effect correlated with a reduction in migration (p=0.0004) and invasion (p<0.0001). SCID-beige mice injected via the tail vein with SUM149 shERK2 cells had a significantly lower lung metastatic burden than control mice (p=0.0034) or mice injected with shERK1 cells (p=0.0012), suggesting that ERK2 is a mediator of metastatic burden. To determine the mechanism by which ERK2 mediates this phenotype, we performed an Affymetrix Human Genome U133 Plus 2.0 array and compared the gene expression levels between SUM149 cells with ERK2 or ERK1 knockdown or transfection with control shRNA. Analysis of microarray data revealed that global gene expression changes associated with ERK2 knockdown predominantly altered regulation of the EMT pathway. Among the genes with ERK2-knockdown-associated expression change was EGR1, an immediate early response transcription factor whose downstream targets affect cell growth and differentiation. EGR1 is down-regulated 6-fold (p=0.00013) compared to control and shERK1 cells. Knockdown of ERK2, but not ERK1, resulted in significantly lower EGR1 at both the mRNA and protein levels, validating our microarray data.

Conclusions and Future Directions: Our findings support our hypothesis, indicating that ERK2 promotes EMT and the CSC phenotype through EGR1 and mediates metastasis in TNBC. Future studies will determine ERK activity and pathway engagement using a novel peptide sensor based on the Sox fluorophore. We will pursue a therapeutic approach using siRNA against ERK2 incorporated in a DOTAP:cholesterol liposome.

#1625

CUB domain-containing protein 1 (CDCP1) promotes metastatic phenotype in pancreatic ductal adenocarcinoma.

Sunnie Kim,1 Brooke Emerling,1 Benjamin Hopkins,1 Ryan Loughran,1 Salva Naranjos,2 Lewis Cantley1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

CUB domain-containing protein 1 (CDCP1) is a transmembrane glycoprotein that is overexpressed and highly tyrosine phosphorylated in multiple cancer types including pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease. CDCP1 has been implicated in the promotion of metastases, namely cancer cell migration, invasion, and resistance to anoikis. In addition, prior studies in other cancer cell lines have shown that hypoxia inducible factors (HIF-1α and HIF-2α), which are key regulators of metastasis, induce both expression of CDCP1 and phosphorylation at tyrosine 734. The role of CDCP1 and the mechanisms that regulate CDCP1 still need further elucidation in PDAC.

In this study, we demonstrated that CDCP1 is highly expressed and tyrosine phosphorylated in PDAC cell lines MiaPaCa2, PaTu89902, and Panc-1 cell lines compared to the non-tumorigenic pancreatic ductal cell line HPNE. shRNA knockdown of CDCP1 showed a reduction in the phosphorylation of Src and PKC-delta. To determine whether HIF regulates CDCP1 in PDAC, we used PDAC cell lines that stably express shRNAs targeting HIF-1α and HIF-2α. Hypoxia induced an increase in CDCP1 expression in a HIF-1α-dependent fashion. Additionally, CDCP1 knockdown in PaTu8902, a highly metastatic PDAC line, impaired cancer cell invasion by transwell invasion assay but did not show resistance to anoikis by soft agar assay. To validate our data in vivo, we performed intra-pancreatic injection of shCDCP1 PaTu8902 cells and scramble control transfected with GFP/luciferase into nude mice. Live imaging demonstrated that 2 weeks after initial injection resulted in decreased tumor establishment in mice injected with the shCDCP1 cell line compared to control. We propose that CDCP1 expression and tyrosine phosphorylation in PDAC is mediated by hypoxia inducible factors and that CDCP1 confers invasive properties in vitro and may play a role in initial tumor establishment in vivo. Based on these results, CDCP1 is a promising novel target for PDAC.

#1626

Krüppel-like factor 8 activates the transcription of C-X-C cytokine receptor type 4 to promote breast cancer cell invasion, transendothelial migration and metastasis.

Debarati Mukherjee,1 Heng Lu,1 Lin Yu,1 Satadru K. Lahiri,1 Tianshu Li,2 Jihe Zhao1. 1 _University of Central Florida, Orlando, FL;_ 2 _Cleveland Clinic, Cleveland, OH_.

Krüppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels, as determined by quantitative real-time PCR and immunoblotting. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Interestingly, a CXCR4-dependent activation of focal adhesion kinase (FAK), a known upregulator of KLF8, was highly induced by CXCL12 treatment in KLF8-overexpressing, but not KLF8 deficient cells. This activation of FAK in turns induced a further increase in KLF8 expression. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. These results collectively suggest a critical role for a previously unidentified feed-forward signaling wheel made of KLF8, CXCR4 and FAK in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies.

#1627

Kinase-independent function of focal adhesion kinase in lung metastasis of breast cancer.

Ming Luo,1 Max S. Wicha,1 Jun-Lin Guan2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Cincinnati College of Medicine, Cincinnati, OH_.

Focal adhesion kinase (FAK) has been shown to promote mammary tumorigenesis, lung metastasis and the maintenance of cancer stem cells in the MMTV-PyMT breast cancer model. Using mammary epithelial cell (MaEC)-specific FAK knockout and kinase-defective (KD) knockin mouse models, we recently demonstrated that loss of FAK kinase activity in MaECs specifically impaired luminal progenitor proliferation and alveologenesis, whereas FAK kinase-independent function was sufficient to support ductal invasion and basal mammary stem cell activity. To further elucidate this kinase-independent role of FAK in mammary tumorigenesis and lung metastasis, we established and analyzed three cohorts of PyMT mice expressing wild type FAK (Ctl-MT), KD-FAK (cKD-MT) and FAK-null (cKO-MT) in MaECs. This study revealed that both FAK deletion and KD knockin mutation in MaECs significantly suppressed mammary tumorigenesis and the content of stem-like tumor cells (CD24+CD29+CD61+). However, in contrast to cKO-MT mice that displayed diminished metastatic nodule formation in the lungs, loss of FAK kinase activity by KD knockin mutation did not affect lung metastatic nodule formation. Using a Boyden chamber assay, we further demonstrated that, unlike cKO-MT tumor cells that displayed diminished cell migration induced by fibronectin or TGFβ1 (as compared to Ctl-MT tumor cells), cKD-MT tumor cells exhibited strong fibronectin-, but not TGFβ1-induced migratory activity. Immunofluorescence of primary tumor sections also revealed that both Ctl-MT and cKD-MT tumors displayed strong ERK activation at tumor periphery, whereas cKO-MT tumors had weak ERK activation in this region. Lastly, when treated with or without paclitaxel in culture, we found that paclitaxel was able to induce strong expression of cleaved caspase 3 and p21 in cKO-MT but not Ctl-MT and cKD-MT tumor cells, suggesting a role for FAK kinase-independent function in the suppression of p21 expression and protection of apoptosis. Together, this study strongly implicates a role for FAK kinase-independent function in promoting lung metastasis.

#1628

MAFF, a new hypoxia target gene involving tumor invasion and metastasis.

Eui Jung Moon,1 Stephano S. Mello,1 Jen-Tsan Chi,2 Adam J. Krieg,3 Amato Giaccia1. 1 _Stanford University, Stanford, CA;_ 2 _Duke University, Durham, NC;_ 3 _University of Kansas, KS_.

Activation of hypoxia‐inducible factor (HIF) under hypoxia is significantly correlated with tumor progression and treatment resistance by regulating target genes involved in invasion, metastasis, angiogenesis, apoptosis , and metabolism. This important role of HIF and hypoxia in promoting tumor progression provides a strong rationale to develop therapies that disrupt or target critical effectors of these pathways.

V-Musculoaponeurotic Fibrosarcoma homolog F (MAFF) is a basic leucine zipper (bZIP) transcription factor that regulates antioxidative responses, hematopoiesis, and inflammation. However, its role in tumor hypoxia has not been well studied. In this study, we demonstrated that hypoxia specifically regulated MAFF in various tumor cell types and it was specifically induced by HIF-1 but not HIF-2.

To determine the role of MAFF in tumor progression, we investigated tumor cells by both gain and loss of function studies. Knocking down MAFF in metastatic cancer cell lines, MDAMB-231 and OVCAR-8, resulted in reduced tumor cell invasion and metastasis both in vitro and in vivo. In contrast, overexpressing MAFF in non-metastatic cell lines, MCF7 and A549 increased tumor cell invasion.

To elucidate the underlying mechanisms of MAFF-mediated tumor progression, we performed RNA sequencing in MDAMB-231 cells, which were exposed to either 20% O2 or 0.5% O2 with or without genetic inhibition of MAFF using siRNA. After performing a functional annotation using DAVID (Database for Annotation, Visualization, and Integrated Discovery) analysis, we observed genes involved in "blood vessel development" and "cell motility" were dysregulated in the absence of MAFF expression. In addition, genes relevant to "apoptosis", "transcription", and "inflammatory response" were altered when MAFF was inhibited. These observations suggest novel aspects of MAFF-mediated gene regulation. To further determine genes which were directly regulated by MAFF, we performed ChIP-sequencing with cells treated under normoxia or hypoxia. By combining profiles from RNA-sequencing and ChIP-sequencing, we highlighted 45 genes under normoxia and 44 genes under hypoxia. Among these, eight genes were regulated both under normoxia and hypoxia. Future studies will determine the set of genes that are responsible for tumor invasion and metastasis as well as other aspects of tumor progression.

In conclusion, our study identifies MAFF as a novel HIF-1 target gene and also demonstrates its role in cell invasion and metastasis, which has not previously been described in cancer. This work may point towards a new therapeutic strategy to target tumor progression.

#1629

Novel interaction of MUC16 with FAK activate EMT process and metastasis of pancreatic ductal adenocarcinoma.

Sakthivel Muniyan, Dhanya Haridas, Satyanarayana Rachagani, Imayavaramban Lakshmanan, Suprit Gupta, Seema Chugh, Parthasarathy Seshacharyulu, Moorthy P. Ponnusamy, Surinder K. Batra. _University of Nebraska Medical Center, Omaha, NE_.

Introduction: MUC16 is a heavily glycosylated, type I transmembrane mucin, which is over expressed in different cancers. We have previously shown that significant overexpression of MUC16 in human PDAC tissues with disease progression compared to normal pancreas. However, the functional consequences of MUC16 and their role in PDAC is poorly understood. Based on this our hypothesis is that MUC16 can drive pancreatic cancer metastasis through FAK-mediated Akt and ERK/MAPK signaling activation and altering EMT markers.

Methods: We have developed MUC16 knockdown Capan1 and Colo-357 PDAC cells to study the functional impacts. Congenic cell survival, soft-agar colony formation, and trans-well chamber assays were performed to determine the in vitro tumorigenicity. Orthotopic implantation was carried out using capan-1 and colo-357 PDAC cells to determine the oncogenic and metastatic potential of MUC16. Binding assay was performed to determine the cell adhesion property of MUC16 in colo-357 cells. The physical interaction between MUC16 and mesothelin, galectin-3 and FAK were evaluated by confocal and immunoprecipitation analysis. Immunoblot analyses were performed to determine the downstream signaling in MUC16 knockdown cells.

Results: MUC16 knockdown in capan-1 and colo-357 PDAC cell lines resulted in significantly decreased cell proliferation (P<0.05), colony formation (P<0.01), and migration (P<0.01) in vitro. Further, MUC16 knockdown capan-1 and colo-357 cells significantly decreases the tumor formation (P<0.05) and metastasis (liver P<0.05, spleen P<0.001, intestinal wall P<0.01, diaphragm P<0.01 and peritoneum P<0.001) in orthotopic xenograft mouse model. Adhesion assay displays decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 proteins. Immunoprecipitation and immunofluorescence studies confirmed that MUC16 interaction with mesothelin and galectin-3 in PDAC cells. Co-immunoprecipitation revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal markers (N-cadherin and Zeb1) and increased expression of epithelial markers (E-cadherin and CK18) in MUC16 silenced PDAC cells, correlating with the decrease in metastasis. Moreover, MUC16 knockdown show decreased FAK-mediated Akt and ERK/MAPK activation in PDAC cells.

Conclusion: Overall our study concludes that MUC16 interacts with FAK leads to the activation of EMT markers for enhancing pancreatic cancer metastasis.

#1630

Dysregulation of Hippo pathway in metastatic prostate cancer.

Jen-Ming Huang, Gina C-Y Chu, Hyung L. Kim, Haiyen E. Zhau, Leland W.K. Chung. _Cedars-Sinai Medical Center, West Hollywood, CA_.

Background: Prostate cancer (PCa) is prevalently diagnosed and more than 80% of late stage PCa patients develop bone metastases. We hypothesized that the selective lodging of the PCa cells in the bone can be contributed by increased RANK-mediated cell signaling network. Cancer cell-derived RANKL orchestrates a complex bone-remodeling program facilitating PCa bone colonization through the activation of specific transcription factors (TFs) promoting PCa-bone microenvironment interaction through increased epithelial to mesenchymal transition (EMT), cell growth, and osteomimicry. Overexpression of RNAKL in indolent LNCaP cells drives cancer bone and soft tissue metastases.

Methods and Results: Microarray gene expression was performed to compare RANKL- and Neo-vector- transfected LNCaP cells, LNCaPRANKL and LNCaPneo , respectively. qRT-PCR and western blot were performed to confirm the expression of RANKL-targeted genes in these cells. We found the new extracellular diffusible signal mediated by RANKL, regulating Hippo pathway in the aggressive PCa cells. Recombinant RANKL-treated indolent PCa cells, LNCaP and LAPC4, were shown to gain increased aggressiveness. The RANKL-treated indolent PCa cells exhibited down-regulation of Hippo pathway core kinases (Mst1/2 and Lats1/2) but up-regulation of YAP/TAZ expressions. By deleting downstream effectors of Hippo pathway, YAP/TAZ, interrupted the aggressive PCa cells to migrate, proliferate and invade and reversed their EMT phenotype. Inhibitions of YAP/TAZ in aggressive PCa cells via genetic knockdown approach resulted in E-cadherin increase and Vimentin decrease. With the identification of crosstalk between Hippo and TGF-β or Wnt pathways, this opened up a new network and targeting opportunity for PCa bone metastasis. The metastatic PCa cells with YAP/TAZ deletions were illustrated for sensitization to other therapeutic agents such as TGF-β inhibitors. These results prompted us to study the mechanism, validate the functional significance, and devise potential targeting strategies of Hippo pathway in PCa bone metastasis.

Conclusions: Our results showed that dysregulated Hippo pathway in the aggressive PCa cells. With deletions of YAP/TAZ, PCa metastatic abilities were inhibited. Our findings might explain why PCa cells prefer bone as primary metastatic sites. In addition, targeting YAP/TAZ could be a potential therapy for PCa bone metastasis.

#1631

FGF2 activation of FGFR1 in head and neck squamous cell carcinoma is associated with more invasive disease and can be attenuated by FGFR inhibition.

Isabel A. English, Jacqueline Martinez, Edward El Rassi, Mark Schmidt, Ellen Langer, Sophia Bornstein, John Gleysteen, Melissa Wong, Brian Druker, Elie Traer. _Oregon Health & Science University, Portland, OR_.

Introduction. Head and neck squamous cell carcinomas (HNSCCs) account for nearly 600,000 deaths worldwide annually and have limited treatment options. Approximately 20% of HNSCCs harbor amplifications of fibroblast growth factor receptor 1 (FGFR1) on chromosome 8p, however FGFR1 amplification by itself does not predict clinical response to FGFR inhibitors. We hypothesized that FGF2, or basic FGF, ligand expression is a better marker of FGFR activation and predictor of response to FGFR inhibitors.

Results. A tissue micro array (TMA) of HNSCC patient biopsies was stained and quantitated for FGF2 expression by Aperio ImageScope software. FGF2 was significantly increased in recurrent tissue samples (p=0.04). We examined a number of immortalized HNSCC cell lines and found that overexpression of both FGF2 and FGFR1 predicted response to the selective FGFR inhibitor PD173074. FGFR inhibition did not cause apoptosis, but rather induced a G0/G1 arrest and growth inhibition. FGFR inhibition also induced a change in cell morphology, with a significant increase in cell size and adherence. The expression of epithelial-to‐mesenchymal transition (EMT) proteins was examined and FGF2-FGFR1 activation was associated with a more mesenchymal phenotype. Accordingly, FGFR inhibition reversed invasiveness as measured using the Incucyte WoundMaker scratch assay, suggesting that HNSCCs with FGF2-FGFR1 activation have more metastatic potential. Invasiveness of these cells in vivo was confirmed using orthotopic injection into the buccal pad of NSG mice. Once primary tumors reached 0.8 cm in size, mice were sacrificed and buccal mucosa, lung, liver, and neck tissue were examined post-mortem. All of the injected animals developed local invasion, and distant metastases in the lungs. 5/7 mice also had metastases in the liver and this model is being used to test the ability of FGFR inhibition to prevent metastasis.

The mechanism of autocrine FGF2-FGFR1 activation was further explored and FGF2 was found to be secreted in association with extracellular vesicles (ECVs). Interestingly, inhibition of FGFR reduced secretion of ECVs and FGF2, providing a novel approach to target autocrine and paracrine FGFR1 activation within the tumor. We further tested a number of small molecule inhibitors in combination with PD173074 to look for synergistic combinations of kinase inhibitors and found significant synergy between EGFR and FGFR inhibitors suggesting this combination may be most effective in patients with HNSCC.

Conclusions. Increased FGF2 in HNSCC patient samples is correlated with recurrent disease. FGF2-FGFR1 activation increases invasiveness through activation of EMT genes both in vitro, and in an orthotopic model. Inhibition of FGF2-FGFR1 reversed the invasive phenotype in vitro and may be an effective therapeutic strategy to reduce metastases in HNSCC patients.

#1632

RelB-mediated EMT activation promotes bone metastasis of prostate cancer.

Yong Xu,1 Yanyan Zhang,1 Fang Fang,2 Chi Wang,2 Xinyu Su,1 William H. St. Clair,2 Daret K. St. Clair2. 1 _Nanjing Medical University, 68 Changle Rd., Nanjing, China;_ 2 _University of Kentucky, Lexington, KY_.

Previously, we demonstrated that the RelB-based NF-kB alternative pathway is essential for tumorigenesis of prostate cancer. Here, we show that RelB contributes to bone metastasis of prostate cancer through an EMT-activating process. High constitutive nuclear levels of RelB were observed in human prostate cancer tissues with high Gleason scores. Up-regulation of RelB in prostate cancer cells led to cell invasion and migration, which was mediated by activation of Snail I and Twist I. ChIP revealed that RelB binds to NF-κB enhancer elements located at the 5'-flanking regions of both Snail I and Twist I genes, which interact with Sp1-based promoters for transcriptional activation of the genes. In addition, the high levels of RelB also increased cell mineralization that was correlated to up-regulation of the bone formation relating protein, S100A4. The RelB-mediated up-regulation of Snail I- and Twist I consequently activate PI3K-AKT pathway through inhibition of PTEN, which was induced by TNFα, but suppressed by an anticancer agent, parthenolide. These results suggest that the activation of NF-kB alternative pathway enhances metastasis of prostate cancer cells by activation of the EMT process.

#1633

Vasodilator-stimulated phosphoprotein promotes β1-integrin-FAK-YAP1/TAZ signaling axis that is required for liver metastasis of GI cancer.

Xiaoyu Xiang,1 Jinhua Piao,2 Selvaraj Muthusamy,2 Lei Wang,1 Yibin Deng,1 Shengbing Huang,3 Raul Urrutia,3 Jinping Lai,2 Ningling Kang1. 1 _The University of Minnesota, Austin, MN;_ 2 _St Louis University, St Louis, MO;_ 3 _Mayo Clinic, Rochester, MN_.

Introduction: Liver metastasis of pancreatic or colorectal tumors is a major cause of cancer-related death worldwide. When cancer cells reach the liver parenchyma and make a direct contact with the surrounding extracellular matrix (ECM), β1-integrin-FAK signaling is activated allowing the cells attach, migration, and proliferate. Thus, the β1-integrin-FAK signaling axis is key for cancer cells to colonize the liver. However, mechanisms governing β1-integrin activation and how this pathway promotes liver metastasis remain incompletely understood. Aim: We sought to test the hypothesis that vasodilator-stimulated phosphoprotein (VASP), an actin binding protein, regulates the β1-integrin-FAK signaling axis, thereby, promoting liver metastasis of GI cancer. Methods: We employed lentiviruses encoding different VASP shRNAs to knockdown (KD) VASP in human GI cancer cells. We used a Matrigel-based 3-dimensional (3D) cell culture model and intrasplenic tumor injection mouse model to assess the role of VASP for cancer cells in vitro and in vivo. Immunofluorescence (IF) was used to study localization of VASP and active β1-integrin (using Huts-4 antibody), and Western blot analysis (WB) was used to quantitate protein levels of active β1-integrin and YAP1/TAZ. Additionally, we performed VASP immunohistochemistry (IHC) to analyze VASP immunoreactivity in 116 colorectal or pancreatic adenocarcinomas of patients. Results: In the Matrigel-based 3D-culture model, a single cancer cell gave arise to a 3D-cancer spheroid after being cultured for 6-8 days. Using KM12L4, HCT116, or HT29 colorectal cancer cells, and L3.6 or MIA PaCa-1 pancreatic cancer cells, we found that VASP KD reduced the sizes of the 3D-cancer spheroids grown on Matrigel (P<0.05 by t-test for each cell line). IF and WB show that VASP KD reduced levels of Huts-4 (active β1-integrin) in KM12L4, HCT116 colorectal cancer cells and in L3.6, MIA PaCa-1 pancreatic cancer cells. VASP KD also reduced the protein levels of P-FAK and YAP1/TAZ in the 4 cell lines tested. Additionally, YAP1/TAZ protein levels were reduced by PF-228, a FAK inhibitor that blocks the β1-integrin-FAK signaling. In the intrasplenic tumor implanation mouse model, VASP KD suppressed liver metastasis of GI cancer as well as reduced Huts-4 and YAP1 signals of their liver metastases in mice (P<0.05 by t-test). IHC of patient's samples reveals that VASP protein levels were upregulated in cancer cells in 81% of colorectal tumors (51/63) and pancreatic tumors (43/53), with higher VASP levels being associated with liver metastasis of colorectal cancer (P<0.01 by Fisher exact test). Conclusions: VASP promotes ECM-mediated β1-integrin-FAK-YAP1/TAZ signaling axis of GI cancer. We propose that VASP and the β1-integrin-FAK-YAP1/TAZ signaling axis represent potential new therapeutic targets for liver metastasis of pancreatic or colorectal cancer.

#1634

The RALA pathway drives invasion and metastasis of soft tissue sarcomas by regulating MMP14 localization and activity.

Steven T. Sizemore, Fen Xia, Arnab Chakravarti. _The Ohio State University, Columbus, OH_.

Soft tissue sarcomas (STS) are a heterogeneous group of tumors originating from mesodermal tissues such as skeletal muscle, adipose, and fibrous tissue. The propensity of STS to metastasize, particularly to the lung which occurs in approximately 20% of all cases, is the major source of mortality associated with this disease. Currently, themolecular mechanisms that drive STS metastasis are poorly understood greatly limiting our ability to determine which STS will metastasize and impeding the development of targeted therapies for the treatment of STS metastases. In this study we utilized analysis of publicly available gene expression data to identify loss of PPP2R1B expression as a strong prognosticator of STS metastasis. In vitro analyses using SW872 liposarcoma (LPS) and HT1080 fibrosarcoma cell lines determined that PPP2R1B plays an essential role in the dephosphorylation and inactivation of RALA, a major downstream effector of the Ras pathway. RALA was found to be essential for invasion of STS cells in modified Boyden chamber assays as knockdown of RALA by either siRNA or shRNA significantly decreased invasion through matrigel. Analysis of publicly available gene expression data revealed that RALA expression correlated with more aggressive LPS subtypes and was prognostic of LPS metastasis. Furthermore, we identify the transmembrane matrix metalloprotease MMP14 (MT1-MMP) as a key downstream effector of RALA mediated invasion in STS. Stable knockdown of RALA in SW872 and HT1080 cells resulted in significant mislocalization of MMP14 and reduced MMP14 enzymatic activity. MMP14 expression was also significantly prognostic of metastasis in a publically available LPS cohort. Finally, we demonstrate that a three gene signature comprised of PPP2R1B, RALA, and MMP14 can retrospectively stratify LPS patients into groups of low, moderate, or high risk for metastatic recurrence. To the best of our knowledge this is the first demonstration of a vital role for PPP2R1A and RALA in STS invasion and metastasis and these data demonstrate a novel role for RALA in the localization and activity of MMP14. In conclusion, we have identified the PPP2R1B-RALA-MMP14 axis as driver of STS metastasis with potential clinical value as both a prognostic marker and therapeutic target.

#1635

The histone demethylase KDM3A in Ewing Sarcoma metastasis.

Marybeth Sechler, Janet Parrish, Paul Jedlicka. _University of Colorado Denver Anschutz Medical Campus, Aurora, CO_.

This study aims to explore the roles and mechanisms of new candidates for therapeutic targeting in Ewing Sarcoma. Ewing Sarcoma is an aggressive pediatric malignancy of the bone and soft tissue, driven by EWS/Ets oncogenic fusions and characterized by high propensity for metastasis and poor clinical outcomes. We have identified the histone demethylase KDM3A as a novel tumor and metastasis promoter downstream of EWS/Fli1, the most common driver in this disease. Moreover, we have identified the cell surface protein, Melanoma Cell Adhesion Molecule (MCAM), as a potential important mediator of KDM3A action. Our data show that both KDM3A and MCAM depletion can inhibit growth and metastatic phenotypes. Additionally we explore the regulation of MCAM, showing that KDM3A may upregulate MCAM expression both through a direct as well as an indirect mechanism via the Ets1 transcription factor. Since both KDM3A and MCAM are pharmacologically targetable, understanding of this pathway could provide at least two new therapeutic targets in Ewing Sarcoma, and, importantly alternatives to drugging of EWS/Ets fusions, which to date has proven very difficult.

#1636

The role of the ROR receptors in ovarian cancer progression and chemoresistance.

Claire E. Henry,1 Estelle Llamosas,1 Neville F. Hacker,2 Viola Heinzelmann-Schwarz,3 Caroline E. Ford1. 1 _Lowy Cancer Research Centre, University of New South Wales, UNSW, Australia;_ 2 _Gynaecological Cancer Centre, Royal Hospital for Women, UNSW, Australia;_ 3 _Department of Gynecology and Gynecological Oncology, Hospital for Women, University Hospital Basel, University of Basel, Basel, Switzerland_.

BACKGROUND: Despite recent advances in our understanding of the underlying genetic profile of distinct subtypes of ovarian cancer, overall survival remains poor. This is partly due to the absence of available targeted therapies, widespread metastatic disease at diagnosis, and the development of resistance to chemotherapy in the majority of patients. Patients with a more mesenchymal gene signature have a shorter overall survival, and are more likely to recur and develop chemoresistance. These patients also have upregulated Wnt signalling, a key developmental pathway associated with metastasis, epithelial to mesenchymal transition (EMT) and chemoresistance.

AIM: To investigate the role of the novel Wnt receptors ROR1 and ROR2 in ovarian cancer progression and chemoresistance, to determine their feasibility as therapeutic targets.

METHOD: Expression of ROR1 and ROR2 was measured in patient tumour (primary and metastatic) samples and ascites fluid taken at multiple time points during disease progression. In addition, the A2780 and cisplatin resistant A2780-cis cell lines were used as a model of ovarian cancer chemoresistance. Profiling of EMT and Wnt genes was undertaken alongside cell proliferation, adhesion, migration, invasion and viability assays after modulation of ROR1 and ROR2.

RESULTS: Expression of ROR1 and ROR2 is increased in ovarian cancer patients compared to benign fallopian tube and ovarian surface epithelium. High ROR1 expression is associated with type I epithelial ovarian cancer, and high ROR2 expression with aggressive type II epithelial ovarian cancer. Expression of each receptor fluctuates in paired metastatic samples compared to primary tumours, depending on the site of metastasis and recurrence time. Both receptors are increased in a model of cisplatin resistant ovarian cancer, and when silenced, cell migration and invasion is significantly inhibited. Silencing ROR1 and ROR2 also sensitizes resistant cells to cisplatin, and is associated with a shift away from a mesenchymal phenotype.

CONCLUSION: Upregulation of ROR1 and ROR2 drives migration, invasion and chemoresistance through regulating EMT via β-catenin independent Wnt signalling. As cell surface regulators of β-catenin independent Wnt signalling, the ROR receptors represent potential therapeutic targets for chemoresistant cancers.

#1637

22(S)-Hydroxycholesterol, an antagonist of LXR, inhibits breast cancer cell migration.

Sewon Hwang, Tae Young Na, Hyelin Na, Minho Lee, Mi-Ock Lee. _Seoul National University, Seoul, Republic of Korea_.

The Liver X Receptor (LXR) is a well-established ligand-regulated transcription factor involved in cholesterol homeostasis, lipid metabolism, and inflammation. Recent studies have reported a strong antiproliferative effect of LXRs in multiple types of cancer including prostate, colon, lung, and breast cancer. However, little is known about its association with cancer metastasis. Therefore we aimed to investigate the potential of LXR ligands in the context of breast cancer cell migration, and its underlying molecular mechanism. We first performed 2D-migration assay in 4T1 breast cancer cells. Treatment of an LXR antagonist significantly reduced the rate of 4T1 cell migration. However, LXR agonists such as T0901317, GW3965 did not have much effect. Next, we investigated the molecular mechanism of cell migration. Since the degree of invasion and metastasis of breast carcinoma is well correlated with elevated expression of metastasis-associated protein 1 (MTA1), we examined whether MTA1 expression could be regulated by LXR ligands. Interestingly, when LXR agonists were treated, increased protein expression of MTA1 and its downstream target protein HIF-1α was observed in MCF7, MDA-MB-231, and 4T1-Luc breast cancer cell lines. Transient expression of LXRα demonstrated the same results as well. Furthermore, the treat of 22(S)-HC down-regulated MTA1 and HIF-1α protein expressions. Together, our findings indicate that LXR influences migratory pattern of breast cancer cells, which may affect metastatic property of breast cancer cells via LXR-dependent regulation of MTA1 and HIF-1α expression.

#1638

Hypoxia-induced WSB1 promotes the metastatic potential of osteosarcoma cells.

Yijie Wang, Ji Cao, Rong Dong, Guanyu Lin, Zibo Chen, Ling Ding, Meidan Ying, Qiaojun He, Bo Yang. _Zhejiang Univ., Hangzhou, China_.

Intratumoral hypoxia occurs in many solid tumors, where it is associated with the development of metastatic character. However, the connections between these phenomena are not fully understood. In this study, we define an integrative role for the E3 ubiquitin ligase subunit WSB1. In primary osteosarcomas, increased levels of WSB1 correlated with pulmonary metastatic potential. RNAi-mediated attenuation of WSB1 or disruption of its E3 ligase activity potently suppressed tumor metastasis. Quantitative proteomic and functional analyses revealed that WSB1 ubiquitylates the Rho-binding protein RhoGDI2 and promotes its proteasomal degradation, thereby activating Rac1 to stimulate tumor cell motility and invasion. Our findings show how WSB1 regulates key steps of the metastatic cascade in hypoxia-driven osteosarcoma, and highlight a candidate therapeutic target to potentially improve the survival of patients with metastatic disease.

#1639

CDCP1 cleavage is necessary for homodimerization-induced migration of triple-negative breast cancer.

Heather Wright, Janahan Arulmoli, Maryam Motazedi, Luke Nelson, Olga Razorenova. _UC Irvine, Irvine, CA_.

Triple negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks the estrogen, progesterone, and HER2 receptors and is resistant to targeted and hormone therapies. TNBCs express high levels of the transmembrane glycoprotein, CUB-domain containing protein 1 (CDCP1), which has been correlated with the aggressiveness and poor prognosis of multiple carcinomas. Full-length CDCP1 (flCDCP1) can be proteolytically cleaved, resulting in a cleaved membrane-bound isoform (cCDCP1). CDCP1 is phosphorylated by Src family kinases in its full-length and cleaved states, which is important for its pro-metastatic signaling. We observed that cCDCP1, compared to flCDCP1, induced a dramatic increase in phosphorylation of the migration-associated proteins: PKCδ, ERK1/2, and p38 MAPK in HEK 293T. In addition, only cCDCP1 induced migration of HEK 293T cells and rescued migration of the TNBC cell line, MDA-MB-231, expressing shRNA against CDCP1. Importantly, we found that only cCDCP1 is capable of dimerization, which can be blocked by expression of the extracellular portion of cCDCP1 (ECC), indicating that dimerization occurs through CDCP1's ectodomain. We found that ECC inhibited phosphorylation of PKCδ and migration of TNBC cells in 2D culture. Furthermore, ECC induced morphological changes, inhibited proliferation, and stimulated apoptosis of TNBC cells in 3D culture, indicating that the cCDCP1 dimer is an important contributor to TNBC aggressiveness. These studies have important implications for development of a therapeutic to block CDCP1 activity and TNBC metastasis.

#1640

SORBS1-related multiprotein complex regulates metastasis of cancer.

Woo-Cheol Cho, Ja-Lok Ku. _Department of Biomedical Sciences, Seoul National University, Seoul, Republic of Korea_.

Purpose: CAP (Cbl-associated protein), also known as Sorbin and SH3 domain-containing protein 1 is encoded by the SORBS1 gene. These proteins contain a conserved sorbin homology (SOHO) domain and three SH3 domains. CAP is an adaptor protein that has been associated with the actin cytoskeleton, signaling through receptor tyrosine kinases and cell adhesion. Although, CAP is proven to have many known functions, this protein is only reported in specific field of signaling pathway. Cell adhesion and actin dynamics which have been supposed to participate in CAP are crucial factors of cancer metastasis. The role of CAP has been described in the literature, although their specific roles remain to be clarified in cancer.

Methods and materials: Colorectal cancer cell lines were transduced by lentivirus containing shRNA plasmid (shSORBS1 plasmid). To elucidate function of SORBS1 as regulator of cancer metastasis, western blotting, cell migration assay and immunoprecipitation assays were performed.

Conclusion: According to the several reports of SORBS1 function, phosphorylation of ERK and mTOR was identified. Although ERK phosphorylation were not changed in normal condition, there were different aspects between control group and SORBS1 knockdown group with insulin treatment. Moreover, mTOR phosphorylation of SORBS1 knockdown group was less in the control group than in the normal condition.

Immunoprecipitation of SORBS1 was performed to search for the binding compounds. We chose the AHNAK protein as the convincing candidate protein related with SORBS1. Several studies reported that AHNAK protein is related to proliferation and EMT by binding of specific signaling molecules. Indeed, there were remarkable differences of invasiveness ability between control group and knockdown group.

#1641

Complex behavior of ALDH1A1 and IGFBP1 in liver metastasis of colorectal cancer.

Jin Cheon Kim,1 Ye Jin Ha,1 Ka Hee Tak,1 Seon Ae Roh,1 Chan Wook Kim,1 Tae Won Kim,1 Dong-Hyung Cho,2 Seon-Kyu Kim,3 Seon-Young Kim,3 Yong Sung Kim3. 1 _Univ. of Ulsan Asan Medical Ctr., Seoul, Republic of Korea;_ 2 _Kyung Hee University, Seoul, Republic of Korea;_ 3 _KRIBB, Seoul, Republic of Korea_.

The present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) using our dataset (GSE50760) previously established by RNA sequencing and to verify their biological behavior. Candidate genes were assessed for their role in tumor proliferation, invasion, and the expression of epithelial-mesenchymal transition/CRC stem cell (EMT/CSC)-related molecules. The role of the identified genes in CLM was verified in mice after intrasplenic transplantation of CRC cells. Of nine candidate genes, ALDH1A1 and IGFBP1 were selected and showed few post-translational changes. The upregulation of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ≤ 0.001-0.003) and suppressed invasiveness by ≥3 fold over control cells (p < 0.001) in the SW480 cell line, whereas it had a mild effect on reducing SW620 cell proliferation. EMT/CSC-related molecules showed different patterns of expression in CLM tissues and treated CRC cells. The cadherin switch, namely, N-cadherin upregulation with E-cadherin downregulation, was not observed in CLM tissues and treated CRC cells. Persistent overexpression of β-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells may have contributed to CLM development in mice implanted with IGFBP1-overexpressing cells (CLM occurrences: SW480/IGFBP1 transfected mice vs. SW480/vector and /ALDH1A1 transfected mice, 4/8 vs. 0/10, p = 0.023). ALDH1A1 and IGFBP1 were differentially overexpressed in CLM and may play a dual role, functioning as tumor suppressors and metastasis promoting genes in CRC. Unidentified behaviors of these genes may facilitate the reassessment of their potential value as CLM modulators and as therapeutic targets.

#1642

Calponin-3, acidic (CNN3) mediates EMT-related genes in human colorectal cancer metastasis.

Soon-Chan Kim, Chang-Won Hong, Sang-Geun Jang, Yeh-Ah Kim, Seung-Yong Jung, Jea-Gab Park, Ja-Lok Ku. _Seoul National University, Medical campus, Seoul, Republic of Korea_.

(a) An introductory sentence indicating the purposes of the study Distant metastasis is the major cause of cancer-related death in colorectal cancer (CRC) patients. Although the Calponin family has emerged as a distinguishing feature in epithelial-to-mesenchymal transition (EMT) in human cancer, the role of CNN3 member in the metastatic CRC has not been investigated.

(b) A brief description of pertinent experimental procedures. Three pairs of primary CRC and corresponding matched metastasis cell lines were analyzed for gene expression by microarray and western blotting. CNN3 was transduced to CRC cell line with lower CNN3 expression level (SW-480), and CNN3 with higher CNN3 expression CRC cell lines (SW-620, HCT-116) was suppressed by lentiviral-induced shRNA. Functional analysis of CNN3 overexpression and suppression was investigated in these CRC cell lines for proliferation and invasion. Expression of several potential CNN3 target genes (ERK1/2) and EMT markers (E-cadherin and vimentin) in primary CRC and matching metastasis CRC cell lines was validated.

(c) A summary of the new, unpublished data. Global CNN3 expression levels were significantly higher in metastasis cell lines compared with the matched primary cell lines in both mRNA (FC=2.3, p=0.017) and protein levels. CRC cell lines with suppressed CNN3 showed decreased cell proliferation and reduced invasive behavior in CRC cell lines. Suppression of CNN3 in CRC cell lines with higher CNN3 expression resulted in increased E-cadherin and decreased vimentin. pERK1/2 were decreased in accordance with suppression of CNN3 in both SW-620 and HCT-116. Overexpression of CNN3 in CRC cell lines caused reduced E-cadherin in SW-480 and HT-29.

(d) A statement of the conclusions CNN3 is involved in mediating EMT and metastatic behavior in the colon. Its expression is accordantly related to EMT markers, and CNN3 may serve as a possible diagnostic marker and therapeutic target for patients with CRC.

#1643

p53 family and oncogenic signaling in cancer metastasis.

Zhi-Xiong Xiao, Tao Lv, Yang Wan, Johann Bergholz, Yujun Zhang. _Sichuan University, Chengdu, China_.

The tumor suppressor protein p53 is mutated in over 50% of human cancers. Multiple lines of evidence have shown that mutant p53 not only loses its ability to inhibit tumorigenesis, but also gains pro-metastatic functions. However, the molecular mechanisms underlying these effects are still not totally understood. In this study, we demonstrate that the p53 hotspot point mutant, p53(R273H), potently induces cell spreading, migration, and invasion. At the molecular level, p53(R273H) regulates the DLX2-NRP2 pathway in promoting cancer cell migration in vitro and metastasis in vivo. Interestingly, p53(R273H) strongly inhibits tumor cell growth in vitro and in xenograft nude mice model. In addition, we show that p53-related p63 inhibits Erk2 signaling in regulating cell migration/invasion.

### Radiation Science

#1644

NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC.

Peter V. Deraska, Colin O'Leary, Jean-Bernard Lazaro, Christopher J. Sweeney, Alan D. D'Andrea, David Kozono. _Dana-Farber Cancer Institute, Boston, MA_.

Background: Despite optimal multimodality therapy, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. A common limitation is our inability to provide sufficient radiotherapy (RT) to eradicate tumor due to risk of toxicities in surrounding tissues. There is thus an unmet need for radiosensitizers that can improve the therapeutic index. Dimethylaminoparthenolide (DMAPT), an orally bioavailable small molecule NF-κB inhibitor, inhibits repair of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) and increases control of subcutaneous mouse NSCLC xenografts. While our prior work focused on inhibition of homologous recombination as a mechanism for radiosensitization, we sought to characterize the effect of DMAPT on a second major DSB repair pathway, non-homologous end-joining (NHEJ).

Methods: NHEJ was assessed in NSCLC lines using the pEJ reporter and flow cytometry. The NF-κB super repressor (IκBαS32A,S36A) was used as a control. Immunofluorescence and Western blotting were used to assess NHEJ biomarkers including 53BP1, DNA-PKS2056, Ku70/80 and XRCC4. Cell fractionation was performed to assess Ku chromatin binding. Quantitative RT-PCR was performed to assess gene transcription. Ku complexes were purified to identify binding partners.

Results: NSCLC cells treated with IR-sensitizing doses of DMAPT (5-15 µM) or the NF-κB super repressor showed significant decreases in NHEJ. DMAPT increased the persistence of 53BP1 foci, indicating a failure to complete NHEJ. Regardless of exogenous DNA damage, there was reduced Ku70 chromatin binding following DMAPT treatment. DMAPT-treated cells produced fewer distinct IR-induced DNA-PKS2056 foci. Further, there was decreased IR-induced XRCC4 chromatin recruitment, suggesting that repair was impaired prior to ligation. There was no change in Ku70/80 transcription following DMAPT and/or IR. However, Western blotting of purified Ku complexes showed that DMAPT treatment decreased Ku association with RNA binding partners including RPL19. We also observed decreased recruitment of DNA-PKcs to the Ku complex, suggesting decreased Ku affinity for DSBs.

Conclusions: These results indicate that DMAPT radiosensitizes NSCLC by perturbing the binding affinity of Ku to RNA, DNA and its complex binding partners, thus blocking NHEJ. Further mechanistic investigation and analysis of NHEJ biomarkers in vivo is needed to identify the precise mechanism.

#1645

Tumor-associated macrophages enhance DNA damage repair and improve survival of murine breast cancers after irradiation.

Luis A. Soto, Marjan Rafat, Marta Vilalta Colomer, Amato Giaccia, Edward Graves. _Stanford University, Stanford, CA_.

Tumor-associated macrophages (TAMs) are known to promote physiological processes that drive tumor progression and survival, including angiogenesis, immunosuppression, and invasion. While most studies on TAMs have focused on the molecular mechanisms by which TAMs drive these processes, less focus has been given to direct effects that TAMs may exert on tumor cells. It is known that monocytes are recruited to tumor sites after radiotherapy and support tumor regrowth. In this study we sought to determine if TAMs are able to exert a direct, protective effect on tumor cells against radiotherapy in vitro. We used murine 4T1 cells, a well-characterized animal model for breast cancer, and murine RAW264.7 macrophages to carry out co-culture experiments. Co-culture of 4T1 cells with RAW264.7 macrophages resulted in increased 4T1 cell survival rates post-irradiation compared to 4T1 cells cultured alone. We further determined that this effect is not contact-dependent but mediated through macrophage secreted factors. Next, we tested whether co-culturing RAW264.7 macrophages with 4T1 cells induced macrophage polarization to an M2/TAM phenotype. Using antibodies against M2 markers and FACS, we found that co-culture of RAW macrophages with 4T1 cells polarizes these macrophages toward an M2 phenotype. We hypothesized that the increased 4T1 cell survival rates were mediated by an enhanced DNA damage repair mechanism induced by TAMs. We tested this via a combination of immunofluorescence against phosphorylated histone H2AX and comet assays. We show that co-culture of 4T1 cells with TAMs after irradiation results in lower 4T1 cell levels of DNA damage and a faster decrease in DNA damage over time compared to 4T1 cells alone. Future work will focus on characterizing the molecular mechanism through which TAMs induce this enhanced DNA damage repair response in 4T1 cells after irradiation and we will test whether such response occurs in vivo after radiotherapy. In conclusion, we show that TAMs confer resistance against ionizing radiation to tumor cells in vitro and that this resistance is driven by an enhanced DNA damage repair mechanism in tumor cells induced by TAM secreted factors.

#1646

A glioblastoma methylation assay (GaMA) developedfrom genomic analysis of glioma spheroid cultures predicts response toradiation therapy in patients with glioblastoma.

Qianghu Wang,1 Ravesanker Ezhilarasan,1 Eskil Eskilsson,1 Joy Gumin,1 Jie Yang,1 Mona Jaffari,1 Ming Tang,1 Kenneth D. Aldape,2 Frederick F. Lang,1 Roel G.W. Verhaak,1 Erik P. Sulman1. 1 _The University of Texas, MD Anderson Cancer Center, Houston, TX;_ 2 _Princess Margaret Hospital, Toronto, Ontario, Canada_.

Radiation therapy (RT) remains one of the most effective treatments for patients with GBM and has been repeatedly demonstrated to improve survival; yet response to RT is variable. We explored the relationship between methylation status and radiation response to develop a predictor of RT response using the epigenetic data of glioma sphere-forming cells (GSCs). The DNA methylomes of 42 GSCs were profiled using Illumina Infinium 450K methylation bead arrays. 15 GSCs were irradiated with 2-, 4-, and 6-Gy RT and response determined using clonogenic assays. We discovered 168 CpG probes capable of distinguishing sensitive from resistant GSCs. To validate, we analyzed 362 TCGA GBM samples, 272 that received standard 60Gy RT and 90 treated with low or no RT. Using the glioblastoma methylation assay (GaMA) signature, we classified the samples as either RT sensitive or resistant. Survival was significantly different between the predicted sensitive vs resistant patients for those treated with standard RT (median 21.0m vs 14.7m, p<0.005). GaMA did not predict a survival difference among patients receiving no/low-dose RT, suggesting a predictive, but not prognostic, role for the signature. Using the ENCODE ChIP-Seq Significance Tool, we observed that the transcription factor EZH2 was significantly associated with the radiation resistant promoters in the GaMA signature. Among the hypermethylated genes with EZH2 binding sites, the NR2F2 promoter had the greatest number of hypermethylated CpG sites correlated to RT resistance. NR2F2 has previously been identified as negatively associated with activation of the wnt/β-catenin, a pathway associated with RT resistance of mammary progenitor cells. Expression of WNT1 in TCGA GBM cohort was negatively associated with NR2F2 expression. Our GSC RT response-based methylome analysis corroborates this association and provides a rationale for the methylation signature as a predictive biomarker of radiation response.

#1647

Radiation response genome-wide analysis using paired pre and post-radiation FFPE human breast tumor samples.

Sharareh Siamakpour-Reihani, Wei Chen, David Corcoran, Chen-Ting Lee, Ying Zhou, Mark W. Dewhirst, Jen-Tsan A. Chi, Janet K. Horton. _Duke University, Durham, NC_.

Background: Breast cancer is the most common female malignancy in the US, and radiotherapy is a routine part of multi-disciplinary breast cancer care. Radiation treatment (RT) tailored to target radiation response pathways could maximize treatment benefit in radiation resistant tumors, while minimizing unnecessary toxicity and cost in more radiation responsive breast tumor subtypes. Here we report on genome-wide analysis, gene expression profiles and biological pathways associated with RT response in breast cancer patients. Methods: Between 2010-2013, a total of 32 breast cancer patients were enrolled at Duke University Medical Center on an IRB approved Phase I clinical trial testing preoperative radiotherapy. Paired pre and post-RT FFPE biopsy samples were available for N=26. Genome wide-analysis was performed using two distinct techniques: i) Microarray analysis using the Affymetrix GeneChip ® Human Transcriptome Array 2.0 (HTA 2.0); ii) RNA-Seq using TruSeq ®RNA Access Library kit (Illumina). All libraries were sequenced on the Illumina HiSeq 2000 generating 50 bp single reads at a sequencing depth of 40 million reads. The RNA-Seq data was processed using the TrimGalore toolkit. For the paired pre and post-RT differential gene expression analysis we used the linear model. Genes that had an FDR ≤ 5% in the RNA-Seq experiment were compared with all microarray probesets that had an FDR ≤ 5%. If at least one probeset that belonged to a gene in the RNA-Seq list was significant and showed the same direction of change between pre and post-RT samples, they were identified as consistent. Gene Set Enrichment Analysis (GSEA) and Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to identify significant pathways and gene ontologies that were differentially regulated between the conditions. Results: 2648 genes showed the same significant results in response to RT in microarray and RNA-Seq including: i) a total of 1401 genes that had significantly higher expression levels, and ii) a total of 1247 genes that had significantly lower expression levels following RT. Principal Components Analysis (PCA) demonstrated distinct separation of pre and post-RT gene expression signatures. Differentially expressed genes were involved in major biological pathways such as cell cycle checkpoint, DNA replication and DNA repair pathways. A subset of these genes also demonstrated differential expression in radiation responsive vs. non-responsive breast cancer cell lines. Conclusion: New laboratory techniques have become available which provide the opportunity to use FFPE samples for microarray and RNA-seq analysis. Using these new techniques and our rare pre and post-RT FFPE breast tumor samples, we have been able to demonstrate distinct differences in gene expression profiles and modulation of biological pathways in response to RT in human breast tumors.

#1648

Ionizing radiation switches the function of tumor-derived exosomes from messengers of tolerance to inducers of antitumor immunity.

Julie M. Diamond,1 Jessica R. Chapman,1 Beatrix Ueberheide,1 Silvia C. Formenti,2 Sandra Demaria2. 1 _NYU School of Medicine, New York, NY;_ 2 _Weill Cornell Medicine, New York, NY_.

Tumor-derived exosomes (TEX) are constantly shed by cancer cells and have been shown to carry tumor-derived antigens to dendritic cells (DCs). However, presentation of tumor antigens derived from TEX in the absence of activation signals for DCs leads to immune tolerance. Our lab has demonstrated that ionizing radiotherapy (RT) can convert the irradiated tumor into an in situ vaccine, leading to anti-tumor immune responses. We hypothesized that TEX derived from irradiated cancer cells play a role in RT-induced anti-tumor immunity by delivering tumor antigens together with pro-inflammatory signals that activate DCs and prime tumor-specific effector T cells. To begin to address this hypothesis we first studied how RT modifies TEX molecular composition and their ability to induce DC activation in vitro.

Mouse carcinoma cells TSA cultured in exosome-free media were treated in vitro with sham RT (control TEX), or 3 fractions of 8Gy (RT-TEX). TEX were isolated from supernatants 48 hr later using differential ultracentrifugation and purified with a sucrose gradient. Electron microscopy was used to confirm TEX size and morphology. Mass spectrometry (LFQ-MS) followed by MS/MS analyses was used to characterize protein composition. miRNA were analyzed by nanoString nCounter Mouse miRNA expression assay kit using a panel of 578 mouse miRNAs. Normalized results were analyzed with MultiExperiment Viewer. Mouse bone marrow-derived dendritic cells (BMDC) were cultured with TEX for 48 hours. Cells were analyzed by flow cytometry for expression of activation markers.

Significant changes in microRNA and protein compositions were seen in RT-TEX compared to control TEX. Specifically, RT-TEX showed increase in proteins involved in the Antigen Processing and Presentation pathway. Additionally, 17 unique proteins were present only in RT-TEX and included proteins involved in T cell development, MHC class I peptide processing, and pro-inflammatory lipid signaling.

Analysis of DCs cultured with RT-TEX, but not control TEX, revealed an increase in cell surface expression of CD80 (1428 MFI vs. 963 MFI) and CD86 (3487 MFI vs. 2578 MFI). Interestingly, culturing BMDC with RT-TEX also resulted in an increase in DCs expressing CD103 and CD8a (2% vs 1.2% of CD11c+ cells), suggesting that RT-TEX may influence differentiation of BMDC towards this subset, which is critical for cross-presentation of tumor antigens to T cells.

Obtained data support the hypothesis that RT-TEX may switch tolerogenic DCs into activated DCs by providing activation signals together with tumor-derived antigens. Further in vivo experiments are ongoing to determine the ability of RT-TEX to stimulate anti-tumor immune responses.

#1649

Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation.

Katharina Röck,1 Alexandra Schütze,1 Janna Morawitz,1 Verena Jendrossek,2 Klaus Pantel,3 Jens W. Fischer1. 1 _Institut für Pharmakologie und Klinische Pharmakologie, Düsseldorf, Germany;_ 2 _Institute of Cell Biology (Cancer Research), Essen, Germany;_ 3 _Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany_.

Despite surgery or chemo radiotherapy the five-year survival rate of esophageal carcinoma (eSCC) patients remains around 15% indicating the need for new therapy regimes. Recently the role of the tumor microenvironment has been recognized as an important determinant of radiation responses. Here we investigate the influence of stromal fibroblasts (hF) on radiation induced tumor cell death.

To evaluate functional consequences of ionizing radiation hF and eSCC cells were either cultured separately and irradiated (2Gy) or co-cultured after irradiation to investigate radiation dependent tumor-stroma interactions. Subsequently cells were monitored by time-lapse microscopy and cell death was assessed visually as well as by PARP cleavage, live cell staining with Hoechst33342/propidium iodide and quantification of sub-G1 cell cycle. The rate of cell death was barely altered in monocultures. Of note significantly increased cell death of eSCC was observed in co-culture with irradiated hF compared to co-cultures with mock-irradiated fibroblasts (16.10±4.25 fold of control). Microscopic evaluation of co-cultures revealed that hF and cancer cells are connected via tunnelling nanotubes (TNT). Furthermore, TNT formation was found to be dependent on the extracellular matrix component hyaluronan. eSCC cell death was prevented by pharmacological inhibition of TNT formation by cytochalasin B as well as inhibition of hyaluronan by 4-MU. As TNTs are known to mediate intercellular protein or mitochondrial transfer we investigated which stromal proteins are shuttled into eSCC cells by trans-SILAC experiments. For this purpose the proteome of fibroblasts was labeled with heavy amino acids. After co-culturing hF and eSCC were separated by FACS-sorting and labeled stromal proteins were identified in the cancer cells by mass spectrometry. Four consistently shuttled proteins were identified in the cancer cells, which were exclusively transferred after irradiation of both cell lines. Knock-down experiments of these proteins in hF were performed and SHANK1, which is not expressed in eSCC cells, was found to be responsible for the hF induced cell death of eSCC cells in response to radiation.

In conclusion, by usage of a novel trans-SILAC approach, we provide evidence that in response to ionizing radiation stromal hF transfer SHANK1 to eSCC cells, thereby inducing cellular death. SHANK1 might therefore serve as potential new pharmacological target to sensitize cancer cells to radiation therapy.

#1650

Whole-genome RNAi screen identified BCAS1 as a novel modifier of non-small cell lung carcinoma radioresistance.

Colin O'Leary, Peter Deraska, Alan D'Andrea, David Kozono. _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Despite optimal chemotherapy, radiotherapy and/or surgery, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. Therefore, there is a need for agents, such as radiosensitizers, that can improve the efficacy of current therapies. Using a whole-genome shRNA screen, we sought to identify and characterize novel gene products that may be involved in NSCLC radioresistance. We hypothesize that these proteins may represent novel targets for NSCLC radiosensitization.

Materials and Methods: A whole-genome pooled retroviral shRNA screen was performed using the Hannon-Elledge library of 74,705 distinct shRNAs directed against over 18,000 genes, to identify gene knockdowns that showed cytotoxicity only in cells treated with fractionated radiation given daily Monday-Friday. To confirm targets identified in the whole-genome screen, we treated A549 and NCI-H460 NSCLC cells with siRNAs targeted against the genes of interest and assessed cell viability following irradiation using the CellTiter-Glo assay and clonogenic survival assays.

Results: We identified six novel genes (BCAS1, c7orf24, CDC45L, KIAA0101, TCN1 and TNC) whose knockdown sensitized NSCLC cells to radiation. Of the six genes, siRNA knockdown of BCAS1 showed the most consistent increases in sensitivity to ionizing radiation. Analysis of cell viability following radiation revealed that BCAS1 knockdown resulted in a 50% additional decrease in cell viability in A549 and NCI-H460 cells following 8 and 4 Gy of ionizing radiation, respectively. We confirmed these results by clonogenic survival assays. To assess for tumor-specific expression in NSCLC, we compared expression data between matched normal tissue and NSCLC biopsy samples and found that BCAS1 is substantially more highly expressed in tumor tissues. Examination of affinity capture-mass spectroscopy data suggested that BCAS1 interacts with RUVBL2, a RuvB-like AAA ATPase, which has known functions in DNA damage and repair.

Conclusions: BCAS1 emerged from a whole-genome RNAi screen as a potential target for improving the efficacy of radiotherapy in NSCLC. We demonstrated that BCAS1 is overexpressed in tumor tissues compared to normal lung, and that its knockdown consistently sensitizes NSCLC cell lines to radiation, validating the screen hit. Ongoing work suggests that BCAS1 may modulate DNA damage repair. Further studies to understand the mechanism by which BCAS1 is involved in radioresistance are necessary.

#1651

Overexpression of hepatoma upregulated protein (HURP) promotes radioresistance in prostate cancer cells.

Mohamed Hassan,1 Abdelouahid El Khattouti,1 Tangeng Ma,1 Ingrid Espinoza,1 W. Andrew Day,2 Srinivasan Vijayakumar,1 Christian R. Gomez1. 1 _University of Mississippi Medical Center Cancer Institute, Jackson, MS;_ 2 _Belhaven College, Jackson, MS_.

Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents major cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by significant resistance in advanced tumor. So far, the mechanisms governing the acquired resistance to radiotherapy have not been discussed in detail. Hepatoma Up-Regulated Protein (HURP) is a cell cycle-regulated and microtubule-associated protein. It functions as a Ran GTPase effector and is involved in the stabilization of the mitotic kinetochore fibers. The expression of this protein is elevated in different tumor types and associated with tumor progression and aggressiveness. We recently reported on the value of HURP as an independent prognostic factor for high-risk PCa. The aim of this study was to address the role of HURP in the modulation of PCa resistance to γ- irradiation. Cell lines (LNCaP, PC3, and DU145) with regulated expression of HURP protein using a Lent viral Tet-On 3G Inducible Expression System were irradiated using a Cs-137 γ-source. Overexpression of HURP suppressed γ- irradiation- induced apoptosis. This suppression was associated with the expression of E2F1, p53, p21 together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Also, inhibition of γ- irradiation- induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) was noted. The observed resistance was consequence of HURP's ability to trigger the ubiquitination of p53 and ATM that, in turn, resulted in the inhibition of downstream pathways, which are essential for the modulation of γ- irradiation-induced apoptosis. Our data provide evidence for the involvement of HURP in the modulation of PCa cell resistance to γ-irradiation via a mechanism mediated by ubiquitination of ATM and p53. Understanding of the biological mechanisms underlying HURP-mediated resistance of PCa to available therapies may help to improve the treatment strategy and emphasize HURP as therapeutic target for PCa treatment.

Funding sources: Department of Defense Grant PC094680 and Prostate Cancer Foundation Creativity Award.

#1652

P16INK4a over-expression sensitizes HPV(-) HNSCC to radiation through down-regulation of USP7.

David Molkentine, Li Wang, Kathleen Bridges, Kathy Mason, Raymond Meyn, Heath D. Skinner. _UT MD Anderson Cancer Center, Houston, TX_.

Background: Head and neck squamous cell carcinoma (HNSCC) tumorigenesis induced by human papillomavirus (HPV) shows a greater clinical response to radiation therapy when compared to HPV negative patients. The underlying mechanism for this more favorable outcome is unknown, but the presumption is that DNA repair response must play a large role. P16INK4a is a known surrogate marker for HPV positivity; however the relationship between radiation sensitivity and P16INK4a has not been completely elucidated in this context. Ubiquitin specific protease 7 (USP7), also known as HAUSP, is a deubiquitylating enzyme that cleaves ubiquitin from substrates to stabilize target proteins. USP7 overexpression is a predictor of poor prognosis in lung carcinomas and correlates with disease progression and lower survival in gliomas. More recent studies have shown USP7 has a role in the stability of DNA damage response proteins such as RNF168, BRCA1 and Chk1; and USP7 knockdown has been shown to radio-sensitize breast cancer cells to radiation. We have investigated the possible role of USP7 in the radio-response of HNSCC cells in the context of HPV status.

Methods: Four HPV(+) and four HPV(-) HNSCC were chosen for this study. The radio-response was determined by clonogenic survival assay. P16INK4a overexpression was generated using lentivirus. Western blot analysis was used for visualizing protein expression. The Cancer Genome Atlas was interrogated to examine the relationship between USP7 and clinical outcome.

Results: Our data suggest P16INK4a negatively regulates USP7 and leads to the more radiosensitive phenotype associated with HPV(+)HNSCC. Radio-resistant HPV(-) HNSCC cell lines show higher levels of USP7 than HPV(+) lines and over-expression of P16INK4a in HPV(-) HN5 cells leads to down-regulation of USP7 and radio-sensitization. Conversely, P16INK4a siRNA knockdown in HPV(+) UMSCC-47 shows an increase of USP7 protein expression and radio-resistance. P22077, an inhibitor of USP7 activity, was also shown to radiosensitize a HPV(-) cell line. This shows proof of principle that inhibiting USP7 can be a viable approach to sensitizing HPV(-) HNSCC to radiation. USP7 overexpression was also associated with poorer overall survival in HNSCC.

Conclusion: These results suggest that USP7 may be a marker of clinical radioresistance in HNSCC and that its inhibition has the potential to improve the treatment outcome of patients with HPV(-) HNSCC when combined with radiotherapy.

#1653

A novel role for Wnt/β-catenin signaling in mediating resistance of colorectal cancer to chemoradiotherapy.

Georg Emons,1 Melanie Spitzner,1 Sebastian Reineke,1 Frank Kramer,2 Margret Rave-Fraenk,3 Jochen Gaedcke,1 Michael Ghadimi,1 Thomas Ried,4 Marian Grade1. 1 _University Medical Center, Department of General, Visceral and Pediatric Surgery, Goettingen, Germany;_ 2 _University Medical Center, Department of Medical Statistics, Goettingen, Germany;_ 3 _University Medical Center, Department of Radiotherapy and Radiooncology, Goettingen, Germany;_ 4 _Genetics Branch, NCI, NIH, Bethesda, MD_.

Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. Previously, we demonstrated that the Wnt transcription factor TCF7L2 (also known as TCF4) was overexpressed in primary rectal cancers that were resistant to chemoradiotherapy (CRT), and that TCF7L2 functionally mediates resistance of CRC cells to clinically relevant doses of ionizing radiation (IR).

Methods: Using siRNAs we silenced CTNNB1 (β-catenin), another key-component of canonical Wnt-signaling, in colorectal cancer cell lines LS1034, SW480, and SW837. To asses influence on CRT, cells were exposed to 0, 1, 2, 4, 6 and 8 Gy of X-rays and 5-FU. Wnt- signaling was stimulated in retinal pigment epithelial cells (RPE) either by adding Wnt-3A, or overexpressing non degradable β-catenin (S33Y-mutated) and analyzed changes in CRT. Finally we repetitively irradiated SW1463 (68Gy) to establish an isogenic radio-resistant cell line and examined changes in protein expression.

Results: Silencing of CTNNB1 resulted in (chemo-) radiation-sensitization of all three CRC-cell lines. To further investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, non-tumorigenic RPE cells were stimulated with Wnt-3A, which significantly increased resistance to CRT. This effect could be recapitulated by overexpression of β-catenin (S33Y-mutated), resulting in a significantly increased resistance to CRT. The effect could be rescued by siRNA mediated knockdown of β-catenin. Consistent with these findings, we observed higher expression levels of active (unphosphorylated) β-catenin as well as increased TCF reporter activity in SW1463 cells that were rendered radiation-resistant due to repeated IR treatment.

Conclusion: Together, these findings strongly support the interpretation that Wnt/β-catenin signaling plays a central role in mediating resistance of CRC cells to CRT. Hence, pathway inhibition may represent a promising strategy to increase therapeutic responsiveness to CRT, which represents the standard treatment for locally advanced rectal cancers. This would have considerable clinical implications.

#1654

p21 WAF1/Cip1 -mediated radiosensitization of bladder cancer cells by mTOR inhibitor, RAD001 disrupts the balance between autophagy and apoptosis.

Jose J. Mansure, Shraddha Solanki, Wael Alamjed, Roland Nassim, Fabio Cury, Simone Chevalier, Wassim Kassouf. _McGill Urologic Oncology Research, Montréal, Quebec, Canada_.

Purpose: Radical cystectomy (RC), remains the "standard" treatment for Muscle Invasive Bladder Cancer (MIBC), but is associated with major impact on quality of life. Radiation therapy (RT) is an alternate treatment as it preserves the bladder. However, lack of local control remains problematic. Therefore, the use of a radio-sensitizer could overcome those challenges and limit side effects. In this sense, inhibiting the mTOR signaling pathway in combination with radiation is a promising therapeutic strategy. As the cyclin dependent kinase inhibitor p21 WAF1/Cip1 has been found to be associated with DNA damage repair and autophagy, which are two major pathways associated with radiation resistance, we sought to investigate the role of p21 in response to combination therapy in bladder cancer.

Methods: Expression of p21 was characterized in different bladder cancer cell lines via Western Blotting. Correlation of p21 expression and response to mTOR inhibitor was done using proliferation assay. Knockdown of p21 was carried out via RNAi silencing. To assess the effect of the p21 expression in response to combination therapy, Clonogenic assay was done. Western blot was utilized to assess the effect of mTOR inhibition in p21 expression. In vivo, athymic mice were subcutaneously injected with bladder cancer cell lines. Treatment consisted of either placebo or combined RAD001 (12 hours prior to radiation) and fractionated IR. Tumor volume was measured at endpoint. Immunohistochemistry was used to detect the levels of p21 in paraffin-embedded mouse xenograft bladder cancer tissues treated with placebo, IR, RAD001 and in combination. Autophagy was investigate by LC3 cytoplasmic localization by immunofluoresecence and LC3 I/II levels were detected by Western Blot . Clevage of caspase-3 was used to determine apoptosis by Western Blot.

Results: There was a significant correlation between the levels of p21 and sensitivity to mTOR inhibitor (P value <005). RAD001 induced a dose response decreased in the level of p21 expression. Addition of the mTOR inhibitor, along with knocking down p21 significantly inhibited colony formation compared to untreated p21-scramble cells. A significant effect in tumor volume was observed in vivo in the group treated with the combination arm of compared to all other groups (P value <005). Levels of p21 increased following the treatment with IR alone and in combination with RAD001, whereas a slight decrease was observed following the treatment with RAD001 alone. Addition of RAD001 induces autophagy, and point to the functional role of p21 in inhibiting autophagy as its knock-down was successful at increasing the levels of LC3 I/II even without RAD001 addition.

Conclusion: Our in vitro and in vivo results support a functional role for p21 in mediating the response to combination therapy, thus promoting p21 as a potential player that can be modulated in the treatment of bladder cancer.

#1655

Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas.

Joel S. Greenberger, Lora Rigatti, Aranee Sivanathan, Shaonan Cao, Xichen Zhang, Donna Shields, Darcy Franicola, Michael Epperly. _University of Pittsburgh Cancer Institute, Pittsburgh, PA_.

Introduction: Transgenic FVB/n mice with the HPV E7 oncogene under the control of the CK14 promoter were bred with Fancd2+/- (129/Sv background) mice to obtain K14E7 Fancd2-/- mice Park, et al. Cancer Research, 70(23): 9959, 2010), in which delivery of 4-nitroquinoline N-oxide in drinking water produces oral and esophageal adenomas. The hematopoietic system, of these mice was studied, using long term bone marrow cultures (LTBMCs) from Fancd2-/- (SV129), K14E7 transgenic (FVB/n), and K14E7 Fancd2-/- mice.

Methods: LTBMCs were established by flushing bone marrow into T-25 flasks from which bone marrow stromal cell lines and IL3-dependent hematopoietic cell lines were developed. The cell lines were analyzed using irradiation survival curves, Western analysis for protein expression. K14E7 Fancd2-/- IL3 dependent clonal cell lines of the IL3-dependent cell lines were established by flow cytometry. Clonal lines were expanded and tested for tumorigenicity by injecting 1 X 106 cells of each of four clones into flanks of K14E7 Fancd2+/+ mice.

Results: K14E7 Fancd2-/- LTBMCs were similar to those from Sv/129 Fancd2-/-: 1) hematopoiesis was shortened when compared to K14E7 Fancd2+/+ or Fancd2+/+ LTBMCs, respectively. Stromal cell line irradiation survival curves with K14E7 Fancd2+/+ and Fancd2-/- cell lines showed (Do = 1.48 ± 0.05 and 1.52 ± 0.15 Gy) they were radiosensitive compared to those from K14E7 Fancd2+/+ or Sv/129 Fancd2+/+ cells (Do = 2.33 ± 0.11 and 2.23 ± 0.01 Gy, p = 0.0043 and 0.0368, respectively). Fancd2-/- stromal cells were more radiosensitive than Fancd2+/+ cells (decreased shoulder on the survival curve of n = 1.5 ± 0.5 compared to 4.7 ± 0.3 (p = 0.0126). K14E7Fancd2+/+ cells had an n of 1.9 ± 0.3 compared to 3.5 ± 0.1 (p = 0.0309) for Sv/129 cells. In contrast, hematopoietic IL-3 dependent K14E7Fancd2+/+ and Sv/129 Fancd2+/+ cell lines were similar (Do = 2.15 ± 0.4 and 1.92 ± 0.06, p = 0.6469, respectively). Confirming prior data, (Berhane et. al, Rad Res 182: 35, 2014) Sv/129 Fancd2-/- IL-3 dependent cell lines were radioresistant compared to Fancd2+/+ cell lines (Do = 2.12 ± 0.12 and 1.92 ± 0.06, respectively, p = 0.0236). In contrast, K14E7 Fancd2-/- cell lines were radiosensitive compared to K14E7 Fancd2+/+ cell lines (1.44 ± 0.13 and 2.15 ± 0.28, respectively, p = 0.0498). Hematopoietic and marrow stromal cell lines from K14E7Fancd2-/- marrow expressed cytokeratin 14 and E7 oncogene by Western analysis. K14E7 Fancd2-/- (but no other genotype derived) IL-3 dependent cell lines transformed in vitro to dense tumor cells lines. Cloned sublines were injected into K14E7 Fancd2+/- mice producing plasmacytomas.

Conclusions: Expression of the E7 oncogene in the K14/E7 Fancd2-/- mouse marrow derived IL-3 dependent cell lines leads to radiosensitivity and tumorgenicity.

Supported by NIAID/NIH U19-A 1068021 and the Fanconi Anemia Research Foundation.

#1656

Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation.

Chen-Ting Lee, Yingchun Zhou, Kingshuk R. Choudhury, Sharareh Siamakpour Reihani, Mark W. Dewhirst, Janet K. Horton. _Duke University, Durham, NC_.

Breast cancer is the most common malignancy diagnosed among women worldwide and represents a heterogeneous group of subtypes. Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, little is known about radiation response among these subtypes. Recent clinical data suggests that distinct patterns of response to systemic therapy may exist in association with each phenotype. Therefore, understanding the intrinsic variation of radiation response in breast cancer subtypes is important in prescribing individualized radiotherapy that maximizes the therapeutic ratio.

Our previous studies have identified marked changes in gene expression in breast patient tumors and breast tumor cell lines in response to radiation. Among candidate genes, FAS was consistently and significantly induced after radiation in luminal breast tumors and cell lines. Modulation of FAS in basal breast cancer cell lines favorably impacted radiation response. In this study, we further investigated the role of FAS modulation and radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7, and to a less extent HCC1954 tumor growth in a dose-dependent manner, while SUM159 tumors were resistant to radiation. The estimated TCD50 value was 19.30 Gy for MCF7 and 44.88 Gy for SUM159. FAS was induced in MCF7 tumors after radiation but showed no change in SUM159 tumors. To investigate whether FAS modulation can affect radiation response, we silenced FAS in MCF7 tumors and activated FAS in SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation delayed SUM159 and HCC1954 tumor growth (p=NS).

In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS concurrent with radiation slows tumor growth in the radiation resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation.

#1657

Mapping mechanisms of radiotherapy resistance in prostate cancer.

Serena T. Bruens, Chiara Milanese, Nicole Verkaik, Pier Mastroberardino, Akos Gyenis, Jiang Chang, Kasper Derks, Erik Wiemer, Dik van Gent, Wytske van Weerden, Guido Jenster, Jan Hoeijmakers, Joris Pothof. _Erasmus MC, Rotterdam, Netherlands_.

Cancer cells that acquire radio-resistance are major problem for treatment outcome. We aimed at identifying mechanisms and networks that control ionizing radiation (IR)-resistance in prostate cancer cells. To this end, we applied to cancer cell lines an identical radiotherapy regiment as performed in the clinic, i.e. a total dose of 78 Gy in steps of 2 Gy per day for 5 consecutive days followed by 2 days rest. We observed that radio-resistance occurs early during treatment as seen in colony survival assays in which 100% of cells that can form colonies are resistant up to 8 Gy. Remarkably, a two-week period of culturing without radiotherapy completely sensitized these resistant cancer cell lines, indicating that resistance is not acquired by permanent mutations or chromosomal rearrangements. Currently, we are characterizing these extreme resistant cancer cell cultures by comparing these to their sensitive parental cancer cell lines and the two-week re-sensitized cancer cell lines. We observe changes in DNA repair pathway utilization, DNA damage checkpoint activation and energy metabolism parameters, but not in PI3-kinase signalling and cancer stem cell markers. We also performed genome-wide gene expression analysis by next generation sequencing and isolated gene signatures that correlate with resistance. Based on our results we applied inhibitors to sensitize these IR-resistant cells. Notably, application of single inhibitors did not lead to sensitivity. Combinations of small molecule inhibitors however, were able to sensitize these cancer cell lines, indicating that IR-resistance is a multi-factorial process. In summary, mapping gene networks and mechanisms will uncover new pathways associated with IR-resistance.

#1658

M3814, a novel investigational DNA-PK inhibitor: enhancing the effect of fractionated radiotherapy leading to complete regression of tumors in mice.

Frank T. Zenke,1 Astrid Zimmermann,1 Christian Sirrenberg,1 Heike Dahmen,1 Lubo Vassilev,2 Ulrich Pehl,1 Thomas Fuchss,1 Andree Blaukat1. 1 _Merck, Darmstadt, Germany;_ 2 _EMD Serono, Billerica, MA_.

Physical or chemical agents that damage DNA such as ionizing radiation are among the most widely used classes of cancer therapeutics today. Double strand breaks (DSB) generated in DNA by radiation induce multitude of cellular responses, including DNA repair, cell cycle arrest or cell death if the damage is left unrepaired. A complex set of molecular events are responsible for DNA repair via two major mechanisms - homologous recombination (HR) or non-homologous end joining (NHEJ). DNA-PKcs with its regulatory protein subunits, Ku70 and Ku80, is an integral component of NHEJ and considered an attractive intervention point to inhibit DNA repair.

We have developed an orally bioavailable, highly potent, and selective inhibitor of DNA-PK, M3814, for cancer therapy in combination with DNA damaging modalities such as radiation, and radio-chemotherapy. Here, we present the preclinical characterization of M3814 using biochemical, cellular and human tumor xenograft models. M3814 sensitized multiple tumor cell lines to radiation therapy in vitro and strongly enhanced the antitumor activity of ionizing radiation in vivo with complete tumor regression applying a clinically relevant fractionated radiation regimen. These effects are due to inhibition of DNA-PK protein kinase activity as demonstrated by the levels of DNA-PK autophosphorylation in human tumor cell lines, and xenograft tumors M3814 is currently investigated in PhI clinical trials.

#1659

Radioprotection of IDH1-mutated solid tumor, but not leukemia cells by the IDH1-mutant inhibitor AGI5198.

Remco J. Molenaar,1 Johanna W. Wilmink,1 Jaroslaw P. Maciejewski,2 William P. Leenders,3 Fonnet E. Bleeker,1 Cornelis J. Van Noorden1. 1 _Academic Medical Center, Amsterdam, Netherlands;_ 2 _Cleveland Clinic, Cleveland, OH;_ 3 _Radboud UMC, Nijmegen, Netherlands_.

Introduction: Activating hotspot mutations in isocitrate dehydrogenase 1 and 2 occur in glioma, acute myeloid leukemia (AML), chondrosarcoma and cholangiocarcinoma and confer an improved prognosis in glioma and cholangiocarcinoma patients, but not AML, compared with IDH1 wild-type counterparts. IDH1/2 mutants catalyze α-ketoglutarate to D-2-hydroxyglutarate at the expense of NADPH, an important cellular antioxidant. IDH1/2 mutations are early events in oncogenesis, rendering them attractive therapeutic targets because the majority of the cancer cells carry IDH1 mutations and this limits the chance of therapy resistance. Inhibitors of mutant IDH1/2 were recently developed and are currently in clinical trials.

Results: In solid tumor cells, IDH1/2 had a large contribution to the total cellular NADPH production, whereas this contribution was small in AML cells. IDH1/2-mutated solid tumor and AML cells exhibited decreased IDH-mediated production of NADPH than control IDH1/2 wild-type cells. After exposure of IDH1-mutated solid tumor cells to irradiation, there were higher levels of reactive oxygen species, DNA double strand breaks and cell death compared with IDH1 wild-type solid tumor cells, whereas we observed no differences between IDH1/2-mutated and IDH1/2 wild-type AML cells in these experiments. Furthermore, IDH1/2-mutated AML cells were not sensitized to cisplatin, 5-azacytidine or daunorubicin compared with IDH1/2 wild-type AML cells.

The antioxidant scavenger N-acetyl-cysteine or the IDH1-mutant inhibitor AGI5198 protected IDH1-mutated, but not IDH1 wild-type, solid tumor cells against irradiation. Exposure to D2HG reduced IDH-mediated NADPH production in solid tumor cells, AML cells and human glioma samples and increased irradiation sensitivity in solid tumor cells, but not AML cells. Irradiation sensitivity of IDH1-mutated solid tumor cells was independent of the well-described global DNA hypermethylation phenotype in IDH1-mutated cancers.

Discussion: Our results offer an explanation for the relatively longer survival of patients with IDH1-mutated glioma and cholangiocarcinoma and the unchanged survival of patients with IDH1-mutated AML. A plausible mechanism is the large contribution of IDH1/2 to antioxidant NADPH in solid tumor, but not AML cells. These data imply that concomitant administration of IDH1-mutant inhibitors and irradiation in patients with IDH1-mutated solid tumors may limit therapy efficacy in this setting. In contrast, IDH1-mutant inhibitors can probably be used safely as adjuvants to conventional antineoplastic regimens in patients with IDH1/2-mutated AML. This is essential for the rational design of further clinical trials with IDH1-mutant inhibitors. We warn against multiagent clinical trials with concomitant use of IDH1-mutant inhibitors and irradiation in patients with IDH1-mutated solid tumors.

#1660

JP4-039/F14 treatment of E13 pregnant mice 24 hours after total body irradiation (TBI) improves survival, growth and development of fetal mice.

Michael W. Epperly, Lora Rigatti, Tracy Dixon, Song Li, Joel S. Greenberger. _University of Pittsburgh Cancer Institute, Pittsburgh, PA_.

Purpose: A practical radiation mitigator must be safe in pregnant females. Irradiation during pregnancy can induce fetal death, stunt growth, and/or lead to teratogenic and carcinogenic effects dependent on stage of gestation. We evaluated the mitochondrial targeted GS-nitroxide mitigator JP4-039 for effects on total body irradiated (TBI) pregnant mice.

Methods: Timed pregnant C57Bl/6NHsd mice at E13 were irradiated to 3 Gy, subgroups were injected IV 24 hr later (E14) with JP4-039 in F14 emulsion (4 ug JP4-039 in 100 ul of F14). Mice were followed for number of pups born, weight of pups at day 5 after birth, and for number of survivors at time of weaning. In other studies of irradiation effects, new- born pups, on the day of delivery, were euthanized, fixed, sectioned and examined microscopically.

Results: Nonirradiated mice showed 97 ± 2% of newborn surviving until weaning. Pups born to 3 Gy irradiated E13 pregnant mice had decreased survival (8.3 ± 8.7%) (p < 0.0001). Control nonirradiated pregnant mice receiving JP4-039/F14 or F14 alone showed no effect on pup survival (85 ± 10% at weaning) (p = 0.1453). The survival of pups from 3 Gy irradiated E13 pregnant mice that received JP4-039/F14 24 hr after irradiation was significantly decreased (45 ± 16.4% compared to nonirradiated controls (p = 0.0230). All newborn pups were weighed at 5 days after birth: those surviving 3 Gy in utero had significantly decreased weight of 1.64 ± 0.04 g compared to 2.73 ± 0.08 g for nonirradiated controls (p < 0.0001). In contrast, while the 3 Gy TBI E13 irradiated pups from mothers that received JP4-039/F14 showed no significant weight change compared to control nonirradiated pups (2.44 ± 0.15 and 2.73 ± 0.08, respectively, p = 0.0799). Their weight was significantly increased compared to the 3 Gy irradiated group (2.44 ± 0.15 and 1.64 ± 0.04 g, respectively, p = 0.0433). Pups from nonirradiated mothers that were administered JP4-039/F14 had a significantly increased weight on day 5 compared to nonirradiated mice (3.32 ± 0.10 and 2.73 ± 0.08 g, respectively, p < 0.0001). Microscopic examination of irradiated pups dying at day of birth, revealed: 1) increased number of hematopoietic precursors in liver and decreased glycogen stores in the hepatocytes; 2)adrenal glands were enlarged and contain severely hypertrophied cortical cells; 3) brain, showed necrosis and loss of parenchyma within the intermediate zone of white matter and cell debris in the lateral ventricles. These changes were not observed in pups from JP4-039/F14 treated mothers, sacrificed on the day of birth.

Conclusions: Treatment of total body irradiated E13 pregnant mice at E14 with JP4-039/F14 was safe and effective as a radiation mitigator, led to increased numbers of surviving newborns, improved growth and development, and after weaning over 21 days, and increased body weight with no late deaths.

Supported by NIAID/NIH U19-AI068021

#1661

TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects.

Li Wang, Peijing Zhang, David Molkentine, Jessica Molkentine, Uma Raju, David Valdecanas, Ramesh Tailor, Howard Thames, Thomas Buchholz, Junjie Chen, Li Ma, Kathy Mason, Raymond Meyn, Heath D. Skinner. _UT MD Anderson Cancer Center, Houston, TX_.

Objective: Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. P16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity.

Methods: HNSCC cells and xenografts were used. P16-overexpressing (HPV/p16-negative HN5 and UMSCC-1) and p16 shRNA knockdown (HPV/p16-positive UMSCC-47 and UCPI-SCC154) cells, TRIP12 shRNA or siRNA knockdown (HPV/p16-negative HN5 and Fadu) cells were generated. The effects of p16 or TRIP12 on HNSCC cell radiosensitivity were determined by clonogenic cell survival. The effects of p16 on tumor xenografts (HPV/p16-negative HN5 and HPV/p16-positive UMSCC-47) radioresponse were evaluated by tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction (PCR); and data from The Cancer Genome Atlas HNSCC project were analyzed.

Results: P16 expression led to downregulation of TRIP12 protein both in vitro and in vivo via decreasing TRIP12 protein's half life, which in turn led to increased RNF168 levels and subsequently repressed DNA damage repair, represented by increased 53BP1 foci and neutral comet moment tails at 24 hours after irradiation. As a result, p16 expression enhanced radioresponsiveness both in vitro and in vivo. Inhibition of TRIP12 expression led to radiosensitization through repressed DNA damage repair, represented by decreased BRCA1 foci at 1 and 5 hours after irradiation; and increased neutral comet moment tails at 24 hours after irradiation. Furthermore, overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC.

Conclusions: The findings of our study reveal that p16 participates in radiosensitization through influencing DNA damage repair. P16 downregulates TRIP12 protein expression via post-translational regulation. Inhibition of TRIP12 sensitized HNSCC cells via prohibition of DNA damage repair. These findings support the rationale of blocking TRIP12 signaling to improve radiotherapy outcomes.

#1662

Development of a novel mouse model to study image-guided radiation-induced gastrointestinal injury and its application in pre-clinical research.

Ioannis Verginadis, Rahul Kanade, Edgar Ben-Josef, Constantinos Koumenis. _University of Pennsylvania, Philadelphia, PA_.

Radiotherapy is a major treatment modality typically used in conjunction with surgery and/or chemotherapy in treating a variety of cancers. While effective in controlling or eliminating localized tumors or preventing recurrence, radiation does induce a host of normal tissue toxicities. Gastrointestinal (GI) side effects from radiation treatment are very common; often this limits the dose of radiation that can be delivered to GI tumors and thereby its effectiveness. Thus, the discovery and clinical implementation of the use of bowel radioprotectors and mitigators could have significant clinical impact.

The purpose of this study was to develop a novel method of inflicting localized radiation-induced GI damage in order to establish a more physiological model of damage and aid the development of radioprotectors. Current models, while useful in elucidating the pathophysiological characteristics of broad field radiation, are hampered by complex surgical procedures (exteriorizing the rat intestine and then irradiating the herniated loop of bowel) which can introduce confounding factors such as infection, inflammation and fibrosis or involve whole abdominal irradiation, recapitulating a pathophysiology that is not relevant to clinical practice.

Here we surgically glued a 2mm diameter radiopaque marker made of bismuth subcarbonate onto the surface of the mouse (C57BL/6) jejunum to serve as a target for radiation treatment. One-week post-surgery the mice were imaged with cone-beam CT to locate the marker, and irradiated with 12Gy on the marker using the Small Animal Radiation Research Platform (SARRP®) using a 5x5mm collimator. Mice were sacrificed at various time points post-RT and intestinal tissue directly under, adjacent to, and distal to the marker were collected to assess both morphological and molecular features of radiation-induced damage and inflammation. These included levels of γ-H2AX, multiple cytokines, EdU incorporation, TUNEL assay and intestinal stem cell marker analysis.

Our findings suggest that our image-guided radiation injury mouse model is feasible since we observed a high survival rate post-surgery (approx. 90%). DNA damage, cell-cycle arrest, intestinal crypt apoptosis and inflammation were all significantly higher in the irradiated mice and confined to the immediately irradiated or adjacent area. However, IR-induced damage at 12Gy over 5mm appeared to be transient, as we observed regeneration of the majority of crypts at later time points post- IR. We are currently extending these findings with higher doses of radiation in the presence and absence of clinically used radioprotectors such as curcumin. Our model constitutes a new tool to rapidly evaluate multiple biological markers of DNA damage in the mouse intestine and we believe it will be valuable in future screens for radiation response modifiers and the study of molecular aspects of IR damage.

#1663

Histone methylation as a target for the radiosensitization of glioblastoma-stem like cells.

Barbara Helen Rath, Kevin Camphausen, Philip J. Tofilon. _National Cancer Institute, Bethesda, MD_.

Radiotherapy is a primary treatment modality for glioblastomas (GBMs). However, while many GBMs initially respond to radiation, even in combination with surgery and chemotherapy, median survival continues to be less than 2 years after diagnosis. Because post-translational histone modifications have been implicated in the regulation of radiosensitivity, as a potential strategy for enhancing the response of GBMs to radiotherapy we have evaluated modifiers of histone methylation on the radiosensitivity of glioblastoma stem-like cells (GSCs). For these studies we initially focused on DZNep (3-deazaneplanocin A), which induces degradation of the methyltransferase EZH2. DZNep treatment (0.5µM) of GSCs resulted in a decrease in the amount of H3K27me3 within 6hrs of exposure. Using clonogenic survival analysis, exposure of GSCs to DZNep was then found to enhance their radiosensitivity in a schedule dependent manner: addition of DZNep immediately after irradiation significantly enhanced radiosensitivity, whereas treatment with DZNep for 24h pre-irradiation had minimal to no effect on radiation-induced cell killing. To begin to investigate the mechanism responsible for this radiosensitization, GSCs were irradiated (2Gy), treated with DZNep and collected at 1-24h later for analysis of γH2AX nuclear foci, a surrogate marker of DNA double strand breaks (DSBs). DZNep had no effect on the initial level of radiation-induced γH2AX foci, yet significantly delayed foci dispersal, suggestive of an inhibition of DSB repair as the mechanism of DZNep mediated radiosensitization. To further investigate the potential role of histone methylation as a determinant of radiosensitivity, GSCs were exposed to GSKJ4, an inhibitor of the histone demethylase JMJD3, which results in an increase histone methylation. Given the results from the DZNep experiments, it was anticipated that GSKJ4 would have no effect on or possibly reduce GSC radiosensitivity. However, based on clonogenic survival analysis, addition of GSKJ4 (4µM) 24h prior to or immediately after irradiation significantly enhanced GSC radiosensitivity. With respect to DSBs, GSKJ4 had no effect on the initial level of radiation-induced γH2AX foci or, in contrast to DZNep, on foci dispersal. The γH2AX data suggest that the two modifiers of histone methylation induced radiosensitization through different mechanisms. The radiosensitization induced by an increase or decrease in histone methylation may be accounted for by the biphasic changes in methylation pattern observed after irradiation. Exposure of GSCs to 10Gy resulted in an initial decrease in H3K27me3 levels at 30min followed by an increase above control at 60min. Taken together, these results suggest that disrupting histone methylation status induces GSC radiosensitization.

#1664

Alpha-tocopheryloxyacetic acid (aTEA) induced immune activation synergizes with radiation therapy to treat primary and metastatic murine mammary carcinoma.

Joshua M. Walker,1 Diego M. Barragan,2 Melissa J. Kasiewicz,2 Charles R. Thomas,1 William L. Redmond2. 1 _Oregon Health & Science University, Portland, OR; _2 _Earl A Chiles Research Institute, Providence Portland Medical Center, Portland, OR_.

Immunotherapy has now emerged as a promising treatment for metastatic cancer. However, immunotherapy for advanced stage breast carcinoma remains an unmet need. Here we demonstrate that the novel immunomodulator alpha-tocopheryloxyacetic acid (aTEA) synergizes with radiation therapy (RT) in a metastatic murine mammary carcinoma model to improve tumor control more than each individual therapy, and we propose a mechanism based on the stimulation of type I interferons by aTEA.

For in-vivo studies, C57BL/6 mice bearing 4T1 murine mammary carcinoma lesions were treated with dietary aTEA, 20 Gy x 2 fractions ionizing radiotherapy, or the combination of aTEA/RT. Immune activation in animals treated with dietary aTEA was determined by analyzing the percentage of proliferating CD4 and CD8 T cells using a Ki-67 assay. Pulmonary metastatic burden was determined using a clonogenic metastasis assay. For in-vitro studies, murine mammary carcinoma cell lines were treated with aTEA or LPS and whole cell lysates were analyzed by Western blot. Expression of interferon regulation factor 3 (IRF3) pathway proteins, including phospho-TANK binding protein kinase 1 (TBK1) and stimulator of interferon genes (STING) were determined.

Our results indicate that treatment with the combination of aTEA and RT significantly improves tumor control compared to either treatment alone. Combination therapy resulted in complete and durable tumor regression in a subset of animals treated, an effect not observed with aTEA or RT alone. Dietary administration of aTEA stimulates proliferation of both CD4 and CD8 T cells and significantly reduces metastatic burden in 4T1 tumor bearing mice. This immune stimulatory effect of aTEA appears to be mediated by reactive oxygen species (ROS) production and release of type I interferons through a STING mediated pathway.

These preliminary results suggest that the combination of aTEA and ionizing radiation may be a viable therapy for the treatment of metastatic breast carcinoma. This therapeutic combination may provide more durable response to radiotherapy while simultaneously reducing further metastatic spread.

#1665

Piperlongumine (PPLGM) has potent tumor-radiosensitizing properties in a mouse model of lung cancer.

Anastasia Velalopoulou,1 Ralph A. Pietrofesa,1 Raymond Luke,1 Katie Reindl,2 Melpo Christofidou-Solomidou1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _North Dakota State University, Fargo, ND_.

Background: Although over half of all lung cancer patients receive radiation therapy as part of their treatment plan, the therapeutic window of radiation is quite small, and it is difficult to deliver tumoricidal doses of radiation without the development of severe adverse effects in the nearby normal tissues. An agent that could sensitize lung cancer tissue to radiotherapy while sparing damage to normal tissues is still an unmet need. Piperlongumine (PPLGM), a natural product of the plant Piper longum has shown potent anti-carcinogenic properties through an ROS-inducing mechanism. Our hypothesis is that PPLGM will sensitize tumor cells to radiation-induced cell death in an orthotopic murine model of lung cancer. Methods: 7-week old female C57BL/6 mice were injected intravenously with 1x106 Lewis lung carcinoma (LLC) cells. Mouse cohorts (n=30/group) were administered PPLGM (1 mg/kg of body weight) or vehicle daily by oral gavage initiated 7 days post-LLC injection. At 14 days post-LLC injection, 15 mice per treatment cohort were exposed to 12 Gy thoracic X-ray radiation treatment (XRT). Administration of PPLGM or vehicle continued 7 days post-XRT. Mice were euthanized 14 days post-XRT and evaluated for total lung weight and tumor burden using morphometric analysis. Additionally, we assessed the potential for a dual role of PPLGM (0.1-2.5µM) as a radiosensitizer of cancer cells (LLC) and a radioprotector of normal lung cells (pulmonary microvascular endothelial cells (PMVEC)) via clonogenic survival assays. Results: Using LC/MS/MS we were able to detect significantly elevated levels of PPLGM above baseline in mouse plasma and lung tissue. We observed a significant (p<0.01) increase in lung weight (from 0.14 g to 0.36 g), an indicator of tumor burden, that was significantly (p<0.01) reduced by exposure to a tumoricidal dose of XRT. Importantly, while thoracic XRT-alone reduced tumor burden by 48%, administration of PPLGM+XRT radiosensitized tumor and enhanced tumor killing by XRT, leading to a significant (p<0.05) 56% reduction in lung weight. Morphometric analysis of lung sections confirmed that PPLGM-alone significantly (p<0.01) reduced tumor area relative to vehicle, irrespective of radiation exposure. While exposure to XRT significantly (p<0.01) reduced tumor volume by 65% (from 44.4 ± 7.6% to 15.4 ± 4.5% tumor area), lung sections from mice exposed to PPLGM+XRT significantly (p<0.05) potentiated radiation-induced tumor eradication (reduction in tumor area to 5.6 ± 3.0%) above XRT-only exposure. Furthermore, PPLGM significantly (p<0.05) radiosensitized LLC dose-dependently to gamma-radiation killing, while trending towards radioprotecting normal lung cells. Conclusion: These findings provide evidence that PPLGM is a potent radiosensitizing agent that could aid in the eradication of irradiated tumors in the clinical setting.

Funding: Funded in part by NIH-1R21CA178654-01

#1666

Biomarkers for early detection of radiation nephropathy.

Feifei Song, Jidhin Jacob, Arnab Chakravarti, Naduparambil K. Jacob. _Ohio State University, Columbus, OH_.

Purpose/Background: Chronic kidney disease is among the common late effects detected in patients receiving chemotherapy and/or radiotherapy. Currently, there are no biomarkers that allow detection of normal tissue toxicity in late responding organs such as kidney. Developing predictive biomarkers could help identify individuals that are at high risk for close follow-up and prophylactic treatments. Our studies show that miRNAs detectable in cell-free body fluids can be developed as biomarkers of acute radiation syndromes. In the present study, we sought to investigate the potential of serum and urine miRNAs as indicators of radiation induced kidney damage in rodent models.

Methods and Results: An amplification-free hybridization based nCounter assay was optimized for evaluating the changes of miRNAs in body fluids. Comparison of cell-free miRNAs in urine as well as serum, collected from control versus irradiated mice, enabled us to identify candidate biomarkers that are potential indicators of radiation response. Over 40 miRNAs were detectable in urine samples collected from mice and over 80 miRNAs were detectable in serum. Molecules such as miR-1224, miR-804, miR-714 and miR-709 showed significant increase in their urine level after radiation, which peaked 6-8 hours after exposure to acute doses. On the other hand, markers such as miR-378 that are also abundant in urine did not show significant change after radiation, hence serving as an internal control. Comparison of urinary response in mice exposed to 2, 4, 6 and 8 Gy TBI revealed good dose response. Furthermore, the same markers exhibited a dose and time dependent changes following exposure to fractionated myeloablative regimen (6 x 2 Gy, bid) that is commonly used in leukaemia patients, prior to stem cell transplantation. Specifically, two of these biomarkers, miR-714 and miR-1224 exhibited a dose dependent increase in their serum level after fractionated or acute single dose exposure. In-situ hybridization displayed an overall increase in signals of miR-1224 and miR-714 at outer medulla after radiation. Increased signals detected in tubular epithelial cells indicate the possible tubular origin of the urinary miRNAs that are responsive to radiation.

Conclusion: We have developed a panel of miRNAs for non-/minimally invasive detection of radiation response in kidney. Several of the urinary miRNA biomarkers exhibited a dose and time dependent changes, suggesting their potential as biomarkers for non-invasive evaluation of radiation response in organs such as kidney for which early detection of damage is a major challenge.

#1667

A novel method to identify radiosensitizers targeting hypoxic cancer cells by high-throughput screening.

Keisuke Tamari, Yuji Seo, Yutaka Takahashi, Osamu Suzuki, Fumiaki Isohashi, Yasuo Yoshioka, Kazuhiko Ogawa. _Radiation Oncology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan_.

Tumor hypoxia correlates radioresistance and poor clinical outcomes after radiotherapy. Radiosensitizers have been studied for a long time especially to overcome the problem of hypoxic tumor cells. Until now, however, radiosensitizers for hypoxic tumors have had only limited success in clinic. High throughput screening is popular method to identify candidate compounds from a large number of compounds for drug discovery, but have not been established method to study new radiosensitizers. The aim of this study was to identify radiosensitizers targeting hypoxic cancer cells by high throughput screening and validate the efficacy of this method. We performed cell-based high throughput screening by using 384 well plates and 1280 compounds. After exposing compounds to HeLa cells, gamma-irradiation was delivered under hypoxic condition and incubated for 4 days. To identify candidate compounds, cell counting was performed by MTS assay. Then, radiosensitizing effect of these compounds was validated by clonogenic survival assay. We performed high throughput screening and identified 22 compounds as candidates. We selected Ro90-7501 and dicyclomine hydrochloride which were not reported as radiosensitizers. Clonogenic survival assay showed their significant radiosensitizing effect in hypoxic condition. In conclusion, we identified novel radiosensitizers by high throughput screening. This method may be useful for identifying radiosensitizers targeting hypoxic cancer cells.

#1668

Bouvardinis a radiation modulator with a novel mechanism of action.

Tin tin SU. _University of Colorado, Boulder, CO_.

Protein synthesis is essential for growth, proliferation and survival of cells. Translation factors are overexpressed in many cancers and their experimental inhibition curbs cancer growth in preclinical models. Differential regulation of translation also occurs upon exposure to cancer-relevant stresses such as hypoxia and ionizing radiation (IR). The failure to regulate translation has been shown to interfere with recovery after genotoxic stress. These findings suggest that modulation of translation, alone or in conjunction with genotoxins, may be therapeutic in oncology. Yet, only two drugs that directly inhibit translation are FDA-approved for oncology use today. We described before the identification of protein synthesis inhibitor bouvardin in a screen for small molecule enhancers of IR in Drosophila melanogaster. Bouvardin was independently identified in a screen for selective inhibitors of engineered human breast cancer stem cells. Here we report the effect of bouvardin in preclinical models of head and neck cancer (HNC) and glioma, two cancer types for which IR is a key therapy choice. Our data show for the first time that bouvardin blocked translation elongation on human ribosomes and suggest that it did so by blocking the dissociation of elongation factor 2 from the ribosome. Bouvardin enhanced the induction of clonogenic death by IR in HNC and glioma cells, although by different mechanisms. Bouvardin enhanced the anti-tumor effect of IR in HNC tumor xenografts in mice. These data suggest that inhibition of translation elongation, particularly in combination with IR, may be a promising treatment option for cancer.

#1669

Inhibition of the Rac1/PAK pathway sensitizes pancreatic cancer cells to gamma-irradiation.

Michel M. Ouellette, Surinder K. Batra, Ying Yan. _University of Nebraska Medical Center, Omaha, NE_.

Radiation therapy is a staple treatment for pancreatic cancers. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both G1/S and G2/M checkpoints. However, cancer cells are often defective in G1/S checkpoint due to mutations/alterations in the key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras mediated transformation. Here, we show that Rac1 and its downstream effector, the PAK kinases, also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1/PAK pathway with pharmacological inhibitors (Ehop-016, AZA1, NSC23766, and FRAX597) or a dominant negative mutant of Rac1 (Rac1T17N) blocks activation of the ATM/Chk2 and ATR/Chk1 pathways, abrogates activation of the IR-induced G2/M checkpoint, delays the repair of IR-induced double-stranded DNA breaks, and increases the radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer.

#1670

Radiosensitization of glioma cells by the ketone body β-hydroxybutyrate is associated with enhanced cell cycle arrest in the G2/M phase.

Helena B. Silva-Nichols,1 Alex P. Rossi,1 Eric C. Woolf,1 Marshall J. Fairres,1 Loic P. Deleyrolle,2 Brent A. Reynolds,2 Adrienne C. Scheck1. 1 _Barrow Neurological Institute, Phoenix, AZ;_ 2 _University of Florida, Gainesville, FL_.

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor with a 5 year survival rate of 25% in children and less than 10% in adults. Improvement in the prognosis of GBM patients requires the development of new therapeutic approaches. One emerging strategy is to target aberrant cell metabolism, a trait shared by virtually all tumor cells. The ketogenic diet (KD), a high fat, low carbohydrate and protein metabolic therapy has been shown to prolong survival in animal glioma models, and when used in conjunction with radiation cured 9 of 11 mice of their implanted tumors. We have also shown that the KD alters hypoxia, angiogenesis, and other hallmarks of glioma progression. To elucidate the underlying mechanisms through which ketones exert their effects on glioma, we are doing analyses of the effect of β-hydroxybutyrate (βHB), the most prevalent ketone body synthesized during ketosis, on glioma cells in vitro. We found that βHB both alone and in conjunction with radiation significantly inhibits proliferation of human and mouse glioma cells and human glioma stem cells (GSC). Alterations in passage through the cell cycle may affect proliferation, and cells are also more sensitive to radiation in the G2/M cell cycle phase. We therefore analyzed the effect of βHB on cell cycle distribution of cells treated with βHB and/or radiation. An analysis of cell cycle status by flow cytometry demonstrated that treating the GSC line L0 with 5mM βHB in combination with 4 Gy of radiation significantly increased the number of cells in G2/M cell cycle arrest. In GL261-Luc2 mouse glioma cells, 5mM βHB alone significantly enhanced G2/M cell cycle arrest, which could lead to radiosensitization. Currently we are analyzing proteins involved in cell cycle progression and apoptosis to better understand βHB mediated changes in growth and radioresistance. In summary, these data provide insight to the radiosensitization and anti-proliferative mechanisms of βHB and may hold implications for the use of the KD in the treatment of GBM.

#1671

Local release of combretastatin A-4 from NIR-light activatable prodrugs overcomes areal and temporal limitations of photodynamic therapy.

Pallavi Rajaputra, Moses Bio, Gregory Nkepang, Pritam Thapa, Sukyung Woo, Youngjae You. _University of Oklahoma, Oklahoma City, OK_.

A unique prodrug strategy for treating localized cancers, in which NIR light-illuminated prodrug effectively ablates tumors through the combined effects of photodynamic therapy (i.e., singlet oxygen [SO]) and locally released anticancer drugs has been proposed. Due to short distance of action (< 0.04 μs) and short lifetime (< 0.02 μm) of SO, direct damage of PDT is both areally and temporally limited. We hypothesized that the locally released anticancer drugs would overcome the areal and temporal limits of SO. Near IR-activatable prodrug of combretastatin A-4 (CA4), Pc-(L-CA4)2, and its pseudo-prodrug, Pc-(NCL-CA4)2, were evaluated in vitro and in vivo. After partial illumination of a 24 well, all the cells in the prodrug-treated well were killed by the released CA4. Limited areal damage was observed in the pseudo-prodrug-treated wells. A time-dependent cell survival study revealed more extensive cell death in the prodrug-treated cells, due to the sustained damage from the released CA4. Cell cycle analysis and microscopic imaging data demonstrated the typical damage patterns of CA4 in the prodrug-treated cells. A time-dependent histological study showed that prodrug-treated tumors lacked mitotic bodies. The prodrug caused broader and more long-lasting tumor size reduction than did the pseudo-prodrug. These data consistently support that the released CA4 overcomes the areal and temporal limits of SO, providing far superior antitumor effects.

#1672

Photodynamic therapy using photosensitizer and light emitting diodes (LEDs) demonstrates oncolytic activity against prostate cancer.

Taher Gheewala,1 Troy Skwor,2 Gnanasekar Munirathinam1. 1 _University of Illinois College of Medicine at Rockford, Rockford, IL;_ 2 _Rockford University, Rockford, IL_.

Prostate cancer (PCa) affects over 2 million Americans and its treatment options include surgery, anti-hormonal drugs for androgen sensitive tumors, and radiotherapy. However, patients undergoing androgen deprivation therapy develop resistance leading to development of castration resistant or androgen independent PCa. It is clear there is an urgent need to find an effective treatment strategy to manage aggressive PCa. An alternative treatment is the use of photodynamic therapy (PDT), which involves the activation of a photosensitizer by a defined wavelength of light in the presence of oxygen, generating transient concentrations of reactive oxygen species. We hypothesize that PDT via a photosensitizer (pheophorbide) in combination with light emitting diodes (LEDs) [670 nm] will demonstrate oncolytic activity against PCa cells, thus suggesting an alternative, less toxic cancer treatment. To test our hypothesis, we used C4-2 and DU-145 PCa cell lines as an in vitro model in this study. To explore the anti-cancer effects of PDT on PCa, cell viability assay and wound healing assay were performed. Western blotting was employed to identify the signaling molecules modulated by PDT. Our cell viability assay results demonstrated significant oncolytic activity against PCa cell lines using PDT with pheophorbide as a photosensitizer combined with 670 nm LEDs in as little as an 88 second pulse with increased lytic activity correlating with elevated energy intensities. We also observed variability in cell line PDT susceptibility; wherein, C4-2 cells were more susceptible than DU-145 PCa cells. The wound healing assay results with pheophorbide and 670 nm LEDs demonstrated significant inhibition of migration abilities of both C4-2 and DU-145 PCa cells. These findings implicate the anti-metastatic activity of PDT on PCa. Further, our mechanistic analysis showed activation of BiP/GRP78, an endoplasmic reticulum (ER) chaperone, in C4-2 as well as DU-145 PCa cells. This suggests that the anti-cancer activity of PDT with pheophorbide and 670 nm LEDs is via ER stress. In conclusion, our results show PDT using pheophorbide and 670 nm LEDs as a promising therapeutic strategy for PCa.

#1673

Optical metabolic imaging of response to radiation in radiation-sensitive and resistant lung cancer cells.

Kinan Alhallak,1 Ruud P.M. Dings,2 Narasimhan Rajaram1. 1 _University of Arkansas, Fayetteville, AR;_ 2 _University of Arkansas for Medical Sciences, Little Rock, AR_.

Tumor resistance to radiation therapy is a significant obstacle that patients and doctors face while treating tumors. Meaningful changes in therapy are contingent upon the ability to identify an unfavorable response very early (first week) in the course of treatment. Optical imaging can perform noninvasive, frequent and quantitative measurements of tumor biology at the point of care. The goal of this study was to identify optically measurable metabolic endpoints in a matched model of radiation resistance before and after treatment. A human lung cancer cell line was induced to develop radiation resistance through repeated exposure to a clinically relevant dose of X-ray radiation (2 Gy). We used a fluorescent glucose analog called 2-NBDG to quantify glucose uptake. We measured the endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), metabolic coenzymes found primarily in the cytoplasm and mitochondria, respectively. We determined the redox ratio [FAD/(NADH + FAD)] in both cell lines. We evaluated the behavior of the cellular antioxidant system by comparing the levels of reactive oxygen species (ROS) and ROS scavengers, such as glutathione (GSH) for each cell group. Finally, we analyzed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a metabolic flux analyzer.

Radiation-resistant A549 lung cancer cells were induced by exposure to 25 fractions of X-ray radiation (2.2 Gy/fraction). Clonogenic assays were performed to confirm radiation resistance. All fluorescence images were obtained using a multiphoton microscope. 2-NBDG, ROS, GSH, NADH, and FAD fluorescence were excited at 960, 930, 780, 755 and 860 nanometers, respectively. The cells were exposed to 2 Gy 60 minutes prior to the Seahorse assay. All experiments were performed in both the parental A549 cell line (A549C) and the radiation-resistant A549 cell line (A549R) at baseline (prior to radiation) and after radiation.

At baseline, the A549R cells revealed a significantly lower level of oxidative stress and a higher level of GSH. Interestingly, glucose uptake, quantified using 2-NBDG fluorescence, was significantly lower in the A549R cells compared to the A549C cells at baseline. The redox ratio was significantly higher in the A549R cells prior to radiation, indicating a preference for mitochondrial respiration. However, after radiation, the OCR of the A549R cells was significantly lower than the parental cell line, indicating a lower use of mitochondrial respiration. There were no significant differences in ECAR levels between both cell lines after radiation. Our results indicate that the radiation-resistant A549 lung cancer cells present very distinguishable metabolic properties at baseline and after exposure to radiation relative to the parental cell line that could potentially be exploited in vivo. 

### Regulators of Epithelial-Mesenchymal Transition and Metastasis

#1674

Tripartite motif containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein.

Chunli Wei,1 Jingliang Cheng,1 Hanchun Chen,2 Dianzheng Zhang,3 Junjiang Fu1. 1 _Sichuan Medical University, Luzhou, China;_ 2 _Central South University, Changsha, China;_ 3 _Philadelphia College of Osteopathic Medicine, Philadelphia, PA_.

Regulation of gene expression by TRIM28 is not only by transcriptional repression but also by post-translational regulation. Here we report that TRIM28 promotes breast cancer cell metastasis through TRIM28-TWIST1-EMT pathway. We find that TRIM28 is highly expressed in metastatic cancer cell lines and advanced breast cancer tissues. The expression of TRIM28 and TWIST1 is positively correlated with most of the invasive breast carcinomas. Notably, we find that overexpression of TRIM28 upregulates TWIST1 protein, whereas TRIM28 depletion downregulates TWIST1 protein, indicating that TWIST1 upregulation likely occurs at post-transcriptional level, not at transcriptional level. Overexpression of TRIM28 in BT549 breast cancer cell line promotes cell migration and invasion functionally; conversely, knockdown of TRIM28 reduces TWIST1 protein level, up-regulates E-cadherin and down-regulates N-cadherin, and consequently inhibits cell migration and invasion. Furthermore, Immunoprecipitation and GST pull down reveal that TRIM28 interacts with TWIST1 directly, thereby preventing from proteasomal degradation. Moreover, TWIST1 degradation rate is reduced after cycloheximide blocking protein synthesis through the depletion of TRIM28 in BT549 cells. Taken together, our findings suggest that TRIM28 functionally stabilizes TWIST1 protein, reveals a novel mechanism, and provides an effective therapeutic strategy to target TRIM28 in breast cancer treatments.

#1675

Activation of phosphatase toward the retinoblastoma protein inhibits invasion in MDA-MB-231 breast cancer cells.

Jacklynn Egger, Maria Lane, Brixhilda Dedi, Nancy A. Krucher. _Pace University, Pleasantville, NY_.

Excessive phosphorylation of the Retinoblastoma protein (Rb) is found in most cancer tumor types. Several cyclin dependent kinase (cdk) inhibitors are in development or in clinical trials at the present time. Our previous studies have focused on the regulation of Rb phosphorylation by the phosphatase, PP1. In proliferating cells, PP1 is regulated by a protein called PNUTS (Phosphatase Nuclear Targeting Subunit). PNUTS is a competitive inhibitor of Rb binding to PP1 and siRNA mediated knockdown of PNUTS in several cancer cell types (breast, colon, ovarian) leads to an increase in Rb directed PP1 activity, Rb dephosphorylation on several sites, and a dramatic induction of apoptosis. In this study we have used MDA-MB-231 breast cancer cells grown in Matrigel. By lentiviral-mediated doxycycline inducible expression of PNUTS siRNA, expression of PNUTS protein is blocked and phosphorylation of Rb cells is reduced. Cells become less invasive and expression of mesenchymal markers N-cadherin, Zeb and Slug is lost. A reversion to the epithelial phenotype is suggested by an increase in E-cadherin expression. PNUTS knockdown has no effect on cells that do not contain highly phosphorylated Rb such as non-transformed MCF-10A cells or Rb-negative MDA-MB-468 cells. Further experiments using highly invasive HT-1080 cells show that reduction of Rb phosphorylation appears to reduce invasiveness in this cell model, supporting the idea that Rb phosphorylation plays a critical role in the regulation of invasion.

#1676

15-Lipoxygenase-2 inhibits epithelial-to-mesenchymal transition (EMT) in metastatic prostate cancer.

Rana K. Alfardan,1 Debasish Boral,1 Daotai Nie2. 1 _Southern Illinois University School of Medicine, Springfield, IL;_ 2 _Southern Illinois University School of Medicine and Cancer Institute, Springfield, IL_.

Introduction

15-lipoxygenase-2 (15-LOX-2) is a lipid oxidizing enzyme which converts the unsaturated fatty acids (Arachidonic and linoleic acid) to bioactive lipids with anti-inflammatory properties. Abundantly expressed in normal prostate, 15-LOX-2 expression and activities were found reduced in prostate tumors. 15-LOX-2 has been suggested as functional tumor suppressor since restoration of its expression was found to reduce prostate tumor formation and growth and induce tumor dormancy. However, it is unknown how 15-LOX-2 modulates prostate tumor progression and metastasis.

Methods

Silico analyses of gene array data, Inducible Tet-on system to express 15-LOX-2 in PC3MM cells in an inducible manner, qPCR to check the expressions, western blot, wound healing assay, immunohistochemistry.

Result

Through in silico analyses of gene array data, we found 15-LOX-2 expression was markedly reduced in metastatic prostate tumors when compared to normal or primary prostate tumor tissues. To determine whether 15-LOX-2 can modulate metastatic potentials of prostate cancer, we used an Inducible Tet-on system to express 15-LOX-2 in PC3MM cells. Treatment with 750ng/ml of doxycycline led to induced expression of 15-LOX-2 in PC3MM cells. Restoration of 15-LOX-2 expression in PC3MM cells caused re-expression of several epithelial markers including claudin, desmocollin, desmoglein, Jam, EPCAM, β-Catenin molecules involved in the tight and desmosomal junctions. At mean time, 15-LOX-2 expression led to a decrease in the levels of mesenchymal markers, vitronectin and fibronectin, in PC3MM cells. Restoration of 15-LOX-2 expression in PC3MM cells reduced cell migration and reduced MMP2 and MMP3 expression.

Conclusion

Our data suggest that 15-LOX-2 is a barrier for prostate tumor cells to acquire metastatic potentials through inhibiting epithelial to mesenchymal transition.

#1677

CLCA2 interaction with EVA1 promotes mammary epithelial differentiation.

Grace N.T. Ramena,1 Yufang Yin,1 Yang Yu,1 Vijay Walia,2 Randolph C. Elble1. 1 _Southern Illinois University School of Medicine, Springfield, IL;_ 2 _National Cancer Institute-Frederick, Frederick, MD_.

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized HMEC caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The TMS primary sequence of CLCA2 was found to be conserved throughout mammals. Moreover, competitive inhibition of the interaction by ectopic expression of the TMS caused partial EMT, suggesting that the interaction is important for differentiation. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.

#1678

DRAM1 regulates EMT of lung cancer A549 cells via SQSTM1/p62 in vitro.

Binfeng He,1 Jiajing Cai,1 Guangcheng Huang,1 Fan Chang,1 Lei Xu,1 Dongsheng Wang,1 Yibin Deng,2 Xiaolan Guo1. 1 _North Sichuan Medical College, Sichuan, P.R. China, Nanchong, Sichuan, China;_ 2 _The Hormel Institute, University of Minnesota, Austin, MN_.

Epithelial-to-mesenchymal transition (EMT) has been linked to the regulation of non-small cell lung cancer (NSCLC) progression, migration, invasion, metastasis, and chemoresistance as well. Recent studies showed that autophagy was a novel target to mediate EMT of cancer cells through regulating the migration and invasion in cancer. The DNA Damage-Regulated Autophagy Modulator 1 (DRAM1) is an evolutionarily conserved transmembrane protein and localizes predominantly to lysosomes, while also colocalizing with the autophagosome marker LC3. Previous investigation discovered that DRAM1 played an important role in regulating autophagy, inducing apoptosis of NSCLC cells. However, the function and mechanism of DRAM1 mediated autophagy in regulating EMT in NSCLC remains poorly understood. In this study, transforming growth factor-β (TGF-β) and starvation were employed to induce autophagy for investigating EMT in NSCLC A549 cells. The results showed that after exposure to TGF-β 24h, the protein levels of DRAM1, autophagy marker SQSTM1/p62, LC3I/II and EMT marker vimentin significantly increased while another EMT marker E-cadherin protein decreased obviously compared with the control by western blot. Also the similar results were observed after starvation 48h in this study. To further illustrate the role of DRAM1 in regulation of autophagy and EMT, wildtype DRAM1 overexpression plasmid and DRAM1 siRNA was transfected into A549 cells respectively to check the autophagy markers and EMT markers expression, meanwhile wound healing test and cell invasion transwell chamber test were assayed after the gene manipulation. These experiments results demonstrated that DRAM1 overexpression dramatically increased the SQSTM1/p62, LC3I/II and vimentin protein levels and inhibited the expression of E-cadherin, promoting the migration and invasion abilities of A549 cells. Conversely, down-regulation of DRAM1 restrained SQSTM1/p62, LC3 I/II and vimentin protein levels and elevated the expression of E-cadherin, leading to the inhibition of migration and invasion abilities of A549 cells. Interestingly, our further study showed that after p62 siRNA transfected into A549 cells E-cadherin upregulated while vimentin reduced compared to the control group. Consistently, knockdown of p62 suppressed the migration and invasion abilities of A549 cells and down regulated the expression of twist, which is an EMT related transcription factor. These data suggested that DRAM1 played an important role in regulating the EMT of lung cancer A549 cells through SQSTM1/p62 modulating, which might be a novel potential target for lung cancer treatment.

#1679

Notch4 mediates mesenchymal-epithelial-like transition in melanoma.

Ehsan BonyadiRad,1 Heinz Hammerlindl,2 Dinoop Ravindran Menon,2 Christine Hafner,3 Meenhard Herlyn,4 Helmut Schaider2. 1 _Medical University of Graz, Graz, Austria;_ 2 _The University of Queensland, Brisbane, Australia;_ 3 _Karl Landsteiner University of Health Sciences, St Poelten, Austria;_ 4 _The Wistar Institute, Philadelphia, PA_.

In melanoma Notch signaling is a well-established oncogenic factor and presumably driver of disease progression. Here we report that overexpression of the constitutively active intracellular domain of Notch4 (N4ICD) directs a mesenchymal-epithelial-like transition that results in an epithelial, low migratory, invasive and proliferative phenotype with decreased tumorigenicity in-vivo. Investigating the expression profile of 50 EMT markers in N4ICD overexpressing cells revealed an epithelial-like gene signature with major EMT markers, including Vimentin, MMP2, Snail1, Snail2 and Twist1, significantly decreased and several epithelial markers, including E-cadherin, Desmoplakin and Occludin significantly increased. N4ICD suppresses the expression of Snail2 and Twist1 in a non-canonical fashion by inducing transcription factors Hey-1 and Hey-2 which directly bind to promoter regions of Snail2 and Twist1 as determined by EMSA and luciferase assays. N4ICD overexpression also results in increased beta-catenin protein levels exclusively limited to the cytoplasm. Immunohistochemical stainings of primary melanomas and subcutaneous metastasis showed a correlation between Notch4, E-cadherin and beta-catenin expression. Our results demonstrate that overexpression of Notch4 ICD results in the emergence of an epithelial-like phenotype with increased E-cadherin and cytoplasmic beta catenin potentially steering the reformation of adherens junctions. This indicates that Notch4 supports a mesenchymal-epithelial-like transition trapping cancer cells in a low proliferative, invasive and migratory epithelial state, suggesting Notch4 to be a tumor suppressor in melanoma.

#1680

Targeting subcellular localization of Kaiso to limit dissemination through MAPK signaling.

Shweta Tripathi,1 Balasubramanyam Karanam,1 Bo Ma,2 Alan Wells,2 Clayton Yates1. 1 _Tuskegee University, Tuskegee, AL;_ 2 _Department of Pathology, Pittsburgh VAMC and University of Pittsburgh, Pittsburgh, PA_.

Breast cancer is the second most commonly diagnosed cancer among American women. The American Cancer Society has estimated over 230,000 new cases of breast cancer, and over 40,000 deaths for the year 2015. Unfortunately, metastasis is the major contributor for the significant number of these deaths. The Epithelial-Mesenchymal Transition (EMT) is promoted by the Master Regulator known as Kaiso, a transcriptional repressor that is a member of the BTB/POZ family of zinc finger transcription factors. We have previously demonstrated that Epidermal Growth Factor Receptor (EGFR) signaling shuttles Kaiso into the nucleus, although, its mechanism is still not understood. EGFR signaling activates multiple downstream signaling cascades including the MAP Kinase cascade, which has a significant influence on proliferation, cell adhesion, gene expression, and cell survival. Mesenchymal cells demonstrate high levels of MAPK activity and disruption or inhibition of the cascade has proven to be a method for decreasing tumor growth. Therefore we hypothesize that subcellular localization and expression of Kaiso is modulated by EGFR induced MAPK activity. Using the TCGA Breast cancer dataset, we observed that Kaiso was altered in about 11% of patients, compared to 15% of E-cad expression and 13% of MAPK3 of 959 patients/cases of Breast cancers. MAPK3 (eRK1); MAPK6, MAPK7, MAPK9 AND MAPK14 (p38) seems to have trends in mutual exclusivity or co-occurrence with Kaiso (ZBTB33). To determine if MAPK3 is responsible for increased Kaiso expression, MCF-7 cells, which express low levels of Kaiso in the cytoplasm, were treated with 10ng/ml of EGF in the presence or absence of MAPK inhibitor, 20µM PD98093. EGF stimulation caused robust increase in Kaiso protein expression, while PD98093 inhibited EGF induced expression. Similarly, in Kaiso overexpressing, MDA-MB-231, blocking EGFR kinase activity with 500nM PD153035 resulted in an opposite effect as EGF stimulation in MCF-7 cells. Furthermore, we observed a significant decrease in p-ERK expression, re-expression of E-cadherin, and low EGFR expression levels in sh-Kaiso MDA-MB-231 cells compared to sh-Scr cells. The decreased p-ERK expression in sh-Kaiso MDA-MB-231 cells was also associated with decreased expression of mesenchymal markers ZEB1, ZEB2, Twist and SNAIL at both the mRNA and protein levels compared to the scrambled control. Similarly, we observed significant decrease in the somatic cell reprogramming factors Klf4, Oct4, Nanog expression in sh-Kaiso MDA-MB-231 as well, which coincide with a reversal of EMT. To our knowledge, this is the first study to investigate the molecular mechanism and/or functional consequences of increased Kaiso expression in breast cancer, and highlights the multiple roles that Kaiso plays in promoting aggressive disease.

#1681

DUOX1 expression in lung cancer disrupts pro-oncogenic activation mechanisms and localization of Src and EGFR.

Andrew C. Little, Karamatullah Danyal, Robert A. Bauer, David E. Heppner, Milena Hristova, Christopher Dustin, Aida Habibovic, Albert van der Vliet. _University of Vermont, Burlington, VT_.

Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is associated with epithelial-to-mesenchymal transition (EMT), a key feature of tumor invasiveness and metastasis, and that RNAi-mediated DUOX1 suppression can promote EMT, but the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. Furthermore, nuclear Src/EGFR localization and phosphorylation of EGFR-Y1101 in lung cancers was recently associated with metastatic cell behavior and poor clinical outcome. Preliminary findings in alveolar lung cancer A549 cells, which possesses some EMT-like features, indicate that DUOX1 overexpression redistributes Src localization to the plasma membrane and decreases its nuclear accumulation. Moreover, DUOX1 overexpression in A549 cells also suppressed EGF-stimulated EGFR internalization and nuclear translocation, in association with reduced EGFR phosphorylation on its Src target, Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292 (which has retained DUOX1 expression) promoted EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Further mechanistic studies will be performed to elucidate the molecular mechanisms by which DUOX1 is able to alter these events. Collectively, our findings indicate that DUOX1 silencing in lung cancer may contribute to EMT and/or tumor invasiveness by altering Src/EGFR localization and activation mechanisms.

#1682

Lyn drives cancer metastasis via post-translational regulation of SNAI proteins.

Daksh Thaper,1 Sepideh Vahid,1 Ka Mun Nip,1 Igor Moskalev,1 Sebastian Frees,1 Morgan E. Roberts,2 Krisi Ketola,1 Kenneth W. Harder,1 Jennifer L. Bishop,1 Amina Zoubeidi1. 1 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada_.

Introduction: Metastasis is the most common cause of death from cancer and occurs when malignant cells discard epithelial restraints and acquire invasive abilities, facilitating their dissemination to permissive micro-environments. This process is enhanced by tumor cell activation of Epithelial Mesenchymal Transition (EMT), a (normally embryonic) developmental program in which epithelial cells assume a mesenchymal phenotype during gastrulation and organogenesis, allowing single cell invasive movement away from the ectodermal layer. Recent evidence strongly implicates EMT induction in malignant progression and treatment resistance. For example, EMT regulatory transcription factors are required for breast cancer metastasis. Several oncogenic pathways (growth factors, Src family, MAPK, AKT) induce EMT. Lyn tyrosine kinase, a member of Src family tyrosine kinase is up-regulated in advanced prostate cancer and has been reported to correlate with aggressive breast cancer. Our objective is to determine the role of Lyn tyrosine kinase in EMT.

Methods: LNCaP (Lymph Node Metastasis of Prostate Cancer), BT-549 (Triple Negative Breast Cancer) and UM-UC-13 (Bladder Cancer) cells were transfected with Lyn siRNA; EMT markers were monitored by western blot and qRT-PCR and immunofluorescence, migration by scratch assay and invasion by Boyden chamber. Sub cellular localization of proteins was examined by IF and nuclear/cyto extraction. In vivo experiments were performed in UC13-luc cells with shRNA of Lyn.

Results: Here we report that Lyn expression is low in epithelial cells and is up-regulated in mesenchymal cells. Targeting Lyn using siRNA decreases EMT markers (Fibronectin, Vimentin and Zeb-1) at both mRNA and protein levels while increasing the epithelial marker (E-cadherin). Moreover, we found that Lyn siRNA decreases cell migration and invasion. Thisis decrease in mesenchymal phenotype can be attributed to the decrease in the amount of Slug and Snail, transcriptional repressors of E-Cadherin and activator of Vimentin and Fibronectin. Interestingly, we found that Lyn knockout induces a decrease of SLUG only at protein levels and not at mRNA levels. We discovered that Lyn triggers a signaling cascade through Vav-Rac-Pak1 pathway to alter sub cellular localization of the SNAI proteins leading to their proteasomal degradation. This effect results in decreased invasion and migration in vitro as well as decreased metastasis in vivo.

Conclusion: Expression of Lyn kinase can be correlated to low prognosis and aggressive/metastatic phenotype. We show that knocking down Lyn by siRNA initiates a switch to a more epithelial phenotype reducing cell migration and invasion.

#1683

Dual role of semaphorin5A in maintaining epithelial and mesenchymal phenotype in metastatic pancreatic cancer.

Sugandha Saxena, Abhilasha Purohit, Michelle Varney, Surinder K. Batra, Rakesh K. Singh. _University of Nebraska Medical Center, Omaha, NE_.

Pancreatic cancer (PC) is a highly aggressive disease and in general has metastasized to distant organs by the time of its clinical diagnosis. Poor five-year survival rate of 7% with an average survival time of 5-8 months highlight the gravity of the situation and necessitate a better understanding of the molecules and mechanisms that lead to metastatic dissemination in PC. One of the key processes that lead to metastasis is the gain of cellular migration ability. Previous reports from our laboratory have identified semaphorin5A (SEMA5A), an axon guidance cue molecule regulating cellular migration, as a putative cell adhesion molecule which is involved in organ-specific homing during PC metastasis. Also, differential expression of SEMA5A was observed in cells derived from metastatic sites in comparison to those arising from primary tumors. On the basis of the these findings, we hypothesize that SEMA5A regulates epithelial to mesenchymal changes necessary to drive cellular migration and further leads to establishment of these cells at secondary sites. We utilized various cellular models such as the L3.3 and L3.6pl cell lines with different metastatic potential, cell lines derived from metastatic sites (Capan-1 and CD18/HPAF) with stable knock down of SEMA5A, and cell lines derived from primary tumors (Panc1) with overexpression of SEMA5A. We evaluated epithelial and mesenchymal markers, in vitro cellular migration and the metastatic potential of these cells. We observed down regulation of E-cadherin expression and re-localization of beta-catenin from the membrane to the nucleus in SEMA5A knockdown cells. Similarly, overexpression of SEMA5A in Panc1 cells resulted in higher E-cadherin expression. We also observed higher SEMA5A and E-cadherin expression in the highly metastatic L3.6pl cells in comparison with L3.3 cells. Furthermore, knock-down SEMA5A in Capan-1 cells resulted in enhanced in vitro cellular migration and higher metastasis when control or SEMA5A knockdown cells were orthotopically implanted in nude mice. Our observations suggest that SEMA5A plays a bi-functional role in mediating epithelial to mesenchymal transition at primary site while regulating mesenchymal to epithelial transition and establishment at secondary sites.

#1684

Mesothelin regulates epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells.

Xiaoqing He,1 Liying Wang,2 Emily Despeaux,1 Yon Rojanasakul3. 1 _Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV;_ 2 _Allergy and Clinical Immunology Branch, NIOSH, Morgantown, WV;_ 3 _Department of Pharmaceutical Sciences and Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV_.

Lung cancer and malignant mesothelioma are two of the most deadly forms of cancers. The prognosis of lung cancer and mesothelioma is poor due to limited treatment modalities and lack of understanding of the underlying disease mechanisms. Mesothelin (MLSN), a plasma membrane differentiation antigen, is expressed at a high level in several human cancers, including 70% of lung cancer and nearly all mesotheliomas. Recent studies have shown that plasma and/or pleural effusion MSLN may be used as a biomarker for certain cancers and malignant pleural mesothelioma. However, the role of MLSN in lung cancer and mesothelioma is relatively unknown. In this study, we found that MLSN plays an important role in controlling epithelial-to-mesenchymal transition (EMT) and stem properties of human lung epithelial and mesothelial cells, which control their tumorigenicity. Firstly, MLSN was found to be highly upregulated in human non-small cell lung cancer tissues and in lung cancer and mesothelioma cells, as compared to normal tissues and non-tumorigenic lung epithelial and mesothelial cells. Secondly, knockdown of MLSN by RNA interference significantly reduced anchorage-independent cell growth, tumor sphere formation, and cell migration and invasion in vitro, as well as tumor formation in vivo. Thirdly, knockdown of MLSN attenuated cancer stem cell marker (ALDH) activity and several EMT markers in lung cancer and mesothelioma cell lines. These findings indicate the essential role of MLSN in EMT and malignant properties of human lung cancer and mesothelioma cells. Since EMT is an important process in tumor progression and metastasis, our finding on the role of MLSN may provide new insights into the disease mechanisms and treatment strategies for aggressive lung cancer and mesothelioma.

#1685

Identification and characterization of EMT/MET signatures in circulating tumor cells isolated from patients with metastatic breast cancer using graphene oxide nano-chip.

Tae Hyun Kim, Hyeun Joong Yoon, Sunitha Nagrath. _University of Michigan, Ann Arbor, MI_.

Disseminated cancer cells identified in the circulatory system of cancer patients has implied to be the key drivers of tumor metastasis. These cells known as circulating tumor cells (CTCs) are emerging as an alternative to tissue biopsies to predict prognostic values, and to monitor evolving tumor heterogeneity through serial drug treatments. Recent technological advances have enabled the detection of CTCs in patient blood samples. However, the enumeration alone seems to be insufficient to obtain information that could benefit any clinical decisions. The limited sensitivity as well as the specificity of current approaches struggle from realizing the full promise of CTCs. We developed a sensitive microfluidic chip using functionalized graphene oxide (GO) nano-sheets to isolate CTCs with improved sensitivity and purity. Blood samples collected from metastatic breast cancer patients were processed through the chip within 6 hours of sampling and the CTCs were identified by the presence of cytokeratin with the absence of CD45 expression. EMT/MET signatures were characterized in the isolated CTCs to investigate its role by assessing gene expression profiles. Protein markers known to be up regulated during the EMT such as Vimentin and N-cadherin was examined along with HER2. mRNA expression

of EMT markers were studied simultaneously using a multiplex TaqMan-based qRT-PCR method and compared to healthy controls. CTCs were successfully detected in >95% of patients examined in this study. Cell-to-cell intrapatient heteroheneity was observed in the isolated CTCs. Markers of EMT/MET phenotypes including Vimentin, EpCAM, HER2, CDH1, CDH2 and cytokeratin in the CTC samples were differentially expressed. These studies demonstrate the feasibility of utilizing the GO nano-chip in CTC isolation and characterization from metastatic breast cancer patients. Given the high sensitivity and the reproducibility to measure protein/gene expression levels of CTC biomarkers, we envision that our GO nano-chip may provide potential tool for real-time monitoring of cancer patients on clinical trials.

#1686

Glypican-3 (GPC3) induces a mesenchymal-epithelial transition in human breast cancer cells lines.

Lilian F. Castillo, Rocio Tascon, Elisa Bal de Kier Joffe, Giselle Peters. _Instituto de Oncologia Angel H. Roffo, CABA, Argentina_.

Glypicans constitute a family of heparan sulphate proteoglycans which are linked to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol anchor. Since GPC3 has been linked to cancer, herein we generated and characterized a pre-clinical human breast cancer cell model to evaluate the role of GPC3 in human mammary tumor progression. GPC3 expression was blocked in MCF-7 cells (poorly-metastatic, GPC3 +) by siRNA (generating MCF-7-shGPC3 sublines), while it was overexpressed in MDA-MB231cells (metastatic, GPC3 -) by viral infection (producing MDA-MB231-GPC3 sublines).

We performed in vitro and in vivo characterization of this model. GPC3 silenced MCF-7 cells acquired F-actin stress fibers, enhanced their clonogenic ability (Colony number 130 -shGPC3 vs. 37 -sh Negative Control (NC)) as well as their migration (Wound coverage (%): 15 -shGPC3 vs. 2 -shNC), were less susceptible to cell death induction (Cell death (%): 21.6 -shGPC3 vs. 34 -shNC), diminished the expression of the epithelial marker E-Cadherin while acquired the mesenchymal marker N-Cadherin, and were more invasive and metastatic in vivo. These cells exhibited an upregulation of the EMT-transcription factor ZEB1, and the canonical Wnt/beta-Catenin signaling pathway was activated.

GPC3 overexpressing MDA-MB231 cells misplaced their fibroblast-like appearance as well as their F-actin stress fibers, repressed their clonogenicity (Colony number: 8 -GPC3 vs. 40 -vector) and migratory capacity (Wound coverage (%): 10 -GPC3 vs. 90 -vector), were more sensitive to death in starving condition (Cell death (%) 30 -GPC3 vs. 6.5 -vector), got the ability to form E-Cadherin dependent-spheroids, reexpressed E-Cadherin in cell-cell junctions and downregulated the mesenchymal markers N-Cadherin and vimentin, while were less invasive and metastatic in vivo. The expression of the E-Cadherin repressor ZEB1 was diminished, as well as the activity of the canonical Wnt/beta-Catenin signaling pathway. However, when canonical Wnt signaling was activated by LiCl, the expression of E-Cadherin did not change.

In summary, we present in vitro and in vivo experimental evidence supporting the hypothesis that GPC3 has a protective role of human breast cancer progression by inducing mesenchymal-epithelial transition (MET). The canonical Wnt/beta-Catenin signaling inhibition would not be required for MET process induced by GPC3.

#1687

KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A.

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

Background & Purpose

In recent, KIF5B-RET gene rearrangement has been discovered as a driver oncogene in non-small cell lung cancers. This fusion gene was demonstrated to be able to induce tumorigenesis in vivo mouse model. Hereby, we investigated how KIF5B-RET fusion gene affect tumor differentiation and which transcription factors and signaling pathway are activated.

Methods

We analyzed cell proliferative activity in transformed HEK293T cells with KIF5B-RET fusion gene (K4) and empty vector (V5) using CCK8 assay. We used light microscopy and H&E staining to observe morphological changes of transformed cells. We evaluated protein and mRNA expressions of four EMT markers (E-cadherin, N-cadherin, Snail, and Twist) and two transcription factors (FOXA2, p-STAT5A) using transcription factor PCR array, Western blot, RT-PCR, immunofluorescence staining.

Results

K4 cells showed decreased cell proliferative activity compared with V5 cells. V5 cells had spreading pattern alike mesenchymal phenotype when their density was lower. However, their morphology changed to epithelial phenotype as they were overgrown with higher density. On the other hands, K4 cells exhibited the island growing pattern similar to epithelial phenotype in lower density of tumor cells, but changed to mesenchymal phenotype as the density of tumor cells gradually increased. On H&E staining, the cytoplasm of K4 cells was much smaller than that of V5 cells. We observed that E-cadherin mRNA was down-regulated, but N-cadherin and Twist were up-regulated in K4 cells. Based on the results of repeated screening assay, we finally selected two transcription factors, FOXA2 and STAT5A, which were significantly overexpressed in K4 cells. In K4 cells, phosphorylated-STAT5A was increased and FOXA2 was decreased compared to V5 cells. In addition, the expression of E-cadherin was reduced, but p-GSK3β, p-AKT, and p-ERK were markedly increased in K4 cells. The pattern of immunofluorescence staining of V5 and K4 cells were similar to the protein expressions of E-cadherin and p-STAT5A in Western blot analysis.

Conclusion

Taken together, FOXA2 and E-cadherin are down-regulated and p-STAT5A is up-regulated in HEK-293T cells stably expressing the KIF5B-RET. Our data suggest that KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A.

#1688

Mutation and gene expression analysis of circulating tumor cells (CTCs) enriched and retrieved by a sensitive microfluidic device.

Yixin Wang. _Celsee Diagnostics, Plymouth, MI_.

Molecular characterization of CTCs is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment devices and assays. We have developed a novel technology to enrich and retrieve CTCs from blood samples using a microfluidic chip. The Celsee PREP100 device captures CTCs with high sensitivity and then allows the captured CTCs to be retrieved for subsequent molecular analysis. The Celsee PREP100 system uses the microfluidic chip which has approximately 56,320 capture chambers. Based on differences in cell size and deformability, each chamber ensures that small blood cells such as red blood cells and most leukocytes escape while larger CTCs are trapped and isolated in the chambers. In the work we used the Celsee PREP100 to capture breast and lung cancer cells spiked into normal donor blood samples, including SKBR3 and NCI-H1975. We were able to show that the device can capture 20 cells with high reproducibility. The captured CTCs were retrieved from the microfluidic chip and further purified with anti-CD45 magnetic beads. Our results suggest that the cell recovery rate of this back-flow procedure is greater than 80% and the level of remaining background leukocytes is very low (about 100 cells). Work in progress to extract DNA and RNA. The resulting DNA samples are subjected to mutation analysis of a panel of cancer markers by using PCR and sequencing. The resulting RNA samples are converted to cDNAs, and gene expression analysis of selected cancer markers are carried out by using RT-QPCR. Specific DNA mutations such as EGFR exon 21 (L585R) in NCI-H1975 and mRNA expression markers including CK-19, TTF1 are tested in the retrieved cells. The sensitive and easy-to-use Celsee PREP100 system represents a promising technology for CTC capturing and molecular characterization. Clinical blood samples from patients with metastatic cancer are being tested on the Celsee PREP100 device and the results will be discussed.

#1689

Resveratrol inhibits epithelial-to-mesenchymal transition and metastasis in colorectal cancer through regulating Snail/E-cadherin expression by TGFβ1/Smads signaling pathway.

Qing Ji,1 Zhifen Han,1 Lihong Zhou,1 Hua Sui,1 Xuan Liu,1 Jianlin Ren,2 Fenggang Hou,2 Ronghua Zhao,3 Qi Li1. 1 _ShuGuang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China;_ 2 _Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai, China;_ 3 _University of Saskatchewan College of Medicine, Shanghai, China_.

Resveratrol has been an ideal alternative drug in the therapy of different cancers including colorectal cancer (CRC). Since the underlying mechanisms of resveratrol on the invasion and metastasis of CRC have not been fully elucidated, and epithelial-to-mesenchymal transition (EMT) is a key process associated with the progression of CRC, here we aimed to investigate the potential mechanism of resveratrol. We investigated the anticancer effect of resveratrol against LoVo cells in vitro and in vivo. In vivo, the impact of resveratrol on invasion and metastasis was investigated by mice tail vein injection model and mice orthotopic transplantation tumor model. In vivo imaging was applied to observe the lungs metastases, and hemaoxylin-eosin (HE) staining was used to evaluate metastatic lesions from lung metastases or hepatic metastases. In vitro, impact of resveratrol on the migration and invasion of LoVo cells was evaluated by transwell assay. Inhibition effect of resveratrol on TGF-β-induced EMT was examined by morphological observation. Epithelial phenotype marker E-cadherin and mesenchymal phenotype marker Vimentin were detected by western blot and immunofluorescence. The promoter activity of E-cadherin was measured using a dual-luciferase assay kit. The mRNA expression of Snail and E-cadherin was measured by RT-PCR. We demonstrated that, resveratrol inhibited the lung metastases of CRC LoVo cells in vivo. In addition, resveratrol reduced the rate of lung metastases and hepatic metastases in mice orthotopic transplantation. In vitro, TGF-β1-induced EMT promoted the invasion and metastasis of CRC, reduced the expression of E-cadherin and elevated the expression of Vimentin, and activated the TGF-β1/Smads signaling pathway. But resveratrol could inhibit the invasive and migratory ability of LoVo cells in a concentration-dependent manner, increased the expression of E-cadherin, repressed the expression of Vimentin, as well as the inhibition of TGF-β1/Smads signaling pathway. Meanwhile, resveratrol reduced the level of EMT-inducing transcription factors Snail and the transcription of E-cadherin during the initiation of TGF-β1-induced EMT. Our new findings provided evidence that, resveratrol could inhibit EMT in CRC through TGF-β1/Smads signaling pathway mediated Snail/E-cadherin expression, and this might the potential mechanism of resveratrol on the inhibition of invasion and metastases in CRC.

#1690

IGF-1 and TGF-β promote EMT and angiogenesis in 3D cultures of lung adenocarcinoma cells: A pilot study.

Imad Tarhoni,1 Gabriela C. Lobato,1 Cristina Fhied,1 Jeffrey Borgia,1 Melissa Pergande,1 Yi Wen Chai2. 1 _Rush University, Chicago, IL;_ 2 _BRTI, Two Harbors, MN_.

Background

Lung cancer is leading cause of cancer related deaths worldwide, with metastasis underlying this high mortality rate. An epithelial to mesenchymal transition (EMT) and angiogenesis are processes well known to increase tumor cell invasive potential. Transforming growth factor - beta (TGF-β) and insulin-like growth factor (IGF-1) are well established inducers of EMT and promoters of angiogenesis. In this pilot study, we tested the effects of IGF-1 and TGF-β on the lung adenocarcinoma cell line, A549, using a novel 3D cell culture method.

Method

A549 lung adenocarcinoma cells were seeded (15 million cells/ 500 µL 3D cocoon) in a Cell Mate 3D matrix (BRTI). After 24 h of recovery the cocoons were maintained in DMEM containing 2.5% FBS and received either IGF-1 (100 ng/mL), TGF-beta (20 ng/mL) for up to 7 days. A fourth group receiving only DMEM medium with 2.5% FBS was used for control purposes. Cocoons were formalin-fixed and paraffin embedded; 5um sections were prepared and stained for vimentin and E-cadherin. Cell-conditioned media was evaluated for angiogenesis factors(MILLIPLEX® Human Angiogenesis Kits (EMD Millipore®, Billerica, MA)) using the Luminex immunobead platform (Luminex Corp., Austin, TX). All data were then analyzed using SPSS.

Results

80% of TGF-β treated cells expressed vimentin compared to 27.5% in the control group. Cell staining strongly positive for vimentin demonstrated increased migration to the periphery compared to cells that were positive for E-cadherin. Both of IGF-1 and TGF-β significantly upregulated VEGF-A, IL-8, sHer2. Tenascin-C and Follistatin were significantly increased with TGF-β only (p<0.01). sVEGFR2 and sVEGFR3 were down regulated by IGF-1 and upregulated by TGF-beta relative to control (p<0.05). G-CSF, FGF-2, sIL6R, sAXLs, HGFR, Neuropillin-1 and suPAR were significantly higher in IGF-1 and lower in TGF-β (p<0.01) stimulated cultures. Other factors, such as VEGF-D, EGF, and Angiopoietin-2, showed significant decrease in both treated groups compared with control (p<0.05).

Conclusion

A549 cells in this 3D model demonstrated increased metastatic potential with associated EMT features and increased expression of angiogenesis factors upon stimulation with IGF-1 and TGF-β. These findings will be contrasted to 2D conventional culture methods for the substantiation for the 3D scaffolds for routine culturing. Evaluation of EMT in a 3D format is a critical tool for cancer research as it emulates dynamic interactions of the cancer cells with surrounding matrix, the tumor microenvironment in a more practical fashion than conventional 2D culture methods.

#1691

TMPRSS2:ERG fusion enhances osteoblastic phenotype of prostate cancer bone metastases.

Carine Delliaux,1 Tian V. Tian,2 Mathilde Bouchet,3 Anaïs Fradet,3 Nathalie Vanpouille,1 Anne Flourens,1 Rachel Deplus,4 Xavier Leroy,5 Yvan de Launoit,1 Edith Bonnelye,3 Martine Duterque-Coquillaud1. 1 _Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T – Mechanisms of Tumorigenesis and Target Therapies, Lille, France;_ 2 _Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T – Mechanisms of Tumorigenesis and Target Therapies, Lille, France; Center for Genomic Regulation, Barcelona, Spain;_ 3 _Unité INSERM U1033; Université Claude Bernard Lyon 1, Lyon, France;_ 4 _Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161 - M3T – Mechanisms of Tumorigenesis and Target Therapies, Lille, France; Institut Laboratory of Cancer Epigenetics, Faculty of Medicine, ULB, Brussels, Belgium;_ 5 _Institut de Pathologie-Centre de Biologie-Pathologie-Centre Hospitalier Régional et Universitaire, Lille, France_.

Background: Bone metastases are the major cause of morbidity and mortality in prostate cancer (PCa) patients. Recently, TMPRSS2:ERG gene fusions produced by rearrangements along chromosome 21 was found in more than 50% of PCa samples, resulting in androgen-dependent aberrant expression of the functional ERG transcription factor. Interestingly, ERG transcription factor has been previously shown to be involved in bone development. This study is therefore focused on investigating whether TMPRSS2:ERG gene fusions are involved in PCa bone metastasis development.

Methods: We previously established cell clones overexpressing TMPRSS2:ERG from two PCa cell lines, PC3 and PC3c. We first studied induced bone lesion phenotype using in vivo intra-tibial injection models. Secondly, we analyzed transcriptional changes induced by ERG transcription factors in PC3c clones to identify potential target genes. Then, direct target genes were further validated and investigated in vitro using RT-qPCR, ELISA, siRNA and ChIP techniques. Importantly, using a cohort of prostate carcinoma samples, we validated the expression correlation between ERG and its target target genes expression in human pathology.

Results: Bone lesions induced in vivo by intra-tibial injections of PC3 or PC3c cells are known to be osteolytic or mixed (osteoblastic/osteolytic) respectively. Interestingly, intra-tibial injections of PC3c clones expressing the fusion showed a statistically significant increase of osteoblastic phenotype compared to control cells. Furthermore, intra-tibial injections of PC3 clones expressing the fusion showed a strong decrease of osteolytic phenotype, reinforcing our previous result in PC3c clones.

Among the genes identified by transcriptomic study and dysregulated in PC3c clones expressing the fusion, we have identified the ERG candidate target Endothelin-1 (ET-1), which is known to be involved in osteoblast proliferation and in osteoblastic metastasis formation in PCa. Indeed, we found that ET-1 expression was up-regulated in PC3c-TMPRSS2:ERG clones, and the up-regulation was dependent on ERG expression levels. Importantly, silencing of ERG resulted in decreased expression of ET-1. In silico analysis of the promoter of ET-1 revealed the presence of several potential binding sites of ERG. ChIP experiments followed by qPCR demonstrated a direct binding to one of them. Moreover, in human PCa samples, there was a significant expression correlation between ET-1 and fusion gene TMPRSS2:ERG, reinforcing the direct functional link between ET-1 and the fusion in PCa.

Conclusion: Taken together, these results strongly suggest that the TMPRSS2:ERG gene fusion contributes to the osteoblastic phenotype of PCa bone metastases and ET-1 is one of the involved factors, directly regulated by the transcription factor ERG.

#1692

Role of snail in cancer-bone microenvironment interactions.

Veronica M. Henderson, Liza J. Burton, Simone M. Howard, Valerie A. Odero-Marah. _Clark Atlanta University, Atlanta, GA_.

Prostate Cancer (PCa) is the second leading cause of cancer death in American men and African Americans are twice as likely to get PCa as their Caucasian counterparts. As with most forms of cancer, PCa patients' mortality is mainly attributed to complications caused by metastasis of the disease to organs critical for survival such as bone. As such, it is important to understand cancer-bone microenvironment interactions in order to develop therapeutics that will slow or halt the process of cancer metastasis. It is also known that African Americans have a higher bone mineral density compared to any other race. Snail1 is a zinc-finger transcription factor that induces epithelial-mesenchymal transition (EMT) which is associated with cell migration and metastasis in cancer cells. In preliminary studies, cancer cells co-cultured with bovine bone discs led to increased calcium release that was higher in cancer cells overexpressing Snail, as well as in bone discs of higher density. We hypothesized that cancer cell-bone interactions would promote higher calcium release from bone by cancer cells overexpressing Snail, which would lead to increased paracrine cell migration. For this study, we utilized prostate (ARCaP) or breast (MCF-7) cancer cells stably overexpressing Snail as well as prostate (C4-2) cancer cells with stable Snail knockdown. Cancer cells were co-cultured with bovine bone disc or Hydroxyapatite (HA; inorganic component of bone) of different densities to represent the African American vs Caucasian bone ratio. The conditioned media was then used for to assay calcium levels, test paracrine cell viability and migration assays using C4-2 parental cells. We observed that calcium levels were elevated in conditioned media from cancer cell-bone co-cultures, compared to media alone or media plus bone, and this could be antagonized by EGTA, a calcium chelator. It was increased with higher bone density as well as with bone co-cultured with MCF-7-Snail, ARCaP-Snail and C4-2 cancer cells as compared to MCF-7 Neo, ARCaP-Neo or C4-2 with Snail knockdown. When we utilized the conditioned media for a paracrine cell viability assay, there was no significant differences. However, C4-2 cancer-bone co-culture conditioned media increased paracrine cell migration which was decreased by Snail knockdown as well as lower bone density. Hence Snail expressing PCa cells co-cultured with HA led to increased paracrine cell migration. We are currently studying the signaling mechanism(s) involved in this cancer-bone interactions. In conclusion, our study shows that Snail can mediate cancer-bone microenvironment interactions that can possibly promote increased cell migration towards bone of high mineral density such as is found in African Americans.

GRANT SUPPORT: 1P20MD002285

#1693

Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases.

Anna Reustle,1 Steffen Rausch,2 Stefan Winter,1 Florian Büttner,1 Stephan Kruck,2 Joerg Hennenlotter,2 Marcus Scharpf,3 Falko Fend,3 Jens Bedke,2 Arnulf Stenzl,2 Matthias Schwab,4 Elke Schaeffeler1. 1 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany;_ 2 _Department of Urology, University Hospital Tuebingen, Tuebingen, Germany;_ 3 _Institute of Pathology and Neuropathology, University Hospital Tuebingen, Tuebingen, Germany;_ 4 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart and Department of Clinical Pharmacology, University Hospital Tuebingen, Stuttgart, Germany_.

Background/Purpose:

Nicotinamide N-methyltransferase (NNMT) is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. It is overexpressed in different cancers and has been shown to enhance the migratory and invasive activity of cancer cells. Additionally, NNMT is an important regulator of the methylation potential in cells by consumption of S-adenosyl methionine (SAM), an important substrate for methyltransferases. By this mechanism NNMT reduces methylation of proteins such as certain histones leading to epigenetic remodeling and induced expression of tumor-promoting genes (Ulanovskaya et al, Nat. Chem. Biol. 2013). In clear cell renal cell carcinoma (ccRCC), the dominant histological subtype of renal cell cancer, NNMT is expressed at high levels compared to normal kidney tissue and could represent a novel therapeutic target, especially for the treatment of metastatic disease which is marked by a five-year survival rate of below 10% with currently available therapies.

Methods:

We analyzed NNMT in a ccRCC patient cohort from The Cancer Genome Atlas (TCGA) concerning mRNA expression (n=463), somatic variations (n=410) and association with clinical parameters and patient survival. In an independent collection of ccRCC tissues (n=64), corresponding normal kidney tissues and metastatic tissues from different organs (n=129), NNMT mRNA and protein expression were quantified via Real-Time PCR and immunohistochemical staining of tissue microarrays, respectively.

Results:

In both cohorts, NNMT mRNA expression was significantly upregulated in tumor compared to non-tumor tissue. Somatic mutations and copy number alterations in the NNMT gene locus were observed in only a small number of patients. NNMT mRNA expression correlated weakly with tumor stage and grading in both cohorts, but not with the presence of lymph node or distant metastases. In Kaplan-Meier analyses high NNMT expression was associated with worse overall and cancer-specific survival. In ccRCC metastases from different organ sites, NNMT mRNA and protein expression were also induced compared to normal kidney tissue and high expression was again associated with worse patient survival in Kaplan-Meier analysis (HR=2.15, Plog-rank=0.05).

Conclusion:

NNMT mRNA and protein are highly expressed not only in primary tissue of ccRCC, but also in metastases derived from different organs, offering it as a potential drug-target for metastatic ccRCC. Although NNMT expression is a good prognostic indicator for patient survival, its molecular function in ccRCC tumorigenesis and metastasis formation has to be further evaluated.

Supported by the Robert Bosch Foundation, Stuttgart, Germany and the ICEPHA Grant Tuebingen-Stuttgart, Germany

#1694

Conjugated bile acids aggravate metastatic pancreatic cancer via sphingosine-1-phosphate receptor 2.

Hiroaki Aoki, Masayo Aoki, Eriko Katsuta, Partha Mukhopadhyay, Jing Yang, Huiping Zhou, Sarah Spiegel, Kazuaki Takabe. _Virginia Commonwealth Univ., Richmond, VA_.

Introduction: "Painless jaundice" is a classic sign of advanced pancreatic cancer, which is often times lethal. Bile drainage for obstructive jaundice by pancreatic cancer has been known to be clinically beneficial, however, the role of bile acids on pancreatic cancer biology has not been thoroughly investigated. We published that conjugated bile acids bind to sphingosine 1-phosphate receptor 2 (S1PR2) (Hepatology 2012) and regulate lipid metabolism in hepatocytes through SphK2 (Hepatology 2015). SphK1 and K2 are the enzymes that generate sphingosine-1-phosphate (S1P), a lipid mediator, which cause cell proliferation and migration via S1P receptors 1-5. We hypothesize that bile acid from obstructive jaundice aggravate the pancreatic cancer progression via S1PR2.

Method: Murine and human pancreatic cancer cell lines were used to examine growth response to CYM5520 (S1PR2 agonist), JTE-013 (S1PR2 antagonist), or FTY720 (functional antagonist of all S1P receptors except S1PR2). Obstructive jaundice model was made by total ligation of middle and left bile ducts with cholecystectomy, and Panc02-luc cells were implanted in the left lobe for liver metastasis, and intraperitoneally injected for carcinomatosis model.

Result: Stimulation by TCA or CYM5520 promoted Panc02-luc and AsPC-1 cell proliferation, which dominantly express S1PR2 among S1P receptors in dose dependent manner, but that was not the case in other cells (PANC-1, BxPC-3 and Miapaca-2) that express other S1P receptors. JTE-013 inhibited Panc02-luc and AsPC-1 cell growth, regardless of additional TCA stimulation. Treatment with FTY720 had no effect on S1P mediated Panc02-luc cell proliferation. Neither TCA nor CYM5520 was able to stimulate growth of Panc-1, BxPC-3, nor Miapaca-2. Obstructive jaundice significantly increased liver metastasis and carcinomatosis tumor burden, and significantly shortened survival. The role of S1P from the host was investigated by using Panc02-luc carcinomatosis model on SphK1 or SphK2 knockout mice. Unexpectedly, carcinomatosis progressed slower with less tumor burden in both SphK1 and K2 knockout mice and significantly prolonged survival compared with their littermate control wildtype. Significantly less proliferation, but no difference in apoptosis was seen in tumors in SphK1 knockout mice. Treatment by FTY720 to carcinomatosis of wildtype mice did not suppress cancer progression nor tumor burden.

Conclusion: Conjugated bile acids from obstructive jaundice aggravated metastatic pancreatic cancer via S1PR2, which is independent from host S1P.

#1695

Vascularization of colorectal cancer liver metastasis: correlation with growth patterns.

Anthoula Lazaris,1 Abdellatif Amri,1 Pablo Zoroquiain,1 Stephanie K. Petrillo,1 Rafif Mattar,1 Zu-Hua Gao,1 Peter Vermeulen,2 Peter Metrakos1. 1 _McGill University Health Centre- Research Institute, Montreal, Quebec, Canada;_ 2 _Translational Cancer Research Unit (TCRU), GZA Hospitals, Antwerp, Belgium_.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in North America. Approximately 50% of patients will be diagnosed with CRC liver metastasis (CRLM) during the course of their disease. Untreated, patients will survive for only a few months, but with chemotherapy, a median survival of 20 months can be achieved. At present, no reliable indicators exist to predict outcome or prognosis in treatable patients. Moreover, no biological parameters are currently being considered for patient stratification into different treatment groups.

We examined CRCLM resected from patients and have identified two major histologic growth patterns (HGP): a desmoplastic (DHGP) pattern and a replacement (RHGP) pattern where tumor cells replace parenchymal cells in the liver plates. These HGP involve distinct modes of angiogenesis and host cell responses. Namely, liver metastases with a RHGP grow by co-opting the stroma, without hypoxia-induced angiogenesis and with little perturbation of the liver architecture. In contrast, metastases with a DHGP show characteristics of ongoing, hypoxia-driven angiogenesis including increased fibrin deposition at the tumour-liver interface and increased endothelial cell proliferation. In addition RHGP is associated with poor clinical response to bevacizumab chemotherapy. These differences suggest that the two patterns trigger distinct microenvironment responses and as a consequence, may initiate and utilize different modes of vascularization and expansion. Our hypothesis is that there are distinct gene expression signatures in the tumor and/or host compartments of different HGPs, which will shed light on the biological mechanisms underlying this diversity of HGPs. To test this hypothesis we: i) have extracted high quality RNA from lesions of chemonaïve patients and through RNAseq analysis identified gene expression differences between DHGP, RHGP lesions ii.) to further understand the role of tumor associated vascularity, we have stained different lesions (chemonaïve, chemo only and chemo + bevacizumab) with vascular markers to look at tumor associated blood vessels (immature, intermediate and mature: aSMA1 & CD31) and the rate of endothelial cell proliferation (Ki67/CD34).

Our preliminary data demonstrates the expression of neo-vessels in the DHGP desmoplastic ring and in the peripheral tumor along the parenchyma tissue have lower vascularity with fewer branches that are supplying the tumor. The RHGP has similar vasculature to the adjacent normal tissue with a sinusoidal network of blood supply and a higher vascularity than DHGP. We are now correlating these findings with RNAseq expression data.

This work will result in the identification of targets in the HGPs that will help stratify patients in terms of treatment. We currently have less treatment options for the RHGP patients and it would be important to utilize the data from this study to develop new treatment strategies for those patients.

#1696

Mitochondrial haplotype alters metastasis in a non-cell autonomous manner.

Amanda E. Brinker, Carolyn J. Vivian, Danny R. Welch. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

Mitochondrial Nuclear Exchange (MNX) mice, created by transferring the nucleus from an oocyte from strain x into an enucleated oocyte of strain y, showed that mammary tumor formation and metastasis can be regulated by inherited mitochondrial polymorphisms (PMID26471915). Besides genetic (cell autonomous) changes observed, we asked whether mitochondrial polymorphisms in non-cancer compartments could exert effects on tumor formation or metastasis. Tumor cells were injected into syngeneic wild type mice and tumor growth and metastasis were compared to similarly injected cells into MNX mice sharing the same nuclear but different mtDNA backgrounds. Orthotopic tumor growth rates were equal for all of the cell lines tested. However, the ability to form experimental lung metastases following i.v. injection were dramatically altered. Compared to injections into wild-type, syngeneic mice, E0771 mammary carcinoma and B16-F10 melanoma cells (both syngeneic to C57BL/6J), formed significantly (P<0.01) more lung metastases in C57BL/6J-mtMNX(C3H/HeN) and K1735-M2 melanoma cells (syngeneic to C3H/HeN) formed significantly fewer lung metastases in C3H/HeNmtMNXC57BL/6J. These results have been replicated at least three times using >10 mice per experiment. Interestingly, C57BL/6J mitochondria confer resistance to metastasis in both cell autonomous and non-cell autonomous experiments. Basal metabolic differences comparing mouse embryonic fibroblasts isolated from wild-type and MNX mice are among mechanisms being explored. Together, our findings highlight the striking influences that mitochondrial haplotypes can exert on tumorigenicity and metastasis via both intrinsic and extrinsic mechanisms. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Steiner Family Fund for Metastasis Research, Kansas Bioscience Authority, CA134981, P30-CA168524

#1697

Label-free separation and concentration of cancer cells by acoustophoresis.

Cecilia Magnusson, Andreas Lenshof, Per Augustsson, Maria Antfolk, Thomas Laurell, Hans Lilja. _Lund Univ., Lund, Sweden_.

Introduction: Circulating tumor cells (CTCs) is a promising tool for disease monitoring and for better-targeted therapies for patients with disseminating tumors. However, isolation of CTCs is challenging due to their scarcity, variation in size, morphology, and expression profile. The lack of a universal marker impairs reliable detection and characterization of CTCs. We are using acoustophoresis, ultrasound standing wave radiation forces to enable label free separation of cancer cells from white blood cells (WBC). The cell separation is based on intrinsic cells properties such as size, density and compressibility.

Materials and Methods: The acoustic setup is a glass/silicon microchip connected to a pressure driven system. The system is temperature controlled and has a pre-alignment step for optimal separation performance. The separation system can process samples up to 10 mL, with a processing speed of 1 mL in 13 minutes without compromising the cell separation capacity. If required the separation step can be complemented with a subsequent concentration channel, which allows at least 20x concentration of the sample, by extracting the cells in a smaller liquid volume compared to the input sample. The prostate cancer cell line DU145 and the breast cancer cell line MCF7 spiked in red blood cell (RBC) lysed blood from healthy donors, are used to evaluate the model system performance for cancer cell separation.

Results and Discussion: The acoustic separation model system is flexible and can be amended to suit different requirements and conditions. The cancer cell recovery after acoustic separation of blood samples spiked with 50 DU145 cells per mL is approximately 80% with a WBC contamination level of less than 0.3%. At this performance level there is a 100x cancer cell enrichment in the collected cancer cell fraction compared to input. However, by changing parameters such as the voltage setting and sample flow rate it is possible to regulate the cancer cell recovery and WBC contamination levels after acoustophoresis. Cancer cell recovery well above 90% is possible with slightly higher WBC contamination levels. If high cancer cell purity is of importance, a 1000x cancer cell enrichment can be achieved at the cost of slightly lower cancer cell recovery. RBC lysing is required before acoustic separation, due to cell crowding in the separation channel when the cell concentration is too high. We have identified eosinophils as the major contaminant in our enriched cancer cell fraction. They constitute 85% of the contaminating WBCs. The eosinophils have similar acoustic properties as the smaller cancer cells, and can therefore not be completely isolated from the cancer cells by acoustophoresis.

This work demonstrates the flexibility with acoustophoresis that allows for integration of three previously established units: pre-alignment, sorting and concentrating, onto the same chip. It also serves as a proof of concept for in line rare cell label free sorting and isolation.

#1698

Novel evidence that blood plasma vitronectin is a major chemoattractant for cancer cells and its pro-migratory activity is suppressed/chaperoned after binding to fibrinogen.

Gabriela Schneider,1 Nichola C. Garbett,1 Ewa Bryndza,1 Agata Poniewierska-Baran,1 Michael L. Merchant,1 Karol Serwin,2 Zachariah P. Sellers,1 Barbara Dolegowska,2 Mariusz Z. Ratajczak1. 1 _University of Louisville, Louisville, KY;_ 2 _Pomeranian Medical University in Szczecin, Szczecin, Poland_.

Background. One of the crucial problems with cancer therapy is migration of cancer cells that leave primary tumor and migrate to vital organs where they form metastases. Throughout the years several factors have been identified that direct metastatic process including growth factors, chemokines, bioactive lipids or extracellular nucleotides. To our surprise however, we noticed that highly diluted plasma (1%) possess remarkable chemokinetic activity against several cancer cell lines that highly exceeds those observed for optimal doses of chemokines (e.g. SDF-1) and growth factors (e.g. HGF/SF), that are considered as a main metastatic factors present in plasma. Aim of the study. Based on this observation our aim was to identify the "factor/s" responsible for this remarkable chemokinetic activity of normal diluted 1% plasma. Experimental strategies. We employed several human cancer cell lines and different dilutions of normal human, murine and bovine plasma and serum in Transwell migration assays, adhesion and cell signaling studies. We tested effect of heat inactivation, protease exposure, dialysis and molecular filtration on chemotactic activity of plasma. We also used gel filtration followed by hydrophobic interaction chromatography (HIC) to identified plasma fractions with the most potent chemotactic activity that were further analyze using MassSpec. Results. We found that remarkable chemokinetic activity of highly diluted 1% plasma against malignant cells, rapidly decreased at higher plasma concentration (>5%). Our initial characterization studies revealed that this "activity" is sensitive to proteolytic treatment, is not removed from plasma by dialysis and is temperature sensitive. The similar effect has been observed for diluted 1% serum however chemotactic responsiveness of serum was maintained with its higher concentrations. Based on this we hypothesized possible involvement of inhibitory effect of fibrinogen, which was subsequently confirmed in experiments where fibrinogen was removed from plasma or it was added to serum. Finally, gel filtration followed by HIC and MasSpec analysis allow us to identify several possible candidates that were further tested in in vitro experiments. These studies revealed that vitronectin (VTN) is a main factor responsible for observed chemotactic response and that its effect can be inhibited by fibrinogen. Conclusions. Our data indicate that VTN present in normal plasma is a main migration inducing factor responsible for metastasis of malignant cells and that VTN is more potent chemoattractant than already known pro-metastatic chemokines or growth factors. Pro-migratory effect of VTN is neutralized/chaperoned by fibrinogen what may explain preference in migration of tumor cells into lymphatic vessels and body cavities, where concentration of fibrinogen is low.

#1699

Protumoral role of trans fatty acid in colorectal cancer.

Hitoshi Ohmori,1 Toshio Kuwai,2 Yasuhiko Kitadai,3 Kiyomu Fujii,1 Rina Fujiwara,1 Yukiko Nishiguchi,1 Tomonori Sasahira,1 Hiroki Kuniyasu1. 1 _Molecular Pathology, Nara Medical University, Kashihara, Japan;_ 2 _Gastroenterology, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure, Japan;_ 3 _Gastroenterology and Metabolism, Hiroshima University Graduate School, Kashihara, Japan_.

Trans fatty acids (TFAs) is a recent focus of health problems. TFA is a definitive risk factor for cardiovascular diseases and the death. TFA is also possible risk factor for Alzheimer's disease, obesity, diabetes, fatty liver, and ovulation infertility. Relation of TFA with carcinogenic risk is controversy; however, TFA is reported to increase the risk of breast cancer and prostate cancer. Elaidic acid (EA), a trans form of oleic acid, enhances cancer cell growth, invasion, and anti-apoptotic survival. By animal models, EA promotes tumor growth and metastasis to the lung, liver, and peritoneum. EA induces stemness in cancer cells through transactivation of EFGR via SRC from GPR40/120 as receptors in EA-integrated cholesterol raft. Activated EGFR relays the signals to activate canonical and non-canonical wnt pathway and to inactivate notch1 pathways. EA also increases miR-494, which inhibits cell differentiation through decrease of target genes. Continuous EA feeding with dosage alteration increased cancer cell stemness. EA diminishes the efficiency of 5-fluorouracil by increase of residual cancer stem cells. These findings suggest that TFA is a relevant cancer promoting factor. Decision of removal of TFA from foods by FDA might provide an impact to cancer clinics.

#1700

Novel microfluidic blood-brain niche to study breast cancer metastasis to the brain.

Megan Altemus, Brendan Leung, Aki Morikawa, Michele Dziubinski, Maria Castro, Sofia Merajver. _University of Michigan, Ann Arbor, MI_.

Metastasis from the primary tumor site to the brain is the most lethal complication of advanced cancer. Once a patient has developed brain metastasis, treatment involves surgical resection or radiation and is often only palliative. Poor prognosis following these therapies as well as the presence of concurrent disease outside of the nervous system emphasize the need for new systemic therapies that can reach and are active within the brain. In order to study the process of brain metastasis and develop potential therapies, models that mimic the human blood-brain niche must be used. Models currently utilized are murine in vivo models and various in vitro methods that do not accommodate all the biophysical characteristics of a blood-brain barrier. In vivo murine models are costly, time intensive, very slow to manifest metastases, and the metastatic process is seldom amenable to monitoring in real time. Current in vitro models are faster and more cost effective; however the models currently used do not have the same micro-environment complexity as "live" models. Here we describe a novel microfluidic device that accurately mimics the physical and cellular components of the human blood-brain niche to study the brain metastatic process.

This device is composed of two chambers separated by a porous membrane. The top chamber and apical side of the membrane is seeded with human brain endothelial cells with and uses flow to mimic shear stress encountered within the vasculature. Cancer cells are introduced into this chamber in which they adhere to and migrate through the endothelium into the bottom chamber. The bottom chamber contains astrocytes suspended in a collagen gel to mimic the brain stroma and provide room for invading cancer cells to colonize and grow. Barrier integrity is monitored using TEER (trans-endothelial electrical resistance), and fluctuates as the tight junctions of the endothelium are compromised by invading cancer cells. Throughout all time points, from introduction into the flow chamber, adherence to the endothelium, extravasation through the barrier, migration into the stroma, and proliferation the cancer cells can be monitored via microscopy or TEER.

This device provides a powerful novel tool to study the brain metastatic process and can be used to determine differences in brain metastatic potential of various cell lines or patient derived material, study the molecular mechanisms that promote brain metastasis, and test potential new therapies.

#1701

CCR6 associates with colon cancer metastasis.

Neeraj Kapur,1 Hina Mir,1 Clarence E. Clark,1 Uma Krishnamurti,2 Derrick J. Beech,1 James W. Lillard,1 Shailesh Singh1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Emory University School of Medicine, Atlanta, GA_.

Despite established benefits of screening, colon cancer remains a leading cause of cancer death in the U.S. Majority of colon cancer deaths result from metastasis. Effective treatments are not available for advanced disease because molecular mechanisms of initiation and progression of this disease are yet to be defined. Chemokine-chemokine receptor interaction plays an important role in cancer progression. In this study, using colon cancer tissue microarray, we have shown that expression of CCR6 was significantly higher in advanced colon cancer (p<0.0001) with distant and regional lymph node metastasis as compared to non-metastatic and adjacent normal tissues. Expression of CCR6 was further confirmed in cell lines derived from Dukes's type C and type D colon cancer patients using flow cytometric analysis. Like tissues, CCR6 expression was significantly (p<0.0001) higher in colon cancer cell lines compared to normal colon epithelial cells. Furthermore, colon cancer cells showed higher migratory potential toward chemotactic gradient of its only known natural ligand, CCL20. Cell proliferation was also inhibited in presence of CCL20. Significant decrease in E-cadherin, increased expression of vimentin, β-catenin, N-cadherin, α-SMA, SNAIL and ZEB1 was observed following CCL20 treatment. These results suggest the importance of CCR6-CCL20 axis in the etiopathogenesis of colon cancer and highlight its potential as therapeutic target.

#1702

Sucrose nonfermenting 1-related kinase (SNRK) expression in ovarian cancer and correlation with clinical features.

Elizabeth E. Hopp,1 Erin Bishop,1 Stephanie Cossette,2 Ramani Ramchandran2. 1 _Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, WI;_ 2 _Department of Pediatrics, Children's Research Institute, Medical College of Wisconsin, Milwaukee, WI_.

Objective: Human omental adipocytes promote migration and invasion of ovarian cancer cells and result in alterations in metabolism including increased AMP-activated protein kinase (AMPK) and b-oxidation, which allow for use of lipids as an energy source. Sucrose nonfermenting 1-related kinase (SNRK) is a member of the AMPK family of serine/threonine kinases, which are activated by starvation and stress conditions. The goal of this study is to describe the expression of SNRK in human ovarian cancer tissue.

Methods: Tissue was collected from patients undergoing surgery for serous ovarian cancer or benign indications. Immunohistochemistry (IHC) was used to detect SNRK expression. SNRK+ cells were compared between benign and malignant tissue, and statistical significance between positivity and clinicopathologic variables was determined using the Mann-Whitney U test.

Results: In the 31 samples analyzed, SNRK demonstrates a nuclear staining pattern. IHC shows increased SNRK+ cells in malignant tissue versus benign tissue (42.0% versus 31.3% positive nuclei, p<0.001). When examining patient characteristics, the malignant samples with the lowest percentage of SNRK+ nuclei were stage IIIB-IV with a high incidence of recurrence and death due to disease.

Conclusions: This is the first study to describe SNRK expression in ovarian cancer tissue. We show that SNRK has a nuclear staining pattern and that expression is greater in malignant ovarian tissue compared to benign ovarian tissue, which may be due to more metabolic stress on cells with higher proliferation rates. SNRK expression appears to be lower in those with a higher disease stage and poor outcome potentially secondary to metabolic changes that occur once the disease metastasizes. We are currently examining SNRK expression in metastatic omental tumors in order to further explore expression differences between primary versus metastatic disease sites.

#1703

Expression of Hedgehog signals and growth inhibition by itraconazole in endometrial cancer.

Kayo Inoue,1 Hiroshi Tsubamoto,1 Hiroaki Shibahara,1 Kazuo Tamura2. 1 _Hyogo College of Medicine, Nishinomiya-shi, Hyogo, Japan;_ 2 _Kinki University, Osaka, Japan_.

Background: There are limited therapeutic opportunities for the treatment of endometrial cancer (EC). Itraconazole, a common antifungal agent and a potent inhibitor of the Hedgehog pathway, has been shown to be clinically effective for various types of cancer, but its clinical efficacy for EC is unknown. Herein, we evaluated the efficacy of itraconazole in treating EC.

Patients and Methods: We reviewed cases of patients with serous endometrial intraepithelial carcinoma (SEIC) and patients who were diagnosed with EC with endometrioid adenocarcinoma grade 1 or 2 at FIGO stage IA with myometrial invasion and negative cytology of ascites and who recurred at our institution from January 2005 to December 2013. We also reviewed cases with endometrioid adenocarcinoma grade 1 or 2 at FIGO stage IA with myometrial invasion and negative cytology of ascites without recurrence over 5 years after surgery during 2006 and 2007. Immunohistochemistry was performed on EC tumour samples including SEIC for sonic Hedgehog (sHh), glioma-associated oncogene homolog 1 (GLI1), Progesterone receptor (PR), and chemokine ligand 18 (CCL18). Furthermore, we examined the effects of itraconazole on human EC cell lines (Ishikawa and HEC-1A cells) using a modified methylthiazol tetrazolium (MTT) assay and a migration assay.

Results: SHh and GLI1 were expressed in SEIC and endometrioid adenocarcinoma. Immunohistochemical evaluation of sHh and GLI1 did not distinguish between recurrent and non-recurrent low-grade FIGO stage IA EC. The MTT assay revealed that itraconazole significantly inhibited the growth of Ishikawa and HEC-1A cells in a dose-dependent and time-dependent manner. However, migration was not significantly effected for either Ishikawa or HEC-1A cells. HEC-1A cells exhibited dose-dependent migration retardation.

Conclusion: Hedgehog signalling plays a role in carcinogenesis and malignant progression in EC. Itraconazole at a physiological dose may suppress progression of EC.

### Stemness Properties of Intestinal, Pancreatic, and Hepatic Cancer

#1704

High mobility group A1 chromatin remodeling protein expands the intestinal stem cell compartment and Paneth cell niche through Wnt/β-catenin signaling and Sox9.

Lingling Xian, Tait Huso, Amy Belton, David Huso, Linda M. S. Resar. _Johns Hopkins University School of Medicine, Baltimore, MD_.

The High Mobility Group A1 (HMGA1) gene is overexpressed in most poorly differentiated cancers and high levels portend adverse clinical outcomes, although the molecular mechanisms through which it functions are poorly understood. HMGA1 encodes the HMGA1a and HMGA1b chromatin remodeling proteins, which modulate gene expression by bending chromatin and orchestrating the assembly of transcription factor complexes to DNA. HMGA1 is highly expressed during embryogenesis, but silenced in adult, differentiated tissues. Postnatally, HMGA1 expression is maintained in adult stem cells, such as intestinal stem cells (ISCs); however, its role in this setting has been unknown. Here, we report that Hmga1 overexpression in ISCs of transgenic mice drives expansion in the ISC compartment leading to hyperproliferation, aberrant crypt formation, and polyposis. Surprisingly, Hmga1 transgenic mice also exhibit marked expansion in terminally differentiated Paneth cells, which comprise an epithelial cell niche for ISCs. To dissect the mechanisms mediating these phenotypes, we generated three-dimensional (3D) intestinal organoids with varied expression of Hmga1. Strikingly, silencing Hmga1 in wildtype crypt cells disrupts their ability to organize into functional 3D organoids with bud formation, while crypt cells expressing ectopic Hmga1 exhibit enhanced organoid formation with increased ISC number, proliferation, and bud development. Because Wnt/β-catenin signaling is central to ISC function, we determined whether Hmga1 modulates this pathway. β-catenin protein is increased in the crypts of the Hmga1 transgenic mice and organoids. Hmga1 amplifies Wnt/β-catenin signaling by inducing both genes that encode Wnt cell surface receptors and target genes downstream of Wnt/β-catenin. Hmga1 also directly up-regulates Sox9, which is required for terminal differentiation to Paneth cells. This is the first example of Hmga1 fostering terminal differentiation to establish a stem cell niche. In human intestinal epithelium, HMGA1 and SOX9 are highly correlated (P=0.008), and both become up-regulated in carcinogenesis. These results reveal a novel role for Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment and suggest that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis.

#1705

CCK2R marks a gastrin-responsive stem cell that gives rise to Barrett's esophagus.

Aleksandra M. Urbanska,1 Yoomi Lee,1 Yoku Hayakawa,1 Julian A. Abrams,1 Michael Quante,2 Timothy C. Wang1. 1 _Columbia University, New York, NY;_ 2 _Technical University of Munich, Munich, Germany_.

The incidence of esophageal adenocarcinoma (EAC) has markedly increased in the United States over the last few decades. Barrett's esophagus (BE) is the most significant known risk factor for this malignancy. Barrett's metaplasia, a condition of esophagus characterized by a columnar-cell metaplasia that replaces the native squamous-cell epithelium is considered to be a complication of gastroesophageal reflux disease (GERD), another risk factor for EAC. The paradigm is that BE arises from an adaptation to the harsh intra-esophageal environment of chronic GERD, and predisposes to EAC. Other established risk factors for EAC include obesity, smoking, poor diet. Moreover, while BE patients often undergo periodic endoscopic examinations, screening for dysplasia has not been shown to decrease mortality and remains controversial.

Thus, a better understanding of the risk factors for BE progression is needed. The precise role of CCK2R in the gastric cardia and Barrett's esophagus has not been defined. CCK2R is upregulated in the esophagus in human BE and EAC, and real-time PCR of BE tissues indicated that the level of expression was 2x higher than that in esophageal tissues from normal control patients. CCK2R is upregulated in the gastric cardia of mice with Barrett's metaplasia, and this effect is enhanced with administration of bile acids, a key component of the refluxate.

In this study we used the L2-IL-1β model to determine whether CCK2R-expressing cells can give rise to BE, and whether inhibition of CCK2R could ameliorate the BE phenotype in our mouse model.

In order to understand the role of CCK2R-expressing cells in normal cardia, we performed lineage tracing studies in CCK2R-CreERT mice. We generated CCK2R-CreERT/TdTomato mice and induced them with tamoxifen at 6 weeks of age. Single Tomato-red positive cells became visible at the base of the cardia at 24 hours after induction. By days 2, 3, and 5 these Tomato-red cells expanded and moved upward in the cardia gland in contiguous fashion until almost all the cells in the gland were labeled. Tomato-red cells persisted for as long as one year in the cardia. Thus, CCK2R labels a progenitor cell in the normal gastric cardia whose progeny populate the gland and persist for at least one year, suggesting the ability to self-renew and consistent with a cardia stem cell.

Since CCK2R marks a proliferating progenitor cell in the cardia, we verified whether CCK2R also has functional importance in BE progression. We hypothesized that overexpression of gastrin, the ligand for CCK2R, could accelerate BE in L2-IL-1β mice. INS-GAS mice, which overexpress amidated gastrin under the transcriptional control of the insulin promoter, were crossed with L2-IL-1β mice. L2-IL-1β/INS-GAS mice treated with bile acids had significantly increased areas of BE involvement compared to L2-IL-1β or INS-GAS controls. No areas of BE were seen in wild type or INS-GAS mice treated with bile acids until the age of 12 months.

#1706

Evaluation of LGR5 protein expression in colon cancer stem cells.

Wei Fu, Rachel Gonzalez, Kehu Yuan, Caiwei Chen, Haitao Wei, Hsiangmin Lu, Qi Ren, Evelin Logis, Sean Kelly, Wei-Wu He, Donghui Ma. _OriGene Tech Inc, Rockville, MD_.

Cancer stem cells are a subpopulation of cancer cells responsible for cancer initiation, development and metastasis. A number of studies demonstrated that Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5) can drive cancer development through triggering canonical Wnt signaling and its downstream gene expression. LGR5 is an important biomarker specifically expressed on colon cancer stem cells. In this study, we have successfully developed an LGR5 antibody with high specificity to detect endogenous LGR5 expression in different immunoassay application.

By screening multiple anti-LGR5 hybridoma clones, antibody generated by clone UMAB212 was proven to be highly specific on our high density protein microarray chip assay. Here our experimental data demonstrated that clone UMAB212 recognizes not only human LGR5 protein but also mouse LGR5 protein in both western blot and flow cytometry applications. No cross-reactivity was observed with the other two LGR family members, LGR4 and LGR6. Furthermore, this antibody also works great on immunohistochemistry application on FFPE tissue blocks. In summary, UMAB212 is great tool for us to study LGR5 protein in different immunoassay setting and it could also be a potential cancer diagnostic reagent.

#1707

Epithelial mesenchymal transition generates cancer stem cells in CD44- colorectal cancer cells.

Michitaka Nakano, Mamoru Tanaka, Taichi Isobe, Kohta Miyawaki, Yoshikane Kikushige, Hitoshi Kusaba, Shigeo Takaishi, Takashi Ueki, Eishi Baba, Koichi Akashi. _Kyushu University, Fukuoka City, Japan_.

Background: EpCAMhigh CD44+colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to acquire a property of cancer stem cells. Epithelial-mesenchymal transition (EMT) is a possible process of acquisition of cancer stem cell (CSC)-like properties. However, it is unclear whether EMT can be induced in primary human CRC cells.

Patients and Methods: We obtained surgical specimens from 51 CRC patients, and cultured isolated cancer cells on matrigel-coated dish with medium containing growth factors. For induction of EMT, TGF-beta was added into the culture medium. Otherwise, TWIST1 expression was enforced in the cells by using lentiviral transduction. Immunocytochemical analysis, flow cytometric analysis and single cell PCR analysis using 24-genes set containing embryonic stem cell (ES)-related and EMT-related genes were performed. PCR analysis was carried out by C1 single cell auto prep system. CD44- CRC cells with or without enforced expression of TWIST1 were injected into immunodeficient mice.

Results: Fifteen out of 51 samples of cancer cells formed sphere (>50 μm in diameter) after one week culture. The sphere-forming ability was related with clinical stage (Stage 1 and 2;16.7%, Stage 3 and 4;41.3%). Sorted single CD44+ cell had higher sphere-forming ability, compared to CD44- cell. The higher expression of ES- and EMT-related genes was observed in short term culture than in long-term culture, suggesting that differentiation occurred in sphere cells after long-term culture. Single cell PCR analysis revealed that sphere-forming cells were classified into 2 different populations on the basis of primary component analysis. Correlation analysis showed expression of TWIST1 and ES-related genes were correlated. In addition, flow cytometric analysis revealed that sphere forming CD44+ cells gave rise to CD44- cells. These results suggest that CD44+ cells have an ability to reconstruct the heterogeneous population, and EMT is involved in acquisition of CSC-like properties. TGF-beta increased the number of CD44+ cells in CD44- cells, and enhanced sphere-forming ability of CD44- cells. TGF-beta also induced expression of ES-related genes and TWIST1. Enforced expression of TWIST1 induced sphere-forming ability and tumorigenicity in CD44- cells.

Conclusions: We established the culture system to observe the differentiation of CSCs and revealed that EMT might be involved in maintenance of CSCs. We firstly demonstrated that primary CD44- CRC cells undergo EMT and become CD44+ cells by TGF-beta treatment or enforced expression of TWIST1.

#1708

Intestinal stem cells are sentinel cells for nutritional exposure.

Leonard H. Augenlicht, Wenge Li, Karina Peregrina, Michele Houston. _Albert Einstein Cancer Ctr., Bronx, NY_.

Lgr5+ CBC cells at the intestinal crypt bottom have been identified as canonical stem cells responsible for mouse intestinal mucosal homeostasis. The data establishing this are derived from mice fed standard diets that generate 25(OH)D serum levels well above the range well documented for the US population, with these higher levels reflecting that of "0" percent of the population. This is important because the Clevers group reported that expression of the vitamin D receptor (Vdr) is high in Lgr5+ cells, and decreased substantially in their immediate daughter cells, suggesting signaling through the Vdr is important for Lgr5+ CBC stem cell functions. Consistent with this, we showed that lower dietary vitamin D3 in the context of a purified western-style rodent diet, or lowering only vitamin D3 in control rodent AIN76A diet, or knockout of the vitamin D receptor specifically in Lgr5+ cells compromised the ability of Lgr5+ cells to lineage trace. Moreover, exposure to lower dietary vitamin D3 characteristic of a large segment of the US population substantially reduced the ability of introduction of a mutant Apc allele into Lgr5+ cells to generate tumors. However, the mucosa of mice exposed to these lower levels of vitamin D3 appears histologically normal and the mice are healthy. Consistent with this, Bmi1+ cells are mobilized to expand and lineage trace in mice in which lower dietary vitamin D3 established 25(OH)D levels similar to those found in the US population. Whether cells from other compartments reported to exhibit intestinal stem cell functions are also mobilized, and the relative contribution of cells from different compartments to intestinal homeostasis across the spectrum of 25(OH)D levels that characterize the human population, are under investigation.

Thus, vitamin D3 is a critical factor in programming intestinal cells from different compartments to express stem cell functions. The extent and nature of this programming has been investigated by RNAseq of specific cell populations from mice fed different diets, and a number of important pathways identified linked to vitamin D exposure level. Moreover, the relationship of change in functional status for cells from different compartments (ie quiescent versus mobilized) to the mutational spectrum and load that accumulate in these cells has been analyzed by single cell exome sequencing.

The sensitivity of intestinal stem cell function to vitamin D exposure suggests that the well-documented plasticity of intestinal stem cells may have evolved to permit adaptation to different environments. Moreover, this raises the questions of whether other nutrients key to maintaining intestinal homeostasis also modulate the relative contribution of different stem cell populations to intestinal homeostasis, and if the multiple cell populations capable of stem cell functions in the intestine can serve as sentinel cells for which analyses of their molecular programming are sensitive and responsive assays of nutritional exposures.

#1709

STRAP mediates the stemness of human colorectal cancer cells by epigenetic regulation of Notch pathway‬‬‬‬‬‬.

Lin Jin, Trung Tam Vu, Pran K. Datta. _University of Alabama at Birmingham, Birmingham, AL_.

Human colon cancer-initiating cells (CCICs) are responsible for the unrestrained cell proliferation and chemoresistance of colorectal tumors. Notch signaling is critical for the maintenance and self-renewal of cancer-initiating cells in colon cancer. We have shown that the serine-threonine kinase receptor-associated protein (STRAP) , a ubiquitous WD40 protein, is involved in promoting tumorigenesis through activated MAPK/ERK pathway and pRb phosphorylation. In addition, our previous studies uncovered that STRAP is up-regulated in 60% colon carcinomas and behaves as an oncogene in a TGF-β-dependent or -independent manner. Based on our new finding that STRAP stabilizes intracellular fragment of Notch3 (ICN3) via post-translational modification, we sought to explore the cross-talk between STRAP and Notch pathway in regulating stemness of CCICs. Here, we have generated STRAP knock down (KD) colorectal cancer cell lines (RKO, DLD1, HCT116, HT29, WiDr, SW480, SW620, and LOVO) to show how STRAP abrogates apoptosis by up-regulating CD44+/CD133+ subpopulations of CCICs. Heterozygous Strap KO inhibits tumorigenesis accompanied by the reduction of stemness phenotype (CD44+, Lgr5+) in a colitis-associated mouse model. Western blot assays and immunofluorescene staining demonstrated that the expression of stemness biomarkers (Nanog, Oct4 and Sox2) is decreased in STRAP KD cells. Furthermore, the transcriptional activation of Notch pathway related genes is downregulated in the STRAP KD cells. EpiTect Chip qPCR Arrays profiled that 17 out of 84 genes key to Notch signal transduction had higher activating (H3K4me3) and lower repressive (H3K27me3) histone modifications in WT cells compared to STRAP KD cells. Among these genes, we identified two transcription factors, Hes1 and Hes5, that are transcriptionally regulated by STRAP as demonstrated by luciferase assays and qPCR. We further demonstrated that Hes1 and Hes5 were transcriptionally activated by STRAP through Polycomb Repressive Complex 2 (PRC2)-dependent mechanism. Immunoprecipitation experiments showed that STRAP interacts with the key components, EZH2 and SUZ12 of PRC2 complex in vivo, which antagonizes the histone methyltransferase activity in H3K27 on the promoters of Hes1 and Hes5, resulting in activating these two genes. Of interest, ectopic expression of either Hes1 or Hes5 can rescue the stemness phenotype in STRAP KD cells. Together, our findings provide a novel mechanistic insight into the regulation of CCICs via STRAP-PRC2-Hes1/5 regulatory axis, which represents a potential target for CCICs, thus overcoming cancer chemoresistance and relapse.

#1710

Novel syngeneic mouse gastric cancer models identified Sca-1 as a gastric cancer stem cell marker.

Jun Won Park, Hark K. Kim. _National Cancer Center Korea, Goyang, Republic of Korea_.

Genetically well-defined syngeneic mouse models for gastric cancer are not available in the scientific community, but critical for the development of immunotherapeutic agents. We generated primary mouse gastric cancer cell line NCC-S1 (S1) established from a Villin-cre;Smad4F/F;Trp53F/F;Cdh1F/wt mouse and its metastatic variant cell line NCC-S1M (S1M). These cells closely recapitulated human diffuse type gastric cancers in histology and molecular profiles. S1M cells showed enhanced in vivo growth and metastatic potentials compared with S1 cells, and developed orthotopic and heterotopic tumors in immunocompetent mice in predictable manner. S1M allograft model was useful for testing the efficacy of an immunotherapeutic agent, anti-4-1BB. Sca-1 was overexpressed in S1M cells compared with S1 cells. Approximately 7% of gastric cancer cells in primary tumors expressed Sca-1. Sca-1high cells were more tumorigenic according to the in vivo limiting dilution assay, and more resistant to cisplatin and fluorouracil. Sca-1high cells overexpressed CD133, CD44, EPCAM and Bcl-xL. A chromatin immunoprecipitation analysis demonstrated that Sca-1 was a β-catenin/LEF1 target gene. Expression profiles of 85 genes overexpressed in Sca-1high S1 cells clustered 123 pretreatment gastric cancer patient samples according to the overall survival following cisplatin/fluorouracil chemotherapy. Thus, our novel metastatic gastric cancer cell lines are useful resources for drug development, and identified Sca-1 as a novel gastric cancer stem cell marker that mediates β-catenin signaling.

#1711

Notch and mTORC1 regulate human gastric stem cell function during normal tissue homeostasis and gastric cancer.

Elise S. Demitrack, Gail B. Gifford, Gabriela Wong, Linda C. Samuelson. _University of Michigan, Ann Arbor, MI_.

Notch and mTOR pathway activation have been observed in human gastric cancers. We have recently shown that chronic Notch activation induces gastric stem cell proliferation and tissue expansion in mouse via gland fission, which can be attenuated by mTORC1 inhibition. These findings suggest that these two pathways may synergize to promote gastric tumorigenesis. Here we tested the requirement of Notch and mTOR signaling for maintenance of human stem cells from control and gastric cancer tissues. Gastric glands were isolated from control (non-cancer) and human gastric cancer tissues, as well as mouse antrum to initiate organoid cultures. Established organoids were treated with vehicle, the Notch inhibitor DAPT, the mTORC1 inhibitor rapamycin, or both, every other day for 5 days, to test epithelial-specific requirements of each pathway for stem cell maintenance. Growth was determined by measuring organoid size, and proliferation was assessed by EdU incorporation. Gastric cancer cell lines AGS, MKN45 and NCI-N87 were treated with pathway inhibitors once per day for 5 days, with cell growth measured each day. Gene expression analysis of Notch pathway components was performed by qRT-PCR. In both human and mouse stomach, Notch1 and Notch2 were the main Notch receptors expressed in tissue and organoids. Notch inhibition with DAPT significantly inhibited growth of both human and mouse gastric organoids; however organoids derived from human gastric cancer tissue required 10-fold increased DAPT for significant growth inhibition. Surprisingly, mTORC1 inhibition with rapamycin significantly increased organoid size and epithelial proliferation of mouse organoids, as well as both control and tumor-derived human organoids, suggesting that active mTORC1 signaling restricts human stem cell proliferation. Combination treatment of human organoid cultures with both pathway inhibitors attenuated the rapamycin-induced increase in organoid size and proliferation. In contrast, treatment of gastric cancer cell lines with DAPT, rapamycin or both inhibitors significantly reduced cell growth compared to vehicle, suggesting that blocking Notch or mTORC1 in gastric cancer cells can be used to reduce cancer cell growth. In conclusion, Notch and mTORC1 signaling regulate mouse and human gastric stem cell function although through opposite mechanisms, with Notch stimulating human stem cell proliferation and mTORC1 inhibiting proliferation. Additionally, mTORC1 may regulate human gastric stem cell proliferation via a different mechanism than gastric cancer cells. Further studies testing the mechanism of Notch and mTORC1 interactions to regulate human gastric stem cell maintenance will inform our understanding of therapeutic strategies for gastric cancer.

NIH F32-DK093349; NIH UL1TR000433; NIH P01-DK062041 and NCI P50-CA130810

#1712

Intestinal stem cell integrity is preserved through modulation of endoplasmic reticulum stress by guanylyl cyclase C.

Crystal Kraft, Jieru Lin, Adam Snook, Gilbert Kim, Scott Waldman. _Thomas Jefferson University, Philadelphia, PA_.

Intestinal stem cells (ISCs) are a long-lived, dynamic cell population at the base of intestinal crypts that adjust their molecular phenotypes as necessary to meet the maintenance and regenerative needs of the epithelium. The long-life, plasticity and proliferative potential of ISCs underlie the necessity for tight regulation, as these cells have been implicated as initiators of colorectal tumorigenesis. In that context, guanylyl cyclase C (GCC) and its effector, cGMP, regulate intestinal epithelial proliferation, differentiation, and susceptibility to intestinal insult and tumor formation. However, the specific role of this signaling axis in ISCs remains undefined. Here, utilizing transgenic mouse models and immunodetection techniques, we reveal that GCC mediates ligand-dependent cGMP signal transduction in ISCs. Further, the absence of GCC alters expression of key ISC markers, decreasing the active ISC marker Lgr5 and increasing the reserve ISC marker Bmi1. Endoplasmic reticulum (ER) stress, which is typically absent from ISCs and has been associated with inflammation and tumorigenesis, is elevated throughout the crypt base in the absence of GCC. Conversely, administration of cGMP or the chemical chaperone taurourdeoxycholic acid eliminates ER stress and restores the normal crypt ISC phenotype. Finally, ISCs exhibit an impaired regeneration response following irradiation in the absence of GCC, highlighting the importance of this signaling pathway in ISC survival and epithelial repair. Together, these observations suggest that GCC signaling modulates ER stress as part of the essential machinery defending the integrity of the ISC niche.

#1713

Macrophage-induced bystander effect activates Wnt/β-catenin signaling and induces cellular dedifferentiation.

Xingmin Wang,1 Yonghong Yang,1 Mark M. Huycke2. 1 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK;_ 2 _Oklahoma City VA Health Care System, Oklahoma City, OK_.

Cancer stem cells (CSCs) in colorectal cancer (CRC) help maintain tumor heterogeneity, promote tumor growth, invasion, and metastasis, and produce resistance to therapeutic agents. The origin of colorectal CSCs, however, is unclear. Our recent studies showed that a macrophage-induced bystander effect (MIBE) induced gene expression of stem/progenitor cell markers including Ly6A/E and Dclk1 in murine colonic epithelial cells, implying that MIBE helps induce colorectal CSCs.

In current studies we show that commensal-triggered MIBE activates Wnt/β-catenin signaling and induces dedifferentiation of colonic epithelial cells during colorectal carcinogenesis, thus contributing to the origin of colorectal CSCs. Exposure of murine primary epithelial cells (YAMC) to commensal-polarized macrophages induced phosphorylated Gsk3β (Ser9), active β-catenin, and Tcf4, suggesting activation of Wnt/β-catenin signaling by MIBE. In vivo, active β-catenin was evident in both stromal and epithelial cells for colon biopsies from E. faecalis-colonized Il10-/- mice compared to sham-colonized mice. In addition, we showed that 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation of ω6 polyunsaturated fatty acids, and tumor necrosis factor α (TNFα) mediated activation of Wnt/β-catenin signaling. Epigenetic analysis revealed that MIBE activated Wnt/β-catenin signaling through DNA methylation in the CpG islands of secreted frizzled-related protein 2 (Sfrp2), a soluble modulator of Wnt. Next, we showed that exposure of YAMC cells to 4-HNE and TNFα induced expression of c-Myc, Klf4, Oct4, and Sox2―factors essential for pluripotent stem cells. Immunofluorescent staining of colon biopsies from E. faecalis-colonized Il10-/- mice showed increased expression of these factors in colonic epithelial cells, suggesting dedifferentiation induced by MIBE. This was confirmed by qRT-PCR and Western blots that showed increased expression of Dclk1 and CD44, two markers for colorectal CSCs. Furthermore, increased expression of DCLK1 was noted in human tubular adenomas and invasive CRCs compared to hyperplastic polyps. Finally, inhibition of β-catenin/TCF4 using FH535, and by silencing CTNNB1, decreased DCLK1 expression in HCT116 human colon cancer cells, supporting the notion that Wnt/β-catenin signaling induces cellular dedifferentiation leading to colorectal CSCs.

In summary, these results demonstrate that commensal-polarized macrophages activate Wnt/β-catenin signaling and induce dedifferentiation of colonic epithelial cells through 4-HNE and TNFα-mediated bystander effects. These findings provide new evidence for the origin of CSCs in colorectal carcinogenesis and should lead to novel strategies for CRC prevention and therapy.

#1714

The role of Hedgehog signaling in the regulation of human colon cancer stem cells.

Joseph L. Regan,1 Stephanie Staudte,1 Dirk Schumacher,2 Ulrich Keilholz,3 Johannes Haybaeck,4 Hans Lehrach,5 Marie-Laure Yaspo,5 David Henderson,1 Andreas Steffen,1 Joern Toedling,1 Ralf Lesche,1 Reinhold Schaefer,2 Christian R. Regenbrecht,2 Dominik Mumberg,1 Martin Lange1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Charité – Universitätsmedizin, Berlin, Germany;_ 3 _Charité Comprehensive Cancer Center, Berlin, Germany;_ 4 _Medical University of Graz, Graz, Austria;_ 5 _Max Planck Institute for Molecular Genetics, Berlin, Germany_.

Recent data support a hierarchical model of colon cancer in which tumor growth is driven by a subpopulation of cancer stem cells (CSCs) that may also be the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumor would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here, as part of the OncoTrack* consortium, we report the use of Matrigel-based 3D in vitro models of patient-derived colon cancer for the characterization of molecular pathways important in the regulation of CSC self-renewal and survival.

Cellular subpopulations were isolated from patient-derived 3D-culture models based on expression of the CSC marker aldehyde dehydrogenase (ALDH) and then functionally tested for tumor initiation and self-renewal capacity by limiting dilution serial xenotransplantation. These studies demonstrate CSCs to be ALDH+. ALDH+ and ALDH- cells from 3D-culture and xenograft models were then subjected to RNA sequencing for whole transcriptome analysis.

These analyses demonstrated ALDH+ CSCs cells to be enriched for stem cell associated genes (ALDH1A1, LGR5, BMI1, CD44, CD166), developmental processes (embryonic, tissue development, EMT) and signaling pathways (Wnt signaling). In particular, the Hedgehog signaling pathway was found to be enriched in both 3D in vitro and xenograft models.

The role of Hedgehog signaling in colon cancer remains controversial. It has been reported as a negative regulator of Wnt signaling and to be inactive in colon cancer cells lines. Here, using small molecule inhibitors and lentivirus mediated gene knockdown, we report on the role of Hedgehog signaling in the regulation of colon CSC self-renewal and identify non-canonical Hedgehog signaling as a positive regulator of Wnt signaling.

*The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234.

#1715

Whole transcriptome analysis of patient-derived 3D in vitro and xenograft models of colon cancer identifies placental genes required for the survival of cancer stem cells.

Joseph L. Regan,1 Stephanie Staudte,1 Dirk Schumacher,2 Ulrich Keilholz,3 Johannes Haybaeck,4 Hans Lehrach,5 Marie-Laure Yaspo,5 David Henderson,1 Andreas Steffen,1 Joern Toedling,1 Ralf Lesche,1 Reinhold Schaefer,2 Christian R. Regenbrecht,2 Dominik Mumberg,1 Martin Lange1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Charité – Universitätsmedizin, Berlin, Germany;_ 3 _Charité Comprehensive Cancer Center, Berlin, Germany;_ 4 _Medical University of Graz, Graz, Austria;_ 5 _Max Planck Institute for Molecular Genetics, Berlin, Germany_.

Growing evidence supports a subpopulation of cancer stem cells (CSCs) as both the drivers of tumor growth and the source of relapse following treatment. The identification of genes and pathways required for the survival of CSCs may therefore lead to better treatment outcomes. Here, as part of the OncoTrack* consortium, we report the use of 3D in vitro and xenograft models of patient-derived colon cancer for the identification of novel regulators of CSCs.

Increased aldehyde dehydrogenase (ALDH) activity has been demonstrated to be a marker of normal intestinal stem cells and colon CSCs. We therefore isolated and functionally tested ALDH+ cells for CSC properties using 3D in vitro models and xenografts of patient derived colon cancer. These studies revealed ALDH+ cells to be highly enriched for CSCs. In addition, there was a positive correlation between CSC frequency in the models and tumor grade at time of surgical resection.

ALDH+ CSCs were subjected to RNA sequencing for whole transcriptome analysis. These analyses demonstrated ALDH+ CSCs to be enriched for stem cell associated genes (ALDH1A1, BMI1, LGR5, CD44, CD166), signaling pathways (Wnt, Hedgehog) and developmental (embryonic, placental, tissue development, EMT) and metabolic processes (retinol metabolism, drug metabolism, hypoxia).

Lentivirus mediated gene knockdown was carried out on a panel of genes that were differentially expressed in ALDH+ cells from 3D in vitro models and xenografts. These studies led to the identification of genes required for the self-renewal and survival of colon CSCs. Interestingly, these genes have been reported as important regulators of early embryonic and placental development but have not previously been reported in the regulation of cancer stem cells.

*The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234.

#1716

The role of CD44v9+ colorectal cancer stem cells in the resistance to cetuximab treatment.

Chia-Nung Hung, Ching-Yu Chang, Jou Hsiao, Wan-Chen Wei, Wei-Ting Chao. _Tunghai University, Taichung, Taiwan_.

Recent studies found that conventional therapy combined with cetuximab treatment (EGFR inhibitor) to treat metastatic colorectal cancer, had no improvement of patient survival rate in some population. One of possibilities might be cancer stem cells (CSCs) population resist to cetuximab treatment and modulate metastasis, however, the detail mechanism has not been addressed in colorectal cancer. Thus, our study is to decipher the mechanism of resistance to cetuximab in CD44v9+ specific colorectal cancer stem cells. First, to reveal clinical significance of CD44v9, the tumor samples of colorectal cancer patients were analyzed by immunohistochemistry to evaluate CD44v9 expression status in patients. To further investigate the biological mechanism, single cell analysis in CD44v9+ population of HT29 cells was performed with Fluidigm Biomark™ HD system. The results showed that 67% patients had highly expressed CD44v9 with lower survival rate. In vitro studies showed that when HT29 cells were in 5uM cetuximab treatment for 48 hours, CD44v9, Oct4, CD133, CD24, Lgr5, CSCs markers, were up-regulated in RNA level. Furthermore, CD44v9+/Oct4+ cancer cell population was increased but CD44v9+/pEGFR+ cancer cells were eliminated according to immunofluorescence results. The results of single cell analysis demonstrated that CD44v9+/Oct4+ cancer cells highly expressed proliferation markers consistently with cetuximab treatment. Trans-well migration results showed that CD44v9+/Oct4+ cancer cells migrated collectively via mediating E-cadherin dynamics. Furthermore, in clinical outcome, CD44v9 and E-cadherin co-expression were associated with recurrence and metastasis and decreased survival rate significantly. In conclusion, cetuximab failed to target CD44v9+/Oct4+ cancer cells, which characterized as cancer stem cells and might modulate collective cell migration. This finding suggested that new therapeutic strategy to against CSCs would be the potential clinical treatment in colon cancer.

#1717

Circulating tumor cells from in vitro 3D culture systems have tumor-initiating capacity in vivo.

Marina C. Cabrera, Elaine Hurt, Xiaoru Chen, Shi Xiaoqing, Haifeng Bao. _MedImmune, Gaithersburg, MD_.

Circulating tumor cells (CTCs) are cells that have detached from the primary tumor and entered the bloodstream with the potential to seed metastatic tumors in distal sites. High CTC numbers correlate with aggressive disease, increased metastasis, and decreased time to relapse. It has also been shown that cancer stem cells (CSCs) represent a proportion of the CTCs present in patients. Given that CSCs are resistant to chemotherapy and are responsible for tumor initiation, it is posited that the CSCs are the seeds of metastasis. However, direct evidence for this hypothesis is limited because there are few methods for culturing and studying these rare cells. We are using a 3D culture chamber system (RealBio D4™) to establish long-term cultures of human-derived breast and pancreatic tumors. We observed that the system's 3D matrix supported culture development and incorporation of tissue organization and microenvironment. Further, the chamber design allowed CTCs generated within the cultures to migrate out of the cell mass into the circulating nutrient medium where they were collected for characterization. The isolated CTCs displayed CSC properties via CellSearch® (CD44+,CD24-; CD44+,CD24+ for breast and pancreas respectively). In addition, these isolated CTCs displayed tumor-initiating capacity when implanted into mice. Future studies will compare CTCs isolated from 3D culture with cells from tumors, blood, and tissue grown on scaffold through RT-PCR, histology, and gene expression assays.

#1718

Characteristics of carbonic anhydrase 9-expressing cells in the human intestinal crypt base.

Yozo Suzuki,1 Hidekazu Takahashi,2 Masahiro Tanemura,1 Junichi Nishimura,2 Naotsugu Haraguchi,2 Masahisa Ohtsuka,3 Susumu Miyazaki,4 Mamoru Uemura,5 Taishi Hata,2 Tsunekazu Mizushima,2 Hirofumi Yamamoto,2 Yuichiro Doki,2 Masaki Mori2. 1 _Osaka Police Hospital, Osaka, Japan;_ 2 _Osaka University Graduate School of Medicine, Osaka, Japan;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Osaka General Medical Center, Osaka, Japan;_ 5 _Osaka National Hospital, Osaka, Japan_.

Background/Aims: In many tissues, stem cells are sequestered in hypoxic microenvironments. However, little is known about the relationship between hypoxia and the intestinal crypt base, the presumed location of intestinal stem cells. This study investigated the expression of carbonic anhydrase 9 (CA9), a hypoxia-inducible membrane-tethered protein, in normal intestinal crypt base cells and in adenoma and early colorectal cancer.

Methods: Using surgically resected human colorectal cancer specimens, we investigated the expression pattern and functional associations of CA9 in human adult normal intestinal epithelia and in adenoma and early colorectal cancer using immunofluorescence, immunohistochemical staining, flow cytometry, and quantitative real-time polymerase chain reaction.

Results: Almost all slender cells in the crypt base in the ileum were CA9+. In the right and left colon, 25% and 40% of all cells, respectively, were CA9+. Notably, CA9+ cells had eosinophilic structures in their basal cytoplasm. Adenomas and T1 colorectal cancer showed broad CA9 expression. Flow cytometry analysis indicated that CA9+ cells had increased mRNA levels of AXIN2, a Wnt signaling factor.

Conclusion: These observations indicate that CA9 expression in normal crypt base cells is associated with intestinal epithelial stemness. CA9 expression may be involved in the carcinogenesis of colorectal cancer.

#1719

CD133 attenuates ROS accumulation via a steady increase in the expression of the cystine/glutamate transporter xCT: Consequence on chemoresistance in hepatocellular carcinoma.

Haengran Seo. _Institut Pasteur Korea, Seongnam-si, Republic of Korea_.

CD133+ cells in hepatocellular carcinoma (HCC) exhibit cancer stem cell (CSC)-like properties as well as resistance to chemotherapeutic agents and ionizing radiation; however, their function remains unknown. In this study, we tried to define new target and compound for enhancement of chemotherapy efficiency in HCC through elucidation of mechanism of CD133-induced chemoresistance.

Here, we show that CD133+ HCC cells exhibit strong resistance to reactive oxygen species (ROS) via persistent maintenance of the ROS-induced increase in xCT expression that upregulates glutathione (GSH) levels, and thereby play a central role in resistance to liver cancer therapy.

ROS are generally known to act as mediators of apoptosis induced by various anti-cancer drugs. We found that increased CD133 expression enhanced the capacity for ROS defense by enhancing cellular GSH levels, and ablation of CD133 attenuated not only the capacity for defense against ROS, but also chemoresistance, in HCC. Sulfasalazine, a potent xCT inhibitor, impaired the ROS defense system and increased the therapeutic efficacy of anticancer therapies in CD133+ HCC but not CD133-HCC in vivo and in vitro. Furthermore, we found that CD133 sustained the ROS stress-induced increase in the expression of xCT, a cystine/glutamate transporter that plays an important role in maintaining GSH levels, and thereby in preventing cell death via anticancer therapies. These results strongly indicate functional roles for CD133 in ROS defense and in evading anticancer therapies in HCC, and suggest that sulfasalazine, administered in combination with conventional chemotherapy, might be an effective treatment for CD133-targeted liver cancer therapy.

#1720

Role of SENP1 in HBx-induced cell migration and stemness-related properties in hepatocellular carcinoma.

Te Sheng Chang,1 Yen-Hua Huang2. 1 _Chang Gung Memorial Hospital, Chiayi, Taiwan;_ 2 _Taipei Medical Univ. College of Medicine, Taipei, Taiwan_.

Hepatocellular carcinoma (HCC) is the sixth most common cancer, and the third leading cause of cancer mortality worldwide. Surgical resection can be curative for early stage HCC, but the risk of tumor recurrence remains high. Tumor stemness properties have been linked to early recurrence of HBV-related HCC (HBV-HCC), which continues to be a great challenge in clinical practice. However, the progress in the strategy of individualized therapy against HBV-HCC is slow and disappointed. Here we demonstrate that HBx regulates the cell migration and cancer stemness properties of HCC cells through Sentrin-specific protease 1 (SENP1) modification. By a large cohort of HCC patients receiving curative heptectomy, we found that SENP1 expression level is highly associated with the gene expression levels of cell migration-related gene SNAIL/TWIST and pluripotency-related gene OCT4 particularly in HBV-HCC. Furthermore, SENP1 expression level is significantly associated with tumor metastasis as well as the early recurrence of HCC using Kaplan-Meier analysis. Further experiments using cell line model, we demonstrated that HBx overexpression not only increases the protein levels of SENP1 and pluripotent transcription factor OCT4, but also enhances cell migration in HepG2 cells. Inhibition of SENP1 significantly suppressed the HBx-induced cell migration and cancer stemness in vitro and in vivo. In summary, in this work, we demonstrate that SENP1 plays a critical role in HBx-induced cell migration- and cancer stemness-related properties in HBV-HCC. Findings in this study will facilitate the potential strategy targeting on SENP1 for individualized adjuvant therapy against HBV-HCC.

#1721

Desmoplasia stem and progenitor cells within the tumor microenvironment.

Tamsin Lannagan,1 Susan Woods,1 Laura Vrbanac,1 Miao Yang,1 Jia Ng,1 Tongtong Wang,1 Yagnesh Tailor,2 Samuel Asfaha,3 Timothy Wang,2 Daniel L. Worthley1. 1 _University of Adelaide, Adelaide, Australia;_ 2 _Columbia University, New York, NY;_ 3 _University of Western Ontario, London, Ontario, Canada_.

All developing and adult organs are supported by connective tissues. We recently demonstrated that Gremlin 1 expressing cells in the bone (osteochondroreticular stem cells) and the bowel (intestinal reticular stem cells) are connective tissue stem cells, during development and healing. The contribution of these stem cells to the desmoplasia surrounding gastrointestinal and skeletal cancers, however, is unknown. In this study we first established that typical markers of bone marrow skeletal stem cells, also identify colony forming unit-fibroblasts (CFU-Fs) in the tumour microenvironment (identified by CD45-Ter-119-CD31-CD1040a+CD105+). Next we tested the local origins of alpha-smooth muscle actin (Acta2) expressing cancer-associated fibroblasts in orthotopic (colonoscopically delivered MC38 and carcinogenesis AOM/DSS) mouse models of colorectal cancer. Using transgenic mouse models to lineage trace and report the connective tissues in the bone and bowel, including Grem1-creERT;R26-LSL-ZsGreen; Acta2-RFP and our Acta2-CreERT line, we found that normal Grem1-expressing and Acta2-expressing cells each contribute to some, but not all, of the reactive cancer-associated fibroblasts surrounding our mouse models of cancer. Whilst, neoplastic cells appear to make a significant contribution to cancer-associated fibroblasts in some other cancers, using an epithelial specific Cre (K19-cre) there was no contribution of epithelium to Acta2-expressing cells in our AOM-DSS colorectal cancer models. We investigated Grem1 and Acta2 derived cells from the tumor microenvironment and found that these cells were clonagenic. Finally, we compared their capacity to support the in vitro growth of colorectal normal and neoplastic organoids compared to other colonic mesenchymal cell types. We are currently examining the role of these cells on expanding intestinal stem cells in normal and neoplastic gastrointestinal glands and examining the secreted factors from these cells that are relevant to tumor initiation and spread.

#1722

Hedgehog and Wnt crosstalk in pancreatic cancer.

Elissaveta Petrova, Arne Scholz, Juliane Paul, Andrea Sturz, Katja Haike, Franziska Siegel, Dominik Mumberg, Ningshu Liu. _Bayer Pharma AG, Berlin, Germany_.

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States. It is characterized by a dense desmoplastic stroma that is thought to limit drug delivery and to contribute to the low response rate to chemo and targeted therapies. There are many pathways involved in the tumor-stroma communication, and Wnt and Hedgehog (Hh) signaling are amongst them. Both pathways are abnormally activated in pancreatic cancer by overexpression of their ligands and receptors, and both, although to a different extent, affect tumor and stromal cells. Disappointingly, in the clinic, Hh pathway inhibition using Smoothened (Smo) inhibitors did not improve patient outcomes, despite the strong anti-stromal activity of the compounds.

During embryonic development, Hh signaling directly crosstalks with the Wnt pathway through the expression of secreted frizzled-related protein 1 (sFRP-1), a negative regulator of Wnt signaling. In cancer, the expression of sFRP-1, however, is frequently suppressed. Here, we show for the first time, that in pancreatic cancer, sFRP-1 is not only expressed, but that its expression is increased in the metastatic disease setting. Furthermore, Hh pathway inhibition using the Smo inhibitor GDC-0449 decreased sFRP-1 expression, and this was paralleled by an increase, or by maintenance of the elevated expression, of β-catenin target genes. In vivo, in a patient-derived xenograft (PDX) model of pancreatic cancer, treatment with GDC-0449 maintained elevated levels of nuclear β-catenin, and had no effect on tumor growth. To confirm the importance of the Hh-Wnt crosstalk in pancreatic cancer we compared the effects of the Smo inhibitior to those of an acetyl-CoA carboxylase 1(ACC1) inhibitor, which we recently developed (BAY ACC002), and which we have shown to block both Wnt and Hh signaling by inhibiting ligand lipidation. As in the case of GDC-0449, treatment of Capan-2 human pancreatic cancer cells in vitro with BAY ACC002, indeed, decreased sFRP-1 expression. However, unlike the Smo inhibitor, BAY ACC002 reduced the levels of β-catenin target genes. Furthermore, in vivo BAY ACC002 showed anti-tumor efficacy in several different xenograft models, whereas GDC-0449 showed no activity. Together, these data build evidence for a crosstalk between Hh and Wnt signaling in pancreatic cancer, and offer a novel approach to overcome the limitations observed with Smo inhibitors in the clinic. These results also highlight the benefit of a simultaneous inhibition of both pathways in order to achieve a sustained effect on both the tumor and the tumor stroma.

#1723

Novel drug candidates CEP1430 and CEP1507 against cancer stem cells and circulating tumor cells in advanced stage pancreatic cancer.

Cristian Sharma,1 Padmini Narayanan,1 Michael Sharma,1 Robert Rodriguez,1 Miriam Navel,1 Donna Stanton,1 Natalee Amezcua,1 Shaleekha Sharma,1 Mandana Amiri,1 Jitesh Jani,1 Sherven Sharma,2 Jay P. Sharma1. 1 _Celprogen, Inc., Torrance, CA;_ 2 _Department of Medicine, UCLA/VAGLAHS Lung Cancer Research Program and Jonnson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Molecular Gene Medicine Laboratory, Veterans Affairs Greater Los Angeles Healthcare System,, Los Angeles,, CA_.

Despite improvements in treatment modalities, pancreatic cancer remains the fourth leading cause of cancer mortality in the US. The 5-year survival rate is less than 6% because of the extent of metastases at the time of diagnosis. The treatment of pancreatic cancer remains challenging however there is much optimism with targeted strategies and trial designs. The interrogation of pathway centric targets that regulate the regenerative and metastatic activities in the tumor, adequate molecular-hypothesis driven trial designs with robust prognostic and predictive markers of response are necessary for the development of effective therapeutic strategies for long term anti-tumor benefit.

Here, we examined the efficacy of CEP1430 and CEP1507 to target cancer stem cells (CSC) and circulating tumor cells (CTC) in human pancreatic cancer. The pancreatic CSCs expressed the following markers(CD133,CD44,CXCR4,SSEA3/4,Oct4,ALDH,Telomerase,& Nestin). In vitro CEP1430 inhibited the growth of CSCs whereas CEP1507 inhibited CTCs that expressed the following markers [CK19, CEA, CA19-9,CK8 /18,h-TERT,C-MET,CK20,EpCAM, Vimentin, E- Cadherin, MUC1, CEA CAM5,CD45 neg]. Gemcitabine on the other hand suppressed the viability of non-CSCs (differentiated tumor Cells) by 70%. In accord, in vivo studies showed that CEP1507 when combined with gemcitabine eliminated the engraftment of human pancreatic cancer CTCs by 85% (n=20,p<0.001). In contrast, CEP1430 in combination with gemcitabine selectively inhibited CSCs by 80% (n=20,p<0.001) and was more effective than the individual agents. This data demonstrates that CEP1430 and CEP1507 that target CSCs and CTCs respectively may change the treatment paradigm and enhance the efficacy of gemcitabine in advanced pancreatic cancer. Further studies will evaluate the full potential of this approach to target the CSCs - and metastasis-initiating cells (CTCs) as combination therapy against locally advanced and metastatic pancreatic cancer.

#1724

Pancreatic cancer stem cell metastasis and mitochondrial respiration is dependent on the the ISG15-mediated ubiquitin-like modification process known as ISGylation.

Bruno Sainz, Sonia Alcala Sanchez, Mireia Vallespinos. _Universidad Autónoma de Madrid, Madrid, Spain_.

We recently discovered that CSCs of distinct solid tumors (e.g. pancreatic, colon, and liver) contain intracellular vesicles coated in the ATP-dependent transporter ABCG2, which act as a sink for the accumulation of riboflavin (vitamin B2). Using autofluorescence and other CSC markers, such as CD133, we have begun to dissect CSCs at the genetic and protein level. Using RNAseq to transcriptionally distinguish CSCs from non-CSCs, we observed that pancreatic CSCs of different primary PDAC tumors exhibit a transcriptional gene activation signature belonging to the Type I interferon (IFN) signaling pathway. In particular, CSCs up-regulate the interferon-stimulated gene 15, a 165 amino acid (17kDa) protein that is induced by Type I IFN treatment and functions to ISGylate proteins, a ubiquitin-like modification process whereby ISG15 can be covalently linked to cytoplasmic and nuclear proteins. We have discovered that CSCs not only upregulate the expression of monomeric ISG15, but numerous proteins within CSCs are ISGylated, the level of ISGylation correlates with their metastatic capacity and knockdown of ISG15 ablates pancreatic CSCs functions, such as self-renewal, migration and invasion in vivo and mitochondrial respiration. Thus, our data indicate that the ISGylayion pathway is active in CSCs and plays an important role in the "stem-like" properties of these cells. We have also elucidated the mechanism underlying the differential regulation of ISG15 in CSCs versus non-CSCs. Specifically, using over expression lentiviral vector systems and pharmacological inhibitors (i.e. BRD4 inhibitor JQ-1), we show that MYC controls the expression of miR22, a micro-RNA that targets, among others transcripts, a number of Interferon Regulatory Factors (IRFs) that are essential for intracellular IFN signaling, including ISG15 expression. CSCs express low levels of MYC and miR-22, relieving the inhibitory block on IRFs and subsequent ISG15 signaling. As expected, over expression of MYC increases miR-22 levels and blocks IRF-mediated ISG15 expression. While the intricacies of these complex and distinct pathways still need to be unraveled at the CSC level, our data suggest that MYC may be a key universal player in these pathways, and targeting ISG15 in CSCs may represent a unique approach to altering the biology of CSCs.

#1725

The significance of c-Kit proto-oncogene in iCSC-derived PDAC model.

Anna Sanchez Calle, Kenta Hoshikawa, Neha Nair, Marta Prieto-Vila, Arun Vaidyanath, Tomonari Kasai, Masaharu Seno. _Okayama University, Okayama, Japan_.

Our study is focused on the generation of a novel pancreatic iPS-derived cancer stem cell line (iCSC), as a cutting-edge model of study for PDAC, and the significance of the proto-oncogene c-Kit in PDAC, using the new model of iCSC-derived PDAC generated in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is a neoplasia that originates within the pancreatic ducts. The mortality rate is high due to its rapid dissemination, the strong resistance to the radio and chemotherapy, along with the lack of prognostic approaches. Therefore, this highlights the urgent need to find novel therapies along with new pancreatic cancer markers towards the early detection of the primary stages of the PDAC.

The iCSC cells were orthotopically transplanted into the pancreas of Balb/c nude mice. After a short-time course of 15 days, PDAC progression was recapitulated not only at the level of the tumour phenotype, but also with the corresponding invasion towards specific hepatic metastatic nodes. iCSC cells were obtained from iPS, following the procedure previously established by our group, where the reprogramming is accomplished exclusively through conditioned medium, without any genetic modification, which may represent a substantial tool to study fairly about the insights on the actual tumour microenvironment. In order to confirm these experiments are reproducible, iPS were cultured with different pancreatic cancer cell line -PK-8, KLM-1 and PK-59-conditioned medium. Histochemistry assay of the tumour developed in vivo demonstrated the effectiveness of the conversion from iPS into iCSC among the cell lines, generating stroma-rich ductal adenocarcinoma structures that resemble the human tumour phenotype.

The tyrosine receptor c-Kit is expressed only during the embryonic pancreas differentiation, whereas adult pancreatic normal tissues lack the expression of the receptor. However in PDAC, its overexpression seems to be tightly correlated with the progression of the disease, and c-Kit has been suggested as a CSC marker.

The overexpression of c-Kit receptor was observed at protein and mRNA expression level, in the primary cultures of iCSC-derived PDAC tumours. Interestingly, this overexpression is directly related to the tumour phenotype, being the most disrupted ductal structures those, which show the c-Kit over-expression. Additionally, CSC from the primary cultures of iCSC-derived PDAC were isolated by puromycin selection as well as sphere formation, and after 7 days of culture the mRNA expression levels of Pax4 appears to be up-regulated, suggesting that iCSC cells indeed may differentiate into a tissue-specific lineage.

Hence, c-Kit might be a prominent candidate as a CSC marker in PDAC, and according to the current results we have generated a suitable PDAC model, which recapitulates the actual disease in a short-time course, and allows the study of the actual tumour microenvironment.

#1726

Targeting pancreatic cancer stem cells by afatinib in organoid culture.

Garima Kaushik, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Muzafar A. Macha, Moorthy P. Ponnusamy, Surinder K. Batra. _UNMC, OMAHA, NE_.

Introduction: Pancreatic cancer (PC) is the third leading cause of cancer related deaths in United States and has a 5 year survival rate of 7.2%. Current front line therapy for PC includes Gemcitabine, 5FU and Abraxane. Cancer stem cells (CSCs), a small subpopulation in cancer comprising of less than 5% of total cancer cells, are responsible for therapy resistance and aggressive metastasis resulting in poor prognosis of cancer including PC. It has already been established that EGFR family members (EGFR and HER2) are involved in CSC maintenance. Therefore, CSCs are important target to reduce the drug resistance and aggressiveness. Afatinib is an FDA approved pan-EGFR inhibitor. Our major goal is to use afatinib to target pancreatic CSC and understand the mechanism of its action.

Methods: After identifying the IC50 by MTT assay, PC cell lines (Capan1 and SW1990) were treated with afatinib and gemcitabine and cell lysates were analyzed by western blotting (WB) for EGFR family members and CSC markers like Shh, SOX2, Aldh1 and CD133.CSCs or Side Population (SP) were analyzed in afatinib and gemcitabine treated Capan1 cells by Hoechst based FACS analysis. Hoechst based FACS was used to isolate SP/CSC and NSP (non-side population) from parental PC cell lines and their IC50 for gemcitabine and afatinib were determined. SP and NSP cells were subjected to drug treatments to determine alterations in EGFR family proteins and stem cell markers by WB. Tumor sphere assay (TSA) was performed to determine tumorigenic potential of SP/CSC and NSP fractions with and without drug treatments. We developed Tumor-Organoids from Kras;PdxCre (KC) mice and used them to test effect of afatinib in 3D culture system.

Results: Our results showed significant decrease in SP/CSC fraction upon afatinib treatment and an enrichment of SP/CSC fraction on gemcitabine treatment. The SP/CSC cells showed higher sensitivity to afatinib and resistance towards gemcitabine when compared to parental cells suggesting specific targeting on SP/CSCs. Upon afatinib administration, we observed a reduction in phospho forms of EGFR family members and CSC markers like Sox2, Aldh1 and CD133 confirming functionality of the drug and its effect on pancreatic CSCs. A significant decrease was also observed in number and size of tumor spheres formed in vitro with both SP/CSC and NSP. We also confirmed a drastic reduction in the size and number of tumor-organoids following nine days of afatinib treatment compared to untreated controls.

Conclusion: Our results demonstrate a novel role of afatinib in targeting pancreatic CSCs and provide a scope to generate targeted therapy towards PC.

#1727

Quinomycin A inhibits pancreatic cancer growth and affects stem cell viability by inhibiting oncogenic YAP1 function.

Dharmalingam Subramaniam,1 Sivapriya Ponnurangam,1 Gaurav Fnu,1 Prabhu Ramamoorthy,1 Animesh Dhar,1 Ossama W. Tawfik,1 Shahid Umar,1 Scott J. Weir,1 Roy A. Jensen,1 Arun Balakrishnan,2 Shrikant Anant1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Piramal Life Sciences Ltd, Mumbai, India_.

Background: Pancreatic cancer (PCa) remains a leading cause of death in the United States. Cancer stem cells (CSC) are responsible for tumor behavior, and therapeutic resistance. Quinomycin (Qui) is an orally administered quinoxaline antibiotic that bifunctionally intercalates with double stranded DNA. As a first step for repurposing this drug, we determined whether Qui affects PCa growth, and if so whether this is by suppressing stem cells.

Method: Growth and apoptosis of PCa lines (MiaPaCa-2, PanC-1, BxPC-3) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, and caspase 3/7 assays, respectively. Pancosphere formation and FACS sorting were used to identify effects on stem cells. For in vivo, MiaPaCa-2 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for stem cell markers and Hippo signaling proteins.

Results: Qui demonstrated a dose- and time-dependent inhibition of proliferation and colony formation in all three PCa lines but not HPNE cells. Qui induced PCa cells to undergo apoptosis. It also significantly reduced the number and size of pancospheres, and flow cytometry and western blot analyses confirmed that Qui suppressed PCa stem cell marker proteins DCLK1, CD44, CD24 and EPCAM. We next determined whether Hippo signaling pathway is affected. For this, we tested the effect of Qui on Hippo signaling pathway, which is an active pathway in CSCs including in PCa. The key effector protein, YAP1 has been shown to be oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm, and therefore no induction of downstream gene expression. Qui significantly decreased the phosphorylation oncogenic YAP1. Furthermore, Qui inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from Qui-mediated growth suppression. To determine the effect of Qui on tumor growth in vivo, mice carrying MiaPaCa-2 tumor xenografts were administered the compound intraperitoneally (20 µg/kg bw) every day for 21 days. Qui treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of CSC marker proteins, oncogenic YAP1 phosphorylation and YAP1 interacting proteins TEAD1 in the Qui-treated xenograft tissues.

Conclusion: Together, these data suggest that Qui suppresses PCa growth that targets that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway.

#1728

DCLK1 promotes tumor growth and invasion through Slug-mediated epithelial-mesenchymal transition in pancreatic neuroendocrine tumors.

Yu Ikezono,1 Hironori Koga,1 Jun Akiba,2 Mitsuhiko Abe,1 Fumitaka Wada,1 Toru Nakamura,1 Hideki Iwamoto,1 Atsutaka Masuda,1 Takahiko Sakaue,1 Hirohisa Yano,2 Osamu Tsuruta,1 takuji Torimura1. 1 _Division of Gastroenterology, Department of Medicine, Kurume university of Medicine, Kurume city, Japan;_ 2 _Division of pathology, Kurume university of Medicine, Kurume city, Japan_.

Introduction: Accumulating evidence suggests that Doublecortin-like kinase 1 (DCLK1) is a putative marker for intestinal and pancreatic cancer stem cells. (Nakanishi et al., Nat Genet 2013, Bailey et al., Gastroenterology 2013). Recently, we have reported that DCLK1 was highly and diffusely expressed in human rectal neuroendocrine tumors (NETs) (Ikezono et al., Oncol Lett, 2015). However, the function of DCLK1 has not yet been investigated in detail. Aims: The aims of the present study were to assess expression levels of DCLK1 in pancreatic NET (PNET) tissues and to identify critical functions of this molecule in PNET cells. Methods: Fifteen patients (8 male, 7 female; mean age, 56) with PNETs were enrolled in this study. The tumors were surgically resected between 1997 and 2012 in Kurume University Hospital. Immunohistochemistry (IHC) was employed to assess expression levels of DCLK1. QGP1, a human PNET cell line, was used in this study, and the cells were transfected with dclk1 cDNA to establish the DCLK1-overexpressing (QGP1-DOE) cells. The QGP1-DOE cells were subjected to dclk1 silencing to confirm acquired cellular characteristics by DCLK1 overexpression. Protein and mRNA expression levels were analyzed by Western blot and real-time PCR (ABI PRISM 7700), respectively. Results: In IHC, all of the 15 PNETs clearly and diffusely expressed DCLK1 in the tumor areas. QGP1-DOE cells robustly expressed full length of DCLK1, showing epithelial-mesenchymal transition (EMT)-like appearance. Of note, extremely high expression of the EMT regulator Slug was found in QGP1-DOE cells compared with control cells at both protein and mRNA levels. Expressions of N-cadherin and Vimentin were also upregulated, in concert with those of phospholylated (p-) FAK, Paxillin, and cyclin D1, in the QGP1-DOE cells. These cells increased cellular motility and proliferation. DCLK1 knockdown restored the above-mentioned expression profile of the EMT-associated molecules. Conclusion: We demonstrated robust expression of DCLK1 in human PNET tissues and PNET cells for the first time. Enforced expression of DCLK1 induced EMT via upregulating Slug expression and promoted proliferation and invasion probably through increasing expression levels of cyclin D1, pFAK, and Paxillin. Therefore, it is speculated that inhibition of DCLK1 expression is a novel therapeutic strategy for PNETs.

#1729

Withaferin A blocks formation of IFN-γ-induced metastatic cancer stem cells through inhibition of the CXCR4/CXCL12 pathway in the UP-LN1 carcinoma cell model.

Andy Shau-Bin Chou,1 Li-Lei Ting,2 Chin-Hsuan Hsieh,3 See-Tong Pang,3 Shuen-Kuei Liao2. 1 _Tzu-Chi General Hospital, Hualien, Taiwan;_ 2 _Taipei Medical University, Taipei, Taiwan;_ 3 _Chang Gung Memorial Hospital, Taoyuan, Taiwan_.

As our understanding of cancer stem cell (CSC) biology improves, there is an urgent need to search for inhibitory agents of CSCs and metastatic CSCs (mCSCs) positive for CXCR4. Withaferin A (WA), a withanolide extracted from the medicinal plant Withania somnifera, has been shown to exhibit anti-cancer effects through multiple mechanisms. Whether WA could selectively target CSCs, mCSCs, or non-CSCs of a gastrointestinal (GI) carcinoma tumor remains unclear. Side population analysis, flow cytometric phenotyping and sorting, non-invasive imaging in conjunction with xenotransplantation, and immunohistology were used in this investigation. Using the lymph node metastatic GI cancer cell line UP-LN1, consisting of CD44high/CD24low grape-like floating (F) and CD44low/CD24high adherent (A) cell subsets, this study demonstrated that, as compared with parental UP-LN1 cells or A cells, WA preferentially reduced F-cell proliferation, tumor sphere formation, and side population cells in vitro in greater efficiencies by apoptosis. This action was mechanistically mediated via the down-regulation of CXCR4/CXCL12 and STAT3/IL-6 pathways, both of which are instrumental in the acquisition of metastatic ability. Attenuation of IFN-γ-induced CXCR4 expression in F cells by knockdown with siRNA or blocking with anti-CXCR4 antibody, followed by Western blot analysis, showed significantly reduced metastatic potential in vitro. The extent of in vitro anti-invasive effect of WA on the IFN-γ-treated F cells was significantly greater than on the F cells without WA treatment, or F cells treated with control siRNA or with control IgG antibody. The observed in vitro effects of WA on the CSC and mCSC targeting were validated by data obtained with non-invasive imaging in NOD/SCID mouse xenotransplantation. We conclude that WA could efficiently block the formation of both CSCs and mCSCs in the UP-LN1 cell line, suggesting that WA may be considered an effective therapeutic agent for GI malignancies exhibiting grape-like tumor sphere.

#1730

Cancer stem cells might sustain stemness by the induction of autophagy in starvation stress.

Go Masuda, Masakazu Yashiro, Kishu Kitayama, Yuichiro Miki, Hiroaki Kasashima, Tamami Morisaki, Tatsunari Fukuoka, Tsuyoshi Hasegawa, Masaichi Ohira, Kosei Hirakawa. _Osaka City University, Osaka, Japan_.

Background: Cancer stem cells (CSCs), which have capacity for self-renewal and cancer cell supply, have been thought to be responsible for tumor initiation, distant metastasis, and chemoresistance. Side population (SP) cells extracted by the fluorescence-activated cell sorting technique using Hoechst 33342 are thought to be enriched for CSCs. Autophagy is an intracellular degradation system that is induced under stress, such as starvation. Although autophagy has been reported to be associated with tumor progression under various stresses, the significance of autophagy in CSCs has still remained to be unknown. The aim of this study is to clarify the role of autophagy of CSCs under starvation stress.

Materials and Methods: Two gastric cancer cell lines, OCUM-2MD3 and OCUM-12 were used. Two side population (SP) cell lines, OCUM-2MD3/SP and OCUM-12/SP were sorted by flow cytometry as CSC-rich cells from the parent OCUM-2MD3 and OCUM-12 cells, respectively. The effects of starvation stress, including low glucose, low amino acid, or low serum, on the stemness of CSCs were examined. The expression level of autophagy associated proteins, LC3A and LC3B, was evaluated by RT-PCR and western blotting. The effects of autophagy on the stemness and the proliferation of CSCs were examined by SP fraction and MTT assay. Chloroquine was used as an autophagy inhibitor.

Results: The percentages of SP cells were significantly higher in the OCUM-12/SP cells (10.2%) and OCUM-2MD3/SP cells (12.3%) than in their parent OCUM-12 cells (2.1%) and OCUM-2MD3 cells (1.2%). The expression level of LC3A and LC3B was high in both OCUM-2MD3/SP cells and OCUM-12/SP cells, in comparison to that in OCUM-2MD3 cells and OCUM-12 cells. Starvation stress increased the autophagy status of OCUM-2MD3/SP and OCUM-12/SP cells. Chloroquine significantly decreased the SP fraction and proliferation activity of OCUM-2MD3/SP cells and OCUM-12/SP cells.

Conclusion: Autophagy acts as a survival pathway in CSCs under starvation stress. CSCs might sustain stemness by the induction of autophagy in sarvation status. The autophagy inhibitor might be a promising therapy for CSCs.

#1731

Overexpression of DCLK1 in pancreatic cancer activates KRAS/PI3K/MTOR pathway signaling and supports tumorigenesis, invasiveness, and stemness.

Dongfeng Qu,1 Nathaniel Weygant,1 Randal May,1 William Berry,1 James Tomasek,1 Parthasarathy Chandrakesan,1 Michael Schlosser,2 Courtney Houchen1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _COARE Biotechnology Inc., Oklahoma City, OK_.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of any major malignancy with less than a 6% 5-year survival rate. Oncogenic KRAS mutation plays a key role in PDAC tumorigenesis with nearly 95% of PDAC harboring mutationally activated KRAS. Cells with cancer stem cell-like (CSC) properties have been identified in PDAC. These cells are often resistant to conventional chemotherapy and radiation therapy, and as such may explain why current treatments do not cure PDAC or prevent recurrences. It is suggested that "driver" mutations in these stem-like cells may be the root cause of PDAC. Doublecortin-like kinase 1 (DCLK1), is overexpressed and marks a population of tumor-initiating cells in PDAC. It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. In this study we evaluated the role of DCLK1 in KRAS-mediated signaling in PDAC. Human pancreatic cancer cells (AsPC-1 and MiaPaCa-2) were infected with Lentivirus containing human DCLK1 cDNA sequence to overexpress DCLK1 or red fluorescent protein (RFP) cDNA sequence as control. The expressing levels of DCLK1, active KRAS, and proteins in KRAS/PI3K/mTOR pathway were analyzed by western blotting. The proliferative and invasive potential of these cells were compared using a MTT assay for proliferation, wound healing assay for migration, soft agar assay for clonogenicity, and Matrigel coated transwell assay for invasion. Gemcitabine, Rapamycin, and PI3K inhibitors were used for evaluation. Analysis of human PDAC was performed using the TCGA PAAD dataset. Here we report that DCLK1 protein levels were increased more than 20 fold in AsPC-DCLK1 and MP2-DCLK1 cells resulting in activation of KRAS which was reversible by DCLK1-inhibitor XMD8-92. Compared to RFP control cells, AsPC-DCLK1 and MP2-DCLK1 cells exhibited a more than 20% increase in proliferation, approximately 30% increase in colony formation, 20% increase in migration, and a 2-fold increase (p < 0.05) in invasion. Evidence from TCGA PAAD demonstrated that pancreatic tumors expressing high levels of DCLK1 had activated PI3K/AKT/MTOR-pathway signaling suggesting greater KRAS activity. These data taken together suggest that DCLK1 promotes KRAS-driven PI3K/AKT/mTOR signaling in PDAC leading to increased migratory, invasive, anti-apoptotic effects, stem-like and tumorigenic properties.

#1732

The analysis of stem cell property and mesenchymal feature of anaplastic pancreatic cancer.

Kenjiro Kimura, Kotaro Miura, Ryosuke Amano, Sadaaki Yamazoe, Naoki Kametani, Kohei Nishio, Masaichi Ohira, Kosei Hirakawa. _Osaka City University, Osaka, Japan_.

Background: Anaplastic pancreatic cancer (APC) is the subtype of pancreatic ductal adenocarcinoma (PDAC). APC is poorer prognosis than ordinary PDAC. However, it is unclear what feature of APC is different from that of ordinary PDAC. Our previous studies suggested that the angiogenesis and the tolerance of hypoxia might related to the higher in vivo proliferation of APC than ordinary PDAC.

Purpose: We tried to estimate the difference between APC and PDAC using SP cells which reflected cancer stem-like cell and drug resistance and western blotting of E-cadherin and vimentin which reflected epithelial-mesenchymal transition.

Materials and Methods: For SP cell analysis, OCUP-A1 and OCUP-A2 were used as APC cell lines and Panc-1 and MIAPaCa2 were used as ordinary PDAC cell lines. These cells were incubated in the medium including Hoechst 33342 with or without verapamil. Then, propidium iodide was added. And the analysis was performed using FACS Aria II. For western blotting, OCUP-A1 and OCUP-A2 were used as APC cell lines. And breast cancer cell line MCF-7 was used as control.

Results: The proportion of SP cells in all cells was as follows; OCUP-A1/ OCUP-A2/ Panc-1/ MIAPaCa2 = 1.8±0.28%/ 1.7±0.12%/ 1.1±0.20%/ 0.6±0.058%. The proportion of SP cells of OCUP-A1 and OCUP-A2 were similar to that of Panc-1. However the proportion of SP cells of OCUP-A1 and OCUP-A2 significantly larger than that of MIAPaCa2. In western blotting, the expression of vimentin was observed and the expression of E-cadherin was lost in OCUP-A1 and OCUP-A2. Inversely, the expression of E-cadherin was observed and the expression of E-cadherin was lost in MCF-7.

Discussion: It was reported that SP cells might contribute to tumorigenesis and drug resistance the same as cancer stem-like cell. Our current study suggested that larger proportion of SP cells in APC might induce higher malignancy of APC. Also, it was reported that mesenchymal feature might related to cancer stem cell properties. In this study, it was suggested that APC cell lines might have mesenchymal feature. However, it has been unclear whether mesenchymal feature of APC would affect the proportion of SP cells of APC from this study. So, we need to confirm the correlation with cancer stem sell property and mesenchymal feature of APC.

Conclusion: The large proportion of SP cells and the mesenchymal feature might contribute to higher malignant property of APC.

#1733

Inhibition of embryonic stem cells gene expression in pancreatic cancer: Effects of metformin and indole-3-carbinol.

Beverly D. Lyn-Cook, Stancy Joseph, Beverly Word, George Hammons. _FDA-NCTR, Jefferson, AR_.

Pancreatic cancer is among the top five deadliest cancers in developing countries and is rapidly increasing in minority populations, particularly African Americans. Understanding molecular mechanisms involved in the etiology and progression of this cancer can greatly improve patient prognosis. Cancer stem cells have been identified in pancreatic tumors and have been shown to contribute to its progression and resistance to standard treatments. Pancreatic cancer stem cells are subject to regulation by key embryonic stem cell transcription factors that are known to be aberrantly expressed in pancreatic cancer, such as SOX2, OCT4 and NANOG. Overexpression of OCT4, SOX2 and NANOG together or separately led to tumor transformation and tumor metastasis. These stem cell factors are known to promote self-renewal by interacting with other transcription factors. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes were expressed in pancreatic cancer cell lines (MIAPaca2, Panc1). Both cell lines expressed high levels of SOX2, NANOG, CD44, GATA2, POU5F1 (OCT4) and other genes. Panc1 expressed high levels of SOX 17, while MIAPaCa2 expressed high levels of SOX 3,15 and 17. Treatment of cells with indole-3-carbinol alone at 100 and 200µM inhibited or decreased the expression of SOX2, NANOG, CD44, SOX15 and STAT3. However, treatment with metformin alone increased expression of SOX 15 and STAT3 in Panc1 cells but not in MIAPaCa2. Combination of metformin and indole-3-carbinol inhibited the expression of SOX2, NANOG, STAT3, and CD44. SOX2, POU5F1(OCT4) and NANOG are thought to be important players in various human cancers. OCT4 has been found to be expressed in 69% of PDAC and expression was shown too correlated to status and clinical state. High levels of OCT4 and NANOG has been found to correlate to worse prognosis and furthermore, contribute to multidrug-resistance and metastasis. This study have shown that the dietary agent, indole-3-carbinol alone and in combination with metformin down-regulate critical stem cell genes involved in maintenance of pluripotency and self-renewal of cancer stem cells.

## EPIDEMIOLOGY:

### Diet, Tobacco, Smoking, and Other Lifestyle Factors in Cancer Epidemiology

#1734

Body mass index, physical activity and risk of colorectal cancer in the Korean Multi-center Cancer Cohort (KMCC).

Sooyoung Cho,1 Aesun Shin,1 Sue K. Park,1 Hai-Rim Shin,2 Soung-Hoon Chang,3 Keun-Young Yoo1. 1 _Seoul National University, College of Medicine, Seoul, Republic of Korea;_ 2 _World Health Organization Regional Office for the Western Pacific, Manila, Philippines;_ 3 _Konkuk University, College of Medicine, Chungju, Republic of Korea_.

Objectives: To examine the association between body mass index (BMI), physical activity and colorectal cancer risk among Korean adults.

Methods: Data from the Korean Multi-center Cancer Cohort (KMCC) between 1993 and 2005 were analyzed. The study population comprised 12,379 subjects aged above 20 years old. The subjects were followed until December 31, 2011 (median follow-up of 10.1 years). Measured weight and height values was used to calculate BMI and self-reported total time spent for physical activity were used. The Cox proportional hazard model was used to estimate the hazard ratio (HR) and 95% confidence intervals (CI) of BMI and physical activity for colorectal cancer risk.

Results: Men who did moderate physical activity showed a lower risk for colorectal cancer (HR 0.35, 95% CI: 0.19-0.65 for 30-419 minutes compared to who spend less than 30 minutes a week doing moderate activities). We did not find any association between the total time of vigorous activities and muscle-strengthening activities and colorectal cancer risk in both men and women. Men with BMI of 25 or higher showed an increased risk for colorectal cancer compared to men with BMI of 18.5 to 22.9 (HR 1.64, 95% CI 0.94-2.88 for 25.0-29.9 kg/m²; HR 1.64, 95% CI 0.94-2.88 for greater than 30.0 kg/m²).

Conclusions: Moderate physical activities were associated with lower colorectal cancer risk among Korean men.

#1735

Fruit and vegetable consumption and risk of gastric cancer: a prospective nested case-control study in China, Japan and Korea.

Tianyi Wang,1 Hui Cai,2 Shizuka Sasazuki,3 Shoichiro Tsugane,3 Wei Zheng,2 Eo Rin Cho,4 Sun Ha Jee,4 Angelika Michel,5 Michael Pawlita,5 Yong-Bing Xiang,6 Yu-Tang Gao,6 Xiao-Ou Shu,2 Weicheng You,1 Meira Epplein2. 1 _Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Cancer Epidemiology, Peking University Cancer Hospital and Institute, Beijing, China;_ 2 _Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN;_ 3 _Epidemiology and Prevention Group, Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo, Japan;_ 4 _Department of Epidemiology and Health Promotion, Institute for Health Promotion, Graduate School of Public Health, Yonsei University, Seoul, Republic of Korea;_ 5 _Division of Molecular Diagnostics of Oncogenic Infections, Research Program in Infection, Inflammation, and Cancer, German Cancer Research Center (DFKZ), Heidelberg, Germany;_ 6 _Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China_.

Introduction: Epidemiological findings on the association between fruit and vegetable consumption and gastric cancer risk remain inconsistent, resulting in the designation of fruits and vegetables as "probable" and "possible" respectively, but not "convincing", protective factors. However, intervention studies provide support for the effect of micronutrient supplementation in the prevention of gastric cancer and its precursor lesions, though the effects remain less substantial than for Helicobacter pylori (H. pylori) eradication. Objective: To ascertain the association between fruit and vegetable intake and non-cardia gastric cancer incidence with adjustment for H. pylori within East Asian cohort studies. Methods: The present analysis includes 1970 participants (810 prospectively ascertained non-cardia gastric cancer cases with 1160 matched controls) from 5 cohort studies in the Helicobacter pylori Biomarker Cohort Consortium. These cohorts collected blood samples as well as demographic, lifestyle, and dietary data at baseline. Pre-diagnostic antibody levels to 15 H. pylori proteins were assessed using multiplex serology. Conditional logistic regression, adjusting for total energy intake, smoking, and H. pylori status, was applied to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for gastric cancer risk across cohort- and sex-specific quartiles of fruit and vegetable intake. Results: Increasing fruit intake was significantly associated with decreasing risk of non-cardia gastric cancer, so that individuals in the highest quartile of fruit consumption had a 29% reduced odds of gastric cancer, compared to individuals in the lowest quartile (OR=0.71, 95% CI, 0.52-0.95, P for trend=0.02). Compared to CagA-positive H. pylori low fruit consumers, the strongest inverse association of gastric cancer risk was amongst those high fruit consumers without evidence of H. pylori antibodies (OR=0.12, 95% CI: 0.06-0.25), whereby the inverse association by increasing fruit consumption was attenuated among individuals infected with CagA-positive H. pylori (OR=0.82, 95% CI: 0.66-1.03). We observed a weaker, non-dose-response suggestion of an inverse association of vegetable intake with non-cardia gastric cancer risk. Conclusions: To our knowledge, this is the largest prospective study in the high-risk region of East Asia to examine the association of fruit and vegetable consumption with non-cardia gastric cancer risk adjusted for H. pylori. We have found that high fruit intake may play a role in decreasing risk of non-cardia gastric cancer, even after adjustment for H. pylori subtype-specific infection. Funding: R01 CA174853

#1736

Physical activity, sitting time and prostate cancer specific mortality: The Cancer Prevention Study II Nutrition Cohort.

Ying Wang, Eric J. Jacobs, Ted Gansler, Marjorie L. McCullough, Victoria L. Stevens, Susan M. Gapstur, Alpa V. Patel. _American Cancer Society, Atlanta, GA_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2016 Official Press Program. It will be posted online following its presentation.

#1737

Reproductive and hormonal factors in relation to lung cancer among Nepali women.

Sanah N. Vohra,1 Amir Sapkota,2 Mei-Ling T. Lee,3 Mia Hashibe,4 Bhola Siwakoti,5 Chin B. Pun,5 Cher M. Dallal3. 1 _FDA, Laurel, MD;_ 2 _Maryland Institute for Applied Environmental Health (MIAEH), University of Maryland, College Park, MD;_ 3 _Department of Epidemiology and Biostatistics, University of Maryland, College Park, MD;_ 4 _Division of Public Health, University of Utah School of Medicine, Salt Lake City, UT;_ 5 _B. P. Koirala Memorial Cancer Hospital, Bharatpur, Nepal_.

Of the 1.8 million global incident lung cancer cases estimated in 2012, approximately 58% occurred in less developed regions (LDRs). Studies conducted in more developed regions suggest potential gender differences in lung cancer risk, whereby women may be more susceptible to the carcinogenic effects of tobacco. However, studies of reproductive and hormonal factors and lung cancer among women have been inconsistent and to date, no prior study has assessed these relationships among Nepali women. Using data from a hospital-based case-control study conducted in B. P. Koirala Memorial Cancer Hospital (Nepal, 2009-2012), relationships between reproductive and hormonal factors and lung cancer were examined among women aged 23-85 years. Lung cancer cases (n=268) were frequency-matched to controls (n=226), on the basis of age (±5 years), ethnicity and residential area. Controls were selected from visitors of non-lung cancer patients within the same hospital. Participants completed an interviewer-administered questionnaire and provided detailed information on demographics, medical history, smoking history, and reproductive and hormonal factors. The main exposures in this analysis included menopausal status, age at menarche, age at menopause, menstrual duration, gravidity, and age at first live-birth. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariable logistic regression. Final models include adjustment for key confounders including age and smoking status. Among postmenopausal women, those with a younger age at menopause (<45years; 45-49years) had an increased odds of lung cancer compared to those with an older (≥50years, referent) age at menopause (OR=2.14, 95%CI=1.09, 4.17; OR=1.93, 95%CI=1.07, 3.51, respectively), after adjusting for age and cumulative active smoking (CAS). No statistically significant associations were observed with the other exposures examined. Ongoing analyses include stratification by key factors including CAS years. Further research is needed to corroborate and expand on these findings in order to better understand hormonal risk factors for lung cancer among Nepali women.

#1738

A pooled analysis of carbohydrate and dietary fiber intake and prostate cancer risk.

Elkhansa Sidahmed,1 Stephen J. Freedland,2 Kana Wu,1 Jeanine M. Genkinger,3 Stephanie A. Smith-Warner1. 1 _Harvard T.H Chan School of Public Health, Boston, MA;_ 2 _Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA;_ 3 _Columbia University Mailman School of Public Health, New York, NY_.

Scientific findings on the association between carbohydrate intake (both quantity and quality) and prostate cancer (CaP) risk are conflicting. We examined this association in the Pooling Project of Prospective Studies of Diet and Cancer, a consortium of 15 cohorts with validated measures of dietary intake. During follow-up of 842,149 men, 52,683 were diagnosed with CaP. We examined associations between carbohydrate and fiber intake and 3 different definitions of advanced CaP: 1) Advanced cases were defined as men with tumors that were advanced at diagnosis (T4, N1, and/or M1) or men who died of CaP during follow-up regardless of diagnosed stage (n=4,934); 2) Advanced (restricted) cases were defined as above, but excluded men diagnosed with localized CaP who later died of Cap (n=3,091); and 3) Fatal CaP was defined as men who died of CaP (n=3,097). We performed Cox proportional hazards models to calculate study-specific multivariable relative risks (RR) and pooled these results using random effects models. Models were adjusted for age, race/ethnicity, education, marital status, body mass index, height, smoking status, family history of CaP, physical activity, history of diabetes, multivitamin use, and energy and alcohol intake. Median total carbohydrate intake across studies ranged from 41% - 64% of total energy intake. Preliminary analyses were suggestive of a modest inverse association between carbohydrate intake and risk of advanced and fatal CaP. The pooled multivariable adjusted RR (MVRR) comparing the highest versus lowest study-specific quintile of carbohydrate intake (percent of energy) was 0.90 (95% confidence interval [CI] 0.81-1.01; trend test p-value 0.06) for advanced CaP and 0.91 (0.97-1.04; trend test p-value 0.16) for advanced (restricted) Cap. For fatal CaP, the pooled MVRR for the same comparison was 0.88 (95% CI 0.77-1.00), trend test p-value 0.05. Findings were similar for energy-adjusted total carbohydrates (g/day) or available carbohydrates (total carbohydrates - dietary fiber). When total carbohydrate intake was categorized using common absolute cutpoints across studies, no significant change in risk was observed for men consuming ≥ 60% vs 45%-<50% energy for carbohydrates for advanced, advanced (restricted) or fatal CaP. Total dietary fiber intake was not statistically significantly associated with risk of advanced, advanced (restricted), or fatal CaP; men in the highest versus lowest quintile of intake had nonsignificant 3%-11% lower risks. No statistically significant between-studies heterogeneity was observed for any of these estimates. Although these preliminary findings suggest a modest inverse association for carbohydrate intake and CaP risk, further analyses on carbohydrate quality (glycemic index and load, refined vs whole grains) and examination of the effect of substituting carbohydrates for other macronutrients are needed.

#1739

Calcium to magnesium intake ratio and colorectal carcinogenesis, results from the Prostate, Lung, Colorectal, and Ovarian cancer screening trial.

Jing Zhao,1 Xiangzhu Zhu,1 Martha J. Shrubsole,1 Todd L. Edwards,1 Edward L. Giovannucci,2 Wei Zheng,1 Qi Dai,1 PLCO Project Team. 1 _Vanderbilt University, Nashville, TN;_ 2 _Harvard Medical School, Boston, MA_.

Background

A recent randomized trial found no effect of calcium supplementation on reducing risk of colorectal adenoma recurrence. However, the role of calcium in carcinogenesis is yet unconfirmed. An earlier trial found calcium treatment only reduced recurrence when the baseline calcium: magnesium intake ratio was <2.6. Nor is it clear whether calcium intake is differentially related to risks of different stages of colorectal carcinogenesis (incident adenoma, adenoma recurrence and incident colorectal cancer). Further, it is unclear if calcium intake or supplementation confers additional protection against incident colorectal cancer in individuals receiving endoscopic screenings. To address these important scientific inquiries, we have conducted analyses within the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial.

Methods

The PLCO is a large, multicenter, population-based randomized trial designed to determine the effects of screening on cancer-related mortality with a median of 12.5 years of follow up. We evaluated the associations between calcium intake and the risks of incident colorectal adenoma (1,147 cases), recurrent adenoma (855 cases) and incident colorectal cancer (697 and 578 cases in intervention and control arms, respectively) among 108,563 PLCO participants aged 55 to 74 years. Odds ratios (OR) and 95% confidence intervals (95%CIs) for incident adenoma and recurrent adenoma, and hazard ratios (HR) and 95%CIs for colorectal cancer incidence were calculated using unconditional logistic regression models and Cox proportional hazard models after adjusting covariates.

Results

Compared to low calcium intake (<600 mg/day), higher intakes of calcium were not related to a reduced risk of incidence adenoma, but significantly associated with a reduced risk of advanced/multiple adenomas (p for trend, <0.05). Further, the significant inverse association only appeared when calcium:magnesiuim intake ratios between 1.7 and 2.5. No significant association was observed between Ca intake and risk of adenoma recurrence. In the control arm, we found, compared to low calcium intake, higher intakes of calcium were associated with significantly reduced risks of incident colorectal cancer, primarily distal cancer. Again, we found the inverse association may primarily appear in those with calcium:magnesiuim intake ratios between 1.7 and 2.5. In contrast, in the intervention arm (i.e. screening with flexible sigmoidoscopy), overall, we did not find any significant associations.

Conclusion

Intake of calcium may be more strongly related to reduced risks of incident advanced/multiple adenoma and incident colorectal cancer among those without active screening with flexible sigmoidoscopy than to recurrent adenoma and incident colorectal cancer risks among those with active screening with flexible sigmoidoscopy.

#1740

Lifestyle determinants of mammographic density in Asian and Caucasian populations: A comparative analysis.

Nadia Rajaram,1 Shivaani Mariapun,1 Mikael Eriksson,2 Jose Tapia,2 Pui Yoke Kwan,1 Weang Kee Ho,3 Faizah Harun,4 Nor Aishah Mohd Taib,4 Per Hall,2 Soo-Hwang Teo1. 1 _Cancer Research Malaysia, Subang Jaya, Malaysia;_ 2 _Karolinska Institutet, Stockholm, Sweden;_ 3 _University of Nottingham, Semenyih, Malaysia;_ 4 _University Malaya Cancer Research Institute, Kuala Lumpur, Malaysia_.

Mammographic density is an independent risk factor for breast cancer and has been shown to differ among populations with varying risk to breast cancer. We sought to compare the distribution of known breast cancer risk factors between an Asian and a Caucasian population, and to determine if the variations in mammographic density between Asian and Caucasian populations can be attributed to the differences in the distribution of these risk factors.

2948 women with no personal history of breast cancer attending an opportunistic screening program in Malaysia (MyMammo) were included in the analysis. Participants were age and BMI matched to 8837 women of the Karolinska Mammography Project for Risk Reduction of Breast Cancer (KARMA) study in Sweden. For analyses involving mammographic density, a subset of Malaysian women with available raw FFDM images (n=1501) and matched Swedish women (n=4501) were included for analysis. Volume-based mammographic density measurements for both cohorts were measured using an automated method (Volpara). Descriptive statistics were used to describe the distribution of breast cancer risk factors and mammographic density measures between the two cohorts. General linear models and stepwise selection method were used to determine the risk factors associated with mammographic density within each cohort.

Most of the anthropometric, reproductive and lifestyle risk factors examined were differentially distributed between the two cohorts. There was a significantly greater proportion of postmenopausal Swedish women (56%) compared to Malaysian women (51%). Hormone replacement therapy and oral contraceptive ever use was substantially higher among Swedish women (24% and 73%) than Malaysian women (14% and 29%). Mean dense volume was not significantly different between the two cohorts, but Swedish women had a significantly higher mean non-dense volume and lower percent volumetric density compared to Malaysian women. However, pre-menopausal Malaysian women had statistically significant higher dense volume than Swedish women (mean dense volume of 73.9cm³ compared to 70.4cm³). Among post-menopausal women, Malaysian women had significantly lower dense volume than Swedish women (mean dense volume of 54.7cm³ compared to 57.5cm³). In multivariable analyses, age, BMI and parity were associated with dense volume in both cohorts, but regular alcohol intake, height, changes in body shape over time, and menopausal status were only significantly associated with dense volume for Swedish women.

In an age and BMI matched cohort, pre-menopausal Asian women had significantly higher dense volume than Caucasian women, whereas, post-menopausal Asian women had lower dense volume than Caucasian women. Population differences in height, body shape changes over time, and alcohol intake could explain, in part, the variation seen in mammographic density, and potentially breast cancer risk, across populations.

#1741

The association between menopause symptoms and risk of postmenopausal breast cancer among non-users of hormone therapy.

Tricia Li,1 Jiali Han,2 Chunyan He2. 1 _Harvard School of Public Health, Boston, MA;_ 2 _Indiana University Richard M. Fairbanks School of Public Health, Indianapolis, IN_.

Background: Hot flashes are a common symptom among women during their menopause transition, likely due to the changes of endogenous hormone levels. Previous research suggested that hot flashes symptoms were associated with a reduced risk of cardiovascular diseases. Few studies have investigated the association of hot flashes symptoms with the risk of breast cancer. Furthermore, the confounding effect by hormone use has not been sufficiently addressed in previous studies.

Methods: We investigated the association between hot flashes symptoms and risk of postmenopausal breast cancer in the Nurses' Health Study (NHS). In order to eliminate the confounding by hormone use, we investigated the association only among women who never used hormone therapy. We calculated relative risk of breast cancer using a multivariate Cox model, adjusting for potential confounders, including age, race, family history of breast cancer, age at menarche, age at menopause, parity, age at first birth, body mass index, smoking, alcohol use, and education. We further investigated the severity of hot flashes symptoms in association with risk of postmenopausal breast cancer.

Results: We documented 1079 incidence of breast cancer during 396,166 person-years of follow-up. Hot flashes symptoms were not associated with risk of postmenopausal breast cancer after adjustment for potential confounders (RR=1.02, 95% CI, 0.90-1.15) among non-users of hormone therapy. Relative risk of breast cancer across symptom severity categories (mild, moderate, severe) were 1.01 (95% CI, 0.87-1.17), 1.04 (95% CI, 0.88-1.22), and 1.05 (95% CI, 0.75-1.46), respectively.

Conclusion: These data suggested that menopausal symptoms were not associated with risk of postmenopausal breast cancer.

#1742

Smoking and risk of mucinous epithelial ovarian cancer among 300,000 women.

Idlir Licaj,1 Bjarne Koster Jacobsen,1 Randi Marie Selmer,2 Gertraud Maskarinec,3 Elisabete Weiderpass,4 Inger Torhild Gram1. 1 _UiT University of Tromsø, Tromsø, Norway;_ 2 _Norwegian Institute of Public Health, Oslo, Norway;_ 3 _University of Hawai'i Cancer Center, Honolulu, HI;_ 4 _Karolinska Institutet Department of Medical Epidemiology and Biostatistics, Stockholm, Sweden_.

Background

In 2012, The International Agency for Research on Cancer classified mucinous epithelial ovarian cancer as tobacco related. In a recent pooled analysis of 51 epidemiological studies, smoking was associated with an increased risk of mucinous epithelial ovarian cancer, but not with other ovarian cancer histological subtypes. The main purpose of this study was to examine impact of smoking related increase in epithelial ovarian cancer according to histological subtypes and invasiveness in a large prospective cohort study.

Methods

We followed 300,398 Norwegian women born between 1899 and 1975, recruited from 1974 to 2003, by linkage to national virtually complete registries through December 2013. The three prospective cohort studies conducted by the Norwegian Institute of Public Health and included in the analysis were the Norwegian Counties Study (1974-1988), the 40 Years Study (1985-1999), and the Cohort of Norway (CONOR) Study (1994-2003). Their data were merged and analyzed altogether. We used Cox proportional hazard models to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between smoking status, smoking-intensity and duration and epithelial ovarian cancer (EOC) histological subtypes. We used multivariable analyses stratified by birth cohort (≤1950>) and cohort study, and tested for heterogeneity by BMI, attained education and physical activity.

Results

During >5.9 million person-years, with a median follow-up of 19 years, 2,336 primary epithelial ovarian cancers (EOC) were identified, of which 1,647 (71%) invasive and 689 (29%) borderline. In our study, 38.0% of women were current, 21.1% former and 40.8% never smokers. Current versus never smokers risk of all histological subtypes of invasive EOC (multivariable HR=0.97 95% CI, 0.86-1.08) was significantly different from the corresponding risk of all histological subtypes of borderline epithelial ovarian tumours (multivariate HR=1.55 95% CI, 1.29-1.85) (pheterogeneity < 0.0001). Compared with never smokers, current smokers had more than doubled risk of mucinous epithelial ovarian cancer (HR=2.09 95% CI, 1.67-2.62). When stratified according invasiveness the corresponding figure was an increased risk of 78% [HR=1.78 95% CI, 1.20-2.64 (ncases=138)] for invasive mucinous epithelial ovarian cancer and more than doubled risk [HR=2.26 95% CI, 1.71-2.97 (ncases= 302)] for borderline mucinous epithelial ovarian cancer (pheterogeneity =0.34). Women who had smoked for more than 10 years had a 42% (HR=1.42 95% CI, 1.07-1.88) increased risk of mucinous epithelial ovarian tumous compared to never smokers. The corresponding risk for those who smoked for longer than 20 years was increased with (HR=2.13 95% CI, 1.68-2.70) (ptrend < 0.001).

Conclusions

In this study smoking increases the risk of mucinous epithelial ovarian cancer in similar extent for invasive and borderline tumours.

#1743

Sedentary behavior and breast cancer in the Black Women's Health Study.

Sarah Nomura,1 Chiranjeev Dash,1 Jeffrey Yu,2 Julie Palmer,2 Lynn Rosenberg,2 Lucile L. Adams-Campbell1. 1 _Georgetown-Lombardi Comprehensive Cancer Center, Washington, DC;_ 2 _Slone Epidemiology Center at Boston University, Boston, MA_.

Background: Rates of breast cancer incidence among African American women are increasingly similar to rates among Caucasian American women. Lifestyle behaviors, such as sedentary time, may contribute to differences in breast cancer incidence, but have not been well studied among African American women. Sedentary activities (physically inactive tasks that require little to no additional energy expenditure beyond basal metabolic rate) account for a large proportion of time per day in the United States and are more prevalent among African American women. Sedentary behavior may contribute to cancer risk independently of physical activity, but has not been well studied among African American women.

Objective: The objective of this study was to evaluate the association between sedentary behaviors over time on breast cancer incidence in the Black Women's Heath Study.

Methods: In this ongoing prospective cohort of African American women (analytic cohort N=55,629), 2,482 incident breast cancer cases were diagnoses between baseline (1995) and 2013. Questionnaire data (collected every two years since 1995) was used to calculate time spent sitting. Time spent sitting at work and sitting doing recreational activities (watching TV, using internet) was summed to total sedentary time. A time-varying analytic approach was utilized (Anderson-Gill method) to reduce within-person variation and better represent long-term habits.

Results: Among women in this cohort, 23.8% reported spending <5 hours/day in sedentary activities, while 8.2% reported spending 10 or more hours. A majority reported spending more than 5 hours/day sitting at work, while most women reported <5 hours sitting watching TV (or related activities). Total time spent sitting was significantly associated with breast cancer incidence overall (≥10 vs. <5 hours/day HR=1.38, 95% CI: 1.16-1.65). Associations were similar for both premenopausal (≥10 vs. <5 hours/day HR=1.44, 95% CI: 1.07-1.93) and postmenopausal (≥10 vs. <5 hours/day HR=1.39, 95% CI: 1.09-1.78) breast cancer incidence. Associations remained regardless of leisure time physical activity levels and body size.

Conclusions: Our findings suggest that high sedentary time may increase risk for breast cancer among African American women. Additionally, leisure time physical activity levels and body size did not change associations, suggesting sedentary time may confer additional risk. Previously reported frequent sedentary behavior among African Americans could contribute to breast cancer disparities and should be explored further in future studies.

#1744

Is adherence to the WCRF/AICR cancer prevention recommendations associated with colorectal cancer in African American women in the Black Women's Health Study.

Sarah J. O. Nomura,1 Chiranjeev Dash,1 Lynn Rosenberg,2 Jeffrey Yu,2 Julie R. Palmer,2 Lucile L. Adams-Campbell1. 1 _Georgetown-Lombardi Comprehensive Cancer Center, Washington, DC;_ 2 _Slone Epidemiology Center at Boston University, Boston, MA_.

Background: African American women are more likely to be diagnosed with colorectal cancer than women from other racial/ethnic groups. Differences in adherence to cancer prevention recommendations may contribute to disparities. Adherence to cancer prevention guidelines has previously been shown to be associated with lower breast cancer incidence, but previous study populations have included few African Americans.

Objective: The purpose of this study was to evaluate adherence to the World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) cancer prevention recommendations and colorectal cancer incidence in the Black Women's Health Study (BWHS).

Methods: In this ongoing prospective cohort of African American women (analytic cohort N=49,103), 354 incident colorectal cancers were diagnosed between baseline (1995) and 2011. Adherence scores for seven WCRF/AICR recommendations (adherent=1 point, non-adherence level 1=0.5 points, non-adherence level 2=0 points) were created using questionnaire data and summed to an overall adherence score (maximum=7). Diet-specific recommendations were additionally summed for a diet only score (maximum=5). Recommendation adherence and incidence of colorectal cancer combined and colon cancer only were evaluated using baseline and time-varying data in Cox regression models.

Results: Guideline adherence was low with 8.5% of women (5.6% among women who later developed colorectal cancer) adhering to more than four recommendations at baseline. Adherence to a greater number of WCRF/AICR recommendations was not statistically significantly associated with colorectal cancer risk in time-varying or baseline models. In time-varying analyses, the HR (per 0.5 point increase) was 0.98 (95% CI: 0.84-1.15) and 0.51 (95% CI: 0.23-1.10) among women who adhered to >4 compared to <3 recommendations. Similarly, colon cancer evaluated alone was not statistically significantly associated with breast cancer incidence in time-varying (>4 vs. <3 recommendations HR= 0.54, 95% CI: 0.23-1.26) or baseline models (>4 vs. <3 recommendations HR= 0.72, 95% CI: 0.39-1.34). Regardless of modeling approach, adherence to dietary recommendations or individual recommendations was not associated with colon only or combined colorectal cancer risk.

Conclusions: Further research in diverse populations is essential to evaluate the validity of existing recommendations, and to assess whether there are alternative recommendations that are more beneficial for colorectal cancer prevention in specific populations.

#1745

Comorbidity factors associated with human papillomavirus infectivity: Implications in cervical cancer health disparity.

Vivek K. Kashyap,1 Sheema Khan,1 Mohammad Sikander,1 Diane M. Maher,2 Santosh Kumar,1 Namita Sinha,1 Murali M. Yallapu,1 Nadeem Zafar,1 Meena Jaggi,1 Subhash C. Chauhan1. 1 _University of Tennessee Health Science Center, Memphis, TN;_ 2 _Sanford Research, Sioux Falls, SD_.

Objective: High-risk strains of human papillomavirus (HPV), HPV E6/E7 cause cervical cancer (CxCa). Certain underserved populations in the United States, such as American Indian and African American women disproportionately suffer from CxCa compared to their Caucasian counter parts. However, precise etiology and comorbidity factors associated with CxCa health disparity are not fully uncovered. Understanding of these factors at molecular level will entail developing novel strategies to reduce this health disparity. In this study, we have investigated the molecular interplay existing between various comorbidity factors, namely, smoking, alcohol and HIV co-infection on the HPV infectivity which are primarily known for the progression of CxCa.

Method: In order to define a molecular association of smoking, alcohol and HIV co-infection with CxCa, Caski and SiHa (HPV infected cervical cancer cells) cells were treated with a smoking carcinogens Benzo[a]Pyrene (BaP) or alcohol (EthOH) or both for different time periods. Effects of these treatment was analyzed on cell proliferation, clonogenicity, cell migration, cell cycle and the expression of HPV E6/E7 was determined by qRT-PCR, immunoblotting and confocal microscopy. The effect of HIV co-infection on the expression of HPV E6/E7 was also investigated by incubating CxCa cells with conditioned medium derived from HIV infected U937 monocytic cells (U1). Additionally, we examined effect of these cofactors on the expression enzymes associated with cellular oxidative stress using immunoblotting and confocal microscopy analyses.

Result: Our results show that the exposure of BaP or EthOH or their combination enhances the expression of HPV E6/E7 oncogenes. Additionally, cells treated with BaP and EthOH alone or in combination show higher oncogenic phenotypes as evident by increased cell proliferation, clonogenicity and cell migration andinvasion. These cofactors in presence of HIV co-infection also augment the expression of HPVE6/E7 oncogenes. Exposure of these cofactors alter cellular oxidative stress via modulation of the expression of PRDX6 enzyme. Interestingly, curcumin and its nanoparticle formulation (Nano-Cur) effectively inhibits BaP/EthOH induced expression of E6/E7 oncogenes, growth, migration of CxCa cells and induces apoptosis.

Conclusions: The study suggests a molecular link between smoking, alcohol and HIV infection with HPV infectivity and their potential association with CxCa health disparity. These events however, can be effectively attenuated by curcumin/nano-curcumin treatment, implying its role in CxCa prevention/treatment. This provides hope for developing a feasible approach to effectively reduce CxCa health disparity among underserved populations.

#1746

Tobacco use among US cancer survivors.

Tainya C. Clarke. _CDC—National Center for Health Statistics, Hyattsville, MD_.

Background: Combustible tobacco use is a known risk factor for several types of cancer including cancers of the head and neck, esophagus, lung, stomach, bladder, pancreas and cervix. Smokeless tobacco use is a known risk factor for cancers of the mouth, tongue, cheek, gum, and esophagus. Continued use of cigarettes, non-cigarette combustible tobacco products, or smokeless tobacco post diagnosis may interfere with treatment as well as increase the chance of developing a second malignancy. In the past, the US Food and Drug Administration (FDA) has designed targeted educational campaigns to prevent and reduce tobacco use among high-risk populations. Information on the prevalence of use among cancer survivors is vital to inform targeted smoking cessation programs.

Methods: We analyzed combined data from the 2012-2014 National Health Interview Survey (NHIS) for tobacco use by cancer status among over 6,000 cancer survivors aged 18 and older. Non-cigarette combustible tobacco products include cigars, pipes, hookahs, bidis or cigarillos. Smokeless tobacco refers to tobacco products which are placed in the mouth or nose and include chewing tobacco, snuff, dip, snus (snoose), or dissolvable tobacco. Current users had used at least 100 cigarettes in their lifetime— or used other non-cigarette combustible tobacco products at least once in their lifetime— and reported current use as "rarely," "some days" or "every day." Survey data were weighted to produce national estimates that are representative of the civilian noninstitutionalized US adult population. Differences between percentages were evaluated using two-sided significance tests at the 0.05 level.

Results: More than 1 in 5 cancer survivors were current cigarette smokers. This was significantly higher than the population of persons without a cancer history. More than 1 in every 15 survivors were current users of some form of non-cigarette combustible tobacco product. However, this prevalence was not significantly different from persons without a history of cancer. The current use of smokeless tobacco products among cancer survivors (under 2%) was just over half the prevalence of that of their peers without a history of cancer.

Conclusion: Tobacco use is an important modifiable risk factor for cancer- and non-cancer related morbidity and mortality of cancer survivors, yet its use remains as prevalent among survivors as among persons with no cancer history. Current cigarette use is higher among US cancer survivors compared with persons without a cancer history, and there is no significant difference in the current use of non-cigarette combustible tobacco products between both groups. Findings from this study may be used to discern differences in use of various types of tobacco products among cancer survivors and their peers without a cancer history.

#1747

Relationships between anthropometric and reproductive factors and risks of triple-negative and HER2-overexpressing breast cancers.

Christopher I. Li,1 Lu Chen,1 Mei-Tzu Tang,1 Peggy Porter,1 Linda Cook2. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of New Mexico, Albuquerque, NM_.

Background: Triple negative (TN, do not express estrogen receptor (ER), progesterone receptor (PR), or HER2) and HER2-overexpressing (H2E, ER-/HER2+) tumors are two particularly aggressive molecular subtypes of breast cancer that disproportionately impact young women. There is still lack of knowledge on factors influencing etiologies of these cancers and how these relationships may vary between premenopausal and postmenopausal women.

Methods: We conducted a population-based case-case study of women 20 to 69 years of age diagnosed with invasive breast cancer while living in the Seattle, WA or Albuquerque, NM areas between 2004 and 2012. It included four case groups defined based on joint ER/PR/HER2 status: TN (n=1,294), H2E (n=489), luminal A (ER+/HER2-, n=778) and luminal B (ER+/HER2+, n=131). Medical records were reviewed at both study sites to ascertain information on risk factors at the time of breast cancer diagnosis, supplemented with data from telephone interviews (Seattle cases only). Polytomous logistic regression was used to estimate the odds ratio (OR) and associated 95% confidence intervals (CIs) for TN, H2E, and luminal B breast cancers relative to luminal A cancers and various risk factors including body mass index (BMI), ever having a full term pregnancy, number of full term pregnancies, and age at first birth.

Results: Obese pre-menopausal women (BMI≥30 kg/m2) had an 82% (95% CI: 1.32-2.51) increased risk of TN breast cancer compared to normal weight women (BMI<25 kg/m2), and those in the highest weight quartile had a 78% (95% CI: 1.21-2.60) increased risk of TN disease compared to those in the lowest quartile. In contrast, among post-menopausal women obesity was associated with reduced risks of both TN (OR= 0.72, 95% CI: 0.53-0.98) and H2E (OR= 0.47, 95% CI: 0.32-0.68) cancers. Earlier age at first full-term pregnancy and age at menopause were positively associated with risk of TN breast cancer (p-values for trend: 0.003 and 0.024, respectively). Parity was associated with a 43% (95% CI: 1.08-1.89) elevated risk of H2E breast cancer, and women who had ≥3 full-term pregnancies had a 63% (95% CI: 1.16-2.29, p-trend: 0.013) increased risk of this subtype. Breastfeeding for ≥36 months was associated with a 3.47-fold (95% CI: 1.17-10.33) increased risk of luminal B breast cancer, but a 49% (95% CI: 0.27-0.99) lower risk of TN breast cancer.

Conclusion: This large population-based study adds to evidence that anthropometric and reproductive factors have divergent impacts on risk of aggressive subtypes of breast cancer. The higher incidence rates of TN cancers observed among certain populations, such as younger African American and Hispanic women in the United States, may be due in part to these relationships. Given the poorer prognoses of these aggressive breast cancer subtypes, clarifying the impact of potentially modifiable factors on their risks is of great public health importance.

#1748

The relationship between nut consumption and risk of colorectal cancer.

Jeeyoo Lee,1 Aesun Shin,1 Jeonghee Lee,2 Ji Won Park,3 Jae Hwan Oh,4 Jeongseon Kim2. 1 _Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Molecular Epidemiology Branch, Research Institute, National Cancer Center, Goyang-si, Republic of Korea;_ 3 _Department of Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 4 _Center for Colorectal Cancer, National Cancer Center, Goyang-si, Republic of Korea_.

Introduction

Nuts consumption has been associated with reduced risk of obesity, diabetes mellitus, and cardiovascular diseases. However, the association between nuts and colorectal cancer risk remains unclear.

Objective

In this study, we aimed to investigate the relationship between nuts consumption and colorectal cancer risk in a case-control study.

Methods

A case-control study was conducted with 923 colorectal cancer patients and 1846 controls recruited from the National Cancer Center in Korea. Information on dietary intake was collected using a food frequency questionnaire (FFQ) with 106 items. Nuts consumption level was classified by sex-specific quartile of control group. Binary and polytomous logistic regression models were used to estimate odds ratios (OR) and their 95% confidence intervals (CI) for the association between nuts consumption and colorectal cancer risk.

Results

High nuts consumption was strongly associated with a reduced risk of colorectal cancer in women (OR, 0.18; 95% CI 0.10-0.32 for highest vs. lowest quartile) and similar inverse association was observed for men (OR, 0.36; 95% CI 0.25-0.52). In subsite analysis, adjusted ORs (95% (CI)) comparing the highest vs the lowest quartile of nuts consumption were: 0.49 (0.24-0.99) for proximal colon cancer, 0.38 (0.23-0.64) for distal colon cancer, and 0.29 (0.18-0.46) for rectal cancer in men. An inverse association was also found in women for proximal colon cancer (0.16 (0.05-0.51)), distal colon cancer (0.16 (0.07-0.37)) and rectal cancer (0.25 (0.12-0.51)).

Conclusion

We found a statistically significant association between higher nuts intake and reduced risk of colorectal cancer and the association was observed for all subsites of colorectum in both men and women.

#1749

Different dietary patterns and reduction of lung cancer risk: a large case-control study in the U.S.

Huakang Tu,1 John V. Heymach,2 Chi-Pang Weng,3 Yuanqing Ye,1 Jeanne A. Pierzynski,1 Jack A. Roth,4 Xifeng Wu1. 1 _Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Institute of Population Health Science, National Health Research Institutes, Zhunan, Taiwan;_ 4 _Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Importance: Reducing lung cancer risk by modifying diet is highly desirable. However, the association of different U.S. dietary patterns with lung cancer risk is poorly understood, especially for regional dietary patterns.

Objective: We investigated whether different U.S. dietary patterns were associated with lung cancer risk and whether the associations could be modified by genome-wide association study identified susceptibility loci.

Design, Setting, and Participants: Study participants were accrued from a large ongoing case-control study of lung cancer initiated in 1995. Cases were newly-diagnosed and histologically confirmed non-small cell lung cancer patients from The University of Texas MD Anderson Cancer Center, and healthy controls without a history of cancer were from the Kelsey-Seybold Clinics. We limited the analysis to non-Hispanic whites, and after exclusion, 2,139 cases and 2,163 frequency-matched controls on age (±5 years), sex, and smoking status (current, former, never) were included in the final analysis.

Exposures: Dietary intake was assessed with a validated food frequency questionnaire. Three dietary patterns (i.e., "Tex-Mex", "fruits and vegetables", and "American/Western") were derived using exploratory factor analysis, and factor scores of derived dietary patterns were categorized into quintiles.

Main Outcome and Measure: The association between dietary patterns and lung cancer risk.

Results: The adjusted odds ratios comparing the highest to the lowest quintile of the factor scores of "Tex-Mex", "fruits and vegetables", and "American/Western" patterns were 0.45 (95% CI=0.37-0.56), 0.68 (95% CI=0.55-0.85), and 1.45 (95% CI=1.18-1.78), respectively. The effects were stronger for squamous cell carcinoma and among former or current smokers for the "fruits and vegetables" pattern, and stronger for other non-small cell lung cancer and among never smokers for the "American/Western" pattern. Additionally, a variant (rs2808630) of the

C-reactive protein gene significantly modified the associations of the "fruits and vegetables" (P for interaction=0.03) and "American/Western" (P for interaction=0.02) patterns with lung cancer risk.

Conclusions and Relevance: Our study provides the first evidence that the "Tex-Mex" dietary pattern is associated with reduced lung cancer risk. Also, the "fruits and vegetables" and "American/Western" patterns affects lung cancer risk, and the effects are further modified by host genetic background. Our results support modifying diet to reduce lung cancer risk.

#1750

Sleepduration across the adult lifecourse and risk of lung cancer mortality in Xuanwei, China.

Jason Y. Wong,1 Robert S. Chapman,2 Wei Hu,1 Wei Jie Seow,1 Bu-Tian Ji,1 Bryan Bassig,1 Nathaniel Rothman,1 Qing Lan1. 1 _National Cancer Institute, Rockville, MD;_ 2 _College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand_.

Previous studies have implicated sleep duration as a risk factor for cancer incidence and all-cause mortality. However, to our knowledge, no study has addressed its relationship to lung cancer mortality among Chinese populations. Our objective was to evaluate the association between average sleep hours during various age periods, and risk of lung cancer mortality in rural Xuanwei, China.

Men and women who were born in 1917-1951 and lived in Xuanwei as of Jan 1 1976 were recruited for this cohort study. A total of 42,421 participants interviewed in 1992 contributed data to this analysis. The exposure was average sleep hours in the age periods of: 21-30, 31-40, 41-50, and 51-60 years, categorized as ≤ 7, 8 (reference), 9, and ≥10 hrs/day. The outcome was time to lung cancer mortality in 1992-2009. Cases diagnosed in the first two years of follow-up were excluded to avoid reporting bias due to early, undiagnosed disease. Fine and Gray models with age as the time scale were used to estimate the hazard ratios (HR) and 95% confidence intervals (CI). Separate models were performed for each age period, and were adjusted for hours of indoor activities, respondent, secondary job, education, ethnicity, number of people and rooms in residence, fuel type and tons used, ventilation, smoking status and duration in men, mining work in men, and cooking age in women. Analyses were further stratified by sex and median age at baseline (≥ 61 and < 61 years).

Non-linear relationships were found between sleep duration and lung cancer mortality. In men ≥61 years of age at baseline, ≥10 sleep hrs/day across adulthood was associated with substantially increased risks of lung cancer mortality compared to the recommended 8 sleep hrs/day [21-30 years: HR=1.44 (95%CI: 1.00-2.07), 31-40 years: HR=1.73 (95%CI: 1.15-2.59), 41-50 years: HR=3.97 (95%CI: 2.64-5.97), and 51-60 years: HR=3.55 (95%CI: 2.41-5.22). Furthermore, ≤ 7 sleep hrs/day in middle and later age was associated with significantly increased risks [41-50 years: HR=1.56 (95%CI: 1.17-2.07) and 51-60 years: HR=1.48 (95%CI: 1.15-1.92)]. Trends were similar in men <61 years of age at baseline for the later age period. In women ≥61 years of age at baseline, ≥10 sleep hrs/day in ages 41-50 and 51-60 years was associated with substantially increased risks [HR=2.45 (95%CI: 1.37-4.37) and HR=2.59 (95%CI: 1.61-4.17)]. However, no statistically significant associations with ≤ 7 sleep hrs/day were found. Similar trends were observed for women <61 years of age at baseline.

These findings suggest that both excessive and insufficient sleep may be risk factors for lung cancer mortality. Sleep deficit was associated with increased risks of lung cancer mortality in men, but less apparent in women. However, overabundance of sleep was found to be detrimental in both men and women, particularly during later ages. This study enriches the body of evidence implicating sleep duration in risk of lung cancer.

#1751

Coffee and tea intake and risk of endometrial cancer in the Epidemiology of Endometrial Cancer Consortium (E2C2).

Marta Crous-Bou, Epidemiology of Endometrial Cancer Consortium (E2C2), Immaculata De Vivo. _Brigham and Women's Hospital - Harvard Medical School, Boston, MA_.

Background: Endometrial cancer (EC) is the most common gynecological malignancy in developed countries. Well-known risk factors for EC include obesity and elevated levels of estrogen and insulin. Coffee and tea are the most widely consumed beverages in the world, after water. Both are a complex mixture of chemicals with potential antimutagenic and antioxidant properties. Previous studies have reported an inverse association between coffee consumption and circulating levels of estrogen and C-peptide, a marker of insulin secretion, both involved in endometrial carcinogenesis. The presence of antioxidants and other chemopreventive compounds in coffee and tea may have an anticarcinogenic effect. Thus, coffee and tea drinking may decrease the risk of EC.

Objective: To examine the association between coffee and tea intake and endometrial cancer risk.

Patients and Methods: We combined individual-level data from 24 epidemiologic studies (10 Cohort studies, 14 Case-Control studies) from the Epidemiology of Endometrial Cancer (E2C2) Consortium and performed a pooled analysis of 11,424 endometrial cancer cases and 28,388 controls. Logistic regression models were used to calculate ORs and the corresponding 95% CI. All models were adjusted for potential confounders.

Results and Conclusions: Increased coffee consumption was associated with a reduced risk of endometrial cancer: compared to non-coffee drinkers, the pooled OR for those who drank more than 1 cup of coffee per day was 0.79 (95%CI=0.71-0.87, p=5.48x10-6). The association is mainly observed in cohort studies and remains significant after adjusting for potential confounders. We found no association between tea consumption and endometrial cancer risk.

#1752

Dietary fat intake during adolescence and breast density among young women.

Seungyoun Jung. _University of Maryland School of Medicine, Baltimore, MD_.

BACKGROUND: Lack of association between fat intake and breast cancer risk in cohort studies might be attributed to disregard of temporal effects during adolescence when breasts develop and are particularly sensitive to stimuli. We prospectively examined associations between adolescent fat intakes and breast density.

METHOD: Among 177 women who participated in the Dietary Intervention Study in Children, dietary intakes at ages 10-18 were assessed on five occasions by 24-hour recalls and averaged. We calculated geometric mean and 95% confidence intervals for MRI-measured breast density at ages 25-29 across quartiles of fat intake using linear mixed-effect regression.

RESULTS: Comparing women in the extreme quartiles of adolescent fat intakes, percent dense breast volume (%DBV) was positively associated with saturated fat (mean=16.4% vs. 21.5%; Ptrend<0.001). Conversely, %DBV was inversely associated with monounsaturated fat (25.0% vs. 15.8%; Ptrend<0.001) and the ratio of polyunsaturated fat to saturated fat (P/S ratio; 19.1% vs. 14.3%; Ptrend:<0.001). When examining intake by pubertal stages, %DBV was inversely associated with intake of polyunsaturated fat (20.8% vs. 16.4%; Ptrend=0.04), long-chain omega-3 fat (17.8% vs. 15.8%; Ptrend<0.001), and P/S ratio (22.5% vs. 16.1%; Ptrend<0.001) before menarche, but not after. These associations observed with %DBV were consistently observed with absolute dense breast volume but not with absolute non-dense breast volume.

CONCLUSIONS: In our study, adolescent intakes of higher saturated fat and lower mono- and polyunsaturated fat are associated with higher breast density measured approximately 15 years later.

IMPACT: The fat subtype composition in adolescent diet may be important in early breast cancer prevention.

#1753

Associations between exercise, diet and inflammation: Results from NHANES III.

Marisa A. Bittoni,1 Steven K. Clinton,1 Randall E. Harris,2 Brian Focht3. 1 _Ohio State University Comprehensive Cancer Center, Columbus, OH;_ 2 _Ohio State University College of Public Health, Columbus, OH;_ 3 _Ohio State University College of Education and Human Ecology, Columbus, OH_.

Background: Chronic inflammation is associated with adverse lifestyle factors, including poor diet, lack of physical activity and smoking. Systemic inflammation also plays an important role in carcinogenesis, and can be measured by C-reactive protein (CRP), an acute phase protein that is elevated in those with cancer. We recently examined associations of CRP and lifestyle factors with fatal lung cancer in the NHANES III cohort and reported significant associations, which varied by smoking status and gender. The purpose of this study was to examine associations between baseline lifestyle factors and CRP levels in this same cohort and to determine if differences existed by smoking status and gender.

Methods: Data from cancer-free individuals >age 40 in the Third National Health and Nutrition Examination Survey (NHANESIII:1988-1994) were examined to assess relationships between exercise patterns, dietary factors (measured by the Healthy Eating Index (HEI)) and inflammation (CRP≥3 versus CRP<3). Baseline demographic and clinical data were obtained from interviews and lab examinations. Logistic regression was performed to estimate odds ratios (ORs) for the entire cohort and by smoking status and gender.

Results: Out of 8,950 participants who were eligible for this study, 52% were female, 73% were white, mean age was 61 years, and over half had ever smoked >100 cigarettes (55%). Significant inverse relationships were found in univariate models between elevated CRP and several types of physical activity performed in the past 30 days (walking, jogging, biking, swimming, gardening, lifting weights; p<0.0001), with the lowest OR=0.43 for walking. In multivariate models stratified by gender and smoking status, the OR for walking increased to 0.78 but remained statistically significant for all but non-smokers (p<0.01). Significant inverse relationships with CRP were also observed for serum selenium (OR=0.50), serum vitamin D (OR=0.85) and HEI score (OR=0.94) in univariate models (p<0.0001). When stratified in multivariate models, however, the OR estimates for selenium remained similar and statistically significant in all groups (p<0.05), but the ORs for serum vitamin D and HEI score were only significant for smokers and males [OR=0.90 (p<0.01) and OR=0.92 (p=0.001), respectively].

Discussion: This analysis showed that healthier diet and increased physical activity were associated with decreased levels of CRP, especially for smokers and males, which few studies have examined. These results are in line with those of our prospective study, which showed reductions in lung cancer deaths from increased physical activity and healthier eating, primarily in smokers and males. Since male smokers comprise the highest risk group for lung cancer, these findings are very important in the context of lung cancer prevention, especially regarding the potential role of inflammation. Future studies should further assess reasons for differences among these subgroups..

#1754

Body powder use and ovarian cancer: the African American Cancer Epidemiology Study.

Lauren C. Peres,1 Sarah E. Abbott,1 Anthony J. Alberg,2 Elisa V. Bandera,3 Jill Barnholtz-Sloan,4 Melissa Bondy,5 Michele L. Cote,6 Ellen Funkhouser,7 Edward S. Peters,8 Ann G. Schwartz,6 Paul D. Terry,9 Sydnee Crankshaw,10 Fabian Camacho,1 Frances Wang,10 Patricia G. Moorman,10 Joellen M. Schildkraut1. 1 _University of Virginia, Charlottesville, VA;_ 2 _Medical University of South Carolina, Charleston, SC;_ 3 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 4 _Case Western Reserve University School of Medicine, Cleveland, OH;_ 5 _Baylor College of Medicine, Houston, TX;_ 6 _Wayne State University School of Medicine, Detroit, MI;_ 7 _University of Alabama at Birmingham, Birmingham, AL;_ 8 _Louisiana State University Health Sciences Center School of Public Health, New Orleans, LA;_ 9 _University of Tennessee Medical Center - Knoxville, Knoxville, TN;_ 10 _Duke University Medical Center, Durham, NC_.

Findings of epidemiologic studies indicate an increased risk of ovarian cancer among women who use powders applied to perineal areas. Although African American (AA) women have a high prevalence of powder use, this relationship has not been thoroughly investigated in this group of women. The objective of the present study was to evaluate the relationship between use of genital and non-genital powder in invasive epithelial ovarian cancer (EOC). Subjects are women enrolled in the African American Epidemiology Cancer Study (AACES), an ongoing, population-based case-control study of EOC in AA women in 11 geographic locations in the U.S. Newly diagnosed EOC cases were identified by SEER and state cancer registries, gynecologic oncology departments or hospitals, and were between the ages of 20-79 years. AA controls were identified through random digit dialing and frequency matched to cases on state of residence and five year age groups. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between genital powder and non-genital powder exposure and risk of EOC, while controlling for several confounders, including age at diagnosis/interview, study site, education, tubal ligation, parity, BMI, duration of oral contraceptive use, first degree family history of breast or ovarian cancer, and interview year. Due to experimental models suggesting a relationship with inert particulates and estrogen, we also examined potential effect modification of this relationship by hormone therapy use among postmenopausal women. Body powder use was common in this study population (62.8% of cases and 52.9% of controls). Any genital powder use was associated with a 44% increased risk of EOC (OR = 1.44, 95% CI = 1.11-1.86) and a dose-response relationship was present for duration of body powder use applied to genital areas, p<0.05. A 31% increased EOC risk was observed for non-genital powder use (OR = 1.31, 95% CI = 0.95-1.79), and this relationship was strongest among non-serous cases (OR = 2.28, 95% CI = 1.39-3.74). Although not statistically significant, hormone therapy may be a potential modifier of the effect of body powder use on EOC risk. Among ever users of hormone therapy, any genital powder use was associated with over a 2-fold increase in risk (OR = 2.68, 95% CI = 1.33-5.40), while an OR of 1.24 (95% CI = 0.87-1.79) was present for never users of hormone therapy. Having an upper respiratory condition was associated with both genital and non-genital powder use suggesting a systemic inflammatory response may explain the associations we observed with EOC for non-genital powder use. In conclusion, body powder use was prevalent among AA women and strongly associated with EOC risk.

#1755

Weight change, prostate cancer-specific mortality, and biochemical recurrence in a prospective cohort study of U.S. men.

Barbra Dickerman, Thomas Ahearn, Edward Giovannucci, Lorelei Mucci, Kathryn Wilson. _Harvard School of Public Health, Brookline, MA_.

Obesity is associated with an increased risk of fatal prostate cancer. However, the relevant timing of obesity for risk has not been established, and the impact of weight change on prognosis has been understudied.

We prospectively investigated the association between weight change and obesity with prostate cancer outcomes among 5,173 men diagnosed with prostate cancer (clinical TNM-stage <T3b) from 1986 to 2012 in the Health Professionals Follow-up Study. Men were followed for biochemical recurrence, development of distant metastasis, and mortality until 2014. Cox proportional hazards models were used to estimate hazard ratios for change in weight from age 21 to diagnosis and in the 4 years prior to diagnosis, adjusting for potential confounding by lifestyle and clinical factors. Because weight, weight change, and mortality are strongly associated with smoking, we conducted the analysis among all prostate cancer patients and among never smokers only (N=2,554).

Among all patients, weight gain from age 21 to the time of diagnosis was associated with risk of lethal prostate cancer (distant metastases or prostate cancer death). Compared to those with stable weight (±10 lbs) from age 21 to diagnosis, those who gained >30 lbs had a 1.44-fold higher risk of lethal cancer (95% CI 1.03-2.01, p-trend=0.06, adjusting for lifestyle factors, stage, grade, PSA at diagnosis, and weight at age 21). Among never smokers, this association was stronger (HR 2.19, 95% CI 1.31-3.68, p-trend=0.005). Weight gain in the 4 years preceding diagnosis was suggestively, but not significantly, associated with lethal cancer among never smokers (HR for >5 lbs gain vs stable weight [±5 lbs] 1.48, 95% CI, 0.96, 2.28). No significant associations were found between weight change and biochemical recurrence.

Our findings suggest a positive association between adult weight gain and risk of lethal prostate cancer. Metabolic changes associated with weight gain may promote prostate cancer progression.

#1756

Marijuana use and serum testosterone concentrations among U.S. males.

Katherine A. McGlynn. _NCI, Bethesda, MD_.

Background: Testicular germ cell tumors (TGCT) of young men can be histologically divided into nonseminomas and seminomas. Although TGCTs are thought to be hormonally-related, very few risk factors have been identified. Recently, however, marijuana use has been linked to risk of nonseminoma, which has peak incidence at a younger age (25 years) than does seminoma (35 years). Whether marijuana may be related to nonseminoma via an effect on testosterone concentrations is unknown. Thus, an analysis of U.S. males was conducted, using data from the 2011-2012 National Health and Nutrition Examination Survey (NHANES) of the NCHS, CDC.

Methods: Data from 2409 males aged >20 years were included in the analysis. Serum total testosterone (T) concentrations, determined using liquid chromatography-tandem mass spectrometry, were log transformed for statistical analyses. Marijuana use was categorized as: never, current (use on at least one day in prior 30 days) and past (no use within prior 30 days). Regular use was defined as use at least once a month for > 1 year. Covariates included age, body mass index, race/ethnicity, cigarette smoking status and time of day of phlebotomy. Adjusted mean T concentrations were compared among marijuana use categories using weighted multiple linear regression analysis accounting for the complex sample design of NHANES. In addition to the overall analysis, models were stratified by age (20-30 years, >30 years).

Results: Mean T concentration did not differ between never- and ever-users of marijuana (p=0.62), but did differ between never-users and regular-users (2.56 ng/mL vs 2.59 ng/mL, p=0.03) and between never-users and current-users (2.60 ng/mL, p=0.007). In the analysis restricted to men >30 years of age, significant differences in mean T concentration were evident among never-users and current-users (2.55 ng/mL vs 2.60 ng/mL, p=0.003). Significant differences were also seen with increasing duration of use as mean T concentrations were higher among men who used marijuana regularly for >5 years. In the analysis of men 20-30 years of age, no significant differences in mean T concentration by marijuana use were seen.

Conclusion: These results suggest that regular use and current use of marijuana are associated with higher T concentrations among U.S. men, particularly men > 30 years of age. As nonseminomas are more common in younger men than in older men, the results may indicate that the association between marijuana use and nonseminoma is not due to an effect of marijuana on circulating T concentrations.

#1757

Male pattern baldness and risk of incident skin cancer in a cohort of men.

Wen-Qing Li, Eunyoung Cho, Jiali Han, Martin Weinstock, Abrar Qureshi. _Brown University, Providence, RI_.

Background: Male pattern baldness (androgenic alopecia) has been associated with scalp skin cancer in case-control studies. A potential androgen basis of melanoma has been hypothesized and baldness has been associated with higher levels of androgens. We examined the association between male pattern baldness and risk of skin cancer, including melanoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) in a prospective cohort study.

Methods: We included 36,032 participants from the Health Professionals' Follow-up Study. In 1992, participants reported their status of male-pattern baldness at age 45 years by choosing from five crown-view pictograms based on Norwood's classification. Diagnosis of skin cancers was reported in the self-reported questionnaires biennially and report on melanoma and SCC was pathologically confirmed. Cox proportional hazard models were used to compute the hazard ratios (HR) and 95% confidence intervals (CI).

Results: We identified 327 cases of melanoma, 1324 cases of SCC, and 8438 cases of BCC during the follow-up. Male-pattern baldness at age 45 was not associated with risk of incident melanoma, but was significantly associated with increased risk of both SCC and BCC. The multivariate-adjusted HR (95% CI) for the highest category of baldness (frontal plus severe vertex baldness) was 1.33 (1.06-1.68) for SCC (Ptrend=0.001) and 1.23 (1.12-1.35) for BCC (Ptrend<0.0001), compared with no baldness. Analyses by body sites of tumors found significant associations between frontal plus moderate to severe vertex baldness and risk of melanoma (HR=1.83, 95% CI: 1.01-3.34) and SCC (HR=1.30, 95% CI: 1.02-1.66) at head and neck. The associations were particularly stronger for scalp melanoma (HR=7.15, 95% CI: 1.29-39.42) and scalp SCC (HR=7.09, 95% CI: 3.84-13.08), but not for non-scalp head and neck sites.

Conclusion: Male pattern baldness was positively associated with increased risk of skin cancer. The associations may only exist for skin cancer at head and neck, particularly those occurring at scalp.

#1758

Circulating insulin-like growth factors: Correlates and responses to calcium supplementation in colorectal adenoma patients.

Caroline Um,1 Veronika Fedirko,1 Christine Höflich,2 W Dana Flanders,1 Roberd M. Bostick1. 1 _Emory University, Atlanta, GA;_ 2 _Ligandis Biomarker Diagnostics GbR, Gulzow, Germany_.

Insulin-like growth factor 1 (IGF-1) and its binding proteins are an important part of the IGF/growth hormone axis needed for normal growth and organ function. IGF-1 signaling has also been implicated in tumorigenesis with higher circulating levels of IGF-1 and the molar ratio of IGF-1 to IGF binding protein 3 (IGFBP-3) being directly associated with colorectal, breast, and prostate cancers, as well as with colorectal adenomas. IGF binding protein 2 (IGFBP-2) also appears to be overexpressed in colorectal and lung cancers and leukemias.

Limited evidence suggests that various dietary and lifestyle factors may influence circulating levels of IGF-1, IGFBP-2, and IGFBP-3. Among these factors, calcium has been statistically significantly associated with IGF-1, IGFBP-2, and IGFBP-3, but its effects on them have not been tested in a clinical trial. To investigate the effects of supplemental calcium (1.0 or 2.0 g/day) on serum levels of IGF-1, IGFBP-2, and IGFBP-3, we analyzed samples from a randomized, double-blinded, placebo-controlled, dose-response clinical trial of patients with previous sporadic, colorectal adenoma. We also investigated associations of various risk factors with the growth factors at baseline. IGF-1 was measured using an enzyme-linked immunosorbent assay, and IGFBP-2 and IGFBP-3 were measured using quantitative Western ligand blot assays.

We found no appreciable effects of calcium over the 4-month treatment period on IGF-1, IGFBP-2, and IGFBP-3. At baseline, lower levels of IGF-1 and IGFBP-3 were associated with being female (P = 0.02 and 0.01, respectively) or older (P = 0.02 and 0.02, respectively); the IGF-1/IGFBP-3 molar ratio was inversely associated with body mass index (BMI) (P = 0.04); and IGFBP-2 was directly associated with age (P = 0.002) but inversely associated with BMI (P < 0.0001). We calculated residuals of non-fat milk intake as a novel method of examining the non-calcium, non-fat component of milk, modeled after the energy adjustment residual method. These residuals were inversely associated with IGFBP-2 (P = 0.05) and directly associated with the IGF-1/IGFBP-3 molar ratio (P = 0.11).

Overall, our results do not support that calcium supplementation can modulate circulating levels of IGF-1, IGFBP-2, or IGFBP-3 among patients with previous colorectal adenoma. However, our findings do suggest potential associations of these growth factors with established risk factors for colorectal cancer (age [inversely associated with IGF-1 and IGFBP-3, directly associated with IGFBP-2] and adiposity [inversely associated with IGFBP-2 and the IGF-1/IGFBP-3 molar ratio]). Finally, our results provide new evidence of potential associations of IGF-1, IGFBP-2, and IGFBP-3 with milk intake independent of its calcium and fat content.

#1759

The association between alcohol, physical activity and breast cancer subtypes in a nested case-control study from the Norwegian Breast Cancer Screening Program.

Merete Ellingjord-Dale,1 Linda Vos,1 Steinar Tretli,1 Solveig Hofvind,1 Isabel dos-Santos-Silva,2 Giske Ursin1. 1 _Cancer Registry of Norway, Oslo, Norway;_ 2 _London School of Hygiene and Tropical Medicine, London, United Kingdom_.

Background:

Breast cancer (BC) is a complex disease, consisting of molecular subtypes with different prognosis and possibly different etiology. We and others have previously reported that hormonal factors such as hormone therapy and pregnancies predominantly affect luminal-like BC, but it is unclear whether alcohol and physical activity are associated only with certain subtypes.

Methods:

We conducted a case-control study nested within a cohort of 457,036 women who participated in the Norwegian Breast Cancer Screening Program (NBCSP) in 2006-2012, and who completed a questionnaire at baseline screening. In all, 5,554 BC cases with information on risk factors and hormone receptor status (i.e. estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) occurred during the follow-up. For each case, 5 controls were randomly selected from among participants who did not develop BC, frequency-matched to the cases on year of birth and year of screening. The following surrogate definitions of BC subtypes were used: ER+PR+HER2- ("luminal A-like"), ER+PR-HER2- ("luminal B-like, HER2 negative"), ER+HER2+ ("luminal B-like, HER2 positive"), ER-PR-HER2+ ("HER2 positive") and ER-PR-HER2- ("triple negative"). We used multinomial logistic regression to estimate odds ratios (ORs), with 95% confidence intervals (CIs), adjusted for age, body mass index (BMI), education, age at menarche, number of pregnancies and menopausal status.

Results:

Weekly amount of alcohol intake was associated with an increased risk of BC overall (p for trend=0.001). Analysis by subtype revealed a positive association between alcohol intake and BC risk for several subtypes, albeit it was statistically significant only for luminal A-like cancer. Compared to never drinkers, women who reported an alcohol intake >6 glasses/week had a moderately increased risk of luminal A-like (OR=1.41, 95% CI 1.14-1.73, p for trend=0.0001), luminal B-like Her2 positive (OR=1.55, 0.87-2.75) and triple negative BC (OR=1.39, 0.81-2.37). In contrast, physical activity (low and high intensity exercise) was inversely associated with risk of breast cancer overall (p for trend=0.002). Relative to women who reported doing no exercise, those who reported physical activity of ≥4 hours/week were at reduced risk for luminal-A like (OR=0.76, 95% CI 0.63-1.01, p for trend=0.01), luminal B-like Her2 positive (OR=0.84, 0.46-1.54), and triple negative BC (OR=0.80, 95% CI 0.45-1.44). The risk estimates for the other subtypes were close to 1.

Conclusions:

Alcohol intake was positively associated, and physical activity negatively associated, with BC overall. Although these associations were statistically significant only for luminal A-like BC, their direction and magnitude were rather similar across several subtypes.

#1760

Saturated fat intake and prostate cancer aggressiveness: Results from the population-based North Carolina-Louisiana prostate cancer project.

Emma H. Allott,1 Lenore Arab,2 L. Joseph Su,3 Laura Farnan,1 Elizabeth T.H. Fontham,4 James L. Mohler,5 Jeannette T. Bensen,1 Susan E. Steck6. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _University of California, Los Angeles, Los Angeles, CA;_ 3 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 4 _Louisiana State University, New Orleans, LA;_ 5 _Roswell Park Cancer Institute, Buffalo, NY;_ 6 _University of South Carolina, Columbia, SC_.

Introduction: Epidemiologic data support a positive association between hypercholesterolemia and aggressive prostate cancer, and an inverse association between statin use and aggressive prostate cancer. Saturated fat intake is an important dietary determinant of serum cholesterol levels. However, epidemiologic evidence supporting a role for saturated fat in prostate cancer aggressiveness is mixed. We hypothesized that high saturated fat intake would be associated with increased prostate cancer aggressiveness, and that this association would be modified by statin use.

Methods: Of 1,854 prostate cancer cases in the North Carolina-Louisiana Prostate Cancer Project (PCaP), 321 (17%) were classified as high aggressive based on clinical criteria. Saturated fat intake was adjusted for total fat intake using the residual method, and categorized into tertiles based on the distribution among all research subjects. Using low and intermediate aggressive cases as the reference group, logistic regression was used to examine the association between tertiles of saturated fat intake and prostate cancer aggressiveness, overall and stratified by race and by statin use. In addition to demographic and screening variables, energy-adjusted total fat intake and energy intake were included as covariates in our models. In secondary analysis, we examined associations for total fat, monounsaturated and polyunsaturated fatty acids (MUFA and PUFA, respectively), trans fat, and cholesterol intake.

Results: High saturated fat intake was associated with an elevated odds ratio (OR) for aggressive prostate cancer (ORupper tertile (T3) vs. lower (T1) 1.44; 95% CI 1.06-1.96; p-trend=0.020). The magnitude of this association was weaker in statin users (ORT3 vs. T1 1.10; 95% CI 0.63-1.91; p-trend=0.790) compared to men not using statins (ORT3 vs. T1 1.63; 95% CI 1.11-2.37; p-trend=0.015). There was a suggestion of a positive association between high cholesterol intake and aggressive prostate cancer among all men (ORT3 vs. T1 1.28; 95% CI 0.94-1.75; p-trend=0.074), and this association was more pronounced in European Americans (ORT3 vs. T1 1.82; 95% CI 1.15-2.88; p-trend=0.012). Elevated PUFA intake was inversely associated with prostate cancer aggressiveness (ORT3 vs. T1 0.66; 95% CI 0.48-0.90; p-trend=0.009), with similar effect estimates in both races. There were no associations between trans fat or MUFA and prostate cancer aggressiveness.

Conclusions: Elevated intake of saturated fat was positively associated, while high intake of PUFA was inversely associated, with aggressive prostate cancer. Weaker associations between saturated fat and prostate cancer aggressiveness in statin users suggest that the impact of saturated fat on serum cholesterol levels may be one potential mechanism by which saturated fat impacts prostate cancer aggressiveness.

#1761

Serous ovarian cancer risk factors by grade: Evidence for etiologic heterogeneity.

Elizabeth M. Poole,1 Nicolas A. Wentzensen,2 Britton Trabert,2 Shelley S. Tworoger,1 The Ovarian Cancer Cohort Consortium. 1 _Brigham & Women's Hospital, Boston, MA; _2 _National Cancer Institute, Washington, D.C., DC_.

Background: Growing evidence suggests that high grade serous ovarian carcinomas may arise from the fallopian tube, while low-grade serous ovarian cancer may develop on the ovaries. Previous studies have suggested differences in ovarian cancer risk factors by histologic subtypes, but few have evaluated differences in risk factors for high- vs. low-grade serous cancers. In the Ovarian Cancer Cohort Consortium (OC3), a pooling project of individual-level data from 23 prospective cohort studies, we evaluated associations of hormonal, reproductive, demographic and lifestyle factors, and family history of cancer with for serous ovarian cancer subtypes.

Methods: Among 16 studies that abstracted grade information from pathology reports, 3,095 serous ovarian cancers were identified during follow-up (125 well differentiated, 506 moderately differentiated, 1671 poorly differentiated, 793 undetermined). We used competing risks Cox proportional hazards regression to compute relative risks (RRs) and 95% confidence intervals (CIs) for differences in association by grade. Models were stratified on study and year of birth and adjusted for age, parity and OC use; subtype heterogeneity was evaluated by likelihood ratio tests.

Results: Although sample sizes were small for low-grade tumors, there was evidence for heterogeneity in the associations for oral contraceptive (OC) use, age at menopause, endometriosis, and family history of ovarian cancer. For example, each 5-year increase in OC use was associated with a 21% decrease in risk of low-grade serous ovarian cancer (RR: 0.79; 95% CI: 0.62-1.00), but only a 10% decrease in risk of high-grade serous cancers (RR: 0.90; 95% CI: 0.84-0.96; p-heterogeneity=0.09). Consistent with a prior report in the Ovarian Cancer Association Consortium, endometriosis was associated with increased risk of low-grade serous tumors (RR: 3.77; 95% CI: 1.24-11.48), but not with high-grade serous tumors (RR: 1.11; 95% CI: 0.70-1.74; p-heterogeneity=0.12).

Conclusion: Our results demonstrate heterogeneous associations of risk factors with subtypes of serous ovarian cancer, supporting the idea that the high- and low-grade serous tumors develop through different pathways. Despite the small sample size for low-grade serous tumors, most risk factors were more strongly associated with low-grade tumors compared to high-grade serous carcinomas, suggesting that risk prediction may be more challenging for the most fatal subtype. Identifying subtype-specific risk factor and biomarkers is important both for better understanding ovarian cancer etiology and for targeted development of novel prevention approaches. These results underscore the importance of consortial projects to evaluate rare subtypes (low-grade serous cancers) for the better understanding of etiologic heterogeneity of this deadly disease.

#1762

Body mass index shows etiologic heterogeneity for risk of diffuse large B-cell lymphoma (DLBCL) subtype defined by cell-of-origin.

Matthew K. Breitenstein, Megan M. O'Byrne, Andrew L. Feldman, Carrie A. Thompson, William R. Macon, Grzegorz S. Nowakowski, Stephen M. Ansell, Susan L. Slager, Thomas M. Habermann, James R. Cerhan. _Mayo Clinic, Rochester, MN_.

Background. DLBCL is the most common non-Hodgkin lymphoma (NHL) subtype in western countries and is known to be clinically heterogeneous. Gene expression profiling has identified two major, biologically distinctive DLBCL subtypes on the basis of their cell-of-origin (COO): the germinal center B-cell (GCB) - characterized by BCL2 rearrangement and C-REL amplification, and activated B-cell (ABC) - characterized by constitutive activation of the NF-kB pathway. Recently, the InterLymph Subtypes Project identified medical history, lifestyle, and family history risk factors for DLBCL using a large pooled analysis of case-control studies. In this study we evaluated these same risk factors for etiologic heterogeneity as defined by DLBCL COO.

Methods. For this analysis we used a clinic-based study of newly diagnosed NHL cases and frequency matched controls, enrolled from 2002-2012, with 474 DLBCL cases and 2203 controls. COO was determined clinically using the Hans algorithm, which is based on immunohistochemistry markers using formalin-fixed, paraffin-embedded tumor tissue. Amongst DLBCL cases 107 were ABC, 207 were GCB, and 160 were missing subtype (tissue unavailable). Risk factor data, including body mass index (BMI), smoking, alcohol use, any allergy, asthma, blood transfusion, diabetes, rheumatoid arthritis, sun exposure, lived on a farm and family history of hematologic malignancy, were collected from self-administered questionnaires. Polytomous logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals for ABC and GCB subtypes, adjusted for age and sex.

Results. The mean age of cases was 60.4 years with 53% male; the mean age of controls was 61.6 years and 53% male. We identified a strong positive association for BMI with risk of ABC but not GBC DLBCL (p-heterogeneity 0.025). Compared to BMI 18.5-24.9 kg/m2, those with a BMI of 25.0-29.9 (OR=1.65; 0.93-2.93) or 30+ (OR=2.61; 1.48-4.62) were at increased risk of ABC DLBCL. There was no association for a BMI of 25.0-29.9 (OR=0.91; 0.63-1.31) or 30+ (OR=1.21; 0.83-1.76) with GCB DLBCL. There was no evidence of significant heterogeneity between ABC and GCB DLBCL for the other risk factors that we evaluated. Further adjustment of the BMI association for educational level, smoking, and alcohol only slightly attenuated these findings.

Conclusions. We observed a strong positive association of BMI with ABC but not GCB DLBCL. In contrast, other evaluated risk factors showed no heterogeneity by COO. While BMI has not been clearly associated with NHL overall, there has been evidence for an association with DLBCL. The etiologic heterogeneity we observed for ABC DLBCL is notable as BMI is also a strong risk factor for multiple myeloma, another post-germinal center B-cell malignancy. While requiring replication, our findings suggest BMI may influence B-cell neoplasia through effect on post-germinal center pathways.

#1763

Body size and incidence of TMPRSS2:ERG fusion-positive and fusion-negative prostate cancer.

Thomas Ahearn,1 Rebecca E. Graff,2 Andreas Pettersson,1 Claire Pernar,1 Sarah C. Markt,1 Kathryn M. Wilson,1 Michelangelo Fiorentino,3 Massimo Loda,4 Edward L. Giovannucci,1 Lorelei A. Mucci1. 1 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 2 _University of California, San Francisco, San Francisco, CA;_ 3 _S.Orsola Malpighi Hospital, Bologna, Italy;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Background: Evidence suggests that genetic, lifestyle, and hormonal factors are differentially associated with the incidence of TMPRSS2:ERG fusion-positive and fusion-negative tumors, suggesting that these molecular subtypes have distinct etiologies. There is scant literature on whether anthropometrics are differentially associated the risk of developing fusion-positive and fusion-negative prostate cancer.

Methods: We examined the associations between adult height, recalled BMI at age 21, and updating BMI over time with risk of prostate cancer defined by TMPRSS2:ERG subtype using a prospective cohort of 49,372 men from the Health Professionals Follow-up Study. Tumor TMPRSS2:ERG status was assessed by ERG immunohistochemistry on tissue microarrays constructed from radical prostatectomy of the prostate specimens. We utilized multivariable competing risks models to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for risk of fusion-positive disease and, separately, fusion-negative disease.

Results: During 23 years of follow-up, we identified 5,847 total prostate cancer cases. Among them, 2,402 were treated with RP, of whom 913 (15.6% of all cases) were assayed for ERG status. Height (per 5 inches) was positively associated with the risk of developing ERG-positive prostate cancer (HR=1.24, 95% CI 1.03-1.50), but not associated with ERG-negative disease (HR=0.98, 95% CI = 0.82-1.18, Pheterogenity = 0.07). Updated BMI (per 5 kg/m2) was inversely associated with the risk of developing ERG-positive disease (HR = 0.86, 95% CI = 0.74-1.00), but not associated with ERG-negative disease (HR = 1.03, 95% CI = 0.90-1.18, Pheterogenity = 0.07). BMI at age 21 was not differentially associated with the risk of developing ERG-positive or ERG-negative disease.

Conclusion: Our results suggest that height and obesity are differentially associated with the risk of developing fusion-positive and fusion-negative prostate cancer.

### Race/Ethnicity and Disparities in Diagnosis, Treatment, and Outcomes

#1764

Predictors of participants' retention among African Americans in the Healthy Eating and Living in The Spirit (HEALS) trial.

Oluwole A. Babatunde, Swann A. Adams, Michael D. Wirth, Jan M. Eberth, Jameson Sofge, Brook Harmon, Lisa Davis, Ruby Drayton, Tom Hurley, Heather M. Brandt, James R. Hebert. _University of South Carolina, Columbia, SC_.

Background: Recruitment and retention of minority racial/ethnic groups is necessary to assess and address cancer health disparities in the United States. The objective of this study was to characterize participants' retention status and identify baseline participant factors associated with retention among an entirely African American (AA) population in a randomized controlled trial (RCT).

Methods: Using data from the Healthy Eating and Living in the Spirit (HEALS) program, an RCT conducted from 2009 to 2012 among AAs in South Carolina we examined participant-level factors associated with retention. We used SAS v9.4 to compute chi square tests and fit logistic regressions in order to compare 220 (53.14%) retained to 194 not-retained participants with the goal of identifying important predictors of retention. Among the entire study population, main predictor variable of interest was network distance in miles from home of participants to the clinic venue (i.e. their church) whereas among participants randomized to the intervention arm, a second predictor was percentage of intervention classes attended.

Results: Baseline characteristics that were significantly associated with retention status included group assignment, age, body mass index (BMI), distance from home to clinic site(s), and partner enrollment in the study. Participants who lived in locations >5 miles from the clinic sites were more likely to be retained in the study (OR = 1.58; 95% CI: 1.04 - 2.4) compared to participants who lived <5 miles away from the clinic. Older participants (>60 years) were 3.3 times as likely (95% CI: 1.59 - 6.81) than those aged < 41 years to be retained while individuals randomized to the control group were more likely to be retained (OR =1.63; 95% CI: 1.06 - 2.50) compared with those randomized to the study group. Those who were obese were less likely to be retained (OR = 0.37; 95% CI: 0.17 - 0.79) compared to those who had normal BMI. Participants who had their partner enrolled in the study were less likely to be retained (OR = 0.59; 95% CI: 0.36-0.95) compared with participants who did not have their partners enrolled. Among individuals randomized to the intervention arm, attending 60% of the classes in the first 3 months of the RCT was strongly predictive of being retained in the study with an odds ratio of 4.31 (95% CI: 2.25 - 8.24) compared with those who did not complete 60% of the classes.

Conclusions: Participants who lived further away (>5 miles) and attending 60% or more of the intervention classes was strongly predictive of being retained in the study. Ensuring that there is a run-in period as part of the screening procedure for all participants before randomization will help project managers to identify participants that are likely to be retained in the study and more

studies need to be done to know why those who lived farther away were more likely to be retained.

#1765

Comorbidities contribute to breast cancer disparities among African-American and Hispanic women.

Marianna Sarkissyan, Yanyuan Wu, Jaydutt V. Vadgama. _Charles R. Drew University of Medicine & Science, Los Angeles, CA_.

Background: Comorbidities may influence health outcomes from breast cancer and can significantly contribute to health disparities. Studies examining the association of specific comorbidities with breast cancer outcomes in underrepresented African-American and Hispanic women have been few. Notably, underrepresented women are more likely to have comorbidity. Presence of comorbidity may impact not only breast cancer risk and progression, but also likelihood to complete treatment and therapy. Therefore, the aims of the present study are to evaluate comorbidity and breast cancer among African-American and Hispanic women. In addition, the study will assess the consequence of comorbidity on therapy completion and breast cancer survival.

Methods: The study included a cross-sectional cohort of 426 African-American and Hispanic women from South Los Angeles. Personal and medical history was obtained during the informed consent process and from post-hoc medical chart. The results were evaluated using the chi-square test and logistic regression with multivariate analysis. Five year disease free survival analysis (DFS) was performed using Kaplan-Meier survival curves with log rank test. The two-sided P-value less than 0.05 was considered significant.

Results: Our study identified a high frequency of comorbidity among women with breast cancer compared with women without breast cancer (55% vs.45%, respectively: P=0.032). Diabetes was also more frequent among breast cancer patients than non-cancer participants (62% vs. 38%; P=0.005). African-American women were more likely than Hispanic women to have comorbidity (P=0.003), particularly hypertension and hypertension with diabetes. Hispanic women were more likely to have diabetes only. In univariate analysis, the presence of any comorbidity was significantly associated with breast cancer (OR =1.536; 95% CI =1.045-2.255; P-value=0.029). In the adjusted multivariate model, this association was borderline significant (P-Value =0.057). The association of compound comorbidity (diabetes with hypertension) was significant in both the univariate and multivariate models (OR=3.333; 95% CI=1.290-8.611; P-Value=0.013). Women with an increasing number of comorbidities were increasingly less likely to complete treatment with chemotherapy (P=0.022). African-American women with compound comorbidity were least frequently able to complete treatment with chemotherapy (P=0.002). Lastly, women who completed treatment with chemotherapy had a significantly higher survival vs. women who did not complete (Log rank=5.09; P=0.024).

Conclusions: The presence of comorbidity can affect risk of breast cancer, likelihood of completion of therapy, and subsequent survival. African-American and Hispanic women have higher frequency of comorbidities and this may place women, especially with compound comorbidity, at a greater risk for breast cancer and lower survival outcomes.

#1766

An evaluation of the post-stroke cancer risk among post-menopausal women: The Women's Health Initiative.

Shawnita Sealy-Jefferson,1 Michele L. Cote,2 Jennifer Beebe-Dimmer,2 Rowan Chlebowski,3 Kathryn Rexrode,4 Michael Simon2. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _Wayne State University, Karmanos Cancer Institute, Detroit, MI;_ 3 _University of California, Los Angeles, Los Angeles, CA;_ 4 _Harvard Medical School, Brigham and Women's Hospital, Boston, MA_.

Links between cancer history and incident stroke have been established, such that 15% of cancer patients experience a stroke at some point during their clinical course. However, we do not know whether stroke history is associated with cancer risk, especially for those cancers with overlapping risk factors with stroke. To address this, we used data from the observational and clinical trial of the Women's Health Initiative (n=145,075), to test whether stroke history was associated with cancer risk and tumor type, and whether variation in cancer risk and tumor type exist by race/ethnicity. Time to incident cancer (in months) was estimated with bivariate and multivariate adjusted cox proportional hazards models, accounting for competing risks, comparing women with incident or prevalent stroke to women without stroke at baseline or at any point during follow-up, and models were stratified by race/ethnicity. Women with a stroke history, compared to those without, had significant differences in the following baseline characteristics: age, body mass index, smoking status, hormone replacement therapy usage, hypertension, hypercholesterolemia and diabetes, and differences were consistent among race/ethnic groups. In unadjusted and adjusted competing risk models of the overall sample, lower cancer risk was observed for women who had a prior stroke, compared to those with no stroke history (adjusted HR: 0.81; 95% CI: 0.75, 0.88). In race-stratified models, Non-Hispanic White (NHW) women experienced significantly lower risk of cancer following stroke, compared to those with no stroke history (adjusted HR: 0.81; 95% Confidence Interval: 0.74-0.89), and the magnitude of the association was similar, although not significant, for African American (AA) and 'Other' racial/ethnic groups. The associations with stroke were stronger for African American women with invasive breast (p=0.02), lung (p=0.04), and 'other' (p=0.001) cancers, compared to NHW women. The results of the current study suggest that overall, women have lower cancer risk post-stroke, but that AA women, compared to their NHW counterparts, have higher risk of certain types of cancers after stroke. Future studies should investigate the mechanisms underlying the lower cancer risk among all stroke survivors, as well as examine why AA women develop certain tumors more often than NHW women.

#1767

Racial/ethnic disparities of obesity in a breast clinic population: Consideration for weight loss program in improving clinical outcomes.

Carolina Puyana Barcha,1 Eunkyung Lee,1 Cristiane Takita,1 Jean L. Wright,2 Wei Zhao,1 Isildinha M. Reis,1 Jennifer J. Hu1. 1 _University of Miami Miller School of Medicine, Miami, FL;_ 2 _Johns Hopkins University, Baltimore, MD_.

Introduction: Accumulating evidence suggest that obesity is associated with breast cancer diagnosis, recurrence, and mortality. Obese subjects with genetic predisposition may need more aggressive weight loss management, such as bariatric surgery. Therefore, the primary aim of this study was to assess the racial/ethnic disparities in obesity and the prevalence of breast cancer study participants who may be eligible for bariatric surgery.

Methods: In three clinic-based studies conducted at University of Miami during 2008 to 2015, we evaluated 462 healthy controls and 975 breast cancer patients. Genotyping data of two obesity-related genes (FTO and MC4R) were also evaluated. Race/ethnicity and obesity-related comorbidities were self- reported. Body Mass Index (BMI) was calculated as kg/m². The classification of BMI was: (1) < 18.5, underweight; (2) 18.5-24.9, normal weight; (3) 25.0-29.9, overweight; (4) 30.0-34.9, class I obesity; (5) 35.0-39.9, class II obesity; and (6) ≥ 40.0, class III obesity. The NIH eligibility for bariatric surgery was for class II obese subjects with obesity-related comorbidities and class III obese subjects.

Results: The study consists of 70.0% Hispanic whites (HW), 21.7% black or African American (AA), and 6.4% non-Hispanic whites (NHW). Mean and SD of age was 54.1± 9.9 years and mean BMI was 29.0±6.1 kg/m². Overall, about 11.1% of study participants met criteria for bariatric surgery and there were significant racial/ethnic disparities (19.7% AA, 8.5% HW, 8.8% NHW, and 17.2% others; p<0.0001). Subgroup analysis observed increased BMI at follow up (29.60 vs. 29.16 kg/m², p=0.00229). Women with the AA risk genotype of rs1121980 (FTO gene) had 3.6-fold higher risk of obesity class II or III compared to those with the GG genotype (p=0.00393).

Conclusion: In addition to cancer risk, obesity is a major risk factor for cancer treatment-related side effects and worse survival. Our results suggest that at least 11% of breast cancer patients may be eligible for bariatric surgery. Considering some other beneficial effects of bariatric surgery on physical quality of life, metabolic syndrome, diabetes, and microbiome, future studies will consider weight loss and bariatric surgery programs in breast cancer patients, particularly in underserved minorities with higher obesity rate and worse survival.

#1768

Racial/ethnic differences in endometrioid endometrial cancer survival among cases identified through the National Cancer Database.

Anna B. Beckmeyer-Borowko,1 Caryn E. Peterson,1 Katherine C. Brewer,1 Mary O. Otoo,1 Faith G. Davis,2 Kent F. Hoskins,1 Charlotte E. Joslin1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Department of Public Health Sciences, University of Alberta School of Public Health, Edmonton, Alberta, Canada_.

OBJECTIVES: Past research suggests that at the local level, Non-Hispanic whites (NHW) have better survival when diagnosed with endometrioid endometrial cancer (EEC) than non-Hispanic blacks (NHB), Hispanics, and American Indians; however, little is known about survival differences at a national level and among other minorities. This study examined whether racial and ethnic differences in 5-year survival are present in U.S. women (NHW, NHB, Hispanics, non-Hispanic Asians (NHA), non-Hispanic Pacific Islanders/Hawaiians (NHPI), non-Hispanic American Indians/Aleutians or Eskimos (NHAI/AN) and non-Hispanics Others (NHO).

METHODS: EEC cases from the National Cancer Database were analyzed to evaluate racial/ethnic differences in 5-year survival among women diagnosed with EEC between 1998 and 2007. Chi-Square test was used to examine whether differences in demographic, clinical, institutional, and treatment variables varied by race/ethnicity. Multivariable Cox proportional hazard regression models were fit to estimate the adjusted hazard ratio (HR) and 95% confidence interval (95% CI) between race/ethnicity and survival.

RESULTS: A total of 178,310 women were diagnosed with EEC between 1998 and 2007. Of these; 74.8% were NHW, 8.3% were NHB, 13.9% were Hispanic, 1.8% were NHA, 0.2% were NHPI, 0.2% were NHAI/AN and 0.8% were NHO. Adjusting for covariates, NHB (HR=1.29; 95%CI 1.24-1.34) and NHPI (HR=1.59; 95%CI 1.50-1.67) had poorer survival than NHW. Yet results show that NHA (HR=0.83; 95%CI 0.74-0.92) and NHO (HR=0.81; 95%CI 0.69-0.94) had a better survival when compared to NHW. No survival differences were detected between Hispanics, NHAI/AN and NHW.

CONCLUSIONS: Results identify racial/ethnic differences in 5-year survival among women diagnosed with EEC cancer in the U.S. between 1998 and 2007. NHA and NHO had a better and NHB and NHPI had a poorer survival when compared to NHW.

#1769

Presence of cysts in benign breast tissue and the risk of subsequent breast cancer in African American women.

Julie J. Ruterbusch,1 Michele L. Cote,1 Sudeshna Bandyopadhyay,1 Baraa Alosh,1 Eman Abdulfatah,1 Vishakha Pardeshi,1 Woodlyne Roquiz,1 Daniel Visscher,2 Rouba Ali-Fehmi1. 1 _Wayne State University, Detroit, MI;_ 2 _Mayo Clinic, Rochester, MN_.

Introduction: Benign breast disease (BBD) is an established risk factor for developing breast cancer, and certain pathologic features in benign breast tissue are more strongly associated with breast cancer risk. Most of the studies evaluating BBD and breast cancer risk have been done in primarily white populations. Our previous work among African American (AA) women with BBD suggested the presence of cysts was associated with increased breast cancer risk. This finding may be unique to AA women as other studies among white populations have not replicated these findings. We sought to further explore the association between breast cysts and breast cancer risk in our expanded AA BBD cohort.

Methods: Biopsies from AA women initially diagnosed with BBD from 1997 to 2009 were examined for 14 pathologic features, including the microscopic presence of cysts, and followed for subsequent breast cancer. The association between cysts and the other pathologic features were compared using chi-square tests, and the risk of developing breast cancer was estimated using logistic regression and summarized with odds ratios (OR) and 95% confidence intervals (95% CI).

Results: A total of 3,360 AA women with BBD have been identified and their benign biopsies reviewed. 190 women have developed a subsequent breast cancer with a mean follow-up time of 11.5 years. Cysts were present on 1,366 (38%) of the biopsies, and were significantly associated with nearly all of the other benign features evaluated: apocrine metaplasia, ductal hyperplasia, calcifications, duct ectasia, fibrodenomas, fibrosis, intra-ductal papillomas, sclerosing adenosis, columnar alterations, mucocelle-like lesions, radial scars, and proliferation with and without atypia ( all p<0.001). Adjusting for age and year of biopsy, cysts were associated with a 36% increase in breast cancer risk (OR: 1.36, 95% CI 1.01 - 1.82). When further adjusted for hyperplasia with atypia, the benign feature with the most established and strongest association with breast cancer risk, the risk associated with cysts was attenuated (OR: 1.20, 95% CI 0.88 - 1.64).

Conclusion: Among AA women, cysts are highly correlated with other BBD features that increase subsequent breast cancer risk, but the risk associated with cysts does not appear to be independent of hyperplasia with atypia. Understanding the etiology of BBD and cyst development may provide insight into breast tumorigenesis, and the interplay between cysts and other BBD features warrant further investigation.

#1771

Metabolic dysregulation and cancer mortality in a national cohort of African-Americans and whites.

Tomi Akinyemiju, Justin X. Moore, Suzanne Judd, Monika Safford, Maria Pisu. _University of Alabama at Birmingham, Birmingham, AL_.

Introduction: Complex biological pathways link metabolic dysregulation (obesity, hypertension, dyslipidemia and diabetes) with cancer tumorigenesis, progression and metastasis, and those include higher circulating insulin, chronic inflammation, and increased visceral fat stores and estrogen. In this study, we examine the association between metabolic dysregulation and cancer mortality in a racially diverse cohort of adults.

Methods: A total of 25,038 African-American and White adults recruited through the prospective Reason for Geographic and Racial Disparities in Stroke (REGARDS) study were included in this study. Socio-demographics, medical history, and biomarkers (including blood pressure, fasting blood glucose, C-reactive protein, HDL and LDL cholesterol, height, weight, waist and hip measurements) were assessed at baseline. Metabolic dysregulation was defined in two ways: 1) based on Alberti et al. (2009) criteria of at least three of: high blood pressure, elevated triglycerides, reduced HDL cholesterol, elevated fasting blood glucose and high BMI; and 2) factor analysis of 15 variables indicative of metabolic abnormalities, including BMI, diabetes, triglycerides, total cholesterol, diastolic and systolic blood pressure, and fasting blood glucose. Cox proportional hazards regression analysis was used to model the hazards of cancer mortality in relation to each individual component as well as the cluster of metabolic dysregulation.

Results: About 51% of African-American females, 38% of African-American males, 38% of White females and 40% of White males met the joint criteria for metabolic dysregulation. White females in the highest quartile of the factor associated with high fasting blood glucose and diabetes experienced a 2-fold increase in cancer mortality (HR: 2.02, 95% CI: 1.27 - 3.20) compared to those in the lowest quartile. In crude analysis, elevated HDL cholesterol (HR: 1.16, 95% CI: 1.02 - 1.31), elevated blood pressure (HR: 1.33, 95% CI: 1.15 - 1.54), and elevated fasting blood glucose (HR: 1.23, 95% CI: 1.09 - 1.40) were each associated with higher mortality, although these associations were attenuated after adjusting for gender, race, age, and SES. However, in the fully adjusted model, having at least three of the five metabolic dysregulation components was associated with a 46-61% increased hazard of cancer-related death compared with having none (HR for 5 components: 1.61, 95% CI: 1.02 - 2.55).

Conclusion: There are marked racial differences in the prevalence of metabolic dysregulation, and the strong association with cancer mortality suggests that this may be a major factor in the observed and persistent disparities in cancer mortality. Metabolic abnormalities are highly modifiable through lifestyle changes and medication; appropriate prevention strategies have significant potential to reduce the risk of tumor metastasis, recurrence and death.

#1772

Cancer prevalence and screening among US Asian adults by nativity.

Tainya C. Clarke. _CDC, National Center for Health Statistics, Hyattsville, MD_.

Background: While heart disease is the leading cause of death in the general United States (US) population, the number one cause of death among US Asian adults is cancer. The cancer burden in the US is higher than that in Asia. However as Asians migrate to the US, acculturation may increase their risk of acquiring cancers. We examine the prevalence of selected cancers and recommended screening behaviors among Asian Americans by US nativity.

Methods: We analyzed combined data from the 2010 and 2013 National Health Interview Survey (NHIS) to examine the prevalence of selected cancers and adherence to the US Preventive Services Task Force (USPSTF) recommended screening behaviors by US nativity among more than 4,000 non-Hispanic Asian adults aged 18 and over. Asian adults include persons who identified their race as "Asian," "Asian Indian," "Chinese," "Filipino," "Korean," "Japanese," "Vietnamese," or other detailed Asian responses, identified no other non-Asian races, and now reside in the US. Nativity was determined by place of birth located within or outside the US or its territories. Survey data were weighted to produce national estimates that are representative of the civilian noninstitutionalized US adult population. Differences between percentages were evaluated using two-sided significance tests at the 0.05 level.

Results: While the overall prevalence of cancer among Asian adults (3.1%) was not significantly different from that of their non-Hispanic white adult peers (3.4%), US born Asian adults were approximately 1.7 times more likely to have had a cancer diagnosis compared with Asian adults born outside the US. Nativity was not significantly related to cancer diagnosis for non-Hispanic white adults. There was no significant difference by US nativity in the prevalence of colorectal cancer among Asian adults aged 50-75 or breast cancer among Asian women aged 40-75. However, US born Asian women aged 21-65 without a hysterectomy were more than 5 times as likely to have a diagnosis of cervical cancer as their peers who were born outside the US. There was no significant difference in adherence to the USPSTF recommended mammography or colorectal screening among US born Asian adults within the recommended screening age groups compared with those born outside the US. However, US born Asian women were more likely to adhere to the USPSTF recommended screening for cervical cancer compared with those born outside the US (79.0% vs. 70.0%).

Conclusion: The higher prevalence of cancer among US Asian adults compared with those not born in the US appears to be driven by differences in cervical cancer prevalence. Although higher than their peers born outside the US, adherence to USPSTF recommended cervical screening among US born Asian women aged 21-65 remains lower than the Healthy People 2020 baseline of 84.5%.

#1773

Racial/ethnic disparities in ovarian cancer treatment and survival.

Elisa V. Bandera,1 Valerie S. Lee,2 Lorna Rodriguez-Rodriguez,1 Bethan Powell,3 Lawrence H. Kushi2. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Kaiser Permanente Northern California, Oakland, CA;_ 3 _Kaiser Permanente Northern California, San Francisco, CA_.

Among ovarian cancer (OC) patients, African American (AA) women are less likely to receive adequate treatment and experience worse survival compared to white women. These differences have been attributed to unequal access to care. AA women are also more likely to be obese with related comorbidities, which are known to affect chemotherapy dosing, and dose reduction has been shown to reduce OC survival. Previous studies have not taken these factors into account and possible disparities among other racial/ethnic groups have received little attention. We evaluated racial/ethnicity differences in chemotherapy dosing and survival in a cohort study of epithelial invasive OC cases who were diagnosed and treated as members of Kaiser Permanente Northern California, and thus had equal access to health care.

Analyses included cases (n=806) diagnosed from January 2000 to March 2013 receiving adjuvant first-line therapy of carboplatin and paclitaxel with curative intent. Outcomes were overall and OC-specific deaths, with median follow-up of 50 months. Relative dose intensity (RDI) was computed for carboplatin and paclitaxel separately as actual dose administered per week divided by expected dose per week, and average RDI (ARDI) was then calculated for the regimen. Early discontinuation was defined as completing less than four scheduled treatments. Treatment delay was defined as delays in scheduled chemotherapy of more than 7 days. Proportional hazards regression was used to calculate HR and 95% CI after adjusting for relevant covariates.

AA women were more likely to be diagnosed with advanced disease (26.2%), to have hypertension (73.9%) and renal disease (50%), not to have surgery, and to have elevated post-treatment CA-125, as marker of residual disease. Both AA and Hispanic women had high prevalence of obesity, with Hispanic women having the highest prevalence of diabetes (33%). Compared to whites, AAs were more likely to have dose reduction (ARDI<85%), treatment delay, and early discontinuation, while Hispanics were also more likely to have dose reduction, but less likely to have early discontinuation or dose delay. After controlling for age at diagnosis, stage, grade, histology, chemotherapy-related toxic effects, G-CSF use, BMI at diagnosis, comorbidities likely to affect dosing (diabetes, hypertension, cardiovascular diseases, renal disease), ARDI, surgery type, and post-treatment CA 125, AA women had the worst survival. Compared to whites, adjusted HRs (95%CI) for overall mortality were 1.59 (1.03-2.44) for AA; 0.89 (0.61-1.30) for Asians; and 1.31 (0.90-1.91) for Hispanics. Findings for OC-specific mortality were similar.

Our study shows that disparities in OC treatment and survival in AA persisted in a setting of equal access to care and after taking into account treatment and other clinical characteristics and warrants further evaluation of biological, personal, and social factors that may be responsible for these differences.

#1775

Differential expression of exosomes-associated onco-microRNAs in African Americans with prostate cancer.

Ahmed A. Moustafa,1 Andrew B. Sholl,1 Koji Tsumagari,1 Mohamed Hassan,2 Emad Kandil,1 Asim B. Abdel-Mageed,1 Zakaria Y. Abd Elmageed1. 1 _Tulane University School of Medicine, New Orleans, LA;_ 2 _University of Mississippi Medical Center, Jackson, MS_.

Background: The mortality rate of prostate cancer (PCa) is two times higher in African American (AA) than Caucasian American (CA) men. The molecular mechanisms underlying these health disparities among AA men have yet to be fully elucidated. Our study aimed to evaluate the role of exosomes-associated onco-microRNAs (miRs) as biological determinant for the racial disparity of PCa patients.

Methods: Exosomes were isolated from the conditioned medium of MDA-PCa-2b (AA-origin), PC-3 (CA origin), C4-2B and RWPE-1 (non-tumorigenic) cell lines to study the endogenous expression of a number of onco-miRs and their anticipated target genes using Real-Time PCR and Western blot analyses. The potential candidates were validated in exosomes procured from plasma of human AA PCa patients and their counterparts. Onco-miRs target genes such as Lats-2, PDCD4, and SMEK1 were validated on cellular and exosomes derived from cells and human specimens. Onco-miRs and their targets were correlated with age and Gleason score of the disease.

Results: Not only onco-miR-125b, miR-155, and miR-3613 were upregulated in PCa versus normal RWPE1 cells but also in AA versus CA PCa cells. Onc-miR-3613 and miR-3680 were 3-4 times upregulated in cells and in C4-2B-derived exosomes compared to RWPE-1-derived vesicles. Also, these miRs were upregulated (~2.5 fold) in MDA-PCa-2b versus PC-3 cells. The expression of tumor suppressor genes, such as Lats2, PDCD4, and SMEK1, were downregulated in PCa cells compared to normal cells. Differential expression of PCa exosomes-associated onco-miRs was also demonstrated in AA PCa patients compared to CA PCa and their age-matched normal subjects. A positive correlation was found between onco-miR-125b and high Gleason score (r=0.763, p<0.01).

Conclusion: Our findings underline the role of exosomes-associated onco-miRs in health disparity of PCa. The differential expression of onco-miRs and their tumor suppressor genes in AA men projects their potential role as prognostic markers and/or future therapeutic targets in the context of racial disparity.

#1776

Aspirin use among African American men reduces prostate cancer risk: Findings from the NCI-Maryland Prostate Cancer Case-Control study.

Cheryl J. Smith, Symone Jordan, Tiffany H. Dorsey, Dean Mann, Chris A. Loffredo, Elise Bowman, Wei Tang, Stefan Ambs. _National Institutes of Health, Bethesda, MD_.

Prostate cancer is a leading cause of cancer death in US men. Yet, the etiology of prostate cancer remains poorly understood, with only older age, African ancestry, family history of the disease, and multiple germline genetic variations being established disease risk factors. Prostate cancer occurs more often in African-American men and Caribbean men of African descent than in men of other race/ethnicities. The causes of these racial and ethnic differences are multifactorial, but it has been proposed that differences in tumor biology contribute to the health disparity associated with prostate cancer.

Previously, our laboratory discovered immunobiology differences between African-American and European-American men where genes involved in the inflammatory response tended to be up-regulated in tumors of African American patients. Accumulating evidence suggests that inflammation may be involved in prostate carcinogenesis. Given these observations, we hypothesized that inflammation may contribute to prostate cancer risk and the survival health disparity observed between African-Americans and European-Americans. To this end, we conducted a case-control study in the greater Baltimore area where we recruited 1000 cases and 1000 controls composed of equal populations of African-American and European-American men. In our survey, we asked about non-steroidal anti-inflammatory drugs (NSAIDs) use categorized into aspirin, Tylenol, and other pain relievers that neither contain Tylenol nor aspirin. We found a significant association between aspirin use and prostate cancer risk—where aspirin use decreased the risk of prostate cancer. Strikingly, the decreased prostate cancer risk as a result of aspirin use was most profound in African-American men. We were also able to measure the levels of C-reactive protein, a marker for inflammation, to further evaluate the relationship between aspirin use, inflammation, and prostate cancer risk. Indeed, we did observe a decrease in C-reactive protein in patients who took aspirin. Interestingly, overall levels in healthy controls and prostate cases were higher in African-American subjects and patients when compared to European-Americans. Although aspirin use decreased C-reactive protein levels in African-American and European-American cases, African-Americans had significantly higher levels of C-reactive protein. The robust association between aspirin consumption and prostate cancer risk suggests that inflammation might be a driver in prostate cancer of African-American men. Additionally, the increased levels of C-reactive protein in African-American controls and cases might contribute to the excessive disease risk and mortality among these men.

#1777

Prostate cancer risk factor profiles in black and white men in the NIH-AARP Diet and Health Study.

Tracy M. Layne,1 Barry I. Graubard,2 Xiaomei Ma,1 Susan T. Mayne,1 Demetrius Albanes2. 1 _Yale School of Public Health, New Haven, CT;_ 2 _National Cancer Institute, Rockville, MD_.

Background: Few well-established prostate cancer risk factors exist, and associations with modifiable factors, such as diet/nutrient intake and other health-related parameters, have primarily been identified in non-Hispanic white populations. In this study we examined whether prior associations explain prostate cancer risk in non-Hispanic black men and/or underlie some of the black-white difference in risk.

Methods: Race-specific associations between diet, nutrient intake, and other factors and prostate cancer risk in black (N=7,030) and white (N=230,968) men in the NIH-AARP cohort were examined. During an average of 9.4 years of follow-up, there were 1,049 incident cases among black men (156 advanced stage), and 21,301 cases among white men (2,677 advanced). Cox proportional hazards regression models estimated prostate cancer hazard ratios (HR) and 95% confidence intervals (CI). We also evaluated the change in the HR for black race following addition of each factor to the model.

Results: Race-specific parsimonious prostate cancer models showed similar black and white associations only for history of diabetes (black men HR=0.79, 95%CI: 0.65-0.95; and white men HR=0.72, 95% CI: 0.68-0.77). For all other risk factors examined, the associations were either similar but did not reach statistical significance in black men (i.e., for body mass index, smoking status, and tomato intake), or differed by race. For example, in white men we observed a null association with non-advanced disease for height (per 10 cm increase, HR=1.01, 95% CI: 0.99-1.03), and an inverse association in black men (HR=0.91, 95% CI: 0.82-0.996; p for interaction=0.03). Similarly, a positive association was evident between dietary vitamin D intake and non-advanced disease in white men (highest versus lowest quintile, HR=1.11, 95% CI: 1.05-1.18), with an inverse association in black men (HR=0.88, 95% CI: 0.71-1.10; p for interaction=0.04). Risk of advanced disease showed a null association with alcohol consumption in white men (≥ 6 drinks/day versus never drinkers, HR=0.96, 95% CI: 0.78-1.20) and a statistically significant direct relation in black men (HR=2.19, 95% CI: 1.04-4.59; p for interaction=0.15). Overall, adjustment for the risk factors increased the hazard ratio for black race by 23% (1.73 to 1.90) for non-advanced and 13% (2.03 to 2.16) for advanced disease.

Conclusions: Our data suggest that many factors thought to influence prostate cancer risk in predominantly non-Hispanic white populations explain only a small proportion of black male risk, or the black-white difference in risk. Dietary vitamin D intake, height, and alcohol consumption associations with prostate cancer risk may vary between black and white men.

#1778

A comparison study of the disparities of cervical cancer excess mortality between Black and Caucasian women in Alabama and the US.

Ehsan M. Abdalla. _Tuskegee University, Auburn, AL_.

Background: The main purpose of this study was to assess the disparity of cervical cancer (CerCancer) mortality rates change through time between Black and Caucasian women residing in Alabama and the US.

Methods: The CerCancer behavioral risk factors and utilization of screening tests data were obtained from CDC's Behavioral Risk Factor Surveillance System (BRFSS) database. Baseline data and target objectives of utilization of CerCancer screening and mortality rates were obtained from Healthy People 2020. The CerCancer mortality rates data were obtained from the Surveillance, Epidemiology, and End Results (SEER). Mortality rates, percentage difference, percentage change and annual percentage change for trends were calculated and compared with the US baseline. Data were stratified by age, gender, race, and geography, using SEER*Stat version 8.4.1 in conjunction with Linear Trendlines analysis to model the racial changes of CerCancer mortality rates through time in Alabama and the US.

Results: Our results depicted in general, that although Blacks had higher CerCancer mortality rates at all times, a decreasing trend in CerCancer mortality rates was noted for both races. However, the degree of disparities in mortality rates fluctuated over time and differed by age. In Alabama, Blacks aged 65 years and older have not experienced significant decline in CerCancer mortality rates in recent years, despite a high screening rate compared to Whites. In contrast, between 2002 and 2012, Whites in Alabama and the US made a significant progress toward the Healthy People 2020 goal.

Conclusions: Conclusively in Alabama, a disparity still exists for the high CerCancer mortality rates in Blacks despite the higher rates of screening for CerCaner as would otherwise be expected. Additionally, the state has not yet achieved the Healthy People 2020 goal. Thus, although the gap in mortality rates in Alabama has narrowed, however the disparity remains, but if the dismal narrowing is sustained, it will reduce the racial disparities in CerCancer mortality though at a very slow rate. Therefore, Public health officials should monitor progress toward reduction and/or elimination of these disparities.

#1779

Risk factors for early-onset ER- and ER+ breast cancer in African American women.

Kimberly A. Bertrand, Lynn Rosenberg, Julie R. Palmer. _Slone Epidemiology Center at Boston University, Boston, MA_.

Introduction: Relative to white women, African American (AA) women have a disproportionately high incidence of early-onset breast cancer, particularly higher incidence of aggressive breast cancer subtypes such as estrogen receptor (ER) negative tumors. Elucidation of risk factors for early-onset of specific subtypes of breast cancer is needed.

Methods: We evaluated associations of reproductive and lifestyle factors with early-onset invasive breast cancer, defined as diagnosis before age 50, in 47,212 AA women ages 21-49 in the Black Women's Health Study (BWHS). The BWHS is an ongoing prospective cohort study in the U.S. that began in 1995. Risk factor information is updated every two years via mailed questionnaires. Incident cases were self-reported on biennial questionnaires or identified through death records and confirmed by medical records and cancer registry data. Through 2013, we identified 192 ER- and 322 ER+ invasive breast cancers diagnosed before age 50. We used multivariable Cox proportional hazards regression, stratified by age, to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of family history of breast cancer, age at menarche, parity, age at first birth, years since last birth, lactation, body mass index (recent and at age 18), waist-to-hip ratio, oral contraceptive use, alcohol consumption, smoking history, and physical activity, with risk of ER- and ER+ early-onset breast cancer, separately.

Results: The strongest associations were observed for family history of breast cancer, early age at menarche, and lower body mass index at age 18, and these factors were associated with increased incidence of both ER- and ER+ breast cancer. Oral contraceptive use within the past 10 years was also associated with increased risk of both subtypes. High waist-to-hip ratio (HR for top tertile vs. bottom: 1.46, 95% CI: 0.98, 2.19) and high parity (HR for 3+ births vs. 1: 1.43, 95% CI: 0.88, 2.32) were associated with increased risk of ER- breast cancer, while breastfeeding was associated with a reduced risk (HR for ever vs. never: 0.68, 95% CI: 0.47, 0.97). In contrast, higher parity was associated with a reduced risk of ER+ cancer (HR for 3+ births vs. 1: 0.68, 95% CI: 0.45, 1.01). Recent pregnancy (HR for <10 years vs. 10 or more years: 1.33, 95% CI: 0.94, 1.88) and greater alcohol consumption (HR for 7+ drinks/week vs. <1/week: 1.33, 95% CI: 0.81, 2.17) were associated with an increased risk of ER+ tumors.

Conclusions: Differential associations of risk factors for ER- vs. ER+ breast cancers in young AA women suggest etiological heterogeneity by tumor subtypes. These differences could explain, in part, disparities in breast cancer incidence and mortality between black and white women. Some identified risk factors are potentially modifiable, suggesting possible opportunities for prevention.

#1780

Differences in pancreatic cancer incidence across five racial/ethnic populations in the Multiethnic Cohort.

Veronica Wendy Setiawan,1 Christopher A. Haiman,1 Daniel O. Stram,2 Stephen J. Pandol,3 Lynne R. Wilkens,4 Loic Le Marchand,4 Kristine R. Monroe2. 1 _University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA;_ 2 _University of Southern California, Los Angeles, CA;_ 3 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 4 _University of Hawaii Cancer Center, Honolulu, HI_.

Background: The pancreatic cancer incidence is steadily increasing and the death-to-incidence ratio approaches one. While disparity in pancreatic cancer incidence between US blacks and whites has been observed, no study has been conducted to examine disparity in other populations. We examined racial/ethnic differences in pancreatic cancer incidence and assessed the extent to which known risk factors for pancreatic cancer account for differences in risk among African Americans, Native Hawaiians, Japanese Americans, Latinos and whites in the Multiethnic Cohort Study.

Methods: During a 16.2 year follow up period, 1,415 incident pancreatic cancer cases were identified among 183,269 at risk participants. Data on risk factors were obtained from the baseline questionnaire (1993-1996). Cox regression was used to calculate the age- and sex-adjusted relative risks (RRs) and 95% confidence intervals (CIs) for pancreatic cancer associated with risk factors and race/ethnicity.

Results: Current smoking status (RR<20 pack-years=1.40; 95% CI: 1.15, 1.71; RR≥20 pack-years=1.72; 95% CI: 1.42, 2.09, P trend<0.0001), obesity (BMI ≥30 kg/m2) (RR=1.26; 95% CI: 1.08, 1.47), history of diabetes (RR=1.38; 95% CI: 1.18, 1.60), family history of pancreatic cancer (RR=1.91; 95% CI: 1.44, 2.54), and intake of red meat (RR highest vs. lowest quartile=1.26; 95% CI: 1.08, 1.48, P trend=0.025) were associated with increased risk of pancreatic cancer. The RRs for pancreatic cancer (vs. whites) were 1.76 (95% CI: 1.42, 2.18) for Native Hawaiians, 1.34 (95% CI: 1.13, 1.58) for African Americans, 1.28 (95% CI: 1.11, 1.48) for Japanese Americans, and 0.93 (95% CI: 0.79, 1.11) for Latinos. After further adjustment for the risk factors above, the RRs were 1.56 (95% CI: 1.26, 1.95) for Native Hawaiians, 1.20 (95% CI: 1.01, 1.43) for African Americans, 1.29 (95% CI: 1.10, 1.50) for Japanese Americans, and 0.87 (95% CI: 0.73, 1.04) for Latinos.

Conclusions: In the MEC, Native Hawaiians were at the highest risk of developing pancreatic cancer, followed by African Americans, Japanese Americans, whites, and Latinos. Interethnic differences in pancreatic cancer risk do not appear to be explained by differences in the distribution of known risk factors. The greater risks in Native Hawaiians and Japanese Americans compared to whites are new important findings and elucidating the causes of these high rates may improve our understanding of the disease.

#1781

Ethnic disparity in pediatric leukemia relapse.

Mirinda Gillespie,1 Greg Hale,2 Ernest K. Amankwah3. 1 _All Childrens Hospital Johns Hopkins Medicine, St. Petersburg, FL;_ 2 _Johns Hopkins University/All Childrens Hospital, FL;_ 3 _Johns Hopkins University, Baltimore, MD_.

Background: Leukemia is the most prevalent pediatric malignancy, representing 25% of all cancer diagnoses among children. Despite recent substantial improvements in therapy that have dramatically increased survival, 10-20% of cases develop relapse and become refractory to treatment. Although racial/ethnic disparity in survival is well documented, limited knowledge currently exists on the disparity in relapse. The objective of this study was to compare the risk of pediatric leukemia relapse between Hispanic Whites and non-Hispanic Whites.

Methods: The study included children (<21 years) diagnosed with leukemia between January 2006 and December 2014 at the All Children's Hospital Johns Hopkins Medicine, St. Petersburg, FL. Patients' demographic and clinical information were obtained from an electronic health record-derived data warehouse. Ethnicity was self-reported. Leukemia diagnosis and relapse were identified using appropriate ICD-9 codes. Relapse-free survival was defined as the time from diagnosis to the first documentation of relapse. Hazard ratios (HR) and 95% confidence intervals (95% CI) for relapse-free survival were calculated using Cox proportional hazard models adjusting for age at diagnosis, gender and type of insurance (commercial vs government).

Results: The study included 66 Hispanic Whites and 202 non-Hispanic Whites. The median age for both groups was 7 years. However compared to non-Hispanic Whites, Hispanic Whites were more likely to be males (59% vs 53%), to have government insurance (71% vs 35%) and had a shorter median follow up time (52 vs 66 months). Approximately 15% of Hispanic Whites developed relapse compared to only 5% non-Hispanic Whites. Kaplan-Meier survival analysis showed a difference in relapse between the two groups (log-rank p=0.006). After adjusting for age at diagnosis, gender and type of insurance, Hispanic Whites were three times more likely to develop relapse compared to non-Hispanic Whites (HR=3.1, 95%CI=1.2-8.0, p=0.02).

Conclusion: Although the findings of this study suggest that ethnic disparity in pediatric leukemia may exist, our findings are limited by the inability to control for potential confounders such as medication adherence, timing of diagnosis, white blood cells at diagnosis and minimal residual disease. Future studies should further investigate this racial/ethnic disparity.

#1782

Racial differences in maternal and cord blood leukocyte telomere length and their correlations.

Kari A. Weber,1 Christopher M. Heaphy,2 Corinne E. Joshu,1 Sabine Rohrmann,3 Jessica L. Bienstock,2 Tanya Agurs-Collins,4 Alan K. Meeker,2 Elizabeth A. Platz1. 1 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 3 _University of Zurich Institute of Social and Preventive Medicine, Zurich, Switzerland;_ 4 _National Cancer Institute, Bethesda, MD_.

Background: Telomere length at birth is hypothesized to set the baseline for lifetime telomere shortening trajectory and influence adult disease risk including prostate cancer, which shows a pronounced racial disparity. Telomere length is heritable, but may also be a marker of exposures in utero, including those impacting adult cancer risk. Thus, we investigated racial differences in leukocyte telomere length in maternal blood and cord blood from their male neonates, and in telomere length correlations between mothers and neonates. The purpose of this work is to inform whether telomere length differences at birth may account for some of the racial disparity in adult disease risk.

Methods: Black and white Baltimore women were recruited at the start of pregnancy in 2006-7 and followed to postpartum. They completed surveys at enrollment and at the first postpartum visit. Medical records were abstracted from each clinic visit, mothers provided blood each trimester, and cord blood was collected at birth. 55 mother-male neonate pairs with complete materials were included. Relative telomere length (telomere/single copy gene) was measured by quantitative PCR in leukocyte DNA. We used linear regression to estimate mean telomere length in mothers and neonates before and after adjusting for assay batch, mother's age and other factors. We calculated the Pearson correlation between mother and cord blood telomere length overall and by race.

Results: Black mothers were younger, less likely to have a graduate degree and to be nulliparous, and more likely to be overweight/obese than white mothers. Adjusting for batch and mother's age, black mothers had shorter mean telomere length (2.18, 95% CI 1.81-2.56) than white mothers (2.65, 95% CI 2.35-2.95) and black neonates had shorter mean telomere length (2.64, 95% CI 2.27-3.01) than white neonates (3.02, 95% CI 2.73-3.31); differences were not significant (p=0.08, 0.1). Differences were attenuated after further adjustment for education, nulliparity, and BMI. Adjusting for mother's age, mother and cord blood telomere lengths were highly correlated overall (r=0.73, p<0.0001), and did not differ between blacks (r=0.77) and whites (r=0.67; p-interaction=0.6). Correlations were similar after further adjustment for education, nulliparity, and BMI.

Conclusion: Leukocyte telomere length was shorter in black compared with white mothers and in cord blood, but racial differences were attenuated after adjustment. Correlation between mother and neonate telomere length was high, did not differ by race, and was not attenuated after adjustment. Our results appear to support the hypothesis that telomere length differs by race at birth in part due to inherent differences and in part due to maternal factors, and thus, may inform the racial disparity in risk of adult diseases like prostate cancer.

Funding: T32 CA009314, U54 CA091409, P30 CA006973, Dept of Epidemiology Doctoral Research Fund

#1783

Heterogeneity of triple negative breast cancer across race and ethnicities: Indian women at higher risk for early onset breast cancer.

Padma P. Tadi Uppala,1 Maheswari Senthil,2 Utkarsh P. Patel,1 Sharon Lum,2 Carlos Garberoglio,2 Larry Beeson,1 John Morgan1. 1 _Loma Linda University School of Public Health, Loma Linda, CA;_ 2 _Loma Linda University School of Medicine, Loma Linda, CA_.

Breast cancer is a heterogeneous disease with several subtypes presenting various morphological and molecular features, and response to therapy. While targeted therapies are available for estrogen receptor positive and HER2-positive breast tumors, triple-negative breast cancer (TNBC) which are negative for ER, progesterone receptor PR and HER2 receptors lack suitable targeted therapies. TNBC is one of the most aggressive breast cancer subtypes and poses a clinical challenge as they lack suitable targeted therapies. While a majority of breast cancer patients in the US are postmenopausal, more than 80% of Indian patients are younger than 60 years of age, with median age being 40-55, presenting with larger tumor size, poor tumor grade, and low rates of hormone-receptor positive status. Literature review suggests that none of the standard risk factors for breast cancer had any significant associations for the early onset breast cancers among Indian women. Recent studies on breast cancer subtypes across culture and geographical regions in India have indicated that incidence of early onset breast cancer, TNBC in particular is rising at alarming rates among Indian women. The interaction of race and ethnicity with age, molecular profiles and lifestyles has contributed significantly to the heterogeneity of TNBC breast cancer. The purpose of this study is to conduct a systematic literature review to identify factors that may increase risk for breast cancer among young Indian women. Methods: All the major databases including Web of Science and PubMed were used to search the literature for early onset breast cancer among Indian women. In a study that analyzed molecular subtypes in early onset breast cancer among various races that included Indian, Chinese, non-Hispanic White (NHW), African American (AA), and Hispanic women, incidence of TNBC was significantly higher (p=0.0369) with early onset (40 years and younger) in Indian women. Incidence of HER2 over-expression subtype was also highest among Indian women. Conclusion: Early onset breast cancer is increasing rapidly among Indian women, along with obesity and diabetes. It is suggested that epigenetic factors that include sedentary lifestyles, lack of exercise, fatty diets and increasing obesity with underlying molecular mechanisms may contribute to early onset breast cancer among Indian women. Future studies that focus on racial and ethnic differences in genetic, reproductive, lifestyle and environmental exposures of TNBC pathways will offer unique biomarkers, targeted therapies and clinical trial design leading to personalized medicine.

#1784

Disparities in pediatric leukemia incidence and survival: a population-based cancer registry analysis comparing Thailand and the United States.

Kathryn Demanelis,1 Hutcha Sriplung,2 Rafael Meza,1 Surapon Wiangnon,3 Laura S. Rozek,1 Michael E. Scheurer,4 Philip J. Lupo4. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Prince of Songkla University, Songkhla, Thailand;_ 3 _Khon Kaen University, Khon Kaen, Thailand;_ 4 _Baylor College of Medicine, Houston, TX_.

Background: Pediatric leukemia incidence and survival varies worldwide. This variability may be due to differences in genetics, environmental factors, and access to treatment and diagnosis. However, more work is needed to elucidate patterns of pediatric leukemia in developing countries including Thailand. Therefore, we analyzed pediatric leukemia incidence and survival trends in children age 0-19 years from 1990-2011 in Thailand and compared these results to United States (US) data. We evaluated the following pediatric leukemia subtypes: acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

Methods: We extracted pediatric leukemia cases from five provincial population-based cancer registries in Thailand (n=1,245). Cases from the US were obtained from the Surveillance, Epidemiology, and End Results program (n=6,738). We computed age-standardized incidence rates (ASIR) using the WHO 2000 standard population and relative survival using the Ederer II method and estimated survival functions using the Kaplan-Meier method. We evaluated temporal changes in incidence and survival by year of diagnosis and estimated annual percent changes using joinpoint regression.

Results: While the ASIR of ALL was lower in Thailand compared to the US (21.1 vs. 31.2 cases per million), the ASIR of AML was similar between Thailand and the US (7.7 vs. 7.7 cases per million). The proportion of ALL and AML diagnosed in Thailand was 60% (n=746) and 23% (n=284), respectively, vs. 75% (n=5,080) and 19% (n=1,267) in the US (p<0.001). The mean age at diagnosis was significantly older in Thailand compared to the US for both ALL (7.2 vs. 6.5 years, p <0.001) and AML (9.9 vs. 9.0 years, p=0.047). ALL incidence increased by 1.5% per year in Thailand (p=0.015) while it increased by 0.9% per year in the US (p=0.003). AML incidence increased by 2.3% per year in Thailand (p=0.047) while it remained constant in the US. Five-year survival significantly improved in Thailand between the first (1990-2000) and second half (2001-2011) of our study period for both AML (19% to 29%, p=0.009) and ALL (45% to 55%, p=0.001). ALL and AML five-year survival was 86% and 54% in the US. ALL survival increased by 1.5% per year in Thailand (p=0.022) while it increased by 0.7% per year in the US (p <0.001). AML survival increased by 1.8% per year in the US (p <0.001). Due to variability in survival by year, no trend was identified in Thailand.

Conclusion: The incidence of both ALL and AML increased more rapidly in Thailand than in the US from 1990-2011. While five-year survival for pediatric leukemia improved in Thailand, it was much lower than in the US. These disparities in incidence and survival warrant: 1) investigating novel population risk factors for pediatric leukemia, especially AML, in Thailand and 2) identifying diagnostic and treatment disparities to address the survival gap between Thailand and the US.

#1785

Sociodemographic correlates of knowledge and risk perception of HPV and HPV-associated oropharyngeal cancer.

Betty Y. Chen,1 Nosayaba Osazuwa-Peters,1 Eric Adjei Boakeye,1 Jennifer Clancy,1 Jonathan Su,1 Patrice Vallot,1 Geoffrey Beck,1 Ronald J. Walker,1 Mark A. Varvares2. 1 _Saint Louis University School of Medicine, St. Louis, MO;_ 2 _Harvard Medical School, Boston, MA_.

Purpose

To analyze how key sociodemographic variables correlate with risk perception and knowledge of HPV-associated oropharyngeal cancer in the community.

Methods

A cross-sectional study was conducted at a drag racing event on September 12-13, 2015 in Madison, Illinois. The independent variables were sociodemographic factors including age, gender, race, income level, education level, drinking history, smoking history, and number of sexual partners. The outcome variables were HPV risk perception and knowledge of HPV-associated oropharyngeal cancer, both derived from combining questionnaire items to form knowledge and perception scores. Multivariable linear regression analysis assessed the predictors of risk perception and knowledge of HPV-associated oropharyngeal cancer.

Results

Final sample population was 301. Mean risk perception score was 2.16 ± 1.37 out of 6, and mean knowledge score was 5.65 ± 4.63 out of 15. In multivariable linear regression, race and education were significant predictors of HPV risk perception. Compared to Caucasians, African Americans had a lower perceived risk of developing oropharyngeal cancer (β = -0.35; 95% CI: -0.69 - -0.02). Participants with ≤ high school diploma had a lower perceived risk of developing oropharyngeal cancer compared to those with ≥ college degree (β = -0.60; 95% CI: -1.05 - -0.15). Age, gender, race and education were significant predictors of HPV-associated oropharyngeal cancer knowledge in multivariable analysis. For every one year increase in age, HPV-associated oropharyngeal cancer knowledge decreased by 5%. Men had lower HPV-associated oropharyngeal cancer knowledge (β = -1.30; 95% CI: -2.35 - -0.26) as were African Americans compared to Caucasians (β = -1.30; 95% CI: -2.36 - -0.25). Participants with ≤ high school diploma had lower HPV-associated oropharyngeal cancer knowledge compared to those with ≥ college degree (β = -3.33; 95% CI: -4.75 - -1.91).

Conclusions

Age and gender were independent predictors of knowledge of HPV-associated oropharyngeal cancer, while race and education predict both knowledge and risk perception of HPV-associated oropharyngeal cancer. The sociodemographic correlates demonstrated in our study should inform future interventions targeted at increasing knowledge of HPV-associated oropharyngeal cancer in the community.

#1786

One in four Hispanic women with early onset breast cancer carry BRCA1, BRCA2, or PALB2 mutations: Results from a population-based study in South America.

Anna Marie Tuazon,1 Mabel Bohorquez,2 Carolina Ramirez,2 Paul Lott,1 Ana Estrada,2 Angel Criollo,2 Cathy Wang,1 Magdalena Echeverry,2 John Suarez,2 COLUMBUS Consortium,2 LaFamilia Consortium,1 Luis G. Carvajal Carmona1. 1 _University of California, Davis, Davis, CA;_ 2 _University of Tolima, Ibague, Colombia_.

Breast cancer is the leading cause of cancer incidence and mortality among Hispanic women, who are often diagnosed with late stage tumors and are more likely to die after diagnosis when compared to non-Hispanic whites. While strides have been made in understanding Hispanic breast cancer genetics, most studies have been limited to investigating cases from high-risk clinics. In this study, we aimed to assess the prevalence of pathogenic mutations in three most important breast cancer genes (BRCA1, BRCA2 and PALB2) in an unselected Hispanic breast cancer cohort. Initially, six hundred and forty-six unselected Colombian breast cancer cases were screened for four known Hispanic BRCA1/2 mutations by genotyping. Subsequently, cases that remained mutation negative, as well as 186 cancer-free controls, were screened for BRCA1, BRCA2 and PALB2 mutations using targeted next generation sequencing. We identified 67 cases with pathogenic mutations, one case with a likely-pathogenic variant, and 16 carriers of variants of unknown significance. Among the pathogenic mutation carriers, 88.1% harbored a founder mutation (n=59), which includes the known BRCA1 3450del4 mutation, strikingly found in 32 unrelated cases. Remarkably, we found that 1 in 4 of the cases diagnosed with breast cancer by age 40 years, regardless of family history of cancer, carried a pathogenic mutation. The high frequency of pathogenic mutations in this unselected cohort (10.4%), in particular those with an early age of onset (25.3%), suggests that population-based genetic testing among Hispanic communities can identify most carriers who would otherwise ineligible for testing. Identifying mutation carriers of these genes has implications in clinical management and surveillance for Hispanic women, a population that is vulnerable to breast cancer disparities in the U.S. and Latin America.

#1787

Adolescent boys and the human papillomavirus (HPV) - Geographical patterns of vaccination uptake.

Christian J. Geneus,1 Kahee A. Mohammed,2 Betelihem B. Tobo,2 Eric Adjei Boakye,3 Nosayaba Osazuwa-Peters2. 1 _Department of Biostatistics and Bioinformatics, Tulane University, New Orleans, LA;_ 2 _Department of Epidemiology, Saint Louis University, St. Louis, MO;_ 3 _Center for Health Outcomes Research, Saint Louis University, St. Louis, MO_.

INTRODUCTION

Human Papillomavirus (HPV) remains the most common sexually transmitted infection (STI) and is associated with multiple cancers. Previous research has examined HPV vaccine initiation and completion among girls. Among adolescent boys who are also at risk for contracting HPV, however, vaccine initiation and completion have not been widely explored.

OBJECTIVE

To examine predictors of HPV vaccine initiation and completion among boys in the United States.

METHODS

Analysis was conducted on 10,866 adolescent boys aged 13-17 using the 2014 National Immunization Survey-Teen. Our outcomes of interest, HPV vaccine initiation and completion, were measured using provider-verified vaccine history. Weighted multivariable logistic regression models assessed predictors of HPV vaccine initiation and completion.

RESULTS

Data stratified by geographic region showed that the prevalence of HPV vaccine initiation and completion ratio was lowest in the South: South (initiation: 38.82%, completion: 18.20%), Midwest (initiation: 38.15%, completion: 19.69%), West (initiation, 48.88%, completion: 26.12%), Northeast (initiation: 48.30%, completion: 26.96%).

Multivariable analysis of geographic patterns indicated that adolescent boys residing in the West had higher odds of vaccine initiation compared to those in the South (OR = 1.41; 95% CI: 1.08 - 1.83).On the other hand, the odds of vaccine completion were higher in adolescents residing both in the Northeast (OR = 1.46; 95% CI: 1.14 - 1.87) and West (OR = 1.42; 95% CI: 1.03 - 1.95) compared to adolescent boys residing in the South.

Adolescent boys who were recommended the HPV vaccine by a physician had 8.49 (95% CI; 7.03 - 10.26) and 5.97 (95% CI: 4.49 - 7.94) higher odds of initiation and completion, respectively. Compared to White adolescent boys, the odds of vaccine initiation (OR = 1.90; 95% CI: 1.47 - 2.48) and completion (OR = 1.49; 95% CI: 1.09 - 2.04) were higher amongHispanics boys. Moreover, boys aged 17 had higher odds of vaccine initiation compared to those who were 13 (OR = 1.41; 95% CI: 1.05 - 1.90). Finally, while mothers' education partially influenced the odds of vaccine initiation in adolescent boys (<12 years; OR = 2.053; 95% CI: 1.43 - 2.95), such was not significant in vaccine completion. Instead, higher odds of vaccine initiation among adolescent boys were associated with mothers who had two children aged<18 years compared to one child (OR = 1.29; 95% CI: 1.07 - 1.56).

CONCLUSION

Our findings highlight the suboptimal HPV vaccine across the United States, as well as the sociodemographic disparities and geographic variation of HPV vaccine initiation and completion. Particularly among adolescent boys residing in the South, tailored interventions are needed to improve HPV vaccine uptake.

#1787A

Puerto Rico BioBank: The first cancer tissue biobank at a US Hispanic-serving institution.

Patricia Casbas-Hernandez,1 Idhaliz Flores,1 Edward Seijo,2 Domenico Coppola,2 Steve Eschrich,3 Rodrigo Carvajal-Pelaez,3 Sonia Abac,1 Dagmar Correa,1 Edna Gordian,3 Teresita Munoz-Antonia4. 1 _Ponce School of Medicine, Ponce, PR, PR;_ 2 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _3 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 4 _H. Lee Moffitt Cancer Center, Tampa, PR_.

There are currently 580 million people worldwide considered to be of Hispanic origin. In the US Hispanics represent 17% of the population and its largest ethnic minority. Funded by NIH, Moffitt Cancer Center (Florida) and Ponce School of Medicine (Puerto Rico) initiated a partnership that conducts high-quality research, training, and community outreach focusing on cancer health disparities among Hispanics. A central component to this partnership is the establishment of the first cancer-focused biobank in the island, the Puerto Rico BioBank (PRBB). The PRBB has actively recruited cancer patients since 2009; over these years we have established essential collaborations and a functional infrastructure. Eligible cancer patients provide informed consent and agree to donate blood samples (for DNA) and solid tissue(fresh-frozen & paraffin embedded). Additionally, patients complete a comprehensive questionnaire related to clinical and lifestyle factors. Biospecimen collection at the PHSU follow standard operating procedures that mirror and often exceed NCI biobaking best practices for biospecimens and ensuring patient data confidentiality. Currently, the PRBB has over 1,100 consented patients (65.8% females, 34.2% males) all of Hispanic origin. The most common banked tumors are Breast, Prostate, Lung and Endometrium, although other tumors are represented. All the information and data are entered into a biospecimen management system (BMS) developed by the partnership, which allows for real-time biospecimen inventory management and reporting functionality. In conclusion, the Puerto Rican patient samples banked in the PRBB are a unique resource that can support multiple molecular and population studies. Also, a Hispanic-specific biobank can foster collaborations among local and international researchers that may facilitate the application of novel molecular techniques to solving cancer health disparities among Hispanics.

## PREVENTION RESEARCH:

### Prevention Clinical Research

#1788

Benefit of low-dose tamoxifen in a large, single-institution cohort of high-risk ER-positive DCIS.

Aliana Guerrieri-Gonzaga,1 Ivana Sestak,2 Matteo Lazzeroni,1 Davide Serrano,1 Nicole Rotmensz,1 Massimiliano Cazzaniga,1 Clara Varricchio,1 Mangesh A. Thorat,2 Giancarlo Pruneri,1 Maria Cristina Leonardi,1 Viviana Galimberti,1 Bernardo Bonanni,1 Andrea DeCensi3. 1 _European Institute of Oncology, Milan, Italy;_ 2 _Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Queen Mary University of London, London, United Kingdom;_ 3 _E.O. Ospedali Galliera, Genoa, Italy_.

Background: Low dose tamoxifen has shown comparable antiproliferative activity to 20 mg/day in biomarker trials, but its clinical efficacy is unclear. We assessed the effect of low dose tamoxifen, 10 mg on alternate day in most, on ipsilateral recurrence in high risk DCIS patients treated in a single institution.

Methods: Following breast conserving surgery, women with DCIS received radiotherapy and/or low dose tamoxifen as per clinical judgment and patient preference. Multivariate Cox regression analyses adjusted for potential confounding variables were performed.

Results: In a cohort of 1,091 women, median age was 53 years (IQR 46-62), 544 received radiotherapy versus 547 no radiotherapy. Of these, 883 had ER-positive DCIS, 467 received low-dose tamoxifen versus 416 no tamoxifen. After 7.7 years of median follow-up (IQR, 5.1-9.7), 235 ipsilateral recurrences and 62 contralateral tumors were observed. Low dose tamoxifen decreased any breast event (HR=0.70, 95% CI, 0.54-0.91) and ipsilateral DCIS recurrence (HR=0.66, 95% CI, 0.49-0.88), but not ipsilateral invasive recurrence (HR=0.78, 95% CI, 0.56-1.09) or contralateral tumors (HR=0.89, 95% CI, 0.51-1.55). Radiotherapy decreased any breast event (HR=0.55, 95% CI, 0.42-0.72). Low dose tamoxifen was more effective in women aged >50 years for all events (HR=0.51, 95% CI, 0.33-0.77) and ipsilateral recurrences (HR=0.43, 95% CI, 0.26-0.72) than in women aged 50 or younger (HR=0.84, 95% CI, 0.60-1.18 and HR=0.80, 95% CI, 0.55-1.16, respectively, p-interaction=0.03). Young age or premenopausal status, positive margins, high Ki67, high grade and low BMI were independent predictors of ipsilateral recurrence. No increase in endometrial cancers was observed and fewer deaths occurred in women on low dose tamoxifen.

Conclusions: In high risk ER-positive DCIS, low-dose tamoxifen seems to be safe and effective in reducing ipsilateral recurrence, and represents a valuable option in women aged >50 years. A randomized clinical trial is underway to confirm these results.

Supported by Lega Italiana per la Lotta contro i Tumori, AIRC, Italian Ministry of Health (RFPS-2006-1-339898), Gruppo bancario Credito Valtellinese.

#1789

Estrogen receptor alpha and beta gene expression in benign breast tissue of high risk premenopausal women treated with the SERM acolbifene.

Bruce F. Kimler, Teresa A. Phillips, Carol J. Fabian. _University of Kansas Medical Center, Kansas City, KS_.

Background

We conducted a pilot study to assess the feasibility of a 4th generation selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women at high risk for development of breast cancer [Cancer Prev Res 8, December 2015]. Favorable modulation of biomarkers was observed in benign breast tissue acquired before and after intervention. This included reduction in proliferation (Ki-67 immunocytochemistry) and decreases in expression of genes that code for ERα, pS2, and PgR. While ERα is over-expressed and stimulatory in breast carcinogenesis, ERβ which would be inhibitory is down-regulated. Thus, a change in the ratio of gene expression might be most informative. We have now analyzed gene expression of ERβ so as to examine whether it, or the ratio of ERα to ERβ, may contribute to assessment of response.

Methods

Premenopausal women at elevated risk for breast cancer were screened by random periareolar fine needle aspiration (RPFNA) performed during the follicular phase of the menstrual cycle. Eligible women (n=25) received 6-8 months of open-label acolbifene (20 mg/d), followed by repeat RPFNA. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed on 16 paired specimens for several genes associated with estrogen activity: estrogen receptor 1 (ESR1) for ERα, trefoil factor 1 (TFF1) for pS2, GREB1 for growth regulation by estrogen in breast cancer 1, PGR for progesterone receptor, and estrogen receptor 2 (ESR2) for ERβ. The quantity of each biomarker transcript was expressed relative to the reference transcript HPRT1. Further normalization by epithelial cell markers (KRT19 for cytokeratin 19 or CDH1 for E-Cadherin) did not materially alter the results.

Results

Significant changes had been previously shown for transcripts that code for pS2 (15 decreases, 1 increases; p=0.002), ERα (11↓, 5↑; p=0.013), and PgR (15↓, 1↑; p=0.001); plus a suggestion of effect of GREB-1α (10↓, 6↑; p=0.074). ERβ exhibited a general increase, but this was not statistically significant (5↓, 11↑; p=0.24). However, the reduction of the ratio of ERα to ERβ was statistically significant (15↓, 1↑; p=0.002).

Conclusions

Assessment of the ratio of gene expression for ERα to ERβ, and possibly their protein products by immunocytochemistry, may provide a useful surrogate endpoint biomarker by which to monitor response to SERMs used for breast cancer chemoprevention in premenopausal women.

Supported by NO1-CN-35135.

#1790

Factors associated with false positive results on screening mammography in a population of predominantly Hispanic women.

William Ueng,1 Julia McGuinness,2 Katherine Infante,1 Meghna S. Trivedi,2 Hae Seung Yi,3 Raven David,2 Alejandro Vanegas,2 Jennifer Vargas,2 Rossy Sandoval,2 Rita Kukafka,2 Katherine D. Crew2. 1 _Mailman School of Public Health, New York, NY;_ 2 _College of Physicians and Surgeons, Columbia University, New York, NY;_ 3 _Teachers College Columbia University, New York, NY_.

Objective - High rates of screening mammography have been reported among Hispanic women in the U.S. However, a potential harm of screening is a false positive result with recall breast imaging or biopsy. Our objective was to identify factors associated with false positive results on screening mammography among a predominantly Hispanic population in New York City.

Methods - We enrolled women receiving mammography at Columbia University Medical Center in New York, NY. They completed a questionnaire on breast cancer risk factors and gave consent to access their medical records for breast imaging and biopsy reports for the past 15 years. Breast cancer risk was assessed using the Gail model and eligibility for BRCA genetic testing was determined using a family history screener. High breast density was defined qualitatively as heterogeneously or extremely dense. Recall breast imaging was based upon a BIRADS score of 0, 3, 4, 5, or 6 on the screening mammogram. False positive breast biopsies were any biopsies that did not yield breast cancer.

Results - From November 2014 to May 2015, 1325 women were enrolled: median age 58 years (range, 29-89); white/black/Hispanic/other (%): 10/10/76/4; 25% met high-risk criteria for breast cancer; 31% had high breast density; 71% were undergoing annual mammography; 53% had at least one recall breast imaging and 6% had at least one false positive breast biopsy. In multivariable analysis, high breast cancer risk, high breast density, and more frequent screening mammograms were associated with recall breast imaging and biopsy.

Conclusions - Based upon our results, a potential strategy to reduce the false positive rates on screening mammography is to target women at high risk for breast cancer or with high breast density for screening with breast tomosynthesis, which has less false positives than digital mammography. Additionally, we may adopt less frequent breast cancer screening in average risk women to further reduce the harms of screening.

Factors associated with Recall Breast Imaging and False Positive Biopsy

---

Variable | OR (95% CI) | P-value

Annual Mammograms vs. Every 2+ Years | 2.42 (1.88, 3.13) | <0.0001

High Breast Density vs. Low Breast Density | 1.46 (1.14, 1.88) | 0.0031

High Breast Cancer Risk vs. Average Breast Cancer Risk | 1.45 (1.09, 1.92) | 0.0098

50+ Years Old vs. <50 Years Old | 1.03 (0.77, 1.38) | 0.8344

#1791

Effects of calcium and vitamin D supplementation on circulating markers of inflammation.

Veronika Fedirko,1 Julia Latash,1 Roberd M. Bostick,1 Elizabeth L. Barry,2 John A. Baron on behalf of Investigators in the PPS4 Study3. 1 _Rollins School of Public Health, Atlanta, GA;_ 2 _Geisel School of Medicine at Dartmouth, Lebanon, NH;_ 3 _University of North Carolina School of Medicine, Chapel Hill, NC_.

Chronic inflammation plays a major role in colon carcinogenesis. Recent evidence from experimental models suggests that vitamin D and calcium may reduce systemic inflammation and local inflammation in the colorectum, thus contributing to reducing risk for colorectal neoplasia. However, human experimental evidence to support these purported anti-inflammatory effects remains inconclusive, coming from very few, small, mainly short-term clinical studies with a variety of doses and administration regimens, with and without calcium supplementation in very different patient populations, and with a limited number of inflammatory biomarkers measured. To address this, we tested the effects of calcium (1.2 g/d) and/or vitamin D3 (1,000 IU/d) supplementation for 3 or 5 years on circulating pro- (IFNγ, TNFα, IL-6, IL-8, IL-12p40) and anti-inflammatory (IL-10) cytokines in 471 participants with previous colorectal adenoma in a recently completed, multi-center, randomized, double-blind, placebo-controlled, modified 2 x 2 factorial chemoprevention clinical trial (NCT00153816). The cytokines were measured in plasma using multiplex electrochemiluminescence detection-based immunoassays. Over a 3- or 5-year treatment period, we found no appreciable effects of calcium and/or vitamin D supplementation on pro-inflammatory cytokines (all P-values > 0.3). However, there was a suggestion that vitamin D may favorably modulate concentrations of IL-10, an anti-inflammatory cytokine. In the calcium, vitamin D, and calcium plus vitamin D groups, the relative increase vs. placebo in IL-10 from baseline to end of follow-up was 3% (P = 0.78), 15% (P = 0.15), 13% (P = 0.35). Our preliminary results indicate no considerable effects of calcium and vitamin D treatment on circulating pro-inflammatory cytokines, and might suggest that vitamin D may increase circulating levels of anti-inflammatory cytokine IL-10, which plays an important role in the Th2-type immune response.

#1792

Phospho-IGF-1R/IR provides independent prognostic information in primary breast cancer but is not associated with prognosis in endocrine-treated patients.

Sofie Björner,1 Ann Rosendahl,1 Maria Simonsson,1 Andrea Markkula,1 Karin Jirström,1 Signe Borgquist,1 Carsten Rose,2 Christian Ingvar,3 Helena Jernström1. 1 _Lund University and Skåne University Hospital, Department of Clinical Sciences, Lund, Division of Oncology and Pathology, Lund, Sweden;_ 2 _CREATE Health and Department of Immunotechnology, Lund University, Medicon Village, Lund, Sweden;_ 3 _Lund University and Skåne University Hospital, Department of Clinical Sciences, Lund, Division of Surgery, Lund, Sweden_.

Introduction

The insulin-like growth factor (IGF) system is implicated in several cancers including breast cancer. Phosphorylated IGF type I receptor/insulin receptor (pIGF-1R/IR) indicates active IGF signaling. The signaling network may interfere with several treatment mechanisms and be involved in endocrine resistance. However, the prognostic value of pIGF-1R/IR in breast cancer is unclear.

Aim and methods

In order to elucidate the significance of activated IGF-IR/IR in breast cancer, the level of receptor phosphorylation was evaluated by immunohistochemistry on invasive breast cancer tissue microarrays from 984 primary breast cancer patients included in a population-based cohort in Sweden. Patients were followed for up to 11 years, the median follow-up for patients still at risk was five years. Phospho-IGF-1R/IR in relation to prognosis was analyzed using Cox regression, adjusted for age, invasive tumor size, axillary lymph node involvement, histological grade, estrogen receptor (ER) status, and body mass index.

Results

Phospho-IGF-1R/IR status was available for 902 patients (91.7%). Any positive cytoplasmic or membranous staining for pIGF-1R/IR was present in 749 patients (83.0%). pIGF-1R/IR+ was associated with ER+ OR (1.76; 95% CI 1.09-2.83). During follow-up, 112 patients had a first breast cancer event. Patients with pIGF-1R/IR positive tumors had lower risk for any breast cancer event compared to patients without pIGF-1R/IR (Log Rank P=0.016), adjusted HR (0.60; 95% CI 0.40-0.91). In patients with ER+ tumors (n=792, 87.9%), pIGF-1R/IR was evaluated in relation to prognosis and endocrine treatment. In patients who had not received endocrine treatment, pIGF-1R/IR was associated with significantly lower risk for any breast cancer event adjusted HR (0.37; 95% CI 0.15-0.92). In contrast, pIGF-1R/IR was not associated with prognosis in endocrine-treated patients adjusted HR (0.90; 95% CI 0.50-1.63).

Conclusions

These results indicate that IGF-1R/IR activation status may add independent prognostic information in primary breast cancer. In contrast to previous studies, pIGF-1R/IR+ was associated with better prognosis overall in this cohort. However, pIGF-1R/IR+ was not associated with prognosis in endocrine-treated patients. Taken together, these results are in line with the negative results from clinical trials with IGF-1R inhibitors in breast cancer patients, and highlight the complexity of the IGF-system.

#1793

POLE gene mutations confer good progression free survival in Asian women with FIGO grade 3 endometrioid endometrial carcinoma.

Adele Wong,1 Chik Hong Kuick,1 Wai Loong Wong,1 Jill M. Tham,2 Sorsiah Mansor,1 Loh Eva,1 Sudhanshi Jain,1 Sock Hoai Chan,3 Sze Huey Tan,3 Shao Tzu Li,3 Nadkarni N. Vikas,4 Sung Hock Chew,1 Joanne Ngeow,3 Wanjin Hong2. 1 _KK Women's and Children's Hospital, Singapore, Singapore;_ 2 _Institute of Molecular and Cell Biology, Singapore;_ 3 _National Cancer Centre Singapore, Singapore;_ 4 _Duke-NUS Graduate Medical School, Singapore_.

Introduction: Endometrial carcinoma (EC) is the most common gynaecological cancer in developed countries. Mortality rates of patients with FIGO grade 3 endometrioid ECs are almost similar to that of uterine serous carcinoma and clear cell carcinoma. In 2013, the Cancer Tumour Genome Atlast (TGCA) identified POLE gene mutations in a subset of ECs, predominantly in the endometrioid subtype with high FIGO grade. While it has been suggested that POLE mutation confers good prognosis, the data remains conflicting. Our study aims to evaluate the association between the POLE gene mutations and progression free survival in Asian women with FIGO grade 3 endometrioid EC.

Methods: Forty-five Asian patients diagnosed with FIGO Grade 3 endometrioid EC between 2009 and 2013 were included. Next generation sequencing (NGS) performed utilizing the Ion Torrent Personal Genome Machine (PGM) and custom designed AmpliSeq panel for formalin fixed embedded (FFPE) tissue was performed. Using the Ion Reporter, mutations with at least 10% allele frequency and present in the COSMIC database were deemed to be pathogenic. All other mutations, excluding silent mutations, were considered as variants of unknown significance (VUS). Survival curves for pathogenic somatic POLE mutated and wild-type tumors were estimated by Kaplan-Meier method and compared using logrank test.

Results: All detected pathogenic and VUS POLE gene mutations were missense mutations. Our results provide further evidence to corroborate the mutation effect of POLE gene with 100% recurrence free survival in FIGO Grade 3 endometrioid EC patients with pathogenic POLE gene mutations, extends to South East Asian women as well (median follow-up POLE vs wild type; 52.5 vs 45; p=0.2). Pathogenic somatic POLE gene mutations were present in 25.5% (12/45) patients with Grade 3 endometrioid EC. All were still alive at the time of censure with no reports of relapse following initial treatment. Three of these women harbored mutations outside exons 9 to 14 with one mutation listed as pathogenic in colorectal carcinomas. Among women without the pathogenic somatic POLE mutation, recurrence was seen in 22% (8/35) with 14.3% (5/35) subsequently dying of the disease.

Conclusion: POLE gene mutations confer a good prognosis for FIGO grade 3 endometrioid EC and will be important in clinical decision making. POLE wild-type patients potentially show have closer follow-up and should be considered for newer targeted chemotherapeutic agents.

#1794

Identification of novel single nucleotide polymorphisms (SNPs) associated with risk and prognosis in patients with castration-resistant prostate cancer (CRPC).

Tristan M. Sissung,1 Deeken John,2 Crystal R. Leibrand,1 Douglas K. Price,1 Sheryl Ehrlich,1 Seth M. Steinberg,1 David J. Liewehr,1 William L. Dahut,1 William D. Figg1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Inova Comprehensive Cancer and Research Institute, Falls Church, VA_.

Background: Liver metabolism plays a major role in life-long exposure to endogenous and exogenous carcinogens. We therefore explored associations between polymorphisms in genes involved in absorption, distribution, metabolism, and elimination (ADME) and the risk and prognosis of castration resistant prostate cancer (CRPC).

Methods: DNA samples from 47 patients (43 Caucasians) with CRPC were genotyped using the Drug Metabolizing Enzymes and Transporters platform v1.0, which tests 1,243 genetic variations in 169 ADME genes. First, the frequency of SNPs in genes previously reported to correlate with PCa risk (CYP17, CYP1A1, NAT2, and PPARgamma) were determined and compared to controls. Second, SNP variants in all tested genes were screened for an association with survival using a Cox regression model and subsequent Kaplan-Meier plot evaluations and comparisons between groups with the log-rank test.

Results: Overall 634 genotypes were ascertained. The rs743572 T>C in CYP17 was associated with decreased risk of CRPC (Fisher's exact test, P=0.009, odds ratio 0.165, 95% confidence limits, 0.03-0.69), and we found no evidence that any other SNPs were strongly related to risk in this study. A combination of Cox model screening and graphical evaluation using Kaplan-Meier plots revealed that 5 were worthy of further consideration for survival. Of these, there was evidence that three SNPs were associated with CRPC prognosis in Caucasians (log-rank test p-values and hazard ratios): ABCB11 rs7602171 G>A (P=0.003, adjusted P=0.006, HR 0.307, 95% CI 0.149-0.714, n=30), GSTP1 rs1799811 C>T (P=0.001, HR 0.254, 95% CI 0.094-0.690, n=38), and SLC5A6 rs1395 (P=0.004, adjusted P=0.008, HR 3.15, 95% CI 1.39-7.09, n=35). Two other polymorphisms were considered interesting trends: ABCB4 rs2302387 C>T (P=0.039), and ABCC5 rs939339 A>G (P=0.036).

Conclusion: This exploratory pilot study is the first to show that polymorphisms in transporters involved in sterol disposition, ABCB11 and ABCB4, may be related to CRPC prognosis. Other potential associations were found in genes that regulate glutathione conjugation of carcinogens (GSTP1), vitamin uptake (SLC5A6), and nucleotide efflux (ABCC5). We also found evidence that CYP17 rs743572 is related to CRPC risk.

#1795

The feasibility study of a randomized cancer screening trial in China.

Ping Hu,1 Min Dai,2 Jufang Shi,2 Jiansong Ren,2 Jiang Li,2 Xianzhen Liao,3 Lingbin Du,4 Yuqing Liu,5 Zhaoli Chen,2 Ning Wu,2 Qian Liu,2 Paul Pinsky,1 Philip Prorok,1 Richard Fagerstrom,1 Martina Taylor,1 Barnett Kramer,1 Jie He2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing, China;_ 3 _Hunan Cancer Hospital, Hunan, China;_ 4 _Zhejiang Cancer Hospital, Zhejiang, China;_ 5 _Gansu Cancer Hospital, Gansu, China_.

Lung cancer has been the leading cause of cancer death in China since the 1990s and the disease burden likely will increase. The US National Lung Screening Trial (NLST) showed a 16-20% mortality reduction in a high-risk population that had undergone Low-Dose Computed Tomography (LDCT). China would like to confirm the main result from the US NLST in the Chinese population. The aim of this study is to obtain necessary information on performance of the organizations involved in designing, conducting, monitoring and managing the study. Another goal is to test colorectal cancer screening strategies in China. This is a first randomized trial on lung and colorectal cancer screening in China. The feasibility study will aid in the development of a practical design for the Randomized Cancer Screening Trial in China.

This study has been carried out in three cites (Changsha city, Hunan province; Lanzhou city, Gansu province; and Haining city, Zhejiang province) since August, 2014. Individuals at elevated risk of lung cancer were randomized into three arms: annual low-dose helical CT exams for three years (T0, T1, T2) and baseline colonoscopy (T0); two low-dose helical CT exams (T0, T2) plus annual faecal immunochemical test (OC-faecal immunochemical tests at T0, T1, T2); and annual InSure-faecal immunochemical tests combined with Septin 9 test (T0, T1, T2). All participants will be followed for at least three years from randomization to yield data relevant to answering the study objectives.

As of March 31, 2015, a total of 2696 eligible participants were enrolled at 3 cancer hospitals and randomly assigned to three study arms (894 in arm 1, 902 in arm 2, and 900 in arm 3). The gender distribution is 53% male and 47% female. Baseline characteristics of the study population by age, gender and total pack-year exposure were balanced. The rate of adherence to LDCT screening at the first round (T0) was 89.0%. 1598 participants received LDCT in the first round. The rate of abnormality was 77.4% with LDCT and 6.5% and 6.1% with suspicious for lung cancer for arm 1 and arm 2 respectively at T0. 2150 participants received colorectal cancer screening. The positivity was 34.1% with colonoscopy screening, 8.0% with OC-FIT test, 4.5% with InsureFIT test, and 5.8% with Septin9 test.

#1796

Metformin may function as an anti-cancer agent of pancreatic cancer via targeting RET signaling pathway.

Xiang-Lin Tan,1 Wen Yue,1 Tao Wang,2 Darren Carpizo,1 Yong Lin,3 Xi Zheng,4 Chung S. Yang,4 Lanjing Zhang,5 Qing Xu,6 Robert S. DiPaola1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Department of Epidemiology & Population Health, Albert Einstein College of Medicine,, Bronx, NY; _3 _Department of Biostatistics, School of Public Health, Rutgers, The State University of New Jersey, Piscataway, NJ;_ 4 _Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ;_ 5 _Department of Pathology, University Medical Center of Princeton, Plainsboro, NJ;_ 6 _Department of Oncology, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai, China_.

Pancreatic cancer (PaCA) is one of the most lethal human cancers and is the fourth leading cause of cancer-related deaths in the United States. However, there are currently no established recommendations for prevention of PaCA using pharmacological agents. Metformin has recently gained attention as an anti-cancer drug and/or a chemoprevention agent because of its effects on inhibiting mTOR, lowering hyperinsulinemia, modulating inflammatory responses, and selectively killing cancer stem cells. However, the key underlying molecular mechanisms for the inhibitory effects of metformin on PaCA progression remain largely unknown. Using RNA sequencing followed by the confirmation of RT-PCR and Western blotting, we found that metformin significantly decreased the mRNA and protein levels of RET (REarranged during Transfection), a single-pass transmembrane receptor tyrosine kinase (RTK). RET and its ligand, glial cell-derived neurotrophic factor (GDNF), were strongly expressed in PaCA and correlated to invasion and worse survival after surgical resection. GDNF, as a chemoattractant for PaCA cells, can activate RET to induce tumor progression, migration and invasion in vitro and in vivo. We further observed that metformin significantly inhibited GDNF-induced migration and invasion of PANC-1 cells. These data indicate that targeting RET with metformin or the combination of metformin and RET inhibitors may be an attractive and novel strategy for the prevention and treatment of PaCA progression and metastasis. To elucidate the molecular mechanisms for the inhibitory effects of metformin on the growth and spread of PaCA in humans, further in vitro and in vivo studies are warranted to investigate how metformin modulates RET signaling to inhibit the progression and metastasis of PaCA. Such studies have potential clinical significance using metformin as adjuvant preventive and therapeutic options for PaCA prevention and treatment. [This work was supported by grants from the National Cancer Institute at the National Institutes of Health (K07CA190541 and P30CA072720) and the National Natural Science Foundation of China (Grant No.: 81470132)]

#1796A

Race and gender differences in lung cancer: examining the effect of smoking history and depression on hospitalization costs.

Baqar A. Husaini,1 Van A. Cain,1 Robert S. Levine2. 1 _Tennessee State University, Nashville, TN;_ 2 _Baylor College of Medicine, Houston, TX_.

Objective: Incidence rates of lung cancer (LC) are reported to have declined in the nation. However, the role of depression and tobacco history relative to hospitalization cost for LC remains unknown. Tennessee, being a tobacco producing State, provides an opportunity to examine two issues: (i) Prevalence of LC, depression, and history of tobacco use among LC patients by race and gender; (ii) Effect of depression and smoking history on hospitalization costs by sub-groups of LC patients.

Methods: We extracted data pertaining to depression, smoking history, and demographics on patients discharged with all lung cancer (adenocarcinoma, ICD-9 codes 160-165) from the 2008 Tennessee Hospital Discharge Data System (HDDS). The sample (n=6,665) had patients that were mostly white (86%) and male (57%), with an average age of 67 years. We computed age-adjusted LC rates for race and gender (per CDC methodology), and compared the costs for LC patients with depression/ smoking history with LC patients without depression/smoking history.

Results: LC rates (per 100K) were higher among blacks than whites (146.6 vs. 131.6, p<.000), and higher among male than female patients (174.9 vs. 118.0, p<.00). While nearly 40% of LC patients had a smoking history (more males than females had a history with no racial variation), 25% had depression (more females and whites were depressed). Further, the total hospitalization cost for 2008 was 50% higher for LC patients with depression plus smoking history compared to peers without depression/smoking history ($122,500 vs. 80,522, p<.000).

Conclusion: LC rates are higher among blacks and males who also have more smoking history. Depression is higher among females and whites with LC. Further, both depression and smoking history adds significantly to the hospitalization costs. These results point to: (i) costs savings might be realized by treating depression first among the LC patients, and (ii) population-based smoking cessation programs need to be vigorously implemented to reduce lung cancer and associated co-morbidities.

___________________

This presentation at the Eight AACR annual Conference, New Orleans, April 16-20, 2016, was partially supported by grants from CDC #U58CCU422782, a sub-contract #ED-07-20811-09, and a NCI/NIH grant (5U54CA163066) to Tennessee State University, B. Husaini, PI.

## ENDOCRINOLOGY:

### Molecular Endocrinology of Hormone-dependent Malignancies

#1797

Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells.

Ali D. Vaziri-Gohar, Kevin D. Houston. _New Mexico State University - Las Cruces, Las Cruces, NM_.

Breast cancer is one of the leading causes of cancer-related deaths in the world. The majority of breast cancers express estrogen receptor alpha (ERα) and tamoxifen, a selective estrogen receptor modulator, is commonly prescribed as adjuvant therapy for ERα-positive breast cancer patients. The development of chemoresistance is a common occurrence in treated patients, however, the molecular mechanisms associated with acquired tamoxifen resistance are not well defined. Previous work from our lab identified a novel mechanism of tamoxifen action that involved the accumulation of extracellular Insulin-like growth factor binding protein-1 (IGFBP-1) and modulates IGF-1-dependent signaling transduction. Tamoxifen-induced IGFBP-1 induction in this model was mediated by G protein-coupled estrogen receptor (GPER)-dependent CREB activation as shown by phosphorylation on serine 133. To determine IGFBP-1 induction is altered during the development of chemoresistance, this mechanism of action was studied in tamoxifen resistant MCF-7 (TamR) cells. Whereas CREB was constitutively phosphorylated in TamR cells, IGFBP-1 levels were not elevated when compared to parental cells and IGFBP-1 was not induced by tamoxifen treatment in TamR cells. These data suggest that another transcription factor(s) are also necessary for the induction of IGFBP-1 in breast cancer cells. FoxO1 is a known regulator of IGFBP-1 transcription and we tested whether FoxO1 contributes to tamoxifen-induced IGFBP-1 transcription. Our results indicate that FoxO1 expression levels were significantly reduced in TamR cells compared with parental cells. Furthermore, FoxO1 knockdown experiments demonstrated this transcription factor mediates tamoxifen-induced IGFBP-1 induction in MCF-7 cells. Consistent with previously published observations, IGF-1R expression was reduced in TamR cells compared to parental MCF-7 cells. To determine if loss of IGF-1R expression in involved in the loss of FoxO1 expression in TamR cells, IGF-1R was exogenously expressed and FoxO1 expression was measured. IGF-1R expression increases FoxO1 levels and partially restores tamoxifen-induced IGFBP-1 induction in TamR cells. These results suggest that FoxO1-mediated IGFBP-1 accumulation is altered during the development of tamoxifen resistance and is associated with decreased IGF-1R expression.

#1799

The effect of dovitinib on angiogenesis in human neuroendocrine tumors.

Tanja Milosavljevic, Elise Juge, Peter Casey, Michael Hall, Eugene Woltering. _LSU Health New Orleans, Department of Surgery, School of Medicine, New Orleans, LA_.

Background: Dovitinib is a potent oral inhibitor of multiple angiogenic factors, including receptor tyrosine kinases (RTKs) such as VEGFR1-3, PDGFRᵦ, and FGFR1-3. Human neuroendocrine tumors (NETs) rely on these RTKs to mediate tumor development, metastasis and neovascularization. They usually originate in the small bowel and metastasize to lymph nodes (LNs) and/or liver (LV). Although antiangiogenic approach is a valid therapeutic option for metastatic NETs, currently there are no clinical trials on Dovitinib in human midgut NETs. We hypothesized that Dovitinib will inhibit angiogenesis in both primary (1) and metastatic human NETs.

Methods: Tissue samples from 126 NET patients were assayed in our in-vitro Human Tumor Angiogenesis model. Neovessels were visually scored and evaluated based on three parameters: percent initiation (%I), angiogenic growth (AG), and overall angiogenic response (OAR). Treatment doses [165 nM, n=126; 82.5 nM, n=94], tested on both 1 and metastatic NETs (LN, LV), were selected based on unpublished physiologic angiogenesis model data. Four normal and tissue-matched LV samples were tested at both doses. Angiogenesis data was analyzed for significance using Z-test (Primer) and paired t-test (MedCalc). Concentration of angiogenic factors in supernatant was measured by Human Angiogenesis/Growth panel (Milliplex, EMD Millipore). FGFR3 gene and protein levels were determined by TaqMan assay and immunohistochemistry staining on tissue-matched NETs.

Results: Both Dovitinib doses achieved over 60% inhibition of %I in 1, LV and LN tumors. Angiogenic growth was significantly (p<0.0001) inhibited in 1 (86.78%, 82.5 nM; 84.74%, 165 nM), LV (97.09%, 82.5 nM; 89.48%, 165 nM), LN (95.27%, 82.5 nM; 89.48%, 165 nM), and all NETs (86.76%, 82.5 nM; 84.74, 165 nM). Overall angiogenic response was reduced by over 88% in all NETs (p<0.0001). Greater than 65% inhibition of OAR was observed in LV tumor (p=0.0141, 82.5 nM; p=0.0198, 165 nM), and matching normal tissue; however, normal tissue response was not statistically significant. Concentrations of VEGF pathway mediators (VEGFA, PlGF, VEGFC) were higher in LV tumor versus normal and down-regulated in response to Dovitinib. Gene and protein FGFR3 levels were increased in all three NET sites compared to normal.

Conclusions: Our preclinical results demonstrate robust efficacy of Dovitinib in inhibiting angiogenesis in human midgut NETs in vitro. Dovitinib effectively inhibits angiogenesis in both primary and metastatic sites by more than 60% at both doses by simultaneously targeting VEGF pathway and FGFR3. Comparatively, dovitinib targets mechanisms of angiogenic growth more effectively than those involved in initiation. This study provides a compelling rationale for future clinical investigation of Dovitinib antiangiogenic therapy in patients with NETs.

#1800

Inhibition of scavenger receptor class B type I suppresses androgen pathway activity and induces cytotoxic stress in C4-2 castration resistant prostate cancer cells.

Jacob A. Gordon,1 Ankur Midha,2 Mitali Pandey,3 Kishor Wasan,4 Michael E. Cox3. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Freie Universität Berlin, Berlin, Germany;_ 3 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada;_ 4 _University of Saskatchewan, Saskatoon, Saskatchewan, Canada_.

Existing therapies for castration-resistant prostate cancer (CRPC) extend life and provide clinical benefit; however, patients continue to develop therapeutic resistance. Androgen pathway activity persists in CRPC even under maximal androgen blockade conditions. A proposed mechanism for continued androgen pathway signaling is de novo intratumoral synthesis of androgen receptor agonists from the precursor cholesterol. The high density lipoprotein(HDL)-cholesterol receptor, scavenger receptor class B type I (SR-BI), is upregulated in CRPC models in vitro and in vivo. Here, we test the hypothesis that SR-BI is a source of cholesterol for de novo steroidogenesis in CRPC cells using the castration-resistant LNCaP-derived cell line, C4-2. Cells were transfected with either non-targeted (NC) or stealth RNAi duplexes (Thermo Fisher) targeting SR-BI to silence protein expression (SR-B1 KD). Prostate specific antigen (PSA) levels in media from SR-B1 KD samples were reduced to 39% of that seen in control cell media (20.9 ± 1.4 ng/mL/µg protein SRBI-KD vs. 34.5 ± 2.4 ng/mL/µg protein NC; n=4, p < 0.05). Intracellular testosterone concentrations measured by LC-MS showed a 2-fold reduction in SRBI-KD samples (0.20 ng/mL/mg) compared to NC (0.41 ng/mL/mg; n=1). Additionally, SR-BI KD cells exhibited reduced cell viability as measured by MTS assay and induction of G1/S cell cycle arrest as assessed by propidium iodide staining cell cycle analysis by flow cytometry (70.9 ± 7.9% G0-G1 phase fraction SRBI-KD vs. 58.3 ± 4.1% NC; n=4, p < 0.05). SR-BI KD cells exhibited evidence of increased cell stress relative to NC cells. SR-BI-KD cells showed elevated induction of autophagy as assessed by measuring changes in LC3-I:LC3-II ratio by immunoblotting and formation of autophagosomes by immunofluorescence, alongside induction of senescence as assessed by measuring senescence associated beta-galactosidase (SA-β-gal) activity using flow cytometry analysis and a fluorogenic substrate. Further, treating C4-2 cells with an established SR-B1 inhibitor, BLT-1, lead to decrease PSA secretion, and co-treatment with the common cholesterol synthesis inhibitor, simvastatin, synergistically decreased PSA secretion from C4-2 cells. These results suggest that under androgen-deprived conditions, SR-BI targeting can decrease androgen pathway signalling and induce cellular stress, leading to G1/S arrest and decreased proliferation in the absence of an apoptotic induction. Lastly, combined inhibition of cholesterol uptake (BLT-1) and synthesis (Simvastatin) lead to a synergistic decrease in PSA secretion indicating the possibility that a multi-faceted cholesterol blockade approach to CRPC treatment may improve response to maximal hormone blockade by disrupting intratumoral steroid production.

#1801

FOXA1, a novel regulator of neuroendocrine differentiation.

Jung Kim,1 Hongjian Jin,1 Jonathan Zhao,1 Vamsi Parimi,2 Ximing Yang,1 Jindan Yu1. 1 _Northwestern University, Chicago, IL;_ 2 _Loyola University, Maywood, IL_.

Neuroendocrine prostate cancer (NEPC) is a subtype of prostate cancer that is highly aggressive and exhibits a neuroendocrine phenotype, being distinct from prostate adenocarcinoma. Cases of NEPC are predicted to increase rapidly following broader use of new-generation hormonal therapies including abiraterone and enzalutamide. Although androgen deprivation and cytokine induction have been previously suggested to induce neuroendocrine differentiation of prostate cancer, little is known regarding the underlying molecular mechanisms. FOXA1 is a forkhead box family transcription factor that has been shown to act as a pioneer factor for androgen receptor (AR), thereby defining the prostatic transcriptional program. We have recently shown that FOXA1 plays an important role in suppressing epithelial-to-mesenchymal transdifferentiation, but is often downregulated in castration-resistant prostate cancer (CRPC). Along this line, in the present study, we showed that FOXA1 loss, achieved by shRNA-mediated knockdown, led to cell morphology changes typical of neuroendocrine differentiation, accompanied by a strong increase of NEPC marker enolase2 (ENO2), while ectopic FOXA1 overexpression reverses these alterations. This was further linked to ERK phosphorylation, which has been previously reported to be up-regulated following neuroendocrine differentiation. Moreover, to understand the mechanism underlying FOXA1 regulation of neuroendocrine differentiation, we performed bioinformatics analysis and nominated interleukin 8 (IL8) as a direct target of FOXA1 and its up-regulation is critical in mediating neuroendocrine differentiation of FOXA1-depleted cells. Finally, we validated a negative correlation between FOXA1 and neuroendocrine differentiation in primary prostate cancer specimens through analysis of publically available RNA-seq data as well as direct IHC staining of various NEPC markers. In summary, we report a new role of FOXA1 as a critical inhibitor of neuroendocrine differentiation and the importance of balanced FOXA1 expressing in the maintenance of the epithelial phenotype of prostate cancer.

#1802

ER-positive breast cancer prevention with the use of hormone replacement therapy.

Anna G. Dembo, Geoffrey L. Greene. _University of Chicago, Chicago, IL_.

Menopause occurs in women between the ages of 45 and 55 and often results in undesirable vasomotor symptoms. Hormone replacement therapy (HRT) alleviates these symptoms, including hot flashes, difficulty sleeping, fatigue, and vaginal atrophy. HRT also prevents osteoporosis. PremPro, a currently available HRT formulation that combines conjugated equine estrogens (CE) with medroxyprogesterone acetate, increases the risk of breast cancer. Due to the perceived risk based largely on the results of the Women's Health Initiative (WHI) trial, the number of women taking HRT has decreased dramatically. Duavee, a new form of HRT that combines CE and bazedoxifene (BZA), a selective estrogen receptor modulator (SERM) and degrader (SERD), has recently been approved by the FDA for treatment of moderate to severe hot flashes and to reduce the risk of osteoporosis. Importantly, this CE/BZA mixture not only relieves symptoms associated with menopause, but it also does not stimulate the breast or uterus. Although several preclinical studies suggest that CE/BZA might be protective in the breast, the mechanism of action of this new combination therapy is not known. Our goal, therefore, is to elucidate the underlying molecular mechanisms by which CE/BZA differentially affects estrogen receptor alpha (ERα) action in the mammary gland, using transcriptome and whole genome occupancy analysis in breast cancer cell lines. We are also studying the effects of CE/BZA on early mammary cancer progression in the polyoma middle T antigen (PyMT) transgenic mouse model, which has been shown to be sensitive to estrogens. In addition, we are studying the effects in an ERα-positive patient-derived xenograft mouse model. We have determined that CE modulates gene expression in MCF7 cells similar to 17β-estradiol (E2), and that BZA is able to inhibit these effects. We have also observed that CE increases ERα occupancy versus E2 at response elements associated with some estrogen target genes, whereas CE/BZA decreases this occupancy. In the PyMT mouse model, we have determined that CE/BZA significantly delays the onset of mammary tumors in ovariectomized mice and prolongs their survival when compared to E2 and CE treatment alone. An improved understanding of the molecular mechanisms of CE/BZA action in hormone sensitive breast cancer cell and animal models should have important implications for women considering HRT.

#1803

Breast cancer cell lines with acquired resistance to selective ERα degraders (SERDs) are dependent on PI3K signaling.

Christy Ong, Anneleen Daemen, Jason Oeh, Ellen Ingalla, Alfonzo Arrazate, Michelle Nanini, Deepak Sampath, Lori Friedman, Thomas O'Brien. _Genentech, South San Francisco, CA_.

Estrogen positive (ER+) breast cancer accounts for the majority of all breast cancers and is primarily treated with endocrine therapy. However, even though current endocrine treatments are effective, a subset of patients inevitably become resistant to these treatments. The novel oral SERD, GDC-0810/ARN-810, is currently in Phase II clinical trials in ER+ breast cancer patients. GDC-0810 induces ERα degradation in ER+ MCF7 and T47D cells and inhibits cell proliferation. Moreover, it is also efficacious in ER+ tumor models in vivo, including in MCF7-ESR1 Y537S xenografts and in WHIM20 ESR1 mutant Y537S PDX tumors. Given that little is known about resistance mechanisms to SERDs, our aim was to understand what mechanisms may underlie resistance after continuous exposure.

We generated cell lines resistant to GDC-0810 and show that all of these lines were also cross-resistant to fulvestrant and tamoxifen. We determined that cells with acquired-resistance were more sensitive than parental cells to inhibitors of the PI3K/AKT signaling pathway. Additionally, in some resistant clones, ERα protein expression was lost, but this loss was transient and could be regained after long-term growth in the absence of SERDs. A range of approaches are being undertaken to explore the underlying mechanisms driving acquired-resistance to SERDs. These preclinical studies may inform future clinical combinations.

#1804

**Expression analysis of adamantinomatous craniopharyngioma suggests two subtypes associated with** CTNNB1 **mutational frequency and highlights potential therapeutic targets.**

John R. Apps,1 Nital Jani,1 Gabriela Carreno,1 Jose M. Gonzalez-Meljem,1 Kyoko Tossell,2 Thomas J. Stone,1 Mark A. Ungless,2 Hywel J. Williams,1 Thomas S. Jacques,1 Juan-Pedro Martinez-Barbera1. 1 _University College London, London, United Kingdom;_ 2 _MRC Clinical Sciences Centre, Imperial College London, London, United Kingdom_.

We conducted RNA-sequencing analysis of Adamantinomatous Craniopharnygioma (ACP), including Laser capture micro-dissection (LCM) of individual cellular compartments, to characterise both disease heterogeneity and targetable deregulated molecular pathways.

RNA sequencing was performed on 18 samples of ACP (17 pediatric, 1 adult) and six control samples (three fetal pituitaries, three non-functioning pituitary adenomas). LCM and RNA sequencing were performed on a subset of cases selecting nuclear β catenin accumulating clusters, and paired non-cluster tumour tissue (palisading epithelium) . Mouse ACP models indicate that these clusters initiate tumorigenesis in a non-cell autonomous manner.

Consensus clustering analysis revealed two subgroups of human ACP, one associated with a high CTNNB1 mutational frequency (>30%), a relatively high tumour content and fibrous reactive tissue (Group H) and another with a lower mutation frequency (<30%) and glial reactive tissue (Group L).

The meta-gene signature of Group H defined by non-negative matrix factorization included secreted factors, such as Sonic Hedgehog (SHH) and Fibroblast Growth Factors (FGFs) 3, 4 and 19, Keratins and Matrix Metalloproteinases and was enriched for genes related to odontogenesis. Weighted gene co-expression network analysis independently showed correlation of expression of such genes with mutational frequency. Glial markers and neural differentiation genes were up-regulated in Group L tumors. Differential expression showed up-regulation in ACPs of potential therapeutic targets including SHH (32x), EGFR (8.9x) and TNF (10x).

Similarly, differential expression analysis of LCM dissected nuclear β catenin accumulating clusters identified up-regulation of SHH, FGFs and other secreted factors including as WNTs and BMPs.

Analysis of the SHH pathway found that SHH is over expressed in clusters (9x) whilst its downstream targets Ptch1 (1.7x), Gli1 (2.1x), Gli3 (2.9x) are over expressed in palisading epithelium, indicating the presence of paracrine signaling. These findings were independently confirmed by in situ hybridisation in human samples and the mouse model.

Clustering of murine expression data with those genes differentially expressed in the human clusters revealed murine and human clusters group together, suggesting they are similar structures functionally and biologically.

Together, these results suggest that a subset of cells, carrying CTNNB1 mutations and showing nuclear β-catenin accumulation, express secreted pro-oncogenic factors and the response may be observed in neighboring cell types. Targeting such signalling offers an attractive therapeutic opportunity, for which the results suggest the mouse model is well

placed for testing. The clinical significance of the two putative ACP subgroups remains to be determined.

#1805

CYP3A4 regulation of androgen receptor signaling in ER+ breast cancer.

Priyatham Gorjala, Oscar Goodman Jr, Ranjana Mitra. _Roseman University of Health Sciences, Las Vegas, NV_.

Purpose: Eighty percent of breast cancers that are estrogen receptor-positive (ER+) express AR. According to American Cancer Society, every two out of three women are ER+ and initially respond to estrogen-receptor targeted therapies but later almost 50% of breast cancers, despite the presence of ER, fail to respond. Acquired resistance to these therapies is common and portends a poor prognosis, frequently requiring the use of cytotoxic therapies. Our preliminary results indicate that endocrine-resistant tumors express higher levels of AR and may depend on AR for their growth. Hence AR targeted therapies may be appropriate for these resistant tumors. As we have recently demonstrated that CYP3A regulates androgen receptor signaling, we wish to ascertain the role of CYP3A in regulating AR in endocrine-resistant breast cancer.

Methods: The ER+ cell lines MCF7 and T47D were used to study the role of AR in breast cancer. To study endocrine resistance in breast cancer, tamoxifen resistant MCF7 and T47D lines were generated with recurrent treatment of IC90 dosage of tamoxifen over 6 months' time period. Cell fractionation, western blotting and q-RTPCR techniques were used to study the role of AR. siRNA inhibition of CYP3A4, the isoform expressed in breast, was used to study its role in regulating AR in breast cancer.

Results: Our results indicate that both MCF7 and T47D express active AR, inducible by DHT and R1881. Similar to prostate cancer we also observe that AR in these lines is regulated by CYP3A4. Cell fractionation studies indicate that inhibition of CYP3A4 by siRNA results in reduced AR migration to the nucleus inhibiting its role as a transcription factor. Our results also indicate that tamoxifen resistant cell lines express increased levels of AR. Tamoxifen resistant cell lines respond to bicalutamide/enzalutamide with a lower IC50 value than the native cell lines, reflecting their AR dependence. The resistant cell lines also show increased AR nuclear migration with R1881 induction as compared to native breast cell lines.

Conclusions: Endocrine-resistant breast cancer is sensitive to antiandrogen therapy. CYP3A4 may play an important role in endocrine-resistant breast cancer by negatively regulating AR activity.

#1806

TRIM24 is an oncogenic transcriptional activator in prostate cancer.

Anna C. Groner,1 Laura Cato,1 Jonas de Tribolet-Hardy,1 Tiziano Bernasocchi,2 Qing Zhong,3 Christian Fankhauser,3 Christine Fritz,3 Cédric Poyet,4 Ulrich Wagner,3 Levi A. Garraway,1 Peter J. Wild,3 Jean-Philippe Theurillat,2 Myles Brown1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Institute of Oncology Research, Bellinzona, Switzerland;_ 3 _Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland;_ 4 _Department of Urology, University Hospital Zurich, Zurich, Switzerland_.

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in SPOP stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly up-regulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease-recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP-mutant and CRPC patients.

#1807

Progestin enrichment of the cancer stem cell-like pool provides novel therapeutic targets for hormone-dependent human breast cancer.

Sandy Goyette, Benford Mafuvadze, Matthew T. Cook, Yayun Liang, Salman M. Hyder. _University of Missouri-Columbia, Columbia, MO_.

Clinical trials in post-menopausal women show that combined hormone replacement therapy (HRT), containing both estrogen (E) and a progestin (P) such as medroxyprogesterone acetate (MPA), is associated with an increased risk of breast cancer compared with E alone (JNCI, 2000, 92:328). We previously showed that both natural and synthetic P accelerate tumor development in vivo, a process that is blocked by anti-progestins (Can Res 2007; 67:9929). Furthermore, P also increases lymph node metastasis (Menopause 2010; 17:1040), leading us to hypothesize that P-induced tumor growth and metastasis may be mediated by an enrichment of the pool of cancer stem cell (CSC)-like cells. This increases the proliferative and metastatic potential of already existing tumors. CSCs are a small and highly tumorigenic cell population present in most tumors, which have self-renewal properties. In tumors of the breast, the CSC subpopulation has a phenotypic signature that is CD24 -/low, CD44 high and aldehyde dehydrogenase-positive (ALDH+) (Cell Stem Cell, 2007; 1:555). Using flow cytometry to measure CD44 density under a variety of experimental conditions we sought to determine whether P influences expression of CSC markers in P-responsive human breast tumors that grow in vivo. Following exposure of T47-D cells for 24 h with physiological levels (10 nM) of natural and synthetic P, CD44 protein expression was increased considerably. Compared with controls, induction of CD44 increased 10-fold in response to MPA (Control 5.9 %, MPA 60.7 %). Alternative steroid hormones failed to induce CD44 in tumor cells. This phenotypic shift was blocked by the anti-progestin RU-486, indicating that the process is mediated via progesterone receptors (PR). Cells treated with P formed mammospheres more easily than their non-treated counterparts, and a subset of the cell population with induced CD44 high also demonstrated high ALDH enzyme activity. RT-PCR analysis showed that MPA specifically induced CD44v3 and CD44v6 transcripts. Importantly, a variety of synthetic P similarly induced CD44 protein expression. Based on our observations we contend that exposure of breast cancer cells to synthetic P, such as MPA, may lead to an enrichment of the CSC-like pool, supporting the development of P-accelerated tumors in vivo. These findings also suggest that a novel means by which to combat P-dependent tumor growth might be through blockade of CD44 functions in the pool of stem cell-like cells. This could be achieved by immunotherapeutic means, using tissue selective antiprogestins, or through a combination approach involving both immunotherapy against CD44 and small molecule targeting of PR.

Supported in part by COR award from the College of Veterinary Medicine, and by generous gifts from donors of Ellis Fischel Cancer Center, University of Missouri.

#1808

Nuclear receptor, ESRRG functions as tumor suppressor by preventing Helicobacter pylori infection in gastric cancer.

Myoung-Hee Kang,1 Yong-Soo Lee,2 Jin-Hak Jung,2 Young Soo Park,2 Jin-Yong Jeong,2 Mi-Na Kweon,2 Chan-Ki Paik,2 Seung-Jae Myung,2 Yun-Yong Park2. 1 _Ulsan College of Medicine / ASAN Medical Center, Seoul, Republic of Korea;_ 2 _ASAN Institute for Life Sciences, ASAN Medical Center, Seoul, Republic of Korea_.

Gastric cancer (GC) is the most common cancer type in Asia. Surgical resection and chemotherapy is standard treatment of GC. Molecular therapeutic targets and mechanism contributing to develop the gastric cancer is poorly understood. To discover the molecular target in gastric cancer, we analyzed gene expression data from gastric cancer patients. Data analysis reveals that nuclear receptor ESRRG is top candidate gene conferring the tumor suppressive property in gastric cancer. ESRRG expression is down-modulated by Helicobacter pylori (H. pylori) infection, which is one of causing factor triggering gastric cancer. In addition, ectopically over-expressed ESRRG protects the cell from H. pylori infection and suppressed cancer cell growth in vitro and in vivo mouse model. Gene expression profile reveals that TFF1, which is well known as tumor suppressor in gastric cancer is down-stream target of ESRRG and ESRRG well reflects clinical outcome in gastric cancer. Our study provides new molecular insights how ESRRG regulates gastric cancer cell growth induced by H. pylori and suggests possibility that ESSRRG can be therapeutic target for the treatment of gastric cancer patients.

#1809

Endothelin causes transactivation of the EGFR and Her-2/Neu in non-small cell lung cancer cells.

Terry W. Moody,1 Irene Ramos Alvarez,2 Samuel Mantey,2 Robert T. Jensen2. 1 _NCI-CCR, Bethesda, MD;_ 2 _NIDDK, Bethesda, MD_.

Endothelin-1 (ET-1) is a 23 amino acid peptide which is present in many lung cancer cell lines (Ahmed et al., Am. J. Respir. Cell Mol. Biol. 2000; 22: 422). Expression of ET-1 or the ETA receptor is associated with poor prognosis of non-small cell lung cancer (NSCLC) patients (Boldrini et al., Eur. J. Cancer 2005; 41: 2828). ET-1 stimulates whereas the ETA antagonist ZD4054 inhibits the proliferation of cancer cells (Bagnato et al., Br. J. Pharmacol. 2011: 163; 220). Here the effects of ET-1 on the transactivation of the EGFR and Her-2/Neu were investigated in NSCLC cells. Twelve of 13 NSCLC cell lines tested had both ETA and ETB mRNA. Addition of ET-1 to adenocarcinoma cell line NCI-H838, which had both ETA and ETB mRNA, elevated cytosolic Ca2+, increased tyrosine phosphorylation of the ERK, EGFR and Her-2/Neu and increased colony number. The ability of ET-1 to increase cytosolic Ca2+, tyrosine phosphorylation of ERK, EGFR and Her-2/Neu and colony number was inhibited by ZD4054 or BQ123 (ETA antagonists). The transactivation of EGFR and Her-2/Neu by ET-1 was inhibited by lapatinib (EGFR and Her-2/Neu TKI), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or N-acetylcysteine (anti-oxidant). ET-1 stimulated whereas lapatinib and ZD4054 inhibited the clonal growth of NSCLC cells. The results indicate that ET-1 may regulate the proliferation of NSCLC cells in an EGFR and Her-2/Neu-dependent manner.

#1810

Heterogeneous activation of PP-1 and Akt determines AR-v7 protein expression in prostate cancer cells under AR inhibition.

Yinan Li, Ning Xie, Martin E. Gleave, Paul Rennie, Xuesen Dong. _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Background: Blocking the androgen receptor (AR) signaling is the first- and second-line treatment for metastatic prostate cancers. Although the newly developed AR pathway inhibitor (ARPI) improves patient survival, tumors invariably recurs and are most often driven by reactivation of the AR signaling. Enhanced expression of the AR splice variant, AR-v7, had been demonstrated to be one of the key mechanisms that confer prostate tumors with therapy resistance. However, molecular mechanisms by which control AR-v7 protein expression remain unclear.

Experimental Design: Multiple prostate cancer lines were challenged with androgen depletion and/or Enzalutamide treatment. Protein phosphatase-1 (PP-1) and Akt kinase activation were measured. AR and AR-v7 serine phosphorylation, ubiquitination, protein degradation as well as subcellular localization were determined.

Results: Enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the genetic background of prostate cancer cell lines. PP-1 activation prevents AR-v7 from serine213 phosphorylation, Mdm2-mediated ubiquitination modification, cytoplasmic localization and proteasomal protein degradation. By contrast, Akt activation functions in the opposite to PP-1 and stimulates AR-v7 degradation. Therefore, the balance of PP-1 and Akt activations under ARPI treatment determines AR-v7 protein expression in prostate cancer cells.

Conclusion: Our studies highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. It also provides an explanation on heterogeneous AR-v7 protein expression in prostate tumor biopsies.

#1811

ERβ sensitizes NSCLC cells to chemotherapeutic agents by regulating DNA damage response.

Igor Bado,1 Fotis Nikolos,2 Jan-Åke Gustafsson,1 Christoforos Thomas1. 1 _University of Houston, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

The expression of wild-type estrogen receptor β (ERβ1) correlates with increased survival in patients with Non-Small Cell Lung Cancer (NSCLC). However, the molecular mechanism that accounts for this association is unknown. ERβ1 was previously shown to sensitize breast cancer cells to chemotherapeutic agents. The role of the receptor in regulating sensitivity of NSCLC cells to chemotherapy, a common treatment option for advanced disease, has not been studied. Here we show that upregulation of ERβ1 decreases the survival of NSCLC cells in response to treatment with doxorubicin. This effect was observed in p53-defective but not wild-type p53-expressing cells. ERβ1 enhanced G2/M cell cycle arrest in NSCLC cells by activating the checkpoint kinase 1 (Chk1) and altering downstream signaling. The expression of the p63 target gene cyclin G2 that regulates the G2/M checkpoint was induced by ERβ1 proposing an ERβ1-p63 transcriptional cooperation. Our findings suggest the involvement of ERβ1 in the regulation of DNA damage response in NSCLC cells and support the potential predictive value of the receptor in clinical management of the disease.

#1812

Targeting nuclear transport pathways to overcome endocrine resistance and recurrence.

Eylem Kulkoyluoglu,1 Kinga Wrobel,1 Yiru Chen Zhao,1 Karen L. Chen,1 Kadriye Hieronymi,1 Jamie Holloway,2 Yosef Landesman,3 Tania Ray,4 Partha S. Ray,1 Alexander E. Lipka,1 Rebecca L. Smith,1 Zeynep Madak Erdogan1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _Georgetown University, Arlington, VA;_ 3 _Karyopharm Therapeutics, Newton, MA;_ 4 _Onconostics Technologies, Urbana, IL_.

Currently, around 75% of patients with breast tumors test positive for estrogen receptor alpha (ERa) and are treated with endocrine therapies, such as tamoxifen. One-third of the breast tumors eventually become refractory, reducing the survival rate for affected patients. A combination of alternative endocrine therapies and kinase inhibitors is currently used in such patients. However, after an initial period of therapy response, these tumors relapse in a more aggressive form. Further, the alternative therapies are not optimal in terms of pharmacological properties, are poorly tolerated, and have side-effects that severely decrease quality of life of the patient. Thus, there is a critical need for novel, targetable, mechanism-based therapeutic strategies that 1) re-sensitize ERa (+) tumors to endocrine therapies, and 2) include diagnostic methods to select patients likely to benefit from this approach.

Our objective in this study is to validate a group of nuclear transport genes as biomarkers for endocrine resistance, and to evaluate their inhibition as a novel means to enhance the effectiveness of endocrine therapies. Our central hypothesis is that high expression of these genes in ERa (+) tumors serve as a viable biomarker for risk of endocrine therapy failure. We focused on XPO1, the main nuclear export protein, which exports ERK5 from the nucleus to the cytoplasm and we used selinexor (KPT-330), the inhibitor of XPO1, which is already used in clinical trials for solid and hematological cancers (clinicalTrials.gov). Our experiments show that estradiol induces nuclear localization of ERK5, which otherwise would contribute to increased invasiveness and metastatic potential in the cytoplasm. Selinexor increases ERK5 nuclear localization in tamoxifen resistant breast cancer cell lines. Our hypothesis is that sequestering ERK5 in the cell nucleus and blocking its recycle into the nucleus by selinexor is directly associated with the improved transcriptional response to endocrine therapies. The nuclear export pathways have not previously been implicated in the development of endocrine resistance, and given the need for better strategies for selecting patients to receive endocrine reagents and improving therapy response of relapsed ERa(+) tumors, our findings show high and significant promise for uncovering the role of these pathways and demonstrating their use in reducing cancer recurrences.

#1813

Insulin-like growth factor receptor and sphingosine kinase co-expression has prognostic and therapeutic potential in breast cancer.

Aleksandra M. Ochnik, Robert C. Baxter. _Kolling Institute of Medical Research/Univesity of Sydney, St Leonards, Australia_.

The search for reliable prognostic markers of breast cancer disease outcome is ongoing. The insulin-like growth factor receptor (IGF1R) and sphingosine kinase (SphK1) signaling pathways are known to contribute to breast oncogenesis and are therapeutic targets [1, 2]. Since IGF-I signaling activates SphK1 [3] we hypothesized that high IGF1R and SphK1 co-expression may be clinically relevant, and potential dual therapeutic targets in breast cancer. Kaplan-Meier analysis was performed to determine the prognostic significance of IGF1R and SphK1 high co-expression on disease free survival (DFS) and overall survival (OS) probability in breast cancer (http://glados.ucd.ie/BreastMark/). Cell viability studies and clonogenic assays were undertaken using the luminal, estrogen receptor (ER)-positive MCF7 and T47D and the basal-like, ER-negative HCC-1806 and HCC70 breast cancer cells to test the effectiveness of the dual IGF1R and insulin receptor inhibitor (OSI-906; 0.1 - 10 µM) and the SphK1 inhibitor (SKI-II; 1 - 20 µM) as single and combined agents. Statistical analysis was completed using a one-way ANOVA (p<0.05), repeated measures (p<0.05) and calculation of drug synergism by a combination index (CI) <1 (CommSyn). Immunoblot analysis was used to show effective drug target inhibition of IGF1R and SphK1. IGF1R and SphK1 high co-expression was associated with decreased DFS (HR; 2.184 (1.007 - 4.735, p=0.042; total 270 patients) and OS (HR; 2.433 (1.136 - 5.214, p=0.018; total 170 patients) in ER-positive, luminal A, lymph-node positive breast cancers compared to no significance using single-gene analysis. A reduction in OS in basal, ER-negative, lymph node negative breast cancers was associated with high IGF1R and SphK1 co-expression (HR; 5.687 (1.534 - 21.084, p=0.003; total 55 patients), compared to high IGF1R expression alone (HR; 4.103 (1.34 - 12.57, p=0.007; total 55 patients). In all four cell-lines 4 µM SKI-II acted synergistically with OSI over the range 0.1 - 6.4 µM (CI<1), and significantly increased sensitivity towards OSI (p<0.05). p-IGF1R and SphK1 protein levels were both reduced by SKI-II and OSI-906 in the HCC-1806 and HCC70 cell-lines, revealing a reciprocal regulation of signaling and effective drug treatment. Based on the limited clinical success of the IGF1R mono-therapies better predictive markers of IGF1R activity are now required for the development of more effective IGF1R-directed combination therapies. Collectively, these studies have identified: i) IGF1R and SphK1 expression are potentially co-dependent and have prognostic significance and ii) a novel IGF1R targeted combination therapy that may be of clinical benefit to specific molecular subtypes of breast cancer.

1. Martin, J.L., et al., Mol Cancer Ther, 2014. 13(2): p. 316-28.

2. Beckwith, H. and D. Yee., Endocr Pract, 2014. 20(11): p. 1214-21.

3. Granata, R., et al., J Thromb Haemost, 2007. 5(4): p. 835-45.

#1814

RAD1901, an orally available SERD, as an effective combination partner in ER+ breast cancer.

Teeru Bihani, Jeffrey L. Brown, Dinesh M. Purandare, Gary Hattersley, Fiona Garner. _Radius Health, Waltham, MA_.

Breast cancer is subdivided into categories based on a patient's estrogen receptor (ER), progesterone receptor (PR) or Her2 expression status. Estrogen receptor positive (ER+) breast cancer makes up approximately 70% of all breast cancers diagnosed and given the dependence on ER signaling in this disease segment, most treatment modalities focus on inhibiting some aspect of this pathway. Indeed, preventing estrogen synthesis (e.g. with aromatase inhibitors) and modulating ER pathway activity (e.g. with tamoxifen) continue to be mainstays in the standard of care for ER+ breast cancer patients. While patients typically respond well to these agents, a majority of patients will relapse, emphasizing the need to understand the specific mechanisms that can contribute to clinical resistance. One such mechanism is the activation of compensatory or concurrent signaling pathways that can stimulate growth (eg. Her2, CDK4/6) and/or confer survival (eg. PI3K, AKT, mTOR). The recent approval of palbociclib (CDK4/6i) or everolimus (mTORi) in combination with an aromatase inhibitor for the treatment of advanced ER+ disease demonstrates the effectiveness of combining anti-hormonals with targeted agents. While these combinations have superior efficacy compared to the use of aromatase inhibitors alone, alterations in ER itself, such as amplification and mutations, have been described as a second mechanism that can drive resistance to aromatase inhibitors in patients. These findings highlight the need for a selective estrogen receptor downregulator (SERD) that can degrade both wild-type and aberrant forms of ER in order to more effectively treat this patient population. We have recently demonstrated that treatment with RAD1901, an orally bioavailable SERD, results in consistent and robust tumor growth inhibition of patient derived xenograft (PDx) models, regardless of ER status. We hypothesized that combining RAD1901 with agents that inhibit compensatory signaling pathways would mitigate both mechanisms of resistance in ER+ breast cancer patients, leading to greater efficacy. We performed an efficacy screen with RAD1901 in patient derived xenograft (PDx) models with varied genetic backgrounds, allowing us to mimic clinical phenotypes and to better represent the heterogeneity within the ER+ breast cancer patient population. These models harbored a wide range of ER expression levels, in addition to other genetic alterations, which accurately represented the patients' diverse treatment history profiles. Based on results from the screen, models were selected for additional studies to determine which targeted agent, if any, can be combined with RAD1901 to achieve maximal efficacy. By doing this, we were able to correlate response with genetic background in clinically relevant models, potentially allowing us to predict treatment strategies that have a higher likelihood of success for specific patients.

#1815

ATM inhibition potentiates death of androgen receptor-inactivated prostate cancer cells with telomere dysfunction.

Sahn-Ho Kim, Vidyavathi Reddy, Min Wu, Evelyn R. Barrack, G. Prem-Veer Reddy. _Henry Ford Health System, Detroit, MI_.

Prostate cancer is the most commonly diagnosed non-skin cancer, and second leading cause of cancer deaths, in American men. The androgen receptor (AR) plays a critical role in the survival of prostate cancer (PCa) cells. Therefore, treatments that inactivate AR, either by decreasing the androgen level or by inhibiting AR directly, are used to treat advanced PCa. However, these treatments are not curative, and disease progression generates castration-resistant prostate cancer (CRPC). Our studies point to a novel role of AR in telomere stability that, in our opinion, can be exploited to enhance the effectiveness of AR-targeted therapies for CRPC.

Telomere stability is essential for cell proliferation and survival. We previously reported that AR plays a role in maintaining telomere stability in prostate cancer cells. Since telomere dysfunction activates the DNA damage response (DDR) signaling pathway which can activate cell cycle checkpoints, repair damaged DNA, and promote cell survival, we tested a hypothesis that AR-inactivated telomere dysfunction activates a DDR signaling pathway protein, called ATM (ataxia telangiectasia mutated) kinase, and ATM inhibitor potentiates cell death by averting telomeric DNA repair in AR-inactivated PCa cells.

ATM pathway was assessed by the phosphorylation of ATM and its downstream target Chk2 through Western blotting, inhibition of telomere DNA repair was evaluated by counting RPA (DNA replication protein A) foci at damaged telomeres, and the cell viability was determined by PARP cleavage and colony formation assays. We observed that telomere dysfunction by AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation of ATM and Chk2 and the presence of phosphorylated ATM at telomeres, indicating the activation of DNA damage response signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation and induced PARP cleavage, suggesting that ATM inhibitors induce apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomere. This was further corroborated by a conspicuous localization of RPA at damaged telomeres; presence of RPA foci is indicative of unrepaired stretch of single-stranded DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone.

These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration- resistant prostate cancer.

#1816

Alpha lipoic acid inhibits proliferation and epithelial mesenchymal transition of thyroid cancer cells.

Hyemi Kwon,1 Min Ji Jeon,1 Won Gu Kim,1 Seonhee Lim,2 Soyoung Sim,2 Tae Yong Kim,1 Young Kee Shong,1 Won Bae Kim1. 1 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Asan Institute of Life Sciences, Seoul, Republic of Korea_.

The short-chain fatty acid, α-lipoic acid (ALA) is a powerful antioxidant used for treatment of diabetic neuropathy. Recent studies suggested the possibility of ALA as a potential anti-cancer agent, because it could activate adenosine monophosphate activated protein kinase (AMPK) and inhibit transforming growth factor-β (TGFβ) pathway. We evaluate the effects of ALA on thyroid cancer cell proliferation, migration, and invasion. We performed in vitro cell proliferation analysis with BCPAP, HTH-83, CAL-62, and FTC-133 cells. ALA suppressed cell proliferation through activation of AMPK and subsequent down-regulation of mammalian target of rapamycin (mTOR)-S6 signaling pathway. Low-dose ALA, which had minimal effects on cell proliferation, also decreased cell migration and invasion of BCPAP, CAL-62, and HTH-83 cells. ALA inhibited epithelial mesenchymal transition (EMT) evidently by increase of E-cadherin and decreases of activated β-catenin, vimentin, snail, and twist in these cells. ALA suppressed TGFβ production and inhibited induction of p-Smad2, and twist by TGFβ1 or TGFβ2. These findings indicate that ALA reduces thyroid cancer cell migration and invasion through suppression of TGFβ production and inhibition of TGFβ signaling pathways. ALA also significantly suppressed tumor growth in mouse xenograft model using BCPAP and FTC-133 cells. This is the first study to show anti-cancer effect of ALA on thyroid cancer cells. ALA could be a potential therapeutic agent for advanced thyroid cancer, as an adjuvant therapy with other systemic therapeutic agents.

#1817

A novel non-secosteroidal VDR agonist, VDRM2, inhibits prostate tumor growth.

Justin Roberts,1 James MacKrell,2 D B. Piyarathna,1 Gary Krishnan,2 Cristian Coarfa,1 David Rowley,1 Nancy L. Weigel1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Lilly Research Laboratories, Indianapolis, IN_.

The role of vitamin D signaling in prostate cancer is controversial. Some studies find an inverse correlation between levels of vitamin D metabolites and PCa risk, but others do not. The active metabolite 1α,25-dihydroxyvitamin D3 (1,25D), a ligand for the vitamin D receptor (VDR), inhibits the growth of PCa cells in vitro. 1,25D or less calcemic analogs also inhibit growth in some pre-clinical models, but clinical trials have been disappointing. A majority of prostate cancers contain a genomic rearrangement that links the androgen and 1,25D regulated TMPRSS2 promoter to the coding region of an ETS transcription factor, typically ERG, termed T/E. VCaP cells are the only commonly used cell line that contains the T/E fusion. We have shown previously that VDR agonists inhibit VCaP cell growth in vitro despite inducing T/E. However, EB1089, a 1,25D analog, which inhibited growth of LNCaP xenografts, had no effect on VCaP xenografts. VDR and ERG cooperate to hyper-induce the vitamin D metabolizing enzyme, CYP24A1. Thus, T/E may limit VDR signaling through inactivation of 1,25D. To counteract the induction of CYP24A1 in VCaP cells, we tested a novel non-secosteroidal VDR agonist, VDRM2, developed by Eli Lilly & Co. VDRM2 inhibits LNCaP and VCaP cell growth and is resistant to metabolism. Using a subcutaneous VCaP xenograft model, we saw a significant reduction in tumor volume and tumor mass in mice treated with 3 ug/kg of VDRM2 compared to vehicle treated animals. We have shown for the first time a VDR agonist inhibits growth of a T/E expressing prostate cancer cell line in vivo and we achieved this effect without causing hypercalcemia as indicated through serum calcium levels. To gain a better understanding of the differences in biology of 1,25D and VDRM2 as well as VDR actions in T/E and non-T/E expressing cells, we used next generation sequencing (RNA-Seq). We compared gene expression in proliferating LNCaP and VCaP cells treated with concentrations of 1,25D and VDRM2 that resulted in similar levels of CYP24A1 gene induction. We had previously found that downregulation of c-Myc was a key factor in VDR mediated growth inhibition. As predicted, we observed significant downregulation of genes enriched in both the Myc and E2F pathways indicating our dataset recapitulates our current understanding of VDR actions in prostate cancer. There was substantial overlap between the 1,25D and VDRM2 mediated genes in each cell line, but substantial differences between the two cell lines, some of which is likely due to induction of T/E and its downstream effects in VCaP cells. Our study demonstrates a positive role for the use of VDR agonists in the treatment of T/E containing prostate cancer.

#1818

Coregulation of progesterone receptor and O-GlcNAc in breast cancer.

Gloria Trinca,1 Merit Goodman,1 Lauren Workman,1 Jason Carroll,2 Clive D'Santos,2 Chad Slawson,1 Christy R. Hagan1. 1 _University of Kansas Cancer Center, Kansas City, KS;_ 2 _Cancer Research UK, University of Cambridge, Cambridge, United Kingdom_.

Progesterone receptor (PR) activity is highly regulated by post-translational modifications. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analysis identified O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) as a PR-interacting protein. OGT catalyzes the addition of N-acetylglucosamine sugar moieties to serine/threonine residues in order to coordinate several cellular processes—including transcription, metabolism, and cell fate. Further investigation revealed that PR is O-GlcNAcylated, and enhanced OGT activity subsequently decreases PR protein stability. Interestingly, PR-expressing breast cancer cells display lower OGT levels in comparison to PR-null counterparts, suggesting that PR may govern its own O-GlcNAcylation or that of other key regulatory proteins. Our lab previously identified a phosphorylation site at Ser-81 in PR that potentiates the expression of PR proliferative target genes. Phosphorylation and O-GlcNAcylation are known to compete for the same residue substrates; and our preliminary data suggests that PR O-GlcNAcylation may also alter its transcriptional activity for a subset of target genes. While the role of PR in breast cancer progression is well characterized, the overall effect of protein O-GlcNAcylation within these tumors remains highly controversial. Collectively, our data supports a potential regulatory mechanism wherein OGT serves to restrain PR function in early PR+ tumors; whereas those that have lost PR expression may exhibit the elevated pro-tumorigenic OGT activity previously associated with more advanced breast cancers. As such, the continued study of O-GlcNAcylation with respect to PR stability, phosphorylation, and transcriptional activity may lead to a clearer understanding of these effectors and their contribution to breast cancer progression.

#1819

Intrinsic determinants of constitutive activity of exon-skipping variants of androgen receptor in castration resistant prostate cancer.

Takuma Uo,1 Heidi Dvinge,2 Cynthia C. Sprenger,1 Robert K. Bradley,2 Peter S. Nelson,2 Stephen R. Plymate1. 1 _Univ. of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

The C-terminally located ligand-binding domain (LBD) of androgen receptor (AR) folds into 12 helices (H1-H12) and its structural integrity permits a strict ligand-dependency of AR to act as a transcription factor. The two most common AR variants (ARVs) V7 and v567es lack LBD and are constitutively active and resistant to the forefront antiandrogen drugs. Moreover, the recent comprehensive splicing landscapes revealed that metastatic castration resistant prostate cancer (mCRPC) harbored multiple forms of ARVs, many of which have diverse patterns of inclusion/exclusion of exons (exon 4-8) corresponding to LBD to produce namely exon-skipping variants. The presence of these previously unreported ARVs adds another layer of complexity to AR signaling. Thus, it crucial to predict the functional outcomes associated with their expression of these ARVs in relation to mCRPC progression. To this end, we characterized these ARVs with respect to their subcellular localization and transactivation capability. Notably, v5es, which skips exon 5 (E5), displayed predominant nuclear localization and constitutive activity as reported for V7 and v567es. v6es, which includes E5 but excludes E6, is mainly localized in cytoplasm and transcriptionally inactive. Exclusion of E5 and 6 introduce premature stop codons in E6 and E7, respectively, which results in deletion of most of the LBD in v5es and v6es. On the other hand, v56es maintains the reading frame resulting in the inclusion of C-terminal half of LBD corresponding to E7 and 8, show nuclear and cytoplasmic distribution, and lack transactivation capability. Both active and inactive ARVs commonly possess E1 through E4. Accordingly, E5 appears to blunt transactivation capability of v6es while both/either of two exons E7 and 8 suppress the activity of v56es. To gain insight into what aspects are different between active and inactive LBD-less ARVs, we first generated and systematically analyzed a series of mutations with deletion of E5 in v6es toward E4-E5 junction. Among the 12 helices in LBD, H4 and H5 reside in E5. While the deletion mutants with intact H5 were similar to v6es in their properties, those with compromise or deletion of residues in H5 exhibited exclusive nuclear localization and constitutive activity. When inhibitory effects of E7 and E8 are considered, v56es without E7 and v56es was found to share similar properties. In contrast, v56es missing E8 was exclusively localized in nucleus and displayed significant but much lower transcriptional activity than that of v5es and the v56es derivative with simultaneous deletion of E7 and 8. These clearly suggest that multiple mechanisms exist to determine subcellular localizations and transactivation activities of ARVs. To develop the treatment based on AR expression profiles, we are continuing to maximize precise prediction of characteristics of ARVs that can singly or in aggregate contribute to mCRPC progression.

#1820

Investigating the role of estrogen receptor β agonists in the prevention and treatment of breast cancer.

Cathy Samayoa, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. _UTHSCSA, San Antonio, TX_.

Breast Cancer is the primary cause of cancer-associated mortality worldwide. In the United States alone, more than 250,000 women are diagnosed every year. Asian women have the lowest incidence rates. However, their rates increase to those of the US within one generation after migration. Suggesting that diet and lifestyle are major contributing factors in breast cancer risk. Since Asian women consume high amount of soy when compared to other populations, perhaps compounds found in soy may have a protective effect that is lost once they adopt a western diet.

Recent studies have identified plant-derived compounds from soy and licorice that activate Estrogen Receptor β. Unlike previous ERβ agonists, these novel compounds are selective for ERβ and have been tested for safety in clinical trials.

Current breast cancer treatment strategies focus on Estrogen Receptor α signaling, given that the majority of cases diagnosed are ERα positive. These treatment strategies include endocrine therapies; such as anti-estrogens or aromatase inhibitors. However, in the last decade, the importance of ERβ has emerged. Unlike ERα, ERβ has been shown to have tumor-suppressive function in various cancers, including breast cancer.

The objective of this study was to investigate the utility of using ERβ agonists in the prevention and treatment on breast cancer. To investigate the significance of ERβ activation in the prevention of breast cancer, we used an immunocompetent transgenic mouse model of breast cancer. Specifically, HER2/neu overexpressing mice develop premalignant lesions at 4-5 months and tumors starting at month 7. HER2/neu mice were treated with ERβ agonists for 3 months and subsequently mammary glands were analyzed using whole mount and IHC. When compared to controls, ERβ agonist treated mice showed a decrease in branching and ductal hyperplasia, with no change in body weight. These results suggest that ER β agonist treatment is chemopreventive and provided protection against premalignant lesion in the mammary gland.

To investigate the utility of ERβ agonists in the treatment of breast cancer, we used in-vitro and in-vivo models systems. Our results demonstrated that treatment with the agonists was able to inhibit the short-term and long-term growth of the breast cancer cell lines; MCF7aro and LTLT which represent post-menopausal breast cancer and letrozole resistant cells, respectively. In addition to growth inhibition, treatment with ERβ agonists also decreased cell migration and invasion. In addition to activating ERβ, we also found that the agonists are also able to increase the expression of ERβ without increasing ERα levels. Our studies also demonstrated that treatment with ERβ agonists modulated key signaling molecules involved in cell death and cell cycle. Our studies suggest that activation of ERβ signaling is a valuable strategy in the treatment and chemoprevention of breast cancer.

#1821

Identifying a novel mechanism underlying the enzalutamide and bicalutamide resistance in African-American prostate cancer patients.

Arsheed Ganaie,1 Badrinath R. Konety,2 Todd Schuster,1 Mohammad Saleem1. 1 _University of Minnesota, Austin, MN;_ 2 _University of Minnesota, Minneapolis, MN_.

Recently, FDA approved the Enzalutamide as a drug for castration-resistant prostate cancer. Recent studies reported patient populations which are non-responsive to the Enzalutamide therapy. Notably, African-American patients do not fare well for the castration-therapies (Bicalutamide and Enzalutamide). To identify a new therapeutic strategy for such populations, it is very important to understand the mechanism underlying the resistance to Bicalutamide or Enzalutamide. This would involve the use of an appropriate Bicalutamide or Enzalutamide-resistant model. We hypothesized that heterogeneous sub-populations contribute to Enzalutamide and bicalutamide resistance. In this study, we generated cell models representing Bicalutamide- and Enzalutamide-resistant phenotypes of primary prostate tumor of African-Americans. We next isolated subpopulations of Enzalutamide RC-Enz; Eht-Enz) and bicalutamide-resistant (RCbc; Eht-bc) cells. The cell sub-populations were ranked into three major classes' viz., (1) highly stem-cell like (expressing several stemness markers BMI1 & CD133), (2) less stem-cell like (express one stemness marker BM1), and (3) non-stem cell like (negative for BMI1 and CD133). Notably, African-American primary CaP cells exhibited higher number of stem cell-like populations than Caucasian primary (22Rν1) and metastatic cells (PC3). When compared, the African-American Enzalutamide/Bicalutamide-resistant stem cell-like (RC-EnzCD133+/BM1+, RC-bcCD133+/BM1+, Eht-bcCD133+/BM1+ & Eht-EnzCD133+/BM1+) cells exhibited increased rate of proliferation, invasiveness and migration than Enzalutamide-resistant Caucasian stem cell-like cells(LNCaP95EnzCD133+/BM1+). We show that RC-EnzCD133+/BM1+ and RC-bcCD133+/BM1+ cells resistant cells exhibit increased (i) promoter activity of BMI1 gene, (ii) localization of BMI1 protein on PTEN gene, and (iii) physical interaction of BMI1 tumor to E4F1 tumor suppressor protein. This was validated in prostatic tumors of African-Americans. We provide evidence that E4F1 under normal conditions negatively regulates the activity of Androgen receptor (AR)-associated signaling and inhibits growth of cells. We show that BMI1 protein sequesters E4F1 protein and inhibits its check-point type or negative regulation of AR-pathway in Enzalutamide and Bicalutamide-resistant cells. This leads to the growth of stem-cell like tumor cells during the Enzalutamide and Bicalutamide therapies. To conclude, we suggest that (1) E4F1-regulated AR-signaling plays an important role in prostate cancer, particularly in African-Americans, (2) ratio of E4F1 and BM1 expression has the potential as a biopsy biomarker for deciding the disease phenotype and (3) E4F1/BMI1 as a therapeutic target could be exploited to increase the sensitivity to Enzalutamide and Bicalutamide therapies in African-Americans.

#1822

Circadian regulation of estrogen receptor alpha protein and phosphorylation in mammary carcinoma cells.

Alix Youngblood, Yifang Zhang, Murali Anbalagan, Dawei Tian, Brian G. Rowan. _Tulane Univ. School of Medicine, New Orleans, LA_.

Circadian regulation of nuclear receptors in peripheral tissues is important in the timing of key physiological processes. However few studies have examined circadian regulation of estrogen receptor alpha (ERa) protein in cancer or normal tissues, and no studies have evaluated ERα phosphorylation during the circadian cycle. In addition to transcription, protein turnover and posttranslational modifications are important regulatory mechanisms in the circadian control of protein function. ERα protein is important in regulation of the peripheral clock proteins Period (PER), Cryptochrome (CRY), CLOCK, and brain and muscle arnt-like protein-1 (BMAL1). Several key ERα phosphorylation sites, serine 118 and serine 167, important in regulating ERα function contain consensus sequences for two kinases, casein kinase 1 and 2 (CK1, CK2), that regulate the peripheral clock proteins levels. The present study examined ERα protein levels and phosphorylation in human and rat mammary carcinoma cells following entrainment by 50% serum shock that results in circadian oscillation of clock genes in vitro. In human MCF-7 breast cancer cells, ERα protein levels exhibited circadian regulation after serum shock with an approx. 60% decrease at 4 hours, recovery to baseline level or above at 16 hours, and a 30% decline from baseline at 24 hours. Estradiol treatment resulted in a longer sustained suppression of ERα protein levels. Rat MAT mammary carcinoma cells exhibited a similar circadian regulation of ERα protein following serum shock. In MCF-7 cells, serine 118 phosphorylation was elevated 2-7 fold 4 hours after serum shock with a subsequent return to baseline levels by 12 hours. Estradiol treatment resulted in a more robust circadian cycle of serine 118 phosphorylation of up to 40 fold with peaks occurring between 4-8 hours and at 16-24 hours. A reverse phosphorylation pattern occurred for serine 167 phosphorylation. Serum shock resulted in a single induction up to 23 fold of serine 167 phosphorylation at 8-12 hours that was suppressed by estradiol. In rat MAT cells, serum shock resulted in circadian oscillation of serine 167 phosphorylation up to 3 fold with peaks at 8 and 20 hours that was similar when cells were treated with estradiol. For both MCF-7 cells and MAT cells, it was noted that peak levels of ERa protein and ERα phosphorylation were out of phase. Estradiol induction of cyclin D1 and c-myc mRNA demonstrated functional ERα signaling following serum shock. These data demonstrate a very significant circadian regulation of ERα protein levels and ERα phosphorylation in mammary carcinoma cells following entrainment by serum shock that likely contribute to time-dependent effects on peripheral clock function and ERα signaling in mammary cancer.[A.Y. and Y.Z. contributed equally to this work.]

#1823

Mechanism of the anterograde transport of the androgen receptor.

Jianzhuo Li, Guanyi Zhang, Hee-Won Park, Haitao Zhang. _Tulane University School of Medicine, New Orleans, LA_.

Androgen receptor (AR) is a ligand-activated nuclear receptor that plays a critical role in normal prostate physiology, as well as in the development and progression of prostate cancer. In addition to the classical paradigm in which AR exerts its biological effects by activating the transcription of its target genes, there is considerable evidence to support a rapid, non-genomic action mediated by membrane-associated AR, which facilitates the activation of kinase signaling cascades, leading to cell proliferation. While the nuclear translocation of AR is well studied, the molecular events controlling the recruitment of AR to the plasma membrane remain poorly understood. In this study, we set out to elucidate the mechanism underlying AR transport to the membrane. We found that a subpopulation of AR was localized to the membrane fraction shortly after androgen stimulation. Treatment with microtubule-targeting agents, but not actin inhibitors, blocked androgen-induced AR membrane localization, suggesting that AR transport to the membrane is mediated by the microtubule cytoskeleton. By utilizing dominant negative mutants, we identified the KIF5B, a member of the kinesin 1 family of motor proteins, is involved in the anterograde transport of AR. We further demonstrated that AR interacts directly with KIF5B, and such interaction is enhanced by androgens. Through a series of deletion constructs, a region includes the N-terminal and DNA binding domains of AR was determined to be involved in the association with KIF5B. Disruption of AR membrane transport decreased AKT phosphorylation. Together, our data suggest that KIF5B mediates AR membrane transport and regulates the non-genomic function of AR. Disruption of AR membrane translocation may represent a novel strategy to target AR signaling in prostate cancer.

#1824

Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes.

Jan Roger Olsen, Waqas Azeem, Margrete R. Hellem, Kristo Marvyin, Yaping Hua, Yi Qu, Lisha Li, Biaoyang Lin, XiSong Ke, Anne Margrete Oyan, Karl-Henning Kalland. _Univ. of Bergen - The Gade Inst., Bergen, Norway_.

Background: Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells. Androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Cancer cell escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation.

Methods: Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.

Results: A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen and AR dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial EP156T-AR cells, but did not exert a positive feedback on endogenous AR expression. Mesenchymal type prostate EPT3-AR cells with exogenous AR expression, in contrast to epithelial type EP156T-AR cells, were androgen non-responsive and were unable to produce detectable PSA in the culture supernatants even with higher levels of exogenous AR protein than in EP156T-AR and LNCaP cells for up to 2 weeks in androgen containing growth medium. The restricted PSA expression in the mesenchymal context suggests that if ADT increases the pool of mesenchymal type prostate cancer cells, then this might go undetected during PSA monitoring of disease progression.

#1825

ONC201 induces cell death in androgen receptor positive prostate cancer cells and shows synergistic effect with anti-prostate cancer drugs.

Avital Lev, Amriti R. Lulla, David T. Dicker, Wafik S. El-Deiry. _Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA_.

Prostate cancer is the most common cancer and the second leading cause of cancer related death among men in the United States. Current therapy for advanced prostate cancer is mainly based on androgen deprivation, though in many cases tumors become androgen-independent and resistant to available treatments. Thus, accessing novel therapies for prostate cancer and resistant tumors remains an important goal in the field. ONC201 is a first-in-class small molecule anti-cancer drug, shown to selectively induce cell death in most cancer cells tested in contrast to matched normal cells. In this study we investigated the single-agent and combination efficacy of ONC201 on a panel of prostate cancer cell lines with varied androgen receptor (AR) and receptor tyrosine kinases (RTKs) expression status. Our preliminary data suggests that the pro-apoptotic effects of ONC201 correlate with expression of androgen receptor and other receptor tyrosine kinases (RTKs). Specifically, high AR and low RTK (c-MET, EGFR, HER2 and IGFR) expression demonstrated higher sensitivity to ONC201 as compared to low AR and high RTK expression as evaluated by cell viability using the Cell-Titer Glo assay. We further modeled these findings in ONC201-sensitive 22Rv1 and ONC201-resistant DU145 cell lines. ONC201 induced robust apoptosis (cleaved PARP) in ONC201-sensitive 22Rv1 cells as compared to ONC201-resistant DU145 cells In addition, ONC201-sensitive 22RV1 cells showed abrogation of total AR expression 72 hours following treatment with ONC201. Thus, AR and RTK status seem to be emerging efficacy markers for response of ONC201 in prostate cancer cells. We are further investigating the involvement of ONC201 in the signal transduction pathway of AR and other RTKs in prostate cancer. Lastly, our initial screening for treatment of cells with a combination of ONC201 and FDA-approved therapies for prostate cancer showed synergistic potential of ONC201 with everolimus and docetaxel in both AR-dependent and independent cells. As expected, synergism of anti-androgen MDV3100 (enzalutamide) with ONC201 was restricted to AR positive prostate cancer cell lines. Our results indicate that ONC201 has therapeutic potential both as a single agent and in combination therapy for prostate cancer. Our long-term goal is to integrate our pre-clinical studies and translate the findings to single agent/combination trials of ONC201 for prostate cancer patients. 

## REGULATORY SCIENCE AND POLICY:

### Regulatory Science and Policy

#1826

IMI's CANCER-ID: Status of liquid biopsy standardization.

Klaus Pantel,1 Leon WMM Terstappen,2 Barbara Baggiani,3 Thomas Krahn,4 Thomas Schlange4. 1 _University Cancer Center Hamburg/Eppendorf, Hamburg, Germany;_ 2 _University of Twente, Enschede, Netherlands;_ 3 _Silicon Biosystems, Bologna, Italy;_ 4 _Bayer Pharma AG, Wuppertal, Germany_.

The CANCER-ID (www.cancer-id.eu) consortium was established in early 2015 with more than 30 partners as part of the Innovative Medicines Initiative (IMI), Europe's largest public-private partnership funded in equal parts by the European Union and the European Federation of Pharmaceutical Industries and Associations (EFPIA). The goal is to develop standard operating procedures (SOPs) and qualify respective mature technologies for pre-analytical sample handling, enrichment, isolation and analysis of Circulating Tumor Cells (CTCs), circulating free tumor DNA (ctDNA) and microRNAs (miRNAs) as novel blood-based biomarkers in cancer liquid biopsy, with a particular focus on Non-Small Cell Lung Cancer (NSCLC) and HER2-treatment refractory breast cancer. Over the 5-year period of the project, CANCER-ID will evaluate different technologies in the field, testing their clinical utility in multicenter clinical studies in support of regulatory approval of these devices. During the first year of the project, a number of different technology platforms have been implemented at several partner sites to establish SOPs with standardized blood samples spiked with selected cancer cell lines as standards. An important outcome of these studies are pre-analytical sample handling procedures, e.g. using different fixatives and tube systems as well as biobanking protocols that allow shipment and standardized long-term storage of liquid biopsy material. Most importantly, the knowledge generated within IMI's CANCER-ID will be used to establish and validate SOPs for the analysis of patient blood samples using different technologies, which represents a promising diagnostic tool holding the potential for optimized treatment decisions and therapeutic monitoring of cancer patients. The international exchange of human patient samples for research purposes poses a number of regulatory challenges linked to the different legal and ethical environments. The diverse national regulations for clinical samples and data require coordinated monitoring of interactions and guidance of the different partner organizations. As close collaboration with regulatory authorities is crucial for CANCER-ID to achieve its objectives, the consortium has established a contact with CDER/FDA via a Critical Path Innovation Meeting (CPIM). We will discuss the CANCER-ID approach to meet these many requirements and will present a matrix for coordinating such international efforts with stakeholders from different organizations.

#1827

The prospective Dutch colorectal cancer cohort: A prospective nation-wide observational cohort study.

Robert R.J. Coebergh van den Braak,1 Geraldine R. Vink,2 Martijn G.H. van Oijen,3 Mirre E. de Noo,4 Sophie A. Kurk,5 Maarten J.P. Burbach,5 Alice M. Couwenberg,5 Anne M. May,5 Helena M. Verkooijen,5 Gerrit A. Meijer,6 Miriam Koopman5. 1 _Erasmus University Medical Center, Rotterdam, Netherlands;_ 2 _Netherlands Comprehensive Cancer Organisation, Utrecht, Netherlands;_ 3 _Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands;_ 4 _Deventer Hospital, Deventer, Netherlands;_ 5 _University Medical Center Utrecht, Utrecht, Netherlands;_ 6 _Netherlands Cancer Institute, Amsterdam, Netherlands_.

The increasingly complex subclassification of tumors based on clinical and molecular characteristics, as well as the large sample sizes needed for clinical trials, pose new challenges to the recruitment of patients for studies. Currently, less than 10% of all cancer patients are enrolled in clinical trials, limiting their external validity, but also leaving room for improvement in the recruitment of patients. Clinical trials are often complicated by low inclusion rates, inadequate data collection, high non-completion rates and costs, and often provide insufficient data for post-hoc subgroup analyses. We believe that a prospective observational cohort study can provide standardized and validated collection of clinical data, tissue and blood samples and patient-reported outcome measures, and can improve patient recruitment for clinical trials. The Prospective Dutch ColoRectal Cancer cohort is a prospective multidisciplinary nation-wide observational cohort study in the Netherlands, a country with a yearly colorectal cancer incidence of 15,000 patients. The goal of the study is to facilitate future basic, translational and clinical research in the field of colorectal cancer for both national and international research groups with short or absent inclusion periods, even for studies with stringent inclusion criteria.

All patients > 18 years with histologically proven colorectal cancer are asked to participate. Four essential features characterize the study: 1) a patient-centered informed-consent approach; 2) a framework to systematically collect long-term clinical follow-up and patient-reported outcomes; 3) an infrastructure for the standardized collection and storage of tissue and blood samples and 4) efficient recruitment for clinical trials which is facilitated by the innovative cohort multiple randomized controlled trial (cmRCT) design. The cmRCT design allows patients to participate in multiple non-conflicting clinical trials. We believe that the key to the development of a sustainable cohort study is the collaboration with existing organizations using their expertise, and preventing duplicate data entry and unnecessary costs. To these ends, close collaborations with other national initiatives, like the national cancer registry (hosted by IKNL), the national pathology registry PALGA, and the national biobanking infrastructure BBMRI-NL have been established. An effective governance structure to secure the privacy of patients and hospitals, and to facilitate research is needed.

This study will provide long-term clinical data, tissue and blood samples, and patient-reported outcome measures collected under a broad informed consent, of a large cohort of patients with colorectal cancer. The available data and tissue will facilitate basic, translational and clinical research. Furthermore, the design of this cohort study can be used as an example for other tumor types.

#1828

Analysis of putative serum biomarker candidates for cardiotoxicity prediction in a cohort of early stage breast cancer patients treated with docetaxel, cyclophosphamide, and bevacizumab.

David W. Murray,1 Alice C. O'Farrell,2 Giuseppe Gullo,3 Ashwini Maratha,4 Patrick Dicker,5 Mairin Rafferty,4 Verena Murphy,6 Bonnie Ky,7 William M. Gallagher,8 Norma O'Donovan,9 John Crown,3 Annette T. Byrne2. 1 _Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland and OncoMark Ltd., Dublin, Ireland;_ 2 _Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland;_ 3 _St.Vincent's University Hospital, Dublin, Ireland;_ 4 _OncoMark Ltd., Dublin, Ireland;_ 5 _Department of Epidemiology and Public Health Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland;_ 6 _All Ireland Co-operative Oncology Research Group, Dublin, Ireland;_ 7 _Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;_ 8 _UCD School of Biomolecular and Biomedical Science, University College Dublin and OncoMark Ltd, Dublin, Ireland;_ 9 _National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland_.

Background: Breast cancer is the leading cause of cancer in women, with 1.8 million patients diagnosed globally each year. In early stage disease, anthracycline regimens are commonly employed, but the risk of cardiotoxicity is high. Replacing anthracyclines with taxanes in combination with cyclophosphamide may improve response [1] and Bevacizumab (BVZ) may provide addtional benefit. However, as BVZ can cause hypertension and increased risk of cardiac failure, a rationale exists for studying biomarkers of cardiotoxicity in this setting.

Methods: We assessed a panel of biomarkers in n=57 early stage breast cancer patients whom underwent docetaxel/cyclophosphamide/BVZ therapy on the TC-Avastin Trial (NCT00911716). Serum was collected prior to treatment and at 3 months. Cardiotoxicity was defined as decline in left ventricular ejection fraction ≥ 10% to ˂55% over 1 year treatment + 2 year follow-up. Cardiac troponin I and T [cTnI, cTnT], Endothelin-1, Galectin-3, Placental Growth Factor [PlGF], Platelet Derived Growth Factor, Vascular Endothelial Growth Factor receptors 1 and 2, total nitrous oxide NO and myeloperoxidase [MPO]) were screened by ELISA in a subgroup (n=16). Pilot and recently published findings [2] implicated PlGF, cTn1 and MPO, which were subsequently assessed in the larger cohort.

Results: Changes in serum MPO or cTnI were not related to cardiotoxicity when analysed in the larger cohort. However, a "cut-off" PlGF fold change of 0.99 could differentiate patients who developed cardiotoxicity from those that did not. 74% of cardiotoxicity patients, compared to only 35% of non-cardiotoxicity fell below the 0.99 cut-off. Conversely, 65% of non-cardiotoxicity patients, compared to 25% of cardiotoxicity patients fell above the cut-off (p=0.0042).

Conclusions: Our data suggest that decreased PlGF at 3 months may predict cardiotoxicity. These observations require further validation.

This work was funded by AngioTox, an EC FP7 IAPP Marie Curie Award (Grant Agreement 251528). [1]Jones et al., J. Clin. Oncol.27, 1177-1183 (2009); [2] Putt et al., Clin. Chem 61, 1164-1172 (2015).

#1829

The validation of a pyrosequencing KRAS mutation detection assay.

Matthew L. Poulin, Ann Meyer, E. Andrew Mead, Jessica Xu, Ryan Drennan, Liyin Yan. _EpigenDx, Inc., Hopkinton, MA_.

Mutations in the KRAS gene, especially affecting the codon 12 and 13 region of the Kras protein, have implications in the treatment of certain cancer types. Because of the current potential for the FDA to oversee laboratory developed procedures (LPDs), it is imminently important that the validation of such tests be published to ensure that it is shown that these LPDs are properly reviewed and properly tested within the CLIA guidelines, thus keeping LPDs within the CLIA regulatory domain. We have developed a pyrosequencing assay that can detect eleven mutations in the codon 12 and 13 position of the KRAS gene. Our validation consisted of a sensitivity study in which a purified mutant PCR product was introduced into a wildtype DNA background and diluted down to undetectable levels. This sensitivity test was done on six different KRAS codon 12/13 mutants to determine the detection limit of the assay. Intra- and inter-assay precision and assay accuracy was determined by comparing assay results of over 40 total samples, both mutant and wild type, over five days carried out by two technicians. The results will show that this KRAS pyrosequencing assay falls within the acceptance criteria for sensitivity, accuracy and precision.

#1830

Answering the challenge of cancer genomic testing.

Michael J. Clark. _Personalis, Inc., Menlo Park, CA_.

Cancer genomes are a hodgepodge of mutations. Single nucleotide somatic variants, complex structural rearrangements, whole and partial gene deletions and amplifications, splice variants and expression changes—these are the potential mutation types in cancer. Tumor heterogeneity is common, tumor samples can be solid or liquid biopsies, and they are often contaminated with adjacent normal tissue. Cancer samples are treated with formalin for archival purposes causing additional genomic variations. All of these factors combine to make genomic analysis in cancer far more complicated than that of normal germline tissue.

Recent efforts to define gold standard human genomes lack many features characteristic of cancer genomes. A single cancer sample may contain a few distinct variant types, but typically not enough to confidently assess the accuracy of cancer variant detection algorithms. The Horizon Quantitative Multiplex, an engineered FFPE cell line mix with variants at allele frequencies (AFs) from 1.0 to 41.5%, and the Acrometrix Oncology Hotspot Control with >500 SNVs and small indels in one sample are available standards. In the Horizon cell line, we detected 18/18 SNVs and 4/4 indels. Meanwhile, we found that the artificial nature of the Acrometrix sample made it incompatible with next generation sequencing. While samples of this type have limited utility, there is a clear need for samples containing the full spectrum of mutation types and frequences.

Since the commercially available standards are of limited utility, we aimed to create a cancer standard set that consists of many cancer cell lines in combination with some real primary cancer samples. We started with a collection of 28 cancer cell lines including 817 known SNVs, 62 small indels, 21 deletions, 23 amplifications, and 14 gene fusions. We simulated tumor heterogeneity by mixing the cell lines at various ratios, generating variant allele frequencies down to 1% and emulated reduced purity by mixing cell lines with paired normal samples at ratios down to 10%. This gave us our platform accuracy metrics for tumor-only somatic analysis.

These experiments required sequencing over 200 samples and mixes. Using augmented exome and cancer gene panel (targeting >1,500 cancer genes), we were able to interrogate substantial quantities of variants. We detected 16136/16146 SNVs at 5% AF, 639/646 indels at 10% AF, 29/30 CNAs at 20% purity, and 14/14 gene fusions using the panel, for example. These high variant counts gave us tighter confidence intervals and accuracy metrics for detection of all major cancer variant types.

After validating our variant calling approaches via this set, we applied our algorithms to primary tumor samples both formalin treated and fresh frozen, and also tested them both with and without a paired normal tissue sample. These gave us the ability to understand the fundamental effects of formalin fixation, and the benefits of including the paired normal sample, which will be covered in this presentation.

#1831

Cancer genomic resources and needs in the Latin American region.

Javier Oliver,1 Sandra Perdomo,2 Felipe Vaca3. 1 _Instituto de Ciencias Básicas y Medicina Experimental, Ciudad Autonoma de Buenos Aires, Argentina;_ 2 _Instituto de Investigación en nutrición, genética y Metabolismo IINGM, Bogota, Colombia;_ 3 _Facultad de Estudios Superiores Iztacala, UNAM; Instituto Nacional de Cancerología, Mexico DF, Mexico_.

In 2012, approximately 1,005,255 new cancer cases and 550,164 deaths from cancer occurred in both sexes in central and South American region, by 2025 and increment of nearly 30% of new cases and 35% of deaths from cancer are predicted. One of the priorities of cancer control within the region is to reduce avoidable deaths from cancer by improving early detection and personalized treatment. The development of cancer genomics and their integration to cancer care has shown a great improvement in cancer control worldwide; however, all these key advances have been mainly concentrated in highly developed nations and little is known about the capacities and needs of cancer genomics in the Latin-American (LA) context.

In order to evaluate the capacity and development of cancer genomic applications in the LA we collected available information for all countries in central, South America and Cuba. Data reviewed included: number of cancer research institutions, number of NGS platforms, research groups working in cancer genetics, publications on cancer genetics for the last 10 years, educational programs on genomics and related health policies.

In average 15 Research groups per country were registered as conducting cancer-genetics related projects. In the las 10 years, 206 publications on cancer genetics including reviews were led by (1st, 2nd or corresponding) authors affiliated to LA institutions and only publications in the last 2 years included cancer genomic analysis. Most publications related to breast, gastric and hematological cancers were underrepresented in general. In the past 5 years physical resources have grown markedly by the acquisition of nearly 150 NGS platforms in 9 countries, the majority installed in medical related services and universities. Participation in large genomic consortia has only included two countries Mexico (as partner) and Brazil (as leading). Educational programs in genomics are scarce, almost exclusive of graduate programs and few are applied to cancer.

Despite the recent advances in introducing cancer genomics knowledge and application in LA, the region lacks development of integrated genomic research projects, improved use of platforms dedicated to cancer, educational programs and health policies that focus on the most frequent cancers and could impact cancer care.

#1832

Large variations in clinical and ethical aspects of genomic sequencing initiatives: A Global Alliance for Genomics and Health (GA4GH) survey.

Jeremy Lewin,1 Daniel J. Vis,2 Mark Lawler,3 Rachel Liao,4 Mao Mao,5 Bin Tean Teh,6 William Sellers,7 Robyn Ward,8 Anamaria Aranha Camargo,9 Fabrice Andre,10 Richard Schilsky,11 Denis Lacombe,12 Tatsuhiro Shibata,13 Stephen Fox,14 Christophe Le Tourneau,15 William S. Dalton,16 Bartha Maria Knoppers,17 Charles Sawyers,18 Emile E. Voest,2 Lillian L. Siu1. 1 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 2 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 3 _Centre for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom;_ 4 _The Global Alliance for Genomics and Health, Toronto, Ontario, Canada;_ 5 _Yonsei Cancer Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 6 _National Cancer Centre Singapore, Singapore;_ 7 _Novartis Institutes for Biomedical Research, Cambridge, MA;_ 8 _University of Queensland, St. Lucia, Australia;_ 9 _Hospital Sírio-Libanês, São Paulo, Brazil;_ 10 _Gustave Roussy and Université Paris Sud, Villejuif, France;_ 11 _ASCO, Alexandria, VA;_ 12 _EORTC, Brussels, Belgium;_ 13 _University of Tokyo, Tokyo, Japan;_ 14 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 15 _Institut Curie, Paris, France;_ 16 _Moffitt Cancer Center, Tampa, FL;_ 17 _Centre of Genomics and Policy, McGill University, Montreal, Quebec, Canada;_ 18 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Although next generation sequencing (NGS) has expanded our understanding of disease prognostication and cancer treatment, there is heterogeneity regarding its implementation. GA4GH is a not-for-profit organization that promotes and harmonizes responsible and effective data sharing, as unconnected data silos unacceptably stall the advancement of precision medicine. The GA4GH Cancer Task Team conducted a survey of international cancer sequencing activities to evaluate variability in these initiatives and report our findings on clinical/ethical aspects.

A total of 108 sequencing initiatives were approached via a web-based survey, of which 59 responded (55%) (Characteristics: Table 1). Most initiatives (61%) were North American or European based. Genomic-based drug matching occurred in 39 initiatives (66%): unplanned opportunistic matching to existent trials e.g. phase I (n=29), specifically designed genomics driven trials (n=10). In matching initiatives, outcome data was collected via RECIST in 24 (62%), time on treatment in 23 (59%), and clinical assessment in 10 (26%). Toxicity data was collected in all clinical trials but in few genomic sequencing programs.

Specific or implied informed consent was identified in 34 (58%) and 7 (14%) initiatives respectively and 36 (61%) allowed re-contacting of patients. However, only 31 (53%) had a protocol for communicating genetic results and 23 (39%) had a policy to handle incidental germline mutations. In total, 63% of initiatives are currently sharing data with an additional 10% partially sharing or planning to share.

In conclusion, there is currently no uniform approach for collecting data for precision medicine application. GA4GH is actively leading harmonization efforts (e.g. standardized outcome data, toxicity data collection, policies for returning genetic results and strategies for data sharing) to maximize the value of the increasingly complex datasets generated from NGS.

|

---|---

Table 1 | N (%)

Number of initiatives | 59

Regional Location

North America

Europe

Asia

Australia

South America

Intercontinental | 20 (34)

16 (27)

7 (12)

3 (5)

3 (5)

10 (17)

Regional Scope

Institutional (local)

Multi-institutional (regional) / National

European Union

International

Unknown | 15 (25)

28 (48)

4 (7)

10 (17)

2 (3)

Patient Samples per Year

1-500

501-5000

>5000

Unknown | 22 (37)

22 (37)

7 (12)

8 (14)

Purpose of test

Diagnostic

Research

Diagnostic /Research

Unknown | 9 (15)

22 (37)

20 (34)

8 (14)

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Assessment of Gene Regulation in the Malignant Context

#1833

Transcriptome characterization of high grade serous and endometrioid epithelial ovarian cancer tumors.

Brooke L. Fridley,1 Junqiang Dai,1 Rama Raghavan,1 Chen Wang,2 Pengcheng Lu,1 Stacey Winham,2 Madalene Earp,2 Kate Lawrenson,3 Simon A. Gayther,3 Kimberly R. Kalli,2 Ellen L. Goode2. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _University of Southern California, Los Angeles, CA_.

Background: Little transcriptomic research has compared epithelial ovarian cancer (EOC) histological subtypes. We set out to characterize the transcriptomes of high-grade serous carcinomas (HGSC) and endometrioid carcinomas (EC), which make up around 70% and 20% of EOC tumors, respectively, and have some histopathological similarities.

Methods: Fresh frozen tumors from EOC patients seen at the Mayo Clinic (30 EC and 62 HGSC) were used. 1ug RNA riboZero was used for library preparation using the Illumina TruSeq kit and sequenced on a HiSeq 2000 machine. Reads were aligned using TopHat2 followed by quantification of abundances using RSEM and differential expression analysis with edgeR. We analyzed transcriptomes, conducted pathway analyses, and summarized key candidate gene sets. Expressed SNVs (eSNVs) from the RNA-seq data were determined using GATK and RVboost.

Results: The analysis found 699 genes with FDR < 1x10-5 for differential expression between HGSC and EC, with most genes being up-regulated in EC. The top most associated genes were TPH1, MAP2K6, KLK2, ADAM23, TESC and TRAF3IP2 (p<10-22). Pathway analysis of the genes up-regulated in EC revealed enrichment of the "basal cell carcinoma signaling pathway" (p = 1.2x10-5). Within 1 Mb of the 25 known EOC risk loci, we observed higher expression in HGSC for RSPO1 and HPSE (p <5x10-7). For genes functionally related to EOC, we observed in HGSC up-regulation (*p < 10-5, ^p<0.003) for FOXM1^, CDKN2A^, CCNE1*, CCND2^, PIK3CA*, BRCA2^, BIRC5^, MMP9^, FANCD2^, and MAML2^. In contrast, we found up-regulation in EC for MDM2*, KLK4*, BCL2^, CCND1*, ANXA4*, CDH1^, MMP7^, and MAML3^. We also identified 204 eSNVs (44 non-synonymous) associated with EC v HGSC subtype (p<10-4); this included an exonic TRAF3IP2 eSNV (66% EC, 13% HGSC, p = 4x10-7, chr6:111877117).

Discussion: Using one of the largest sets of identically processed fresh-frozen EOC tumors, some patterns emerged among the numerous EC v HGSC transcriptomic differences. TPH1, up-expressed in EC, is regulated by SOX4 which was also up-regulated in EC. Two sets of genes related to Kallikreins serine proteases were differentially expressed, including KLK2 which is known to regulate EGFR and pro-inflammatory cytokines and is regulated by MYC. Lastly, TRAF3IP2 encodes for a protein involved in regulating cytokines through members of the NFKB pathway.

Conclusions: These findings suggest important biological insights into one of the rarer EOC histologies and may aid in the development of targeted treatment options. Research is on-going to incorporate additional features (e.g., DNA methylation, copy number) into a "systems biology" framework to better understand the molecular differences between EOC histologies.

#1834

Semaphorin 3C is an androgen receptor-regulated gene.

Kevin J. Tam,1 Kush Dalal,2 Michael Hsing,2 Chi Wing Cheng,2 Yan Ting Chiang,1 Aishwariya Sharma,2 James W. Peacock,2 Artem Cherkasov,2 Yuzhuo Wang,2 Martin E. Gleave,2 Paul S. Rennie,2 Christopher J. Ong2. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in North America. The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is heavily implicated in PCa progression. First-line therapies frequently target the AR axis and are initially met with favourable response but restored and aberrant AR signaling fuel disease progression to stages for which treatment is palliative. Transcriptional targets of the AR include genes involved in cell growth and cell fate but are not completely described. The semaphorin family of signaling proteins is a large grouping of cell-surface or secreted signaling proteins that function in neurogenesis and development. The roles of semaphorins in cancer are becoming increasingly evident however mechanistic details surrounding their involvement in cancer are poorly defined. One member of the class 3 semaphorins, SEMA3C, has been shown to confer invasive phenotypes in prostate cancer cells. Using the AR-positive LNCaP cell line we show that SEMA3C is an androgen-responsive gene. Additionally, using RSAT DNA analysis software we identify an androgen response element (ARE) in intron 2 of SEMA3C and show that AR is recruited to this genomic region in an androgen-dependent manner in chromatin immunoprecipitation assays. Furthermore, the isolated ARE binds to the AR-DNA binding domain in gel shift assays and can be transactivated by AR in reporter gene assays. Finally we show that the pioneering factor, GATA2, is necessary for AR-mediated expression of SEMA3C. Collectively, our work identifies SEMA3C as a direct transcriptional target of AR in support of our hypothesis that dysregulated AR signaling drives inadvertent upregulation of SEMA3C and PCa disease progression. Deregulated AR signaling underpins prostate cancer progression and underscores the need to elucidate transcriptional targets of AR. Identification of SEMA3C as a novel target of AR provides the rationale for targeted therapies directed against SEMA3C.

#1835

NFATC1 regulates the long non-coding RNA MALAT1 in breast cancer.

Joshua K. Stone, Jung-Hyun Kim, Ming Tan, Eun-Young E. Ahn. _University of South Alabama, Mobile, AL_.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA overexpressed in many solid tumors such as breast, colorectal, liver, lung, and pancreatic. Its expression progressively increases, first in comparison of adjacent normal tissue to the primary tumor and again from the primary tumor to secondary metastatic sites. It has been reported that MALAT1 controls gene expression through regulation of alternative mRNA splicing as well as chromatin binding and histone modification. These processes result in cell cycle advancement, cell proliferation and migration. In tumor cells, loss of MALAT1 prevents epithelial-mesenchymal transition (EMT), reduces proliferation, invasion, migration, and increases apoptosis. Even though MALAT1 is overexpressed in many tumors, the signaling pathways and transcription factors which regulate its expression are poorly understood.

To identify positive activators of MALAT1, we first selected candidate transcription factors for screening which were identified by ENCODE ChIP-Seq as bound to the MALAT1 promoter and were linked to metastasis. Through small inhibitory RNA (siRNA)-mediated knockdown, we identified NFATC1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1) as a transcription factor that activates MALAT1 expression in multiple breast cancer cell lines. Overexpression of NFATC1 demonstrated an increase in MALAT1, verifying the siRNA results. Our luciferase reporter assay demonstrated that the MALAT1 promoter region is indeed controlled by NFATC1. Furthermore, chromatin immunoprecipitation using an NFATC1 antibody demonstrated enrichment of NFATC1 at multiple sites within the MALAT1 promoter region.

Examination of endogenous levels of NFATC1 and MALAT1 in five breast cancer cell lines indicated those which were HER2-positive had highest expression of both NFATC1 and MALAT1. To determine the effect of HER2 on NFATC1 activity and MALAT1 expression, we ectopically expressed HER2 in HER2-negative cell lines. Interestingly, this increased total and nuclear NFATC1 protein as well as MALAT1 transcript levels.

In conclusion, these results demonstrate that NFATC1 is a positive activator of MALAT1 in breast cancer and HER2 expression can modulate this activity. Notably, NFATC1 is associated with breast cancer EMT, invasion, and migration which match well with those phenotypes ascribed to MALAT1. Furthermore, HER2-positive status is associated with increased tumor proliferation and risk of metastasis. Therefore, our findings demonstrate that the HER2-NFATC1-MALAT1 signaling axis is a novel driver of breast cancer tumorigenesis and represents a potential therapeutic target.

#1836

Global gene expression profiles from bladder tumor FFPE samples.

Varun Bagai,1 Jeoffrey Schageman,1 Kelli Bramlett,1 David J. McConkey,2 Woonyoung Choi2. 1 _Thermo Fisher Scientific, Austin, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Cancer is a disease characterized by uncontrolled cell growth and proliferation. Recent advances in molecular medicine and cancer biology have changed the way clinicians evaluate and consider treatment. Selected tumor biomarkers have been utilized as targets for drug therapy leading to better more effective treatment. Gene expression profiling has been used for identifying new biomarkers for tumor classification and driving decision making for better patient outcome in different tumor types. DNA microarrays have become a key method to acquire a comparative snapshot of the gene expression profile from test samples in a high throughput manner. Quantitative PCR and newer sequencing techniques are popular alternatives offering highly accurate gene expression measurements, but with limitations due to cost, complex instrumentation and analysis needs. RNA extracted from formalin fixed paraffin embedded tissue (FFPE) creates considerable additional challenges in acquiring accurate gene expression measurements due to the highly fragmented and compromised integrity of FFPE RNA due to the fixation process.

To address the challenges of current sequencing based methods and take advantage of the simplicity of analysis that comes with using technologies such as microarrays; we have tested the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit using RNA isolated from bladder tumor FFPE specimens. This targeted RNA sequencing approach allows profiling the global mRNA expression of human RNA in a highly multiplexed fashion using the Ion AmpliSeq™ technology. 10ng of total RNA extracted from FFPE tissue was reverse transcribed followed by automated library preparation on the Ion Chef™ system using the new Ion AmpliSeq™ Kit for Chef and the Ion AmpliSeq™ Transcriptome Human Gene Expression Panel. Eight pooled libraries were then sequenced on the Ion S5™XL System with Ion 540™ Chip. Libraries were also prepared with well characterized control RNAs, Universal Human Reference RNA (UHR) and First Choice Human Brain Reference RNA (HBR) using both the manual and automated library generation protocol for validation and comparison studies.

The results show detection of more genes than popular microarray platforms with comparable differential gene expression measurements to quantitative PCR (r = 0.96) and RNA-Seq methods (r = 0.94). Gene expression values correlated with R>0.99 for all technical replicates and R>0.95 between manual and automated library preparation methods using well characterized samples. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit is a simple method to measure global gene expression profiles from human RNA samples in a timely, cost effective, and high throughput manner resulting in sensitive and accurate gene expression measurements. The new S5™XL System combined with automated library and template preparation on the Ion Chef™ system enable a simple RNA to gene expression data workflow requiring only 45 minutes of hands on time from 10ng of FFPE RNA.

#1837

Comprehensive RNA-seq transcriptome interrogation of paired hepatocellular carcinoma and cirrhosis tissues revealed significant molecular features of disease evolution and modulation of tumor immunity.

Bonnie P. Liu,1 Kwame Okrah,1 Jeff Cheng,2 Maipelo Motlhabi,1 Charlie Sun,2 Teiko Sumiyoshi,1 Shoji Ikeda,1 Hartmut Koeppen,1 Zineb Mounir,1 Craig Cummings,1 Nadia Haque,1 Garret Hampton,1 Lukas Amler,1 Mark Lackner,1 Shih-Min A. Huang1. 1 _Genentech, South San Francisco, CA;_ 2 _Genentech, China_.

First 2 authors contributed equally

Hepatocellular carcinoma (HCC) is the second most common cause of death from cancer worldwide with extremely poor prognosis. HCC is known to be closely associated with liver injury-induced cirrhosis caused by various etiologies, including HBV infection. The prolonged timeline and heterogeneous nature of HCC adds complexity to dissecting the biology of this disease in humans. While Sorafenib is approved for the first-line treatment of metastatic HCC, most patients rapidly progress on treatment with Sorafenib. Consequently, alternative therapeutic options for HCC are much needed. The identification of molecular subtypes and reliable biomarkers associated with disease evolution is critical in facilitating development of new therapeutic agents in HCC.

To understand the manifestation of early molecular events in HCC disease progression in human, we analyzed genome-wide RNA-seq data derived from 100 paired samples consisting of HCC tumors (most with 60-80% tumor content) and adjacent cirrhotic tissues from early stage patients (TNM system: T1N0M0, T2N0M0, and T3N0M0; similar to BCLC stage A and B). Differential expression analysis revealed a cluster of genes that significantly differentiated HCC from cirrhotic tissues and illustrated a widespread deregulation of cell cycle machinery modulated by probable molecular abnormalities represented by Polo-Like Kinase, Checkpoint kinases, G2/M DNA damage checkpoint regulation, DNA damage-induced 14-3-3σ signaling, ATM signaling, and estrogen-mediated S-phase entry. Prominent down-regulation of FXR/LXR/RXR activation was also observed in HCC tumors. Strikingly, the unsupervised hierarchical clustering of both cirrhotic and HCC tissues revealed 3 groups of genes with mRNA expression closely correlating with disease progression stage-wise from cirrhosis to T1, T2, and T3 stages. Specifically, we made a novel observation illustrating the stage-wise activation of Wnt signaling pathway, but de-activation of MAPK pathway. Upon in-depth analysis, our data also suggests that as HCC progresses, translation machinery and embryonic morphogenesis are stimulated, while angiogenesis, negative regulation of apoptosis, and mesenchymal cell differentiation are possibly impinged. In addition, we found that components of processes crucial for activating immune response appear to be impaired as disease progresses from cirrhosis to stage T3. To confirm the aforementioned finding through focused assessment of immune-microenvironment by gene expression, we utilized Fluidigm platform and corroborated the down-regulation of effector T cell signature.

In conclusion, data presented provides a holistic depiction of evolution of HCC and the associated tumor immunity, thus paving a way for future detailed subtyping and therapeutics discovery.

#1838

Differential CXCL13 and CXCR5 expression mediated by down-regulation of select miRNAs in prostate cancer.

Denise E. Hinton, James Lillard. _Morehouse School of Medicine, Atlanta, GA_.

Chemokines and their receptors are involved in the inflammatory response by attracting immune cells to sites of infection and wound healing. Chemokines have also been implicated in the progression of prostate cancer (PCa). We have previously shown a positive correlation between the expression of CXCL13 and CXCR5 proteins with PCa progression; in particular, the CXCR5 protein is expressed to a greater extent by prostate tumors isolated from African Americans than compared to those from European Americans. In the present study, we sought: (i) to determine whether CXCL13 and CXCR5 mRNA expression patterns correlated with overall and/or disease-free survival in PCa cases and (ii) to identify the possible molecular mechanisms that govern this association. CXCL13 and CXCR5 mRNA levels and PCa survival rates were evaluated using The Cancer Genome Atlas (TCGA) data sets and the CbioPortal, respectively. CXCR5 and CXCL13 mRNA expression did not correlate with prostate tumor stage or grade and was not associated with overall PCa survival. However, CXCR5 mRNA expression was significantly associated with poor disease-free survival. Gene map analysis using the UCSC Genome Browser revealed consensus sequence binding sites for four miRNA's (miR-192/215, miR-486, and miR-494) that could bind the 3' UTR of the cxcr5 transcript. Our results showed that miR-494 was frequently deleted or down-regulated, confirming other reports that miR-494 is down-regulated in high-grade PCa cases. Of note, miR-494 also targets cxcr4 mRNA and suppresses migration of PCa cells. Others have shown that miR-486 is associated with p53 and PI3K-Akt pathways and miR-186 has a putative binding site in the 3' UTR region of the cxcl13 transcript. This miRNA was down-regulated in PCa patients with initial metastasis. These results confirm our previous studies demonstrating a correlation between tumor expression and PCa progression and reveal new mechanisms of post-translational regulation of this chemokine receptor.

#1839

Analysis of cell cycle-related gene expressions in MDM2-transfected LNCaP-MST cells after inhibition of HSP90.

Thiagarajan Venkatesan,1 Ali Alaseem,2 Khalid Alhazzani,2 Priya Dondapati,2 Saad Alobid,2 Appu Rathinavelu1. 1 _Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, Plantation, FL;_ 2 _College of Pharmacy, Health Professions Division, Nova Southeastern University, Davie, FL_.

MDM2 gene is a cellular proto-oncogene amplified in 25-40% of all human cancers and regulates cell proliferation, senescence, and apoptosis through targeting p53. It is widely speculated that MDM2 protein is not only responsible for tumorigenesis via p53 inactivation, but also for increasing the metastatic ability of originally non-metastatic tumor cells. HSP90 is a molecular chaperone that regulates the maturation, activation and stability of critical signaling proteins that drive the development and progression of prostate cancer. 17-Allyamino-17-demethoxygeldanamycin (17-AAG) is an inhibitor of HSP90, which causes proteasomal degradation of client proteins such as AR, HER2, Akt etc., leading to cell cycle arrest and substantial growth inhibition. HSP90 is also believed to be involved in the stabilization of MDM2 and therefore, inhibition of HSP90 is expected to accelerate degradation of MDM2 and disrupt its function. Hence, our study was designed to determine the expression profile of the genes in the cell cycle pathway following treatment of MDM2 overexpressing LNCaP-MST prostate cancer cells with 17-AAG (10 µM at 24 hrs). In the present study, Human cell cycle PCR array was used to examine the alterations in gene expression pattern following exposure to the 17-AAG in MDM2 overexpressed prostate cancer cells. In our experiments we observed that the genes impacted by MDM2 overexpression were reversed by HSP90 inhibition. The expression of genes such as CDC25A, CDC25C, AURKB and Survivin levels were significantly down-regulated after 17-AAG treatment, while p21 and GADD45A were up-regulated compared to the control LNCaP-MST. A heat map and scatterplot analysis clearly confirmed the alterations in the expression levels of these genes following 17-AAG treatment. It is expected that more thorough understanding of the consequences of HSP90 blockade, particularly those that are independent of its role as a regulator of p53, will reveal its therapeutic significance. Our results offer convincing evidence to suggest that the inhibition of HSP90 can induce cell cycle arrest in LNCaP-MST cells. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

#1840

Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays.

Milos Babic,1 Peter Shepard,1 Joanne Yeakley,1 Ruchir Shah,2 Deepak Mav,2 Raymond B. Nagle,3 Bruce Seligmann1. 1 _BioSpyder Technologies, Inc., Rancho Santa Fe, CA;_ 2 _Sciome, LLC, Research Triangle Park, NC;_ 3 _University of Arizona Cancer Center, Tucson, AZ_.

Highly sensitive and quantitative whole transcriptome and surrogate transcriptome targeted sequencing TempO-Seq assays were used to profile gene expression from 1 mm sq focal areas of FFPE sections (5 µm thick) of normal and cancerous tissue within the same prostate. The whole transcriptome assay directly measures changes within the whole transcriptome. The surrogate assay targets fewer genes yet permits in silico extrapolation to identify changes across the whole transcriptome. Both approaches identified differentiating biomarkers and mechanistic pathways in prostate cancer, both corroborating what has been reported and adding new mechanistic insights which will be discussed. These new observations likely result from the sensitivity of the TempO-Seq assays and their robust performance measuring gene expression from focal areas of FFPE, which in turn permit improved isolation of histology. Benefits derived from the TempO-Seq platform which will be discussed are:

1) Insensitivity to RNA degradation (measurements from intact RNA RIN=9.1 correlate to measurements from degraded RNA RIN=3.0, R2=0.97).

2) Does not require RNA extraction. Measures total RNA from FFPE samples, both soluble RNA in the lysate and crosslinked insoluble RNA.

3) Simple capture-free ligation-based assay protocol, without 3' or 5' bias to the probes used to measure each gene. No requirement for poly-adenylation or capture of the RNA (typically required in ligation-based assays).

4) Single base specificity derived from the specificity of probe ligation.

5) Misligation rates <0.01% (unlike other ligation-based assays where misligation can be as great as 30%) enable no sample background controls to be run with typical counts of 0 for most genes and 1 to 3 counts for a few genes.

6) High signal-to-noise provides single cell sensitivity and ability to measure differential gene expression from small focal areas of tissue.

7) Excellent reproducibility (average ~5% CV) between biological replicates across all expressed genes results in high significance of even small fold change differences.

8) Sequencing only the ligated probes complementary to a 50-base sequence of each gene eliminates the need for bioinformatics to analyze the sequencing data. Pooling of samples into a single sequencing run permits 42 or 865 surrogate samples and 8 or 153 whole transcriptome samples to be sequenced at the same time on the MiSeq or NextSeq, respectively, at an average of 250 counts/expressed gene. In combination, this results in low sequencing cost/sample without sacrifice of information or quality.

9) Simple, robust protocol requires only a PCR or qPCR instrument, and access to a sequencer.

#1841

Dissecting gene expression programs that define tumor aggression and patient outcome in pancreatic cancer.

Danielle J. Fassler, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. _Stony Brook University School of Medicine, Stony Brook, NY_.

By the year 2020, pancreatic cancer (PDAC) is projected to be the second leading cause of cancer deaths in the United States. Current systemic therapies offer limited survival advantages in subgroups of patients, suggesting underlying biological differences in this deadly cancer. Our lab has recently identified keratin-17 (K17) gene expression as a prognostic and predictive biomarker in PDAC. Furthermore, we found that K17 acts as an oncoprotein by promoting degradation of tumor suppressor p27KIP1, while knockdown of K17 limits tumor size and sensitizes cancer cells to chemotherapy agents. Together, these findings suggest that targeting K17 expression in PDACs may increase patient survival and response to treatment. The factors that lead to increased K17 gene expression in only a subset of PDAC cases, however, are unknown. K17 is expressed in embryonic ectoderm and aberrantly induced in keratinocytes from psoriasis patients through paracrine signaling from the microenvironment. To determine how K17 gene expression is upregulated in PDAC, we tested the effects of two major contributing factors in PDAC pathogenesis: driver mutations and inflammatory mediators present in tumor microenvironment. Using in vitro and in vivo loss- and gain-of function approaches, we modulated oncogene expression in and performed acute cytokine treatments on cells to test for changes in K17 gene expression. K17 expression was evaluated by qRT-PCR and western blot. We found that forced expression of oncogenes like KRASG12D, Q61K or TP53R248W significantly increase K17 expression, yet these cell-autonomous factors are not sufficient to explain K17 gene expression in PDACs. In addition, we found that pro-inflammatory cytokines, typically released by helper T cells, induce a strong increase in K17 gene expression. Together these findings suggest that K17 gene expression across otherwise clinically identical PDAC cases may be explained by early transformation events and maintained by positive feedback loops associated with patient specific adaptive immune response. Ongoing studies are seeking to test the combined effects of genetic and paracrine events in K17 expression, and understand the signaling pathways and transcription factors downstream of these inducing factors. Ultimately, insight into the mechanisms that mediate K17 overexpression in PDACs could help guide future research to develop K17 as a potential therapeutic target.

#1842

Overexpressing human endogenous retroviral elements (HERVs) in chemorefractory colorectal adenocarcinoma cells are repressed by antiviral drugs.

David Diaz-Carballo. _Marienhospital Herne, Ruhr University of Bochum, Herne, Germany_.

Background: HERVs account for ca. 8 % of all transposable elements found in the genome of humans and other animals. They represent a genetic footprint of ancestral germ-cell infections of exoviruses that is transmittable to the progeny by Mendelian segregation. Traces of human endogenous retroviruses are physiologically expressed in ovarial, testicular and placental tissues as well as in stem cells. In addition, a number of these fossil viral elements have also been related to carcinogenesis. However, a relation between endoretroviruses expression and chemoresistance has not been reported yet. Methods: Twenty colorectal carcinoma patient samples were analyzed for HERV-WE1 and HERV-FRD1 endoretroviruses using immunohistochemical approaches. In addition, to examine for differential expression of HERVs in chemotherapy refractory cells, a resistant HCT8 colon carcinoma subline was developed by serial etoposide exposure. Endoretroviral elements were detected by immunocytochemical staining, qPCR and ELISA. IC50-values of antiviral and cytostatic drugs in HCT8 cells were determined by MTT proliferation assay. The antivirals-cytostatics interaction was evaluated by the isobologram method. Results: In this work, we show for the first time that HERV-WE1, HERV-FRD1, HERV-31, and HERV-V1 are a) simultaneously expressed in treatment-naïve colon carcinoma cells from patients and b) upregulated after cytostatic exposure in colon-cancer cell lines, suggesting that these retroviral elements are intimately related to chemotherapy resistance. A number of antiviral drugs showed not only cytotoxic activity but also downregulation of HERV proteins in vitro. We also demonstrate that the use of different antiviral compounds alone or in combination with anticancer agents present synergistic antiproliferative activity and downregulation of different endoretroviral elements in highly chemotherapy-resistant colorectal tumor cells. Conclusions: Enhanced HERV-expression is associated with chemo-resistance in colon carcinoma cells which can be repressed by antiviral drugs alone or in combination with anticancer drugs. Therefore, the introduction of antiviral compounds to antiproliferative chemotherapy regimens potentially improves patient outcomes.

#1843

Identification of peritoneal dissemination-related genes in gastric cancer.

Qingjiang Hu,1 Shiya Kidogami,1 Sho Nambara,1 Hisateru Komatsu,1 Kuniaki Sato,1 Yushi Ogawa,1 Tomoko Saito,1 Shotaro Sakimura,1 Hidenari Hirata,1 Ryutaro Uchi,1 Naoki Hayashi,1 Tomohiro Iguchi,1 Takaaki Masda,1 Shuhei Ito,1 Hidetoshi Eguchi,1 Yoshihiko Maehara,2 Koshi Mimori1. 1 _Department of Surgery, Kyushu University Beppu Hospital, Beppu City, Oita Prefecture, Japan;_ 2 _Department of Surgery and Science, Kyushu University Hospital, Fukuoka City, Fukuoka Prefecture, Japan_.

INTRODUCTION:

Peritoneal dissemination (PD) is highly frequent and incurable metastasis in gastric cancer (GC). The mechanism causing PD is still poorly understood. In this study, we aimed to identify the genes responsible for PD using several large-scale public microarray databases.

MATERIALS AND METHODS:

First, we identified the candidate genes, which satisfied the all-4 outlines using microarray data as follow: 1) overexpressed in GC cell lines with high potential of PD; 2) overexpressed in GC patients with PD in Singapore from Duke-NUS Graduate Medical School; 3) overexpressed in tumor tissues in GC from TCGA database; 4) poor prognostic factor in GC from TCGA database.

Next, we measured the expression of candidate genes by Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and performed clinicopathological studies of the expression in 146 Japanese GC patients who underwent surgery in our hospital.

Finally, we performed gene set enrichment analysis (GSEA) with TCGA database to elucidate the correlation between candidate genes and gene sets that are associated with tumorigenesis or tumor progression in GC. Then, we examined the correlation between the candidate genes and one of the gene sets that were identified through GSEA by qRT-PCR in 5 GC cell lines (MKN7, KATO3, AGS, HSC39, 39As).

RESULTS:

1. We identified Arl4c (ADP-ribosylation factor-like 4c) as a PD candidate gene by above our screening system.

2. The expression of Arl4c was significantly higher in tumor tissues than in corresponding normal tissues in Japanese GC patients (p<0.0002). In clinicopathological analysis, the high expression group of Arl4c was significantly associated with depth of invasion (p<0.01) and PD (p<0.05). Multivariate analysis indicated that high expression of Arl4c was an independent prognostic factor (hazard ratio 2.20; 95 % CI 1.06-4.97; p< 0.03) among all clinicalpathological factors in GC.

3. GSEA revealed that the expression of Arl4c positively correlated with epithelial-mesenchymal transformation (EMT) gene set. In the 5 GC cell lines, the expression of Arl4c negatively correlated with the expression of cdh1, which was well known as a down-regulated gene in EMT.

CONCLUSION:

Arl4c is a member of the ADP-ribosylation factor family of GTP-binding proteins and plays a role in cholesterol transport. Recent study revealed that Arl4c regulates YAP/TAZ pathway that is well known to induce EMT and promotes tumorigenesis in lung or colorectal cancer. Our results suggest that Arl4c should be involved in PD via EMT induced by the YAP/TAZ pathway, resulting in a poor prognostic marker in GC. Arl4c may be a novel biomarker and a therapeutic target for PD in GC.

#1844

MDM2 overexpression promotes angiogenesis and metastasis through dysregulation of MMP-9 and TIMP-1 pathway.

Ali Alaseem,1 Thiagarajan Venkatesan,2 Priya Dondapati,1 Saad Alobid,1 Appu Rathinavelu1. 1 _College of Pharmacy, Nova Southeastern University, Plantation, FL;_ 2 _Nova Southeastern University, Plantation, FL_.

MDM2 is a multifunctional oncogene that has been associated with highly prevalent and aggressive cancers such as colon and prostate. MDM2 promotes a negative regulation of p53 activities and thus can prevent several of p53 protective effects including DNA repair, cell cycle control and apoptosis. Several pharmacological blockers that target the N-terminal domain of MDM2 leading to a disruption of MDM2-p53 interaction are being developed. For instance, nutlin-3 is the prototype of this family and has been shown to confine the tumor growth in many cancer types. Recently, it has been confirmed that MDM2 also promotes metastasis and angiogenesis through activation of number of mediators such VEGF and HIF-1α. However, mounting evidence suggests that MDM2 has p53 independent functions. Therefore, understanding the mechanisms by which MDM2 promotes the metastatic ability of tumors might provide an invaluable therapeutic target and a potential prognostic biomarker. Particularly, MMP-9/TIMP-1 dysregulation has been known to be associated with cancer progression and metastasis. This system is involved in the digestion of extracellular matrix and thus a release of several sequestered cytokines and growth factors such as TGFβ1. Among the release cytokines, TGFβ1 has been proven to be involved in promoting the MMP-9 expression and consequent metastatic events. Therefore, we hypothized that MDM2 promotes angiogenesis and metastasis through dysregulation of TIMP-1/MMP-9 pathway. Hence, the goal of our study was to evaluate the influence of MDM2 on tumor angiogenesis and metastatic ability through dysregulation of TIMP-1/MMP-9 pathway. To test our hypothesis, LNCaP prostate cancer and MDM2-transfected LNCaP-MST cell lines were used. On the other hand, nutlin-3 at the dose of 20 μM for 24 hours were used to examine the influence of MDM2 in regulating MMP-9/TIMP-1 in LNCaP and LNCaP-MST cells. Utilizing qPCR and western blotting techniques, we obtained data which suggest that MDM2 positively regulates the expression of MMP-9 and negatively regulates TIMP-1. We observed that MMP-9 overexpressed in LNCaP-MST, and this overexpression was reversed after nutlin-3 treatment in LNCaP-MST by 0.40 fold. Conversely, the results showed a complete abolishment of TIMP-1 protein level in LNCaP-MST, and nutlin-3 does not seem to impact TIMP-1 level. The data suggest that MDM2 positively impacts MMP-9 in a p53 dependent fashion as opposed to TIMP-1, which is negatively regulated by MDM2 independent of p53. In the same connection, TGFβ1 overexpression was reversed by 0.51 fold in LNCaP-MST after nutlin-3 treatment as shown by the qPCR results. Altogether, this project would provide a significant insight into understanding cancer metastasis processes with respect to MDM2 activity (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged).

#1845

Development of a quantitative targeted RNA-Seq methodology for use in differential gene expression analysis.

Eric Lader, Melanie Hussong, Matthew Fosbrink. _QIAGEN, Frederick, MD_.

RNA Sequencing (RNA-Seq) uses the capabilities of Next Generation high-throughput sequencing (NGS) methods to provide insight into the transcriptome of a cell as it generates millions of reads. Whole transcriptome sequencing can be used to quantify gene expression on a transcriptome-wide scale, identify splice variants, quantify allele specific expression, and characterize fusion transcripts. Development of a highly reproducible and sensitive targeted quantitative sequencing method would aid in facilitating a deeper understanding and characterization of the roles of a specific set of genes, while enabling much higher sample throughput and significant cost savings relative to whole-transcriptome sequencing. In this study, we report a targeted RNA-Seq technology, QIAseq RNA, which makes use of several methodologies to deliver an extremely flexible, highly precise tool for characterizing gene expression. QIASeq RNA incorporates 12-base random molecular barcodes into each unique target strand which benefits quantifying gene expression in a given multiplexed sample. Counting the number of molecular tags allows one to determine the sequence coverage per target and adjust experimental conditions to use the read budget of any sequencing platform most efficiently. Using either the Illumina or Ion Torrent platforms, users can choose to multiplex up to 96 RNA samples from 12 to 1000-plex expression panels. No mRNA selection or rRNA removal or blocking is required. The entire protocol, from cDNA synthesis to finished library, which is ready for sequencing, can be accomplished in under one day. Custom assays for a specific target site can add the ability to distinguish between isoforms or identify allele specific expression. We explore the capabilities of this system by profiling large numbers of genes in a cell model system's response to small molecule treatment. Changes in gene expression by these treatments were measured by targeted RNA NGS, and fold-changes in gene expression due to chemical perturbation were characterized. Complex gene relationships in perturbed pathways were mapped using QIAGEN's Ingenuity Pathway Analysis (IPA) tool. The IPA tool also facilitated the elucidation of the impact of gene expression changes in the context of biological processes, molecular interactions, cellular phenotypes and disease. This article will provide application data for GRRC including a discussion of technical challenges faced when profiling large numbers of genes in a large cohort.

#1846

Gene expression analysis of P-glycoprotein and LMWPTP in chronic myeloid leukemia cells treated with metformin and imatinib mesylate.

Ligia P. Oliveira, Rafael G. Ferro, Ana CSS Galvão. _UFABC, Santo André, Brazil_.

Chemotherapy is an effective treatment for many tumors; however, some tumors begin to exhibit a wide range of resistance after a few cycles of chemotherapy, which is known as phenotype of resistance to multiple drugs (multidrug resistance - MDR). The MDR phenotype arises through various mechanisms, but often is associated with an increased efflux of hydrophobic cytotoxic drugs. Therefore, the search for more selective and efficient antitumor compounds in the elimination of cancer cells boosts the research in oncology area. Studies of genes and pathways that are deregulated in tumors and that are essential to their growth may provide new strategies that lead to the development of therapeutic agents that target these pathways. Previous data from our research group have shown potent toxic activity of metformin, an oral hypoglycemic used for more than 30 years for the treatment of type II diabetes, on chronic myeloid leukemia cells (CML), potentiating the effects of imatinib mesylate, the standard chemotherapy used in the treatment of CML. CML cells treated with these agents showed changes in expression of proteins associated with the MDR phenotype. In this way, this project aimed to evaluate the expression of ABCB1 (Pgp) e ACP1 (LMWPTP) by Polymerase Chain Reaction in real time (qPCR) in LCM K562 cells and Lucena before and after treatment with metformin and/or imatinib mesylate, seeking to verify the effects of metformin, alone or in combination with Imatinib mesylate in the expression of these genes in Lucena in K562 cells. We found statistically significant differences in expression between treatments compared to control and between K562 and Lucena cells. It is possible to conclude that these genes may be associated with MDR phenotype.

#1847

The mRNA-binding protein HuR, regulates mutant and wild type IDH1 expression in IDH1-mutated cancer.

Mahsa Zarei, Shruti Lal, Nicole C. Mambelli-Lisboa, Edwin Cheung, Saswati N. Chand, Charles J. Yeo, Jonathan R. Brody, Jordan M. Winter. _Thomas Jefferson University, Philadelphia, PA_.

Introduction: Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancers, predominantly an arginine to histidine amino acid change at codon 132. This structural alteration drives the catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate (2HG), which enhances DNA and protein hypermethylation and cellular dedifferentiation. Pharmacologic inhibitors of the mutant IDH1 protein show promise in clinical trials, yet the regulation of IDH1 has not been fully elucidated. We recently discovered in wild type IDH1 tumors that the regulatory RNA-binding and stress response protein, HuR (ELAVL1), protects cancer cells under nutrient deprivation by regulating the expression of core metabolic enzymes, including IDH1. We mapped the regulatory HuR binding site on the IDH1 transcript to the 3'-untranslated region. Since wild type and mutant IDH1 alleles both contain this binding sequence, we hypothesize that HuR is an important regulator of both isozymes and is biologically important in mutant IDH1 tumors.

Methods: HuR expression was suppressed by siRNA in a fibrosarcoma cell line (HT-1080) harboring a natural heterozygous IDH1 mutation and cell viability was determined by PicoGreen and Trypan blue exclusion assays. Sensitivity to pharmacologic inhibition of the mutant IDH1 protein was assessed. Sanger sequencing and mRNP-IP were performed to determine HuR's impact on the expression of each IDH1 allele. To further characterize the post-transcriptional regulation of IDH1 by HuR, CRISPR/CAS 9 editing of the HuR gene was performed.

Results: Sanger sequencing of IDH1 after depletion of HuR in HT1080 cells, as well as after HuR mRNP-IP, revealed that HuR regulates both the mutant and wild type IDH1. Drug sensitivity assays to a mutant IDH1 inhibitor (AGI-5198) under varying glucose concentrations revealed glucose deprivation to be a novel driver of chemo-resistance. However, HuR silencing abrogated HT1080 resistance to AGI-5198 under these conditions. Targeted knockout of HuR using the CRISPR/CAS9 system resulted in potent suppression of mutant and wild type IDH1, at both the mRNA and protein levels when compared to CRISPR/Cas9 control.

Conclusions: These findings reveal that harsh metabolic conditions present in the tumor microenvironment induce chemo-resistance in an IDH1 mutant cell line to a mutant IDH1 inhibitor, in an HuR dependent manner. Moreover, both mutant and wild type IDH1 alleles are potently regulated by HuR. Therapeutic strategies that target the HuR-IDH1 axis and block this resistance mechanism may enhance the efficacy of mutant IDH1 inhibitors for relevant tumors.

#1848

Total RNA discovery: Analyzing mRNA, miRNA and lncRNA as a strategy to identify novel biomarkers.

Brian M. Dugan, Jonathan Shaffer. _Qiagen, Germantown, MD_.

Identifying biomarkers is not limited to only novel sequence variation. For multiple RNA species, differential expression between tissues and pathologies offer distinctions that can be used to identify disease. Many gene expression studies historically only analyzed messenger RNA; however, key regulatory RNA have grown specific targets for novel biomarker identification. By looking at a more total RNA, gene expression cannot only be observed, but the underlying regulatory factors can be determined.

Total RNA, once thought to be only mRNA, has evolved to include not only the coding, but the non-coding transcripts as well. These non-coding RNA specifically regulate the expression of mRNA and the production of proteins. Two major RNA species, microRNA and long non coding RNA, are particular targets of interest. miRNA and lncRNA are noted to be key indicators of cellular dysregulation and can be identified not only in tissue but in exosomes and in serum testing.

The immune system represents a key opportunity to not only understand disease progression through immune-surveillance, but also as a mechanism to induce innate anti-tumor response. To identify key biomarkers associated with directed response, treated and untreated immune cells were isolated and multiple RNA types were analyzed. By applying an integrated approach to pan RNA quantification, we were able to demonstrate that multi-analyte analysis of coding and non-coding RNA yields important markers that may drive anti tumor responsiveness. mRNA, miRNA and lncRNA were quantified using qPCR. Using a systems biology analysis, the RNA types were analyzed to demonstrate not only differential expression across treatment modalities, but observe that non-coding RNA changes affect mRNA expression.

The analysis demonstrates that a combined multi-RNA approach on specific sample types allows for not only identification of novel expression but identification of significant regulatory affects unique to different treatments.

#1849

CXXC5 is overexpressed in human prostate epithelial cancer cell lines in culture and human prostate cancer tissue.

Ines Benedetti, Alfonso Bettin, Niradiz Reyes. _Universidad de Cartagena, Cartagena, Colombia_.

Background: CXXC5 encodes a retinoid-inducible nuclear factor that plays an essential role in human hematopoiesis (1, 2). We have previously identified the differential expression of this gene in rat cancer cell lines with different cancer phenotypes (3). However, knowledge on the expression of this gene in human prostate cell lines or tissue is lacking, both at the transcript and protein level.

Methods: Prospective study conducted between January-2012 through June-2015 at the University of Cartagena, Colombia. Expression of CXXC5 was evaluated by quantitative PCR, both in prostate cancer cell lines and tissue biopsies from patients undergoing transrectal ultrasound-guided prostate needle biopsy for suspected prostate cancer (PCa). A total of 65 prostate tissue samples, consisting of 24 benign prostatic tissue biopsies and 41 prostate cancer tissue biopsies were included in the study. Transcript levels in individual biopsies from each group were normalized to transcript levels of beta-actin of the corresponding sample and compared. The protein expression level of CXXC5 was analyzed by immunohistochemistry in benign and malignant prostate tissue. A final score was determined by multiplying the highest intensity of staining by the mean percentage of positive cells stained to yield a value between 0 and 300 for each case. For statistical analysis we compared parameters between group of cases with PCa and without PCa. This study was approved by the Ethical Review Board of the academic institution.

Results: CXXC5 transcript expression levels were significantly lower in immortalized non-tumoral prostate cell line PWR-1E compared to PCa cell lines: LNCaP (Fold change: 10.5, p=0.001), and PC3 (Fold change: 2.8, p = 0.014). In prostate tissue we observed a significant higher expression in low grade prostate cancer compared to benign prostate tissue (p = 0.034). IHC evaluation showed that CXXC5 staining was higher in tissue samples from patients with prostate cancer compared to patients with benign disease (p= <0.001). In the prostate cancer group, immunostaining was also stronger in malignant acini than in adjacent, benign acini, within the same patient sample.

Conclusions: CXXC5 exhibits differential expression in prostate cell lines with different cancer phenotypes and between cancer and benign prostate tissue. Additional studies are required to determine the biological and clinical significance of CXXC5 in prostate cancer.

References

1. J Biol Chem, 2009. 284(6): p. 3672-81.

2. Oncotarget, 2013. 4(9): p. 1438-48.

3. Biomedica, 2007. 27(2): p. 190-203.

#1850

Transcription factor E2F3 up-regulates the expression of Gadd45b gene in the mouse liver.

Jung-Hwan Kim,1 Frank J. Gonzalez2. 1 _Gyeongsang National University, Jinju, Republic of Korea;_ 2 _National Cancer Institute, MD_.

Growth arrest and DNA damage-inducible beta (GADD45b) plays an important role in many intracellular events, such as cell survival- and cell death-related signaling. In normal condition, Gadd45b is suppressed by the negative regulator, such as STAT3, however, when it induced by the PPARa agonist, Wy-14,643. Thus, we investigated the positive regulator of Gadd45b. Based on the Gadd45b-reporter assay, we assumed that E2Fs could regulate the Gadd45b. As results, E2F3 mRNA was induced only in the liver of Wy-14,643-treated mice. In addition, deletion of E2Fs consensus sequence in the Gadd45b reporter gene showed significant decrease of luciferase activity. Furthermore, overexpression of E2F3 significantly increased the Gadd45b-luciferase activity and this was inhibited by the siE2F3 in the Hepa1c1c7 cells. Taken together, E2F3 may play a pivotal role in the Gadd45b induction as a positive regulator.

#1851

E6-associated protein-dependent estrogen receptor modulation of protein kinase A regulatory subunit R2A expression in neuroblastoma.

Jean-Pierre Obeid, Nawal Zafar, Jimmy El Hokayem. _University of Miami, Miami, FL_.

Purpose/Objective(s): Steroid hormone receptors, upon hormonal activation, act to modulate gene expression in receptive cells through interactions with coregulator proteins. Protein Kinase A (PKA) is a particularly potent downstream effector, and is conservatively regulated in a number of biochemical pathways. The current project aims to elucidate the mechanisms of action of E6AP, a known critical player in endocrine cancer on the expression/activity of PKA in neuroblastoma.

Materials/Methods: A microarray genetic analysis yielded a decreased PKA-R2A expression dependent upon decreased levels of estradiol (E2) or E6AP. This correlation was validated via a PCR determined downregulation of PKA-R2A mRNA with E6AP knockdown in Neuro2a cells. Bioinformatics provided four potential Estrogen Response Element (ERE) sequences in the PKA-R2A promoter region. These sequences were cloned to a Luciferase reporter gene vector. HeLa cells harboring these constructs were incubated with and without E2. Transcriptional activity was surrogated by bioluminescence via a Luciferase-Luciferin reaction, normalized to Renilla luminal output. Electrophoretic mobility shift assay (EMSA) was performed to detect direct ER binding to biotin-labeled ERE in the presence and absence of E2. ER-specific antibodies generated a super-shift. Unlabeled and consensus ERE allowed for competitive binding. Chromatin immunoprecipitation (ChIP) investigated recruitment of the complete transcriptional complex E6AP-ER onto the promoter region of the PKA-R2A gene and its association with active expression of the gene in Neuro2a cells.

Results: A Luciferase luminal output to unit Renilla luminal output ratio was obtained in the absence and presence of E2. The scrambled control yielded ratios of 2.77 and 1.97, respectively. The consensus control yielded 9.55 and 85.37. ERE-1 yielded significant ratios of 8.57 and 55.7. EMSA evaluation of ERE-1 demonstrated a biotin-labeled ERE band shift in the presence of ER incubated with E2 relative to that without E2, and a super-shift with addition of an Anti-ER antibody. Competitive saturation with both the unlabeled and consensus ERE sequences negated these observed shifts. ChIP results show, upon E2 treatment in Neuro2a cells, increased recruitment of the E6AP-ER complex to the same ERE-1 associated with increased H3K14 acetylation and p300 recruitment at the same site which was coupled with the recruitment of pRNAPolII to the transcriptional start site of the PKA-R2A gene indicating active transcription and expression of the gene.

Conclusion: ERE sequences exist and appear to function in response to E2 within the PKA-R2A promoter region, modulating the transcriptional status of a ubiquitous and crucial protein. Novel therapies targeting this hormonal foundation may potentially play a role in ameliorating the symptomatic manifestations associated with neuroblastoma.

#1852

Multiple PI3K pathway mutations predominate in low stage endometrial carcinomas.

Dario R. Roque,1 Will R. Jeck,2 David N. Hayes,1 Arthur M. Dizon,1 Leslie Clark,1 Stuart Pierce,1 Weiya Z. Wysham,1 Tara Castellano,1 Paola A. Gehrig,1 Victoria Bae-Jump1. 1 _University of North Carolina, Chapel Hill, NC;_ 2 _Massachusetts General Hospital, Boston, MA_.

Objectives: Endometrial cancers (ECs) frequently harbor mutations in PI3K pathway genes. Our objective was to determine the mutation frequency of the PIK3 pathway in the tumors of EC patients prospectively enrolled on an IRB-approved institutional tumor sequencing initiative (UNCseq) and correlate these with clinical factors.

Methods: Snap-frozen and FFPE tissue samples were collected from patients enrolled on UNCseq (NCT01457196). DNA was isolated using the Puregene DNA purification system, and Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets, were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Statistical analysis of clinical correlates was performed in R (v 3.1.1).

Results: Data was collected from 109 EC patients. Thirty-six tumors (33.0%), 34 of which were endometrioid, had >2 mutations between PIK3CA and PIK3R1. 20 patients had multiple PIK3CA mutations (18.3%) and 11 had multiple PIK3R1 mutations (10.1%). In all but 5 cases, multiple mutations of PIK3R1 or PIK3CA were coincident with PTEN mutation (p = 0.018). There were strong correlations between PI3K pathway mutations and tumor grade and stage. High stage tumors (stage >2) were less likely (5/36) than stage 1 tumors (31/73) to have multiple PI3K mutations (p=0.0056). Though not reaching significance, grade 3 tumors (8/37) were less likely than low-grade tumors (28/72) to demonstrate multiple PI3K mutations (p=0.11). Of the 10 serous ECs, none exhibited multiple PI3K mutations.

Conclusions: There is a strong relationship between mutated PIK3 pathway genes and clinically relevant tumor biology in ECs. Multiple mutations in the PIK3 pathway are common, suggesting that PI3K pathway inhibition in EC may need to be directed against PIK3CA and PIK3R1 simultaneously. These multiply mutated tumors are strongly linked to lower stage and grade tumors with concurrent PTEN mutations. We posit that initially PTEN mutant clones may give rise to subclonal populations with various PI3K mutations which may be relatively indolent until additional driver mutations are acquired such that one subpopulation predominates, resulting in higher grade and stage ECs.

#1853

Single-cell multiplexed profiling of protein-level changes induced by EGFR inhibitor gefitinib.

Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Benjamin Liu, Marc Unger, Ramesh Ramakrishnan, Haibiao Gong. _Fluidigm Corporation, South San Francisco, CA_.

Single-cell molecular analysis has become increasingly important in biomedical research, especially in the field of cancer research, where cell heterogeneity is a characteristic feature. We developed a multiplexed single-cell protein detection assay for the C1™ system that can isolate and process 96 individual cells in parallel. The assay uses oligonucleotide-conjugated antibodies, which produce a detectable DNA amplicon only when they bind to the target protein. The resulting DNA amplicons are quantified by qPCR on the Biomark™ HD system. Data are analyzed using the Singular™ Analysis Toolset. A total of 50 antibody binders were developed for this assay to target proteins related to apoptosis, cell cycle, cell proliferation, tumor suppressors, biomarkers, stem cells, growth factors and proteins associated with specific cancers, such as breast and prostate cancers. The assay can be performed in a 48-plex format, which yields over 4,600 datapoints in two days. Assay sensitivity and specificity were assessed using recombinant proteins and various cell lysates. To demonstrate the usefulness of this assay, we used it to profile the protein level changes in A431 cells treated with epidermal growth factor receptor (EGFR) inhibitor gefitinib. EGFR is a member of ErbB family of type I tyrosine kinases, whose overexpression and mutations are involved in a variety of cancers. While much effort has been made to investigate the consequences of EGFR activation or inhibition, hardly any information is available regarding how EGFR regulation changes protein expression in single cells. Using a multiplexed protein assay, we discovered some interesting co-expression patterns of proteins in single cells. We also demonstrated that TNFRSF10B was upregulated, while MET, GATA3, BRCA1, CCNB1, Ki-67 and CCNA2 were downregulated by gefitinib treatment. Notably, the reduction of the cell cycle-related proteins CCNB1 and CCNA2 and the cell proliferation marker Ki-67 was consistent with the inhibited cell growth by gefitinib. In addition, we measured mRNA expression to investigate the association between protein and mRNA levels in single cells. Single-cell analysis—determining protein changes and analyzing the correlation between different proteins at the single-cell level—yields valuable information that is not achievable using bulk-cell analysis.

#1854

The regulation of angiogenin expression and the genes regulated by angiogenin.

Jinghao Sheng,1 Guo-fu Hu,2 Zhengping Xu1. 1 _Zhejiang University School of Medicine, Hangzhou, China;_ 2 _Tufts Medical Center, Boston, MA_.

Angiogenin (ANG) is first isolated and identified by its ability to stimulate the growth of blood vessels. As a member of the vertebrate-specific secreted ribonuclease (RNase) family, ANG is encoded by a single exon and is usually located in the middle of its family gene cluster. Particularly, ANG gene locus has a unique gene arrangement, featured by shared promoters and 5'-untranslated regions (5'-UTR) which direct two distinct exons enconding ANG and ribounclease 4 (RNASE4, the 4th member of RNase family) respectively. However, the gene structure and regulation of these genetic regions are largely unknown. Here, we have characterized the promoters, defined the transcription start site, and identified a unique mechanism of transcription regulation. We demonstrated that two Pol III elements within the promoter regulate ANG and RNASE4 expression in a position- and orientation-dependent manner. Moreover, an intragenic chromatin loop formed between the two CCCTC-binding factor (CTCF)-binding sites located in two introns flanking ANG coding exon, which preferentially enhances ANG transcription. These results suggest a multilayer transcriptional regulation of ANG and RNASE4 gene locus. The data also add more direct evidence to the notion that Pol III elements are able to directly influence Pol II gene transcription. Furthermore, our data indicate that a CTCF-dependent chromatin loop is able to differentially regulate transcription of genes that share the same promoters.

On the other hand, the secreted ANG undergoes a receptor-mediated endocytosis from the cell surface to the nucleus and accumulates in the nucleoli. The nucleoli ANG can promote 47S pre-rRNA transcription by binding the ABE (Angiogenin Binding Element) and UCE (Upstream Core Element) region on the promoter of ribosomal DNA (rDNA), where ANG increases the number of actively transcribing rDNA and promotes the assembly of initiation complex by epigenetic activation through promoter methylation and histone modification. We have also shown that the surplus ANG in nucleus also related to mRNA transcription. ANG binds the first exon region of estrogen receptor-related receptor gamma (ERRγ) and inhibits the ERRγ expression. To screen and identify the mRNAs regulated by ANG at a genome-wide level, we carried out chromatin immunoprecipitation-chip assay (ChIP-on-chip) and found a total of 699 genes that may be regulated by ANG. These genes were significantly enriched to tumorigenesis, Wnt and TGF-beta pathways by the KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. Based on the findings that ANG is able to remodel the histone modification through direct binding to the histone protein, we propose that ANG might act as a chromatin remodeling activator to regulate RNA transcription. However, there is still a long way to go before fully disclosing the roles and mechanisms of ANG in gene transcription.

#1855

Role of mTOR inhibition in triple-negative breast cancer.

Daniela Massihnia,1 Giuseppe Bronte,2 Marta Castiglia,3 Nadia Barraco,2 Antonina Cangemi,1 Alessandro Perez,1 Daniele Fanale,1 Gianni Pantuso,1 Salvatore Vieni,1 Valentina Calò,1 Christian D. Rolfo,4 Viviana Bazan,1 Antonio Russo1. 1 _University of Palermo, Palermo, Italy;_ 2 _Univeristy of Palermo, Palermo, Italy;_ 3 _Univeristy of Palermo, palermo, Italy;_ 4 _Antwerp University Hospital, Edegem, Belgium_.

BACKGROUND:

Triple-negative breast cancers (TNBC) represent the 10-17% of all diagnosed breast cancers (BC) and are characterized by the absence of ER/PgR expression, HER2 amplification and often show a basal-like phenotype. TNBC are often diagnosed in patients with BRCA1 germline mutation and unfortunately treatment options are still limited. The mTOR (Mammalian Target Of Rapamycin) pathway seems to play an important role in BC pathogenesis and it is possible to target this pathway by inhibitors such as rapamycin. In human BC cross talk between ER/PgR receptors signaling and the mTOR pathway is believed to be responsible for resistance to hormone therapy probably due to a down regulation of hormone receptors. Based on these evidences we have hypothesized that the inhibitors of mTOR pathway may lead to the up-regulation of ER, PgR and HER2 in TNBC cell lines.

METHODS:

For this study we used TNBC cells (MDA-MB-231 and BT20) cultured in DMEM:F12 (Dulbecco's Modified Eagle's Medium) with 10% bovine serum (FBS), 100 U/mL penicillin (1%) and 50 mg/mL of streptomycin under standard conditions (37°C in an atmosphere composed of 16% O2, 79% N2 and 5% CO2).

Cells were initially treated with Rapamycin (1, 5, 10, 15 microM) for 24, 48, and 72h in order to verify if the drug determines a blockade of cell proliferation. Before drug administration, the cells were subjected to serum starvation by eliminating serum from culture medium. The evaluation of cell viability following the administration of the drug was carried out using the MTT assay. Real time PCR analyses were carried out in order to evaluate gene expression modifications of ER, PgR and HER2 receptors, through Taqman probe chemistry.

RESULTS:

The preliminary cell viability experiments conducted on different TNBC cell lines (MDA-MB-231 and BT20), showed no significant cytotoxic effects by increasing Rapamycin concentrations (1, 5 and 10 microM) after 72h treatment, except for the higher concentration (15 microM) for which a cytotoxic effect was observed.

The following qPCR approach highlighted significant variations in estrogen and progesterone receptor gene expression for the TNBC cell lines after 24, 48, 72h with 1, 5, 10 microM Rapamycin. In particular their expression level resulted up-regulated. Unlikely no association between mTOR inhibition and HER2 expression level were identified, suggesting no effects of mTOR inhibition on HER2 expression.

CONCLUSION AND FUTURE PERSPECTIVES:

To these preliminary results suggest that the mTOR inhibition leads to re-expression of hormone receptors. This finding supports a potential clinical application of mTOR inhibition in TNBC. The perspective of phenotype change upon rapamycin treatment prompts new therapeutic scenarios. However, further investigations are needed to explain the biological mechanisms driving these changes.

#1856

Transcriptional regulation of the UDP-glucuronosyltransferases (UGTs) by SAFB1 and SAFB2: Strategy to reduce DHT levels in prostate cancer cells.

Mirja Rotinen, Julie S. Yang, Sungyong You, Jayoung Kim, Beatrice Knudsen, Michael R. Freeman. _Cedars-Sinai Medical Center, Los Angeles, CA_.

Backround: Androgen deprivation therapy (ADT) is a first-line option for prostate cancer (PC) treatment. While there has been some progress inhibiting AR activation by focusing on DHT synthesis, other mechanisms to reduce intracellular DHT have not been exploited for drug development. The UDP-glucuronosyltransferase (UGT) enzymes UGT2B15 and UGT2B17 inactivate DHT in the prostate by adding a bulky UDP-glucuronic acid moiety to the hormone. These reactions are irreversible and therefore represent a prominent mechanism for depletion of intratumoral androgen in vivo. Here we show evidence that the chromatin scaffolds SAFB1/2 activate expression of UGT2B15/17. This is a novel activity for these proteins, which are known primarily as inhibitors of nuclear receptors (Oesterreich, S. et al. Molecular Endocrinology, 2000 and Debril, MB. et al. Molecular Endocrinology, 2005). We previously showed that SAFB1 can act as an AR suppressor that cooperates with the MST1 kinase and the EZH2 methyltransferase to modify chromatin at AR-responsive genes (Mukhopadhyay et al. Oncogene, 2014).

Methods: Enforced expression, silencing, RNA profiling, quantitative PCR, chromatin immunoprecipitation (ChIP) and ChIP-Seq, mass spectrometry and luciferase gene reporter assays.

Results: SAFB1/2 is genomically inactivated in a high percentage (~28%) of castration-resistant prostate cancer (CRPC). SAFB1/2 silencing, which emulates SAFB1/2 downregulation/gene inactivation, potently down-regulates UGT2B15/17 in LNCaP and 22RV1 cells. Conversely, enforced expression of SAFB1 upregulates UGT2B15/17. Genomic effects of SAFB1 loss on the AR cistrome in LNCaP cells by ChIP-seq are shown. Computational analysis indicates that SAFB1 regulates AR and EZH2-regulated genes, but not exclusively, indicating that gene regulation by SAFB1 independently of AR and EZH2 also occurs in prostate cells. SAFB1 silencing resulted in increases in several androgen species in LNCaP cells. Finally, SAFB1 is recruited to the UGT2B15/17 promoters and its effect on promoter luciferase constructs is analyzed.

Conclusions: This is the first identification of a molecular mechanism of regulation independent of androgen of the UGT2B15/17 genes in prostate cells. Our findings indicate that SAFB1 is an essential node where several oncogenic pathways converge onto chromatin to regulate gene expression in PC in a manner that can affect androgen degradative mechanisms.

#1857

Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis.

Paul Johnson,1 Ches'Que M. Phillips,1 Carl Grim,1 John R. Zatarain,1 Aakash H. Gajjar,1 Suimin Qiu,1 Rui Wang,2 Celia Chao,1 Iryna V. Pinchuk,1 Mark R. Hellmich1. 1 _University of Texas Medical Branch, Galveston, TX;_ 2 _Lakehead University, Thunder Bay, ON_.

Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC.

Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed.

Results:

Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p<0.001) and number (p<0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified.

Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future.

#1858

Analyzing the impact of MDM2 overexpression on cell cycle pathway-related genes using polymerase chain reaction array.

Priya Dondapati,1 Thiagarajan Venkatesan,2 Appu Rathinavelu2. 1 _College of Pharmacy, Health Professions Divison, Nova Southeastern University, Davie, FL;_ 2 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern University, Plantation, FL_.

The human homologue of the mouse double minute 2 (MDM2) oncogene has been well characterized as a negative regulator of the tumor suppressor p53. It has been established that p53 regulates genes that are involved in cell cycle arrest, apoptosis, DNA repair and senescence. MDM2 interacts primarily with p53 in an autoregulatory negative feedback loop and targets it for ubiquitylation and proteasomal degradation there by attenuating p53 cell cycle arrest and apoptosis functions. Studies have shown that MDM2 also exerts its effects via p53 independent pathways. These p53-independent functions of MDM2 may have a role in cancer etiology and progression, yet the exact function and significance of these interactions are not fully understood. MDM2 is overexpressed in more than 40 different types of malignancies, including wide varieties of carcinomas and sarcomas, which is believed to be associated with worse clinical prognosis and increased likelihood of distant metastases. Hence, MDM2 is proving to be a key player in human cancer and thus an important target for therapeutic intervention. In this study, we aimed to investigate the effects of MDM2 overexpression on cell cycle pathways by using LNCaP prostate cancer cells and MDM2 transfected LNCaP-MST. The LNCaP-MST cells expressed at least 10-folds higher levels of MDM2 mRNA due to transfection. We used the Human Cell Cycle PCR array to compare the gene expression profiles of LNCaP and LNCaP-MST cells. Our experimental results clearly indicated that MDM2 modulates the expression of cell cycle related genes. The following genes were found to be significantly down-regulated in LNCaP-MST cells compared to LNCaP: CCND2, GADD45A, RB1, RBBP8, CDKN1A and TP53. Genes such as BIRC5, CDK1 were found to be significantly up-regulated. Heat map and scatter plot analysis further confirmed the alterations in the expression levels of these genes following the MDM2 overexpression. In addition, cluster analysis further denoted that the gene expression patterns positively correlated with cell proliferation and tumor growth. Our experimental results offer conclusive evidence that MDM2 can impact the cancer growth and metastatic potential by altering the gene expressions in the cell cycle pathway. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged).

#1859

Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes.

Jan Sevcik,1 Leslie Burke,2 Gaetana Gambino,3 Brooke L. Brewster,2 Emma Tudini,2 Philip J. Whiley,2 Siranoush Manoukian,4 ENIGMA Consortium,5 Thomas van Overeem Hansen,6 Marta Santamariña Pena,7 Ana Vega,7 Maria A. Caligo,3 Paolo Radice,4 Paolo Peterlongo,4 Etienne Rouleau,8 Amanda B. Spurdle,9 Melissa A. Brown2. 1 _Charles University in Prague, Prague, Czech Republic;_ 2 _The University of Queensland, Brisbane, Australia;_ 3 _The University of Pisa, Pisa, Italy;_ 4 _Foundazione IRCCS Instituto Nazionale dei Tumori, Milano, Italy;_ 5 _ENIGMA, Queensland, Australia;_ 6 _Rigshospitalet - Copenhagen University Hospital, Copenhagen, Denmark;_ 7 _El Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Santiago de Compostela, Spain;_ 8 _Curie Institute, Paris, France;_ 9 _Queensland Institute of Medical Research Berghofer, Brisbane, Australia_.

Many germline DNA sequence variants in gene regulatory elements are associated with an increased risk of cancer. These include large genomic deletions of the promoter of the BRCA1 gene, through to single nucleotide variants in the promoter, UTRs and long-range enhancers of multiple other cancer related genes. Next generation sequence analysis of early onset and familial breast cancer cases is currently identifying an escalating number of variants in non-coding regions of the genome, however for most of these the significance in unknown. This project aims to determine the functional and clinical significance of breast cancer associated variants in regulatory regions of BRCA1 and BRCA2, as part of an international collaborative project arising from the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To date over two hundred variants mapping to promoter, UTR and deep intronic regions have been identified in early onset or familial breast cancer patients that have no identifiable coding or splicing changes in BRCA1 or BRCA2. Through a pipeline of bioinformatics analyses using ENCODE datasets, these variants have been prioritized for experimental evaluation. This has lead to the identification of several variants that significantly alter the regulation of BRCA1 or BRCA2 expression, through mechanisms including altered binding of transcription factors and microRNAs. These studies will lay the groundwork for comprehensive statistical analyses, in which bioinformatics and experimental data is combined with clinical and genetic data to establish multifactorial risk prediction models that can ultimately be used in a clinical setting.

#1860

MDM2 stabilizes and induces HIF-1α levels during reoxygenation of cancer cells.

Thanigaivelan Kanagasabai,1 Rohin Chand,2 AmyAman Kaur,2 Sivanesan Dhandayuthapani,1 Olena Bracho,1 Appu Rathinavelu1. 1 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern University, Plantation, FL;_ 2 _College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL_.

Hypoxia stimulates several pathways that are critical to cancer cell growth and survival, including activation of vascular endothelial growth factor (VEGF) transcription. Overexpression of VEGF and the extent of neo-angiogenesis are closely correlated with increase in tumor size and cancer metastases. The main regulator of hypoxia-induced angiogenesis is the hypoxia inducible factor (HIF)-1α which regulates the transcription of various target genes including VEGF. Stabilization of HIF-1α in cancer cells could enable tumor progression and results in poor survival. In this respect, the multifunctional mouse double minute 2 homolog (MDM2) oncoprotein has been gaining significant amount of attention. The MDM2 oncoprotein has been shown to exert both p53-dependent and p53-independent roles in oncogenesis. It is also well established that MDM2 gene amplification can occur in diverse human malignancies that include soft tissue sarcomas, neuroblastoma, cancers of the brain, breast, ovary, cervix, lung, colon, prostate, and bone. Mechanisms underlying induction and stabilization of HIF-1α in normoxic conditions are less clearly understood. Our present data indicated that the normoxic expression level of HIF-1α was significantly higher (p<0.0001) in LNCaP-MST as compared with LNCaP cells. When we treated the cells with the MDM2 inhibitor nutlin-3, the levels of HIF-1α was significantly down regulated. These results were further correlated with the differences in subcellular compartmentalization and degradation of HIF-1α. Findings from our lab shows the degradation rate of HIF-1α protein in both the nuclear and cytoplasmic compartments of LNCaP (non-transfected) and LNCaP-MST (MDM2 transfected) cells. In LNCaP cells, the degradation of HIF-1α protein occurs within 8 min in cytoplasmic (99%) and 10 min in nuclear compartments (75%). Whereas, in LNCaP-MST cells, the degradation rate of HIF-1α protein was slower even at 10 min of reoxygenation in both cytoplasmic (70%) and nuclear compartment (40%). Our results suggest that, MDM2 could play the role of master regulator in stabilizing HIF-1α even after reoxygenation. Further studies in this direction will shed more light on the in-depth mechanisms involved in the regulation of HIF-1α in MDM2 positive cancers. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida).

#1861

The role and regulation of MTA1-ETS2 axis in prostate cancer.

Avinash Kumar,1 Swati Dhar,1 Nasir Butt,1 Agnes Rimando,2 Luis Martinez,3 Anait Levenson1. 1 _University of Mississippi Medical Center, Jackson, MS;_ 2 _United States Department of Agriculture, University, MS;_ 3 _Stony Brook School of Medicine, Stony Brook, NY_.

Overexpression of the chromatin modifier protein, metastasis associated 1 (MTA1) in prostate cancer contributes to tumor aggressiveness, but the molecular mechanism involved has not been fully elucidated. MTA1 ChIP-Seq analysis in prostate tissues from the prostate-specific Pten heterozygous mice, which exhibit elevated levels of MTA1 revealed MTA1 recruitment onto the ETS2 promoter. Loss of function studies with MTA1 in various prostate cancer cell lines exhibited reduced expression for ETS2 at both mRNA and protein levels. We also observed increased expression of ETS2 at both mRNA and protein levels in prostate tissues from the prostate-specific MTA1 transgenic mice. Meta-analysis of prostate cancer samples in the GEO database exhibited a positive correlation between MTA1 and ETS2 levels. These results suggested that MTA1 functions as a coactivator for regulating ETS2 expression in prostate cancer. We found direct interaction between MTA1 and ETS2 in cells expressing ectopic MTA1 and ETS2. Interestingly, the prostate-specific Pten heterozygous mice, when fed with diet supplemented with pterostilbene, a natural pharmacological inhibitor of MTA1, showed a decrease in ETS2 mRNA and protein levels, perhaps due to decreased recruitment of MTA1 onto the ETS2 promoter. These results suggest that MTA1 functions as a coactivator for regulating ETS2 expression in prostate cancer at the transcriptional level and co-operates with ETS2 to promote prostate cancer progression. At the same time, pterostilbene may become a novel chemopreventive and therapeutic candidate for clinical development in prostate cancer due to its ability to target the MTA1-ETS2 axis.

### GTPase, RAF, and Growth Factor Pathways

#1863

Inactivation of RASA1 promotes melanoma tumorigenesis via R-Ras activation.

Kristen S. Hill,1 Hyeran Sung,1 Krishna L. Kanchi,2 Jane L. Messina,1 Ji-Hyun Lee,3 Youngchul Kim,1 Li Ding,2 Richard K. Wilson,2 Jeffrey S. Weber,1 Minjung Kim1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Washington University St. Louis, St. Louis, MO;_ 3 _University of New Mexico, Albuquerque, NM_.

Hyperactivation of the Ras/Raf/Mitogen-activated protein kinase (MAPK) pathway has been commonly observed in melanoma via frequent activating mutations in NRAS and BRAF. Novel mutations in other components of this pathway such as MEK1, MEK2, MAP3K5, and MAP3K9 have been reported recently by high-throughput sequencing efforts. In addition, Ras GTPase activating proteins (RasGAPs) that negatively regulate Ras, such as NF1 (neurofibromatosis type 1) and RASA2, have been shown to be mutated or suppressed in melanoma. However, importance of other RasGAPs in melanoma has not been addressed.

To obtain a comprehensive view of melanoma genomes, we conducted whole genome sequencing (WGS) of 15 metastatic melanomas and matched normal PBMC genomes from 13 melanoma patients. All melanoma genomes from these 13 patients contained at least one mutation in genes of Ras-Raf-MAPK pathway (MAPK1, MAP3K1, MAP4K2, MAP3K14, NRAS, and BRAF). In addition, we identified two novel, clustered somatic missense mutations (p.Tyr472His and p.Leu481Phe) in RASA1 (RAS p21 protein activator 1, p120RasGAP). In this study, we addressed functional roles of RASA1 in melanoma tumorigenesis. The RNAi-mediated down-regulation of RASA1 promoted, while ectopic expression of wild type RASA1 decreased, anchorage-independent colony formation, tumor growth, and RAS activation. Interestingly, RASA1 Y472H mutant enhanced soft agar colony formation and tumor growth, while RASA1 L481F mutant lost its tumor suppressive activity. Mechanistically, RASA1 required RasGAP activity to suppress colony formation and showed higher activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform among the Ras superfamily of small GTPases. Moreover, RASA1 consistently suppressed Ral-A among Ras downstream effectors. Reduced R-Ras or Ral-A expression via siRNAs suppressed anchorage-independent growth induced by RASA1 loss. Interestingly, RASA1 expression was frequently down-regulated in metastatic melanoma samples (11.4% (4/35) of lymph node metastasis and 3.4% (1/29) of distal metastases) compared to primary melanomas (33.3% (21/63)) and dysplastic nevi (44.1% (15/34)). We also observed significantly shorter overall survival of melanoma patients with BRAF mutations when RASA1 mRNA expression is low, which may be explained by possible cooperative interactions between activation of BRAF/MAPK/ERK and RASA1/R-Ras/Ral-A pathways. Taken together, these data support that RASA1 is a novel melanoma tumor suppressor that is inactivated by suppressed expression or by mutation.

#1864

In vivo **efficacy of the PAK4 allosteric modulator KPT-9274 against a triple-negative breast cancer model.**

Chetan Rane,1 William Senapedis,2 Erkan Baloglu,2 Sharon Shacham,2 Audrey G. Minden1. 1 _Rutgers University, Piscataway, NJ;_ 2 _Karyopharm Therapeutics, Newton, MA_.

The p21-activated kinases (PAK) belong to a family of serine threonine kinases that promote cell survival and play an important role in cell proliferation, cell cycle regulation and cell shape determination. There are six mammalian PAK proteins which can be subdivided into two groups by sequence homology and mode of activation- Group A PAKs consisting of PAK 1, 2 and 3 and Group B PAKs consisting of PAK 4, 5 and 6. We have found that PAK4 protein levels are elevated in breast cancer, including Her2 positive and triple negative breast cancers, while it is expressed at low levels in normal mammary tissue, making it an attractive drug target. PAK inhibitors are being tested for effectiveness against solid tumors, but generation of highly specific PAK4 inhibitors has been a challenge. Furthermore, PAK4 has been reported to have kinase-independent functions. Therefore inhibiting its kinase activity alone might not be sufficient in blocking its tumorigenic potential. Our lab has previously reported the effectiveness of PAK4 allosteric modulators (PAM; KPT-8752 and KPT-9274) against multiple breast cancer cell lines. These novel PAK4 inhibitors reduce steady state protein levels and were able to block cell growth, cell migration and induce apoptosis in breast cancer cell lines, without affecting the control cells. Here, we tested the efficacy of the orally bioavailable PAM, KPT-9274 against tumors formed by the triple negative breast cancer cell line, MDA-MB-231. Following six weeks of treatment with orally administered KPT-9274 (150mg/kg bidx4), there was almost a five-fold reduction in tumor volume and tumor weight in the treatment group as compared to the control group. The treatment did not significantly affect mice body weight. After six weeks of treatment, the tumors were excised and analyzed for PAK4 levels. We observed a significant decrease in PAK4 levels in excised tumors from the treatment group as compared to those from the control group. PAK1 levels were monitored to see any off-target effects, but their levels were unchanged. Our results indicate that PAK4 plays a key functional role in triple negative breast cancer and treatment with an orally administered KPT-9274 was capable of specifically binding and inhibiting PAK4, and consequently reducing tumor growth. Future studies analyzing the effects of KPT-9274 in blocking PAK4 mediated functions that promote tumorigenesis are ongoing. Additional studies of the effectiveness of KPT-9274 on mammary fat pad tumors formed by MDA-MB-231 and the ER positive cell line, MCF7 are under investigation.

#1865

Areca nut-induced JNK/ATF2/Jun axis regulates TGF-beta signaling contributing to pre-cancerous oral submucous fibrosis.

Ila Pant, Paturu Kondaiah. _Indian Institute of Science, Bangalore, India_.

Oral Submucous Fibrosis (OSF) is an inflammatory, pre-cancerous condition of the oral submucosa. It affects individuals with prolonged areca nut chewing habit and therefore areca nut is regarded as the etiological agent for OSF manifestation. Transforming Growth Factor beta is an established promoter of cancer and fibrosis. Up-regulation of TGF-β and its target genes has also been reported in OSF. However, the underlying mechanism for the regulation of TGF-β by areca nut has not been elucidated.

The present study demonstrates that TGF-β pathway is activated in epithelial cells after two hour exposure to areca nut extract. Areca nut increases TGF-β transcript as well as protein and thereby the downstream canonical Smad2/3 signaling. Pre-treatment with various pathway inhibitors showed JNK pathway activation as a pre-requisite to TGF-β pathway activation by areca nut. Areca nut activated JNK pathway within half an hour of treatment and this was reversed by atropine treatment suggesting involvement of muscarinic acid receptor signaling. Second messengers upstream to JNK activation and regulated by areca nut were calcium/CAMKII and reactive oxygen species. Further, areca nut treatment resulted in JNK dependent phosphorylation of ATF2 and c-Jun transcription factors. These were found to bind TGF-β promoter, as revealed by chromatin immunoprecipitation experiments. Expectedly, knockdown of these transcription factors abrogated areca nut induced TGF-β pathway activation.

Based on the data obtained a model is proposed to elucidate activation of TGF-β pathway by areca nut extract involving activation of JNK and ATF2, c-Jun resulting in the over expression of TGF-β in epithelial cells which may contribute to the manifestation of OSF. Thus, this study highlights ATF2/c-Jun as the critical nodes for therapeutic intervention to prevent TGF-β over production in OSF.

Funding by DST and CSIR is acknowledged.

#1866

HER3 targeting sensitizes HNSCC to cetuximab - evidence from cell line and patient derived xenograft (PDX) models.

Dongsheng Wang, Guoqing Qian, hongzheng zhang, kelly R. Magliocca, sreenivas nannapaneni, zhengjia chen, mihir R. patel, Mark W. El-Deiry, J.trad Wadsworth, dong M. Shin, fadlo R. khuri, Nabil F. Saba, Zhuo G. chen. _Winship cancer institute of Emory University, Atlanta, GA_.

Background and Objectives: Our previous study demonstrated that a combination of EGFR-targeted antibody cetuximab and HER3-specific antibody MM121 could significantly inhibit growth of head and neck squamous cell carcinoma (HNSCC) as compared with either of the single drugs. The current study aims to understand the role of HER3 in cetuximab resistance and the anti-tumor mechanisms of dual targeting of EGFR/HER3 in HNSCC. Materials and Methods: A cetuximab resistant HNSCC cell line SCC1-C, its parental cell line SCC1-P, and a HER3 knockdown counterpart SCC1-C/H were treated with cetuximab, MM121 and their combination. SRB and colony formation assays were performed to investigate the combined effect of MM121 and cetuximab on cancer cell growth. Activations of EGFR, HER2, HER3, AKT, and ERK signaling pathways were evaluated by Western blotting. The SCC1-C xenograft animal model and PDX models from 6 patients with HNSCC were utilized to determine the combined efficacy of MM121 and cetuximab. Results: Cetuximab induced HER3 activation and HER2/HER3 dimerization in HNSCC cell lines. The combined treatment blocked both EGFR and HER3 activation and inhibited both PI3K/AKT and ERK signaling pathways as well as HNSCC cell growth more effectively compared to either single antibody treatment in vitro. Furthermore, knock down of HER3 re-sensitized the resistant cell line to cetuximab. The anti-HER3/EGFR combination also demonstrated better efficacy than either single agent antibody in the cetuximab resistant HNSCC xenograft model and the PDX animal models. In PDX models from patients 1-4, both cetuximab and the combination significantly inhibited tumor growth in nude mice compare to the control (p<0.001 for both treatments) and there was no difference between single cetuximab and the combination. In 1 of the 4 PDXs, tumors treated with cetuximab grew back while no relapse could be detected in the combination group. In the PDX from patient 5, only the combination treatment significantly inhibited tumor growth (p<0.004) which was also more effective than cetuximab treatment (p<0.032). In the PDX from patient 6, both cetuximab and the combination significantly inhibited tumor growth in nude mice as compared with the control (p<0.001 for both treatments). However, the combination was significantly more effective than either of the single agents (p<0.0004 and p<0.003, respectively). Conclusion: Our study indicates that dual targeting of EGFR and HER3 is more effective than cetuximab alone in both cetuximab resistant HNSCC cell line and PDX models. These results pave the way for further clinical investigations using multiple targeting strategies in patients who have failed cetuximab based therapy. (The current study is supported by NIH grant R21 CA182661 to NFS and ZGC).

#1867

Activation of RHO-DIAPH signaling impairs RAS-driven exosome biogenesis.

Julie Davis-Turner, Andrew M. Howard, Arthur S. Alberts. _Van Andel Research Institute, Grand Rapids, MI_.

Like oncogenic RAS, the RAS homologues RHOA and RHOB are conventionally thought to support the malignant platform by governing cell structure remodeling by effectors that include the Diaphanous (DIAPH) family formins. RHO-activated DIAPHs generate linear actin filaments (F-actin) that physically support membrane protrusions at the leading edge of migrating cells and the trafficking of growth factor receptors, which are processes that may be required for malignant progression. However, our cellular, genetic, and pharmacological studies argue that both RHO and DIAPHs function as tumor suppressors whose loss of function triggers or augments malignant progression. DIAPH1 and DIAPH3 inhibition, for example, reduces cortical F-actin sufficiently to cause non-apoptotic blebbing. Combined with oncogenic RAS, DIAPH inhibition and blebbing accelerate exosome release. These exosomes have the capacity to foment oncogenesis by seasoning cells of the tumor microenvironment in ways that promote inflammation, survival, immune escape, and metastasis. We have identified a mechanism whereby RAS uses its own guanine-nucleotide releasing protein, SOS, to inhibit DIAPHs and facilitate its own escape within exosomes. Consistent with this idea, pharmacological activation of RHO and DIAPH signaling inhibits tumorigenesis and invasion. RHO and DIAPH agonist/activation strategies may prove to be an effective alternative for impairing RAS-driven oncogenesis where conventional inhibitors of RAS-MAPK signaling have failed or have been met by poorly understood resistance mechanisms.

#1868

Glycodelin-producing endometrial adenocarcinoma cells are resistant to TGFβ.

Laura Hautala, Hannu Koistinen. _University of Helsinki, Helsinki, Finland_.

Glycodelin is a secreted glycoprotein belonging to lipocalin protein family. It is expressed mainly in reproductive and glandular tissues. Glycodlin is involved in the regulation of reproductive and immune systems and it drives epithelial differentiation. In endometrium, the expression of glycodelin is abundant in secretory phase of the menstrual cycle, while in proliferative phase it is not expressed. Most studies agree that glycodelin is not expressed in endometrial cancer. To study the effects of glycodelin in endometrial cancer cells, we have made stable glycodelin-producing HEC-1B endometrial adenocarcinoma cells. Previously, we have shown that glycodelin differentiated HEC-1B cells towards less malignant phenotype and the cells formed significantly smaller tumors in preclinical xenograft mice. In addition, glycodelin-producing cells were resistant to phorbol ester-stimulated migration and phenotypic differentiation, which was mediated by repressed activation of PKCδ. Here we studied the effect of TGFβ on glycodelin-induced differentiation of the HEC-1B cells.

Glycodelin-producing and control cells were plated on Matrigel and grown with or without TGFβ. The media was changed every second day and the cells were monitored up to one week. For Western blot analysis the cells were grown on Matrigel and TGFβ was added for 0-2h, after which the cells were detached from Matrigel and lysed.

TGFβ induced control cells to form net-like structures, similar to those found after phorbol ester exposure. However, the glycodelin-producing cells remained unresponsive. PKCδ siRNA and PKCδ inhibitor Rottlerin inhibited the effects of TGFβ. Western blot analysis showed equal phosphorylation levels of SMAD2 in glycodelin-transfected and control cells. In contrast, PKCδ and ERK1/2 were differently phosphorylated, indicating the involvement of MAPK-pathway in the TGFβ-induced changes of the control cells.

Endometrial cancer is the most common cancer of the female reproductive tract in developed countries. However, the molecular mechanisms affecting the pathogenesis of the disease are still poorly understood. It has been suggested that TGFβ is involved in carcinogenesis of endometrial cancer. Therefore, our results showing that glycodelin-producing HEC-1B endometrial adenocarcinoma cells are resistant to the effects of TGFβ, due to the repressed PKCδ phosphorylation, may be important to understand the pathogenic mechanisms of endometrial cancer.

#1869

Dislodgement of K-Ras from plasma membranes, induction of apoptosis and tumor regression by PCAIs, a novel class of polyisoprenylated small molecules.

FELIX AMISSAH, ELIZABETH NTANTIE, ROSEMARY A. POKU, AUGUSTINE T. NKEMBO, OLUFISAYO O. SALAKO, HERNAN FLORES-ROZAS, NAZARIUS S. LAMANGO. _FLORIDA A &M UNIVERSITY, TALLAHASSEE, FL_.

Although mutation-induced dysregulation of Ras signaling constitutes the biochemical change that drives some of the most difficult-to-manage cancers, directly targeting the constitutively active mutant Ras GTPases has not resulted in clinically useful drugs. Therefore, modulating Ras activity for targeted treatment of cancer remains an urgent healthcare need. In the current study, we investigated a novel class of compounds, the polyisoprenylated cysteinyl amide inhibitors (PCAIs), for their anticancer molecular mechanisms using the NSCLC cell panel with K-Ras and/or other mutant genes. Treatment of the lung cancer cells with PCAIs, NSL-RD-035, NSL-BA-036, NSL-BA-040, NSL-BA-055 and NSL-BA-040 resulted in concentration-dependent cell death in both K-Ras mutant (A549 and NCI-H1573), N-Ras mutant (NCI-H1299) and other (NCI-H661, NCI-H460, NCI-H1975, NCI-H1563) NSCLC cells. The PCAIs at sub- to low micromolar 1.0 -10 μM concentrations induced the degeneration of 3D spheroid cultures, inhibited, clonogenic cell growth, and induced marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 (p<0.001). NSL-BA-040 at 5 μM prominently dislodged YFP-tagged K-Ras, but not H-Ras and N-Ras from the plasma membranes of transfected A549 and NCI-H661 cells. NSL-RD-035 attenuated the tumor growth of A549 cells in xenografted athymic mice in vivo. Taken together, PCAIs likely suppress NSCLC cell growth by disrupting growth signaling through the dislodgement of K-Ras the plasma membrane.

#1870

Inhibition of Galectin-1 sensitizes oncogenic H-Ras to Rapamycin treatment.

James V. Michael, Jeremy G T Wurtzel, Lawrence E. Goldfinger. _Temple University School of Medicine, Philadelphia, PA_.

Specific plasma membrane (PM) localization is essential for H-Ras signaling, and relies on post-translational modifications on the C-terminal targeting domain. H-Ras shuttles from the lipid ordered (Lo) domain to the lipid ordered/lipid disordered (Lo/Ld) border upon activation, which is dependent on Galectin-1. We have previously found that H-Ras, which is sequestered in the Lo domain by swapping the C-terminal targeting domain with the Lo-sequestering targeting domain of R-Ras, is deficient in MAPK signal propagation, while having no effect on PI3K activation, nor on H-Ras-driven tumor progression. We have further found that inhibition of PI3K with LY294002 inhibited tumor progression by H-Ras with or without Lo sequestration. Here we show that Lo sequestration of H-Ras attenuated, but did not completely block, H-Ras-induced mTOR signaling (S6kinase phosphorylation) despite similar activation of PI3K as H-Ras. Interestingly, MEK inhibition with U0126 diminished S6kinase phosphorylation by H-Ras, as well as by Lo-sequestered H-Ras. Here we demonstrate that H-Ras-driven allograft tumor growth in mice was substantially blunted upon treatment with mTOR inhibitor Rapamycin, and this effect of Rapamycin was further enhanced in tumors driven by Lo-sequestered H-Ras. Moreover, Rapamycin treatment ablated ERK phosphorylation in H-Ras tumors as well as in tumors with Lo-sequestered H-Ras (in which ERK phosphorylation was already greatly reduced). Together these findings indicate that Lo sequestration of H-Ras inhibits MAPK pathway activation, and that the MAPK pathway engages in crosstalk with mTOR pathways by regulating S6kinase phosphorylation downstream of H-Ras, whereas mTOR activity is required for H-Ras-induced MAPK signaling. These data further indicate that mTOR activation downstream of H-Ras is sufficient to drive tumorigenesis, but this pathway also requires H-Ras-MAPK activation. To recapitulate H-Ras Lo sequestration by the C-terminal targeting domain swap genetic model, we used a Galectin-1 inhibitor, OTX008, to disrupt H-Ras from transitioning between the Lo and Ld domains. OTX008 treatment alone inhibited H-Ras-driven allograft tumor growth to a similar extent as Rapamycin. However, a combination of OTX008 and Rapamycin resulted in nearly complete ablation of H-Ras-dependent tumor growth. These findings indicate that blockade of H-Ras targeting to the lipid ordered/disordered plasma membrane microdomain border, coupled with blockade of mTOR signaling, could provide a novel therapeutic approach to treat H-Ras-associated cancers.

#1871

Alpha and beta isoforms of fibroblast growth factor receptor 1 in prostate cancer.

Estefania Labanca,1 Xinhai Wan,1 Jun Yang,1 Matthew Iyer,2 Christopher Logothetis,1 Arul Chinnaiyan,2 Nora Navone1. 1 _UT MD Anderson Cancer Center, TX;_ 2 _University of Michigan, MI_.

Castrate-resistant progression and bone metastases are hallmarks of advanced prostate cancer (PCa). The fibroblast growth factor (FGF)/FGF receptor (FGFR) complex mediates tumor-stromal interactions and is commonly altered in PCa. FGFR1, FGFR2, FGFR3, and FGFR4 genes encode alternatively spliced variants of FGFRs that vary in the extracellular ligand-binding and intracellular kinase domains. A published study from our group implicated FGFR1 as a therapy target for PCa bone metastases (STM 2014; 6:252ra122). Further, our studies of FGFR1 transcripts by RNA sequencing of 183 human PCas identified eight different protein coding transcripts as the most abundantly expressed with different human PCas expressing different FGFR1 isoforms (Abstract #3913 AACR 2015). These results suggest that different FGFR1 isoforms in PCa cells may partially underlie the biological heterogeneity of PCa. The studies presented here focus in two of the best-characterized isoforms: FGFR1alpha (R1alpha), with 3 Ig-like domains, and 822 aa in length; and FGFR1beta (R1beta), with only 2 Ig-like domains and 733 aa in length. We assessed whether these isoforms induce activation of the same pathways using PC3 cells transiently transfected with empty vector (EV), R1alpha (NM_023110.2) or R1beta (NM_023105.2) and treated with vehicle, FGF2 or FGF9. By Western blot analysis we found that total FGFR1 expression (relative to a loading control) was similar in cells transfected with R1beta or R1alpha. Levels of p-FGFR1 were high in untreated cells transfected with R1alpha, but no further induction was observed after treatment with FGF2 or FGF9. However, p-FGFR1 expression was almost undetectable in untreated cells expressing R1beta and was slightly induced by FGF2 but not by FGF9. p-PLCγ expression was found only in cells expressing R1alpha. We subsequently stably transfected these isoforms in PC3 cells and discovered that while no significant difference in R1alpha and R1beta transcript levels was detected, the levels of R1alpha protein were higher than those of R1beta suggesting that these isoforms may undergo different translational regulation, We also performed in vivo studies by subcutaneous injection of R1alpha or R1beta expressing PC3 cells in immunocompromised mice and weekly monitored tumor volume. We found that PC3 cells expressing R1alpha developed significantly larger tumors than PC3 cells expressing R1beta. We are now in the process of analyzing at the molecular level these tissue specimens. In conclusion, R1alpha and R1beta isoforms trigger different biological effects in PCa cells. Because different isoforms are expressed in different prostate tumors, the expression of these isoforms could be associated with the typical PCa heterogeneity and could explain differences in therapy responses to FGFR1 blockade. These results warrant further studies to fully understand the biological implications of FGFR1 isoforms in the pathogenesis of PCa.

#1872

Development of reference reagents to accelerate research on the RAS pathway.

Dominic Esposito, Jennifer Mehalko, Vanessa Wall, Carissa Grose, William Gillette, Robert Stephens. _Frederick National Laboratory for Cancer Research, Frederick, MD_.

The RAS Reference Reagents program within the NCI RAS Initiative is produces and distributes qualified DNA vectors, cell lines, and viruses to the external community to facilitate research into RAS biology and development of RAS cancer therapeutics. DNA vectors representing a variety of RAS mutations, as well as a complete collection of 180 genes in the RAS pathway are available as Gateway-compatible Entry clones. These clones represent the most commonly expressed transcript forms of the 180 genes, many of which were not previously available in full-length, wildtype forms. The vectors can be used with the FNLCR combinatorial cloning library to easily generate a large number of different types of expression clones with various promoters, fusion tags, and plasmid backbones. Additional reagents under construction include validated plasmid and lentiviral reagents for shRNA and CRISPR delivery, qualified cell lines for use in research into the RAS pathway, and systems for the generation of highly purified and characterized Ras and Ras-related proteins. A highlight of these reagents are materials for the high yield production of properly processed, prenylated KRAS protein using an engineered insect cell expression system. This system could be used to produce other prenylated small GTPases at levels not previously attainable. All of the resources of the RAS Reference Reagents program are readily available to the academic community through simple mechanisms, and can be licensed to industry scientists as well. The program is also interested in identifying the needs of the community for additional RAS-related resources, and is developing methods for vetting requests from the outside to provide additional reagents.

#1873

The role of p21-activated kinase 4 (PAK4) in cancer stemness and epithelial-to-mesenchymal transition.

Asfar S. Azmi,1 Irfana Muqbil,2 Amro Aboukameel,2 William Senapedis,3 Erkan Baloglu,3 Yosef Landesman,3 Sharon Shacham,3 Michael Kauffman,3 Philip A. Philip,2 Ramzi M. Mohammad2. 1 _Wayne State University, Westland, MI;_ 2 _Wayne State University, Detroit, MI;_ 3 _Karyopharm Therapeutics, Newton, MA_.

The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho family GTPases. PAK4 is found to be over-expressed in many oncogenic Ras-driven cancers and cell lines but not in their normal counterparts. Mesenchymal pancreatic ductal adenocarcinoma (PDAC) stem cells (CSCs) that are triple positive for stemness markers (CD133+, CD44+, and EpCam+) as well as Ras and snail transduced human mammary epithelial cells (HMLER-snail) showed enhanced expression of PAK4 along with activation of Rho, Rac1 and CDC42. PAK4 RNA interference resulted in disruption of PDAC CSC spheroids, reduction of mesenchymal markers (i.e. vimentin and snail) and suppression of CSCs potential to form subcutaneous tumors in mice. These findings point to a novel role of PAK4 in promoting oncogenic Ras-driven EMT and stemness. Using PDAC CSCs (MiaPaCa-2 and L3.6pl) and human mammary epithelial cells (HMECs) that are transduced with Ras and the EMT promoter snail (HMLE-Snail and HMLER-Snail), we investigated the impact of PAK4 allosteric modulators [PAMs (KPT-7189, KPT-7523, KPT-9274 and KPT-9307)] on the reversal of EMT and stemness. The toxicity and efficacy of PAMs were evaluated in vitro and in multiple sub-cutaneous xenograft models. PAMs show anti-proliferative activity in vitro against different PDAC CSCs and HMECs while sparing normal cells. Cell growth inhibition was concurrent with apoptosis induction, suppression of colony formation and reversal of mesenchymal morphology to epithelial (MET). PAMs not only reduced PAK4 RNA and protein steady state levels but also inhibited proliferative and anti-apoptotic signals downstream of PAK4. PAMs caused suppression of EMT markers (EpCAM, vimentin and snail) and re-expression of an epithelial promoter E-cadherin. PAM treatment inhibited RhoA, Rac1 and CDC42 activation in GLISA assays. KPT-9274 showed remarkable anti-tumor activity in sub-cutaneous xenograft models of CSCs and residual tumors showed suppression of EMT markers that was concurrent with inhibition of PAK4 signaling. These studies open a novel therapeutic opportunity against EMT and cancer stem cells through PAK4 inhibition.

#1874

Oncogenic EGFR signaling inhibits the Spred1-NF1 interaction to sustain constitutive Ras signaling.

Evan Markegard, Ellen L. Mercado, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. _UCSF, San Francisco, CA_.

Spred proteins negatively regulate Ras/MAPK signaling following mitogen stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Loss-of-function Spred1 and NF1 mutations occur across multiple cancer types including melanoma, non-small cell lung carcinoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of oncogenic EGFR may disrupt Spred1-NF1 binding. Our findings provide one potential mechanism by which oncogenic EGFR signaling disrupts negative feedback to allow for constitutive Ras signaling. Furthermore, this work may elucidate a novel kinase therapeutic target for restoring NF1 mediated inhibition of Ras.

#1875

Identification of LASEP3 as a serological and tissue biomarker and a therapeutic target for lung cancer.

Atsushi Takano,1 Yusuke Nakamura,2 Yataro Daigo3. 1 _The University of Tokyo, Tokyo, Japan;_ 2 _The University of Chicago, Chicago, IL;_ 3 _The Shiga University of Medical Science, Otsu, Japan_.

Lung cancer is the leading cause of cancer deaths worldwide. The prognosis of patients with lung cancer remains poor, because of its occult metastasis even at earlier stage of the disease and its resistance to conventional multimodal treatments. Therefore, this study aims to identify novel serum and tissue biomarkers for personalized therapy and/or therapeutic targets for lung cancers through screening and functional analyses of cancer-specific oncoproteins. We used cDNA microarrays for screening genes encoding transmembrane/secretory proteins that are up-regulated in lung cancers, and identified a secreted protein, LASEP3 (lung cancer-associated serum protein 3) as a candidate. Immunohistochemical staining of LASEP3 showed that LASEP3 expression was observed in 198 (54.8%) of 361 NSCLCs (non-small cell lung cancer) that had undergone curative surgery. High level of LASEP3 expression was associated with poor prognosis for NSCLC patients. (P=0.0183 by log-rank test). Serum LASEP3 levels were higher in NSCLC patients than in healthy volunteers. The proportion of serum LASEP3-positive cases was 160 (61.8%) of 259 NSCLCs (49.4% for stage I-II, 67.4% for stage III-IV), while 6 (5.5%) of 109 healthy volunteers were falsely diagnosed. Moreover, reduction of LASEP3 expression by siRNAs suppressed lung cancer cell proliferation, probably through apoptosis as suggested by flow cytometric analysis and caspase-3 and 7 assays. Furthermore, subsequent microarray analysis of these cancer cells identified several candidate downstream genes of LASEP3 that relate to cell growth and invasion signals. LASEP3 is a possible diagnostic and prognostic biomarker and a therapeutic target for lung cancer.

#1876

Reduced Pak1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition.

Olga Villamar-Cruz,1 Tatiana Prudnikova,2 Neil Johnson,2 Jonathan Chernoff,2 Luis E. Arias Romero1. 1 _Universidad Nacional Autonoma de Mexico, Tlalnepantla, Mexico;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Cells that are deficient in homologous recombination, such as those that have mutations in any of the Fanconi Anemia (FA)/BRCA pathway genes, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, FA/BRCA-deficient tumors represent only a small fraction of breast cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. p21-activated kinase 1 (Pak1) is a serine/threonine protein kinase located in the chromosome 11q13 and is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme controls many cellular processes by phosphorylating its downstream substrates; in addition to its role in the cytoplasm, Pak1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that Pak1 activation is a component of the DNA damage response. Here, we show that depletion or inhibition of Pak1 down-regulated the expression of several genes involved in the FA/BRCA pathway and compromised the ability of cells to repair DNA by homologous recombination, induced cell cycle arrest, promoted apoptosis and resulted in reduced colony formation. Combined inhibition of Pak1 and PARP in pak1 amplified breast cancer cells had a synergistic effect, enhanced apoptosis, resulted in reduced colony formation and delayed tumor growth in a xenograft setting. Inhibition of Pak1 did not sensitize non-transformed or Pak1 non-amplified cells to inhibition of PARP. Because reduced Pak1 activity impaired FA/BRCA function and consequently, repair by homologous recombination, inhibition of this kinase in pak1 amplified and/or overexpressing breast cancer cells represents a plausible strategy for expanding the utility of PARP inhibitors to FA/BRCA-proficient cancers.

#1877

A new H-ras specific feedback loop from the TOR-pathway impacts on tumorigenicity.

Itziar M. D. Posada, Benoit Lectez, Christina Oetken-Lindholm, Daniel Abankwa. _Åbo Akademi University, Turku, Finland_.

Introduction:

The lack of direct Ras inhibitors, has led to the development of inhibitors of downstream components of the Ras pathway albeit with mixed success. Unexpected mechanistic complexity, feedback loops and tumor specific synthetically lethal requirements continue to be major obstacles. In the past we investigated the mechanisms of the Ras membrane signaling system on the nanoscale (nanoclustering). Ras nanoclustering on the plasma membrane is critical for its signaling activity and we and others recently showed how it is exploited in a number of physiological and pathophysiological conditions, including cancer. Importantly, Ras isoform specificity seems to emerge at the level of laterally segregated nanocluster. This is supported by isoform specific nanocluster scaffolds, such as galectin-1 (Gal-1), which specifically augments GTP-H-ras nanocluster.

Methods:

The effect of compounds and siRNAs targeting the TOR pathway and associated proteins on H- vs. K-ras4B (hereafter K-ras)-nanoclustering was evaluated using FRET-measurements in BHK and HEK cells, or point pattern analysis of electron micrographs from BHK cell plasma membrane sheets. Ras specific effects of treatments were furthermore assessed in PC12 differentiation assays, driven by oncogenic H- or K-ras. Changes in downstream signaling were evaluated using Western Blotting of treated BHK and HEK cells. The effect of treatments (compounds or siRNAs) on mammosphere formation was assessed in MDA-MB-231 cells.

Results:

Here we describe a new feedback mechanism from the TOR-pathway specifically to H-ras, but not K-ras. Inhibition of FKBP12 inhibitors cycloheximide (CHX), Rapamycin (Rapa) and FK506, as well as knockdown of FKBP12 specifically increased H-ras nanoclustering. While all compounds could also specifically increase differentiation of H- but not K-rasG12V transfected PC12 cells, their effect on cellular signaling pathways differed in BHK cells. Both, CHX and Rapa increased Erk phosphorylation, but only Rapa blocked S6K and S6 phosphorylation, while CHX did the opposite. Surprisingly, siRNA-mediated ablation of FKBP12 upregulated Gal-1 protein levels, suggesting that the observed increase in H-ras nanoclustering is due to this response. This was however not observed for FKBP12 inhibitors. On the other hand overexpression of Gal-1 also increased FKBP12 levels. Intriguingly, only FKBP12 ablation (with concomitant Gal-1 upregulation) was sufficient to increase the mammosphere forming ability, but not Gal-1 overexpression alone. This effect could be mimicked by FKBP12 inhibition with CHX or Rapa, but not FK506.

Conclusions:

Our data indicate a H-ras specific feedback from inhibition of the TOR pathway, which can lead to increased mammosphere formation. These data suggest a tumor promoting effect by TOR pathway inhibitors via H-ras probably in a specific cellular and genetic context.

#1878

Inhibition of the Transforming Growth Factor beta pathway in human glioblastoma cell lines induces apoptosis and inhibits anoikis escape.

Gabriel Gallo-Oller,1 Javier Dotor,2 Xing Fan,3 Javier S. Castresana1. 1 _University of Navarra, Pamplona, Spain;_ 2 _BIOHOPE, SL, Madrid, Spain;_ 3 _University of Michigan, Ann Arbor, MI_.

Glioblastoma Multiforme (GBM) is the most prevalent malignant brain tumor accounting for 60-70% of all gliomas. Current achieved median patient survival ranges only 12-15 months, and improvements in survival over the last century can be measured only in weeks. A hallmark of this malignancy is the intrinsic resistance to current therapies. The Transforming Growth Factor beta (TGF-beta) signaling pathway plays a key role in GBM. Several inhibitors of different elements and regulators of the TGF-beta pathway have entered to clinical trials.

Here, we analyzed the potential effect of P144, a TGF-beta pathway inhibitor peptide, over cell proliferation and apoptosis in commercial GBM cell lines (A172 and U-87 MG). We found that treatment with P144 significantly decreased cell proliferation of both cell lines analyzed. In addition, different apoptosis determinations such as ELISA and Acridine Orange/Ethidium Bromide (AO/EB) staining, confirmed a significant increase in apoptosis in both cell lines.

Quantification of SMAD2 phosphorylation status and its nuclear translocation, by western blot, confirmed the inhibition of TGF-beta signaling by P144. These data support the correlation between TGF-beta pathway inhibition and the apoptotic process induction.

Anoikis escape sustains invasiveness and metastatic potential in GBM, and has been established as a clear target against tumor progression. Due to P144 clear effect on apoptosis, we analyzed its influence on anoikis escape. The inhibition of anoikis escape by P144 was confirmed in A172 and U-87 MG cell lines under in vitro anchorage-independent culture conditions. The significant upregulation of BAX, Lamin A and Lamin C produced by P144 confirmed the induction of this apoptotic subtype process in the analyzed cell lines.

We can conclude that P144 increases apoptosis and induces anoikis through inhibition of the TGF-beta signaling pathway. Our results highlight the therapeutic potential of P144 for the treatment against GBM. However, previous to further clinical development, additional studies are required in order to confirm the effect of P144 over GBM in the brain environment, as well as to explore the therapeutic potential of P144 in combination with current and emerging molecular based therapies.

#1879

Structural and biochemical characterization of farnesylated and methylated KRAS-membrane interactions.

Que Van,1 William K. Gillette,1 Dominic Esposito,1 Rodolfo Ghirlando,2 Frank Heinrich,3 Andrew G. Stephen1. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD;_ 3 _NIST, Gaithersburg, MD_.

KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed in several enzymatic steps that results in the addition of a farnesyl and a methyl group to the terminal cysteine residue. Plasma membrane localization of KRAS4b requires this processing along with a polybasic stretch of lysines within the hypervariable region. KRAS4b can bind to RAF-RBD in solution but membrane bound KRAS4b is required for RAF-kinase activation. In order to determine why membrane interaction of KRAS4b is required for MAPK signal transduction we under took a structural and biochemical analysis of KRAS4b-membrane interactions. We have developed methods using an engineered baculovirus for the insect cell production of farnesylated and methylated KRAS4b (KRAS4b-FME) and this material was used in these studies. We have demonstrated that the stable interaction of KRAS4b-FME to lipid Nanodiscs and tethered lipid bilayers is dependent on the presence of anionic phospholipids. Using analytical ultracentrifugation we have evaluated the binding stoichiometry of KRAS4b-FME with Nanodiscs. In addition we have used neutron reflectivity to determine the orientation of KRAS4b-FME on tethered lipid bilayers.

#1880

Determining the role of E-cadherin in regulating IGF1 signaling in breast cancer: An interaction predicted by large-scale modeling.

Alison M. Nagle,1 Cemal Erdem,2 YuFen Wang,3 Kevin Levine,4 Tiffany Katz,1 D. Lansing Taylor,2 Adrian V. Lee,1 Timothy R. Lezon2. 1 _Dept. Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA;_ 2 _Dept. Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA;_ 3 _Breast Center, Baylor College of Medicine, Houston, TX;_ 4 _Dept. Pathology, University of Pittsburgh, Pittsburgh, PA_.

Insulin-like growth factor I (IGF1) plays an important role in breast cancer initiation and progression due to its regulation of cell proliferation, migration, and invasion. These characteristics make IGF1R an attractive therapeutic target. While numerous clinical trials have sought to inhibit IGF1R action, these were largely unsuccessful due to a lack of biomarkers for positive therapeutic response, poor patient selection, and potential compensatory signaling by the highly similar insulin receptor (InsR). To identify biomarkers of response, our laboratory identified a set of genes regulated by IGF1 (IGF-sig) that revealed a correlation between activation of the IGF-sig and poor prognosis in estrogen receptor (ER)-negative breast cancer. Further, we showed that ER-negative breast cancer cells are sensitive to IGF1R inhibition both in vitro and in vivo. In an effort to better understand the IGF1 and insulin signaling networks in breast cancer, we performed a reverse phase protein array (RPPA) using 134 antibodies on lysates from twenty-one breast cancer cell lines stimulated with a six point time-course of IGF1 or insulin. We developed a time-dependent model to predict differential mediators of IGF1 and insulin signaling using perturbation analysis. The model predicted that alterations in levels of E-cadherin (CDH1), a major component of the adherens junction, affect IGF1 induced Akt activation. In breast cancer, E-cadherin is genetically lost in invasive lobular cancer (ILC), a subtype that accounts for ~10-15% of breast cancers. Alternatively, invasive ductal cancers may lose E-cadherin via EMT. I confirmed this in silico prediction, showing that shRNA reduction of E-cadherin enhances the ability of IGF1 to induce Akt signaling, and additionally IGF1R, ERK and S6 ribosomal protein activation. Supporting the clinical relevance of our observations, we found a correlation between loss of E-cadherin (CDH1) mRNA expression and the activation of the IGF-sig in ER-negative tumors within The Cancer Genome Atlas (TCGA). Therefore, we hypothesize that loss of E-cadherin potentiates IGF1 signaling to enhance breast cancer progression, and that loss of E-cadherin expression in ILC and ER-negative tumors may highlight those susceptible to IGF1R inhibition. These studies will investigate how E-cadherin modulates IGF1 signaling and the interaction with EMT with the goal of better defining breast cancers that may respond to IGF1R inhibitors.

#1881

Insulin receptor targeting in breast cancer through yeast surface display.

Jie Ying Chan, Kelly LaPara, Benjamin Hackel, Douglas Yee. _University of Minnesota, Minneapolis, MN_.

Selective estrogen receptor modulators (SERMs) such as tamoxifen are used to treat estrogen receptor (ER) positive breast cancers, the most common intrinsic subtype. The development of secondary resistance to SERMs is an unsolved clinical dilemma, leading to the exploration, and approval, of therapies targeting growth factor signaling pathways. The insulin-like growth factor (IGF) system is well documented to cooperate with ER and is implicated as a contributor to endocrine resistance. Unfortunately, antibodies directed against Type I IGF receptor (IGF1R) failed to demonstrate clinical efficacy in endocrine resistant breast cancer. We developed a tamoxifen resistant (TamR) model to further identify the role for this system in endocrine resistance. In these cells, IGF1R levels are low but the level of insulin receptor (InsR), a closely related receptor of IGF1R still remains functional. Thus, we hypothesize that InsR may serve as a compensatory pathway to the loss of IGF1R in TamR cells and InsR may be a target in the therapy of endocrine resistant breast cancer. Indeed, our results showed that TamR breast cancer cells are more sensitive to insulin stimulation than wild-type cells. As compared to the parental cells, insulin-stimulated TamR cells showed enhanced PI3K/AKT and MAPK/ERK activation, greater monolayer and anchorage-independent growth. Suppression of InsR expression with either lentiviral shRNA or pharmacological methods in TamR cells was able to attenuate their sensitivity towards insulin-regulated PI3K/MAPK activation and growth, suggesting InsR targeting may be necessary. To target InsR, we are developing InsR-selective small protein scaffolds - the 10th type III domain of human fibronectin (Fn3) and T7 phage gene 2 protein (Gp2) using yeast surface display. This technique has arisen as an alternative candidate to bind cells surface proteins. Compared to antibodies, protein scaffolds are smaller (<12kDa), thermally more stable, lack disulfide bonds, tolerance to mutations and have the ability to bind a large variety of proteins. Using error-prone polymerase mutagenesis, we have identified improved Fn3 and Gp2 binders with increased affinity, specificity, and stability for InsR. Yeast surface displayed (YSD) Gp2 and Fn3 libraries yield affinities for recombinant InsR in the high nanomolar range and some specificity with an ability to differentiate binding from IGF1R. Purified Gp2 binder was able to distinguish InsR expression levels between InsRlow and InsRhigh mammalian cells, verifying its specificity for cell surface InsR. Its affinity for cell surface InsR, however was much lower compared to YSD affinity. Preliminary analyses also indicated that Gp2 binders have minimal effect in InsR-regulated signaling and short-term biological functions in vitro. These InsR binding engineered protein scaffolds will further be improved and evaluated as a potential imaging, diagnostic, and therapeutic tools.

#1882

Manumycin-A suppresses exosome biogenesis and chemosensitizes CRPC cells to enzalutamide through inhibition of Ras/Raf/ERK1/2 signaling pathway.

Hogyoung Kim,1 Amrita Datta,1 Madhu Lal-Nag,2 Adedoyin Johnson,1 Ahmed Moustafa,1 Debasis Mondal,1 Marc Ferrer,3 Asim B. Abdel-Mageed1. 1 _Tulane University School of Medicine, New Orleans, LA;_ 2 _National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, MD;_ 3 _National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda, MD_.

Background: Emerging evidence suggests that there is an urgent need for new strategies to combat prostate cancer (PC) that is unresponsive to therapy. In recent years major emphasis has been placed on the role of exosomes (Exo) in cancers. Exosomes are membrane-bound vesicles produced by all cell types under physiologic and pathological states. The objective of the present study was to identify via high throughput library screens compounds that selectively inhibit Exo biogenesis and release by PC cells and further examine if they chemosensitize castration-resistant PC (CRPC) cells to Enzalutamide (ENZ), an FDA-approved second generation antiandrogen drug. The ultimate goal is to incorporate functionally validated compounds for advanced PC therapy. Manumycin-A (MA), a natural, well-tolerated microbial metabolite and a potent experimental tumoricide, has been identified as one of the lead compounds in the initial screens.

Results: A time-course, dose-dependent analysis revealed that MA up to 250 nM is not toxic to C4-2B or RWPE1 cells. The MA-mediated inhibition of Exo secretion was evident by decreased expression of Exo markers (ALIX, TG6101, and tetraspanins; CD9, CD63, CD81) as well as early (Rab5) and late (Hrs) endosome markers in C4-2B cells, with no effect observed in RWPE1 cells. Interestingly, the MA-mediated inhibition of Exo biogenesis was coupled with inhibition of Ras activation. MEK selective inhibitor (U0126), but not the p38 MAPK (SB203580) or JNK (SP600125) inhibitor, suppressed the expression of Exo markers, indicating that Ras-MEK pathway is involved in MA's mediated inhibition of exosome biogenesis and secretion. Subsequent analysis revealed that MA inhibited p-cRaf, pMEK and pERK in C4-2B cells, but not in RWPE1 cells)--further attesting to direct role of Ras/Raf/ERK1/2 pathway in mediating MA inhibition of Exo biogenesis and secretion by C4-2B cells. Treatment of C4-2B cells with ENZ and MA combination further inhibited cell survival than either drug alone, suggesting that MA may chemosensitize CRPC cells to ENZ.

Conclusions: Our findings suggest that at low, non-cytotoxic concentrations, Manumycin is a suitable candidate to suppress exosome biogenesis and secretion and enhances sensitivity of CRPC cells to ENZ cytotoxicity. Further preclinical analysis is required to examine its in vivo therapeutic efficacy of MA against CRPC when administrated in combination with antiandrogen drugs.

#1883

Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells.

Luca Hegedus,1 Tamas Garay,2 Eszter Molnar,2 Karolina Varga,2 Rita Padanyi,2 Katalin Paszty,3 Balazs Hegedus,4 Mathias Wolf,5 Michael Grusch,5 Eniko Kallay,1 Agnes Enyedi6. 1 _Department of Pathophysiology and Allergy Research, Medical University of Vienna, Wien, Austria;_ 2 _Semmelweis University, Budapest, Hungary;_ 3 _Hungarian Academy of Sciences, Budapest, Hungary;_ 4 _Division of Thoracic Surgery, Comprehensive Cancer Center, Medical University of Vienna, Wien, Austria;_ 5 _Institute of Cancer Research, Medical University of Vienna, Wien, Austria;_ 6 _Molecular Oncology Research Group of the Hungarian Academy of Sciences, Budapest, Hungary_.

The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on the expression and activity of the plasma membrane Ca2+ pump isoform 4b (PMCA4b) in BRAF mutant melanoma cell lines. Remodeling of Ca2+ homeostasis during malignancy is caused by the rearrangement of the Ca2+ signaling machinery, including Ca2+ pumps, Na+/Ca2+ exchangers and channels. Plasma membrane calcium ATPases (ATP2B - or PMCA) maintain the resting low intracellular calcium concentration by pumping out excess calcium from the cytosol. Changes in PMCA expression during malignant transformation have been described previously in colorectal and breast cancer cells, and most recently by our group in melanomas. We found that in BRAF mutant melanoma cells the PMCA 4b protein level was markedly elevated by BRAF inhibitor treatment. Overexpression of PMCA4b suppressed motility and metastatic potential. Previously, it was shown that HDAC inhibitors-induced differentiation up-regulated PMCA4b expression in gastric, colon and breast cancer cells. In the present study we treated BRAF mutant and BRAF wild type melanoma cells with HDAC inhibitors (SAHA (suberanilohydroxamic acid) or valproic acid) and tested the changes in abundance, localization and function of PMCA4b. Expression levels of the PMCA proteins were analyzed by qRT-PCR and Western Blotting. Subcellular localization of the PMCAs and the effects of treatments on cytosolic Ca2+ signaling were analyzed by confocal microscopy. We found that treatment with the HDAC inhibitors increased the level of PMCA4b expression at both mRNA and protein levels in both BRAF wild type and BRAF mutant cell types. The increased PMCA4b level was coupled with enhanced plasma membrane localization and with a faster Ca2+ clearance after stimulation. Our results show that in melanoma cells the expression of PMCA4b is under epigenetic control and HDAC inhibitors efficiently enhanced PMCA4b expression independent of the BRAF status of cells.

#1884

A PKCα-RAF-ERK signaling axis for downregulation of Id1 and Id3 in colorectal cancer cells.

Michelle A. Lum, Robert E. Lewis, Adrian R. Black, Jennifer D. Black. _University of Nebraska Medical Center, Omaha, NE_.

Members of the Id family (Id1-Id4) are emerging as attractive therapeutic targets in multiple tumor types. Id proteins are dominant negative antagonists of basic helix-loop-helix transcription factors, with recognized functions in development, stem cell maintenance/self-renewal, and cell fate determination. Recent studies have identified Id proteins as master regulators of cancer-initiating cells and tumor aggressiveness, with critical roles in regulation of central hallmarks of cancer, including cell proliferation, survival, senescence, angiogenesis, migration, metastasis, and chemoresistance. Deregulated Id1 and/or Id3 expression has been observed in more than twenty human cancers, including colorectal cancer (CRC). Based on their important roles in tumors, Id proteins are being actively explored as therapeutic targets, with promising results in mouse models and human tumor cell lines. Id1 and Id3 appear to have redundant functions in CRC; thus Id-based therapy would ideally involve a strategy that targets Id1 and Id3 simultaneously.

We have identified a novel pathway of Id1 and Id3 repression involving the signal transduction molecule PKCα. Activation of PKCα in non-transformed intestinal cells (IEC-18 cells) and human CRC cells that retain PKCα potently suppresses Id1/Id3 mRNA and protein. Restoration of the enzyme in PKCα-deficient CRC cells also results in Id1/Id3 downregulation. Suppression occurs at the transcriptional level and is mediated by the proximal 932 bp of the promoter for Id1. Notably, the effects of PKCα were observed in CRC cell lines with diverse genetic backgrounds, differing in the status of APC, β-catenin, K-RAS, the PI3K/AKT pathway, and/or TP53.

PKCα activates a growth inhibitory MEK/ERK signaling pathway in intestinal cells that is required for Id1/Id3 downregulation. Our demonstration that PKCα can activate ERK and suppress Id1/Id3 in KRAS-mutant CRC cells suggested that the effects of PKCα occur downstream of RAS activation. Notably, PKCα was unable to downregulate Id1/Id3 in CRC cells harboring V600E mutations in BRAF, indicating that RAF activation is a key mediator of PKCα-induced Id downregulation. This conclusion was further supported by the ability of the RAF inhibitor, sorafenib, to block the effects of PKCα on Id1/Id3 in BRAF-wild type cells. KSR1 is an important regulator of RAF function; however, PKCα retained its ability to downregulate Id1 in KSR1 knockdown CRC cells, excluding a role for altered KSR1-RAF interactions in the effects of PKCα. Collectively, these data indicate that PKCα regulates a novel signaling pathway for downregulation of Id1/Id3 that intersects the MEK/ERK pathway at or upstream of RAF, but downstream of RAS. Manipulation of this pathway for coincident downregulation of Id1 and Id3 offers a promising therapeutic strategy for treatment of CRC and other cancer types.

Supported by NIH grants CA036727, CA016056, CA191894 and DK60632.

#1885

Integrated epigenomic profiling reveals widespread demethylation in melanoma and points to the role of CSF1R-RUNX1 axis in resistance against BRAF inhibition.

Orsolya Giricz,1 Yongkai Mo,1 Caroline H. Hu,1 Kimberly Dahlman,2 Nandini Ramachandra,1 Matthias Bartenstein,1 Kith Pradhan,1 Tushar Bhagat,1 Yiting Yu,1 Hoa Nguyen,3 Elizabeth Burton,3 Bernice Matusow,3 Gaston Habets,3 Rafe Shellooe,3 Gideon Bollag,3 Brian West,3 John Greally,1 Jeffrey Sosman,2 Paraic Kenny,1 Amit Verma1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Plexxikon Inc., Berkeley, CA_.

Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. To study this at a genome-wide level in melanoma we analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it to melanocyte controls. We saw widespread demethylation in melanoma occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways aberrantly expressed and regulated by loss of DNA methylation.

The colony stimulating factor-1 receptor (CSF1R) was aberrantly expressed and hypomethylated in nearly all cases. The expression of CSF1R was validated by immunohistochemistry on primary tumors and by Western blotting in BRAF V600E mutant and WT melanoma cell lines. Expression of its ligand IL34, but not of CSF1 was also shown in the melanoma cells by both ELISA and qPCR. The effects of a small molecule inhibitor, PLX3397 as well as shRNA-mediated knockdown of the receptor were investigated in traditional and 3D cell culture. We saw inhibition of cell growth, smaller colony size, increased apoptosis and decreased invasiveness - suggesting a functional role for CSF1R in melanoma.

Treatment of melanoma with small molecule inhibitors of BRAF V600E is effective for a time, but resistance invariably develops. The feedback activation of EGFR, BRAF amplification, BRAF splice variants and others are known to aid in the acquisition of resistance and lead to rebound activation of the MAPK-pathway. In Western blotting experiments, the rebound of ERK phosphorylation after BRAF inhibitor treatment was accelerated with the addition of the CSF1R ligands CSF1 and IL34, or delayed with PLX3397, also attenuating AKT phosphorylation. Melanoma cells stably expressing CSF1R shRNA recapitulated the effects of the inhibitor. Assaying the cells at different time points during a long-term V600E inhibitory experiment, we saw increasing levels of the transcription factor RUNX1, followed by increasing levels of IL34 and of the CSF1R protein, as well as its maturation, evidenced by the appearance of the high MW form. Utilizing shRNA-mediated knockdown of RUNX1 resulted in lower levels of the CSF1R and IL34 transcripts and delayed the rebound. Analysis of primary RNA-Seq data showed an increase in RUNX1, CSF1R and IL34 expression as resistance was acquired. Co-inhibition of CSF1R and BRAF was also tested and resulted in synergistic blockade of cell growth in vitro and xenograft growth in vivo. The CSF1R inhibitor, PLX3397, is in clinical trials for breast and other cancers, and these data present a preclinical rationale for its study in malignant melanoma.

#1886

A crucial role of the small GTPase ARF1 in the MAPK activation and prostate tumorigenesis.

Jason E. Davis, Yong Teng, Guangyu Wu. _Georgia Regents University, Augusta, GA_.

Prostate cancer is one of the most common cancers in men and is the second leading cause of cancer-related mortality in men. It is well known that enhanced activation of the MAPK pathway Raf-MEK-ERK1/2 directly correlates with the progression, androgen independence and poor prognosis of prostate cancer. However, the molecular mechanisms underlying the hyper-activation of the MAPK pathway remain poorly elucidated. Here, we demonstrated that the expression and activation of ADP-ribosylation factor 1 (ARF1), a Ras-related small G protein, were significantly elevated in human prostate cancer cells and tissues. Overexpression and siRNA-mediated depletion of ARF1 dramatically influenced MAPK activation in prostate cancer cells. Furthermore, ARF1 can mimic Ras to directly and activation-dependently interact with Raf1, specifically its Ras-binding domain. Mutations that disrupt ARF1 activation considerably attenuated ARF1-Raf1 interaction and MAPK activation. More importantly, we found that depletion of ARF1 significantly inhibited prostate cancer cell proliferation in vitro and xenograft tumor growth in vivo, whereas overexpression of ARF1 produced opposing effects. These data have revealed an unappreciated function of ARF1 in the MAPK activation and prostate tumorigenesis and suggest that ARF1 may represent a key molecular target for prostate cancer therapeutics.

#1887

RHBDF1 assists intracellular trafficking of pro-TGFα via Clathrin/AP1-coated vesicles to mediate EGFR transactivation by GPCR.

Jie Li, Zhuan Zhou, Zhi Song Zhang, Lu Yuan Li. _The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin, China_.

Epidermal growth factor receptor (EGFR) can be transactivated by ligands of G protein-coupled receptors (GPCR), and GPCR signaling pathways often involve the formation of clathrin-coated recycling endosome and secretion vesicles. It contains EGFR ligands precursor protein such as pro-TGFα, which is proteolytically processed and released from the cell surface to activate EGFR in an autocrine manner. We showed previously that human rhomboid family-1 gene RHBDF1 is essential for GPCR-EGFR transactivation by assisting the secretion of TGFα.

Herein, we found increased expression of RHBDF1 in breast invasive ductal carcinoma tumor tissues was significantly correlated with a higher EGFR phosphorylation rate (r=0.853,p < 0.001). Silencing RHBDF1 gene of breast cancer cells MDA-MB231 with shRNA suppresses cell migration and invasion, and also leads to inhibition of ligand-dependent activation of EGFR induced by GPCR ligands, rather than EGFR ligands. These are mainly caused by blocking regulated shedding and secretion of TGFα. RHBDF1 can promote pro-TGFα to dissociate with its Golgi membrane anchor protein GRASP55, and increase its sorting and trafficking to the plasma membrane by Clathrin-coated vesicles. RHBDF1 protein co-localizes with pro-TGFα at the Golgi complex in starved, quiescent cancer cells, but will translocate to the cell surface upon stimulation by GPCR ligands, and assist Src to phosphorylate β1-AP1, an adaptor protein that mediates vesicular protein sorting between the trans Golgi network (TGN) and endosomes, and this will lead to the dissociation of Clathrin from coated vesicle and trafficking cargo protein to the cell surface. GPCR ligands can also activate pro-TGFα protease TACE (TNFα-converting enzyme, encoded by ADAM17) to cleave and shed TGFα ectodomain. However, silencing RHBDF1 will diminish TACE maturation rate, and the ability of Src to be recruited to plasma membrane and binding to TACE is RHBDF1-dependent.

In summary, out results suggest that in response to GPCR activation, RHBDF1 critically assists pro-TGFα intracellular trafficking to the cell surface through clathrin/AP1 coated vesicles by regulating activity of Src,and is related to TACE maturation and function,all these factors give rise to an important influence of RHBDF1 on transactivation of EGFR.

#1888

Protein kinase C beta II suppresses colorectal cancer by regulating IGF1 mediated cell survival.

Catriona M. Dowling,1 James Phelan,2 Julia A. Callender,3 Mary Clare Cathcart,2 Brian Mehigan,4 Paul McCormick,4 Tara Dalton,1 John C. Coffey,1 Alexandra C. Newton,3 Jacintha O'Sullivan,2 Patrick A. Kiely1. 1 _University of Limerick, Limerick, Ireland;_ 2 _Trinity College Dublin, Dublin, Ireland;_ 3 _University of California San Diego, San Diego, CA;_ 4 _GEMS St.James Hospital, Dublin, Ireland_.

Despite extensive efforts, cancer therapies directed at the Protein Kinase C (PKC) family of serine/threonine kinases have failed in clinical trials. These therapies have been directed at inhibiting PKC and have, in some cases, worsened disease outcome. Here we examine colon cancer patients and show not only that PKC Beta II is a tumour suppressor, but patients with low levels of this isozyme have significantly decreased disease free survival. Specifically, analysis of gene expression levels of all PKC genes in matched normal and cancer tissue samples from colon cancer patients revealed a striking down-regulation of the gene coding PKC Beta in the cancer tissue (n = 21). Tissue microarray analysis revealed a dramatic down-regulation of PKC Beta II protein levels in both the epithelial and stromal diseased tissue (n=166). Of clinical significance, low levels of the protein in the normal tissue of patients is associated with a low (10%) 10 year survival compared with a much higher (60%) survival in patients with relatively high levels of the protein. Consistent with PKC Beta II levels protecting against colon cancer, overexpression of PKC Beta II in colon cancer cell lines reveals that PKC Beta II reverses transformation in cell based assays. Further to this, activation of PKC Beta II in an IGF-1 dependent manor showed a downregulation of AKT, indicating a role for PKCs in regulating IGF-1 mediated cell survival. Thus, PKC Beta II is a tumour suppressor in colon cancer and low levels serve as a predictor for poor survival outcome.

#1889

AXL and EGFR signaling mediate resistance to Crizotinib in non-small cell lung cancer cells harboring the ROS1 fusion gene.

Yuka Kato,1 Kadoaki Ohashi,2 Eiki Ichihara,2 Shuuta Tomida,1 Hiroe Kayatani,1 Kenichiro Kudo,1 Daisuke Minami,2 Takashi Ninomiya,2 Toshio Kubo,2 Toshiyuki Kozuki,3 Katsuyuki Hotta,2 Nagio Takigawa,4 Mitsune Tanimoto,2 Katsuyuki Kiura2. 1 _Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Okayama., Okayama city, Japan;_ 2 _Department of Respiratory Medicine, Okayama University Hospital, Okayama., Okayama city, Japan;_ 3 _Department of Respiratory Medicine, National Hospital Organization Shikoku Cancer Center, Matsuyama, Japan;_ 4 _Department of Internal Medicine 4, Kawasaki Medical School, Okayama., Okayama city, Japan_.

Background: The multi-tyrosine kinase inhibitor (TKI) crizotinib elicits a dramatic response in patients with non-small cell lung cancer (NSCLC) harboring the c-ros oncogene 1 (ROS1) fusion gene (Shaw et al. NEJM 2014); however, these patients inevitably develop resistance to crizotinib within a year, which limits the efficacy of crizotinib. Although reported molecular changes include ROS1 tyrosine kinase mutations, epidermal growth factor receptor (EGFR) activation, and epithelial-to mesenchymal transition, the detailed mechanism of crizotinib resistance has not been elucidated.

Methods: To explore the molecular mechanisms of acquired crizotinib resistance in detail, we used a cell line model. HCC78 cells harboring the SLC34A2-ROS1 fusion gene, which are sensitive to crizotinib, were exposed continuously to increasing concentrations of crizotinib in a step-wise manner. A crizotinib-resistant cell line designated HCC78R was established and assessed by MTT assay, Western blotting, a receptor tyrosine kinase (RTK) array, quantitative PCR (RT-PCR), ELISA, and an RNA kinome targeted kinome panel (612 genes) with next-generation sequencing.

Results: Crizotinib-resistant HCC78R (50% inhibitory concentration [IC50] 4085.9 nmol/L) was 47-fold more resistant to crizotinib than was parental HCC78 (IC50 85.8 nmol/L) (p = 0.0145) according to MTT assay. The RTK array revealed that EGFR phosphorylation was upregulated in HCC78R cells. Western blotting confirmed the activation of EGFR and its downstream signaling pathways. RT-PCR screening of EGFR ligand family member expression showed increased heparin-binding EGF-like growth factor (HB-EGF) in HCC78R compared with HCC78 cells. Consistently, addition of the HB-EGF or conditioned medium from HCC78R cells rendered the HCC78 parental cells moderately resistant to crizotinib. EGFR overexpression or activating mutations were not detected. Furthermore, RNA kinome sequencing revealed 16-fold higher AXL mRNA expression and 1.68-fold lower ROS1 fusion gene expression in HCC78R compared with HCC78. Overexpression of the AXL protein and downregulation of the ROS1 protein were confirmed by Western blotting. Monotherapy with gefitinib (EGFR-TKI) or R428 (AXL inhibitor) or cabozantinib (AXL inhibitor) moderately inhibited the growth of HCC78R cells, while combined therapy suppressed proliferation more significantly compared with each monotherapy.

Conclusions: AXL and EGFR signaling mediate resistance to crizotinib, and combination treatment of an EGFR-TKI and AXL inhibitor may be an alternative strategy in ROS1 fusion gene-driven lung cancer cells.

#1890

IKBKE is a substrate of EGFR and a therapeutic target in NSCLCs with activating mutations of EGFR.

Sridevi Challa,1 Jian-Ping Guo,2 Cheng-xiong Xu,1 Yajuan Li,1 Donghwa Kim,3 Douglas Cress,1 Eric Haura,1 Domenico Coppola,1 Jin Cheng1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Beth Israel Deaconess Medical Center, Tampa, FL;_ 3 _University of South Florida, Tampa, FL_.

EGFR is one of key driver pathways of non-small cell lung cancer (NSCLC). EGFR inhibitors have shown significant response in patients with EGFR mutation. However, patients develop resistance in short period of time, which results from secondary EGFRT790M mutation and other mechanisms. Here, we demonstrated that activating mutations of EGFR significantly activated IKBKE, an IκB kinase family member previously shown to activate Akt and NF-κB pathways. Furthermore, we showed that EGFR directly interacts with and phosphorylates IKBKE on tyrosine 153 and tyrosine 179 residues. IKBKE-Y153F/179-F, a mutant that could not be phosphorylated by EGFR, largely reduced its kinase activity and failed to activate Akt and NF-κB as well as inhibited EGFR signaling. Furthermore, phospho-IKBKE-Y153 correlated with EGFR activation in NSCLC patients. Notably, depletion of IKBKE by either small molecule inhibitor amlexanox or shRNA selectively inhibited cell viability in NSCLC cells with EGFR mutations. Moreover, we observed that inhibition of IKBKE led to feedback activation of the MAPK pathway. Combination of amlexanox with MEK inhibitor AZD6244 significantly inhibits cell survival and tumor growth in NSCLC cells driven by activating EGFR mutations including EGFRT790M. Thus, our findings not only identify IKBKE as a direct downstream target of EGFR but also provide a potential therapeutic strategy for EGFR-TKI resistant NSCLC driven by secondary EGFR mutation.

#1891

PDGF signaling maintains survival of proneural glioma cells by regulating USP1 to stabilize ID2.

Gilbert J. Rahme, Zhonghua Zhang, Alison L. Young, Chao Cheng, Eric J. Bivona, Steven N. Fiering, Yasuyuki Hitoshi, Mark A. Israel. _Norris Cotton Cancer Center, Lebanon, NH_.

Glioblastoma (GBM) is the most aggressive primary brain tumor and responds poorly to currently available therapies. Transcriptomic characterization of GBM has identified distinct molecular subtypes of GBM with the latest report identifying four molecular subtypes: proneural, neural, classical, and mesenchymal. Gain-of-function alterations leading to enhanced platelet-derived growth factor (PDGF) signaling are commonly observed in proneural GBM and can drive proneural gliomagenesis. However, little is known about the critical determinants of malignancy mediated by PDGF signaling in proneural glioma. Using a mouse model of proneural glioma and comparative transcriptomics, we determined that PDGF signaling upregulates ubiquitin specific peptidase 1 (Usp1) to promote survival of murine proneural glioma cells. Further experimentation revealed that PDGF-mediated expression of USP1 post-translationally stabilized Inhibitor of DNA-binding 2 (ID2), which we found to be required for glioma cell survival. Deletion of the Id2 gene delayed tumor-induced mortality. Pharmacological inhibition of USP1 decreased ID2 levels, and delayed tumorigenesis in mice. Importantly, decreased USP1 expression is associated with prolonged survival in patients with proneural GBM but not with other subtypes of GBM. Our data describe a signaling cascade downstream of PDGF that is required for the survival of proneural GBM cells and suggest that inhibition of the PDGF-USP1-ID2 axis could serve as a therapeutic strategy for the treatment of proneural GBM with increased PDGF signaling.

#1892

Investigating the role of Aurora Kinase A as a positive regulator of MAPK signaling.

MaKendra Umstead, Jinglin Xiong, Zenggang Li, Andrei Ivanov, Yuhong Du, Haian Fu. _Emory University, Atlanta, GA_.

Aurora Kinase A (Aurora A), a protein canonically known to facilitate mitosis, has emerged as a promising drug target for cancer therapy. Aurora A is amplified in several cancer types, including glioblastoma, breast, and ovarian cancer, and increased expression is correlated to a worse prognosis for patients. In spite of these observations, the impact of increased Aurora A expression on cell signaling and cancer development remains unclear. Aurora A is also mis-localized to the cytoplasm in cancer and engages in oncogenic functions mediated through protein-protein interactions (PPIs). Thus, we hypothesized that through novel PPIs, Aurora A may be promoting oncogenic signaling and cancer cell growth.

The acquired ability of cancer cells to sustain proliferative signaling is a cancer hallmark. One critical pathway through which mutations drive the development of several cancer types is the Ras-Raf-MEK-ERK signaling cascade, which is also known as the mitogen-associated protein kinase (Ras-MAPK) pathway. When we co-expressed Aurora A and H-Ras in both HEK-293T cells and the glioblastoma cancer cell line, 8-MG-BA, ERK phosphorylation increased compared to expression of Aurora A or H-Ras alone. However, pharmacological inhibition of Raf-1 and MEK was able to block the increase in ERK phosphorylation by Aurora A and H-Ras co-expression. In addition, Aurora A was not able to promote ERK phosphorylation when co-expressed with dominant negative H-Ras (S17N mutant). Together, these findings demonstrate that Aurora A requires the MAPK signaling cascade to potentiate ERK activation. Interestingly, we discovered that this effect correlates to a novel interaction between Aurora A and H-Ras. The Aurora A/H-Ras interaction was confirmed using the lysate-based assay, Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET), the affinity-based assay, GST-pull down, and in live cells using the Venus Protein-fragment Complementation Assay (PCA). Through deletion analysis, the binding domains critical to mediate the interaction of Aurora A and H-Ras were characterized. We also determined that H-Ras is not an enzymatic substrate for Aurora A using an in vitro kinase assay and found that cells treated with an Aurora A kinase inhibitor are able to maintain the Aurora A/H-Ras the interaction, suggesting that the Aurora A/H-Ras interaction is kinase-independent.

In conclusion, our studies reveal a role for Aurora A as a positive regulator of Ras-MAPK proliferative signaling. As Aurora A gene expression is downstream of the Ras-MAPK signaling pathway, our data also provides a mechanism by which Aurora A forms a positive feedback loop, linking Aurora A to sustained proliferative signaling in cancer cells. Finally, the kinase-independence of the Aurora A/H-Ras interaction underscores the consideration that Aurora A kinase inhibitors currently in clinical development may not be sufficient to block all oncogenic functions of Aurora A.

### MicroRNAs in Metastasis and Cancer

#1893

The effect of kinase signaling for miR-205 regulation in gefitinib-resistant lung cancer cell lines.

Toshihiro Suzuki,1 Ikuko Nagasawa,1 Toshimitsu Yamaoka,2 Tohru Ohmori,2 Kazuto Nishio,3 Kiyotaka Koyama,1 Yuki Ogasawara1. 1 _Meiji Pharmaceutical Univ., Tokyo, Japan;_ 2 _Showa University, School of Medicine, Tokyo, Japan;_ 3 _Kinki University School of Medicine, Osaka, Japan_.

Lung cancer is the leading cause of cancer related death in the world. Platinum-based combination regimen or molecular target drugs, such as EGFR tyrosine kinase inhibitors (EGFR-TKIs) and ALK inhibitors are used for first line chemotherapy of NSCLC. Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have been identified in NSCLC and those mutations confer sensitivity to the EGFR-TKIs such as gefitinib. Unfortunately, it is well known that the almost cases are developed to drug resistance after chemotherapy. Several EGFR-TKI resistant mechanisms have been reported, including gatekeeper mutation (T790M), c-MET amplification and ErbB3 activation. Re-biopsy is generally required for determine the resistance mechanism and second line chemotherapy. However, re-biopsy is invasive test for cancer patients. Recently, microRNAs (miRNAs) are paid attention as biomarkers for cancer diagnosis and sensitivity to chemotherapy in various cancers. In the previous study, we revealed that the miRNA biosynthesis pathway is critical for maintain the cisplatin resistant phenotype. Further, it was reported that EGFR modulated microRNA maturation through the phosphorylation of AGO2. Protein phosphorylation is one of most important post transcriptional modification mechanisms and miRNA is also associated with fine tuning of mRNA expression. Thus, we focused on the relationship between miRNA expression and kinase signaling in gefitinib resistance. In this study, we performed characterization of gefitinib resistant cell lines, PC-9/MET200 and PC-9/MET1000 established from NSCLC cell line, PC-9, which were confirmed to have a c-MET amplification using miRNA microarray and quantitative PCR. Furthermore, we analyzed miRNA activity and kinase signaling using a luciferase reporter assay system, siRNA, RTK/phosphokinase protein array and kinase inhibitors. We revealed that miR-205 was significantly elevated in both gefitinib resistant cell lines. By the computational analysis using miRNA target prediction database, we found that ErbB3 was a target of miR-205. However, ErbB3 expression levels were rather up-regulated and highly phosphorylated in both resistant cell lines compared with a parental cell line. Under ErbB3 knockdown condition, miR-205 expression levels were slightly down regulated. Furthermore, under c-MET knockdown or Foretinib treatment condition, miR-205 activity were down regulated and ErbB3 dephosphorylated in resistant cell lines. This down-regulation of miR-205 appears to be mediated by inhibition of kinase signals. Moreover, we found that some kinase inhibitors suppressed miR-205 activity in gefitinib resistant cell lines. In conclusion, our results suggest that alterations of kinase signals are more effective for miR-205 regulation than protein expression of target molecule itself, such as ErbB3.

#1894

Understanding the role of microRNA-206 in breast cancer.

Valery Adorno-Cruz,1 Huiping Liu2. 1 _Case Western Reserve University, Cleveland Heights, OH;_ 2 _Case Western Reserve University, Cleveland, OH_.

MicroRNAs are short non-coding RNA sequences that that can modify genes in post transcriptional manner and are often de-regulated in cancer. Cancer stem cells, a subset of cancer cells with stem cell properties, are considered the root of tumorigenesis and seeds of metastasis. In this study we are interested in elucidating the role of miR-206 in breast cancer stem cell-mediated tumorigenesis and metastasis. We have observed that microRNA-206 (miR-206) regulate the metastatic traits of breast cancer cells, both in vitro and in vivo. Our studies have elucidated a signaling cascade that culminates in the silencing of cytokine (such as interleukin) and integrin expression. Knockdown of the interleukin and integrin genes by siRNAs mimics the phenotype of miR-206 expression in breast cancer cells. DNA content analysis using propidium iodide staining revealed that knockdown of miR-206 targets as well as over-expression of miR-206 in MDA-MB-231 cells regulates cell cycle arrest. Our results indicate that miR-206 mediates its regulatory effects through inhibiting multiple targets. MiR-206 may serve as a new therapeutic candidate for the future treatment of breast cancer.

#1895

Metastasis-associated protein 1 drives prostate cancer progression and metastasis through regulation of onco-epimicroRNAs.

Swati Dhar, Nasir A. Butt, Avinash Kumar, Xu Zhang, Anait S. Levenson. _University of Mississippi Medical Center, Jackson, MS_.

Our recent studies in prostate-specific tumor suppressor Pten gene compromised transgenic mice, revealed a strategic role for Metastasis Associated protein 1 (MTA1), a component of the chromatin remodeling NuRD complex and a key regulator of metastasis, in the progression of prostate cancer. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) analyses from the mouse prostate tissues showed MTA1 occupancy of several potential oncogenic miRNA target promoters including miR-22. Retrospective investigation of microRNA microarray experiments revealed downregulation of these candidate miRNAs in LNCaP cells silenced for MTA1 expression (shMTA1). Real time PCR evaluation of shMTA1 cell lines, LNCaP, DU145 and PC3M attested the microarray findings. Using miRNA-mRNA target prediction algorithms, miRanda, miRDB and Targetscan, E-cadherin (CDH1) was identified as a potential target with high context score for miR-22. Interestingly, E-cadherin, which has a pivotal role in preventing Epithelial to Mesenchymal Transition (EMT) of tumor cells was also identified as a MTA1 target gene by ChIP-Seq analysis. Further, qRT-PCR and western blot experiments validated that MTA1 levels had an inverse correlation with E-cadherin expression in these cell lines. Transient transfection of miR-22 mimics diminished E-cadherin levels while this expression was restored by miR-22 sponge inhibitors showing that miR-22 can control E-cadherin expression. Ongoing experiments aim to address the direct targeting of E-cadherin 3′UTR by miR-22 using Dual-Glo luciferase reporter assay and the functional consequences of miR-22 promoter regulation by MTA1 utilizing chromatin and RNA immunoprecipitation assays. Our studies reveal a novel and previously unrecognized role for MTA1-mediated miR-22 regulation of E-cadherin.

#1896

miR-500a is involved in breast cancer-related gene expression pathways and associated with patients survival.

Vasily N. Aushev,1 Davide Degli Esposti,2 Eunjee Lee,1 Hector Vargas,2 Zdenko Herceg,2 Jun Zhu,1 Jia Chen1. 1 _Mount Sinai School of Medicine, New York, NY;_ 2 _International Agency for Research on Cancer, Lyon, France_.

MicroRNAs act as negative regulators of target genes by reducing their mRNA level. Despite numerous algorithms of target prediction, many microRNAs targets are still not known with enough confidence, making unclear their actual role in biological processes. In this study, we used an integrative approach combining bioinformatics and in vitro experiments to identify hsa-miR-500a as a key player in breast cancer survival.

The Cancer Genome Atlas (TCGA, dataset release of 2012) data were analyzed using recently developed bioinformatics algorithm (Lee et al., 2015) that considers not only expression levels of microRNAs, but also its "activity", determined by the relative changes of expression of potential target genes. High "activity" of miR-500a (hsa-miR-500a-5p, MIMAT0004773) was significantly correlated with poor survival of patients with ER-positive breast cancer. Noteworthy, in both TCGA and GEO (Gene Expression Omnibus) datasets, this form was typically expressed at very low levels (~26th percentile in ranking among detectable microRNAs) while another strand, miR-500a* (hsa-miR-500a-3p, MIMAT0002871) was hundred times more abundant (~81th percentile ranking), but did not correlate with the survival.

We carried out functional validation of miR-500a in a set of breast cancer cell lines including estrogen receptor (ER)-positive MCF-7, ER-negative MDA-MB-231 and their derivatives. In order to define actual targets (direct and indirect), MCF-7 cells were transfected with miR-500a mimics or inhibitors (miRCURY LNA, Exiqon), followed by analysis of gene expression with Illumina Human HT-12 microarray. Genes that displayed the strongest suppression upon transfection with miR-500a mimics are involved in hormone signaling: members of aldo-keto reductase family known for steroid hormones processing and thioredoxin reductase that can regulate transcriptional activity of estrogen receptors. We found that expression of miR-500a is increased in highly tumorigenic derivative of MDA-MB-231 cell line comparing to its low tumorigenic parental cell line. D3H2LN cell line (a MDA-MB-231-derived line by introducing luciferase construct) displayed higher proliferation rates and enhanced spheroids formation capacities comparing to the parental cell line; RNA-seq (by Illumina HiSeq) also revealed that this cell line has higher level of miR-500a expression and lower level of some genes detected as potential miR-500a targets.

Our results indicate potential role of miR-500a in regulation of signaling pathways involved in breast cancer progression. Experimental data lend further support in its functional role in breast cancer survival, which may help develop future intervention strategies.

#1897

P53-induced microRNA-30e suppresses colorectal cancer cell migration and invasiveness by regulating integrins.

Sara Laudato, Nitin Patil, Jörg H. Leupold, Mohammed Abba, Heike Allgayer. _Dept. of Experimental Surgery, Medical Faculty Mannheim, Centre for Biomedicine and Medical Technology, Ruprecht Karls University of Heidelberg, Mannheim, Germany_.

Alterations in miRNA expression have been implicated in the pathogenesis of colorectal cancer (CRC). Importantly, the p53 tumor suppressor is not only one of the most commonly altered genes in CRC, but it also regulates the expression of several protein and non-protein coding genes. In this study, we investigated a potential function of p53 in regulating miRNAs that mediate human CRC progression.

MiRNA microarrays were performed on human isogenic CRC cell line pairs (p53 wt and ko, a generous gift by Bert Vogelstein) to identify deregulated miRNAs attributable to p53 loss. The analysis revealed members of the miR-30 family, miR-30e particularly, as the most significantly downregulated group in the p53 knock-out cells compared to the wild type. To elucidate the impact of p53 loss on the expression of miR-30e, we also performed p53 overexpression and silencing in CRC cells harboring wt p53. Indeed, p53 silencing resulted in a reduction of miR-30e expression, whereas an increase of mature miR-30e was observed in CRC cells treated with Nutlin-3a, a specific MDM2 antagonist which is known to increase p53 expression or activity.

Next, we determined the miR-30e gene promoter region and found putative TP53 transcription factor binding motifs approximately 1 kb upstream the miR-30e genomic sequence by using in silico tools. The regions containing the predicted TF binding sites were cloned (pGL3-30e) and luciferase reporter gene assays were performed to determine if p53 indeed regulates the miR-30e promoter.

Interestingly, promoter reporter activity of pGL3-30e was significantly transactivated in cell lines carrying a wt p53, and this response was further enhanced in the presence of Nutlin 3a. However, the activity of pGL3-30e was not affected in p53 ko cells. Our observations indicate that the tumor suppressor p53 is able to induce the expression of miR-30e in colorectal cancer cell lines.

In addition, we identified two promising candidate targets of miR-30e within the integrin family. We demonstrated that miR-30e directly interacts with the 3' UTRs of the mRNAs of these two candidates and downregulates their expression at both the mRNA and protein level. Functionally, we found that the forced expression of miR-30e led to an integrin-mediated reduction of cell motility, invasion, cell proliferation, and adhesion to the extracellular matrix (ECM).

These results were corroborated by an inverse correlation of miR-30e and the identified integrins in a panel of resected CRC tissues. Taken together, our findings indicate that miR-30e is a p53 induced miRNA whose loss/downregulation in CRC orchestrates an integrin driven enhancement of in vitro tumor migration, invasion, proliferation and adhesion to the ECM. Ongoing experiments seek to recapitulate the above findings in vivo and also explore the impact of the p53/miR-30e/ITGs axis on metastasis.

#1898

microRNA-137/DCLK1 axis: A novel mechanism regulating tumorigenicity of colon cancer stem cell.

Masazumi Sakaguchi, Shigeo Hisamori, Nobu Oshima, Yoshiharu Sakai. _Kyoto University, Kyoto, Japan_.

Background: The understanding of colon cancer stem cell (CSC) biology is essential to developing new treatments, however, little is known about the molecular mechanisms underlying the acquisition of colon CSC properties. Furthermore, the similarity of the predisposition, including the surface markers, between colon CSCs and normal colon stem cells makes it difficult to develop a practical approach that targets colon CSCs. MicroRNA (miRNA) is one of the important factors regulating CSC properties. In addition, doublecortine-like kinase 1 (DCLK1), a microtubule-associated kinase, has been proposed to be a distinctive marker for colon CSCs. In the present study, we aimed to identify miRNAs that potentially target DCLK1 and unveil the role of the miRNA/DCLK1 axis in colon CSC properties.

Material and Methods: Human colon cancer and normal colon specimens were dissociated into single cells and sorted by flow cytometry to separate the stem cell enriched population (EpCAM+/CD44+/CD66a-) and stained with anti-DCLK1 antibody. The expression profile of 384 miRNAs and the expression of DCLK1 in the colon CSC and normal colon stem cell populations were determined using a qRT-PCR. A luciferase activity assay and Western blotting were performed to evaluate the relationship between the miRNA and DCLK1. A lentiviral expression system was designed to investigate the miRNA phenotypes. We adopted an organoid assay and in vivo tumorigenicity assay to examine the influence of the miRNA on CSCs.

Results: MiRNA-137 was highly expressed in the normal colon stem cell population whereas the DCLK1 mRNA expression was significantly upregulated in the colon CSC population. Actually, DCLK1-positive colon cancer cells were widely distributed in the colon cancer specimens, while DCLK1-positive epithelial cells were rarely detected in normal colon tissues, including the crypt bottoms. We confirmed that the activity of the luciferase gene linked to the 3'UTR of DCLK1 was decreased and that the protein level of DCLK1 was suppressed in the miRNA-137 transduced SW480 cells. The transduction of exogenous miRNA-137 suppressed the organoid development of colon cancer cells as well as shRNAs against DCLK1. The defect in organoid development by the transduction of miR-137 or shRNAs were substantially rescued by co-expression of the exsogenous DCLK1. Although miRNA-137 overexpression did not affect the organoid development of the normal intestine, miRNA-137 knockdown promoted the organoid development of normal colon cells. Xenograft tumor formation and growth were markedly suppressed in the miRNA-137-transduced SW480 cell-injected mice.

Conclusion: These results suggest that miRNA-137 has the potential to suppress the tumorigenicity of colon CSCs through the inhibition of DCLK1 and that the dysregulation of the miRNA-137/DCLK1 axis plays an important role in colon CSCs.

#1899

Let-7b overexpression is associated with higher nucleotide excision repair pathway in women with breast cancer.

Jarline Encarnacion,1 Carmen Ortiz,1 Ralphdy Vergne,2 Wanda Vargas,1 Jaime L. Matta1. 1 _Ponce Health Sciences University, Ponce, PR;_ 2 _University of Puerto Rico at Ponce, Ponce, PR_.

Nucleotide Excision Repair (NER) is a critical pathway involved in breast cancer (BC). We have previously published that a low DNA repair capacity (DRC) is associated with a higher risk of BC in Puerto Rican women. In recent years we have focused our investigations on microRNAs (miRNAs) that are differentially associated with a low and a high DRC in women with BC and controls. A discovery experiment with 29 BC cases and 27 controls produced 12 candidate miRNAs associated with DRC including let-7b. The main objective of this study was to elucidate if there is a correlation between let-7b expression and specific DRC levels. Let-7b belongs to a miRNA family with tumor suppressor activity that targets oncogenic genes such as Ras, Myc, and HmgA2. DRC was measured in lymphocytes by means of a host cell reactivation assay with a luciferase reporter gene. We isolated miRNAs from plasma of 145 Puerto Rican women with and without BC (recently diagnosed, untreated cases and controls) using the miRNeasy kit (Qiagen). These women were divided into four groups according to their DRC level: high (>3.8%) and low (<3.8%). The four groups consisted in BC cases with high (n=32) and low (n=41) DRC, and controls with high (n=38) and low (n=34) DRC. Let-7b expression was measured using TaqMan microRNA assay. In addition, epidemiological data of these women has been collected at their initial BC diagnosis (before treatment) and almost five years after diagnosis. Within the BC group with low DRC, 9 women had presented recurrence. We found no significant difference in let-7b expression between controls without BC when compared to women with BC with both high and low DRC levels. A significant difference in let-7b expression was found in BC cases with high DRC when compared with the remaining groups (p<0.001). The down-regulation of let-7b family miRNAs is associated with poor prognosis in various types of cancers, including breast, lung, and ovarian, among others. Thus, our data reveal a possible role of let-7b involving DRC intrinsically when the malignancy is developed. Our findings support the association previously reported between a poor prognosis in several cancers and low levels of let-7b expression. Our study is innovative because it provides the first evidence that let-7b might play role in DRC (through the NER pathway) regulation in BC. Also, our study suggests that the overexpression of let7-b in women with BC and a high DRC is associated with a better prognosis. Supported by grants from the NCI Center to Reduce Health Disparities and NIH-NIGMS MBRS Program grants #S06 GM008239-20, 9SC1CA182846-04, GM082406, and PSM-MCC Partnership grant #5U54CA163071-04.

#1900

MiR-222 accelerates the progression of inflammatory breast cancer by targeting tumor suppressor gene p27kip1 (CDKN1B).

Joshua Were Ogony, Erin Hayes, Jennifer Knapp, Joan Lewis-Wambi. _University of Kansas Medical Center, Kansas City, KS_.

MicroRNAs (miRNAs) are small non-coding RNAs about 18-22 nucleotides in length, whose main role is to regulate gene expression by altering mRNA stability and translation. MicroRNAs have been shown to play a role in many types of cancer, including breast cancer by targeting tumor suppressor genes such as p53, p27, PTEN, BRCA1, BRCA2 and many others . The goal of this study was to investigate the role of miRNAs in inflammatory breast cancer (IBC). IBC is the most aggressive and metastatic form of breast cancer, that is characterized by very rapid progression, poor prognosis, with a 5 year survival outcome of 30-40 %. The mechanism that underlies the rapid progression of IBC is currently under intense investigation, and novel approaches are needed to better understand this disease. To understand the role that microRNAs might play in IBC, we conducted next generation sequencing of the miRNAs from two IBC cell line, SUM149 which is a triple negative IBC (ER-, PR-, HER2-), and SUM190 which is HER2 overexpressing, but ER and PR negative (ER-, PR-, HER2+). We found that 463 miRNAs were differentially expressed in the two cell lines, with 243 miRNAs being overexpressed in SUM149 compared to SUM190, while 220 miRNAs were overexpressed in SUM190. Notably, our qPCR analysis confirmed the raw read counts, showing that miR-155, miR-221, and miR-222 were overexpressed in SUM149 as compared to SUM190 cells by 15, 10, and 3 fold respectively. Furthermore, the inhibition of miR-222 in SUM149 resulted in significant reduction in cell proliferation, tumorsphere formation, and migration in these cells as compared to SUM190 cells. Our flow cytometry data showed that there was cell cycle arrest at the G1 phase in SUM149 cells due to miR-222 inhibition, while SUM190 cells showed little or no change. Additionally, Western blot and immunofluorescence analyses showed that the protein levels of p27, which is one of the targets of miR-222, significantly increased in SUM149 cells, but not in SUM190 cells when miR-222 inhibitor was used in these cells. These results suggest that miR-222 may accelerate the progression of some IBCs by targeting tumor suppressor genes, resulting in rapid cell proliferation, and may be a potential therapeutic target for triple negative inflammatory breast cancers.

#1901

Leptin modulation of PCSC, HDAC, and microRNA in pancreatic adenocarcinoma.

Cynthia M. Tchio, Adriana Harbuzariu, Tia Harmon, Derrick Beech, Ruben Gonzalez-Perez. _Morehouse School of Medicine, Atlanta, GA_.

Background: Obesity is an important risk factor of Pancreatic Adenocarcinoma (PA) and it is characterized by the accumulation of excessive body fat and a Body Mass Index (BMI) value greater than 30. Obesity is also characterized by high levels of leptin, which has been consistently associated with the development of many different cancer including PA. Leptin can induce the proliferation of Pancreatic Cancer Stem Cells (PCSC), which are responsible for chemoresistance, invasiveness and reoccurrence of PA. PCSC express specific cell markers and can form tumorspheres in vitro. Obesity can alter the DNA acetylation and microRNA activity which are also linked to PA progression. We hypothesized that high level of leptin could modulate Histone Deacetylase (HDAC) and microRNA activity in PA cells, which induce PCSC changes and tumor progression.

Methods: The PA cell lines were cultured in mammocult media that contained heparin and hydrocortisone, which will allow the proliferation of tumorspheres enriched with PCSCs. The cells were cultured with leptin, chemotherapeutic drug, and leptin signaling inhibitor IONP-LPrA2. Tumorspheres formation (number and size) was determined after 1 week of treatment. Additionally, the levels of PCSC markers, HDAC1, HDAC2, HDAC3, and HDAC8 in tumorspheres were determined using flow cytometry, western blot, and RT-PCR. In addition, the effects of treatments on miR21 and miR200a/c levels were determined using RT-PCR.

Results: Leptin induced PA tumorspheres formation and size, which was accompanied by higher level of PCSC markers (CD24, CD44, ESA, and ALDH1). Moreover, leptin affected the level of HDACs, miR21 and miR200a/c in PA tumorspheres. IONP-LPrA2 abrogated leptin effects and decreased PCSC which were spared by chemotherapeutics.

Conclusion: Obesity signals via leptin could be involved in the increase PA aggressiveness and chemoresistance, which may be linked to the increase of PCSC. Leptin could induce PA progression and chemoresistance via modulation of HDAC and miRNA.

Acknowledgement: This work was supported by the DOD W81XWH-13-1-0382; NIH/SBIR1R41CA183399-01A1; Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center Partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP; Calvin Johnson Jr. Foundation Pancreatic Cancer Research Scholarship to CITM, and facility and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR03034 and 1C06 RR18386).

#1902

Downregulation of MYC by miR-3189-3p in glioblastoma.

Duane Jeansonne, Krzysztof Reiss, Francesca Peruzzi. _Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA_.

Glioblastoma is a devastating malignancy with a median survival of approximately 15 months. This aggressive tumor originates from the glial cells and accounts for nearly 50% of adult brain tumors. Glioblastomas are characterized by rapid proliferation and resistance to standard treatment of surgery, temazolamide and radiation therapy. MicroRNAs are short single-stranded non-coding RNAs that regulate gene expression in both normal and pathological conditions. We have previously found downregulation of miR-3189-3p in glioblastomas and astrocytomas. Using in vitro models, we demonstrated that ectopic expression of this miRNA inhibited glioblastoma cell growth and migration through downregulation of the splicing factor SF3B2 and the guanine nucleotide exchange factor p63RhoGEF, respectively. The miR-3189-3p-mediated inhibition of glioblastoma growth in vivo further confirmed the anti-cancer activity of this microRNA.

Given the strong biological effect of this miRNA on glioblastoma cells, we sought to investigate the implication of other gene targets in miR-3189-3p-mediated effects. The elevated expression of MYC oncogene is found in a variety of cancer types, including glioblastoma. Here, we demonstrated that overexpression of miR-3189-3p results in downregulation of MYC. Since this gene is not a predicted target for miR-3189-3p, we hypothesized that this modulation of MYC expression is mediated through the translational repressor, TIA1 cytotoxic granule-associated RNA binding protein-like 1 (TIAL1, also called TIAR). We tested the levels of TIAR in cells treated with miR-3189-3p and found no changes in TIAR expression. However, the downregulation of MYC was dependent on TIAR expression, as determined through siRNA-mediated downregulation of TIAR. Further studies are needed to elucidate molecular mechanisms of TIAR-dependent downregulation of MYC by miR-3189-3p. In addition, a putative binding site for miR-3189-3p is located within the open reading frame of MYC; therefore, studies will be performed to determine whether miR-3189-3p downregulates MYC by directly binding outside the 3'-UTR of this transcript.

#1903

A genetic variation in microRNA target site of ETS2 is associated with clinical outcomes of paclitaxel-cisplatin chemotherapy in non-small cell lung cancer.

SON JI WOONG,1 Shin Yup Lee,2 Chang Ho Kim,2 Jae Yong Park2. 1 _Konyang University College of Medicine, Daejeon, Republic of Korea;_ 2 _Internal Medicine, School of Medicine, Daegu, Republic of Korea_.

Background: Recently, crosslinking, ligation, and sequencing of hybrids (CLASH) provided direct observation of transcriptome-wide miRNA-target pairs. The present study was performed to investigate the association of single nucleotide polymorphisms (SNPs) located in the miRNA target sites, which were experimentally verified by CLASH, with the clinical outcome of first line paclitaxel-cisplatin chemotherapy in advanced non-small cell lung cancer (NSCLC).

Methods: Eighty SNPs in miRNA target sites of cancer related genes selected from 18,500 miRNA:target interactions in CLASH data were investigated in 379 advanced NSCLC patients using a sequenom mass spectrometry-based genotype assay. Quantitative reverse transcription-polymerase chain reaction and luciferase assay were conducted to examine functional relevance of potentially functional SNPs in miRNA target sites.

Results: Of the 80 SNPs analyzed, 16 SNPs were significantly associated with the clinical outcome after chemotherapy. Among these, ANAPC1 rs3814026C>T, ETS2 rs461155A>G, SORBS1 rs7081076C>A and POLR2A rs2071504C>T were found to be significantly associated with both chemotherapy response and survival. Notably, the relative expression level of ETS2 was significantly associated with rs461155A>G genotypes in both tumor and paired normal lung tissues (Ptrend = 4 x 10-7,and3x10-4,respectively). Consistently, a decreased expression of the reporter gene for the G allele of rs461155 compared with the A allele was observed by luciferase assay.

Conclusion: These findings suggest that the four SNPs, especially ETS2 rs461155A>G, could be used as biomarkers predicting the response and survival of NSCLC patients treated with first-line paclitaxel-cisplatin chemotherapy.

#1904

MiR641 regulates EMT, ovarian cancer stem cell and angiogenesis by targeting p63/miR200 axis.

Yajuan Li, Edward Richards, Mark Coppola, Cheng-Xiong Xu, Jin Q. Cheng. _Moffitt Cancer Center, Tampa, FL_.

MiR641 is an uncharacterized miRNA which locates intron 1 of AKT2, a gene residing in the chromosome 19q13.2 amplicon and frequently amplified/overexpressed in refractory ovarian cancer. Here, we demonstrated that frequent upregulation of miR641 was observed in human ovarian cancer tissues and in ovarian cancer cell lines. Knockdown of miR641 inhibited cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) as well as angiogenesis and ovarian cancer stem cell growth. Enforced expression of miR641 had opposite effect. In addition, ectopic expression of miR641 promoted VEGF protein expression and secretion but had no effect on VEGF mRNA level. Mechanistically, we found that miR641 directly targeted the 3'UTR of p63 and reduced p63 expression, which leads to a decrease of miR200a/b. As a result, ZEB1 and VEGF, two validated targets of miR200, were upregulated by miR641 overexpression. Furthermore, expression of miR641 induced anchorage-independent growth. Taken together, these findings suggest that miR641 is an onco-miRNA and plays a pivotal role in ovarian oncogenesis.

#1906

Inhibition of miR-186 and repression of aggressive prostate cancer phenotype using a metastatic cell model.

Dominique Z. Jones, M. Lee Schmidt, Katharine R. Hobbing, Geoffrey Clark, LaCreis R. Kidd. _University of Louisville, Louisville, KY_.

MicroRNA (miR) dysregulation alters the expression of cancer related genes and contributes to disease states in many cancers. For example, ectopic miR-186 expression leads to enhanced cell proliferation and migration in pancreatic cancer. However, the role of miR-186 in prostate cancer (PCa) remains unclear. Previously, we observed significant upregulation of miR-186-5p in PCa patient serum (stage III/IV) compared to controls. Furthermore, miR-186 was significantly up-regulated in metastatic PCa (PC-3) compared to normal prostate epithelial cells (RWPE1). We hypothesized miR-186 inhibition will reduce aggressive PCa in vitro.

Consequently, miR-186 was transiently and stably inhibited in PC-3 cells. Cell proliferation and colony formation were evaluated for 7 and 21 days via Trypan Blue exclusion and soft agar assays. Aberrant gene expression was evaluated in transfected cells to identify miR-186 targets. Candidate miR-186 targets were selected using published reports on 'miR-186 and cancer', availability of robust antibodies, and statistical filtering (false discovery ≥0.05 and ±1.2 fold change).

Mir-186 inhibition in PC-3 cells significantly repressed proliferation and colony formation by 34-64%. Following miR-186 inhibition in PC-3 cells, 2,343 identified mRNA targets were differentially expressed compared to scramble controls (p < 0.05). The target list was reduced to 11 up-regulated candidates. Predicted miR-186 targets are undergoing validation via qRT-PCR, western blots, and luciferase reporter assays. In addition, other studies are in progress to evaluate the impact of miR-186 on cellular proliferation, migration and invasion. Such efforts may lead to the identification of novel biomarkers to improve detection and clinical management strategies for aggressive PCa.

#1907

Multi-step microRNA control of pancreatic neuroendocrine tumors metastatic cascade.

Iacovos P. Michael, Douglas Hanahan. _Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland_.

MicroRNAs (miRs) are implicated in the pathogenesis of various malignancies, including pancreatic cancer, and may represent key players in the metastatic process. In a previous study (Olson et al., 2009), we identified two unique miR signatures associated with tumour metastasis in the mouse model of pancreatic neuroendocrine tumour (PNET), RIP-Tag2. The first signature was characteristic of rare liver metastases. Intriguingly, the second signature, identified in a subset of primary PNET, had overlap with the liver metastasis signature. This signature was denoted "met-like primary" (referred to as MLP-miRs). Bioinformatic analysis revealed that three MLP-miRs (miR-181, miR-137, and miR-132), as well as three metastasis-specific miRs (miR-23b, miR-27b, and miR-24-1) are also upregulated in human PNET MLP samples.

To examine the potential pro-metastatic role of the aforementioned miRs, we generated PNET cancer cell lines (β-TC) that stably express single miRs or combinations. We first audited their effect using orthotopic studies. MiR-181 caused a significant delay in the tumor progression, while the rest of the miRs did not affect tumor growth. Notably, the orthotopic tumors overexpressing miR-137 and miR-132 were characterized by markedly enhanced invasion into adjacent normal tissue. Then, using experimental metastasis assays we examined potential roles in distant metastasis. Overexpression of the miR-23b cluster (miR-23b, -27b and -24-1) caused a dramatic increase in the number of liver metastatic foci, indicating that they play a significant role in seeding distant metastases. The aforementioned pro-invasive miRs had little activity in this assay. Using an inducible system we went on to demonstrate that the three miRs comprising the 23b cluster act together in a synergistic manner during the early stages of distant colonization.

Transcriptome analysis of miRNA isolated from β-TC expressing cell lines revealed enrichment of genes involved in axonal guidance, actin nucleation and integrin signaling in the case of miR-137, and of glutamate receptor signaling and EMT in the case of miR-23b cluster.

Overall, our results suggest a multi-step miRNA control of the PNET metastatic cascade, during which miR-137 and miR-132 enables PNET cancer cells to invade,, while the miR-23b cluster plays an important role in distant metastasis. Using miR gene target prediction algorithms applied to mouse and human PNET transcriptome datasets, we are in the process of identifying and validating possible effector genes involved in distinctive aspects of the invasion-metastasis cascade, potentially guiding the design of new therapeutic strategies to target metastasis.

#1908

Lung tumorigenesis and metastasis is suppressed by the miR-200-GATA4/6-BMP4 axis.

Jeong Seon Kim,1 Jonathan M. Kurie,2 Young-Ho Ahn1. 1 _Ewha Womans University School of Medicine, Seoul, Republic of Korea;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

MicroRNA-200 (miR-200) family members suppress the epithelial-mesenchymal transition of various cancer cells, including lung adenocarcinoma cells. Previously, we found that forced expression of miR-200 decreased bone morphogenetic protein 4 (BMP4). In this study, we explored the mechanism and role of BMP4 depletion by miR-200 in murine lung adenocarcinoma cells. miR-200 down-regulated BMP4 via direct targeting of the GATA4 and GATA6 transcription factors that stimulate Bmp4 transcription. Bmp4 knockdown suppressed cancer cell growth, migration, and invasion and inhibited tumorigenesis and metastasis of lung cancer cells when injected into syngeneic mice. In addition, BMP4 was required for normal sphere formation in Matrigel 3-D culture, which might be mediated by MYH10, a downstream target of BMP4. These findings suggest that BMP4 functions as a pro-tumorigenic factor in a murine lung cancer model, and its transcription is regulated by miR-200 and GATA4/6.

#1909

miR-30c blocks tumor cell homotypic aggregation and promotes anoikis to prevent breast cancer metastasis.

Xia Liu, Beijie Yu, Wenjing Chen, Huiping Liu. _Case Western Reserve Univ., Cleveland, OH_.

Circulating tumor cells (CTCs) are considered the seeds of distant metastasis, and may be a predictor of poor prognosis in patients with metastatic breast cancers. Compared to single CTCs, clusters of multiple CTCs possess higher metastatic capacity in mouse breast cancer models, indicating that aggregation increases the survival of CTCs in the circulation. However, the molecular mechanisms by which clustered CTCs mediate tumor cell survival are unclear. In this study, we found that miR-30c, which has been identified as an anti-metastatic microRNAs, inhibited primary breast cancer cell homotypic aggregation in vitro by real-time monitoring. Interestingly, only aggregated but not single tumor cells can survive during culture. The death of dissociated primary tumor cells in vitro is caused by anoikis, a form of programmed cell death which is induced by detachment of anchorage-dependent cells from the surrounding extracellular matrix. Therefore, the role of miR-30c in anoikis was determined by culturing breast cancer cells in poly-hema coated plates. We found that miR-30c promoted anoikis, evaluated by Annexin-V staining and caspase3/7 activity. Mechanistic studies indicated that inhibition of Vimentin (Vim), a positive regulator of epithelial-to-mesenchymal transition (EMT), was required for miR-30c induced anoikis. In vivo, lung metastasis was inhibited by miR-30c after tail vein injection of miR-30c over-expressed cells. In summary, our findings provide the new insights into the mechanisms of miR-30c-mediated metastasis inhibition and suggest that targeting CTC clusters could be a novel strategy to prevent metastasis.

#1910

Tumor-suppressive microRNAs (miR-26a/b, miR-29a/b/c and miR-218) concertedly regulate metastasis-promoting LOXL2 in prostate cancer.

Mayuko Kato,1 Akira Kurozumi,1 Yusuke Goto,1 Ryosuke Matsushita,2 Ichiro Fukumoto,1 Atsushi Okato,1 Rika Nishikawa,1 Shinichi Sakamoto,3 Tomohiko Ichikawa,1 Naohiko Seki1. 1 _Department of Functional Genomics, Chiba University Graduate School of Medicine, Chiba, Japan;_ 2 _Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan;_ 3 _Department of Urology, Chiba University Graduate School of Medicine, Chiba, Japan_.

BACKGROUNDS:

Most patients with prostate cancer (PCa) initially respond to androgen-deprivation therapy, but eventually becomes hormone-insensitive and progress to castration-resistant prostate cancer (CRPC). CRPC is difficult to treat, and most clinical trials for advanced PCa have shown limited benefits, with disease progression and metastasis to the skeleton or other sites. Therefore, understanding the molecular mechanisms of CRPC and the metastatic pathways underlying PCa using genomic approaches would help to prevent and improve therapies for the disease. Our previous studies showed that three tumor-suppressive microRNAs (miRNAs), miR-26a/b, miR-29a/b/c and miR-218, significantly inhibited cancer cell migration and invasion. Therefore, we hypothesized that these miRNAs-regulated target genes deeply contributed to cancer metastasis.

METHODS:

Genome-wide gene expression analysis and in silico analysis were applied to investigate molecular targets regulated by miR-26a/b, miR-29a/b/c and miR-218 in PCa cells. A luciferase reporter assay was carried out to determine whether 3' UTR of target genes have actual biding sites for these miRNAs. To investigate the functional role of target genes in PCa cells, loss-of-function studies by using siRNAs of targets genes were performed.

RESULTS:

Our in silico analysis and luciferase reporter assays showed that lysyl oxidase-like 2 (LOXL2) was directly regulated by tumor-suppressive miR-26a/b, miR-29a/b/c and miR-218 in PCa cells. Overexpression of LOXL2 was observed in PCa clinical specimens by immunohistochemical staining. Moreover, downregulating the LOXL2 gene significantly inhibited cell migration and invasion in PCa cells. The function of the LOXL2 is covalent crosslinking of collagen and/or elastin in the ECM. Aberrant expression of LOXL2 has been reported in several diseases, including cancers.

CONCLUSIONS:

Our present data showed that the gene promoting metastasis, LOXL2, was epigenetically regulated by tumor-suppressive microRNAs, miR-26a/b, miR-29a/b/c and miR-218 providing important insights into the molecular mechanisms of PCa metastasis. Elucidation of tumor-suppressive miRNAs regulated molecular pathways and targets could provide new information on potential therapeutic strategies in PCa.

#1911

ΔNp63α promotes TGFβ-induced metastasis by silencing a microRNA network restraining wound healing.

Lidia Rodriguez Calleja,1 camille Jacques,1 Francois Lamoureux,1 Marc Baud'huin,1 Thibault Quillard,1 Franck Verrecchia,1 Dominique Heymann,1 Leif Ellisen,2 Benjamin Ory1. 1 _INSERM UMR957, Nantes, France;_ 2 _MGH, Harvard Medical School, Boston, MA_.

Primary cancer cell dissemination is the key event in metastasis, yet context-specific determinants remain largely undefined. Multiple reports have suggested an anti-metastatic role for the p53 family member p63 linked to its minor epithelial isoform, TAp63. However, the role and contribution of the major p63 isoform, ΔNp63α, remain largely undefined. Here we report a distinct and TAp63-independent mechanism in which ΔNp63α-expressing cells, in a TGFβ-rich microenvironment, are positively selected for metastatic dissemination. We define the molecular mechanism of this effect, through ΔNp63α-mediated repression of miR-527 and miR-665 and the subsequent upregulation of two TGFβ pathway major effectors, SMAD4 and TβRII. Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound healing response. We show that ΔNp63α and miR-527/665 control the TGFβ-dependent signaling that switches off anti-migratory miR-198 by downregulating the regulatory factor KSRP. Collectively, these findings reveal a novel microRNA network orchestrated selectively by ΔNp63α that is involved in the physiological regulation of wound healing but is hijacked by tumors to promote metastatic dissemination.

#1912

Combinatorial targeting of PI3K and MAPK signaling pathways using microRNAs to inhibit tumor growth and metastasis in breast cancer.

Merve Mutlu, Ozge Saatci, Erol Eyupoglu, Umar Raza, Ozgur Sahin. _Bilkent University, Ankara, Turkey_.

PI3K/Akt and MAPK signaling pathways, regulating cancer cell proliferation, apoptosis and metastasis, are among the top most deregulated pathways in cancer. Current kinase inhibitors used in clinics have limited success since one of these pathways gets activated when the other is suppressed. Alternatively, simultaneous silencing of both pathways results in high toxicity. Here, we aimed to establish a microRNA-based, non-toxic approach to inhibit these pathways simultaneously in breast cancer. By analyzing our previously published Reverse Phase Protein Array (RPPA) data showing differential expression of 26 proteins upon overexpression of 733 different miRNAs, we selected top miRNAs significantly deregulating PI3K and MAPK pathways as well as cell cycle. We narrowed down the list by selecting miRNAs whose predicted targets were among the PI3K and MAPK pathway elements. We came up with a tumor suppressor miRNA affecting both in vitro viability and migration/invasion of breast cancer cells. We identified the set of differentially expressed genes in breast cancer patients under low and high expression of the miRNA. A significant association was observed between the expression of the miRNA or its identified gene signature and clinico-pathological properties of breast cancer patients. Combinatorial knockdown of targets of the miRNA that are in PI3K and MAPK pathways as a cocktail phenocopied the tumor suppressive effects of the miRNA in breast cancer cell lines. Notably, low expression of the target cocktail predicts a better outcome in breast cancer patients. Overall, we identified a tumor suppressor miRNA to be used an alternative to current kinase inhibitor-based therapies for major subtypes of breast cancer by suppressing both PI3K and MAPK pathways and as a result inhibiting proliferation and migration/invasion in breast cancer. 

### Non-coding RNAs and Mechanisms in Cancer

#1913

Inhibition of S-Adenosylmethionine-dependent methyltransferase resists TGF-β1-induced EMT of pancreatic cancer via microRNA alterations.

Rajgopal Govindarajan, Hardik Modi. _The Ohio State University, Columbus, OH_.

Identification of epigenetic reversal agents for use in combination chemotherapies for human pancreatic ductal adenocarcinomas remains an unmet clinical need. Pharmacological inhibitors of EZH2 are emerging as potential epigenetic reversal agents in various solid tumors and leukemia, however, the surprisingly small set of mRNA transcripts identified with EZH2 knockdown suggests novel mechanisms to govern their anti-tumorigenic effects. Here, we report 3-deazaneplanocin-A, an inhibitor of S-adenosyl-L-homocysteine hydrolase and EZH2 histone lysine-N-methyltransferase, to significantly reprogram the noncoding miRNA expression, including a subset of PDAC-downregulated miRNAs, to dampen TGF-β-induced EMT signals in pancreatic cancer. Particularly, we identify epigenetic silencing of miR-663a and -4787-5p in PDAC tissues and cell lines that are reactivated by 3-deazaneplanocin-A to directly target TGF-β1 for RNA interference. Lentiviral overexpression of miR-663a and -4787-5p partially phenocopied 3-deazaneplanocin-A's EMT resisting effects in pancreatic cancer cells, whereas locked nucleic acid antimiR-663a and -4787-5p mitigated them. In vivo, 3-deazaneplanocin-A, miR-663a and -4787-5p significantly reduced tumor burden and metastasis in an orthotopic mouse pancreatic tumor model with miR-663a displaying more potency than miR-4787-5p in curtailing TGF-β1 signaling pathway. These findings reveal tumor miRNA reprogramming as a novel approach for controlling TGF-β-induced EMT in human PDAC and uncover a list of putative miRNA targets that may guide further selection and development of synthetic histone methylation reversal agents for clinical use.

#1914

CtBP1 and metabolic syndrome modulate cell adhesion and target multiple miRNAs in breast cancer cells.

Paola De Luca, Paula Lucía Farré, Nicolás Dalton, Cristian Pablo Moiola, Juliana Porretti, Cintia Massillo, Georgina Scalise, Adriana De Siervi. _Instituto de Biología y Medicina Experimental (IByME) - CONICET, Buenos Aires, Argentina_.

Breast cancer is the leading cause of cancer death among women, after skin cancers. Although genetic susceptibility clearly influences cancer risk, non-inherited factors determine most of the differences in cancer risk across populations and among individuals. Metabolic syndrome (MeS) increases the incidence and aggressiveness of breast cancer. C-terminal binding protein 1 (CtBP1) is a co-repressor of tumor suppressor genes that is activated by low NAD+/NADH ratio. Previously, we found that both, CtBP1 and MeS modulated breast carcinogenesis and tumor growth using a MeS experimental mice model by chronically feeding animals with high fat diet (HFD). The most severe form of breast cancer is metastatic and at this stage the disease is often fatal. The aim of this work was to investigate CtBP1 and MeS role on cell adhesion, a key process for the establishment of metastasis. We found that CtBP1 protein diminished adhesion of MDA-MB-231 breast cancer cells. More important, serum from MeS animals diminished breast cancer cell adhesion compared to control serum. In addition nude mice fed with CD or HFD were subcutaneously injected with MDA-MB-231 breast tumor cells with CtBP1 depleted expression or control cells. We found that CtBP1 and MeS modulated expression of cell adhesion targets in the xenografts, such as Vimentin, Slug, ITGB4, Col17A1, FABP4 and PRSS2.

Interestingly, miRNA expression profile obtained by RNA from CtBP1 depleted or control xenograft tumors hybridization to GeneChip miRNA 4.0 (Affymetrix) identified 42 CtBP1 regulated miRNAs. We found 77 predicted miRNAs target genes up- and 30 genes down-regulated by this set of 42 differentially expressed miRNAs, using miRecords data base. Gene ontology (GO) analysis of all these genes revealed an enrichment of localization, metabolic processes, cellular process and biological regulation categories, among other biological functions. Examining processes within these GO functions; we found important categories overrepresented, such as cell cycle, cell communication, vesicle-mediated transport and primary metabolic process.

These results clearly show that CtBP1 and MeS diminish adhesion of breast cancer cells and suggest a key role for these components in the begining of metastasis. In addition, understanding CtBP1 and MeS regulation of miRNAs could be crucial as markers for the prevention, follow up and treatment of breast cancer.

#1915

microRNA-320a inhibits cancer cell migration and invasion through targeting LAMP1 in castration-resistant prostate cancer.

Atsushi Okato,1 Yusuke Goto,1 Akira Kurozumi,1 Mayuko Kato,1 Ryosuke Matsushita,2 Satoko Kojima,3 Shinichi Sakamoto,1 Tomohiko Ichikawa,1 Naohiko Seki1. 1 _Chiba University, Chiba, Japan;_ 2 _Kagoshima University, Kagoshima, Japan;_ 3 _Teikyo University Chiba Medical Center, Chiba, Japan_.

BACKGROUND:

In early-stage prostate cancer (PCa), more than 90% of patients initially respond to the therapeutic use of androgen deprivation; however, many cases become refractory and progress to castration resistant prostate cancer (CRPC). CRPC is difficult to treat, and most clinical trials for advanced PCa with metastasis to bones or other sites have shown limited benefits. Therefore, understanding the molecular mechanisms of metastatic pathways of CRPC underlying current genomic approaches would help to improve therapies for this disease.

Our recent studies of microRNA (miRNA) expression signatures of CRPC revealed that microRNA-320a (miR-320a) was significantly reduced in cancer tissues, suggesting miR-320a acts as a tumor-suppressor in CRPC cells. The aim of this study was to investigate the functional significance of miR-320a in CRPC cells and to identify miR-320a-mediated target genes involved in oncogenesis and metastasis of the disease.

METHODS:

Gain-of-function studies were performed using transfection of mature miR-320a into cancer cells (DU145 and PC3). Genome-wide gene expression analysis and in silico analysis were applied to identify molecular targets regulated by miR-320a in PCa cells. A luciferase reporter assay was carried out to determine regulation of miR-320a. Loss-of-function studies using siRNA were performed to investigate the functional role of target genes.

RESULTS:

The expression levels of miR-320a were significantly reduced in PCa tissues and the cell lines. Restoration of mature miR-320a in cancer cells led to significant inhibition of cell migration and invasion activities in PCa cell lines. Lysosomal-associated membrane protein 1 (LAMP1) was identified as a direct target of miR-320a in this assays. Silencing of LAMP1 significantly inhibited cell proliferation, migration and invasion in PCa cells. Moreover, overexpression of LAMP1 was observed in PCa and CRPC clinical specimens.

CONCLUSIONS:

To the best of our knowledge, this is the first report demonstrating that tumor-suppressive miR-320a directly regulated LAMP1 in PCa cells. The identification of novel target gene regulated by tumor-suppressive miRNAs may lead to a better understanding of molecular mechanisms of CRPC metastatic pathways.

#1916

Targeting a MAPK-miR-124-Sox9 axis radiosensitizes human glioblastoma cells.

Hanna Sabelstrom,1 Rahul Jandial,2 Ksenya Shchors,1 Selma Masic,1 Edith Yuan,1 Trenten Fenster,1 Supna Saxena,1 Allen Ho,3 Theodore P. Nicolaides,1 Robyn Wong,1 Stanislava Yakovenko,1 Mitchel H. Berger,1 Evan Y. Snyder,3 Johan Jakobsson,4 William A. Weiss,1 Anders I. Persson1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _City of Hope Cancer Center & Beckman Research Institute, Los Angeles, CA; _3 _Sanford-Burnham Institute for Medical Research, La Jolla, CA;_ 4 _Lund University, Lund, Sweden_.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor. Although genetic alterations and overexpression of genes in the RTK/RAS/MEK/ERK pathway (MAPK) is known to drive GBM aggressiveness, less is known about down-stream effector genes. We confirmed that EGFR amplified human GBMs display high levels of the transcription factor SOX9, a known barrier to neurogenesis and target of microRNA-124 (miR-124). Surprisingly, a subpopulation of SOX9-negative tumor cells expressed markers of mature neurons. Here, we demonstrate that the MEK inhibitor PD325901 depletes SOX9, reduces proliferation, and induces neurogenesis in a mouse GBM model and human GBM cells. We find that MAPK signaling regulates a large cluster of miRNAs in mouse neurosphere cultures, including miR-124. In addition, PD325901 effectively induced miR-124 levels in SOX9-expressing human GBMs. Using a doxycycline-regulable approach, we demonstrate that miR-124 overexpression depletes SOX9 levels and induces neurogenesis in a concentration- and time-dependent manner in human GBM tumorsphere cultures. Doxycycline treatment of athymic nude mice intracranially xenografted with human GBM cells resulted in reduced tumor growth, robust neuronal differentiation, and increased overall survival. Finally, we demonstrate that a brief 3-day treatment with PD325901 or doxycycline effectively radiosensitizes human GBM cells and extends overall survival of xenografted mice. Our work shows that the miR-124-SOX9 axis is a down-stream effector pathway that drives GBM aggressiveness. Long-term exposure to PD325901 is associated with adverse side-effects in patients. We propose that a priming regimen of the clinically relevant MEK inhibitor PD325901 should radiosensitize SOX9-expressing tumors in patients and result in reduced side-effects compared to continuous treatment protocols.

#1917

Suppression of miR-101a contributes to leukemogenesis by regulating epigenetic and pro-survival pathways in MLL AML stem cells.

Estrella Gonzales-Aloy, Murray D. Norris, Jenny Y. Wang. _Children's Cancer Institute, University of New South Wales, Sydney, Australia_.

The occurrence of relapse in acute myeloid leukemia (AML) is attributed to the persistence of leukemic stem cells (LSCs), which possess self-renewal and proliferative ability and contributes to the initiation and maintenance of leukemia (Nature 2001, 414:105-111). LSCs are also inherently drug-resistant (Oncotarget 2010, 1: 552-566). Recently, microRNAs (miRNAs), a class of small noncoding RNAs, have been implicated in regulation of cancer stem cells (CSCs). Several studies have shown that depletion of miRNAs leads to cancer and miRNA overexpression represents a promising strategy for cancer treatment (Nature Rev. 2010, 9: 775-789). Our study aims to develop an LSC-targeted therapeutic strategy by identifying miRNAs regulating gene and epigenetic pathways activated in Mixed-Lineage Leukemia (MLL)-mediated AML stem cells.

We performed miRNA microarray analysis of MLL-AF9 mediated pre-leukemic stem cells (pre-LSCs) compared to Hoxa9/Meis1a-derived pre-LSCs and identified a novel tumor suppressor, miR-101a, that is downregulated in MLL-AF9 pre-LSCs (P<0.05, fold change>1.25). Quantitative real-time PCR confirmed the results and further analysis revealed that miR-101a is suppressed in LSCs compared to normal hematopoietic stem cells, suggesting a tumor suppressor role of miR-101a. Serial replating assay showed that overexpressing miR-101a in MLL-AF9 pre-LSCs impaired proliferation as we have observed 60-70% reduction in cell and colony number (p<0.01). Our preliminary in vivo survival studies also showed that primary transplantation of miR-101a overexpressed pre-LSCs in C57BL/6 mice causes a significant delay of MLL AML development. Annexin V and 7-AAD staining followed by flow cytometric analysis revealed a significant increase in apoptosis (p <0.001). To explain the marked reduction in colony forming ability and concomitant increase in apoptosis, we examined the expression of several apoptosis-associated proteins by Western blotting and found a decrease in expression of pro-survival proteins, Myeloid Cell Leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2). We also investigated whether miR-101a regulates epigenetic pathways as epigenetic events in cancer lead to aberrant expression of miRNAs. Strikingly, our result revealed a significant reduction in Enhancer of zeste homolog 2 (Ezh2), a crucial epigenetic regulator in maintenance of MLL AML and a known target of miR-101a. This finding is also accompanied by a global decrease of histone H3K27Me3 mark, implicating that miR-101a may contribute to leukemogenesis via regulating epigenetic pathways in LSCs.

Our data show that miR-101a is supressed in MLL AML stem cells and its forced expression impairs LSC functions via reversing the epigenetic landscape and inhibiting pro-survival pathways, leading to apoptotic cell death. Overexpression of miR-101a may provide a means to eradicate drug-resistant LSCs.

#1918

The role of miR17-92 in the miRegulatory landscape of Ewing sarcoma.

Raphaela Schwentner,1 Maximilian Kauer,1 David Herrero-Martin,1 Heinrich Kovar2. 1 _Children's Cancer Research Inst., Vienna, Austria;_ 2 _Children's Cancer Research Inst, Department of Pediatrics, Medical University, Vienna, Austria_.

MicroRNAs (miRNA) serve to fine-tune gene expression and thus play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a given tissue still poses a significant problem since the presence of a seed sequence in the 3´UTR of an mRNA and its expression modulation upon forced ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in ES. Recently, we reported on the EWS-FLI1 microRNA (miRNA) signature based on knockdown experiments in ES cell lines and on differential expression in primary tumors versus mesenchymal stem cells. Comparison of both datasets revealed the miR17-92 cluster to be the top EWS-FLI1 activated miRNA, which is one of the most potent oncogenic miRNAs. The whole cluster is among the small number of miRNAs down-regulated upon knockdown of EWS-FLI1 in multiple ES cell lines. We now report the use of a combination of AGO2 pull-down experiments by PAR-CLIP - to enrich and sequence RISC-associated RNAs - and of RNAseq upon miRNA depletion by ectopic sponge expression to identify the targetome of miR17-92 in Ewing sarcoma (ES) cells

We found a significant enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3´UTRs of genes up-regulated in response to mir-17-92 but not miR9 and 10b specific sponge expression. Among them, we consistently identified known (i.e. CTGF, MXD1, CYLD1) and a multitude of so far unknown targets of the oncomir-1 cluster in ES in three PAR-CLIP experiments. Interestingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling.

Gene reporter assays using 3'UTRs of select novel candidate genes showed enhancement of luciferase activity upon transfection of a sponge specific to miR-17-92 and 20a, but not to unrelated miR-9 and 10b. Site directed mutagenesis of seed sequences in these 3'UTR confirmed the direct regulation of these genes by miR17-92. Current research focuses on the functional validation of TGFB pathway modulation by miR17-92. This study provides a paradigmatic approach to the comprehensive definition of the targetome for a defined group of miRNAs under physiological conditions.

This study was supported by the 7th framework program of the European Commission (FP7-259348, "ASSET") and the Austrian Science Fund FWF (24708-B21)

#1919

Nitric oxide stimulates expression of miRNA-155 through the BRCA1-dependent pathway.

Justin Hawley,1 Vasily A. Yakovlev2. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _VCU Massey Cancer Center, Richmond, VA_.

Previously we demonstrated that nitric oxide (NO), generated in macrophages or released from the NO-donors, inhibits expression of the breast cancer type 1 susceptibility protein (BRCA1) in the different cell lines (Yakovlev, Cancer Res., 2013). BRCA1 protein contributes to cell viability in multiple ways, including homologous recombination repair (HRR) of DNA double-stand breaks (DSBs), cell-cycle checkpoint control, mitotic spindle assembly, and regulation of chromosome segregation. In addition BRCA1 protein (in association with histone deacetylase 2) epigenetically suppresses oncogenic microRNA-155 (miR-155) expression (Chang, Nature Med., 2011). miR-155 is an established multifunctional oncomiR, which regulates many pro-oncogenic pathways. miR-155 gene was found to be over expressed in several solid tumors, such as thyroid carcinoma, breast cancer, colon cancer, cervical cancer, pancreatic ductal adenocarcinoma, and lung cancer, where it is considered to be a marker of poor prognosis.

We hypothesized that overproduction of NO can stimulate expression of miR-155 through the BRCA1-dependent pathway. A-549 (lung cancer) and MCF-10A (normal breast epithelial) cell lines were incubated with different concentrations of NO-donor DETA NONOate (0 - 300μM). After 24 or 48 hours of incubation cells were analyzed for the BRCA1 and miR-155 expression. Both cell lines demonstrated significant dose- and time-dependent downregulation of BRCA1 expression along with significant stimulation of miR-155 expression. Cell lines infected with adenovirus vector expressing the wild type BRCA1 abolished stimulation of miR-155 in response to NO-donor treatment, compared with the cell lines infected with an empty adenovirus vector.

To analyze the effect of NO-dependent stimulation of miR-155 expression, we measured the protein levels of miR-155 targets: DNA mismatch repair (MMR) proteins - MSH2, MSH6, MLH1; SH-2 containing inositol 5'-polyphosphatase 1 (SHIP1); and suppressor of cytokine signaling 1 (SOCS1). Western blot demonstrated time- and dose-dependent amount decrease for all targeted proteins with the one exception: expression level of SHIP1 protein in A-549 cell line was undetectable even in the non-treated control.

In future studies we will investigate the role of NO-BRCA1-miR-155 pathway on the MMR capacity and stimulation of microsatellite instability.

#1920

miR-211 directly targets pyruvate dehydrogenase kinase 4 to inhibit cellular growth and glucose metabolism in triple negative breast cancer.

Maheedhara R. Guda,1 Swapna Asuthkar,1 Soumen Das,2 Sudipta Seal,2 Andrew J. Tsung,3 Kiran K. Velpula1. 1 _University of Illinois College of Medicine at Peoria, Peoria, IL;_ 2 _University of Central Florida, Orlando, FL;_ 3 _Illinois Neurological Institute, Peoria, IL_.

Deregulated metabolism and aerobic glycolysis are common to many kinds of tumors including, breast cancers driven by a plethora of transcription factors. One such is hypoxia-inducible factor-1α (HIF-1α) known to drive the expression of glycolytic target genes. Here, we identify

PDK4 (pyruvate dehydrogenase kinase 4) as a key regulator of HIF-1α signaling. In triple-negative human breast cancer (TNBC) cell lines, we investigated the contribution of PDK4 and HIF-1α to experimental tumor growth, core glucose metabolism and ionizing radiation effects. When these TNBC cells were exposed to 4, 6 and 8Gy treatment, we observed a linear increase in the expression levels of PDK4 and HIF-1α genes. In this study we also report that miR-211, a microRNA predicted to target PDK4 is suppressed in TNBC patient specimens as evidenced by in situ hybridization analysis. The expression levels of PDK4, HIF-1α and RICTOR were reduced with miR-211 over expression while the levels of PDH and FH were observed to be increased. Further, we observed that miR-211 conjugated to cerium oxide nanoparticles (nanoceria) showed increased cytotoxicity. Similar effects were observed when these TNBCs were treated with 5mM DCA, a potent metabolic inhibitor of PDK. Combinatory suppression of PDK4 and HIF-1α exerted synergistic inhibition of lactate released suggesting of reversal of aerobic glycolysis. Alternatively, TCGA datasets were predictive of patient survival, with a PDK4/miR-211 signature robustly predicting poor patient prognosis. Overall, our findings suggest that combined targeting of PDK4 signaling cascade with miR-211 and DCA in combination specifically regulate core glucose metabolism in TNBC.

#1921

Low doses of metformin increase expression of microRNAs that regulate innate immune genes in breast cancer cells.

Jonne S. Woodard, Susan Yeyeodu, Kevin S. Kimbro. _North Carolina Central University, Durham, NC_.

Background: Metformin (MF), the most widely used anti-diabetic drug, has also been shown to impact the incidence of breast cancer (BrCa), although the molecular mechanisms responsible for the therapeutic effects of MF are poorly understood. Unfortunately, until now in vitro studies have used high doses of MF (5-20 mM) and may not accurately model MF mechanisms of action at clinical doses.

Objectives & Hypothesis: Metformin is thought to inhibit cancer cell growth by decreasing available cellular energy, creating a fatal metabolic deficit in cancer cells. Recent evidence suggests that innate immunity may play a role in the regulation of metabolism. We hypothesize that: 1) metabolic pathways targeted by MF impact innate immune pathways modulated by IRAK4 in BrCa cells and 2) metabolic and innate immune pathways in BrCa are regulated by common microRNAs (miRs). Therefore, we examined the effects of a) pharmacologically relevant MF concentrations on the expression of selected miRs and proteins associated with innate immunity and metabolism, and b) various concentrations of MF on BrCa cell lines that differ in their metastatic potential.

Methods: Highly metastatic 231s and moderately metastatic 468s were cultured in low glucose medium and 15uM MF for 0, 2, 6, 12 and 24hrs. Changes in the expression of 43 miRs associated with innate immune and/or metabolic pathways were determined from total RNA by qRT-PCR using a miR array. MF-induced changes in IRAK4 protein expression were compared with known MF targets AMPK and its upstream regulator LKB1. The effects of MF concentrations up to 1 mM on BrCa cell growth and migration were also tested.

Results: MF enhanced levels of hsa-miR-124-3p (which targets LKB1and AMPK) >2-fold at 2 and 6hrs in 468s. In contrast, hsa-miR-302c (which targets IRAK4) was up-regulated (>2-fold ) by MF in 231s but not 468s at 2 and 6hrs. Expression of total IRAK4 protein was reduced after 12hrs of exposure to MF in 231s and after 6 and 12 hours in 468s. As reported previously, LKB1 was not expressed in 231s, whereas LKB1 was constitutively expressed in 468s. AMPK activation was inhibited at 2 and 12hrs in 231s, while in 468s AMPK activation was highly variable. Neither cell line showed changes in proliferation or migration in the presence of MF.

Conclusions: Low concentrations of MF were sufficient to increase levels of two miRs known to regulate innate immune transcripts. MF treatment reduced IRAK4 levels by 6hrs (468s) to 12hrs (231s). Cell-specific differences in AMPK and LKB1 activation likely reflect differences in their metabolic rates and programs. Our data suggests that MF-sensitive metabolic pathways are involved in IRAK4-dependent innate immunity in breast cancer cells. Future studies will continue to unravel the effects of low doses of MF on BrCa.

#1922

miR-301 expression is deregulated in rhabdomyosarcoma.

Rossella Rota, Alberto Gualtieri, Cristina Cossetti, Silvia Pomella, Laura Adesso, Franco Locatelli. _Ospedale Pediatrico Bambino Gesù, Roma, Italy_.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myogenic derivation. Conversely to normal myoblasts, RMS cells are unable to differentiate, thus they proliferate indefinitely. MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression by affecting both the stability and translation of mRNAs. They are fundamental regulators of myogenesis and their deregulation has been involved in the pathogenesis of RMS. We evaluate here the expression and regulation of miR-301, which has been related to tumorigenesis in other types of cancers. We report here that the expression of miR-301 is upregulated in primary samples of both the alveolar RMS, which bears the fusion protein PAX3-FOXO1 (P3F) (ARMS), and the fusion-negative embryonal RMS (ERMS) compared to normal muscle tissues. Similarly, it appears higher in RMS cell lines versus normal skeletal myoblasts. Using an inhibitor of miR-301 (CONTRAmiR) we demonstrate that this miRNA favors RMS cell mobility and down-regulates p21Cip1, PTEN and DICER1 levels. However, the inhibition of miR-301 with the CONTRAmiR does not reduce tumor cell proliferation suggesting that the regulation of cell cycle is not under the control of this miRNA in RMS. Interestingly, the expression of SKA2 (Spindle And Kinetochore Associated Complex Subunit 2), whose gene hosts miR-301, is also upregulated in our setting. Experiments are ongoing to determine whether miR-301 and SKA2 are associated with RMS pathogenesis and could be targets for an anti-RMS therapy.

This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC).

#1923

Claudin 1 expression levels affect miRNA dynamics in human basal-like breast cancer cells.

Anne AA Blanchard,1 Anna Majer,1 Sarah Medina,2 Stephanie A. Booth,1 Yvonne Myal1. 1 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 2 _Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada_.

MiRNAs have been shown to regulate numerous cellular functions including metastasis of cancer cells, and are considered promising biomarkers of disease. The major tight junction protein of epithelial cells, claudin 1, is down regulated or absent in human invasive breast cancer and is therefore considered a putative tumor suppressor. However, claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype and in vitro studies revealed that claudin 1 can regulate breast cancer cell motility and proliferation. To gain further understanding of how claudin 1 mediates its activities in breast cancer, we examined changes in global micro RNA (miRNA) gene expression when claudin 1 was over-expressed in the phenotypically basal-like human breast cancer (HBC) cell line MDA-MB231.

Using next generation sequencing, followed by qPCR validation, we identified seven miRNAs (miR-9-5p, miR-9-3p, miR- let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) whose expression were altered as a consequence of claudin 1 over expression. Most of these miRNAs were down regulated and associated with tumor suppression in a variety of cancers including breast. Additionally, using gene expression profiling analysis of the claudin 1 over expressing clones, we identified a number of down regulated EMT related genes, including PDGFRB and CDH1 (E- cadherin). Expression of these genes is frequently lost during breast tumor progression. Interestingly, SPP1 and SERPINE 1, genes often associated with tumor cell migration and motility, were up regulated in the claudin 1 over expressing clones. Employing the miRNA target prediction program miRWalk, a number of validated miRNA target sites were identified on some of these deregulated genes. A target site for miR-9-5p was identified on the CDH1 sequence, whereas a target site of miR-99-5p was identified on SERPINE 1. Altogether, over 500 target genes have been validated as targets for the deregulated miRNAs.

Overall, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Moreover, our data provides further evidence to suggest that claudin 1 has the potential to be a useful biomarker that identifies a subset of tumors, within the aggressive but currently poorly characterized basal-like subtype. Our study also suggests that miRNAs may be used in conjunction with claudin 1 to better characterize these cancers. Further studies are now warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer.

#1924

miR-142-3p is a candidate mediator of chloride intracellular channel 4 (CLIC4) loss during squamous cancer progression.

Brandi L. Carofino, Stuart H. Yuspa. _National Institutes of Health, Bethesda, MD_.

Chloride intracellular channel 4 (CLIC4) is a broadly expressed protein that has been implicated in multiple cellular processes, including cellular differentiation and apoptosis. As a soluble factor, CLIC4 translocates to the nucleus following exposure to a variety of stimuli, including TGF-β, TNF-α, and etoposide. Nuclear CLIC4 potentiates TGF-β signaling by preventing the dephosphorylation of phospho-SMAD2 and 3 and promotes growth arrest. Although its primary function is as a tumor suppressor, CLIC4 expression is progressively diminished during cancer progression and CLIC4 protein is excluded from the nucleus of many human epithelial neoplasms and squamous cell carcinoma (SCC) cell lines. To date, no gene deletions or mutations have been identified to account for this phenomenon; thus, identifying the mechanism of CLIC4 loss is our primary focus. To determine if epigenetic gene silencing is responsible, we evaluated the methylation status of the CLIC4 promoter in a progression panel (normal, papilloma, SCC) of epithelial cell lines by performing bisulfite sequencing. No differential promoter methylation was found between the cell lines, suggesting that the regulation may be post-transcriptional or post-translational. Because microRNAs (miRNAs) are known to regulate gene expression by mediating the degradation or translational repression of target mRNAs, we performed a bioinformatic analysis of the CLIC4 3' UTR, which revealed a large number of putative targeting miRNAs. One such miRNA, miR-142-3p, has been predicted to target CLIC4 by multiple algorithms and has been shown to interact with the CLIC4 3' UTR by high-throughput sequencing and crosslinking immunoprecipitation. We confirmed the functional activity of miR-142-3p against the CLIC4 3' UTR by using a 3' UTR luciferase reporter assay and a miR-142-3p mimic. miR-142-3p is high in the tumor tissue and plasma of patients with head and neck SCC and is correlated with worse prognosis, where CLIC4 expression is known to be low. Thus, miR-142-3p may be responsible for mediating CLIC4 loss in squamous lesions and warrants further investigation.

#1925

Regulation of metabotropic glutamate receptor 5 expression by the miR-30 downregulation induced by the SDF-1/CXCR4 system in oral cancer cells.

Nobuyuki Kuribayashi,1 Daisuke Uchida,1 Makoto Kinouchi,1 Chikako Koshiji,2 Sayaka Izumi,1 Kembun Hakata,1 Yuske Komiyama,1 Shuji Tsuchida,1 Tomonori Hasegawa,2 Hitoshi Kawamata1. 1 _Dokkyo Medical University School of Medicine, Tochigi, Japan;_ 2 _Kamma Memorial Hospital, Tochigi, Japan_.

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system enhances the metastases of oral cancer cells via induction of metabotropic glutamate receptor (mGluR) 5. However, the mechanism(s) of mGluR5 expression induced by the SDF-1/CXCR4 system are largely unknown. Recently, small non-coding RNAs, microRNAs (miRNAs) are involved in the metastatic process of several types of cancers. In this study, we examined the miRNA association involved in the mGluR5 expression using oral cancer cells, B88, which express functional CXCR4 and exhibit highly metastatic potentials. We examined the metastasis-related miRNAs in SDF-1 stimulated B88 cells by use of a miRNA microarray analysis. Consequently, we isolated miR-30 family which has predictive binding sites in 3'-UTR of mGluR5 mRNA in silico analysis. Among these miRNAs, we confirmed the downregulation of all miR-30 family in SDF-1 stimulated B88 cells by the real-time PCR analysis. Next, we transfected these miR-30 families in SDF-1 stimulated B88 cells. We confirmed the downregulation of mGluR5 by the miR-30a-5p and miR-30c-5p overexpression. These results indicated that SDF-1/CXCR4 system might regulate the metastases of oral cancer by the upregulation of mGluR5 via downregulation of miR-30 family.

#1926

Hippo pathway effectors YAP/TAZ integrate tissue mechanics and Wnt signaling to regulate microRNA biogenesis.

Siyang Han, Celeste M. Nelson. _Princeton University, Princeton, NJ_.

Although mechanical cues including extracellular matrix (ECM) stiffness are known to regulate cell and tissue behavior, it remains unclear how these mechanical cues integrate with biochemical signals to control these processes. The Hippo pathway and its transcriptional coactivators YAP and TAZ, which have emerged as major regulators of organ size, proliferation and cancer development, are thought to be involved in this integration. In addition to being regulated by the canonical Hippo pathway, YAP/TAZ have been found to mediate responses to both biochemical and mechanical signals including ECM stiffness and Wnt signaling. Moreover, recent studies have shown that microRNAs (miRNAs) are sensitive to mechanical cues and YAP/TAZ may regulate miRNA biogenesis through mechanotransduction. Although misregulation of YAP/TAZ and miRNA expression are frequently observed in multiple cancers, which appears to be associated with the altered mechanics of the tumor microenvironment, the signaling mechanisms controlling these processes remain poorly understood and controversial. Here we examine how YAP/TAZ integrate biochemical and mechanical signals to regulate miRNA biogenesis. We found that Wnt3A and stiff ECM synergistically lead to nuclear accumulation and activation of YAP/TAZ. Surprisingly, ECM stiffness induces miRNA-18a expression while Wnt3A signaling has an opposite effect on miRNA-18a expression depending on the stiffness of the cellular microenvironment. Thus, YAP/TAZ activity may define a mechanism by which cells respond to ECM stiffness and Wnt signaling to control miRNA biogenesis.

#1927

Myc-induced suppression of the RNA-binding protein ZAR2 in breast cancer cells.

Smita Misra, Gautam Chaudhuri. _Meharry Medical College, Nashville, TN_.

Our objective is to verify the hypothesis that c-Myc-regulated oncomiR miR-130a-5p suppresses the level of the RNA-binding tumor suppressor protein ZAR2 in the cMyc-high aggressive breast cancer cells. We experimentally validated the binding of miR130a-5p miRISC to ZAR2 mRNA in vivo and inhibition of the translation of this mRNA by this binding. We used three commercially available c-Myc-high ZAR2-low breast cancer cell lines BT549, MDA-MB-453 and MDA-MB-231 and two c-Myc-low ZAR2-high breast cancer cells MCF7 and HCC38 for our studies. We cloned the full length 3′-UTR (179 bp) of human ZAR2 mRNA immediately downstream of the firefly luciferase open reading frame sequence contained in the reporter plasmid. We also have created different mutants of the 3'-UTR, each having mutation in each of the putative miR130a-5p binding sites. We transiently transfected the recombinant plasmid and miR130a-5p miRNA mimic into breast cancer cells and assessed luciferase activity or fluorescence 24-48 hours after transfection. We also have transfected just the reporter construct into breast cancer cells which express the endogenous miR130a-5p miRNAs, along with vectors which express mutant versions of the miRNA binding sites. In this experimental system the wild-type reporters had less activity than their respective mutants. To evaluate the functional importance of miR130a-5p miRNA/ZAR2 mRNA pair, we transiently over-expressed a miR130a-5p miRNA mimic in the cMyc-low breast cancer breast cancer cells and subsequently analyzed the level of ZAR2 protein levels. Our data showed that ZAR2 protein levels are decreased by the mimic increasing the in vitro invasiveness of these cells. Our results thus reveal a novel mechanism for the induction of aggressiveness by cMyc in the breast cancer cells. This research is supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and NIH grant 1U54RR026140 TO SM

#1928

Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis.

Juan Barajas. _The Ohio State University, Columbus, OH_.

miR-122, a conserved liver-specific miRNA, is critical for maintaining liver homeostasis. Loss of miR-122 is associated with loss of hepatic phenotype, metastasis and poor prognosis. Recent in vivo work has established miR-122 as a bona fide tumor suppressor, with poorly differentiated, Afp-positive, high grade, spontaneous HCC observed in liver-specific (LKO) and germ-line (KO) miR-122 knockout mice. We hypothesized that the pathophysiology exhibited by KO mice would be mediated through de-repressed targets in KO livers and therefore, dysfunctional regulation of specific pathways involved in liver homeostasis and carcinogenesis. Although the role of miR-122 in the maintenance of liver homeostasis is well established, systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Ago-dHITS-CLIP analysis in livers of 6 week-old wild type (WT) and KO mice to identify miRNA targetome in vivo, which revealed ~1800 miR-122 binding sites, with over 30 fold enrichment for the miR-122 seed sequence sites in WT compared to KO livers. Surprisingly, a majority of miR-122 binding sites were observed on coding exons (40%) followed by 3'-UTRs, introns and 5'-UTRs. Motif analysis of miR-122 dependent binding sites lacking the seed sequence revealed a novel G-bulged motif in addition to the canonical miR-122 seed sequences. Moreover, the hepatic gene expression profile of these mice measured by RNA-seq showed up regulation of ~2500 genes and down regulation of ~1200 genes in KO livers compared to WT livers. A comparison of HITS-CLIP and RNA-seq data revealed that majority of miR-122 targets harboring canonical binding sites at their 3'UTRs as well as some coding regions, are significantly upregulated in miR-122 KO livers without notable alteration in the expression of or G-bulged targets at the RNA level. HITS-CLIP analysis of several human HCC and matching benign liver tissues also identified both canonical and G-bulged targets; where the majority of both are located in the coding regions followed by 3'UTRs. Association of miR-122 targets with Argonaute was precipitously reduced in HCCs expressing lower miR-122 levels than respective benign livers. Notably, though miR-122 is completely conserved and highly expressed in human and mouse livers, a large number of identified targets seem to be species-specific. Ingenuity Pathway Analysis of enriched targets revealed conserved pathway specific regulation in mouse and human livers. Pathways commonly associated with critical roles in liver carcinogenesis and liver metabolism, such as AMPK signaling and PI3/AKT pathways, were the major targets of miR-122-mediated regulation. This study suggests that miR-122 functionality is conserved across species.

#1929

The novel role of microRNA-128 in proneural to mesenchymal subtype transition in glioblastoma stem cells by targeting components of pro-oncogenic Polycomb Repressor Complex.

Arun K. Rooj, Marco Mineo, Franz Ricklefs, Agnieszka Bronisz, Ennio Chiocca, Jakub Godlewski. _Brigham and Women's Hospital/Harvard Medical School, Boston, MA_.

Heterogeneous glioblastoma multiforme (GBM) was categorized based on transcriptional signatures into four subtypes (proneural (PN), neural, classical, and mesenchymal (MES)). In order to develop effective targeted therapeutic strategies, understanding the heterogeneous gene expression and molecular features of these subtypes is crucial. De-regulation of microRNA expression and activity has been shown to play an important role in tumor initiation and progression, including gliomagenesis. We have previously reported that expression of microRNA-128 (miR-128) is significantly down regulated in GBM and it diminishes self-renewal of GBM stem-like cells (GSCs) and sensitizes them to irradiation. Proneural-to-mesenchymal transition (PMT) manifested by concomitant up regulation of MES markers and down regulation of PN markers, is associated with increased malignancy, therapy-resistance and worse prognosis, but the underlying causes of PMT have not been convincingly characterized yet. In this study, we have demonstrated that miR-128 can regulate the PMT in GSC subsets. We showed that the expression of miR-128 in PN GSCs was significantly higher compared to MES subtype. As a tumor suppressive microRNA, miR-128 inhibited the MES GSC-specific high expression of Bmi1 and Suz12, two components of Polycomb Repressor Complexes (PRC) 1 and 2, respectively. In both GSC subtypes, miR-128 driven targeting of PRCs suppressed their epigenetic activity measured by ubiquitination of H2AK119 and tri-methylation of H3K27. Stable down regulation of miR-128 in PN GSCs significantly increased the expression of MES-specific gene signature (BCL2A1, CD44, WT-1, LYN, and MET) while its stable up regulation in MES GSCs resulted in the restoration of PN specific gene signature (CD133, SOX2, NES, OLIG2, and NOTCH1). We also showed that stable expression of miR-128 in GSCs could regulate the process of irradiation-induced PMT. Our in vivo studies showed the anti-tumorigenic role of miR-128 in both PN and MES GSC-derived intracranial tumor models. Taken together, we demonstrated that altering levels of miR-128 was sufficient to cause or reverse PMT, most likely by targeting the level/functions of PRCs and their target genes in GBM.

#1930

Impacts of the KRAS-variant on breast cell biology.

Song-Yi Jung, Joanne Weidhaas. _UCLA, Los Angeles, CA_.

Background: The KRAS-variant is a germ-line, microRNA binding site mutation in the KRAS oncogene, which is associated with an increased risk of cancer and altered response to cancer treatment. We investigated the hypothesis that normal cells with the KRAS-variant would be biologically unique.

Methods: We created perfectly matched, isogenic normal breast epithelial lines with (MCF10AKRAS+/-, MT) and without (MCF10AKRAS-/-, WT) the KRAS-variant. We also created isogenically matched MCF10A cells with p53 gene-specific stable knockdown with (MLPKRAS+/-) and without (MLPKRAS-/-) the KRAS-variant. We evaluated phenotypic alterations in appearance, gene and microRNA expression and growth in the KRAS-variant vs the parental lines. We used 3-D culture on Matrigel™ (acini formation), and soft agar colony formation (anchorage independent growth) to evaluate growth and transformation. We also investigated EMT and stem cell analysis by performing targeted qRT-PCR, western blotting and flow cytometry.

Results: We found that KRAS-variant normal breast epithelial lines (MT) exhibited a mesenchymal phenotype, exhibiting a spindle-like morphology vs the parental (WT) cells, consistent with a baseline epithelial to mesenchymal transition (EMT). In agreement with their appearance, MT cells had significantly lower E-Cadherin and Occludin, and significantly higher Fibronectin and Vimentin than the WT line. We also found that mir-200c was dramatically down-regulated in the MT line, being 828-fold suppressed. miR-200c has been shown to regulate EMT induction and self-renewal and proliferation of stem cells. We thus next investigated whether the MT line had an increased stem cell component. By sorting cells for CD44+/CD24low/- fraction and ALDH1 positivity, we found that MT cells had a significantly higher proportion of CD44+/CD24low/- cells, and higher levels of ALDH1positive cells. In 3-D culture, MT cells formed small irregularly shaped spheroids, whereas WT cells formed well-organized, polarized spheroids, supporting a mesenchymal phenotype. In the MLPKRAS+/- cells we also found a higher stem cell component, however we did not see an EMT phenotype. However, MLPKRAS+/- cells formed disorganized acinar structures, similar to transformed cells, while the MLPKRAS-/- cells formed single, normal round acini. We next plated these cell line in soft agar to test for baseline transformation. While the KRAS-variant normal breast epithelial cells were not transformed at baseline, MLPKRAS+/- cells exhibit colony formation in soft agar, consistent with transformation.

Conclusions: These findings indicate that KRAS-variant normal cells are biologically different than cells without the KRAS-variant, but are not transformed. In contrast, p53 suppression in addition to the KRAS-variant is associated with cellular transformation, similar to findings with mutated KRAS. Studies are ongoing to better characterize baseline and oncogenic changes in the presence of the KRAS-variant.

#1931

MiR182 mediated control over myeloid differentiation provides novel mechanism of Imatinib resistance in chronic myeloid leukemia.

Deepak Arya,1 P. Sasikala,1 Shang Li,2 Dasaradhi Palakodeti,3 Cecil Ross,4 Sudhir Krishna1. 1 _National Centre for Biological Sciences, Bangalore, India;_ 2 _Duke-NUS, Singapore, Singapore;_ 3 _InStem, Bangalore, India;_ 4 _St. John's Medical College and Hospitals, Bangalore, India_.

MiR182 is an evolutionarily conserved miR, present in a cluster with miR183, and miR96 on human chromosome 7q32.2. This cluster is over-expressed in hESCs, and iPSCs. Developmentally, miR182 is over-expressed during erythropoiesis from CD34 cells. Erythroid differentiation of CML cells induced by Imatinib treatment provides an alternate mechanism of Imatinib escape, and also emphasizes the need to explore miR182 function in this context. In cancerous cells, miR182 regulates metastasis in melanoma cells by regulation of MITF and FOXO3, and DNA repair in breast cancer by targeting BRCA1. These reports provide some understanding of miR182 function by transient in-vitro assays. To refine functional importance, targeted deletion of MIR182 has been done on mice retinal cells where it is shown an over-expressed candidate by quantitative methods; nevertheless MIR182 deletion does not show any phenotypic change indicating challenges to study their functions along with better model systems. We focus to study K562 cells, CML blast crisis cell line with wild type Bcr-Abl tyrosine kinase, partially differentiated cells able to differentiate in different lineages upon induction. We exploit use of CRISPR/Cas9 mediated knock-out approach to find out miR182 function in K562 cells. We show that we have successfully deleted MIR182 loci in K562 cells by CRISPR. We hypothesize if miR182 regulates proliferation or differentiation in bi-potent K562 cells. Using phenotypic characterization, we studied MIR182 deleted cells. We show that myeloid differentiation is augmented by MIR182 deletion. Homozygous MIR182 deleted cells display decreased proliferation. We find striking inverse correlation with notch signalling genes in the context of Imatinib resistance. Notch signalling has been documented in cancer cells where its role has been shown in context dependent which require more explanation of its regulation. Hes1, one of main transcriptional regulator of notch signalling pathway, is targeted by miR182 shown by both bio-informatics, and in-vitro assays. Hes1 manipulation confirms it as down-stream target of miR182 in CML cells. We next explored Imatinib resistance phenotype in the context of miR182 given its expression in CD34 cells. We demonstrate miR182 is over-expressed in CML cells towards Imatinib treatment in ex-vivo, and in-vitro. We find miR182 essential for proliferation of K562 cells, Imatinib resistance. Taken together, our studies highlight miR182 targets notch signalling genes in CML cells implication of which we have shown in the context of Bcr-Abl independent Imatinib resistance mediated by differentiation deregulation.

#1932

The importance of RNA editing enzymes in glioblastoma.

Angela Gallo. _Ospedale Pediatrico Bambino Gesù, Roma, Italy_.

This study was supported by AIRC.

Glioblastoma (or grade IV astrocytomas) is a highly heterogeneous and deadly malignant brain tumor and one of the most incurable form of cancer in human that urgently needs new therapeutic targets. There is great interest in elucidating all the molecular basis and functional importance of genetics and epigenetic mechanisms occurring in glioblastoma. In our laboratory, we investigate whether essential post-transcriptional event (such as the A-to-I RNA editing) is a possible molecular mechanism involved in the appearance/progression of glioblastoma.

A-to-I RNA editing modifies the nucleotide sequence of RNA target molecules. The ADARs (Adenosine Deaminases that Act on RNA) are the enzymes responsible for this conversion acting on pre-mRNAs or non-coding RNAs (such as microRNA). Inosine, is recognized by the translation and splicing machineries, as Guanosine. Therefore, ADARs can play a direct and critical role in generating alternative protein isoforms (by codon changes and splicing pattern alterations) and modulating gene expression levels (by editing microRNAs and/or 3'-UTRs). In mammals, there are three different ADAR enzymes (ADAR1-3). The deregulation of ADAR2 activity has been connected with several neurological human diseases and Adar2 knockout animals show a lethal neurological phenotype. We demonstrated a direct correlation between a progressive decrease of ADAR2 editing activity and the increased grade of malignancy in paediatric astrocytomas (from grade I to grade IV). We show that the rescue of ADAR2 in glioblastoma cells is able to significantly inhibit in vivo glioblastoma tumor growth. Differently, when we decrease the expression of ADAR2 in malignant but not tumorigenic astrocytoma cell line (A172) we observed a boost of several cancers cell features such as cell proliferation, migration and colony formation.

By integrating deep-sequencing and array approaches with bioinformatics analyses and molecular studies, we observed that ADAR2 is essential to edit a small number of mature miRNAs and to significantly modulate the expression of about 90 miRNAs in glioblastoma cells that may contribute to cancer progression.

Our findings disclose an additional layer of complexity in miRNome regulation and provide information to better dissect the impact of ADAR2 editing enzyme in glioblastoma.

#1933

The miR-888 cluster: a new oncogenic cluster involved in aggressive prostate cancer.

Tsuyoshi Hasegawa, Mary Pahuski, Garrison Glavich, Holly B. Lewis, Aurora Esquela-Kerscher. _Eastern Virginia Medical School, Norfolk, VA_.

Prostate cancer (PCa) is the second leading cause of cancer-related death among men in the U.S. No accurate biomarkers exists that can discriminate for aggressive PCa and therapeutic options are limited for metastatic disease. MicroRNAs (MiRNAs) have emerged as promising clinical tools for cancers. These small non-coding RNAs negatively regulate gene expression post-transcriptionally. Widespread miRNA dysregulation has been noted in PCa. However, it is poorly understood how they function in the prostate to promote cancer progression and metastasis. Our lab recently identified miR-888 as a novel oncogenic miRNA up-regulated in specimens from patients with advanced PCa and acts to increase prostate cell proliferation, migration, & colony formation in vitro (Lewis et al, Cell Cycle 2014). MiR-888 belongs to a genomic cluster of seven miRNAs on human chromosome Xq27.3, an area linked to hereditary prostate cancer (HPCX1). We hypothesize additional members of miR-888 cluster (miR-892c, -890, -892a, -892b, -891b, -891a) function to promote PCa progression. Our studies employed paired syngenic human PCa cell lines differing in their metastatic status and response to androgen (i.e., androgen-sensitive, non-malignant RWPE-1 & aggressive WPE1-NB26; androgen-sensitive LNCaP & aggressive C4-2; hormone-refractory PC3-N & aggressive PC3-ML) to examine the miR-888 cluster's ability to modulate prostate cell proliferation (WST-1 assays), colony formation (Soft agar assays), migration, and invasion (Boyden chamber assays). Profiling miRNA expression in these cancer lines, we found that all miR-888 cluster members were preferentially elevated in highly aggressive PC3-ML and extensively down-regulated in non-aggressive PC3-N cells. Furthermore, we noted certain members of the miR-888 cluster, most notably miR-891a, acted in vitro to promote prostate cell proliferation, colony formation, migration & invasion activities. Interestingly, our results also suggested that other miR-888 cluster members act conversely to inhibit these activities - implying the miR-888 cluster functions as a unit to fine tune and coordinate prostate cancer pathways. Xenograft experiments are currently ongoing to evaluate in vivo effects of the miR-888 cluster during tumorigenesis. Preliminary data indicated that miR-888 could accelerate tumor formation of PC3-N cells subcutaneously implanted into flanks of NOD/SCID male mice. MiR-888 cluster members are now being evaluated for their non-cell autonomous effects in the prostate by exosomal transport. We have successfully isolated exosomes from human PCa cell lines and are working towards developing these secreted membrane-bound vesicles as novel miRNA delivery vehicles for PCa. Taken together, this study offers a more detailed understanding of PCa and insight into the complexity of miRNA networks involved in cancer progression.

#1934

MicroRNA-383 located in frequently deleted chromosomal locus 8p22 regulates prostate cancer stem cell marker CD44.

Nathan Bucay, Kiran Sekhon, Varahram Shahryari, Yozo Mitsui, Guoren Deng, Z. Laura Tabatabai, Shahana Majid, Soichiro Yamamura, Rajvir Dahiya, Yuichiro Tanaka, Sharanjot Saini. _UCSF VA Medical Center, San Francisco, CA_.

A major genomic alteration in prostate cancer (PCa) is frequent loss of chromosome (chr) 8p with a common region of loss of heterozygosity (LOH) at chr8p22 locus. Genomic studies implicate this locus in the initiation of clinically significant PCa and with progression to metastatic disease. However, the genes within this region have not been fully characterized to date. Here we demonstrate for the first time that a microRNA component of this region -miR-383- is frequently downregulated in prostate cancer, plays a critical role in determining tumor initiating potential and is involved in prostate cancer metastasis via direct regulation of CD44, a ubiquitous marker of PCa tumor initiating cells (TICs)/ stem cells. Expression analyses of miR-383 in PCa clinical tissues established that low miR-383 expression is associated with poor prognosis. Functional data suggests that miR-383 regulates PCa tumor initiating/ stem-like cells via CD44 regulation. Ectopic expression of miR-383 inhibited tumor initiating capacity of CD44+ PCa cells. Also, 'anti-metastatic' effects of ectopic miR-383 expression were observed in a PCa experimental metastasis model. In view of our results, we propose that frequent loss of miR-383 at chr8p22 region leads to tumor initiation and prostate cancer metastasis. Thus, we have identified a novel finding that associates a long observed genomic alteration to PCa stemness and metastasis. Our data suggests that restoration of miR-383 expression may be an effective therapeutic modality against PCa relapse and metastasis. Importantly, we identified miR-383 as a novel PCa diagnostic biomarker with potential that outperforms that of serum PSA.

#1935

A role for microRNAs in the regulation of transcription factor ATF5.

Kari A. Gaither, David X. Liu, Bhanupriya Madarampalli. _Washington State University, Spokane, WA_.

ATF5 is a widely expressed transcription factor that modulates survival, proliferation, and differentiation and plays a role in homeostasis and cellular stress response. In unstressed conditions, ATF5 has a short half-life and is rapidly degraded due to post-translational modifications. Conversely, ATF5 is upregulated in cells under cellular stress. Furthermore, we have found that ATF5 is elevated in transformed C6 glioma and MCF7 breast cancer compared to nontransformed cells and is a survival factor for both transformed cell lines. Although it is known that ATF5 mRNA can be stabilized at the 5' untranslated region (UTR) and protected from degradation via interactions with other proteins, regulation of ATF5 expression is not fully understood. We hypothesize that microRNA (miRNA) play a role in regulating the expression of ATF5 at the 3' UTR. MiRNAs are endogenous small non-coding RNAs 20-25 nucleotides in length that contribute to regulation of gene expression at the translational level. Herein, we sought to identify the presence of specific miRNA and their levels and ability to downregulate ATF5 during cellular stress and other physiological conditions in vitro. To date, no studies have examined the regulation of ATF5 by miRNA. Binding sites of miRNA are typically found in the UTRs of target mRNAs. We used TargetScan, miRanda, and other in silico modeling programs to identify miRNAs that were predicted to bind to the 3' UTR of ATF5 and selected miRNAs 129-5p, 433-3p, and 520b as initial candidates for their potential in regulation of ATF5. Here, we have employed multiple strategies to investigate a potential role of miRNAs in the regulation of ATF5. Luciferase reporter assays were used as an initial method of validation for the miRNA candidates. Subsequently, transfections were carried out with precursor microRNA and expression levels of ATF5 were measured under varying physiological conditions via Western blot analysis. Additionally, immunoprecipitations were carried out with ATF5 3' UTR mRNA and HeLa cell lysate to assess the presence of miRNAs and quantify the amount of specific miRNA bound to the 3' UTR under varying physiological conditions via qPCR analysis. Preliminary data show that miRNA are bound to the 3' UTR of ATF5 and that miRNAs 129-5p, 433-3p, and 520b help to regulate the expression of ATF5 under varying stress conditions and at steady state. Better understanding of the regulation of ATF5 could have implications in a broad range of human malignancies, including glioma and potentially several other types of cancer.

#1936

Comprehensive long non-coding RNA expression profiling from the TCGA HNSCC RNA-sequencing data.

Nijiro Nohata,1 Martin Abba,2 Panomwat Amornphimoltham,1 J. Silvio Gutkind1. 1 _University of California, San Diego, CA;_ 2 _CINIBA, School of Medical Sciences, National University of La Plata, La Plata, Argentina_.

Objectives; Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer types in the world. Despite of considerable advances in multimodality therapy, including surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients with HNSCC is only 40% - 50%. Although a growing body of evidence indicates that long non-coding RNAs (lncRNAs) contribute to the initiation and development of various types of cancers, the role of lncRNA expression in human HNSCC remains unknown. In this study, we aimed to establish the onco-lncRNAome profiling of HNSCC from The Cancer Genome Atlas (TCGA) project.

Materials and Methods; The Atlas of Noncoding RNAs in Cancer (TANRIC) database (http://ibl.mdanderson.org/tanric/_design/basic/index.html) was employed to retrieve the lncRNA expression information from the TCGA HNSCC RNA-sequencing data. Our recently generated RNA-sequencing data from 22 representative HNSCC cell lines (OPC-22 panel) were also included in this study. Bioinformatics approaches, such as differential gene expression analysis, survival analysis, principal component analysis, and Co-LncRNA enrichment analysis were performed.

Results; Using TCGA HNSCC RNA-sequencing data from 426 HNSCC and 42 adjacent normal tissues, we identified 728 lncRNA transcripts significantly and differentially expressed in HSNCC. Among the 728 lncRNAs, 55 lncRNAs were significantly associated with cancer prognosis (overall survival and/or recurrence free survival). We also identified 140 lncRNA transcripts significantly and differentially expressed between Human Papilloma Virus (HPV) positive tumors and HPV negative tumors. 30 lncRNA transcripts were distinctly expressed between TP53 mutated tumors and TP53 wild type tumors. Among them, there were 19 overlapping genes up-modulated in HPV positive and TP53 wild type tumors. Co-LncRNA enrichment analysis suggested that protein-coding genes that are co-expressed with cancer-associated lncRNAs might be involved in malignant progression. Of interest, many differentially expressed lncRNAs in HNSCC were reflected in the HNSCC cell line panel.

Conclusion; LncRNAs profiling could provide novel insights into the potential mechanisms of HNSCC oncogenesis. A unique set of lncRNAs may represent potential targets for diagnosis, therapy and prevention of HNSCC.

#1937

Dysregulation of lincRNAs is a major driver of aberrant epigenomes during tumorigenesis.

Callie R. Merry, Megan E. Forrest, Sanford Markowitz, Ahmad M. Khalil. _Case Western Reserve University, Cleveland, OH_.

It has now been unequivocally demonstrated that the process of tumorigenesis is driven by both genetic and epigenetic alterations, but the mechanisms of epigenetic dysregulations remain to be elucidated. Work in our laboratory has demonstrated that numerous long intergenic non-coding RNAs (lincRNAs) become dysregulated in human cancers, and a significant subset of these lincRNAs are associated with histone-modifying enzymes and DNA methyltransferases in healthy, non-tumor cells. Based on these observations, we tested an exceptionally novel hypothesis that dysregulation of chromatin-associated lincRNAs is a major mechanism driving cancer-associated aberrant epigenomes. To test this novel concept, we have performed experiments to modulate the expression of specific chromatin-associated lincRNAs in cancer cells, and assessed their impact on the epigenome. Consistent with our hypothesis, modulating the expression of lincRNAs in cancer cells resulted in massive reprogramming of their epigenomes. These epigenomic changes impacted the growth of cancer cells and global gene expression. Mapping the genomic occupancy sites of lincRNAs, and intersection of those sites with altered genomic regions in tumors, further supported direct roles of lincRNAs in modulating chromatin structure. These findings demonstrate key roles of chromatin-associated lincRNAs in suppressing tumorigenesis in normal human tissues by regulating the epigenome.

#1938

The identification of p53 target genes and noncoding RNAs through the combined analysis of RNA-seq and ChIP-seq data.

Masashi Idogawa, Tomoko Ohashi, Yasushi Sasaki, Takashi Tokino. _Sapporo Medical Univ., Sapporo, Japan_.

p53 is inactivated in approximately half of human cancers. p53 execute various functions by modulating transcriptional regulation. To identify direct p53 transcriptional targets including non-coding RNAs, we performed gene expression analysis with next-generation sequencing (RNA-seq) in osteosarcoma U2OS cells treated with Nutlin-3a which activates endogenous p53. We identified 261 genes increased more than 4-fold and 373 long non-coding RNAs (lncRNAs) increased more than 2-fold. Next, we analyzed the data of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) and then searched for p53 binding sites in the whole human genome. p53 binding sites were located in the neighborhood of 126/261 (48.3%) genes including 28 known target genes and 122/373 (32.7%) lncRNAs including 7 lncRNAs we already identified in previous study. Recent reports have revealed that lncRNAs play an important role in various biological and pathological processes such as development, differentiation, stemness and carcinogenesis. Although all functions of lncRNAs have not been elucidated, many lncRNAs are involved in transcriptional regulation like transcriptional factors. Therefore, we performed a gene network analysis of cancer transcriptome including lncRNAs using public RNA-seq data of cancer tissues. As a result, we identified several hub lncRNAs which regulate the transcription of many downstream genes. Our results suggest that p53 and lncRNAs comprise a complex transcriptional network for various biological functions and tumor suppression.

#1939

A novel lncRNA/microRNA circuit regulates colon cancer stem cell asymmetry and plasticity.

Xiling Shen. _Duke University, Durham, NC_.

Emerging reports show that microRNAs, such as miR-34a, can act as cell fate determinants to initiate asymmetric division in colon cancer stem cells (CCSCs) (Bu et al, Cell Stem Cell, 2013, Hwang et al, Nature Cell Biology, 2014). However, this has raised three questions: (1) what initiates microRNA asymmetry? (2) how microRNA and canonical protein cell fate determinants coordinate with each other? (3) Does microRNA play any role in regulating normal colon stem cells?

(1) Here we show that a long non-coding RNA (lncRNA) regulates CCSC asymmetric division by directly targeting miR-34a. Segregated in the daughter stem cell compartment, the lncRNA recruits epigenetic regulators to methylate and deacetylate the miR-34a promoter simultaneously. The lncRNA promotes CCSC self-renewal and tumor formation in xenograft models, and is upregulated in late-stage clinical colorectal cancers (CRC), contributing to miR-34a silencing and CRC proliferation. The fact that lncRNA can target microRNA for spatial asymmetry highlights the regulatory complexity of non-coding RNAs (ncRNAs), which occupy the bulk of the "dark" genome.

(2) We further discovered that the lncRNA, miR-34a, the cell fate determinant Numb, and Notch form both feedforward and feedback loops that commits to cell fate asymmetry. Quantitative analyses revealed that this unique regulatory scheme enforces separation of cell fates. Perturbation to this motif leads to a new population of cells with more plastic and ambiguous identity, displaying both CCSC and differentiation behaviors. RNA-seq analysis revealed that they reside in a new, intermediate transcriptional state between stem and non-stem states.

(3) We show that proinflammatory stress caused by DSS or TNFα treatment can silence the lncRNA and activate miR-34a in Lgr5+ intestinal and colon stem cells, causing some of them to switch to asymmetric division to curb excessive proliferation. miR-34a deletion does not generate any phenotype in normal conditions, but exacerbates stem cell proliferation under stress. Therefore, miR-34a mediated asymmetric division provides a safeguard against excessive stem cell self-renewal due to stress.

#1940

Non-coding RNA regulation of eribulin response in neuroblastoma.

Xiuye Ma,1 Xiaojie Yu,1 Harsh Patolia,2 Zhenze Zhao,1 Liqin Du,3 Alexander Pertsemlidis1. 1 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _Wake Forest University, Winston-Salem, NC;_ 3 _Texas State University, San Marcos, TX_.

Introduction: Neuroblastoma (NB) is the most common cancer in infants and the most common extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. The overall prognosis for those with high-risk or relapsed disease remains poor despite the standard therapies of surgery, radiation, and high dose chemotherapy. We have previously shown that altering levels of specific microRNAs (miRNAs) can improve cellular response to microtubule-targeting agents (MTAs) and that exposure to MTAs results in dramatic changes in microRNA expression.

Methods: Libraries of chemically synthesized ncRNA mimics and inhibitors, including both miRNAs and lncRNAs, are screened to identify ncRNAs that regulate neuroblastoma cell viability or sensitize neuroblastoma cells to specific microtubule-targeting agents. Candidate targets are validated using qRT-PCR, protein quantification, and luciferase reporter assays. The response of neuroblastoma cells to perturbations in candidate ncRNA levels is assessed through flow cytometric analysis of cell cycle phase distribution, density and anisotropy of microtubule bundles, and through colony formation and caspase activation assays, and validated in mouse xenograft models. Networks of miRNAs, mRNAs and lncRNAs are derived from combining complementary cellular response with potential regulatory interactions.

Results: We previously reported the identification of miRNAs and lncRNAs that significantly decrease or increase neuroblastoma cell viability. Here, we extend those observations to drug response. We first demonstrate that the microtubule-targeting agent eribulin has both short-term and long-term effects, as reflected by a significant alteration in microtubule structure in response to high doses for short times and arrest at the G(2)/M phase of the cell cycle in response to low doses for long times. We next show that altering intracellular levels of ncRNAs that sensitize cells to eribulin both alters microtubule density and anisotropy and recapitulates the effect of eribulin treatment. Finally, we show that complementarity of effect between miRNAs and lncRNAs is accompanied by potential regulatory interaction.

Conclusions: Taken together, our results suggest that the response of neuroblastoma cells to eribulin is mediated by a regulatory network that includes different coding and non-coding RNA species. While these RNAs may have intrinsic value as biomarkers or therapeutic agents, either individually or in combination, the vulnerabilities that they uncover may be exploited with pathway-specific perturbations.

This project was supported by NIH R01 CA129632 and CPRIT Training Grant RP140105.

### Profiling MicroRNA Expression in Cancer

#1941

Plasma levels of miRNA-155 as a powerful diagnostic marker for dedifferentiated liposarcoma.

Aleksandar Boro, David Bauer, Walter Born, Bruno Fuchs. _University Hospital Balgrist, Zurich, Switzerland_.

PURPOSE: Atypic lipomatous tumors (ALT) and dedifferentiated liposarcomas (DDLS) are closely related liposarcoma subtypes, often difficult to distinguish but they exhibit an entirely different clinical outcome. Recently discovered regulatory functions of miRNAs in liposarcoma progression prompted us to investigate miRNAs as potential diagnostic biomarkers in liposarcoma with a main focus on circulating miRNAs for fast and reliable differential diagnosis.

PATIENTS AND METHODS: Tumor and blood samples of 35 patients with lipomatous lesions collected between June 2011 and September 2014 were analyzed by qRT-PCR. They included 10 lipomas, 7 ALT, 5 DDLS and 13 myxoid liposarcomas (MLS). Ten samples of normal fat tissue and blood from 20 healthy volunteers were used as controls.

RESULTS: A meta-analysis of public data on miRNA expression in liposarcoma revealed 9 miRNAs with potential diagnostic power. Out of these, miRNA-155 was found significantly elevated in the circulation of DDLS patients as compared to the plasma levels detected in all other liposarcoma subtypes and in healthy subjects. miRNA-155 levels in the plasma samples correlated significantly (r=0.41,p=0.02) with those in corresponding tumor extracts. This correlation was even more pronounced in an analysis of plasma and tumor extracts of malignant liposarcoma subtypes alone (r=0.51,p=0.02). Receiver operating characteristic analysis indicated that plasma miRNA-155 levels have a high diagnostic accuracy for distinguishing DDLS from healthy subjects (AUC=0.91,p=0.005) and from lipomas (AUC=0.86,p=0.02), MLS (AUC=0.92,p=0.006) and most importantly ALT (AUC=0.91,p=0.01) patients.

CONCLUSION: In conclusion, this study identified miRNA-155 as a first blood biomarker for the differential diagnosis of DDLS.

#1942

Change in serum microRNA expression during neoadjuvant chemoradiation for rectal cancer.

Stephanie H. Lim,1 Paul De Souza,1 Wei Chua,1 Weng Ng,1 Benjamin Harris,2 Mark J. Cowley,3 Kevin Spring4. 1 _Liverpool Hospital, Liverpool, Australia;_ 2 _University of Oxford, Oxford, United Kingdom;_ 3 _Kinghorn Centre for Clinical Genomics, Darlinghurst, Australia;_ 4 _Ingham Institute for Applied Medical Research, Liverpool, Australia_.

Introduction

MicroRNAs (miRNA) expression may change with treatment and influence responses in locally advanced rectal cancer (LARC) making them potentially useful predictive and prognostic biomarkers. We investigated 112 miRNAs in serum during chemoradiation for LARC.

Methods

Twenty-one patients with LARC treated with neoadjuvant chemoradiation were prospectively recruited for the study. Serum was collected at three time-points: 1) at diagnosis (baseline), 2) at three weeks into treatment and 3) at completion of chemoradiation (pre-surgery). Serum was also collected from 10 healthy controls. RNA extraction was performed using the Norgen™ total RNA purification kit. Reverse transcription and pre-amplification were performed as per the Taqman™ OpenArray MicroRNA Panels manufacturer's instructions. miRNA array qPCR was performed on 112 miRNA targets using the QuantStudio™ 12K platform. Differentially expressed miRNAs were identified using the delta-delta-Ct method, using the endogenous U6 snRNA as the control. Analysis was performed in R, using paired t-statistics, and the Benjamini-Hochberg False Discovery Rate for multiple hypothesis testing adjustment, with a threshold q<0.05 for significant differential expression. Enriched KEGG pathways were identified using DIANA, based on verified gene targets of each miRNA from Tarbase.

Results

We identified 12 miRNAs that were significantly differentially expressed between patients and matched controls. The strongest differences were observed between patient samples at baseline and completion of chemoradiation, where eight miRNAs decreased in expression: hsa-miR-101, hsa-miR-130a, hsa-miR-130b, hsa-miR-223, hsa-miR-27a, hsa-miR-628-5p, hsa-miR-630 and hsa-miR-720, all with fold change > 3-fold. These genes are known to target key rectal cancer genes such as SMAD4, BCL2, MSH2 and TGFBR2. An additional three miRNAs changed significantly between baseline and week 3: hsa-miR-135b*, hsa-miR-375 and hsa-miR-629. All except hsa-miR-135b* became less abundant.

Conclusions

Ten differentially expressed miRNAs appear downregulated during chemoradiation in LARC. These miRNAs have been implicated in cell proliferation, the epithelial-mesenchymal transition and radiation resistance. Further work will be undertaken to understand the functional implications of these changes.

#1943

A microRNA signature in urinary exosomes for diagnosis of prostate cancer.

Thorarinn Blondal,1 Anne I. Rasmussen,1 Anni R. Thomsen,1 Michael Borre,2 Jacob Fredsøe,3 Ditte Andreasen,1 Torben Falck Ørntoft,3 Karina D. Sørensen,3 Peter Mouritzen1. 1 _Exiqon A/S, Vedbaek, Denmark;_ 2 _Department of Urology, Aarhus University Hospital, Aarhus, Denmark;_ 3 _Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark_.

MicroRNAs constitute a class of small cellular noncoding RNAs (typically 19-23 nt) which function as post-transcriptional regulators of gene expression. The involvement of microRNA in both normal development as well as disease is well established and microRNAs are considered important biomarkers in several diseases including cancer. Surprisingly cell free microRNAs are also found in several biofluids where they have been shown to be very stable, most likely because they are protected from degradation by protein complexes and other particles including exosomes.

Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy.

We have developed different technologies enabling the detection of prostate cancer microRNA biomarkers in exosomes derived from urine from non-prostate massaged men. The first of these technologies is a highly sensitive LNA™-based qPCR platform for microRNA detection, which enables profiling in biofluids where microRNA levels are extremely low. At this point we have already applied this platform on thousands of biofluid samples including serum/plasma and urine to establish normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. The second technology is a simple exosome precipitation system which only requires low speed centrifugation to harvest urine exosomes.

Our developed technologies was applied to cell-free urine from three different cohorts including individuals with prostate cancer (PCa) and benign prostate hyperplasia, to identify several differentially regulated microRNAs in urine from PCa bearing individuals. PCa specific signatures were obtained by different combinations of these differentially regulated microRNAs. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer. Data showing that small miRNA signatures (down to two miRNAs) hold strong diagnostic potential in PCa will be presented.

#1944

Integrated analysis of copy number and miRNA profiling in triple negative breast cancer of Latina women.

Bruna M. Sugita,1 Mandeep Gill,2 Silma R. Pereira,3 Yara R. Zabala,4 Aline S. Fonseca,4 Selene E. Esposito,5 Catalin Marian,6 Iglenir J. Cavalli,1 Enilze MF Ribeiro,1 Yuriy Gusev,4 Luciane R. Cavalli4. 1 _Federal University of Parana, Curitiba, Brazil;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _Federal University of Maranhao, Sao Luis, Brazil;_ 4 _Georgetown Lombardi Comp. Cancer Center, Washington, DC;_ 5 _Pontificia Universidade Catolica, Curitiba, Brazil;_ 6 _Ohio State University, Columbus, OH_.

Triple negative breast cancer (TNBC) is a clinically and molecularly heterogeneous disease, which incidence and outcome vary among the different ethnic and racial groups. These tumors are more commonly seen in younger women of African and Latinas/Hispanic descents, usually diagnosed at more advanced stages, with non-localized disease. Molecular studies have shown differences in the biology of these tumors in Latinas as key contributors for high mortality. The main objective of our study was to identify a miRNA signature associated with TNBC of Latina patients by integrating miRNA and array-CGH data. Archived paraffin samples of 32 TNBC and 25 non-TNBC cases from Latina patients, obtained from the Clinical Hospital (UFPR) in Brazil, were profiled for miRNA and array-CGH using the Nanostring and the Agilent SurePrint G3 Human array-CGH platforms, respectively. The miRNA and array-CGH data, obtained in the same samples, was directly integrated and combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems were performed to identify the most relevant miRNAs and their corresponding gene targets. Eight-nine miRNAs were observed differentially expressed between the TNBC and non-TNBC lesions. Using miRBase and MiRDB target prediction databases 3,378 miRNAs targets were identified. After integration with array-CGH, a number of 15 miRNAs presented concomitant DNA copy number and miRNA alterations, reducing the number of targets to 1,242. Ingenuity pathway analysis identified the most affected canonical pathways, including IL-8, TGF-beta, PI3K-AKT and HER2 signaling pathways. Using our integration approach we were able to identify a robust miRNA signature associated with TNBC of Latina patients and to identify the potential molecular mechanism (s) that underline the observed miRNA deregulation in these patients. This signature revealed miRNAs and corresponding targets biologically relevant, involved in critical cancer signaling pathways and gene networks. Once this signature is functionally validated, its direct role in the aggressive clinical phenotype of these tumors will be determined. In summary, the findings of our study contributes to the identification of race/ethnic specific molecular targets that can set the basis for new TNBC treatments and for the future design of the most appropriate clinical trials and cancer control interventions for Latina women.

Funding:This project was supported by the Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI; U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration to promote regulatory science through innovative research and education. This research does not necessarily reflect the views of the FDA.

#1945

Identification of a miRNA/mRNA network driving non-small cell lung cancer (NSCLC) dissemination.

Margarita González-Vallinas,1 Marco Albrecht,2 Adriana Pitea,3 Manuel Rodríguez-Paredes,4 Damian Stichel,2 Steffen Sass,3 Julian Gutekunst,5 Jennifer Schmitt,1 Thomas Muley,6 Michael Meister,6 Arne Warth,1 Peter Schirmacher,1 Fabian J. Theis,3 Nikola S. Müller,3 Franziska Matthäus,2 Kai Breuhahn1. 1 _Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany;_ 2 _Center for Modeling and Simulation in the Biosciences (BIOMS), University of Heidelberg, Heidelberg, Germany;_ 3 _Institute of Computational Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany;_ 4 _University Tumor Center Düsseldorf, University of Düsseldorf, Medical Faculty, Düsseldorf; and Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany;_ 5 _Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany;_ 6 _Translational Research Unit, Thoraxklinik at the University Hospital Heidelberg, Heidelberg, Germany_.

Background:

Non-small cell lung cancer (NSCLC) is one of the most aggressive tumor entities and first data indicate that microRNAs (miRNAs) are central regulators of NSCLC dissemination. Since each miRNA is able to modulate the expression of several transcripts, they are promising targets for the development of drugs that cause efficient antitumor effects and low resistance. However, the relevant network of miRNA/mRNA driving NSCLC metastasis has not been identified yet.

Methods:

The differential expression of miRNAs was compared between NSCLC samples from patients with and without lymph node metastasis (N1, N2 and N3 vs. N0) in a cohort of The Cancer Genomic Atlas (TCGA) database (n=449). The dysregulation of the miRNAs in tumors versus normal lung samples (n=39) was also analyzed. For validation, fresh-frozen samples from an independent patient cohort (n=108) were analyzed by qRT-PCR. The role of selected miRNAs in tumor dissemination was assessed by migration and invasion experiments after transfection of respective antagomirs and agomirs in NSCLC cells (time-lapse microscopy). The novel algorithm "miRlastic" was used to identify potential miRNA targets through the integration of miRNA-mRNA expression data by negative multiple linear regression analysis. Moreover, differential methylation of the miRNA genomic locations was studied as a possible mechanism of miRNA dysregulation by analyzing Illumina Infinium 450 k DNA methylation TCGA data (n=29).

Results:

By using a stringent selection process, we identified 135 miRNAs differentially induced or reduced in NSCLCs with lymph node metastasis (p≤0.05). Interestingly, 22/135 (16.3%) of the selected miRNAs were located in the chromosomal cluster 14q32.31. Elevated expression of miR-323b, miR-487a and miR-539, which are located in 14q32.31, significantly correlated with poor patient survival. Time-resolved and quantitative analysis of lateral migration illustrated that these miRNAs increased tumor migration without affecting cell viability. Moreover, miRlastic identified several metastasis-related genes as potential downstream targets of these miRNAs. The connection between miRNAs encoded in 14q32.31 and candidate targets was confirmed in NSCLC cell lines (e.g. Pumilio RNA-Binding Family Member-2; PUM2). Lastly, hypomethylation of the 14q32.31 cluster in tumor tissues might explain increased expression of these miRNAs.

Conclusions: Our results demonstrate that miRNAs located in the chromosomal cluster 14q32.31 are driving NSCLC dissemination. Therefore, we hypothesize that the coordinated overexpression of these miRNAs is part of a genetic network supporting cancer progression and that they represent promising cancer biomarkers and therapeutic targets.

#1946

Urinary cell-free microRNA panel in detection of urothelial carcinoma of the urinary bladder.

Jaroslav Juracek,1 Barbora Peltanova,1 Hana Mlcochova,1 Michal Stanik,2 Tana Machackova,1 Michal Fedorko,3 Robert Iliev,1 Jitka Mlcochova,1 Jiri Sana,1 Zuzana Ozanova,1 Jan Dolezel,2 Ondrej Slaby1. 1 _CEITEC - Central European Institute of Technology, Brno, Czech Republic;_ 2 _Masaryk Memorial Cancer Institute, Brno, Czech Republic;_ 3 _The University Hospital Brno, Brno, Czech Republic_.

Urothelial carcinoma of the urinary bladder (UCUB) is the most common malignancy of the urinary system. Although about 80% of cases is a non-muscle invasive form of UCUB a high rate of local recurrence and progression to invasive form is observed. Early-stage tumors have very good prognosis, but current diagnostic methods (cystoscopy and urine cytology) suffer from low sensitivity. This reflects a large number of relapse, which occurs in almost 70% of superficial UCUB. This has led to the development of multiple molecular urinary biomarkers, but none are sufficiently robust to enter clinical practice. In this study we aimed to develop a clinically applicable, specific and sensitive panel of urine microRNAs enabling early detection of UCUB and prediction of risk of progression to muscle-invasive form.

In the first phase of study we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 UCUB patients (6 invasive, 5 high-grade non-invasive, 5 low-grade non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI) using Affymetrix miRNA microarrays. MicroRNAs able distinguish between UCUB and control groups were further validated using specific TaqMan assays and qRT-PCR method on independent cohort of 100 UCUB patients, 40 controls and 25 RCC patients (training phase - 40 UCUB, 15 controls, 10 RCC; validation phase - 60 UCUB, 25 controls, 15 RCC, 20 UTI).

Global expression profiling revealed set of 76 miRNAs significantly differentially expressed in urine of UCUB patients (P < 0,01) compared to healthy controls, thereof 64 highly up-regulated and 12 down-regulated. These miRNAs showed specificity for UCUB even when compared to other examined cohorts (RCC, UTI). Moreover 23 miRNAs were able distinguish invasive and non-invasive forms of UCUB (P < 0,01) and 18 miRNAs high-grade and low-grad non-invasive (p < 0,01). Set of 12 miRNAs with highest specificity and expression level was validated in training phase of study. Based on training phase results the panel of 3 miRNAs was profiled. Validation phase confirmed diagnostic potential and ability of this urine miRNA-based panel to differentiate between UCUB patients and controls with high sensitivity and specificity (AUC = 0,857, sensitivity = 82%, specificity = 80%).

Our data have shown that urinary microRNAs could serve as sensitive and specific biomarkers of UCUB and could be useful tool to increase sensitivity of standard cytological examination and to decrease high costs for long-term follow-up of UCUB patients. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved.

#1947

miRNA expression differentiates RCC molecular subtypes and predicts metastasis.

Andre R. Jordan,1 Soum Lokeshwar,1 Martin Hennig,2 Travis Yates,3 Michael Garcia-Roig,3 Nicholas Ortiz,3 Axel Merseburger,4 Manoharan Murugesan,3 Vinata Lokeshwar5. 1 _University Of Miami, Miami, GA;_ 2 _University of Lübeck, City of Lübeck, Germany;_ 3 _University Of Miami, Miami, FL;_ 4 _University of Lübeck, LÜbeck, Germany;_ 5 _Georgia Reagents Univerisity, Augusta, GA_.

Introduction and Objective: Renal cell carcinoma (RCC) is a molecularly heterogeneous disease. Among the small renal masses (<4cm), that are increasingly diagnosed with high−resolution imaging, 16−28% are benign and are largely indistinguishable from malignant lesions on imaging alone. 1/3rd of RCC patients either have or will develop metastasis despite treatment. We examined the differential expression of microRNAs (miRs) in RCC and normal kidney specimens and correlated their expression with histologic types and clinical outcome.

Methods: Normal kidney and RCC specimens were collected from 86 patients undergoing nephrectomy. miR enriched total RNA was successfully isolated from 46 RCC and 59 normal kidney tissues. Clinical data was collected including tumor histology (oncocytoma: 6, chromophobe: 4, papillary: 6, clear cell (CC): 25, sarcoma: 4, and collecting duct: 1), stage(T1(a,b): 20, 9; T2: 9; T3: 10; T4: 1), age (mean 63.7 years), sex (31 M,14 F). miR microarray analysis was performed on 30 samples. Expression of differentially expressed miRs was validated by q-PCR and normalized to miR−23a levels. Statistical analysis was performed using the Mann−Whitney-U−test and the Cox-model.

Results: Microarray analysis identified up-regulation of 65 and downregulation of 47 miRs (mean fold change > 2-fold; P<0.05). Q−PCR validated 4-fold up-regulation of miR155, miR21 and 6-fold downregulation of miR192 and 194 in RCC specimens (P<0.0001). When compared to oncocytoma, miR150 and miR155 levels were 5.8−8.2 fold up−regulated in chromophobe (25.8±21; 2.3±2.9; P=0.038) and miR21, miR142-3P, -5P and miR155 were 3−10−fold upregulated in CC−RCC (P ≤0.01). Only miR155 was 5.5−fold upregulated in papillary tumors when compared to oncocytoma (P=0.015). miR142-3p and -5p levels were 6.8−13.7−fold elevated high-grade (3p: 52.97; 5p: 6.2) as compared to low−grade tumors (3p: p=0.028; χ2=4.8; 5p: p=0.003; χ2: 8.7). In multivariate analysis, which included TMN, renal vein, lymphovascular invasion, age, gender and miRs levels, only miR-194 expression was independently associated with metastasis (p=0.008; chi-sq: 7.0). Combined miR194 and miR142-5P expression had 79% accuracy in predicting RCC metastasis.

Conclusion: miRs− 21, 150, 155, 142−3p, 142−5p, 192, and 194 are associated with the pathophysiology of RCC and can distinguish between RCC subtypes. miR−142 (3p, 5p) associates with tumor grade, whereas miR−194 expression independently associates with metastasis.

#1948

MiRNA profiling of dogs with transitional cell carcinoma of the bladder using blood and urine samples.

Michael Kent,1 Allison Zwingenberger,1 Jodi Westropp,1 Laura Barrett,1 Paramita Ghosh,2 Ruth L. Vinall3. 1 _University of California, Davis, Davis, CA;_ 2 _University of California, Davis, Sacramento, CA;_ 3 _California Northstate University, Elk Grove, CA_.

Early symptoms of canine transitional cell carcinoma (TCC) are frequently assumed to be caused by other lower urinary tract diseases (LUTD) such as urinary tract infections, resulting in late diagnosis of TCC and decreased rates of survival. To determine whether miRNA can be used as non-invasive diagnostic biomarkers in this setting, we assessed miRNA expression in RNA extracted from urine and blood from dogs with clinically normal bladders (n= 28), LUTD (n=25), and TCC (n= 18). Expression levels of 5 miRNA associated with TCC pathophysiology (miR-34a, let-7c, miR-16, miR-103, and miR-106b) were assessed by quantitative real-time PCR. Statistical analyses using ranked ANOVA identified significant differences in miR-103 and miR-16 levels between urine samples from LUTD and TCC patients (miR-103, fold change=2.55, p=0.015; and miR-16, fold change=3.02, p=0.002). However, logistic regression analyses determined miR-103, but not miR-16, can distinguish between canine LUTD and TCC patients (OR=0.78 p=0.031, AUC=0.85). No statistically significant differences in miRNA levels were observed between blood samples from LUTD versus TCC patients. Expression levels of miR-34a trended with miR-16, let-7c, and miR-103 levels in normal urine samples, however, this coordination was completely lost in TCC urine samples. In contrast, cooordination of miR-34a, miR-16, let-7c, and miR-103 expression levels was maintained in blood samples from TCC patients. Our combined data indicate further investigation of miR-103 as a diagnostic urine biomarker for TCC, and investigation of the role of miR-103 as well as dysregulation of coordinated miRNA expression in bladder carcinogenesis, is warranted.

#1949

MicroRNA-137 is a putative predictive biomarker and can inhibit cell proliferation and induce apoptosis in colorectal cancer.

YU-CHUAN HUANG. _Institute of Bioinformatics and Biosignal Transduction, NCKU, Tainan, Taiwan_.

MicroRNA-137 (miR-137) is a potential tumor suppressor in many kinds of cancers, including colorectal cancer (CRC). However, the role of miR-137 in CRC remains unclear. Our study showed that miR-137 is silenced in human colorectal cancer tissues and colon polyps. The methylation of miR-137 promoter is determined by methylation-specific PCR (MSP) and quantified by pyrosequencing analysis. By bioinformatics analysis and Q-PCR assay, we confirmed that miR-137 can target COX2, CDC42, CDK6, and Aurora-A. The negative expression of miR-137 and Aurora-A is confirmed in human colorectal cancer tissues and colon polyps, as well as colon polyps from the AOM/DSS-treated mouse model. Enforcing expression of miR-137 in colorectal cancer cells resulted in G2/M arrest, reduced cell proliferation, and finally led to apoptosis. Those effects can be rescued by overexpressed Aurora-A. By xenograft animal model, the growth of tumors was attenuated in miR-137-overexpressing colorectal cancer cells, in which the tissues show an increased Annexin V signal. Additionally, the AOM/DSS-induced colorectal adenocarcinoma can be restored by demethylation drug treatment. Taken together, our study suggests that epigenetic silence of miR-137 may contribute to early CRC formation through inhibiting the expression of Aurora-A, and loss of miR-137 expression in colon polyps may serve as a potential biomarker to predict the tendency towards to CRC formation. The investigation of the regulatory mechanism of miR-137 in CRC progression may shed new light on early prognosis or cancer therapy for CRC in the future.

#1950

Whole genome microRNA profiling in functionally validated invasive GBM cells identifies a novel set of microRNAs driving GBM invasion.

Lin Qi,1 Yu-lun Huang,1 Hua Mao,1 Mari Kogiso,1 Xiumei Zhao,1 Yuchen Du,1 Holly Lindsay,1 Xiaojun Yuan,2 Patricia Baxter,1 Jack Su,1 Chris Man,3 Laszlo Perlaky,3 Zhong Wang,4 You-Xing Zhou,4 Xiao-Nan Li1. 1 _Laboratory of Molecular Neuro-oncology, Baylor College of Medicine, Houston, TX;_ 2 _Department of Pediatrics, Shanghai, China;_ 3 _Texas Children's Cancer Center,Texas Children's Hospital, Baylor College of Medicine, Houston, TX;_ 4 _Department of Neurosurgery, the First Affiliated Hospital, Soochow University Medical School, Suzhou, China_.

Glioblastoma multiforme (GBM) is the most malignant brain tumor that occurs in both adults and children. One of the key features that makes GBM particularly difficult to treat is the diffuse invasion of tumor cells into surrounding normal brain tissue. We hypothesize that direct comparison of matched invasive (GBMINV) and tumor core GBM cells (GBMTC) would facilitate the discovery of drivers of GBM invasion. However, GBMINV cells that have migrated deep into normal brain tissues are extremely difficult to obtain from patients as resection of normal human brain tissue can result in debilitating morbidities. To overcome this barrier, we utilized a panel of 6 pediatric patient tumor-derived orthotopic xenograft (PDOX) mouse models to isolate matched pairs of GBMTC cells from the visible tumor mass and GBMINV cells from the "normal" mouse brains. Global profiling of 768 human microRNA using a real-time PCR-based Taqman system identified 23 microRNAs were upregulated in the GBMINV cells in at least 5 of the 6 GBM models as compared with the matching GBMTC cells. Functional validation was performed by lentivirus-mediated silencing of miR-126, miR-487b and miR-369-5p, the top three overexpressed microRNA in the GBMINV cells. Compared with the untreated parental cells and tumor cells transduced with non-target control lentiviruses, silencing of miR-126, miR-487b or miR-369-5p suppressed GBMINV cell (both as neurosphere and monolayer) migration in vitro without affecting tumor proliferation and significantly inhibited the in vivo invasive growth of GBMINV cells, which were pre-enriched through 3 rounds of in vivo selections, into normal mouse brains. The depth of the invasive fronts were remarkably decreased/eliminated, exhibiting sharp, well-demarcated margins between tumor and normal mouse brain. To identify target genes of the 3 microRNAs, whole genome gene expression profiling from the same pairs of GBMINV and GBMTC cells was performed and identified a subset of genes suppressed by miR-126 (n=74), miR-487b (n=46) and miR-369-5p (n=2) that occurred in at least 4 of the 6 pairs. Among them, RDX was inhibited by all three microRNAs, MANEA by miR-126 and miR-369, and a 32 genes (including PDK1, CREBZF, PRKAA2, TMF1 and HSPA13) by miR-126 and miR-487b. In conclusion, our novel strategy of utilizing functionally validated pairs of GBMINV and GBMTC cells has identified a set of microRNAs that are selectively over-expressed in GBMINV cells and allowed functionally confirmation of miR-126, miR-487b and miR-369-5p as drivers of GBM invasion.

#1951

Expression profiling, cellular and clinical implications of mitochondrial miRNAs in colorectal carcinomas.

Faeza Ebrahimi, Vinod Gopalan, Alfred Lam. _Griffith University, Brisbane, Australia_.

Aim: The aim of this study was to determine the expression profiling and cellular implications of mitochondrial miRNAs (Mitomirs) such asmiR-145, miR-15a, miR-335 and miR-126)in colorectal cancer tissues. Expression changes in matched metastatic tissues were also examined.

Method: Colorectal tissues from163 primary cancers, 96 lymph node, 48 metastatic, 40 adenoma and 42 non-neoplastic tissues were recruited from 389 patients. The expression of Mitomirs in tissues and colon cancer (SW-480, SW-48) and normal (FHC) cell lines were investigated by qRT-PCR. Functional assays including cell proliferation, cell cycle analysis and apoptosis assays were performed with an exogenous miR-126 mimic to detect its in-vitro changes on cancer cell biology. In addition, Western blot was used to detect the targets of miR-126.

Result: Expressions of Mitomirs were down-regulated in colorectal cancer tissues compared to non-tumour colorectal tissues. Higher expression of miR-145 was predominantly noted in colorectal cancers with tumour recurrence (P = 0.058). Expression of miR-335 was significantly reduced distal colorectum compared to proximal colon (P = 0.025). Reduced expression of miR-335 also showed significant correlation with occurrence of lymphovascular permeation (P = 0.019), and late T stage (P = 0.004) of colorectal cancer. Moreover, in microsatellite instability (MSI) positive adenocarcinomas, notable downregulation of miR-15a (P = 0.018) was observed and this reduced expression was significantly correlated with size of tumour (> 40 mm) (P = 0.014). Expression of miR-15a, and miR-145 was reduced in metastatic cell line (SW48). Overexpression of miR-126 in-vitro showed increased apoptosis, reduced cell proliferation and suppression of synthetic phase in cell cycle. Also, overexpression of miR-126 has induced reduced expression of BCL-2 protein and increased expression of P53 protein.

Conclusion: This study suggests that expression of Mitomirs significantly reduces in advanced stages of cancer and even in early stages in comparison to normal tissues. In vitro studies indicated that ectopic expression miR-126 has the capability of inducing apoptosis and reducing viability of cancers cells potentially via modulating its target partners such as BCL-2 and p53.

#1952

Diagnostic potential of serum and exosomal microRNAs for patients with pre-malignant lesions of colorectal cancer: Detection of adenoma.

Ryo Uratani,1 Yuji Toiyama,1 Takahito Kitajima,1 Hiroyuki Fujikawa,1 Jyunichiro Hiro,1 Minako Kobayashi,1 Koji Tanaka,1 Yasuhiro Inoue,1 Yasuhiko Mohri,1 Takao Mori,2 Toshio Kato,3 Ajay Goel,4 Masato Kusunoki1. 1 _Mie University, Tsu city, Japan;_ 2 _Moriei Hospital, Kuwana city, Japan;_ 3 _Tohyama Hospital, Tsu city, Japan;_ 4 _Baylor University Medical Center, Dallas, TX_.

Background: MicroRNAs (miRNAs) are dysregulated during colorectal cancer (CRC) development and progression. Profiling of CRC tissue has elicited interest in the potential use of miRNAs as non-invasive biomarkers for early detection of CRC. Exosomes have capacity to envelop specific miRNAs, and maintain the integrity of contents in circulation. Therefore, circulating miRNAs in exosomes are more stable than other forms. Thus miRNAs extracted from exosome in serum may have significant potential as disease specific biomarker; however there is no report to assess whether whole serum or exosome-derived miRNAs from serum is a more accurate diagnostic marker and indicator of tumor progression in CRC. This study aimed to evaluate the diagnostic value of serum and exosomal miRNAs for adenoma patients, respectively.

Methods: Following a literature review, we investigated the expression of candidate miRNAs in 20 normal colonic mucosa, 27 adenoma, and 19 CRC tissue samples. Then, we quantified their expression in serum and in exosome from 26 adenoma patients and 47 healthy controls to evaluate their clinical significance. Additionally, we compared the expression levels of serum miRNAs with of exosomal miRNAs to investigate their potential as biomarker.

Results: We selected four miRNAs, miR-21, miR-29a, miR-92a, and miR-135b, for further investigation. MiR-21, miR-29a, miR-92a, and miR-135b expression in adenoma were significantly higher than in normal colonic mucosa. Regarding the miRNAs expression in serum, miR-21, miR-29a, miR-92a levels in adenoma patients were significantly higher than in healthy controls, and significantly correlated with adenoma size and total adenoma counts in the colorectum. MiR-21, miR-29a and miR-92a levels in serum could significantly discriminate patients with adenoma, and in the patients with advanced adenoma (>1.0 cm), the capacity to discriminate advanced adenoma improved. On the other hand, exosomal miR-21 and miR-29a levels in serum from adenoma patients were significantly higher than that from healthy volunteer. Only exosomal miR-21 significantly correlated with adenoma size and total adenoma counts in the colorectum, and could differentiate patients with adenomas significantly. In the patients with advanced adenoma, the capacity of exosomal miR-21 for biomarker did not improve, and significance disappeared.

Conclusion: Our results revealed that although both serum and exosomal miRNAs could be non-invasive biomarkers for the early detection of CRC, but exosomal miRNAs were unfavorable for the identification of high-risk adenomatous polyps. Serum miR-21, miR-29a and miR-92a are potential diagnostic biomarkers in high-risk adenomatous polyp patients.

#1953

MiRNA and piRNA expression profiles in renal cell carcinoma tissue detected by next generation sequencing.

Robert Iliev,1 Petra Faltejskova-Vychytilova,1 Zuzana Ozanova,1 Silvia Rybecka,1 Jaroslav Juracek,1 Jitka Mlcochova,1 Lenka Radova,1 Michal Stanik,2 Jan Dolezel,2 Michal Fedorko,3 Dalibor Pacik,3 Ondrej Slaby1. 1 _CEITEC, Brno, Czech Republic;_ 2 _Masaryk Memorial Cancer Institute, Department of Urologic Oncology, Brno, Czech Republic;_ 3 _University Hospital Brno, Department of Urology, Brno, Czech Republic_.

MicroRNAs (miRNAs) are the class of small non-coding RNAs, about 21-25 nucleotides in length, that play an important role in regulation of transcription. They affect gene expression at post-transcriptional levels through binding to complementary mRNAs and mediate their degradation in RISC complex. Piwi-interacting RNAs (piRNAs) is newly discovered class of non-coding RNA. PiRNAs are short single-stranded RNAs with 26-31 nucleotides in length. They are involved in silencing of transposable elements and it is assumed that also participate in sequence-specific chromatin modifications. It was repeatedly shown that piRNAs are present also in somatic cells and are dysregulated in kidney, bladder, gastric, breast, pancreatic and liver cancers. According to small non-coding RNA databases there are about 2600 mature miRNAs and more than 24 000 piRNAs in humans. There were extensive studies aiming to discover miRNAs and to analyze their functions. However, there are only few published studies of miRNA and piRNA profiles in renal cell carcinoma (RCC) using next generation sequencing (NGS) technology.

In our study we used the tumor tissue and the paired adjacent non-tumor renal parenchyma of 12 patients (8 males and 4 females) with RCC treated in Masaryk Memorial Cancer Institute (Brno, Czech Republic). RNA was isolated with mirVana™ miRNA Isolation Kit. For preparing RNA library was used TruSeq Small RNA Sample Preparation Kit from Illumina and then the miSeq sequencing technology was employed to detect small RNAs.

In our 12 paired samples of tumor tissue and the paired adjacent non-tumor renal parenchyma we detected 283 miRNAs with over 1 read in at least 7 samples. Expression levels of 55 miRNAs were significantly different expressed (p < 0.05) in tumor tissue and adjacent non-tumor parenchyma. Among miRNAs with the most significantly altered expression (p < 0.01) were for example, miR-129, miR-138, miR-142, miR-149, miR-154, miR-155, miR-200b, miR-210, miR-218, miR-340, miR-584, miR-885, miR-891a, miR-1270, miR-3690 and miR-7641. After analyzing piRNA sequences we found 440 piRNAs with over 1 read in at least 7 samples. From these piRNAs were 38 piRNAs significantly deregulated (p<0.01) in RCC tissue, whereas the most significantly different expression levels were determined in piR-1207, piR-2107, piR-2155, piR-12487, piR-12488, piR-21508, piR-23230, piR-26525, piR-26527and piR-28131.

In our pilot study we found altered expression patterns of miRNAs and piRNAs in tumor tissue of RCC and paired adjacent non-tumor renal parenchyma. For the first time, we have described piRNAs expression profile in RCC tissue by NGS approach.

This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved.

#1954

microRNA expression in bronchial epithelium for lung cancer detection.

Ana Brandusa Pavel,1 Joshua Campbell,1 Gang Liu,1 Sherry Zhang,1 Hanqiao Liu,1 Ji Xiao,1 Kate Porta,2 Duncan Whitney,2 Steven Dubinett,3 David Elashoff,3 Marc Lenburg,1 Avrum Spira1. 1 _Boston University School of Medicine, Boston, MA;_ 2 _Veracyte, South San Francisco, CA;_ 3 _University of California Los Angeles, Los Angeles, CA_.

Introduction

We have previously shown that gene expression alterations in the cytologically-normal mainstem bronchus can be leveraged as a biomarker for lung cancer detection (Silvestri et al. NEJM 2015), a test that is now used clinically. Extending this approach, we hypothesized that bronchial microRNA (miRNA) expression is altered in patients with lung cancer and that incorporating miRNA expression into the mRNA classifier may improve its performance.

Methods

Using bronchial brushes collected prospectively from current and former smokers undergoing bronchoscopy for suspect lung cancer across 28 medical centers as part of the AEGIS 1 and 2 clinical trials, we profiled miRNA expression via small RNA sequencing of 341 patients for which gene expression data was also available on the same bronchial brush sample. Patients were followed for up to one year post-bronchoscopy until a final diagnosis was established. 138 patients from AEGIS 1 (88 cancer-positive and 50 cancer-free) served as a discovery set, while other 203 patients from AEGIS 1 and 2 (103 cancer-positive and 100 cancer-free) were used as an independent test set. First, we identified miRNAs whose expression is associated with cancer by linear modeling in the discovery set. We next explored the relationships between the expression of these miRNAs and their predicted mRNA targets. Lastly, using logistic regression, we incorporated a cancer miRNA feature into our bronchial gene-expression classifier (Silvestri et al., NEJM 2015) and validated its performance in the test set.

Results

We found that expression profiles of 42 miRNAs were associated with cancer status in the discovery set (p<0.05, 23 miRNAs expected by chance). The top four differentially expressed miRNAs (q<0.2) were all decreased in the bronchial epithelium of cancer patients. The expression values of these miRNAs were significantly negatively correlated with the expression of their predicted mRNA targets (p<0.05). The predicted gene targets of the top miRNAs were significantly enriched (p<0.05) for cancer-related processes including the PDGF signaling pathway, insulin/IGF pathway-mitogen and activated protein kinase/MAP kinase cascade. Moreover, we found that the addition of one of the top differentially expressed miRNAs in the discovery set to the existing gene expression biomarker significantly improved the classifier's performance (ROC AUC) in the test set (p=0.025).

Conclusions

We have established that there are alterations in miRNA expression in the cytologically normal mainstem bronchus of smokers with lung cancer. Importantly, we demonstrated the potential of these miRNA alterations to improve the performance of an existing bronchial gene expression biomarker for lung cancer detection.

#1955

Computational correlation among microRNAs and mRNAs specifically expressed in pancreatic cancer stem cells.

Dawoon E. Jung,1 Hyungseok Choi,2 Jee Yeon Heo,2 Hee Seung Lee,3 Kyong Joo Lee,3 Si Young Song3. 1 _Institute of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _LGE Advanced Research Institute, LE Electronics, Seoul, Republic of Korea;_ 3 _Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea_.

The objective of this study was to analyze and identify pancreatic cancer stem cell-specific microRNAs (miRNAs) and messenger RNAs (mRNAs) and investigate their correlations to cancer stem cell biology. More, pancreatic cancer stem cell-specific miRNAs in cancer patients' serum were analyzed to determine as a diagnostic application. We used sphere cultivation methods to enrich the stem cell population from the pancreatic cancer cell line, Capan-1 with the normal pancreatic cell line, YGIC-6, and analyzed the overall miRNA and mRNA expressions using with microarray analysis. Total RNA from normal and patient's serum was extracted and the expression individual miRNAs in serum was analyzed by real-time PCR. Fifty two miRNAs including mir-26a, miR-99a, miR-106a, mir-125b, miR-192 and mir-429 were differentially expressed in the pancreatic cancer stem cells. From the miRNA analysis, 170 miRNAs were differentially expressed in the normal cell line YGIC-6, 126 miRNAs in the cancer cell line Capan-1 and 12 miRNAs in both the normal and cancer cell line originated stem cell populations. Examining both the miRNA and mRNA profiles, 52 miRNAs and 111 stem cell-associated mRNAs were highly correlated (both p values<0.01) that were differentially expressed in the pancreatic cancer stem cells. These miRNAs and mRNAs were further investigated with cross-correlation analysis, which showed a number of highly correlated miRNAs and mRNAs that are either directly or indirectly linked based on the target prediction software. The correlation degree is shown on the heatmap, and individual miRNAs with their highly correlated individual mRNAs were analyzed to predict possible mechanisms for miRNAs in pancreatic cancer stem cells. To examine miRNAs level in serum, total RNA was extracted from normal and cancer patient's serum and the expression level of individual miRNAs were analyzed. The expression level (Ct value) of mir-106a, mir-125b and mir-429 was differentially expressed in cancer patient's serum than normal and mir-99a was differentially expressed in cancer patients with poor prognosis. Differentially expressed miRNAs in pancreatic cancer stem cells provide insights into possible linkages between individual miRNAs and stem cell-associated target mRNAs in cancer stem cells and have broad implications in our understanding of cancer stem cells and cancer stem cell-targeted cancer therapy. Expression level of miRNAs in serum was differed in normal and cancer patient suggests miRNAs as a diagnostic and prognostic marker.

#1956

Mirnas hsa-miR-214-3p, -378a-3p e -34a-5p as biomarkers for uterine mesenchymal neoplasms.

Laura G. dos Anjos,1 Gustavo A. Maciel,1 Isabela W. Cunha,2 Edmund C. Baracat,1 Kátia C. Carvalho1. 1 _Univ. of São Paulo Faculty of Medicine, São Paulo, Brazil;_ 2 _A C Camargo Cancer Center, São Paulo, Brazil_.

Background: Uterine mesenchymal tumors have specific mechanisms of development that are largely unknown. One of the molecular mechanisms which may be involved in the appearance and progression of these tumors is the regulation of gene expression by miRNAs. These molecules are small non-coding RNAs that play important roles in several biological pathways. The identification of miRNAs that show particular differences in expression profile can help broaden the diagnostic methods available for these tumors. Objectives: To evaluate the expression profile of microRNAs described as sequences related to tumors development in uterine leiomyosarcomas samples, comparing to leiomyomas and normal myometrium. Methods: We selected 37 samples of uterine leiomyosarcoma (LMS), 16 unconventional leiomyomas (LMA), 10 conventional leiomyomas (LM) and 2 myometrium (used as reference samples). Samples were obtained from the Discipline of Gynecology of the Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo and Department of Pathology of the A C Camargo Cancer Center, both in Sao Paulo, Brazil. The total RNA was obtained and used to perform the synthesis of cDNA.The real time PCR reactions was performed using the Kit miScript SYBR Green PCR (Qiagen) for analysis from the 84 miRNAs sequences already described in the literature as tumor development related sequences. Results: The data analysis from the 84 sequences of microRNA showed wide variation in the regulation of these molecules expression. After computational analysis, we selected only those miRNAs with different expression profile between LM, LMA and LMS. The goal was to obtain a molecular signature that differentiate and identify each tumor using normal tissue as a reference. We found that hsa-mir-214-3p and hsa-miR-378-3p (fold regulation of -2.32 and -2.97; respectively) presented down-regulation in samples from the uterine LMS. In LMA we found a down regulation of hsa-miR-34a-5p (-3.12 fold regulation). Conclusions: We identified three miRNAs (hsa-mir-214-3p, and -378-3p and -34a-5p) differentially expressed in LMA and LMS. Analyzes of target genes of these miRNAs may identify specific proteins for use in the differential diagnosis, prognosis and patients management in these tumors.

#1957

MicroRNA analysis paired with a novel live cell viability assay: a complete epigenetic workflow in human cancer cell lines.

Samantha Lewis, Brad Hook, Don Smith, Douglas Horejsh, Trista Schagat. _Promega Corporation, Madison, WI_.

The requirements for nucleic acid purification for use in RNA profiling have expanded with the growing interest in the role of microRNAs (miRNAs) and other small non-coding RNAs in cancer cell growth and metastasis. This evolution of expression analysis has highlighted the need for more sophisticated tools for total RNA isolation from cancer cells and tissues, beyond traditional translated messenger RNAs (mRNA). To address this need, we describe tools to isolate total RNA, for mRNA and miRNA analyses, in the context of an epigenetic workflow with cancer cell lines.

In these experiments, histone deacetylase (HDAC) inhibitor treatment of different cancer cell lines was performed and expression of miRNA and mRNAs measured in parallel with assays for cell viability, cytotoxicity, and HDAC activity. This workflow multiplexes RNA and miRNA expression profiling with assays for viability and cytotoxicity to efficiently capture a more complete understanding of expression results.

With this improved cell viability workflow, one experiment from the same well can yield information about cell health, cytotoxicity, HDAC activity, and mRNA plus miRNA expression profiling. Our workflow incorporates monitoring of cell health in real-time during experimental treatments, simple and effective total RNA purification, and robust quantitative PCR reagents for a comprehensive experimental design.

#1958

Developing an online tool to validate survival-associated miRNAs utilizing expression data from 2,061 breast cancer patients.

Balázs Győrffy,1 András Lánczky,1 Giulia Bottai,2 Ádám Nagy,1 András Szabó,3 Libero Santarpia2. 1 _MTA TTK, Budapest, Hungary;_ 2 _Humanitas Research Centre, Rozzano, Milan, Italy;_ 3 _Semmelweis University, Budapest, Hungary_.

Background. MicroRNAs (miRNAs) are small non-coding RNAs capable of simultaneously regulating multiple gene networks, and affecting breast cancer patients' outcome. Here we present the development of an online tool for the real-time meta-analysis of published miRNA datasets to identify novel prognostic biomarkers in breast cancer.

Methods. First, a comprehensive database was set up by searching GEO, TCGA, and PubMed repositories to identify datasets with published miRNA expression and clinical data. Due to platform differences, each dataset was processed separately. Kaplan-Meier and Cox regression analyses were performed to investigate the prognostic value of a set of dysregulated miRNAs in breast cancer. The complete analysis tool can be accessed online at: www.kmplot.com/mirna.

Results. Overall, 2,061 samples from four independent datasets were integrated into the tool including the expression signature of 1,302 distinct miRNAs. In addition to overall survival data, ER status (n = 2,039), histological grade (n = 1,396) and lymph node status (n = 1,933) are also available. By using our online tool, we demonstrated the prognostic value of some of the previous reported miRNAs implicated in breast cancer. For instance, miR-29c, miR-34c, miR-101, miR-125, and miR-190b, showed a consistent prognostic value in at least three out of four breast cancer datasets.

Discussion. In summary, we established the first online integrated platform for the rapid identification of the prognostic value of miRNAs in breast cancer.

#1959

Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells.

Jiri Sana,1 Marek Vecera,1 Romana Butova,1 Pavel Fadrus,2 Leos Kren,2 Jaroslav Juracek,1 Robert Illiev,1 Jitka Mlcochova,1 Zuzana Ozanova,1 Petra Vychytilova,1 Ondrej Slaby1. 1 _Masaryk University, Brno, Czech Republic;_ 2 _University Hospital Brno, Brno, Czech Republic_.

Introduction:

Glioblastoma multiforme (GBM) is the most frequent intracranial malignity of astrocytic origin in adults. This tumor is characterized by infaust prognosis, which is primarily caused by resistance to the therapy and early relapses relate to the presence of glioblastoma stem cells (GSCs). These cells form neurospheres in vitro and express markers of stemness such as Sox-2, Oct-4, and nestin. Targeting of GSCs could be a novel promising therapeutic approach leading to the overcome of therapy resistance and better prognosis of GBM patients. One of the approaches how to successfully regulate GSC is a targeted regulation of microRNAs (miRNAs). These small non-coding RNA molecules post-transcriptionally regulate an expression of more than 2/3 of all human genes that are also involved in stem cell associated signaling pathways. Moreover, deregulated expression of some miRNAs has been observed in many cancers including GBM.

Cell lines and Methods:

We have prepared eleven Sox-2 positive and negative paired primary GBM cell lines, which have been cultured under DMEM/F12 medium supplemented with bFGF, EGF, and B12 supplement and DMEM medium supplemented with 10% FBS, respectively. The global miRNA expression analysis was performed using GeneChip miRNA 4.0 Array (Affymetrix). Targeted regulation of miRNA levels have been carried out by the transient transfection of specific anti-miRNAs or miRNA mimics in GSC cell lines NCH 601 acquired from Interdisciplinary Center For Neurosciences (Heidelberg, Germany). The sphere formation assay was analyzed using IncuCyte ZOOM instrument (Essen BioScience). Sox-2 and nestin expressions were analyzed on both protein and transcriptional levels using combination of PAGE with Western blotting and real-time PCR, respectively.

Results:

Analysis of Sox-2 positive and negative paired GBM cell lines revealed 29 differentially expressed miRNAs, from which miR-93-3p, miR-95-5p, miR-106b-5p, miR-22-3p, and miR-3195 showed high significance (adjust. P value < 0.05) and association with both Sox-2 (Spearman R; p < 0.002) and nestin (Spearman R; p < 0.02) expressions. MiR-22-3p and miR-3195 showed lower expression whereas other miRNAs higher expression in Sox-2 positive GBM cells. The in vitro analyses also suggest that miR-22-3p and miR-106b-5p affect the sphere formation potential and Sox-2 expression in GSC cells.

Conclusion:

Taken together, our data suggest that miR-22-3p and miR-106b-5p are probably involved in GSC biology and, thus, should be promising molecular targets to overcome GBM therapeutic resistance. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved.

#1960

Identification of differentially expressed miRNAs of breast cancer among African American and European American women.

Zhihong Gong,1 Dan Wang,1 Allyson Espinal,1 Qiang Hu,1 Xuan Peng,2 Lara Sucheston-Campbell,1 Li Tang,1 Jo Freudenheim,2 Peter Shields,3 Carl Morrison,1 Steven Belinsky,4 Song Liu,1 Kitaw Demissie,5 Michael Higgins,1 Christine Ambrosone1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _University of Buffalo, Buffalo, NY;_ 3 _Ohio State University, Columbus, OH;_ 4 _Lovelace Respiratory Research Institute, Albuquerque, NM;_ 5 _Rutgers, the State University of New Jersey, Piscataway, NJ_.

African American (AA) women are more likely than European American (EA) women to be diagnosed with aggressive, estrogen receptor (ER) negative breast cancer. Differences in microRNA (miRNA) expression patterns have not been well studied as potential mechanisms underlying this racial disparity. In this study, we performed a whole-genome miRNA expression profiling in 58 (29 AA and 29 EA) fresh-frozen breast tumors, with clinical characteristics (e.g., ER status, histological grade, stage) comparable between AA and EA samples, and in 10 (5 AA and 5 EA) normal breast tissues obtained from women undergoing reduction mammoplasty, with pathology determined free from any abnormalities. Unsupervised hierarchical clustering showed that miRNA expression patterns clearly distinguish breast cancer from normal breast tissue. In tumors, a number of miRNAs are significantly differentially expressed, i.e., DEmiRs defined as >2-fold change in expression and FDR<0.05, between tumor subtypes and by race. We identified 61 DEmiRs between ER negative and ER positive breast tumors; of these, 14 miRNAs were differentially expressed regardless of race; 29 miRNAs were specific in EA tumors only; and 18 were in AA samples only. Specifically, the top most differentially expressed miRNAs from EA women include: several members of miR-17-92 cluster (miR-17,-18a,-19a, -20a), miR-508, -509,and -514a that are up-regulated, and miR-1, -133a, -133b, and -206, which are down-regulated, in ER negative compared to ER positive tumors. In AA women, however, up-regulated miRNAs include miR-105, -106b, -135b, and -520b; down-regulated ones include miR-216a, -217, -1303, and -378a. We also identified 14 DEmiRs between AA and EA tumor samples, with 5 miRNAs specific in ER- tumors, such as miR-9, -106b, and 9 miRNAs in ER+ tumors, such as miR-1, -133a, -133b, and -206. Further, several of these miRNAs, such as miR-17, miR-18a, miR-133a, miR-206, miR-9, have been shown to regulate various target genes involved in apoptosis, cell cycle, invasion, or angiogenesis. We also found that several miRNAs, such as miR-1, -133a, -216a, are associated with improved or reduced recurrence-free or overall survival. In summary, in this genome-wide miRNA expression profiling analysis by next generation sequencing, our results suggest that miRNA expression patterns may differ by tumor subtypes between AA and EA breast samples. These initial results will provide the basis for the functional analysis of the identified miRNAs, and findings could contribute to a better understanding of the biology of breast cancer disparities and more targeted preventative and therapeutic strategies.

#1961

Characterization of MicroRNA Expression during the Development of Resistance to a mTOR-Inhibitor in renal cell carcinoma.

Joana Heinzelmann, Franziska Bauer, Gerhard Unteregger, Darja Schendel, Anne Weiland, Michael Stöckle, Kerstin Junker. _Saarland University Medical Center, Homburg, Germany_.

Introduction:

Second line therapy of metastatic renal cell carcinoma (RCC) using the mTOR inhibitor RAD001 (everolimus) is hampered by the appearance of drug resistance. The cellular mechanisms leading to drug resistance are not yet clarified. Although the influence of miRNAs is proven for other systemic treatments, their impact on mTOR inhibitors is not investigated. Therefore, we analyzed miRNA alterations in correlation to development of resistance to RAD001 in RCC cell culture models.

Material and Methods:

The RCC cell line 786-O was treated with 1µM RAD001 for up to 20 passages after developing sunitinib resistance. TotalRNA was isolated at defined intervals. Resistance-development as well as drug efficacy during the treatment was monitored by proliferation assays. MiRNA Microarrays (Agilent Technologies) were performed using RAD001 sensitive and RAD001 resistant cells to identify differently expressed miRNAs. Results were validated by qRT PCR. Functional analyses are performed after transfection of RAD001 sensitive cells with miRNA candidates.

Results:

The Microarray analyses revealed a miRNA signature including 9 differently expressed miRNAs in RAD001 resistant cells compared to sensitive cells. QRT-PCR results showed that especially miR-302e was strongly up-regulated by the factor of four. At present, functional analyses of miR-302e are ongoing.

Conclusion:

To our knowledge this is the first study analyzing miRNA alterations during the development of resistance to mTOR inhibitors. We have shown that miR-302e expression is associated with RAD001 resistance in RCC cells. Since miR-302e is a known tumor suppressor modulating radio sensitivity and drug sensitivity in several tumors we are proving now if miR-302e can regulate the mTOR inhibitor resistance of RCC.

#1962

Identifying microRNA panels specifically associated with hepatocellular carcinoma and its different etiologies.

Jing Shen, Abby B. Siegel, Helen Remotti, Qiao Wang, Regina M. Santella. _Columbia University, New York, NY_.

Hepatocellular carcinoma (HCC) incidence in the US has increased dramatically due to hepatitis C virus (HCV) infection and the epidemic of obesity-related nonalcoholic fatty liver disease (NAFLD). Many microRNAs (miRNAs) have been identified as deregulated in HCC, but few results are consistent in previous studies. In the current study, we integrate clinical and etiologic data, as well as miRNA profiles from HCC and other 8 types of solid tumors from The Cancer Genome Atlas (TCGA) and Columbia University Medical Center (CUMC) data to investigate "HCC tumor type specific" and "tumor common" miRNA panels. Aberrant miRNA panels associated with HCCs of different etiology were also explored. Levels of 33 miRNAs were significant different between HCC tumor and paired non-tumor tissues (over 2-fold changes) after Bonferroni correction for multiple comparisons, and most (28 miRNAs) were down-regulated in HCC tumors. Using this panel, we well classified HCC tumor tissues with 4 misclassifications among 48 paired tissues. Validating this panel in an additional 302 HCC tumor tissues, we almost perfectly distinguished tumor from non-tumor tissues with only two misclassifications (99% of HCC tissues correctly classified). Evaluating miRNA profiles in 32 independent HCC paired tissues from CUMC, we observed 40 miRNAs significantly deregulated in HCC with over 2-fold changes; 14 overlapped with those identified in TCGA. Eight miRNAs were further examined by quantitative RT-PCR in 66 paired HCC tissues from CUMC; 7 out of the 8 miRNAs showed consistent fold-changes. Subgroup analyses by HCC etiology found that 4 upregulated and 8 downregulated miRNAs were significantly associated with alcohol-related HCC. There were 7 and 4 miRNAs significantly associated with HBV- and HCV-related HCC, respectively. By comparison with the other 8 types of solid tumors, our data for the first time revealed that miR-24-1, miR-130a and miR-505 were significantly down-regulated only in HCC tumors; miR-142 and miR-455 were significantly down-regulated in HCC, but up-regulated in 5 other solid tumors, suggesting their HCC "tumor type specific" characteristics. A panel of 8 miRNAs were identified as "tumor common" markers including up-regulated miR-21, miR-182, miR-183, and down-regulated miR-139, miR-144, miR-101-2, miR-451, miR-486. These findings indicate that aberrant miRNA panels have HCC "tumor type specificity" and may be affected by different etiologies. These candidate miRNAs can be used as markers to improve clinical early diagnosis of HCC subtypes with specific etiologies and for more precise prevention and therapy.

#1963

Characterization of microRNA profilings in tumor tissues and serum from myxofibrosarcoma patients.

Takuya Morita,1 Tomohiro Fujiwara,1 Koji Uotani,1 Aki Yoshida,1 Toshihumi Ozaki,1 Tsukuda Kazunori2. 1 _Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Okayama, Japan;_ 2 _General Thoracic Surgery and Endocrinological Surgery, Okayama University Graduate School of Medicine, Okayama, Japan_.

Introduction:

Myxofibrosarcoma (MFS) is a common type of soft tissue sarcoma in elderly patients that exhibits a propensity for local recurrence. The local recurrence rates of MFS was reported to range from 22% to 79% even after wide resection. Despite difficulty in pathological diagnosis and evaluation of residual tumor cells after surgery, there are no useful biomarkers to solve these problems. Emerging reports have indicated the possibility of microRNAs (miRNAs) as promising novel biomarkers in malignant tumors. To date, several miRNAs were shown to be dysregulated in various malignant tissue specimens. However, little has been known about the miRNA dysregulations of tumor tissues as well as blood samples in MFS patients. In this study, we investigated miRNA dysregulations in tumor tissues and serum of MFS patients in order to develop novel biomarkers for diagnosis and monitoring MFS progression.

Methods:

The serum samples were collected from patients treated at our institute, which included 5 MFS patients, 5 age-matched non-MFS patients and 9 healthy controls. The tissue samples were collected from 11 MFS patients and normal tissue (bone, muscle, fat, and nerve). Human MFS cell lines (NMFH-1, NMFH-2), human mesenchymal stem cell (hMSC) and human skeletal muscle cell (SKMC) were also included and used in the validation analysis.

Total RNA was isolated using a miRNeay Serum/plasma Kit (QIAGEN) for serum from MFS patients, and miRNeasy Mini Kit (QIAGEN) for tissue from MFS patients. Serum levels of miRNA in MFS patients were compared with those in non-MFS patients and/or healthy controls by using miRNA microarray analysis, and compared with the results of tissue samples. The miRNA expressions in serum from MFS patients and in culture medium from human MFS cell lines, hMSC and SKMC were profiled by quantitative real-time PCR (qRT-PCR).

Results:

miRNA microarray analysis based on the extracted RNA from tumor tissue and serum collected from MFS patients revealed marked upregulation of five miRNAs in MFS serum samples compared with non-MFS or healthy controls (p < 0.05), which were included as the upregulated miRNAs in MFS tissue specimens. One of six miRNA expression levels in the serum from MFS patients were higher than in that from healthy controls, and also upregulated in the MFS tissues compared with control normal tissues. The expression of the miRNA was higher in all MFS cell lines than in hMSCs and SKMCs. Expression of the miRNA was higher in all MFS cell lines than in hMSCs and SKMCs. The expression of this miRNA in the culture media from MFS cell lines increased with time (24 and 48 hours) and numbers of tumor cells, indicating that the detected miRNA are certainly secreted to the extracellular fluids.

#1964

**NcRNAs fingerprints in colon cancer** in vitro **model by EGCG, genistein and their combinatorial effects.**

Ioana Berindan Neagoe,1 Valentina Pileczki,1 Cornelia Braicu,1 Cristina Ivan,2 Xinna Zhang,2 George Adrian Calin2. 1 _University of Medicine and Pharmacy Iuliu Hatieganu, Cluj Napoca, Romania;_ 2 _M.D. Anderson Cancer Center, Houston, TX_.

INTRODUCTION

Epidemiological studies have associated green tea intake with reduced risk in cancer. Up to date, thirty-five clinical trials use green tea polyphenol epigallocathechin-3-O-gallate (EGCG) in cancer treatment. The mechanism responsible for apoptosis and the perturbation of the noncoding transcriptome after polyphenols are largely unknown. Our aim was to examine the combined effects of two compounds, EGCG and Genistein alone or in various combinations, on two colon cancer cell lines on the expression of noncodingRNAs.

MATERIAL AND METHODS

The experiments were performed on two colon cancer cell lines RKO (p53+ wild type) and HCT116 (Ras mutated and transforming growth factor beta 1 (TGF beta 1) and beta 2 (TGF beta 2) expression positive) In order to establish the IC50 dose we performed MTT assay. For future analysis, cells were treated for 48 h either with Genistein and EGCG with established IC50 doses for both cell lines or the following combinations: 30 μM EGCG and 10 μM Genistein and 30 μM Genistein and 10 μM EGCG. We performed genome-wide profiling with a non-codingRNA array composed by probes for about 1500 human miRNAs and about 3500 lncRNAs, and ncRNA microarray analyses were performed.

RESULTS

After 48h exposer, both compounds effectively reduced cell viability in a dose-dependent manner in RKO and HCT116 cells. In the combined treatment the most interesting results were obtained at 30 μM EGCG and 10 μM Genistein and 30 μM Genistein and 10 μM EGCG, in both cell lines. Were we observed a 80% decrease of cell viability in the first situation and more than 80% of the cells maintained their viability in thesecond one.

Bioinformatic analysis was performed using R (version 3.0.1) and Bioconductor (h>p:// www.bioconductor.org/). The raw intensity for each probe is the median feature pixel intensity with the median background subtracted.

We found eighteen miRNAs and six lncRNAs commonly DE in HCT 116 cells and 56 miRNAs and 14 lncRNAs commonly DE in RKO cells by various treatments. Among the former genes we identified the oncogenic miR-18 and miR-222, as well as the long-non-codingRNAs CCAT2 that we previously identified as oncogenic in colon cancer, and MALAT-1. Furthermore, we recognized multiple ncRNA transcripts specifically disregulated by one specific type of treatment.

The expression of specific miRNAs was negatively correlated with some lncRNAs supporting a potential direct interaction by sequence complementarity, while for other miRNA-lncRNA pairs the expression was positively correlated suggesting common transcriptional mechanisms. We confirmed the array data by qRT- PCR and a restricted panel of confirmed ncRNAs was tested on the phenotypic effects in colon cancer cells.

CONCLUSION

This is the first study to prove that EGCG and /or genistein alone or in combination affect the expression of multiple types of noncodingRNAs in a correlated manner; their transcriptomic effects are more extensive as initially expected.

#1965

CRISPR/cas9, an innovative genomic tool to knock down microRNA.

Hong Chang, Bin Yi, Ruixia Ma, Hongyou Zhao, Yaguang Xi. _Univ. of South Alabama Mitchell Cancer Inst., Mobile, AL_.

MicroRNAs are a set of small and non-coding RNA molecules showing the "master" role in regulation of coding-gene expression at the post-transcriptional/translational levels. Several methods have been developed for microRNA loss-of-function study, such as antisense inhibitors and sponges; however, their robustness, specificity, and stability cannot be highly satisfied with the increased demand on microRNA functional research. CRISPR/cas9 system is emerging as a novel genome editing tool that has been exclusively studied in coding-genes knockdown, but its indication in microRNA research has not been explored yet. In this project, we clone CRISPR/cas9 constructs with designated single guide RNAs targeting biogenesis processing sites of the selected microRNAs, miR-200b and miR-17. Our results demonstrate that CRISPR/cas9 can robustly reduce the expression of these microRNAs up to 96%. More significantly, our data support high specificity and low off-target effect of CRISPR/cas9 in editing microRNA genes. Given this feature, it possesses an advantage to knockdown microRNAs in same family or with highly conserved sequences superior to antisense inhibitors and sponges. In addition, for the first time, we demonstrate the long term stability of microRNA knockdown phenotype by CRISPR/cas9 in both in vitro and in vivo models. In conclusion, we develop a novel method by adapting CRISPR/cas9 system to downregulate microRNA expression, which shows improved robustness, specificity, and stability than the previously established methodologies.

#1966

Automated miRNA purification without the use of organic solvents.

Jami D. English, Doug Horejsh, Chris Moreland, Marjeta Urh. _Promega Corporation, Madison, WI_.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs 18-24 nucleotides in size that play important roles involving gene regulation associated with cancer, disease control and gene silencing. Because of the impact on disease progression, miRNA research is rapidly shifting towards biomarker discovery. Many of the commercially available miRNA purification methods involve organic extraction during pre-processing. Here, we describe a novel chemistry that enables total RNA purification including smaller RNA such as miRNA. This chemistry offers significant advantages with a simple automated workflow, no organic extraction and minimal pre-processing.

In this study, we successfully show purification of miRNAs from a range of solid and liquid sample types including frozen tissue, whole blood, plasma, exosomes, mammalian cell lines, saliva, bone and FFPE samples. Absorbance yield, absorbance ratios, RINs, and amplification of miRNA show equivalence with organic solvent-based purification methods. The miRNA studied included miR-21, let-7a, miR-141, and miR-125b, depending on starting sample type. Next generation sequencing (NGS) analysis shows equivalent sequence diversity when compared pairwise with a manual, organic pre-processing method.

This novel chemistry and method enables purification of small and large RNA from a range of sample types. The purified RNA has little or no detectable DNA contamination and does not require phenol extraction, thus improving safety. Also, the automation of this workflow decreases manual hands on time, saving scientist time and reducing the risk of RNase contamination.

#1967

MicroRNA-1290 promotes tumor progression and acts as a novel biomarker for poor prognosis in human colorectal cancer.

Yuka Nagano, Yuji Toiyama, Hiroki Imaoka, Hiroyuki Fujikawa, Junichiro Hiro, Susumu Saigusa, Minako Kobayashi, Toshimitsu Araki, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Kenichiro Ishii, Masato Kusunoki. _Mie University Graduate School of Medicine, Tsu City, Japan_.

Background:

MicroRNA (miRNA)-1290 is known to activate Wnt signaling pathway and increase the reprogramming-related transcript factors c-Myc and Nanog. Additionally, miR-1290 promotes the epithelial-mesenchymal transition and interferes with cellular differentiation. Previous report demonstrated that overexpressing miR-1290 shows extensive atrophy and intestinal metaplasia of the gastric mucosa, indicating that miR-1290 is involved in the precancerous lesions of the gastric epithelial cells. However, to the best of our knowledge, miR-1290 expression pattern, its clinical significance and molecular mechanism in colorectal cancer (CRC) have not been investigated.

Materials and Methods:

We screened miR-1290 expression in a subset of 36 tissue samples from normal colonic mucosa (n = 12), adenoma (n = 12) and CRC (n = 12). Next, we further validated miR-1290 expression in a larger, independent cohort, which included normal colonic mucosa (n = 21), adenoma (n = 26) and CRC (n = 179), and investigated the clinical significance of miR-1290 in CRC. In addition, CRC cell lines were transfected with anti-miR against miR-1290 for the assessment of its function.

Results:

In screening step, miR-1290 expression stepwise increased according to adenoma-carcinoma sequence, and its expression in adenoma and CRC was significantly higher than that in normal colonic mucosa, respectively (adenoma: p<0.0001, CRC: p<0.0001). In validation step, we confirmed miR-1290 expression in both adenoma and CRC was significantly higher than that in normal colonic mucosa (adenoma: p<0.0001, CRC: p<0.0001). In addition, miR-1290 expression in CRC gradually increased according to TNM stage and significantly higher in stage IV. High expression in CRC were significantly associated with large tumor size (>40mm), high grade of T stage (T3/4), lymphatic and venous invasion positive, lymph node metastasis and liver metastasis. In addition, Kaplan-Meier survival curves revealed that the CRC patients with high miR-1290 expression were significantly poorer overall survival than that with low expression, respectively (p= 0.0082). Our in vitro study demonstrated that attenuated miR-1290 expression resulted in inhibition of proliferation, invasion and migration capacity of CRC cell lines.

Conclusions:

We, for the first time, demonstrated that miR-1290 in CRC acts as onco-miRNAs that is supported by several evidences from our in vitro analysis and clinical data. MiR-1290 is significantly associated with malignant potential of CRC and can be the biomarker for predicting poor prognosis in patients with CRC.

#1968

Systematic validation of microRNA based prognostic biomarkers for stage II, III and IV colorectal cancer.

Dongling Ma,1 Yaqi Xue,2 Andrew Fesler,2 Haiyan Zhai,2 Jie Yang,2 Apisri Ieamniramit,1 Wenzhe Li,1 Jingfang Ju2. 1 _NMS Labs Inc, PA;_ 2 _Stony Brook University, Stony Brook, NY_.

microRNA (miRNA) based biomarkers have great potential due to their critical regulatory functions, superior stability, and relative small number compared to mRNAs. We have previously discovered a number of miRNAs that play key roles in colon cancer stem cell resistance and demonstrated their clinical potential as prognostic biomarkers. In this study, we have validated the prognostic potential of a panel of miRNAs in 204 stage II, III, and IV colorectal cancer specimens by qRT-PCR analysis. Many of the miRNAs have been functionally validated to be important in contributing to chemoresistance to 5-FU based chemotherapy. We also determined that miR-16 is the most consistent miRNA for expression normalization. We have validated several miRNAs that are significantly associated with disease free survival (DFS) and overall survival (OS) of stage II, III and IV colorectal cancer patients. This study, together with some of the previous studies, establishes a solid foundation for miRNA based precision management of colorectal cancer.

#1969

miRNA expression as potential biomarker for leiomyosarcoma and undifferentiated pleomorphic sarcoma.

Laura Pazzaglia, Chiara Novello, Amalia Conti, Irene Quattrini, Serena Pollino, Piero Picci, Maria Serena Benassi. _Experimental Oncology Lab. Rizzoli Orthopedic Institute, Bologna, Italy_.

Rare and highly aggressive adult soft tissue sarcomas (STS), leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS), have complex genomics characterized by a multitude of rearrangements, amplifications and deletions. Our previous data (Guled et al., Genes Chromosomes Cancer, 2014) showed 21 common differentially expressed miRNAs in UPS and LMS samples when compared to mesenchymal stem cells. On the base of these results, we performed target analysis by using bioinformatic tools (Diana-microt v3.0, Targetscan Vert release 5.2 and Pictar-Vert) and we identified 12 miRNAs potentially implicated in tumor development and progression.

The expression of the selected miRNAs was evaluated by TaqMan microRNA Array in 59 high grade STS primary samples (27 LMS and 32 UPS) stored at Rizzoli biobank. Ten tissues from non oncologic patients were used as control. Our results showed a significant lower expression of miR-1, miR-133b and miR-152 (respectively p=0.0045, p=0.0027 and p=0.0016) in tumors when compared to nontumor tissue. Three different target prediction databases (miRBase, TargetScan, miRanda) recognized MET as common target gene of miR-1, mir-133b and miR-152 and KIT as target of miR-152. MET and KIT gene expression was validated in our series of STS by RT-PCR by using 2-∆∆CT comparative method. The data demonstrated higher mean expression levels in STS than in control (respectively 2.08 and 0.15 for KIT; 18527.9 and 2324.9 for MET) without significant differences between two histotypes.

Our preliminary results confirmed MET and KIT as miRNA targets. Correlation with clinical data and in vitro studies with miRNA precursor transfected cell lines are on-going to better investigate their role as potential prognostic and therapeutic markers.

#1970

miRNA differential profiling between fibroblasts originated from breast cancer and normal adjacent tissue from benign disease.

Patricia Bortman Rozenchan,1 Cristina V. de Carvalho,1 Tatiana Bonetti,1 Carina Melo,1 M Mitzi Brentani,2 Manoel JBC Girão1. 1 _Universidade Federal de São Paulo, São Paulo, Brazil;_ 2 _Universidade de São Paulo, São Paulo, Brazil_.

The importance of tumor-stromal cells interactions in breast tumor progression and invasion has been recognized; therefore, modifications in the stromal fibroblasts can play a significant role in overall cancer development and progression. Accumulating evidence indicates that epigenetic phenomena such as microRNAs are extremely important and were already recognized as key regulators of gene expression, as well as expression of these small RNAs has been linked to human disease, including those related to estrogen-dependent disorders and in cancer homeostasis. So, deciphering molecular alterations in breast tumor microenvironment such as miRNAs differential expression could lead not solely to target for therapeutic intervention as well as to novel early diagnosis markers. Stromal fibroblasts were isolated from four invasive breast carcinoma samples (CAFs) and from five women with benign breast disorders (NAFs). RNA was extracted from low-passage cultures and transformed into cDNA and used for the Human Breast Cancer miScript® miRNA PCR Array expression analysis on StepOne Plus Real Time. This Array profiles the expression of 84 mirRNAs known or predicted to alter their expression during breast cancer initiation or progression. We could find 05 differentially expressed miRNAs in CAFs in comparison to NAFs; 04 down-regulated: mir107 (fold: -1,56, p=0,04), miR-125b-1 (fold:-1,83, p=0,03), miR-200b (fold: -2, p=0,02), miR-93 (fold: -2,25, p=0,02) and 01 up-regulated: miR-424 (fold: 5,73, p=0,01). After bioinformatics analysis, these miRNAs were predicted to regulate genes involved in important cancer related pathways like, Cyclins and Cell Cycle Regulation; EGF Signaling Pathway; MAPKinase Signaling Pathway and Apoptotic Signaling in Response to DNA Damage. All together, our results showed that there are important differences regarding miRNAs expression profiling between CAFs and NAFs that could be determinant for breast cancer tumorigenesis. 

### Regulation of Chromatin State and Gene Expression

#1971

BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer.

Garrett W. Rhyasen,1 Yi Yao,1 Austin Dulak,1 Lillian Castriotta,1 Kelly Jacques,1 Maureen Hattersley,1 Gordon B. Mills,2 Michael Zinda,1 Stephen Fawell,1 Paul Lyne,1 Edwin Clark,1 Huawei Chen1. 1 _AstraZeneca, Waltham, MA;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

The bromodomain and extra-terminal (BET) protein, BRD4 functions as a transcriptional co-activator through recruitment of regulatory complexes to acetylated chromatin. Among all malignancies, the most focal, and recurrent BRD4 gene amplification occurs in high-grade serous ovarian cancer (HGSOC). Despite the elucidation of BRD4 dependencies in cancer, it is unclear whether BRD4 gene amplification can result in oncogenic activity, and hence serve as a patient selection strategy for BET bromodomain inhibitors. To assess the oncogenic potential of BRD4 amplification in HGSOC, we assayed enforced BRD4 expression in non-transformed immortalized surface ovarian cells (IOSE). Physiologically relevant expression of either long or short BRD4 spliced isoforms in IOSE (BRD4-IOSE) resulted in marked phenotypic changes indicative of cellular transformation, including altered growth kinetics, chromatin reorganization, and acquisition of colony formation potential. Transcriptional profiling of BRD4-IOSE and BRD4-amplified HGSOC patients revealed shared expression patterns, including enriched MYC, E2F1, and VEGF gene signatures. Interrogation of BRD4 inhibitor pharmacodynamic markers, and transcripts upregulated through enforced BRD4 expression uncovered Neuregulin-1 as a critical BRD4 transcriptional effector. RNAi-mediated Neuregulin-1 depletion was capable of abolishing the oncogenic activity of BRD4-IOSE, and exogenous Neuregulin-1 was sufficient to transform IOSE without BRD4. A novel BRD4-SWI/SNF interaction was required for driving the oncogenic activity of BRD4-IOSE through maintaining NRG1 expression. The sensitivity of patient-derived HGSOC xenografts to a novel clinical candidate BRD4 inhibitor, AZD5153, correlated with BRD4 amplification and a pharmacodynamic NRG1 response. This study demonstrates the oncogenic potential of BRD4 amplification, defines a relationship between BRD4 and NRG1, and further establishes HGSOC as an appropriate patient population in which to test the therapeutic potential of BET bromodomain inhibitors.

#1972

Changes in the architecture, interactions and chromatin remodeling of SWI/SNF due to loss of Snf5.

Blaine Bartholomew,1 Jim Persinger,1 Payel Sen,2 Mekonnen Dechassa Lemma,3 Jie Luo,4 Jeff Ranish4. 1 _MD Anderson Cancer Center, Smithville, TX;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _FDA National Center for Toxicological Research, Jefferson, AR;_ 4 _System Biology, Seattle, WA_.

The mammalian SWI/SNF complex is a family of an estimated ~100 complexes, each with different combinations of related subunits. Efforts in sequencing many cancer genomes has revealed that mammalian SWI/SNF complexes is one of the most frequently mutated epigenetic factors found in a broad range of cancers. In order to understand more about the subunit organization of SWI/SNF and because yeast and mammalian SWI/SNF complexes share many conserved subunits and domains; we have studied the role of the Snf5 subunit in the yeast SWI/SNF to better understand the role of the mammalian homolog SNF5 that is a known tumor suppressor gene. The basis for the loss of mammalian SNF5 being a driver mutation in pediatric rhabdoid tumors is currently not well understood or how the loss of SNF5 effects the composition, recruitment and nucleosome remodeling activity of the SWI/SNF complex. We have assessed the effects on deletion of Snf5 on the compositional integrity of SWI/SNF and have evidence for Snf5 forming a sub-complex with two other highly conserved subunits of SWI/SNF that are lost from the complex when Snf5 is deleted. In addition we have mapped the inter-subunit and intra-subunit interactions of the yeast SWI/SNF complex with lysine specific homo bi-functional crosslinkers and mass spectrometry. These data confirm the formation of a tri-subunit Snf5 sub-complex and show that this module uniquely interacts with the ATPase domain of the catalytic subunit of SWI/SNF (Snf2). These observations lead to a further investigation into the regulation of the remodeling activity of SWI/SNF by the Snf5 subunit. We have found that Snf5 is required for the ATPase domain to make stable interactions with nucleosomal DNA. And the loss of these interactions in turn also causes a reduction in the remodeling efficiency of the SWI/SNF complex. We have done additional mapping of the interactions of Snf5 as part of the SWI/SNF complex and find that the highly conserved region of yeast Snf5 corresponding to the human SNF5 protein associates with the surface of the nucleosome near the histone H2A-H2B dimer. We will discuss the relevance of these findings in highlighting how an aberrant form of SWI/SNF could be created by loss of Snf5 and provide an example of how it alters the normal function and structure of SWI/SNF.

#1973

HER2 promotes super enhancer formation in breast cancer.

Farah Rahmatpanah,1 Zhenyu Jia,1 Yuanjie Hu,1 Frank Jones,2 Michael McClelland,1 Dan Mercola1. 1 _UC Irvine, Irvine, CA;_ 2 _Tulane University, New Orleans, LA_.

Background. HER2 positive (HER2+) breast cancer (BCa) occurs in 25-30% of cases and is associated with an aggressive phenotype. Multiple HER2-stimulated pathways are known but the genes that are specifically controlled by HER2 are poorly understood. We previously used RNA polymerase II (POL II) immunoprecipitation (ChIP) to identify 737 genes that bind POL II in HER2+ but not in HER2- BCa cell lines. 51 of these genes were differentially expressed in a HER2+ dependent manner and 113 genes were differentially expressed in a HER2-dependent manner in breast tumors from 812 patients. The 113 genes are not differentially expressed in cell lines but expressed in BCa cases can be considered "poised" for expression, and activated when in the context of a breast tumor.

Methods. The 737 genes were examined using pathway finding tools and the MIT database for Super-enhancers and the dbSUPER database (2). The expression of certain genes in attached cell cultures and in mammosphere cultures were examined by qPCR.

Results. Some of the 113 genes that are "poised" for expression in cell lines in a HER2-dependent manner, and then expressed in breast cancers in a HER2-dependent manner, encode proteins that commonly occur in Super-enhancers, which are a variety of DNA regulatory structures formed by looping that associate multiple bound transcription and DNA-modifying factors in proximity to target promoters. Examples include Mediator12 (MED12) and the Bromdomain protein 2 (BRD2), and DNA binding factors such as HDAC and CREB1-cofactors. Moreover, many pluripotency genes are also found in the list of 113 genes; NANOG, OCT3/4, and SOX2 (NOS genes). These genes commonly interact with SEs as associated factors and as targets. We confirmed by qPCR that the three NOS genes exhibit increased expression in mammosphere of HER2+ MCF7 cells but not in control cells.

Using the MIT and dbSUPER (2) database we determined that 70 genes of the 113 genes (62%) that are located in or near SEs, including some present in the HER2+ BCa cell line HCC1954. Similarly, 33 (65%) of the 51 differentially expressed genes in HER2+ cell lines were associated with SEs in many diverse cell types. For comparison, the class of 573 genes that are bound to POL II in HER2+ cell lines, but are not differentially expressed in cell lines or breast cancer tissues, have a much lower fraction of genes, 213 or 37%, that encode potential members or targets of SE. The difference is highly significant, p = 0.008.

Pathway analyses of the 113 genes indicates that major pathways enriched in these genes (p < 0.001) include EGFR and Map Kinase signaling, notch pathway signaling, hedgehog, Wnt, Integrin-mediated cell and cytokine signaling Interleukin 1 and Interleukin 14.

Conclusion. These results suggest that the pathways associated with HER2 over-expression in BCa include genes associated with Super-enhancer assembly, as well as many genes encoded close to the location of Super-enhancers.

1.Rahmatpanah et al,. (2015) Oncotarget

2. Aziz Khan, Nucleic Acids Res., 2015

#1974

Consequence of the tumor-associated deletion of CHD1 on transcriptional output and tumor progression.

Michael A. Augello, Mirjam Blattner-Johnson, Mark Rubin, Christopher Barbieri. _Weill Cornell Medical College, New York, NY_.

CHD1 is a member of the super-family of chromatin modifier proteins, and is characterized by the presence of several chromo-domains as well as an ATP dependent helicase domain. A multitude of studies across several model systems have demonstrated that CHD1 functions as a transcriptional co-activator, responsible for efficient RNA polymerase II (RNA pol II) ejection from the transcriptional start site as well as maintenance of RNA pol II processivity across the gene body. This is accomplished largely though nucleosome remodeling ahead of RNA pol II, which efficiently reorganizes nucleosome position to allow for optimal RNA pol II function. Consequently, as CHD1 plays a critical role in transcriptional competency, tumor associated deletions and/or mutations of the CHD1 gene that compromise its function are rare. Interestingly, prostate cancer proves to be the exception, where the CHD1 locus is deleted with high frequency in primary disease. Tumors harboring homozygous deletion of CHD1 belong to a subclass of prostate cancer that are enriched for either SPOP or FOXA1 mutations, suggesting a prostate-specific, tumor suppressive role for CHD1 in this genomic context. Importantly, while the oncogenic functions of both FOXA1 and SPOP mutations have been largely defined, little is known as to how CHD1 loss contributes to tumor initiation or progression. Here, using 3D organoid cultures derived from GEM models of inducible CHD1 loss, prostate specific cells were found to have dramatically altered transcriptionally competency upon CHD1 depletion. These events were correlated with an aberrant, tumor-associated transcriptome, resulting in changes in morphology and growth. Furthermore, while genome wide assessment of CHD1 occupancy in human prostate cancer models uncovered an enrichment of CHD1 at sites of active transcription, loss of CHD1 did not dramatically affect expression of genes associated with tumor progression. Inversely, genomic loss of CHD1 was associated with changes in lineage dependent markers, suggesting that a major function of CHD1 is to regulate the differentiation state of prostate cells. Collectively, these data help to define the tumor suppressive role for CHD1 in prostate cancer tumorigenesis, and suggest that loss of CHD1 could alter the function of key transcriptional regulators associated with prostate cancer initiation and progression.

#1975

The 3D chromatin structure of the PTHLH region comprises a dynamic hierarchical looping complex that approximates the protein-coding genes and facilitates promoter and enhancer promiscuity.

Adam N. Freeman,1 Michael A. Henderson,2 Miroslaw K. Kapuscinski,3 John Hopper,4 T John Martin5. 1 _St Vincent's Hospital, Melbourne, Australia;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 3 _Centre for Epidemiology and Biostatistics, University of Melbourne, Melbourne, Australia;_ 4 _Centre for Epidemiology and Biostatistics, University of Melbourne, Australia;_ 5 _St Vincent's Institute, Melbourne, Australia_.

Parathyroid Hormone-Like Hormone (PTHLH) is an important regulatory gene encoding the product Parathyroid Hormone-related Protein (PTHrP). Among many biological roles, it has been demonstrated to be essential to the development of multiple tissue-types, the regulation of foetal calcium levels, and the metastasis of breast cancer to bone. Its region has GWAS associations with breast cancer, breast size, height, type 2 diabetes mellitis, neural development, cardiac arrest, and several immunological phenotypes. They extend both up- and down-stream of PTHLH, however their driving molecular mechanisms remain obscure.

Analysis of datasets including Hi-C, ChIA-PET, IM-PET and ChIP-seq suggests PTHLH sits within a 1.3Mb Topologically Associating Domain (TAD) containing 4 other protein-coding genes (pc-genes) CCDC91, MRPS35, MANSC4 and KLHL42. PTHLH itself rests between two 220kb inducible sub-TADs that likely comprise regulatory archipelagos directed primarily at its regulation. There are several other inducible loops in the TAD.

All protein-coding genes within the TAD feature an Activated Chromatin Hub (ACH) nearby their 5'. The 3D-structure of the TAD appears to be supported by a system of permanent and dynamic tether points producing primary and inducible chromatin loops. These result in all pc-genes' ACHs being 30 to 60kb from each other. RNA Pol II (RNAPII) ChIA-PET data suggests there is an inclusive hierarchical regulatory structure within the TAD, with substantial interaction between the ACHs of respective pc-genes that is likely facilitated by the 3D conformation.

#1976

Androgen receptor activity is reprogrammed by lysine-specific demethylase 1 in prostate cancer.

Shuai Gao,1 Dong Han,1 Yanfei Gao,2 Hansen He,3 Wanting Han,1 Steve Balk,2 Changmeng Cai1. 1 _University of Massachusetts-Boston, Boston, MA;_ 2 _BIDMC Cancer Center/Harvard Medical School, Boston, MA;_ 3 _Princess Margaret Cancer Center/University Health Network, Toronto, Ontario, Canada_.

In the recurrent PCa treated with CYP17A1 inhibitor (abiraterone) and more potent AR antagonist (enzalutamide), high levels of AR and AR regulated genes indicate that AR activity has been restored. One major mechanism that contributes to the disease recurrence is reprogramming of AR cistrome by collaborations of other key transcriptional factors and chromatin modifiers. In this process, AR will be recruited to a subset of newly opened enhancers that can drive the expression of genes promoting tumor cell proliferation.

Lysine Specific Demethylase 1 (LSD1) is well known for repressing transcription by removing the methyl group from histone 3 lysine 4 (H3K4me1/2). However, our genome-wide integrated analysis in prostate cancer cells reveals that LSD1 is broadly associated with AR regulated enhancers that are marked by high levels of H3K4me2 and functions as a coactivator of AR. Significantly, LSD1 also tightly associates with pioneer factor FOXA1, and LSD1-FOXA1 interaction enhances binding of both proteins at AR-mediated enhancers. As increased demethylase activity of LSD1 has been reported in several types of cancers including prostate cancer, we hypothesize that LSD1 can reprogram AR cistrome via the interaction with FOXA1 to regulate the availability of enhancers. To test this hypothesis, we performed ChIP-qPCR analyses of LSD1, FOXA1, AR, and histone marks for active enhancers such as H3K4me1,2 and acetylated H3K27 in prostate cancer cells stably expressing wild type LSD1 versus catalytic-deficient mutant, and in cells treated with LSD1 inhibitors. FOXA1 binding on AR-regulated enhancers was significantly decreased by LSD1 inhibition. Importantly, the active histone marks were also decreased at those sites, indicating inactivation of enhancers. These results clearly demonstrated that the availability of AR-mediated enhancers could be dynamically tuned by LSD1-FOXA1 to facilitate prostate cancer development. We are currently carrying out the whole genome ChIP-seq analyses on FOXA1 and enhancer marks to examine the global impact on enhancer availability by inhibiting LSD1.

Mechanistically, by immunoprecipitation of methyl-lysine antibody we further showed that methylated FOXA1 is a direct substrate of LSD1 and the demethylase activity of LSD1 is absolutely required for regulating the FOXA1 binding to chromatin. By liquid chromatography-tandem mass spectrometry (LC/MS/MS), we are currently trying to identify and characterize the methylated-lysine sites on FOXA1 in cells treated with LSD1 inhibitor and cells overexpressing the catalytic-deficient LSD1 mutant. Overall, our study suggests that the LSD1-FOXA1 interaction plays important function in determination of active enhancer prior to AR recruitment and targeting LSD1 in conjunction with AR antagonists may be a promising therapeutic approach to treat PCa.

#1977

Interaction between long noncoding RNA EZR-AS1 and SMYD3 promotes EZR expression in ESCC cells.

Xiaodan Zhang, Guo-Wei Huang, Lian-Di Liao, Lin Long, En-Min Liao, Li-Yan Xu. _Shantou University Medical College, Shantou, China_.

EZR, a member of the ezrin-radixin-moesin (ERM) family, is involved in multiple aspects of cell migration by acting both as crosslinkers between the membrane, receptors and the actin cytoskeleton, and as regulators of signalling molecules that are implicated in cell adhesion, cell polarity and migration. SMYD3 (SET and MYND Domain-containing protein 3), an H3K4 histone methyltransferase, plays important roles in EZR gene transcription activity. However, the molecular mechanisms of how to recruit this epigenetic modifier to the EZR locus still remain ill-defined. In this study, we identify a natural antisense transcript (NAT) EZR antisense AS1 (EZR-AS1), which is transcribed from the opposite strand on the EZR gene locus, is involved in the SMYD3-mediated transcription of EZR. We find that EZR-AS1 is expressed in esophageal squamous cell carcinoma (ESCC) cells and is positive correlated with EZR expression in both ESCC cells and human ESCC tissues. Silenced EZR-AS1expression decreased EZR expression at both RNA and protein levels, resulting in an apparent inhibitory effect on cell motility. Overexpression of EZR in the EZR-AS1 depleted cells partly restored the mobility of cells. Through RNA-binding protein immunoprecipitation (RIP) assay and RNA pull down assay, we further demonstrate EZR-AS1 directly interacts with SMYD3, and it depends on an RNA/DNA hybrid formation. EZR-AS1 knockdown led to a significant decrease in both SMYD3 occupancy and histone H3K4me3 at the EZR promoter region, whereas EZR-AS1 overexpression results the opposite effects. Together, our results suggest a novel mechanism of interaction between a NAT EZR-AS1 and SMYD3 drives the EZR transcription in ESCC cells.

#1978

SON and its splice variants regulate MLL1/2 complex-mediated H3K4me3 and transcription of leukemia-associated genes.

Jung-Hyun Kim,1 Eun Young Park,1 Joshua K. Stone,1 Thomas W. Butler,1 Steve Lim,2 Eun-Young Erin Ahn1. 1 _Mitchell Cancer Institute / University of South Alabama, Mobile, AL;_ 2 _Department of Biochemistry and Molecular Biology / University of South Alabama, Mobile, AL_.

SON is a poorly characterized nuclear protein that is particularly abundant in hematopoietic cells/organs and embryonic stem cells. This protein was recently identified as a splicing co-factor required for proper cell cycle progression and maintenance of stem cell pluripotency. Although SON's function in RNA splicing was recently highlighted, SON was originally identified as a DNA-binding protein and a potential regulator of transcription. However, the mechanism by SON controls transcription and its disease relevance are completely unknown.

To investigate SON's function in genome-wide gene regulation, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) in K562 human leukemia cells using SON antibodies. SON ChIP-seq results demonstrated that most of SON-binding sites are located within the promoter of target genes which include signaling mediators, transcription factors and cell cycle regulators. Our ChIP-qPCR revealed that knockdown of SON causes enhanced recruitment of the mixed lineage leukemia (MLL)1/2 complexes, but not MLL3/4 and SET1A/B complexes, to the SON target chromatin, resulting in significant increase of tri-methylation of histone H3 lysine 4 (H3K4me3) levels. Surprisingly, the C-terminus of SON directly interacts with MLL-binding region of menin and interrupts menin interaction with MLL1/2. These findings demonstrate an inhibitory effect of menin-SON interaction on menin-MLL1/2 interaction, reducing H3K4me3 and MLL1/2 complex assembly.

In addition to full-length SON (SON F), two C-terminally truncated splice variants of SON (SON B and E) have been predicted in genome databases. To address the clinical significance of SON splice variants, we examined whether SON splice variants are differentially expressed in the condition of acute myeloid leukemia (AML). Interestingly, the expression levels of alternatively spliced SON isoforms, but not full-length SON, were significantly increased in human AML patient bone marrow/blood samples and mouse models of leukemia. The short isoforms of SON retain its DNA-binding ability, thereby competing with the full-length SON for target chromatin interaction. However, the short isoforms lack the menin-binding ability and could not inhibit MLL1/2 complex assembly. Importantly, overexpression of a short isoform of SON increased MLL complex-mediated H3K4me3 and markedly enhanced replating potential of hematopoietic progenitors.

Taken together, our study reveals that the MLL1/2 complex activity is competitively regulated by full-length SON and its alternatively spliced isoforms, and that target genes of MLL1/2-menin are aberrantly controlled by overexpressed "short SON" in AML patients. Furthermore, our findings strongly suggest the significant roles of SON splice variants in aberrant transcriptional initiation in leukemia and leukemic stem cell maintenance.

#1979

Comprehensive analysis of the dynamics of aberrant WNT signaling on global gene expression and higher order nuclear structure in human cells.

Markus Brown, Darawalee Wangsa, Yue Hu, Thomas Ried. _National Institutes of Health, Bethesda, MD_.

The organization of the genome in the nucleus is complex and dynamic. Features of the nuclear architecture, including the spatial arrangement of genomic sequences, the structure of chromatin, and the accessibility of regulatory DNA elements modulate nuclear processes such as gene transcription DNA replication, DNA repair, RNA processing, and mRNA transport. However, the interplay between nuclear architecture and gene expression is poorly understood. We propose to elucidate the impact of silencing the prominent transcription factor, TCF7L2, the major effector of the WNT signaling pathway, on the transcriptional equilibrium and nuclear architecture of the nucleus in human colorectal cancer cells. Our multidisciplinary approach combines targeted silencing of TCF7L2, 3D-FISH, RNA-seq, and Hi-C chromosome conformation capture, in parallel with mathematical modeling to delineate structure/function relationships in the interphase nucleus. This approach will elucidate the intricacies of a dynamic cellular signaling pathway in three-dimensional space.

#1980

Long-range regulation of HOTAIR identifies novel biomarkers of breast cancer outcome and suggests a role in genome instability.

Michael J. Milevskiy,1 Fares Al-Ejeh,2 Jodi M. Saunus,1 Korinne S. Northwood,1 Amy E. McCart-Reed,1 Eloise Dray,3 Kenneth Nephew,4 Peter J. Bailey,1 Joshua A. Betts,2 Andrew Stone,5 Julia M W Gee,6 Annette M. Shewan,1 Juliet D. French,2 Stacey L. Edwards,2 Susan J. Clark,5 Sunil R. Lakhani,1 Melissa A. Brown1. 1 _The University of Queensland, Brisbane, Australia;_ 2 _Queensland Institute of Medical Research, Brisbane, Australia;_ 3 _Queensland University of Technology, Brisbane, Australia;_ 4 _Indiana University, Bloomington, IN;_ 5 _Garvan Institute of Medical Research, Sydney, Australia;_ 6 _Cardiff University, Cardiff, United Kingdom_.

Long-range regulators of gene expression are emerging as promising new biomarkers and therapeutic targets for human diseases including cancer. As current breast cancer biomarkers have limited power for predicting disease progression and response to therapy, we have explored the potential of long-range regulators of non-coding RNAs to be useful in the prognostication of breast cancer. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis, and is predictive of poor prognosis in breast cancer. Here we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments HOTAIR transcription. This enhancer is dependent on FOXA1 and FOXM1 transcription factors and functionally interacts with a novel alternate HOTAIR promoter. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in this subtype of breast cancer. The combination of HOTAIR and FOXM1 also enables better predictions of response to endocrine therapy for ER+ breast cancer. FOXM1 is a member of the recently described chromosome instability module and consistent with this, the expression of HOTAIR and FOXM1 is associated with increased frequency of copy number alterations and somatic non-synonymous mutations. Our study corroborates the importance of enhancers in breast cancer, elucidates the transcriptional regulation of HOTAIR, suggests HOTAIR as a novel biomarker of patient response to endocrine therapy, and implicates HOTAIR in chromosome instability.

#1981

Histone demethyalase JMJD1A promotes the DNA damage response of prostate cancer cells.

Lingling Fan,1 Guihong Peng,1 Natasha Sahgal,2 Ladan Fazli,3 Martin Gleave,3 Yuji Zhang,1 Arif Hussain,1 Feyruz Rassool,1 Jianfei Qi1. 1 _University of Maryland, Baltimore, MD;_ 2 _Queen Mary University of London, London, United Kingdom;_ 3 _University of British Columbia, Vancouver, British Columbia, Canada_.

DNA damage is a common feature of prostate cancer (PCa) therapies (e.g. radio-, chemo- and androgen deprivation therapies). Thus, the DNA damage response (DDR) is a key factor in determining the therapeutic response of PCa patients. JMJD1A regulates gene expression by demethylating H3K9. Here, we identified a new role for JMJD1A in the DDR of PCa cells. Inhibition of JMJD1A led to increased r-H2AX foci (marker for the double strand DNA breaks) in the non-irradiated cells, and the delayed clearance of r-H2AX foci in the irradiated cells. JMJD1A knockdown in PCa cells inhibited the DNA strand break repair in the reporter assays. We further found that phosphorylation and ubiquitination were key modifications that regulate the activity of JMJD1A in the DDR. Ectopic expression of the mutant JMJD1A at the phosphorylation or ubiquitination site attenuated the expression of DNA repair factors, DNA strand break repair, and PCa cell proliferation under irradiation and androgen deprivation conditions. Our results suggest that targeting JMJD1A may sensitize the response of PCa to the major anti-PCa therapies.

#1982

BRD4-FOXO axis maintains differentiated mammary epithelial phenotype by regulating p63 and GRHL3 expression.

Sankari Nagarajan,1 Upasana Bedi,2 Anusha Budida,3 Wanhua Xie,3 Feda Hisham Moh'd Hamdan,3 Vivek Kumar Mishra,3 Zeynab Najafova,3 Malik Alawi,4 Daniela Indenbirken,5 Stephan Knapp,6 Cheng-Ming Chiang,7 Adam Grundhoff,5 Christina H. Scheel,8 Vijayalakshmi Kari,3 Florian Wegwitz,3 Steven A. Johnsen3. 1 _Department of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, Germany; Cancer Research Centre UK, University of Cambridge, Cambridge, United Kingdom;_ 2 _Institute for Molecular Oncology, University Medical Center Göttingen, Goettingen, Germany; Institute for Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;_ 3 _Department of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, Goettingen, Germany;_ 4 _Bioinformatics Core, University Medical Center Hamburg-Eppendorf; Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany;_ 5 _Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany;_ 6 _Structural Genomics Consortium and Target Discovery Institute, University of Oxford, Oxford, United Kingdom; Institute for Pharmaceutical Chemistry, Goethe Universität, Frankfurt, Germany;_ 7 _University of Texas Southwestern Medical Center, Simmons Comprehensive Cancer Center, Dallas, TX;_ 8 _Institute of Stem Cell Research, Helmholtz Center for Health and Environmental Research, Munich, Germany_.

Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader which promotes gene transcription to modulate cell-specific functions and is under intensive investigation for its potential as an anti-tumor therapeutic target. However, the role of BRD4 in non-transformed cells remains unclear. Using RNA sequencing and chromatin immunoprecipitation-sequencing analyses in normal basal-like breast epithelial cells, we demonstrate that BRD4 is required for the expression of epithelial-specific genes and suppression of stem cell-like properties by binding to the distal regions of epithelial-related genes. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription of these genes. Interestingly, we show that BRD4 perturbation regulates the expression of p63 and Grainy Head-like transcription factor-3, GRHL3, whose depletion partially mimics BRD4 inhibition and impairs a differentiated phenotype. By binding to the distal regions of p63 and GRHL3, BRD4 promotes RNA polymerase-II occupancy and thus affects eRNA transcription. Motif analyses on cell-specific BRD4 binding sites predict the involvement of FOXO transcription factors. Consistently, inhibition of EGFR-AKT signalling activates FOXO1 function and promotes the expression of p63 and GRHL3. Activation of Src kinase signaling decreases RNA polymerase-II occupancy on BRD4 target genes and enhancers. Altogether, these findings provide evidence that BRD4 promotes a differentiated epithelial phenotype in non-transformed mammary cells at least in part through the activation of p63 and GRHL3 expression by associating with FOXO factors.

#1983

Altered regulation of CEBP transcription factor family in normal bronchial epithelial cells of subjects with lung cancer or COPD.

Jiyoun Yeo,1 Erin Crawford,1 Xiaolu Zhang,1 Albert Levin,2 James Willey1. 1 _University of Toledo, Toledo, OH;_ 2 _Henryford Health System, Detroit, MI_.

Background: Antioxidant (AO), DNA repair (DNAR), and cell cycle control (CCC) genes play a role in protecting normal bronchial epithelial cells (NBEC) from damage, and sub-optimal function is associated with risk for COPD and lung cancer. CEBP transcription factors regulate key AO and DNA repair genes in NBEC. In this study, we investigated the association between CEBP transcription factor function and regulation of these gene pathways in NBEC of cancer (CA) and non-cancer (NC) COPD subjects. Methods: NBEC samples were obtained by bronchoscopy from 66 CA cases and 60 matched NC controls, and 30 NC COPD and 30 NC non-COPD controls defined by spirometry (FEV1 <0.7, FEV1/FVC <80% expected). RNA was extracted and reverse transcribed into cDNA with M-MLV using oligo dT primer. A targeted competitive multiplex next generation sequencing method was used to quantify expression of CEBPA, CEBPG, CEBPD, and 30 AO, DNAR, and CCC target genes. We assessed difference in inter-gene correlation of log-transformed transcript abundance by Pearson, difference in correlation by Fisher Z-transformation, dispersion difference by F-test and difference in mean by t-test. Results: Among NC controls, CEBPG transcript abundance was highly correlated with CEBPA (r=0.78) and NFE2L2 (r=0.86) but correlation was significantly lower in cancer (r=0.45, 0.52, respectively). Correspondingly, CEBPA and CEBPG expression was more disperse among NC compared to CA (p-value =1.6E-04, 4.2E-12, respectively). Further, correlation with CEBPG and/or CEBPA was significantly lower in cancer compared to controls for 13 AO, DNAR, or CCC genes, and significantly higher for only 2 genes. Moreover, CEBPA, CEBPG, CCND2, KEAP1, MYC, NFE2L2, RB1, TP53, and TP73 mean expression was lower in cancer compared to control while BRCA1, GSTM1, and GSTP1 expression was higher (p-value <0.01). Among non-COPD NC controls, CEBPG was positively correlated with OGG1, CCND2 and ERCC5 and correlation with each gene was significantly lower among COPD NC subjects (p-value of Z-score <0.01). In contrast, CEBPD correlation with GSTM4, ERCC5 and EGFR changed from positive in COPD to negative in control (p-value of Z-score <0.01). Further, CEBPA expression was more disperse in COPD compared to non-COPD control (p-value =0.04). Conclusion: CEBPG and CEBPA expression regulation was different in CA compared to NC, and CEBPG, CEBPA and CEBPD regulation was different in NC COPD compared with NC non-COPD subjects. We hypothesize that inherited variation in NBEC regulation of these CEBP transcription factors and respective AO, DNAR, and CCC pathway genes in CA and/or COPD contributes to genetic risk. Exploration of mechanisms for variation in regulation of these genes will be the focus of additional studies in NBEC samples from over 500 subjects at risk for development for lung cancer in effort develop tests that better determine the group at genetic risk for lung cancer.

#1984

A regulatory triad consisting of BMI1, let-7i miRNA and ERK3 kinase in controlling cancer cell invasiveness.

Lobna Elkhadragy, Minyi Chen, Weiwen Long. _Wright State University, Dayton, OH_.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase, whose biological activity is tightly regulated by its cellular abundance. ERK3 expression is upregulated in multiple cancer types, including head and neck cancers. Importantly, our recent finding revealed a critical role for ERK3 in promoting cancer cell invasiveness. However, little is known about the molecular regulation of ERK3 level in cancers. Here we identify a pathway which regulates ERK3 expression and cell invasiveness of head and neck cancers, comprising the oncogenic polycomb group protein BMI1 and the tumor suppressive miRNA let-7i.

To study the regulation of ERK3, we performed several loss-of-function and gain-of-function assays in head and neck cancer cell lines, including OECM1 and Fadu. Stable knockdown of BMI1 greatly reduced ERK3 protein level, whereas overexpression of BMI1 significantly increased ERK3 level, demonstrating a positive regulation of ERK3 by BMI1. Interestingly, we found that this regulation is mediated by the miRNA let-7i, whose transcription is repressed by BMI1, a component of the polycomb repressive complex 1. ERK3 mRNA is directly targeted by let-7i miRNA, which was demonstrated by the assay using a luciferase reporter containing the 3'UTR of ERK3 mRNA downstream of luciferase gene. Mutation of the let-7i binding site in ERK3 3'UTR abolished the negative regulation of let-7i on ERK3. Further, let-7i mimic decreased ERK3 expression and let-7i inhibitor increased ERK3 expression in head and neck cancer cells. To determine the functional significance of this regulation, we investigated their role in cancer cell invasiveness by performing trans-well migration/invasion assays. Importantly, ERK3 depletion abolished BMI1-induced increase in cancer cell migration and invasion, so was by let-7i mimic. Finally, we examined by immunohistochemistry the expression of both BMI1 and ERK3 in head and neck tumor specimens and found that ERK3 expression is positively correlated with BMI1.

All together, we unravel a novel regulatory mechanism for ERK3 by the BMI1/let-7i axis, which together controls head and neck cancer cell invasiveness.

#1985

Oncogenic activation of RNA binding proteins and c-Myc signaling in hepatocellular carcinoma.

Dang T. Hien,1 Atsushi Takai,2 Marshonna Forgues,1 Xin Wei Wang1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _, Kyoto University Graduate of Medicine, Kyoto, Japan_.

Global transcriptomic alterations of both coding and non-coding RNA species are a ubiquitous feature associated with human cancers including hepatocellular carcinoma (HCC). Dysregulation of RNA-binding proteins (RBPs), the key regulators of RNA processing, is one mechanism in which cancer cells select to promote tumorigenesis. We analyzed genomic alterations amongst a family of more than 800 mRNA RBPs (mRBPs) in 1,225 clinical specimens from HCC patients and found that RBPs are significantly activated through gene amplification in a subset of tumors with poor prognosis, suggesting their potential oncogenic roles in HCC progression. Amongst the top candidates, RD binding protein (RDBP) was further characterized for its oncogenic role and effects on the HCC transcriptome. While the activation of RDBP induced an oncogenic phenotype, the abrogation of RDBP in HCC cells significantly decreased cancer associated phenotypes such as cell proliferation, migration/invasion and tumorigenicity in vivo. Further analyses revealed that RDBP-dependent genes were tumor-related with a significant enrichment for c-Myc targets, suggesting interplay between RDBP and c-Myc signaling. Similar data were also found in HCC clinical specimens where c-Myc amplification was uncommon. Consistently, the RDBP-dependent c-Myc target gene signature was able to predict HCC patient survival in two independent cohorts of more than 400 patients. Our results demonstrate that oncogenic activation of RDBP is a novel mechanism that contributes to global transcriptome imbalance, which is selective for the activation of c-Myc oncogenic signaling in HCC. Concurrent with the current model that indicates that c-Myc can promote tumorigenesis through transcription dysregulation, our current work suggests that therapies focused on targeting RDBP may be valuable for clinical treatment of many different tumors with activated c-Myc signaling, including HCC.

#1986

CEBPD promotes stemness in the pathogenesis of gliomas.

Shao-Ming Wang,1 Chiung-Yuan Ko,2 Ju-Ming Wang3. 1 _The institute of basic medical sciences, National Cheng Kung University, Tainan, Taiwan;_ 2 _Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan;_ 3 _Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan_.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain tumor with a median survival of 12-15 months despite optimal therapy. Nowadays, an increasing number of studies has indicated the connection between malignancy and stemness. The molecular mechanisms controlling the stem cell-like properties and tumorigenic potential of glioblastoma stem cells (GSCs) remain less investigated. CCAAT/Enhancer binding protein delta (CEBPD) is a C/EBP family of transcription factors. In this study, CEBPD was highly expressed in GBM specimens and GBM patients with low levels of CEBPD show a significantly better overall survival. The transcription factor OCT4 is known to play an important role in maintaining stem cell self-renewal within the central nervous system (CNS) and this activity is present in gliomas as well. Further, our data showed that CEBPD contributes to stem-like characteristics and promoted cancer cells toward stem cell traits through direct activation of OCT4 gene expression. In conclusion, this study highlights the involvement of CEBPD in enchaining stem-cell property in GBM.

#1987

Recurrent chromosomal translocations mediate reprogramming of Myb dependent regulatory networks in adenoid cystic carcinoma.

Kathryn Brayer, Scott A. Ness. _University of New Mexico, Albuquerque, NM_.

Adenoid Cystic Carcinoma (ACC), the second most common malignancy of salivary glands, is a rare tumor with bleak prognosis for which therapeutic targets are unavailable. Recently, we used RNA-sequencing (RNA-Seq) to analyze low-quality RNA from archival, formaldehyde-fixed, paraffin-embedded samples (1). In addition to detecting the most common ACC translocation, t(6;9) fusing the MYB proto-oncogene to NFIB, we also detected previously unknown t(8;9) and t(8;14) translocations fusing the MYBL1 gene to the NFIB and RAD51B genes, respectively. Mutated versions of c-Myb are potent oncoproteins, and Myb proteins are over- or highly-expressed in a wide variety of human tumors. Interestingly, ACC tumors with MYB and MYBL1 translocations displayed similar gene expression profiles, and the combined MYB and MYBL1 expression correlated with outcome, suggesting that the related Myb proteins are interchangeable oncogenic drivers in ACC.

While RNA-Seq provides information about gene fusions, alternative RNA splicing and gene expression signatures, this type of data does not address how translocations alter transcription factor function. Previous studies in our lab, and others, have demonstrated that Myb proteins contain C-terminal regulatory domains and are known to interact with several proteins via C-terminal motifs (2). These regulatory and interaction sites are lost upon chromosomal translocation in ACC; however, the downstream affect of this loss is not understood. Do the translocations merely relieve Myb transcription factors of repressive regulatory signals, thereby globally increasing Myb-dependent gene expression or, does the altered protein allow novel protein-protein interactions which re-target Myb transcription factors? Furthermore, the most frequent translocations involve the relocation of a large enhancer regions. What role does the recruitment of novel enhancer regions play in up-regulating Myb protein expression? Using a combination of ChIP-Seq and digital genomic footprinting, we are attempting to understand the mechanism of regulatory rewiring observed in ACC tumors.

References

1. Brayer et al. Recurrent Fusions in MYB and MYBL1 Define a Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma. Cancer Discovery. In press.

2. George and Ness (2014) Situational Awareness: Regulation of the Myb Transcription Factor in Differentiation, the Cell Cycle and Oncogenesis. Cancers: 6, 2049-2071.

#1988

MYB overexpression is associated with castration-resistance in prostate cancer.

Sanjeev K. Srivastava,1 Arun Bhardwaj,1 Seema Singh,1 Sumit Arora,1 Nikhil Tyagi,1 James E. Carter,2 Ajay P. Singh1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _Department of Pathology, University of South Alabama, Mobile, AL_.

Clinical progression of prostate cancer (PCa) is characterized by a transition from castration-sensitive (CS) to castration-resistant (CR) phenotype. We previously demonstrated that MYB overexpression sustained the growth of PCa cells in androgen-depleted growth environment as well as promoted aggressive tumor phenotypes. Here, we examined its role in PCa progression and castration-resistance using appropriate mouse models and delineated underlying molecular mechanisms. Luciferase-tagged MYB-overexpressing (LNCaP-MYB) or -silenced (C4-2-shMYB) PCa cell lines along with their respective control sublines (LNCaP-Neo and C4-2-NTScr) were orthotopically implanted in nude mice and tumor growth was monitored by non-invasive bioluminescence imaging using Xenogen-IVIS optical system. Our data demonstrated significantly enhanced tumor growth in mice implanted with high MYB expressing (LNCaP-MYB and C4-2-NTScr) PCa cells as compared to low MYB expressing (LNCaP-Neo and C4-2-shMYB) cells. To study the role of MYB in castration-resistance, we castrated the mice after tumors from all groups reached comparable size as determined by in vivo imaging. Tumors in LNCaP-Neo and C4-2-shMYB groups exhibited drastic regression following castration. In contrast, only slight initial reduction in the tumor growth was reported in mice from LNCaP-MYB and C4-2-NTScr groups following castration, and they later regained tumor growth. Furthermore, Kaplan-Meier survival analysis revealed that the median survival of testis-intact (non-castrated) animals of LNCaP-Neo and C4-2-shMYB groups was significantly lower than that of castrated group mice. However, castration in high MYB-expressing tumor bearing mice could barely improve their median survival. Interestingly, MYB expression levels in orthotopic tumors correlated with serum PSA levels. Furthermore, castration of mice in LNCaP-Neo and C4-2-shMYB groups resulted in a rapid reduction in serum PSA levels; while it sustained and continued to rise in MYB overexpressing groups. In additional studies, we demonstrated that MYB interacts with androgen-receptor (AR) in prostate cancer cells, and facilitates the nuclear localization of AR in a ligand-independent manner. In essence, these results suggest a critical role of MYB in the CR progression of PCa.

#1989

Multiple functional implications of MYB in ovarian cancer.

Sanjeev Kumar Srivastava,1 Seema Singh,1 Arun Bhardwaj,1 James E. Carter,2 Rodney P. Rocconi,1 Jennifer Scalici,1 Ajay P. Singh1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _Department of Pathology, University of South Alabama, Mobile, AL_.

Ovarian cancer (OC) is the most common and deadliest gynecologic malignancies in the United States, with an estimated 21,290 new cases and 14,180 deaths in 2015. Therefore, it is highly imperative to identify novel molecular targets driving OC progression, so that an effective treatment strategy can be developed against this devastating disease. In the present study, we investigated the role of MYB in OC pathogenesis. MYB/c-MYB is a cellular progenitor of v-MYB oncogenes, which encodes for a transcription factor, and confers its oncogenic activity by regulating the expression of several target genes. First, we examined the expression of MYB in clinical specimens and OC cell lines by immunohistochemical and immunoblot analysis, respectively. We observed intense staining of MYB in the tissues of all the subtypes of OC, while no or negligible staining was observed in normal ovarian tissues. Moreover, we found MYB to be expressed in all the OC cell lines examined, with its heightened expression in highly aggressive (TOV112D, SKOV3-ip) and chemo-resistant (A2780-Cip) OC cell lines. To examine the pathological significance of MYB in OC, we silenced its expression in TOV112D, SKOV3-ip and A2780-Cip OC cell lines. Functional studies demonstrate that silencing of MYB led to decrease in growth and clonogenic ability in TOV112D, SKOV3-ip and A2780-Cip cells, as compared to their scrambled sequence-transfected (Scr) control cells. Furthermore, MYB-silencing reduced motility and invasion, as well as resulted in the loss of mesenchymal and gain of epithelial markers. In addition, our data showed that TOV112D-shMYB, SKOV3-ip-shMYB and A2780-Cip-shMYB were more sensitive to cisplatin-cytotoxicity as compared to their respective controls. Altogether, our studies provide the first experimental evidence for a functional role of MYB in altered growth, malignant behavior and chemoresistance of ovarian cancer.

#1990

**Unique** in vitro **transcriptional activities of oncogenic Myb fusions identified in salivary gland adenoid cystic carcinoma.**

Candace A. Frerich, Scott A. Ness. _University of New Mexico, Albuquerque, NM_.

Chromosomal translocations involving the MYB and NFIB (t(6;9)) genes are the sole recurrent cytogenetic aberration in Adenoid Cystic Carcinoma (ACC), a rare cancer that frequently manifests as salivary gland tumors. Since ACC has poor prognosis and treatment is accompanied by high morbidity, there is a need for additional investigation of this cancer with the goal of improving patient outcomes. Recurrent translocations create fusions involving the N-terminus of MYB, located on chromosome 6, and the C-terminus of NFIB, located on chromosome 9. Our lab also recently identified translocations involving the related MYBL1 gene fusing it to NFIB t(8;9) and RAD51B t(8;14) genes. Further, recent work in our lab demonstrated tumors with MYB or MYBL1 translocations have similar gene expression (1). The presence of recurrent translocations involving both the MYB proto-oncogene and the related MYBL1 gene suggests that the resulting novel fusion proteins are likely oncogenic drivers in these tumors. In order to directly address the transcriptional activities of the resulting fusion proteins MYB and MYBL1 fusion genes, identified in primary ACC tumors, were cloned and expressed in vitro. The fusion proteins transcriptional activities were examined using reporter gene assays and trancriptomics. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to interrogate promoters bound by these proteins. Reporter genes specific for normal vs. oncogenic MYB variants have been constructed. These data demonstrate that the proteins predicted to be produced by MYB fusion genes after chromosomal translocation are transcriptionally active. Further, Myb fusion proteins bind and activate gene sets distinct from their wild type c-Myb and A-Myb counterparts. These data indicate Myb fusion proteins have unique transcriptional activities, perhaps underlying their ability to drive tumorigensis in ACC.

1. Brayer et al. Recurrent Fusions in MYB and MYBL1 Define a Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma. Cancer Discovery. In press.

#1991

Mutant GATA3 actively promotes the growth of normal and malignant mammary cells.

Natasha Chandiramani,1 Esther Peterson,1 Paraic A. Kenny2. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Gundersen Medical Foundation, La Crosse, WI_.

GATA3 is a transcription factor expressed in luminal breast epithelial cells and is required for mammary gland development. Analysis of TCGA data reveals that somatic heterozygous mutations in GATA3 occur in up to 15% of estrogen receptor positive breast tumors, and that these tumors are diagnosed a median of eight years earlier than other estrogen receptor positive tumors, suggesting a more aggressive phenotype. These mutants have been proposed to be null alleles resulting in haploinsufficiency, however the mutation spectrum of GATA3 in breast cancer is in sharp contrast to that found in HDR syndrome, a true GATA3 haploinsufficiency disease. Based on this disparity, we propose that there is a selective pressure to mutate and retain a portion of the GATA3 in breast cancer. Here we focus on the GATA3 mutants which lack the second zinc finger which is responsible for GATA motif binding. Expression of these mutants accelerated xenograft tumor growth by ZR751 cells, and transgenic expression in mouse mammary glands promoted precocious lobuloalveolar development. We have used integrated gene expression and ChIP-Seq profiling to demonstrate that these zinc-finger deleted proteins retain the ability to associate with the genome by tethering to complexes associated with FOXA1 and AP-2gamma recognition motifs, where they modulate the expression of adjacent genes. These data support a model in which the GATA3 mutations recently observed in breast cancer encode for active transcription factors which elicit proliferative phenotypes in normal mammary epithelium and promote the growth of estrogen receptor positive breast cancer cell lines.

#1992

Exploring target genes of eukaryotic initiation factor 5A in cancer using RNA sequence analysis.

Tomoki Muramatsu, Kousuke Tanimoto, Johji Inazawa. _Tokyo Medical and Dental University, Tokyo, Japan_.

Protein synthesis has a key role of maintaining cellular homeostasis such as growth and metabolism. However, dysregulation of translational process is a frequent feature of cancer and contributes to cancer progression. Since oncogenes and tumor suppressor genes are regulated by the translation machinery, therapeutic drugs which target the translation machinery have the possibility for overcoming cancer. We then focused on the hypusine cascade which is related to translational process. In our previous study, we showed that overexpression of the deoxyhypusine synthase (DHPS), an enzyme contained in the hypusine cascade, promoted cancer progression such as migration and invasion ability through RhoA signaling pathway. A DHPS inhibitor, GC7, showed significant decrease in the tumor formation in vivo. In particular, eukaryoutec initiation factor 5A (eIF5A) which is activated by hypusination has important roles of the translational regulation such as initiation and elongation of protein synthesis and nucleocytoplasmic transport of mRNA. We tried to identify the target genes of eIF5A by combining RNA-ChIP method with RNA sequencing analysis. As a result, candidate genes which bound to eIF5A were extracted by bioinformatics analysis, and redundant binding motifs for eIF5A in mRNAs emerged. These findings and future analyses would reveal the regulation of translational machinery by eIF5A which could be an important target for cancer therapy.

#1993

IL-18 induces an anti-apoptotic effect through stabilizing COX-2 mRNA expression in leukemia.

Yu Yi Chu,1 Chiung Yuan KO,2 Ju Ming Wang1. 1 _Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Taiwan, Tainan, Taiwan;_ 2 _Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, and Center for Neurotrauma, Taipei Medical University, Taipei, Taiwan_.

Leukemia is a cancer of bone marrow or blood cells. The acute myeloid leukemia (AML) is a common subtype occurred in adults to result in death. Forcing malignant cells to undergo differentiation instead of killing cancer cells is one of AML therapeutic strategy. However, the AML patients with poor outcome due to drug resistance is still a challenge. In this study, the abundances of the pro-inflammatory cytokine IL-18 and its receptor (IL-18R) were found to be correlated with the occurrence of drug resistance in AML patients. Importantly, we found that IL-18 induced an anti-apoptotic effect on U937 and THP-1 cells. Cyclooxygenase 2 (COX-2) has been suggested to have an anti-apoptotic role in chemo-resistant cancer cells. IL-18 increased COX-2 expression through post-transcriptional regulation in differentiated U937 cells. We found that two RNA binding proteins, HuR and IMP-3, stabilized COX-2 mRNA expression. In addition, IL-18 induced their interaction and facilitated this complex shuttle from nucleus to cytoplasm. Subsequently, HuR and IMP-3 bound to COX-2 mRNA 3'UTR. Two chemical inhibitors of JNK and ERK1/2 attenuated IL-18-enhanced COX-2 transcripts and the shuttle of HuR from nucleus to cytoplasm. These results suggested that IL-18 stabilized COX-2 mRNA through the JNK- and/or ERK1/2-regulated HuR nucleocytoplasmic shuttling and contributed to an anti-apoptotic effect in AML.

#1994

Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells.

Kevin Brasseur, Pascal Adam, Laurence Tardif, François Fabi, Sophie Parent, Éric Asselin. _Univ. of Québec at Trois-Rivières, Trois-Rivieres, Quebec, Canada_.

Prostate Apoptosis Response-4 (Par-4) is a tumor suppressor protein whose expression level in cancer is frequently decreased; however, it is rarely mutated nor suppressed. Par-4 is not only regulated at the expression level, but also post-translationally. Most recently, we reported the cisplatin-induced caspase3-dependent cleavage of Par-4 resulting in the accumulation of a 25kd cleaved-Par-4 (cl-Par-4) fragment. In the present study, we investigated the mechanisms regulating this fragment using cl-Par-4-expressing stable clones derived from ovarian and endometrial cancer cell lines.

Briefly, we used cancer cell lines in which we produced stable clones expressing the cleaved form of PAR-4 (cl.PAR-4) using lentiviral particles. Using these models, we studied the regulation of cl.PAR-4 at the protein level using Western blots.

Intriguingly, cl-Par-4 protein was weakly expressed in all stable clones despite constitutive transgene expression. However, upon cisplatin treatment, cl-Par-4 levels increased up to 50-fold relative to baseline conditions. We assessed the subcellular localization of cl-Par-4 in baseline and cisplatin-treated conditions using cytoplasmic/nuclear fractionation. Results showed that cl-Par-4 is localized in both the cytoplasm and nucleus compartments, but cisplatin treatment did not alter its localization. Treatment of stable clones with proteasome and translation inhibitors revealed that cisplatin exposure may in fact protect cl-Par-4 from proteasome-dependent degradation. To confirm proteasome involvement in this process, potential ubiquitination sites were predicted using bioinformatic tools. Interestingly, we showed that PI3K and MAPK pathways are also implicated in this post-translational regulation, as evidenced by an increase of cl-Par-4 in the presence of PI3K inhibitors (Wortmannin and NVP-BEZ235) and a decrease of cl-Par-4 using MAPK inhibitors (U0126 and PD98059). Finally using bioinformatics resources, we found diverse datasets showing similar results to those we observed with the proteasome and cl-Par-4. Indeed, we have showed, using data from a published study, that the use of Bortezomib (a potent proteasome inhibitor) significantly increased Par-4 expression in MCF7 cancer cells.

These new findings add to the complex mechanisms regulating Par-4 expression and activity, and justify further studies addressing the biological significance of this phenomenon in gynecological cancer cells.

#1995

HuR dependent inhibition of PARG enhances PARP inhibitor therapy for DNA repair proficient and deficient pancreatic cancer cells.

Saswati N. Chand, Mahsa Zarei, Akshay R. Kamath, Matthew J. Schiewer, Carmella Romeo, Joseph A. Cozzitorto, Nicole Meisner- Kober, Eric Londin, Isidore Rigoutsos, Karen Knudsen, John M. Pascal, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. _Thomas Jefferson University, Philadelphia, PA_.

Introduction: Despite our deep understanding of genetic drivers of the disease, pancreatic ductal adenocarcinoma (PDA) continues to be associated with dismal survival rates. Targeting the DNA repair machinery has emerged as a promising therapeutic strategy to treat pancreatic cancer patients carrying DNA damage repair (DDR) mutations. Such mutations promote tumorigenesis, but also paradoxically render tumor cells particularly susceptible to platinum-based agents and PARP inhibitors (PARPi). However, despite promising preclinical and clinical results, early data demonstrate that eventually most tumors, regardless of DDR status, become resistant to PARPi-therapies.

The mRNA-binding protein HuR, predominantly expressed in the nucleus, translocates to the cytoplasm upon tumor-associated stress where it post-transcriptionally regulates select mRNA cargo, resulting in resistance to DNA damaging agents in a harsh tumor microenvironment. Here, we sought to evaluate the role of HuR in regulating PARPi efficacy.

Results: In response to cellular stress induced by IC50 dosages of a panel of PARPis such as Veliparib, Olaparib, Rucaparib, Talazoparib and Niraparib, nuclear localized HuR undergoes cytoplasmic translocation. Silencing of HuR via siRNA, CRISPR and a DOX-inducible system resulted in significant decrease in long and short- term PDA cell survival, irrespective of DDR status. To complement and validate in vitro findings, we employed a heterotropic mouse xenograft model using Mia.sh290 cells wherein DOX induction significantly reduced HuR expression. Olaparib mediated PARP inhibition (50mg/kg, 5 days a week) combined with DOX-induced HuR silencing resulted in significant reduction in tumor volumes, compared to Olaparib alone or DOX alone.

Mechanistically, we demonstrate that the pro-survival protein HuR facilitates PDA cells to recover from PARPi insult by, in part, regulating poly ADP ribose glycohydrolase (PARG), the major enzyme responsible for hydrolyzing poly-ADP ribose (PAR) polymers, on chromatin and associated proteins. HuR binds to two 41- 43bp long sites in the 3' untranslated region (3'UTR) of PARG, increasing its mRNA stability and protein expression. Increased PARG activity, further validated via exogenous overexpression, promotes DNA repair efficiency and increases PDA cell survival. Functional analysis indicate that such inhibition of HuR and/or PARG significantly enhances PARPi sensitivity in PDA cells, via increased accumulation of DNA damage γH2AX foci, preventing efficient removal of PAR polymers, and enhancing detrimental trapping of PARP1 on chromatin.

Conclusions: Taken together, our results indicate that HuR- mediated upregulation of PARG acts as a universal pro-survival mechanism and HuR inhibition could significantly potentiate PARPi therapy in PDA, irrespective of DNA repair status.

#1996

Regulation of RNA splicing of the ErbB family receptors by the splicing cofactor SON.

Joshua Phillips, Jung-Hyun Kim, Sangbin Lim, Erin Ahn, Ming Tan. _Mitchell Cancer Institute, Mobile, AL_.

For women, breast cancer has the highest number of annual diagnoses and causes the second highest number of cancer related deaths per year. Receptor tyrosine kinases like those in the ErbB family play a crucial role in breast cancer progression by providing cancer cells proliferative and anti-apoptotic advantages. By targeting the ErbB family with monoclonal antibodies and kinase inhibitors, the prognosis of patients has improved dramatically, but resistance to these therapies does occur which requires novel therapies to be developed. One promising approach to circumvent drug resistance and inhibit tumor growth is to interrupt the elements that regulate the ErbB family protein expression such as RNA processing. The RNA transcripts of the ErbB family members must be spliced to be functional due to their immense size and the number of introns in these genes. Therefore, any alteration to this process could adversely affect tumor cells. SON, a splicing cofactor, has emerged as a protein of interest in cancer research because of its ability to impact the cell cycle and apoptosis by aiding in pre-mRNA maturation. Based on our preliminary data, we hypothesize that SON is required for ErbB family receptor-driven breast cancer by promoting the expression of not only the ErbB family proteins but also certain ErbB downstream effectors through enhanced RNA splicing. Our studies show that knocking down SON with two independent siRNAs significantly lowers the protein expression of ErbB family proteins ErbB1, ErbB2, and ErbB3 in cancer cells. At the mRNA level, the functional ErbB2 and ErbB3 transcripts decrease because introns are inappropriately retained which causes RNA degradation. These changes lead to a decrease in proliferation and increased apoptosis in breast cancer cells. After examining multiple major signaling pathways with a phospho-kinase array, we found that the activation of several critical ErbB family receptor downstream cascades that respond to stress (p38/JNK) and ensure survival (Akt) are suppressed especially in cells that have high levels of ErbB2. Patient samples were also analyzed which showed that breast tumors tend to have higher levels of SON, and the five year survival for these patients is decreased. Taken together, these data indicate that we have discovered SON as a novel ErbB family receptor splicing factor that may play an important role in breast cancer progression. Our future studies will focus on investigating more in-depth mechanisms by which SON regulates the RNA splicing of the ErbB family and evaluating whether SON can serve as a therapeutic target for breast cancer treatment by using drug and in vivo studies.

#1997

Inhibition of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia.

Qimei Han,1 Austin Young Shull,1 Fang Shi,1 Chandraiah Lagisetti,2 Thomas Webb,2 Justin Choi,1 Huidong Shi1. 1 _Georgia Regents University, Augusta, GA;_ 2 _SRI International, Menlo Park, CA_.

Splicing factor SF3B1 is frequently mutated in about 10-15% of CLL patients. SF3B1 mutations are predominately associated with faster disease progression and poor overall survival, and mutations are typically enriched in approximately 20% relapsed and chemorefractory CLL patients. Therefore, there is a growing interest in using SF3B1 as a therapeutic target for CLL. In this study, we investigated the effectiveness of Sudemycin D6 (SD6), a small molecule SF3B1 inhibitor, in reducing cell viability and promoting apoptosis in CLL cells. We then isolated RNA from both the SD6-treated and control cells at 2, 6, and 24hr post-treatment and performed RNA sequencing analysis to investigate the global impact. We demonstrated that SD6 effectively inhibits cell growth and induces apoptosis in CLL cells at a nanomolar concentration in both a time and dosage dependent manner. Furthermore, global analysis of the RNA sequencing data revealed that treatment with SD6 causes an aberrant splicing signature with significant increase in global intron retention. The increase in global intron retention peaked at 6hr then significantly diminished at 24hr post-treatment. Ontology analysis of the intron-retaining transcripts using the Ingenuity Pathway Analysis (IPA) software package suggested that B-cell receptor (BCR) signaling, protein ubiquitination, and PI3K signaling were among the top canonical pathways affected by SD6 treatment. Using RT-PCR analysis, we confirmed intron retention events in several components of the BCR pathway, such as BTK, BLNK, and PIK3CD, and an exon-skipping event in the exons encoding the kinase domain of Bruton's Tyrosine Kinase. The increase in intron retention significantly correlated with a decrease in overall mRNA and protein expression levels of the affected downstream BCR pathway proteins like AKT, PI3Kδ, and PLCγ2. Furthermore, SD6 treatment induced a time-dependent exon-skipping event in the apoptotic regulator MCL1, resulting in alternative splicing of the long, anti-apoptotic isoform of MCL1 into the short, pro-apoptotic isoform. SD6 treatment also caused downregulation of anti-apoptotic gene TRAF1 at both the mRNA and protein level, which could contribute to SD6-induced apoptosis. Cumulatively, these results strongly suggest that SF3B1 is a promising target for therapeutic treatment in CLL. Our study also provides solid rationale for future clinical development of spliceosome inhibitors and combination therapies based on spliceosome inhibitors.

#1998

**Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence** in situ **hybridization (smRNA-FISH).**

Hans E. Johansson,1 Raymund H. Yin,1 Swati Mandal,2 Myung-Hee Park2. 1 _Biosearch Technologies, Inc., Petaluma, CA;_ 2 _NIDCR, NIH, Bethesda, MD_.

Normal expression of the minor oncogene EIF5A2 in the hypusine pathway is restricted to brain and testis. However aberrant expression has been noted in multiple cancers, including ovarian gastric, colorectal and pancreatic, and artificial overexpression causes tumor growth in naked mice. Unlike cDNAs from the homologous gene EIF5A1, which are readily translated into protein upon transient transfection, EIF5A2-mRNAs only associate with monosomes without production of eIF5A-2 protein.

Here we present data from multiple EIF5A2-positive cell lines on the steady state levels of the four different EIF5A2-mRNAs that arise through differential polyadenylation. While considering qPCR and RNA-Seq, we chose to apply Stellaris single molecule RNA fluorescence in situ hybridization with four unique probe sets, each reporting on the dependent A, B, C, or D segments. The probe sets were designed with multiple tiled singly labeled 18-22-mer oligonucleotides with balanced GC content, and hence similar hybridization characteristics, which in turn enabled the simultaneous quadruplex smRNA FISH.

Stellaris RNA FISH leaves the cell intact, and also does not require RNA isolation or nucleic acid amplification that may introduce an otherwise hidden experimental bias. The single molecule spots revealed by widefield microscopy and subsequent image analysis co-localized with a high degree of confidence. Taking advantage of the added spatial dimension that RNA FISH affords, we also determined the localization of each mRNA variant and recorded the cell-to-cell variability and the actual range of mRNA copy numbers per cell.

Our data is consistent with previous gene expression analysis data, but also provide actual counts of each mRNA variant.

The results presented here have direct applications for the expression of the minor oncogene EIF5A2, and the regulated protein synthesis of its mRNAs, as well as of other mRNAs in polyamine and hypusine pathways. Further, the techniques and probes developed here can be directly transferred to the analysis of other mRNA variants that may arise through alternative transcription start site use, alternative splicing or alternative 3' end processing in normal and aberrant cells.

### Transcriptional Regulation and Gene Expression

#1999

Regulation of the oncogenic, invasion-promoting transcription factor myeloid zinc finger-1 (MZF1) in breast cancer by microRNAs.

Tuula Kallunki,1 Siri A. Tvingsholm,1 Knut K. Bundgaard Clemmensen,1 Ditte M. Brix,1 Bo Rafn,1 José Moreira,2 Lisa Frankel,3 Anders Lund,3 Riku Louhimo,4 Sampsa Hautaniemi,4 Irina Gromova,5 Marja Jäättelä1. 1 _Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark;_ 2 _Translational Cancer Research, Danish Cancer Society Research Center, Copenhagen, Denmark;_ 3 _BRIC, Copenhagen University, Copenhagen, Denmark;_ 4 _Systems Biology Laboratory, Genome-Scale Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland;_ 5 _Genome Integrity, Danish Cancer Society Research Center, Copenhagen, Denmark_.

INTRODUCTION: Myeloid Zinc Finger 1 (MZF1) is a transcription factor that is normally involved in myeloid differentiation. MZF1 can also be found in epithelial cancers, where it functions as an invasion and metastasis promoting oncogene. MZF1 is a central node of the invasive signaling network activated by HER2/ErbB2 in breast cancer, where MZF1 mobilizes and activates lysosomes and increases the expression of cysteine cathepsin B to promote invasion and metastasis. Thus, maintaining the sufficient expression level of MZF1 is important for the invasive signaling of breast cancer cells.

RESULTS AND EXPERIMETAL PROCHEDURES: Here we show that MZF1 is essential for the invasion of breast cancer cells in 3-dimensional cultures. We also describe a possible mechanism that regulates MZF1 levels in breast cancer. By using available micro-RNA (miRNA) binding site prediction programs (www.microrna.org/), specific microRNA mimics, corresponding miRNA inhibitors e.g. locked nucleic acids, luciferase reporter assays, immunofluorescense, quantitative PCR and 3-dimensional invasion assays we show that some breast cancer specific miRNAs can regulate MZF1 RNA and protein levels and revert the malignant and invasive cellular phenotype of breast cancer cells by altering the invasion promoting functions of lysosomes. These results are supported by the analysis of breast cancer patient data from the cancer genome atlas (http://cancergenome.nih.gov) showing that MZF1 expression correlates negatively with the expression of the identified miRNAs, when using data collected from over 600 primary breast cancer tumors.

SUMMARY: This study shows that MZF1 is an oncogene whose sufficient expression levels are central for breast cancer cell invasion and whose specific miRNA-mediated downregulation can inhibit breast cancer malignancy by modifying the expression of its lysosomal target genes.

CONCLUSION: Cancer-associated upregulation of MZF1 increases the breast cancer malignancy. Thus finding ways to downregulate MZF1 expression or inhibit its activity can be beneficial for breast cancer therapy.

#2000

ETV6 represses Pax5 in early B-cell development.

Courtney L. Jones,1 Courtney J. Fleenor,2 Seth Welsh,2 Leila Noetzli,1 Susan Fosmire,1 Dmitry Baturin,1 Jorge Di Paola,1 James R. Hagman,2 Christopher C. Porter1. 1 _Department of Pediatrics, University of Colorado Denver, Aurora, CO;_ 2 _Department of Biomedical Research, National Jewish Health, Denver, CO_.

The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia.

Methods: B cell fractions- B cell progenitors from wild type mice were purified using flow cytometry. Gene Expression- RNA was collected and reverse transcriptase quantitative PCR was performed to quantitate Pax5, Etv6, Ebf1 and actin transcripts. Chromatin Immunoprecipitation- Cells were collected; proteins were cross-linked to DNA with formaldehyde, cell lysates were sonicated and immunoprecipitation was performed using 5µg indicated antibody or species-matched IgG as a negative control. DNA was recovered and assayed by quantitative PCR.

Results: Recent studies have revealed a role for ETV6 germline mutations (P214L amino acid change) in the predisposition to Acute Lymphoblastic Leukemia (ALL). These mutations impair the transcriptional activity of ETV6 in a dominant negative fashion. Here, we demonstrate that Etv6 expression is inversely correlated to Pax5 and Ebf1 expression during B cell development (r2=.9993; P= 0.0167). In a murine lymphoid progenitor line (Ba/F3), ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). In addition, Pax5 expression was increased by overexpression of EBF1, a known Pax5 transcriptional activator, in cells expressing the ETV6 P214L mutant (P≤0.05). This data suggests that loss of functional ETV6 is necessary for EBF1 to induce Pax5 expression. To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Aberrant expression of Pax5 leads to myeloid lineage skewing and an increase in biphenotypic and myeloid leukemias. Patients with ETV6 germline mutations have a higher percentage of monocytes compared to unaffected family members, which we hypothesize, is due to aberrant PAX5 expression (P=0.008).

Conclusions: ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis.

#2001

Elucidate the functions of TBX3 in AR positive prostate cancer.

sinan zhu. _MSKCC, New York, NY_.

TBX3 is a transcriptional repressor belonging to the T-box gene family. It is involved in lineage specification in mammary gland, limb and heart during embryonic development. Mutations in TBX3 can cause ulnar-mammary syndrome, affecting limb, apocrine gland, tooth, hair and genital development. TBX3 is amplified/mutated in a variety of cancers, including melanoma, bladder, breast and prostate cancer. Recent findings suggest there is a strict correlation between estrogen receptor and TBX3 expression in mammary gland and TBX3 is mutated in 9% of invasive lobular breast cancer. However, little is known about the functions of TBX3 in hormone-dependent tumors. Here we screened several AR+ and AR- prostate cancer cell line and we found that the expression of TBX3 and AR is positively correlated. Moreover, in benign human prostate tissue, TBX3 localization is overlapped with that of AR. Knocking down TBX3 by siRNA resulted in decreased cell proliferation and cell cycle arrest in AR+ cell line Lncap and Vcap, but there was no effect on AR- cell line PC3 and DU14 upon TBX3 knock-down. TBX3 ChIP-seq in both Lncap and Vcap revealed co-binding of TBX3, ETS transcriptional factors and FOXA1 on AR targeted genes. Gene Set Enrichment Analysis (GSEA) of RNA-seq data from Lncap cells infected with lentivirus expressing siSCR or siTBX3 indicated that TBX3 suppresses AR and ETV1 signaling. In conclusion, our data suggests that TBX3 is essential for AR+ prostate cancer cell growth and may play a role in regulating androgen signaling.

#2002

MAX mutations in endometrial cancer are associated with poor patient outcome, altered E-box binding, and increased tumor vascularity.

Christopher J. Walker,1 Craig Rush,1 Paola Dama,1 Maggie Stein,1 Reena Shakya,1 Matthew O'Hern,1 David G. Mutch,2 David E. Cohn,1 Paul J. Goodfellow1. 1 _The Ohio State University, Columbus, OH;_ 2 _Washington University School of Medicine, St. Louis, MO_.

In contrast to aberrant MYC-signaling, MYC associated factor X (MAX) mutation is not common in many cancers but occurs in a limited number of specific tissue types, where it may be important. We sequenced MAX in 540 endometrioid endometrial cancer (EEC) DNAs. Mutations were seen 5.37% of samples with two hotspots, H28R (n=12) and R60Q/W (n=4 R60Q and n=1 R60W), which accounted for ~60% of the mutations seen. MAX mutation was associated with reduced recurrence-free survival (RFS) (P=0.03; HR [95% CI]=3.798 [1.10-13.0]) and with advanced stage (P=0.024) and high tumor grade (P=0.048). The association with reduced RFS remained significant in multivariable analysis (P =0.05 HR=2.40 [1.00-5.74]), strongly suggesting MAX mutation contributes to clinical aggressiveness in EEC.

Focusing on the H28R hotspot mutation, we performed whole transcriptome expression profiling using AN3CA endometrial cancer cell lines stably expressing exogenous long and short isoforms of flag-tagged MAX-WT and MAX-H28R. Unsupervised hierarchical clustering and principle component analysis proved that the expression profiles for the MAX-H28R long cells were strikingly different than the MAX-H28R short, MAX-WT and unmodified cells.

The MAX:DNA interface contains a predicted hydrogen bond between his28 and a guanine residue in the E-box motif. We hypothesized that the his-arg mutation seen in EECs would alter DNA binding, and assessed altered binding via chromatin immunoprecipitation/qPCR (ChIP). MAX-H28R had higher affinity for the NPM1 and CDK4 promoter regions, both of which include E-boxes that are well-established MYC/MAX targets. EMSAs for the same regions confirmed ChIP results, and showed that the supershifted MAX-WT and MAX-H28R complexes had different motilities, consistent with altered confirmation. We showed that an anti-MYC antibody was not able to supershift the EMSA fragment, indicating MYC is not part of the MAX:DNA complex. Co-IP, however, showed MAX-H28R retains MYC binding ability. Based on these findings, we conclude the differences seen in EMSA were with MAX homodimers. RNA-seq revealed differential gene expression in MAX-H28R mutated tumors, and ChIP showed enhanced MAX-H28R affinity for E-boxes in several of the differentially expressed genes.

In vivo, MAX-H28R AN3CA xenografts were markedly hemorrhagic compared to MAX-WT. MECA-32 (pan-endothelial marker) staining proved that the MAX-mutant tumors had significantly more vessels per field (2.4-fold increase, P=0.002). Work is ongoing to test for paracrine factors elaborated by the H28R-mutant AN3CA cells that might contribute to the vascular phenotype.

Taken together these data implicate MAX mutation as a novel driver of endometrial tumorigenesis, contributing to tumor aggressiveness through enhanced tumor vascularity and potentially other mechanisms.

#2003

C-terminal binding protein (CtBP): An emerging oncogene and small molecule drug target in solid tumors.

Evan T. Sumner,1 Sudha Korwar,1 Benjamin L. Morris,1 Martin M. Dcona,1 Brendan J. Hilbert,2 William E. Royer,2 Keith C. Ellis,1 Steven Grossman1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _University of Massachusetts Medical School, Worcester, MA_.

C-terminal binding proteins 1 and 2 (CtBP) are transcriptional coregulators whose overexpression has been linked to poor prognosis and/or chemoresistance in most common solid tumor types. CtBP exerts oncogenic activities through modulation of gene expression programs governing cell survival, epithelial/mesenchymal transitions, migration/invasion, and cell cycle, and is also required for colon cancer tumor initiating cell function. CtBP, however, has never been formally characterized as an oncogene. We now show that Ctbp2 exhibits transforming activity in immortalized NIH 3T3 as well as mouse and human primary cells. Ctbp2 alone, or in cooperation with SV40 large T antigen (LT) transformed NIH 3T3 cells and MEF's, respectively, to anchorage independence with similar efficiency to mutant H-Ras. Human BJ foreskin fibroblasts were also transformed to anchorage independence by CtBP2 in cooperation with LT, SV40 small T antigen, and h-TERT with efficiency similar to H-Ras, indicating that CtBP overexpression, as found in the majority of human colon, ovary, breast, and prostate cancers, may be a key oncogenic driver gene. To confirm the physiologic role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 hemizygote (Ctbp2+/-) mice, which are otherwise healthy. CtBP is a known target of the APC E3 ligase and is thus stabilized in APC mutated human colon cancers and is found in high levels in APCmin polyps. Remarkably, survival to humane endpoint at 37 weeks for Apcmin/+ vs. Apcmin/+-Ctbp2+/- mice was 0% vs. 100% (p <0.001). As CtBP also encodes a targetable intrinsic dehydrogenase that forms an NADH-dependent oligomerization surface, our cell and mouse model data supports the idea that CtBP could be an attractive small molecule drug target in tumors where it is upregulated. We have therefore synthesized a series of dehydrogenase substrate competitive inhibitor compounds based on CtBP's natural substrate 4-methylthio-2-oxobutyric acid (MTOB) that utilize a phenylpyruvate backbone and demonstrate nanomolar enzymatic IC50's with micromolar cellular GI50's, while exhibiting minimal off target inhibition of lactate dehydrogenase. Phenylpyruvate—based CtBP inhibitors biochemically caused dissolution of CtBP oligomers, which are the active transcriptional conformation, and exhibited on-target transcriptional effects in cells, with reversal of CtBP transcriptional repression of key cancer target genes, such as E-cadherin, Bik, and BRCA1. In summary, we have characterized CtBP2 as a cellular proto-oncogene that can transform primary mouse and human cells in a manner similar to activated H-Ras, genetically validated Ctbp2 as a therapeutic target in the Apcmin/+ mouse model, and identify promising small molecule lead inhibitors for future therapeutic development.

#2004

Nucleation of transcriptional super-enhancers at tumor oncogenes by small insertions.

Brian J. Abraham,1 Denes Hnisz,1 Abraham S. Weintraub,1 Nicholas Kwiatkowski,1 Charles H. Li,1 Sunniyat Rahman,2 Zhaodong Li,3 Tong Ihn Lee,1 A. Thomas Look,3 Marc Mansour,2 Richard A. Young1. 1 _Whitehead Institute, Cambridge, MA;_ 2 _University College London Cancer Institute, London, United Kingdom;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Transcriptional super-enhancers drive expression of oncogenes in many cancers and are being targeted with novel transcriptional and epigenetic therapeutics[1,2,3,4]. Super-enhancers are acquired by cancers through multiple mechanisms, including DNA translocation of an extant super-enhancer and focal amplification. We recently discovered a novel mechanism by which super-enhancers are nucleated in T cell acute lymphoblastic leukemias (T-ALLs)[5]. In this case, a small, monoallelic insertion creates a DNA binding site for a master transcription factor protein, which binds and recruits additional factors to nucleate the super-enhancer, which in turn drives high levels of the TAL1 oncogene. We describe here a method for unbiased identification of similar genomic insertions that nucleate potentially oncogenic regulatory elements in cancers. This approach uses data from genome-wide ChIP-Seq studies that map locations of enhancer-binding proteins to identify short DNA sequences missing from reference genomes. We have found and catalogued many additional genomic insertions in 80 additional cancers. An additional insertion has been functionally characterized and determined to be a bona fide enhancer-nucleating insertion that exists in patient genomes. I will describe new insights into the regulation of cancer that occur due to nucleation of novel regulatory elements.

[1] Hnisz, Abraham, Lee, et al., Cell 2013

[2] Loven, Hoke, Lin, et al., Cell 2013

[3] Kwiatkowski, et al., Nature, 2014

[4] Wang, et al., Cell, 2015

[5] Mansour et al., Science 2014

#2005

Global binding pattern of the Wilms' tumor gene 1 (WT1) +17AA -KTS isoform in leukemic cells.

Tove Ullmark, Linnea Järvstråt, Giorgia Montano, Helena Jernmark-Nilsson, Carl Sandén, Björn Nilsson, Karina Vidovic, Urban Gullberg. _Lund Univ., Lund, Sweden_.

The aim of this study was to investigate the global DNA-binding pattern of Wilms' tumor gene 1 (WT1) in leukemic cells. Clinical and preclinical data indicate the zinc finger transcription factor WT1 as an oncogene, but the full target gene repertoire of WT1 in leukemic cells has not been previously characterized. The -KTS isoforms (excluding the three amino acid (KTS) insert between zinc finger three and four) are considered as the most efficient DNA-binders. Among these, the 17AA isoform (including 17 amino acids encoded by exon 5) is the most abundant one. To specifically analyze the DNA-binding of WT1(+17AA/-KTS) in leukemic cells, we generated a K562 clone that stably expressed BIO-tagged WT1(+17AA/-KTS), as well as the biotinylating enzyme Bir A. From the cells chromatin immunoprecipitation (ChIP) by streptavidin capture was performed followed by sequencing with a minimum of 50 million reads per sample. After alignment to the genome and peak calling, peaks were characterized and compared to available K562 tracks in the ENCODE database. We found that 45% of identified WT1(+17AA/-KTS) peaks are in the proximity of transcription start sites (promoter area, first exon or first intron) of target genes, whereas only 11% of randomized peaks were found here. Within the peaks we show strong enrichment for three different previously published WT1-binding motifs. Comparison to ENCODE tracks showed that WT1(+17AA/-KTS) peaks are in close proximity to binding sites of other transcription factors, to histone marks for actively transcribed genes, and to binding sites of chromatin modifiers. Considering peaks within promoters and gene bodies only (for safe assignment to a target gene), Gene Ontology (GO) analysis revealed enrichment of GO groups important for proliferation, cell death, embryonic development, and cell motility. In conclusion, WT1(+17AA/-KTS) binds close to transcription start sites in areas of active transcription. The target genes implicated in proliferation, cell death, cell signaling and motility adds to the growing evidence of WT1 as an effector gene in leukemia.

#2006

WT1 transactivates the corepressor Nab2, creating a negative feedback loop.

Helena Jernmark Nilsson,1 Giorgia Montano,1 Linnea Järvstråt,1 Tove Ullmark,1 Andreas Lennartsson,2 Karina Vidovic,1 Urban Gullberg1. 1 _Lund Univ., Lund, Sweden;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

The aim of this work was to identify and characterize Nab2 as a novel target gene of WT1. The transcription factor Wilms tumor gene 1 (WT1) is implicated as an oncogene in leukemia, but mechanisms downstream of WT1 are incompletely understood. Several DNA-binding motifs for WT1 have been characterized and WT1 share some of them with the related transcription factor Egr1. The co-repressor Nab2 (NGFI-A-binding protein 2) is transcriptionally induced by Egr1. The Nab2 protein then acts in a negative feedback loop by direct interaction with Egr1, inhibiting its transcriptional activity. Given the similarities in the DNA-binding specificity of WT1 and Egr1, we hypothesized that Nab2 is a direct target gene also of WT1 and asked whether Nab2 binds to WT1 as well, acting as a transcriptional co-regulator. Upon overexpression of WT1 in normal CD34+ progenitor cells or in leukemic U937 cells levels of Nab2 mRNA and protein were raised, but not after overexpression of zinc-finger deleted WT1. When endogenous levels of WT1 in leukemic K562 cells were suppressed by shRNA, expression of Nab2 was decreased. Within a compendium of gene expression profiles of primary myeloid leukemias, we observed a highly significant correlation between the expression of Nab2 and WT1. A GC-rich NAB2 proximal promoter has been defined, containing several Egr1 response elements. From CAGE-analysis we identified the transcription start site utilized in leukemic cells, positioned within the previously defined exon 1. In this area, bioinformatic analysis revealed several potential WT1 binding sites. Chromatin immunoprecipitation (ChIP) experiments in K562 cells confirmed binding of WT1 to the Nab2-promoter in vivo. Cotransfection of promoter-luciferase reporter and WT1 resulted in Nab2-promoter activation. Coimmunoprecipitation followed by immunoblotting demonstrated nuclear binding of WT1 and Nab2 to each other, consistent with functional interaction in transcriptional regulation. To investigate the effect of Nab2 on WT1-mediated transcription, a luciferase reporter containing the IRF8 promoter with a WT1-responsive element was cotransfected with WT1 and increasing amounts of Nab2. While WT1, as expected, repressed promoter activity, Nab2 partially reversed the WT1-mediated repression. In conclusion, we demonstrate that WT1 binds to the Nab2 promoter, increasing the expression of Nab2 mRNA and protein. We also show that the Nab2 protein binds to WT1, inhibiting its transcriptional activity, forming a negative feed back mechanism.

#2007

Transcriptional regulatory program controlled by the oncogenic transcription factor LMO1 in neuroblastoma.

Takaomi Sanda,1 Koshi Akahanse,2 Brian J. Abraham,3 Nina Weichert,2 Adam Durbin,2 Lars Anders,3 Shi Hao Tan,1 Alice Wei Yee Yam,1 Lee N. Lawton,3 Richard A. Young,3 John M. Maris,4 A Thomas Look2. 1 _Cancer Science Institute of Singapore, Singapore, Singapore;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Whitehead Institute for Biomedical Research, Cambridge, MA;_ 4 _Children's Hospital of Philadelphia, Pennsylvania, PA_.

Neuroblastoma is an embryonal tumor of the peripheral sympathetic nervous system, accounting for 15% of all childhood cancer deaths. Both overexpression of the transcription factor LMO1 and the polymorphisms within this gene locus are associated with the susceptibility to neuroblastoma, but the oncogenic roles of LMO1 in neuroblastoma pathogenesis have not been elucidated. The roles of LMO1 in T-cell acute lymphoblastic leukemia (T-ALL) are better understood, and these suggest that some of its effects may be similar between the two malignancies. Here we identify the transcriptional regulatory program controlled by LMO1 in neuroblastoma and T-ALL cells. Knockdown of LMO1 induces apoptotic cell death in both tumor types. ChIP-seq and microarray analyses demonstrate that LMO1 frequently co-occupies its target genes with GATA3 and regulates gene expression in a tissue-specific manner. LMO1 positively regulates genes involved in neuronal development, tumor invasion and metastasis in neuroblastoma cells, whereas it regulates genes involved in lymphopoiesis and immune function in T-ALL cells. Gene set enrichment analysis reveals that many genes bound by LMO1 and GATA3 are significantly downregulated upon MYCN knockdown in the MYCN-amplified neuroblastoma cells. Importantly, LMO1 binds at the CDK6 gene locus, which is associated with a super-enhancer both in neuroblastoma and T-ALL cells. The mRNA expression of CDK6 is positively correlated with LMO1 expression in primary neuroblastoma samples. Knockdown of LMO1 downregulates CDK6 protein expression, whereas overexpression of LMO1 upregulates CDK6 expression. CDK6 knockdown induces apoptosis both in neuroblastoma and T-ALL cells. Our results indicate that CDK6 is a critical downstream target that is directly activated by LMO1 and is required for cell survival in neuroblastoma and T-ALL cells.

#2008

Ewing sarcoma cells harboring a translocation that retains EWSR1 exon 8 require HNRNPH1 to express the in-frame oncogenic fusion transcript EWS-FLI1.

Suntae Kim,1 Christiane Olivero,1 Guillermo O. Rangel Rivera,1 Nirmalya Sen,1 Sara Haddock,1 Konrad Huppi,1 Lee J. Helman,1 Patrick J. Grohar,2 Natasha J. Caplen1. 1 _NCI-CCR, Bethesda, MD;_ 2 _Vanderbilt University School of Medicine, Nashville, TN_.

The primary oncogenic event in ~85% of Ewing sarcomas is a t(11:22)(q24:q12) translocation. This translocation generates a fusion gene containing the 5' end of the EWSR1 gene and the 3' end of the FLI1 gene referred to as EWS-FLI1. The exact genomic breakpoints within the EWSR1 and FLI1 genes vary, but typically occur within introns and require the splicing machinery to generate an in-frame EWS-FLI1 transcript. In an estimated 40% of EWS-FLI1 driven tumors, the generation of an in-frame EWS-FLI1 fusion transcript requires alternative splicing. In particular, translocations that retain exon 8 of EWSR1 generate an out-of-frame transcript unless this exon is removed. In this study, we demonstrate that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require HNRNPH1 to express an in-frame EWS-FLI1 mature mRNA.

Using a genome-wide RNAi screen, we identified several proteins involved in RNA processing as required for the activity of EWS-FLI1, including the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1). The role of HNRNPH1 in alternative splicing led us to hypothesize that ES cells are dependent on HNRNPH1 for the expression of the EWS-FLI1 transcript. Analysis of the expression of EWS-FLI1 following HNRNPH1 silencing in Ewing sarcoma cell lines representing different translocation breakpoints and transcript isoforms showed that only Ewing sarcoma cell lines retaining EWSR1 exon 8 at a genomic level (TC32 and SKNMC cells) are dependent on HNRNPH1 expression. Silencing of HNRNPH1 in TC32 or SKNMC cells, results in the expression of an out-of-frame EWS-FLI1 transcript that cannot express the EWS-FLI1 oncogenic transcription factor. This leads to the reversal of expression of EWS-FLI1 gene targets and cell death. Ewing sarcoma cell lines that harbor a translocation upstream of EWSR1 exon 8 (TC71 and RD-ES) exhibit none of these molecular or phenotypic changes upon HNRNPH1 silencing.

We next employed an RNA pull-down and PCR strategy to identify putative binding sites for HNRNPH1 on the EWS-FLI1 pre-mRNA. This analysis showed enrichment for the binding of EWSR1 exon 8 by HNRNPH1. Towards the 3' end of EWSR1 exon 8 we identified two G-rich sequences, a motif typically bound by HNRNPH1. To determine if HNRNPH1 binds one or both of these sites, we developed an in vitro protein-RNA-oligomer binding assay. This assay confirmed the binding of HNRNPH1 to both G-rich sites in EWSR1 exon 8. Current studies are focused on using the protein-RNA-oligomer binding assay to fully map the interaction of HNRNPH1 with sequences within EWSR1 exon 8 and understand the molecular mechanism to target it. These results demonstrate a sequence-specific, breakpoint-dependent vulnerability in Ewing sarcoma that has the potential to be exploited as a therapeutic target and suggests a novel strategy to target fusion oncogenes.

#2009

Role of FOXO1 in oncogenic program of B cell precursor acute lymphoblastic leukemia (BCP-ALL).

Fan Wang, Alexey Ushmorov, Thomas Wirth. _University of Ulm, Ulm, Germany_.

Although treatment of B cell precursor acute lymphoblastic leukemia (BCP-ALL) in children has shown eminent progress over the past decades, about 15% of patients can still not be cured, however. Therefore, new therapeutic strategies are warranted.

FOXO1 together with other transcription factors including EBF1, PAX5 and TCF3 plays a central role in differentiation of pre-B-cells. In cooperation with PAX5 and other B-cell transcription factors, FOXO1 induces expression of SYK, BLNK, RAG1, RAG2, AICDA and IL7RA genes. Of note, SYK and IL7RA signaling contribute to the oncogenic program of BCP-ALL, which utilize pre-B cell survival and proliferation program.

In this study, we demonstrate that FOXO1 is highly expressed in most of the BCP-ALL cell lines. By using immunofluorescence assays we show that FOXO1 is localized in the nucleus of BCP-ALL cells. Overexpression of constitutively active AKT1 (inactivating FOXO1) negatively regulates the growth of BCP-ALL cells. Inhibition of FOXO1 by shRNA results in cell death in the RS4;11 BCP-ALL cell line. BCP-ALL cells are more sensitive to growth arrest and apoptosis induced by a small molecular FOXO1 inhibitor AS1842856 than other classical Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Gene expression profiling of BCP-ALL cells after treatment with the FOXO1 inhibitor for 24h shows the significantly down-regulation of FOXO target genes including VPREB3, IL1B, KLHL24 and BNIP3L. Interestingly, at the same time, some FOXO targets like NOXA, RUNX2 and PRDM1 were found upregulated, indicating a complex effect of the FOXO1 inhibitor.

We conclude that FOXO1 expression is critical for the BCP-ALL oncogenic program and its targeting represents a novel therapeutic approach.

#2010

Charactering the interactomes of the Myc family of oncogenes.

Diana Resetca,1 Dharmesh Dingar,2 Manpreet Kalkat,1 Brian Raught,3 Linda Z. Penn3. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;_ 3 _Princess Margaret Cancer Centre, University Health Network and University of Toronto, Toronto, Ontario, Canada_.

The Myc family of transcription factors, c-Myc, N-Myc and L-Myc, are known to be deregulated in a large variety of cancers. Mechanisms responsible for the deregulation of the activity of Myc family members in cancer are not well understood. A number of protein-protein interactions and post-translational modifications have been suggested to promote the oncogenic activity of Myc. Traditional biochemical approaches have not been successful at mapping the interactors of Myc family members due to their tight association with chromatin and the labile nature of Myc proteins. Mapping the protein-protein interactions that support the aberrant activity of Myc family of oncoproteins in cancer cells is of high interest, particularly in a more natural context in xenograft models in vivo.

A new mass spectrometry-based technique, BioID-MS, which relies on proximity-based biotin labeling, has recently emerged as a key advance for the characterization of hard-to-detect protein-protein interactions in living cells. Herein, we are reporting a new application of the BioID-MS technique for the characterization of c-Myc interactors in human cell line in vivo, in mouse tumor xenografts. Using the in vivo BioID assay, we were able to identify more than 30 known and validated c-Myc interactors, some of which include the components of the STAGA complex and SWI/SNF chromatin remodelling complex. We were further able to identify more than 100 novel high-confidence c-Myc interactors, which include components of the DNA repair and replication machinery, general transcription and elongation factors, and the co-regulator of transcription-like DNA helicase protein chromodomain 8 (CHD8). Using ENCODE ChIP-seq datasets we were able to map some of the high-confidence interactors to coincident binding sites with c-Myc throughout the genome. This provided further credibility that the newly identified putative interactors could co-occupy sites on chromatin with c-Myc and could be functionally important for activity. Furthermore, we validated the Myc-CHD8 interaction using a number of approaches, including yeast two hybrid and proximity-based ligation assays.

These findings suggest that the BioID-MS technique can be used to extend the mapping of the Myc interactome and contribute to a greater understanding of Myc regulation by protein-protein interactions. Furthermore, we are currently in the process of employing this technique to map the interactomes of two other Myc family members, N-Myc and L-Myc. We are interested in identifying common interactors of the Myc family members that contribute to their oncogenic activity, validate them, and explore whether these interactors could be potential therapeutic targets in cancers with deregulated Myc activity.

#2012

SPDEF enforces tumor quiescence by shifting the transcriptional targets of activated β-catenin.

Yuan-Hung Lo,1 Taeko Noah,2 Min-Shan Chen,1 Winnie Zou,1 Noah Shroyer1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH_.

Background: Colorectal cancer (CRC) carcinogenesis is driven by a series of genetic and epigenetic changes that results in the oncogenic transformation of normal colonic mucosa. Canonical Wnt/β-catenin signaling pathway activation, with resultant high β-catenin transcriptional activity, is frequently implicated human CRC (~ 90% of CRC); however, there are currently no treatments targeting this pathway. We have previously reported that SAM Pointed Domain Ets transcription Factor (SPDEF) is a colonic tumor suppressor that negatively regulates canonical Wnt/β-catenin signaling. In agreement with the tumor repressor role of SPDEF, the absence of SPDEF enhances intestinal tumor formation, while re-expression of SPDEF inhibits colon adenocarcinoma proliferation in both genetic (Apcmin/+) and chemically colitis-associated (AOM/DSS) CRC models. However, the molecular mechanism by which SPDEF mediates colorectal tumor repression is still largely unknown. Here we aim to elucidate the molecular mechanism by which SPDEF mediates repression of canonical Wnt/β-catenin signaling in CRCs.

Material and Methods: To achieve our goal, we directly analyzed the effects of SPDEF expression in β-catenin-driven intestinal tumors in vivo using a new inducible mouse model (Lgr5CreERT2; β-cateninexon3; Rosa26rtta-ires-EGFP; TRE-Spdef) and human colon cancer xenografts. Moreover, wildtype or truncated SPDEF mutants were used for β-catenin transcriptional activity assay, co-immunoprecipitation, and chromatin immunoprecipitation in human colon cancer cell lines.

Results: In this study, we find that SPDEF is sufficient to inhibit β-catenin-driven intestinal tumorigenesis and shrink established tumors in both transgenic mice and xenografted human colon cancer cells. SPDEF inhibits canonical β-catenin transcriptional activity through protein-protein interaction, independent of its DNA binding capacity. We find that SPDEF disrupts the binding between β-catenin and its DNA binding partners TCF1 and TCF3, but not LEF1 and TCF4, selectively displacing β-catenin from the promoter/enhancer regions of cell cycle genes without affecting stem cell signature genes. Consistent with this observation, re-expression of SPDEF enforces a quiescent state on β-catenin-driven tumor cells in vivo. Taken together, for the first time, we unveil a novel mechanism in which SPDEF shifts the transcriptional targets of activated β-catenin to regulate the active to quiescent switch in tumor initiating cells. These findings provide insights into the mechanisms that can regulate tumor cell quiescence, and offer a novel approach for targeting canonical Wnt/β-catenin transcriptional machinery as a therapeutic strategy.

#2013

The PAX3-FOXO1 oncogene drives aneuploidy and overrides aneuploidy-associated proliferative defects in alveolar rhabdomyosarcoma.

Andrew D. Hollenbach,1 Jacob M. Loupe,2 Patrick J. Miller,3 Benjamin P. Bonner,1 Elaine C. Maggi,1 Jyothi Vijayaraghavan,1 Jovanny Zabaleta,1 Christopher M. Taylor,1 Fern Miller,1 Judy S. Crabtree1. 1 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Tulane University Medical School, New Orleans, LA_.

Alveolar Rhabdomyosarcoma (ARMS) is primarily defined by the t(2;13)(q35;q14) translocation, which generates the PAX3-FOXO1 oncogene. Despite the fact that ARMS are frequently aneuploid, like a majority of other solid tumors, it is unknown whether PAX3-FOXO1 contributes to the development and/or persistence of aneuploidy. In this study we show that PAX3-FOXO1 serves as the driver mutation to promote aneuploidy by globally altering gene regulatory networks essential for maintaining proper chromosome number and structure. Further, we demonstrate that PAX3-FOXO1 overrides aneuploid-dependent growth arrest by altering the expression of growth factor related regulatory networks. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these gene regulatory network changes. This is the one of the first studies describing how an oncogene and post-translational modifications of the corresponding oncoprotein drive the acquisition of aneuploidy and override proliferation defects in malignant transformation. The mechanism for PAX3-FOXO1 described in this work has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens.

#2014

Alternative splicing is a frequent event in mouse PTEN-deficient prostate cancer.

Marco A. De Velasco,1 Yurie Kura,1 Kazuko Sakai,1 Yoshihiko Fujita,1 Yosuke Togashi,1 Masato Terashima,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kinki University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Nagakute, Japan_.

Castration-resistant prostate cancer (CRPC) remains an incurable disease and presents a major challenge for the development of novel treatment strategies. Alternative splicing occurs as a common natural phenomenon and promotes diversity, however, cancer-related alternative splicing events have been shown to contribute to disease progression and promote therapeutic escape. In prostate cancer, key genes required for normal biological function are frequently alternatively spliced resulting in altered phenotypes. Novel therapeutic strategies targeting the alternative splicing machinery are being developed and tested. Here, characterized the conditional PTEN-deficient mouse model of prostate cancer to determine its relevancy as preclinical tool for the development and efficacy determination of novel therapeutic strategies targeting aberrant alternative splicing. For this, we used the Affymetrix GeneChip mouse transcriptome assay to perform comparative analysis of the transcriptomes of normal prostate tissue and PTEN-deficient castration-naïve, castration-sensitive (4 weeks post-castration), and castration-resistant (10 weeks post-castration) prostate cancers. Clustering analysis revealed genes enriched with the functions involved in mRNA splicing and processing in mice with prostate tumors. Moreover, alternative splicing events were more prevalent in castration-sensitive tumors (>2.5-fold) compared to castration-naïve and castration resistant tumors. Exon skipping (cassette exon), was the most common splicing event observed in all conditions. Additionally, alternative splicing was observed in several genes frequently associated with human prostate cancer including CD44, AR, p53, Bcl2l1 and Klf6 among others. Collectively, our data shows that alternative mRNA splicing is a frequent event in mouse PTEN-deficient prostate cancer and supports a role for alternative splicing dysregulation and the pathogenesis of the disease. Furthermore, this mouse model may also provide a suitable platform to study current therapeutic approaches targeting alternative splicing.

#2015

Novel oncogenic roles of EVI1 in breast carcinoma.

Hui Wang,1 Thorsten Schaefer,1 Martina Konantz,1 Selina Reich,2 Martin Braun,3 Sven Perner,4 Zsuszanna Varga,5 Holger Moch,5 Tanja Fehm,6 Lothar Kanz,2 Klaus Schulze-Osthoff,7 Claudia Lengerke8. 1 _University Hospital Basel, Department of Biomedicine, Basel, Switzerland;_ 2 _University of Tuebingen, Department of Internal Medicine II, Tuebingen, Germany;_ 3 _University Hospital Bonn, Department of Prostate Cancer Research, Bonn, Germany;_ 4 _Pathology of the University Hospital Luebeck and the Leibniz Research Center Borstel, Luebeck / Borstel, Germany;_ 5 _University Hospital Zurich, Institute of Surgical Pathology, Zurich, Switzerland;_ 6 _University Hospital Duesseldorf, Women's Hospital, Duesseldorf, Germany;_ 7 _Interfaculty Institute of Biochemistry, University of Tuebingen; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Tuebingen / Heidelberg, Germany;_ 8 _University Hospital Basel, Department of Biomedicine and University Hospital Basel, Clinic for Hematology, Basel, Switzerland_.

Ecotropic viral integration site1 (EVI1) is a transcriptional regulator that is essential for the maintenance of hematopoietic stem cells and, when overexpressed in leukemia, associates with particularly aggressive disease. EVI1 expression has also been detected in a number of solid tumors including breast carcinoma (BC) where it was recently shown to predict poor survival in estrogen receptor negative (ER-) but not positive (ER+) tumors. The functional roles and molecular regulation of EVI1 in human BC are largely unexplored.

Here we confirm that EVI1 is widely expressed in both ER- and ER+ BC in a tissue microarray containing 373 individual patient samples. Unlike in leukemia, overexpression of EVI1 in BC rarely resulted from gene amplification or translocation events at its chromosomal locus 3q26. Functionally, lentiviral knockdown of EVI1 reduced proliferation, motility and in vivo tumorigenicity of human BC cells while enhancing their apoptosis sensitivity. Interestingly, estrogen supplementation rescued growth and tumorigenicity in EVI1-knockdown ER+ (but not ER-) cells. Since microarray analyses of EVI1 knockdown cells revealed alterations of several regulators of cell cycle progression (e.g. CDKN1A, CDKN1C, CDK1), apoptosis (e.g. BIK, BMF, BBC3) and growth factor signaling (e.g. NRG2, EREG, PTK2B), we further investigated the canonical MAPK pathway as a major effector axis of EVI1 in BC. Indeed, knockdown of EVI1 in BC cells effectively impaired ERK1/2 phosphorylation (i.e. MAP kinase activation), which was phenotypically reflected by impaired in vitro proliferation and in vivo tumorigenicity rates. In ER+ BC cells, these effects were functionally restored by exogenous addition of estradiol, explaining why EVI1 gains particular clinical significance in the absence of estrogen signaling (i.e. in ER- BC). Supporting this notion, ectopic expression of EVI1 and estradiol treatment co-induced p-ERK1/2. Our microarray analysis further revealed GPCR signaling molecules (e.g. CCR1, GCGR, PTGFR) as a fourth category of genes significantly modulated by EVI1 in BC. Among these, the GPR54-ligand KISS1, previously implicated in cell migration, was most strongly modulated by EVI1 knockdown and also overexpression. A corresponding promoter analysis indicated several potential EVI1-binding sites within KISS1 regulatory elements, of which two showed robust recovery rates in chromatin immunoprecipitation (ChIP) assays. Functionally, EVI1 knockdown impaired BC cell migration, which was restored by addition of Kisspeptin, a soluble gene product of KISS1.

Overall, our data characterize EVI1 as a novel oncogene in BC that impacts growth and migration via pERK and the GPR54-ligand KISS1, which we identified as a novel direct transcriptional target of EVI1-driven oncogenesis.

#2016

Paired box 2 promotes progression of endometrial cancer via cell cycle pathway.

Jieyu Wang,1 Weiwei Feng,1 Yinhua Yu,1 Kwong-Kwok Wong,2 Nan Jia,1 Tianjiao Lyu1. 1 _obstetrics and gynecology hospital of Fudan university, shanghai, China;_ 2 _Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Human paired box 2 (PAX2) plays a key role in cell fate, early patterning and organogenesis. To investigate the function of PAX2 on the biological behavior of endometrial cancer and the regulation mechanism, stable knocking-down and over-expression PAX2 endometrial cancer cell lines were established. CCK-8 and transwell assays were applied to determine proliferation, invasion and migration ability. Cell cycle distribution was analyzed by flow cytometry. Nude mice were used for in vivo experiments and Affymetrix GeneChip® human Exon 1.0 ST arrays was used to screen the downstream target genes of PAX2. The upstream regulation mechanism of PAX2 was explored by dual luciferase reporter gene assay and bioinformatics analysis.Results showed that,no matter in vivo or in vitro,PAX2 significantly enhanced tumorigenicity and invasiveness of endometrial cancer cells, and promoted liver metastasis in vivo. In addition, PAX2 influenced the expression of cyclin-dependent kinase 1(CDK1) and YWHAZ, which play pivotal roles in cell cycle pathway. Knocking down CDK1 in PAX2 over-expressing cells attenuated cell viability.A myeloid zinc finger gene-1(MZF-1)binding site was found in a negative regulation region of PAX2 upstream and down-regulation of MZF-1 reduced the transcriptional activity of PAX2, as well as its protein level.Together, PAX2, through influencing the activity of CDK1 and YWHAZ, promotes proliferation,invasion and progression of endometrial cancer and can be regulated by MZF-1. PAX2 may be a target therapeutic site for endometrial cancer.

#2017

The CEBPD transcription factor, a marker of good prognosis in breast cancer, becomes a signaling hub for promotion of cancer cell stemness by hypoxia and interleukin-6.

Kuppusamy Balamurugan,1 Daniel Mendoza-Villanueva,1 Barbara Rath,2 Philip Tofilon,2 Shikha Sharan,1 Esta Sterneck1. 1 _NCI-CCR, Frederick, MD;_ 2 _NCI-CCR, Bethesda, MD_.

The transcription factor C/EBPdelta (CEBPD) is expressed in normal mammary epithelial cells and high expression in breast cancer correlates with hormone receptors and good prognosis (Mendoza-Villanueva et al., submitted). In the MMTV-Neu mouse model, deletion of CEBPD increases tumor multiplicity, but "paradoxically" reduces metastatic progression, suggesting that CEBPD has dual tumor suppressing and metastasis promoting functions (Balamurugan et al., 2010). Here, we identify a molecular mechanism for the tumor promoting function of CEBPD that may reach beyond breast cancer. We show that CEBPD serves as a signaling hub that integrates and amplifies pathways that promote stemness in breast cancer, glioblastoma and embryonic cancer cells. This work identifies a mechanism by which hypoxia and inflammation promote cancer stem-like cells (CSCs), which are implicated in tumor progression and resistance. Our earlier studies showed that CEBPD promotes inflammatory signaling and hypoxia adaptation by inhibiting the expression of FBXW7 (Balamurugan et al., 2010 and 2013), which can target several oncoproteins for degradation. Using a variety of established breast and glioblastoma tumor cell lines/xenografts, primary mouse mammary tumor cells and tissues, recent patient-derived glioblastoma cell lines, as well as embryonic stem/cancer cell lines, we find that deletion or depletion of CEBPD reduced the number of stem cells as determined by surface markers, sphere formation, limiting dilution xenografts, and by expression of stemness genes. Mechanistic studies in culture models show that CEBPD is expressed in CSCs and connects and amplifies both hypoxia (HIF-1) and inflammation (IL-6/STAT3) pathways to increase Notch1/NICD expression and cell stemness. CEBPD induces NICD expression transcriptionally through HIF-1 and at the level of the protein by inhibition of FBXW7-mediated degradation. In addition to its known role as a target of STAT3 and activator of IL-6 gene expression, we identified an additional role for CEBPD in promoting expression of the IL-6 receptor. Furthermore, we found that CEBPD also directly activates the expression of stemness-promoting genes such as OCT4, Sox2, KLF4, Myc, Nanog, CD44 and CD133. Taken together, this study provides novel insights into the molecular mechanisms that promote cancer cell stemness. Because CEBPD integrates and amplifies multiple stemness promoting pathways, CEBPD may represent a unique point of vulnerability in cancer stem cells, strongly suggesting that pharmacological inhibition of CEBPD signaling may effectively target CSCs. Results from current efforts to pharmacologically downregulate CEBPD in cancer stem cells will also be presented.

#2018

Investigation of ITAF's role in the pathophysiology of breast cancer.

Jessica Bockhorn,1 Toby M. Ward,1 J. Chuck Harrell,2 Xiaofei Liu,1 Gaby Fuchs,3 Mark D. Pegram1. 1 _Stanford Cancer Institute, Stanford, CA;_ 2 _Virginia Commonwealth University, Richmond, VA;_ 3 _University of Albany, Albany, NY_.

A subset of cellular mRNAs can initiate cap-independent translation via internal ribosome entry sites (IRES) structures, thus maintaining translation when cap-dependent translation is compromised. Many of these IRES harboring mRNAs encode canonical oncoproteins such as c-MYC, VEGF and EGFR. Cells use IRES-mediated translation under conditions that are common to the tumor microenvironment, including nutrient deprivation, hypoxia, inflammation, and DNA damage, leading to the hypothesis that cancer cells use cap-independent translation to survive pro-apoptotic pressure. To investigate this possibility, we profiled IRES trans-activating factors (ITAFs), which are accessory proteins that regulate IRES activity in the cells. ITAFs are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that participate in diverse nuclear functions, including RNA splicing, DNA repair, and transcription. During cap-independent translation, ITAFs egress into the endoplasmic reticulum (ER) to form a complex with the 40S ribosome and other accessory proteins to regulate translation of client IRES harboring mRNAs. We hypothesize that ITAFs could influence the pathophysiology of breast cancer by controlling the translation of onco-and antiapoptotic proteins. In this study, we profiled the expression of two ITAFs, interleukin enhancer binding protein 2 (ILF2) and splicing factor proline/glutamine-rich (SFPQ) in normal and breast cancer specimens, and investigated the biological effects of these ITAFs in both cancer cell lines and preclinical in vivo models using knockout studies. Additionally, we investigated the impact of their subcellular localization in cancer cells and the translation of their client mRNAs under ER stress using immunoblots as well as polysome and immunofluorescence analysis. Finally, we investigated the impact of inhibiting nuclear pore translocation on the function of ITAFs in breast cancer cells, using a selective inhibitor of nuclear export (SINE) compound.

Analysis of microarray datasets from cancer and normal tissue specimens (The Cancer Genome Atlas, METABRIC, and University of North Carolina datasets) revealed that expression of several ITAFs is upregulated in malignant tissues compared to normal controls (p<0.05) and associated with survival outcome (p<0.03). Silencing of ITAFs led to significant proliferation impairment in breast cancer cells in 2-D culture (p<0.001), soft agar assays (p< 0.01) and pre-clinical in vivo models (p<0.05). In cancer cell lines, treatment with an ER stressor led ITAFs to egress to the ER, with increased translation of client IRES-harboring mRNAs despite impaired global cap-dependent translation. Finally, the SINE compound prevented ITAFs from translocating to the ER and decreased translation of client mRNAs. These data suggest that ITAFs impact the pathophysiology of breast cancer, and may represent novel therapeutic or diagnostic targets.

#2019

SOX2 silencing upregulates CDKN1A and suppresses growth of lung squamous cell carcinoma.

Takuya Fukazawa,1 Tomokoki Yamatsuji,1 Munenori Takaoka,1 Etsuko Yokota,1 Minoru Haisa,1 Naomasa Ishida,1 Masakazu Yoshida,1 Noriko Miyake,2 Nagio Takigawa,1 Yutaka Maeda,3 Yoshio Naomoto1. 1 _Kawasaki Medical School, Okayama, Japan;_ 2 _Kawasaki Hospital Research Unit, Okayama, Japan;_ 3 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH_.

SOX2 is a master pluripotency controller that was recently identified as a novel major oncogene, recurrently amplified and activated in lung squamous cell carcinoma (lung SCC). Then, transcriptional downstream targets of SOX2 have been actively investigated; however, such targets are often cell line specific. Here, in order to identify highly consensus SOX2 downstream genes in lung SCC cells, we used RNA-seq data from 178 lung SCC specimens (containing tumor and tumor-associated cells) and analyzed the correlation between SOX2 and previously-reported SOX2-controlled genes in lung SCC. In addition, we used another RNA-seq dataset from 105 non-small cell lung cancer cell lines (NSCLC; including lung SCC cells) and again analyzed the correlation between SOX2 and the reported SOX2-controlled genes in the NSCLC cell lines (no tumor-associated cells). We combined the two analyses and identified genes commonly correlated with SOX2 in both datasets. Among the 99 genes reported as SOX2 downstream and/or correlated genes, we found 4 negatively-correlated (e.g., CDKN1A) and 11 positively-correlated genes with SOX2. We used biological studies to demonstrate that CDKN1A was suppressed by SOX2 in lung SCC cells. G1 cell cycle arrest induced by SOX2 siRNA was rescued by CDKN1A siRNA. These results indicate that the tumorigenic effect of SOX2 in lung SCC cells is mediated in part by suppression of CDKN1A.

#2020

Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer.

Susheel K. Khetarpal. _Carnegie Mellon University, Pittsburgh, PA_.

Lung cancer is the leading cause of cancer death in the United States and in the world. Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1, in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce either oncogene induced senescence or apoptosis. However, the key functions of TWIST1 that are required for its transcriptional activity in cancer are unknown.

In the current study, we engineered domain-specific mutations in TWIST1 to determine the impact of altered DNA binding, dimerization and post-translational modifications on its transcriptional activity by luciferase reporter assays. Testing the promoter activity at three TWIST1-regulated loci involved in tumorigenesis, YBX1, SNAI2, and FLIP, we found that DNA binding and nuclear localization were uniformly required for TWIST1 transcriptional function. However, phosphorylation, TWIST1 box function, and ability to form homo- versus hetero- dimers impacted TWIST1 activity in a locus-specific manner. Previous studies have demonstrated that TWIST1 dimerizes with the E2A proteins, E12 and E47. We have shown that silencing of E2A phenocopies loss of TWIST1 in NSCLC and that formation of the TWIST1-E2A heterodimer results in a reciprocal stabilization of the binding partner. Therefore, we sought to determine how differential dimerization by TWIST1 might modulate tumorigenic gene expression. We utilized tethered TWIST1 dimers with either TWIST1 or the E2A proteins to form TWIST1-TWIST1 (homodimer), TWIST1-E12 or -E47 (heterodimers), and tested their transcriptional activities at several TWIST1-regulated promoters. We found that TWIST1-E2A heterodimers could enhance TWIST1 transcriptional activity compared to that of the TWIST-TWIST homodimer. In addition, we determined that the TWIST-E2A heterodimer is degraded by harmine, a harmala alkaloid that we have shown degrades TWIST1, whereas the TWIST-TWIST homodimer is resistant to harmine degradation. This suggests that the TWIST1-E2A heterodimer is the key target of harmine. Furthermore, we have found that overexpression of the TWIST1, E2A proteins, or the TWIST1-E2A heterodimer is able to partially rescue harmine induced cytotoxicity in KRAS mutant NSCLC. Taken together, these data suggest that E2A is essential for TWIST1 mediated tumorigenesis and that targeting of the TWIST1-E2A axis may be an effective therapeutic strategy against oncogene-driven NSCLC.

#2021

Identification of novel brachyury target genes in lung cancer.

Yunping Hu, Wesley Hsu. _Wake Forest University School of Medicine, Winston Salem, NC_.

Lung cancer is the leading cause of cancer-related death worldwide. Previous studies have shown that the T-box transcription factor brachyury is commonly expressed in advanced lung cancer and is associated with a poor prognosis. The activation of brachyury promotes lung cancer progression, aggressiveness and resistance to conventional antitumor therapies. However, the mechanism by which brachyury drives lung cancer is unknown. Here we have sought to identify novel gene targets of brachyury to better understand the molecular pathogenesis of lung cancer. We used a bioinformatics approach and found a shortlist of putative potential brachyury target genes. We subsequently explored the modulation of candidate brachyury target genes in lung cancer. RNA interference analysis for lung cancer cell line H460 overexpressing brachyury demonstrated that fibroblast growth factor 8 (FGF8), MAS1 Proto-Oncogene G Protein-Coupled Receptor (MAS1) and Ran GTPase Activating Protein 1 (RANGAP1) played essential roles in cell growth, whereas transmembrane protein 37 (TMEM37) was involved in cell migration and invasion. ChIP-qPCR experiments confirmed the strong binding of brachyury to these functional target genes. Analysis of datasets derived from patients with lung cancer and a panel of lung cancer cell lines revealed that brachyury expression was positively correlated with FGF8, MAS1, RANGAP1 and TMEM37. In addition, the expression levels of FGF8, MAS1 and RANGAP1 were significantly associated with non-metastasis (early-stage and locally advanced lung cancer), whereas TMEM37 was associated with metastasis. Taken together, our findings suggest that brachyury may influence lung cancer progression and/or metastasis via direct regulation of FGF8, MAS1, RANGAP1 and TMEM37 expression. Identification of potential brachyury transcriptional targets provides novel insights into the molecular pathobiology of lung cancer.

#2022

Twist1 regulates UVB-induced epidermal cell proliferation in non-melanoma skin cancer.

Jaya Srivastava, Okkyung Rho, John DiGiovanni. _University of Texas at Austin, Austin, TX_.

Nonmelanoma skin cancer (NMSC) is the most common cancer and consists of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). SCC metastases account for up to 20% of all deaths from skin cancer. The major risk factor for the development of NMSC is solar ultraviolet radiation (UV). UVA and UVB both function as initiators and promoters in carcinogenesis and possess immunosuppressive activity. Given the prevalence of this disease, there remains a pressing need for the development of novel prevention and treatment strategies for NMSC.

In a recent study (Srivastava et al. 2015. Mol. Carcinog. doi:10.1002/mc.22335), we evaluated the effect of Twist1, a transcription factor that is known to play a role in tumor progression, during epithelial carcinogenesis. Using the two-stage chemical carcinogenesis model, we identified that BK5.Cre x Twist1flox/flox mice, which have a keratinocyte-specific Twist1 knockout (Twist1 KO), developed significantly reduced numbers of papillomas (70% reduction) and SCCs (75% reduction) as well as delayed tumor latency compared to wild-type (WT) mice. Twist1 KO mice also exhibited significantly reduced epidermal proliferation in response to chemical promotion that correlated with reduced levels of cell cycle regulators and increased levels of p53 and p21.

In further studies, we have begun analyzing the effects of UVB irradiation in Twist1 KO mice. Specifically, Twist1 KO mice exhibited heightened sensitivity to UVB-induced apoptosis and decreased epidermal proliferation as compared to similarly exposed WT mice. In addition, the accumulation of mast cells in the skin was significantly decreased in Twist1 KO mice as compared to WT mice. Further preliminary experiments showed that the protein expression levels of c-Myc, phospho-p65 (S536) and phospho-p50 (S337) decreased following UVB irradiation whereas levels of the cell cycle inhibitors p21 and p27 exhibited an increased expression in Twist1 KO mice as compared to WT mice. Further experiments have suggested that Twist1 may play a pivotal role in the proliferation/migration of bulge region keratinocyte stem cells (KSCs) following exposure to UVB. These and other results from ongoing experiments will be presented. Collectively, these findings suggest that Twist1 may have a novel role in UVB-induced epithelial carcinogenesis by regulating proliferation and migration of keratinocytes and KSCs. Research supported by CPRIT RP150346, CPRIT RP140108, and NIH T32 ES007247.

#2023

SOX4 is essential for PTEN-mediated prostate tumorigenesis in vivo.

Birdal Bilir,1 Adeboye O. Osunkoya,1 W. Guy Wiles,1 Soma Sannigrahi,1 Veronique Lefebvre,2 Daniel Metzger,3 Demetri D. Spyropoulos,4 W. David Martin,1 Carlos S. Moreno1. 1 _Emory University, Atlanta, GA;_ 2 _Cleveland Clinic, Cleveland, OH;_ 3 _Université de Strasbourg, Illkirch, France;_ 4 _Medical University of South Carolina, Charleston, SC_.

Prostate cancer is the most common cancer and the second leading cause of cancer mortality in American men, underscoring the significance of unraveling the molecular mechanisms involved in the initiation and progression of the disease. The sex-determining region Y-box 4 (SOX4) gene is overexpressed in many types of human cancers, including prostate cancer, suggesting that SOX4 plays a fundamental role in tumorigenesis. In this study, we demonstrate that SOX4 is critical for PTEN-mediated prostate cancer progression in vivo. We show that homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibits tumor progression initiated by homozygous loss of the Pten tumor suppressor, demonstrating the key role of SOX4 in the development of prostate cancer. Homozygous deletion of Sox4 also reduces the activation of AKT and β-catenin in Pten-null mice, resulting in inhibition of an invasive cancer phenotype. We also show that SOX4 expression is induced by loss of PTEN, and that PI3K-AKT-mTOR signaling activity is critical for SOX4 expression, suggesting a positive feedback loop between SOX4 protein and PI3K-AKT-mTOR activity. Our findings indicate that SOX4 is a critical component of the PTEN-PI3K-AKT pathway in prostate cancer, suggesting that SOX4 may be a promising molecular target for novel combinatorial therapies for both primary and advanced prostate cancers.

#2024

BP1 homeoprotein induces tumor cell growth and estrogen-independent tumorigenesis in mice.

Sidney W. Fu,1 Saurabh Kirolikar,1 Erika Ginsburg,2 Arnold Schwartz,1 Samuel Simmens,1 Patricia E. Berg1. 1 _George Washington University School of Medicine and Health Sciences, Washington, DC;_ 2 _National Cancer Institute, National Institutes of Health, Bethesda, MD_.

BACKGROUND. BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 overexpression increases cell survival, aggressiveness and metastasis in breast cancer cells. BP1 protein is expressed in 80% of invasive ductal breast tumors, including 100% of ER negative (ER-) tumors and 73% of ER positive (ER+) tumors. Therefore we hypothesize that BP1 may regulate ER in breast cancer. A mouse model was developed to test the regulatory effects of BP1 in ER expression. MATERIALS AND METHODS. MCF-7 breast cancer cells transfected with BP1 or vector control were njected into the fat pads of nude mice. Regulation of ER by BP1 was studied using ChIP assays; EMSA assays were used to test binding of BP1 to ER DNA. Tamoxifen assays were used to evaluate the effects of BP1 overexpression on tamoxifen resistance. RESULTS. MCF-7 cells overexpressing BP1 or control cells containing an empty vector were injected into nude mice. Approximately half of the estrogen-supplemented mice developed tumors. Although MCF-7 cells require exogenous estrogen to form tumors in nude mice, approximately 20% of the mice implanted with cells overexpressing BP1 were able to form tumors in the absence of added estrogen. To understand the molecular mechanisms of this observation, we carried out in vitro experiments. Our data suggest that BP1 regulates ER both directly and indirectly: (1) Direct: ChIP assays and EMSA assays showed that BP1 binds to the first intron of the ER alpha gene and transcriptionally activates it, leading to increased ER protein. (2) Indirect: regulation of ER also occurs indirectly by BP1 upregulation of p300, an activator of ER, and also by repression of BRCA1 by BP1; BRCA1 destabilizes ER protein. Furthermore, MCF-7 cells overexpressing BP1 are more resistant to growth in 3uM tamoxifen compared with empty vector containing cells. CONCLUSIONS. BP1 overexpression in MCF-7 cells injected into mice not only increases tumor size but also promotes estrogen-independent tumorigenesis, and leads to resistance to tamoxifen. Therefore, BP1 could serve as a novel therapeutic target for ER+ breast cancer.

#2025

SIX1 overexpression extends the lifespan of normal human keratinocytes and promotes epithelial-mesenchymal transition.

Maria Hosseinipour,1 Aspasia Amiridis,2 Diego Altomare,2 Kim E. Creek,2 Lucia Pirisi1. 1 _University of South Carolina School of Medicine, Columbia, SC;_ 2 _University of South Carolina, Columbia, SC_.

SIX1, a homeodomain-containing transcription factor, has a critical role in the expansion of progenitor cells during embryogenesis. The overexpression of SIX1 contributes to tumorigenesis, promoting malignant transformation and metastasis. Six1 promotes tumor progression by induction of epithelial-to-mesenchymal transition (EMT). In HPV16-transformed human keratinocytes (HKc), SIX1 overexpression produces EMT and a differentiation-resistant phenotype at early stages of in-vitro progression, and transforms cells to malignancy at late stages. We transfected normal HKc with a plasmid encoding human-SIX1 in order to determine to what extent SIX1 overexpression may induce growth, differentiation and EMT changes in normal cells. We also studied the effects of expressing the HRas-V12 oncogene while downregulating the p53 tumor suppressor gene in normal HKc expressing SIX1, in order to push these keratinocytes to a precancerous state. Normal HKc overexpressing SIX1 (HKc/SIX1) or a combination of SIX1, HRas-V12 and p53i (HKc/ALL3) grow continuously while their vector-transfected controls senesced. Continuous growth past the end of the normal lifespan was also observed in normal human fibroblasts overexpressing SIX1. HKc/SIX1 and HKc/ALL3 exhibit a spindle-shape and fibroblastic appearance rather than the cuboidal morphology of epithelial cells. They are also morphologically distinct from SIX1-overexpressing human fibroblasts. HKc /SIX1 and HKc/ALL3 grow best in KSFM supplemented with 10% FBS and 1mM CaCl2, while normal HKc grow best in KSFM with 0.1 mM CaCl2. RT-PCR confirmed that mRNA levels of SIX1 were significantly increased and p53 levels were significantly decreased, and that HRasV12 was expressed in HKc/ALL3. The growth rates of HKc/SIX1 and HKc/ALL3 are comparable to those of early-stage HPV16-immoratlized cells (HKc/HPV16). Invasion assays demonstrated that HKc/SIX1 are more invasive than normal HKc, and their invasiveness is comparable to that of HKc/HPV16. The invasiveness of HKc/ALL3 is greater than that of late-stage, differentiation-resistant HPV-immortalized cells (HKc/DR), which are about 2.5-fold more invasive than HKc/HPV16. In conclusion, SIX1 promotes continued proliferation and invasiveness, signs indicative of cellular transformation, even in the background of normal HKc. Currently, we are exploring the gene expression changes induced by SIX1 in normal cells. Additionally, we plan to investigate whether these cells express stem cell markers. The results of these studies suggest that SIX1 facilitates the establishment of primary human keratinocytes into immortal cell lines, or perhaps into stem-like cells with extended proliferation potential.

#2026

DEC1 negatively regulates AMPK activity via LKB1.

Fuyuki Sato, Yasuteru Muragaki. _Wakayama Medical University, Wakayama city, Japan_.

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in metabolism and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, how the circadian rhythm of AMPK activity is regulated is not known. The aim of this study is the direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. A medium change and serum shock led to rhythmic patterns of DEC1 protein and AMPK activity levels in WI-38 cells, and their peak levels occurred at different times. However, changing the medium did not induce circadian rhythms of DEC1 protein or AMPK activity levels in MCF-7 and U2OS cells, in which the intensities of these levels had inversely correlated patterns. Knocking down DEC1 expression increased AMPK activity, whereas DEC1 overexpression decreased it. DEC1 bound to the E-box of the LKB1 promoter, decreasing the LKB1 activity and total protein levels. In addition, knocking down DEC1 expression inhibited cell death under the condition of glucose depletion, increasing the activity of both AMPK and LKB1. Taken together, our results showed that the levels of DEC1 protein and AMPK activity were affected even long after the medium change and that the rhythmic patterns of these levels were inversely correlated. We concluded that DEC1 negatively regulated AMPK activity via LKB1.

#2027

Global analyses of HOXB13-regulated transcription reveal a potential link between HOXB13 G84E and prostate cancer risk.

Dorhyun Johng, Michael C. Haffner, Steven M. Mooney, David M. Esopi, Charles M. Ewing, Shuangling Chen, William B. Isaacs. _Johns Hopkins University, Baltimore, MD_.

Prostate cancer (PCa) is one of the most heritable cancers. However, germline genetic variations that are consistently and highly associated with PCa have remained elusive. Recently, a variety of non-synonymous SNPs in HOXB13 have been identified in prostate cancer patients from distinct ethnic populations. Among the HOXB13 variants, HOXB13 G84E has been consistently shown to associate strongly with increased PCa risk in men of European descent. HOXB13 is a prostate-specific transcription factor that plays a role in prostate development. HOXB13 has also been implicated in various aspects of PCa biology. However, to this date, the exact role of HOXB13 in normal prostate physiology and in PCa biology remains obscure. Our lab previously reported that neither HOXB13 WT nor G84E alone can transform prostate cells and that the G84E variant does not behave differently from HOXB13 WT in interactions with cofactors (AR, MEIS2) and protein half-life. To further delineate the function of HOXB13 in the prostate and to identify G84E-induced alterations that subject G84E carriers to PCa susceptibility, we performed RNA-seq of the LAPC4 prostate cancer cell line overexpressing a control vector, HOXB13 WT or G84E. Comparisons of the RNA-seq datasets were made using Ingenuity Pathway Analysis (IPA) by selecting genes whose expression was altered by 2 standard deviations or higher. Among pathways altered by HOXB13 WT or G84E compared to the vector control dataset, the VDR/RXR activation pathway was negatively regulated by both WT and G84E. When the G84E dataset was compared to the WT dataset, the IPA analysis revealed that G84E can up-regulate the e-NOS signaling pathway. Interestingly, the top contributor to the difference imparted by HOXB13 G84E compared to WT was identified to be a set of genes regulated by HOXB13 itself. Overall, this preliminary analysis suggests that HOXB13 G84E may be a gain-of-function mutation whereby potential tumor-promoting functions of HOXB13 are over-activated. Finally, to further identify targets directly regulated by HOXB13 WT and G84E, we combined the RNA-seq datasets with HOXB13 overexpression ChIP-seq datasets we previously reported and a HOXB13 knockdown dataset in LAPC4 (Norris et al. 2009). We focused initially on genes that were shown to be up- or down-regulated in the same direction in the RNA-seq and HOXB13 knockdown datasets. Those genes were screened for nearby HOXB13 binding sites as annotated in our ChIP-seq analyses, followed by validation with droplet digital PCR. Among the genes that were down-regulated by HOXB13 WT and G84E include INPP4B, TNFSF10, IGFBP3, KLF5 and SOX9, which all have tumor suppressing functions in PCa. Among HOXB13-upregulated genes was BCHE, which is also implicated in the suppression of PCa initiation and progression. These results provide us with potential areas of further investigation in which HOXB13 G84E may differentially regulate transcription of these genes.

#2028

Transcriptional modules involving EWS-FLI1 and SRF in Ewing sarcoma.

Anna M. Katschnig,1 Maximilian O. Kauer,1 Dave N t Aryee,1 Raphaela Schwentner,1 Elizabeth Lawlor,2 Heinrich Kovar1. 1 _Children's Cancer Research Institute, Vienna, Austria;_ 2 _Department of Pediatrics, University of Michigan, MI_.

Ewing Sarcoma (ES) is an aggressive pediatric bone tumor with a high tendency to metastasize. The primary genetic aberration responsible for ES pathogenesis is EWS-FLI1, a chimeric gene, derived from the chromosomal translocation t(11;22)(q24;q12). EWS-FLI1 encodes an aberrant ETS transcription factor. However, due to its lack of enzymatic activity and intrinsically disordered structure it has yet not been successfully targeted. EWS-FLI1 is prone to rapidly engage in protein-protein complexes, enhancing its oncogenic activity. It has been reported to be involved in interactions with regulators of the basal transcriptional machinery, RNA processing and DNA repair. Furthermore, it has been shown that, similar to the ternary complex factors (TCF) SAP1a and ELK1, EWS-FLI1 is able to form a complex with the global transcriptional regulator Serum Response Factor (SRF) on ETS containing promoter elements. SRF binds to CArG motifs in serum response elements (SRE) and requires a transcriptional co-activator, which is commonly provided upstream via the Rho- or the Ras-axis. Upon serum induced Rho activation, polymerization of globular (G-) actin into filamentous (F-) actin takes place, thereby releasing the G-actin bound myocardin-related transcription factors A and B (MRTFA/B) to translocate to the nucleus and concertedly with SRF regulate transcription of target genes. Binding of MRTFs to SRF is mutually exclusive and competed by TCFs, which are activated downstream of Ras, and require presence of an ETS motif adjacent to CArG. In this study we aim to investigate a potential competition of EWS-FLI1 with MRTFs, which might uncouple SRF dependent transcription from upstream G-protein linked regulation and hence interfere with processes such as proliferation, migration and differentiation. To address this question, we performed gene expression profiling in A673 and SKNMC ES cell lines where we combined EWS-FLI1 knockdown with MRTFA/B knockdown under serum starved and serum induced (SI) conditions. We found that transcriptional activity of MRTFA/B is largely repressed by EWS-FLI1 and that upon combined knockdown conditions, EWS-FLI1 transcriptional effects are rescued by MRTFA/B. To further elucidate the underlying mechanisms of EWS-FLI1 and MRTFA/B gene regulation, we performed chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-seq) of MRTFA, MRTFB, SRF and EWS-FLI1 in A673 ES cells. Preliminary results corroborate EWS-FLI1 and MRTF sharing a large number of target genes and indicate that MRTFA binds to DNA motifs, which are associated with activated and repressive EWS-FLI1 signatures. Ongoing experiments aim at a proteomic understanding of the EWS-FLI1 containing SRF complex as a basis for potential therapeutic intervention.

This study was supported by the Liddy Shriver Sarcoma Initiative and the 7th framework program of the European Commission (FP7-259348, "ASSET"). 

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Clinical Pharmacology and Pharmacogenomics

#2029

A high ratio of tamoxifen metabolite E to tamoxifen is associated with an increased risk of breast cancer recurrences in premenopausal women.

Janina Johänning,1 Diana M. Eccles,2 Bryony Eccles,2 Michel Eichelbaum,1 Matthias Schwab,3 Hiltrud Brauch,1 Thomas Mürdter,1 Werner Schroth1. 1 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart and University of Tuebingen, Tuebingen, Germany;_ 2 _Faculty of Medicine, Cancer Sciences Academic Unit and University of Southampton Clinical Trials Unit, University of Southampton, Southampton, United Kingdom;_ 3 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart and Department of Clinical Pharmacology, Institute of Experimental and Clinical Pharmacology and Toxicology, University Hospital Tuebingen, Tuebingen, Germany_.

Tamoxifen (Tam), a standard therapy for estrogen receptor (ER)-positive breast cancer, is excessively metabolized mainly by enzymes of the CYP450 family to anti-estrogenic and estrogenic compounds. Whereas in breast cancer cells the anti-estrogenic metabolites endoxifen and 4-hydroxy-Tam inhibit the ER, Tam bisphenol (Bis) and both isomers (E)- and (Z)- of metabolite E (Met E) show full estrogenic properties, with (E)-Met E being the most potent ER agonist. Contrary to known clinical effects of ER antagonists, the influence of Tam Bis, (E)-, and (Z)-Met E on treatment outcome is not thoroughly explored. Therefore we analyzed whether interindividual differences in plasma levels of estrogenic Tam metabolites interact with Tam therapy in vivo.

Plasma concentrations of estrogenic metabolites Bis, (E)-, and (Z)-Met E and their metabolic ratios (MR) to Tam were quantified in 306 premenopausal breast cancer patients mainly of European origin who were treated with 20 mg/day of Tam (Eccles D, et al. BMC Cancer 2007; 7: 160) using recently published LC-MS/MS methods (Johänning et al. Anal Bioanal Chem 2015; Mürdter et al. Clin Pharmacol Ther 2011). The endpoints recurrence-free interval (RFI) and event-free survival (EFS) were analyzed in hormone-receptor positive patients (N=296) by Kaplan-Meyer and multivariate Cox regression adjusted for age, nodal status, tumor size, grade and plasma concentrations of endoxifen (as MR of desmethyl-Tam to endoxifen).

Plasma concentrations were as follows: Bis 13-832 pM (median 187 pM), (E)-Met E 15-1029 pM (median 213 pM), (Z)-Met E 128-2484 pM (median 889 pM), and Tam 47-1061 nM (median 360 nM). Patients with higher (E)-Met E to Tam ratios showed a higher risk of breast cancer recurrence when classified into quartiles: compared to patients of the lower quartile, patients had shorter RFI when they grouped to the interquartile (P = 0.037) and the upper quartile (P = 0.005). There was a trend of higher recurrence risk for patients belonging to the upper quartile compared to the interquartile (P = 0.063). These findings were confirmed in multivariate Cox analyses: patients belonging to the upper quartile had a significantly increased hazard ratio (HR) of 2.77 (95% confidence interval (CI) 1.34-5.71; P = 0.006) and patients belonging to the interquartile showed a trend for increased HR of 1.75 (CI = 0.88-3.46; P = 0.1). For EFS, a linearly increased HR was confirmed for patients with higher (E)-Met E to Tam ratios (HR = 1.44, CI = 1.06-1.97; P = 0.021). Bis, (Z)-Met E and their MRs to Tam showed no significantly altered risk of cancer recurrence or death.

These findings suggest that higher formation rates of the most potent estrogenic Tam metabolite (E)-Met E influence Tam outcome in breast cancer patients and may be linked to therapeutic failure.

#2030

The role of genetic variation in calcium-activated potassium channels in breast cancer patients treated with tamoxifen.

Wing-Yee Lo,1 Corinna Mohr,2 Friederike Steudel,2 Marjanka Schmidt,3 Douglas Easton,4 Reiner Hoppe,1 Werner Schroth,1 Peter Ruth,2 Robert Lukowski,2 Hiltrud Brauch,5 in collaboration with the Breast Cancer Association Consortium. 1 _Dr. Margarete Fischer-Bosch - Institute of Clinical Pharmacology, Stuttgart and University of Tuebingen, Tuebingen, Germany;_ 2 _Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tuebingen, Tuebingen, Germany;_ 3 _Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam, Netherlands;_ 4 _Centre for Cancer Genetic Epidemiology, University of Cambridge, Cambridge, United Kingdom;_ 5 _Dr. Margarete Fischer-Bosch - Institute of Clinical Pharmacology, Stuttgart and University of Tuebingen, Tuebingen and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Potassium channels control membrane voltage and are essential for cell proliferation. Abnormal regulation and/or expression of these channels have been reported in cancer and result in dysregulated cell cycle progression, cell proliferation and migration. In breast cancer, clinical correlations of potassium channels have been associated with brain metastases, high stage, high grade, high proliferation, nodal status and poor prognosis. Interestingly, they have been associated with increased estrogen receptor (ER) expression. The selective ER modulator, tamoxifen, blocks estrogen from binding to the ER and is commonly used to treat patients with ER-positive breast cancer. However, up to 33% of patients treated with tamoxifen relapse or dies at 15 years of follow up. Both estrogen and tamoxifen have been demonstrated to activate potassium channels which then cause an increase in cell proliferation. In preliminary animal in vivo studies, we observed a better breast cancer prognosis in those with a knockout in potassium channel genes. Therefore, any alterations in the potassium channels may help to explain why patients treated with tamoxifen fail therapy. We hypothesized that genetic variation in potassium channels may affect breast cancer risk as well as tamoxifen treatment outcome. Genotyping was conducted on 45,290 breast cancer patients and 41,880 controls of European ancestry from 41 studies with the custom Illumina Infinium array (iCOGS). More than 200,000 SNPs were genotyped with over 11 million SNPs estimated using genotype imputation with SHAPEIT and IMPUTEv2 and the 1000 Genomes Project March 2012 release as the reference panel (Michailidou et al. Nature Genetics 2015). Variants from 13 potassium channels including KCNMA1, KCNMB1-4, KCNN1-4, LRRC26, LRRC38, LRRC52 and LRRC55 were analyzed. Of these, 3 imputed SNPs from KCNN4 have shown decreased breast cancer risk with GWAS significance (rs12463319, rs12609846, rs1685191; OR 0.94, P < 1.0E-08). In particular, these SNPs were significant in ER-positive breast cancer (OR 0.93, P < 1.0E-09), but was not observed in ER-negative patients (P > 0.05). These findings encouraged us to now further investigate the outcomes of patients with ER-positive breast cancer treated with tamoxifen. Survival data for 49,751 patients with a median follow-up of 8 years has been completed and released in November 2015. Of these, 32,571 were ER-positive and 10,383 have been treated with tamoxifen. Current efforts are underway in order to determine the effects of genetic variations in potassium channels on the survival of patients treated with tamoxifen. Our work will provide a better understanding of the potential clinical relevance of potassium channels in tamoxifen treatment in breast cancer.

#2031

Association between CYP2D6 genotype and response to tamoxifen in a prospective multicenter study in Japan.

Hitoshi Zembutsu,1 Seigo Nakamura,2 Sadako Akashi-Tanaka,2 Takashi Kuwayama,2 Chie Watanabe,2 Tomoko Takamaru,2 Hiroyuki Takei,3 Kana Miyahara,3 Hiroshi Matsumoto,4 Yoshie Hasegawa,5 Goro Kutomi,6 Hiroaki Shima,6 Fukino Satomi,6 Hideki Maeda,6 Minoru Okazaki,7 Hisamitsu Zaha,8 Mai Onomura,8 Ayami Matsukata,9 Yasuaki Sagara,9 Shinichi Baba,9 Akimitsu Yamada,10 Kazuhiro Shimada,10 Daisuke Shimizu,10 Koichiro Tsugawa,11 Arata Shimo,11 Tan Ern Yu,12 Mikael Hartman,13 Chan Ching Wang,13 Soo Chin Lee,13 Yusuke Nakamura14. 1 _National Cancer Center, Research Institute, Tokyo, Japan;_ 2 _Department of Breast Surgery, Showa University, Tokyo, Japan;_ 3 _Tokyo Medical University, Tokyo, Japan;_ 4 _Department of Breast Surgery, Saitama Cancer Center, Saitama, Japan;_ 5 _Department of Breast Surgery, Hirosaki Municipal Hospital, Hirosaki, Japan;_ 6 _1st Department of Surgery, Sapporo Medical University, Sapporo, Japan;_ 7 _Department of Surgery, Sapporo Breast Surgical Clinic, Sapporo, Japan;_ 8 _Nakagami Hospital, Okinawa, Japan;_ 9 _Sagara Hospital, Kagoshima, Japan;_ 10 _Department of Breast and Thyroid Surgery, Yokohama City University Medical Center, Yokohama, Japan;_ 11 _Department of Breast and Endocrine Surgery, St. Marianna University School of Medicine Hospital, Kawasaki, Japan;_ 12 _Tan Tock Seng Hospital, Singapore, Singapore;_ 13 _National University of Singapore, Singapore, Singapore;_ 14 _Department of Medicine and Surgery, The University of Chicago, Chicago, IL_.

Purpose:

CYP2D6 is key enzyme responsible for the generation of the potent active metabolite of tamoxifen, "endoxifen". We previously reported that reduced- or null-function alleles of CYP2D6 were significantly associated with poor clinical outcome of breast cancer patients treated with tamoxifen. However, there are still discrepant reports questioning the association between CYP2D6 genotype and tamoxifen efficacy. Hence, we carried out prospective multicenter studies to evaluate the value of CYP2D6 genotyping in tamoxifen therapy.

Patients and Methods:

We studied 279 patients with hormone receptor-positive and Her-2 negative, invasive breast cancer receiving preoperative tamoxifen monotherapy for 14 - 28 days. Ki-67 response in breast cancer tissues after tamoxifen therapy was used as a surrogate marker of response to tamoxifen. We investigated the effects of allelic variants of CYP2D6 on Ki-67 change in breast cancer tissues, histological response, breast conservative operation and hot flash.

Results:

Ki-67 labeling index in breast cancer tissues significantly decreased after preoperative tamoxifen monotherapy for 14-28 days (P =0.00000000024). Moreover, proportion of estrogen receptor positive cells in breast cancer tissues were significantly associated with Ki-67 change after tamoxifen therapy (P =0.0099). CYP2D6 variants were not significantly associated with histological response, breast conservative operation and hot flash (P = 0.25, P = 0.28 and P =0.34, respectively). However, CYP2D6 variants were significantly associated with Ki-67 decrease after the preoperative tamoxifen therapy (P = 0.000014; in patients with two variant alleles v patients carrying one or two wild-type alleles).

Conclusion:

Our result suggest that genetic variation in CYP2D6 is a key predictor for the prognosis of patients with breast cancer treated with tamoxifen.

#2032

Germline genetic variants in GATA3 and breast cancer treatment outcomes in SWOG 8897 trial.

Victoria L. Larsen,1 William E. Barlow,2 Jun J. Yang,3 Qianqian Zhu,4 Laura F. Hutchins,5 Susan A. Kadlubar,5 Kathy S. Albain,6 Robert B. Livingston,7 James M. Rae,8 I-Tien Yeh,9 Peter M. Ravdin,9 Silvana Martino,10 Alan P. Lyss,11 C. Kent Osborne,12 Gabriel N. Hortobagyi,13 Daniel F. Hayes,8 Christine B. Ambrosone,1 Song Yao1. 1 _Roswell Park Cancer Institute, Department of Cancer Prevention and Control, Buffalo, NY;_ 2 _SWOG Statistical Center, Seattle, WA;_ 3 _St. Jude Children's Research Hospital, Pharmaceutical Science, Memphis, TN;_ 4 _Roswell Park Cancer Institute, Department of Biostatistics and Bioinformatics, Buffalo, NY;_ 5 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 6 _Loyola University Chicago Stritch School of Medicine, Maywood, IL;_ 7 _University of Arizona Cancer Center, Tucson, AZ;_ 8 _University of Michigan Comprehensive Cancer Center, Ann Arbor, MI;_ 9 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 10 _The Angeles Clinic and Research Institute, Santa Monica, CA;_ 11 _Heartland NCORP, Missouri Baptist Medical Center, St. Louis, MO;_ 12 _Baylor College of Medicine, Houston, TX;_ 13 _University of Texas M.D. Anderson Cancer Center, Houston, TX_.

Purpose: GATA3 is involved in estrogen signaling and mammary cell differentiation, and is frequently mutated in breast cancer. Germline variations of GATA3 are prognostic in childhood acute lymphoblastic leukemia (ALL). Thus, we sought to examine their prognostic and predictive role in breast cancer.

Methods: SWOG S8897 comprised two groups of breast cancer patients. In the first high-risk group, women were randomly assigned to CAF vs. CMF, and secondarily randomized to tamoxifen or not. In the second low risk group, women did not receive any adjuvant therapy after surgery. Germline DNA was extracted from uninvolved axillary lymph nodes and 12 candidate GATA3 SNPs were genotyped. Associations of genotypes with treatment outcomes were evaluated in 441 women in the treated and 799 in the untreated groups separately. Hazard ratio (HR) for disease free (DFS) and overall survival (OS) for each SNP was estimated using multivariate Cox hazard regression. Further stratified analyses by tamoxifen were performed in the treated group.

Results: After correcting for multiple testing, we identified significant associations of two variants (rs3802604 and rs568727) with DFS and OS for patients who received adjuvant chemotherapy. Women carrying the variant GG genotype at rs3802604 had significantly poorer DFS (HR=1.95, 95% CI: 1.27-2.99, p=0.002) and OS (HR=2.45, 95% CI: 1.48-4.05, p=0.0005), compared to women carrying the common AA genotype. Associations of similar magnitude were found for rs568727. In contrast, no association with either SNP was found in the untreated group. Subgroup analysis suggested that these two SNPs more strongly influenced treatment outcomes in the patients who also received tamoxifen. Seven additional variants were also associated with OS and DFS in the tamoxifen treated group. Only 3 of the 12 SNPs analyzed in GATA3 were not associated with survival in this subgroup; however, these three SNPs (rs3781093, rs3824662, and rs3802600) associated with OS in the group not treated with tamoxifen. Functional annotation revealed that several GATA3 SNPs, e.g., rs3802604, rs369421, and rs3824662, are likely to function by affecting transcription factor binding and/or altering regulatory chromatin states at this locus.

Conclusions: GATA3 harbors common germline genetic variants that are related to survival following adjuvant chemotherapy and endocrine therapy in breast cancer patients.

#2034

Personalized chemotherapy regimen selection for breast cancer.

Jinfeng Zhang,1 Kaixian Yu,1 Qingxiang Amy Sang,1 Winston Tan,2 Mayassa B. Dargham,1 Jun S. Liu,3 Ty Lively,1 Cedric Sheffield1. 1 _Florida State University, Tallahassee, FL;_ 2 _Mayo Clinic, Jacksonville, FL;_ 3 _Harvard University, Cambridge, MA_.

Despite the rapid progress in personalized cancer therapy (PCT) for breast cancer, no previous studies have used genomic predictors to choose among multiple chemotherapy regimens. It is unclear that given the current regimens how much PCT can improve the response rate for patients who will receive chemotherapy. In this study, we reanalyzed data from published stud¬ies of 1111 breast cancer patients who were treated with neoadjuvant chemotherapies. Those patients were divided into three regimen groups: an anthracycline alone, anthracycline plus paclitaxel, and anthracycline plus docetaxel. We developed a new strategy called PRES (Personalized REgimen Selection) to reassign the optimal regimen to each of the patients. First, a variable selection scheme was developed to identify significant genetic predictors for chemotherapy response. The selected genetic variables were then combined with clinical variables to build random forest models to predict the response of a patient to each regimen using pCR (pathological complete response) as the measure of response. The models were used to assign an optimal regimen to each patient to maximize the chance of pCR. We found that the expected rate of pCR was improved from 21.2% to 39.6% (95% CI: 34.6% - 43.0%). We also found that 31.1% of the patients may have been overtreated and 8.2% patients undertreated. A validation study on 21 cell lines showed that our prediction agrees with their paclitaxel-sensitivity profiles. We performed additional analysis on the Cancer Genome Atlas (TCGA) data and found that 18 of the 19 genes identified are significantly differentially expressed between normal and tumor tissues, and 2 of them, TAF6L and METRN (meteorin), are associated with overall survival. In conclusion, PRES could substantially increase response rates for breast cancer patients who will receive one of the widely-accepted chemotherapy regimens at present.

#2036

A phase Ib pharmacokinetic drug-drug interaction evaluation of oral buparlisib in combination with lapatinib in HER2+/PI3K-activated, trastuzumab-resistant locally advanced, recurrent and metastatic breast cancer (MBC).

François Lokiec,1 Anthony Goncalves,2 Fanny Bret,1 Jihane Pakradouni,2 Magali Provansal,2 Renaud Sabatier,2 Jean-Marc Extra,2 Carole Tarpin,2 Nicolas Isambert,3 Mario Campone,4 Keyvan Rezai1. 1 _Institut Curie, Saint Cloud, France;_ 2 _Institut Paoli Calmettes, Marseille, France;_ 3 _Centre Georges-François Leclerc, Dijon, France;_ 4 _Institut de Cancérologie de l'Ouest, Nantes, France_.

Background: A novel combination of oral lapatinib (LPT), a selective dual ErbB1/ErbB2 targeted drug, + oral buparlisib (B) a pan-class I PI3K inhibitor, could provide a synergestic antitumor activity in trastuzumab resistant disease. LPT has been shown to be a potent CYP3A4 inhibitor which is also mainly involved in BKM120 metabolism. In this study we investigated the potential pharmacokinetic (PK) drug-drug interactions (DDI) related to the association of B and LPT. Methods: Trastuzumab-resistant HER2 + metastatic breast cancer (MBC) patients were treated with a continuous once daily dosing schedule of LPT + B. Dose levels [DL, LPT (mg)/B (mg)] ranged from 750/40 to 1,250/80. For PK analysis, 2 time point samples were collected on D1 and D8 and 10 time point samples were collected on D15 of cycle 1 for LPT and B assays. Plasma concentrations of B and LPT, were measured using UPLC coupled with tandem mass spectrometry validated methods. Population PK was modeled using a non linear mixed effect model program (Monolix version 4.3s) by computing the maximum likelihood estimator of the parameters without any approximation of the model (no linearization). Results: 343 and 322 plasma concentrations were available for PK analysis of LPT and B respectively. A two-compartment open model adequately described B concentration versus time courses. The inter-individual variabilities (ISV) could be well estimated for all stuctural parameters (clearance: CL, volume of distribution: V, inter-compartmental clearance: Q) except for absorption constant: Ka. The population PK parameters obtained for the structural model were: Ka = 0.985 h-1, CL/F = 10.5L/h, V1/F = 54.8 L, Q/F = 46.3 L/h, V2/F = 582 L. Mean AUC0-24h(CV%) values for B were 10130 (34%), 15450 (29%), and 20560 (38%) ng*hr/mL for 40 mg, 60 mg and 80 mg dose level respectively. A one-compartment model adequately fitted the LPT plasma concentration-time data. The population PK parameters were CL/F = 25.7 L/h, V/F = 291 L and the absorption constant, Ka = 0.214 h-1. B has no significant effect on the PK of LPT and vice versa. Conclusions: B AUC increased proportionally with increasing dose. LPT PK parameters are consistent with those already published. There is no significant evidence for drug-drug PK interaction between LPT and B. Intra-occasion variabilities will be discussed.

#2037

A discovery study to identify clinical and genetic risk factors for bevacizumab (BEV)-related gastrointestinal (GI) hemorrhage (HEM) in metastatic castration-resistant prostate cancer (mCRPC) patients (pts) treated on CALGB 90401 (Alliance).

Jai N. Patel,1 Chen Jiang,2 Kouros Owzar,2 Daniel L. Hertz,3 Flora A. Mulkey,2 William K. Kelly,4 Susan Halabi,2 Yoichi Furukawa,5 Cameron Lassiter,6 Susan G. Dorsey,6 Paula N. Friedman,7 Eric J. Small,8 Michael A. Carducci,9 John F. Mahoney,1 Michael J. Kelley,10 Yusuke Nakamura,7 Michiaki Kubo,11 Mark J. Ratain,7 Michael J. Morris,12 Howard L. McLeod13. 1 _Levine Cancer Institute, Charlotte, NC;_ 2 _Alliance Statistics and Data Center, Duke University, Durham, NC;_ 3 _University of Michigan College of Pharmacy, Ann Arbor, MI;_ 4 _Thomas Jefferson University, Philadelphia, PA;_ 5 _The University of Tokyo, Institute of Medical Science, Japan;_ 6 _University of Maryland School of Nursing, Baltimore, MD;_ 7 _University of Chicago, Chicago, IL;_ 8 _University of California San Francisco, San Francisco, CA;_ 9 _Johns Hopkins School of Medicine, Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD;_ 10 _Durham VA Medical Center/Duke University Medical Center, Durham, NC;_ 11 _Riken Center for Integrative Medical Sciences, Japan;_ 12 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 13 _Moffitt Cancer Center, Tampa, FL_.

Background: Treatment-related GI HEM is a major health concern with few known predictive risk factors. The objective of this analysis was to discover clinical and genetic factors that modulate GI HEM risk in a large randomized phase III study.

Methods: Chemotherapy-naïve mCRPC pts were randomized 1:1 to receive docetaxel and prednisone ± BEV once every 21 days for up to two years (N=1008). Cause-specific time-to-event analysis using a Cox regression model was used to investigate the association between grade 2+ GI HEM (designated as at least "probably" related to therapy) and BEV, age, history (hx) of peptic ulcer disease (PUD), hx of HEM, antiplatelet/anticoagulant use, hx of smoking, and hemoglobin. Genetically-defined Caucasian pts who provided consent for the genomic companion study (CALGB 60404) were genotyped using the Illumina HumanHap610-Quad platform (N=616). Log rank test was used to investigate the association of single nucleotide polymorphisms (SNPs) and GI HEM, and results were adjusted for significant clinical covariates.

Results: The overall incidence of grade 2+ GI HEM was 9.5% (48/503) and 3.8% (19/505) in the BEV and placebo arms, respectively. Of the clinical covariates, only BEV (HR=5.77; 95% CI 2.20-15.11; P<0.001) and age (HR=1.06; 95% CI 1.01-1.11; P=0.01) were significantly associated with GI HEM in the multivariable analysis, while a trend was noted for hx of PUD (P=0.08). Of 498,081 SNPs tested, one intergenic SNP (rs1478947; HR 6.26; 95% CI 3.00-14.4; P=9.40 x 10-8) surpassed Bonferroni-corrected significance (1.0 x 10-7) for association with GI HEM (minor allele frequency = 0.06). The incidence of GI HEM in the BEV arm was 33.3% (13/39) and 6.2% (17/275) for pts with the AA/AG and GG genotypes, while the incidence in the placebo arm was 2.9% (1/35) and 1.9% (5/267), respectively.

Conclusion: BEV, age, and one putative intergenic SNP (rs1478947) were associated with cause-specific GI HEM risk in CALGB 90401. The effect of rs1478947 appears to be specific to pts receiving BEV. Although the mechanism by which rs1478947 increases GI HEM risk remains unclear, rs1478947 is in complete LD (r2 = 1) with rs1478948, variations of which may alter the binding motif for transcription factor hepatocyte nuclear factor-4 (HNF4). HNF4 exerts a major regulatory effect on clotting factor VII (fVII) expression and function. Altered binding of HNF4 to fVII promoter may result in reduced fVII function and an increased risk of bleeding. It is unclear how much weight each identified risk factor contributes to the overall incidence of GI HEM, which in absolute terms was not dramatically different between arms. Exploratory studies from large trials of BEV-treated pts are needed to better understand the genetic contribution to and biological basis of GI HEM. Support: U10CA180821

#2038

Sunitinib impact on kinome profiles of peripheral blood mononuclear cells from renal cell carcinoma patients: Do molecular effects correlate with clinical data.

Audrey Bellesoeur,1 Gaelle Noe,1 Audrey Thomas-Schoemann,1 Olivier Huillard,1 Faris Naji,2 Savithri Rangarajan,2 Alicja Puszkiel,1 Jerome Alexandre,1 François Goldwasser,1 Benoit Blanchet,1 Michel Vidal1. 1 _Cochin Teaching Hospital, Paris, France;_ 2 _PamGene International BV, Netherlands_.

Introduction: Sunitinib, a potent multi-tyrosine kinase (TK) inhibitor, is a standard first-line treatment for metastatic renal cell carcinoma (mRCC). In addition to its antiangiogenic activity, sunitinib is known to have immune-modulating properties especially on regulatory T-cells and tumor-infiltrating lymphocytes. However, data is sparse about sunitinib impact on peripheral lymphocytes and new data is needed to gain insights into the angiogenesis and immunity bidirectional link. This study aims to fill such a gap by investigating the clinical and intracellular modifications of sunitinib on peripheral blood mononuclear cells (PBMC) from naïve mRCC patients.

Methods: Initially, a retrospective study was conducted in 88 mRCC patients treated with first-line sunitinib therapy to assess the evolution of lymphocyte count (expressed as a ratio between Day 21 and Day 0 i.e. D21/D0) during the first cycle of treatment. A new prospective study was carried out to determine kinomic profiles in PBMC from 21 naïve mRCC patients and 12 healthy volunteers. TK activity profiles of PBMC lysates were generated on TK PamChip® microarrays. The ex vivo effect of sunitinib and its active metabolite SU12662 were also determined in PBMCs. All data were analyzed using BioNavigator software.

Results: The retrospective preliminary study showed that an increased D21/D0 lymphocytes ratio was significantly associated with a shorter Progression Free Survival (PFS) in multivariate analysis (p=0.0023). In the prospective study, the phosphorylation level in PBMCs from mRCC patients was significantly lower than in healthy volunteers for 74 peptides (p<0.05). Ex vivo exposure to sunitinib or SU12662 led to a decreased phosphorylation level for majority of peptides in PBMCs from mRCC patients. Moreover, sunitinib had a stronger inhibitory profile than SU12662 for 80 peptides (p<0.05). The ex vivo sunitinib effect was statistically correlated with the IMDC ("Heng") prognostic model and D21/D0 lymphocytes ratio and 53 and 16 peptides, respectively were found to be significant. Less ex vivo inhibition was associated with both a poor prognosis according to Heng and an increased D21/D0 lymphocytes ratio.

Conclusions: Our retrospective study shows a decreased lymphocyte count on D21 after sunitinib initiation is a favorable prognostic factor in mRCC patients. The kinomic analysis of TKs in PBMCs after ex vivo exposure to sunitinib correlates with both Heng prognostic score and lymphocyte D21/D0 ratio, suggesting that PBMCs could be an interesting biological matrix to seek future biomarkers regarding clinical efficacy of sunitinib. Further investigations are underway to determine the involvement of signaling pathways contributing to the inter-individual variability in kinomic profiles of PBMCs from mRCC patients treated with sunitinib.

#2039

Exploring the longitudinal transcriptomic landscape of tyrosine kinase inhibitor treatment response in chronic myeloid leukemia patients.

Aritro Nath, Fan Wang, Divya Lenkala, Bonnie LaCroix, Nancy Glavin, Kristen Kipping-Johnson, Paul Geeleher, Michael Thirman, Lucy Godley, Gordana Raca, Richard Larson, R. Stephanie Huang. _University of Chicago, Chicago, IL_.

The interpretation and implementation of large-scale genetic profiles into clinical practice remains a challenge despite substantial growth in our understanding of genetic contributors to drug response. Most current omic studies focus on identifying genetic features that are distinct between normal and tumor samples, but fail to capture the dynamics of association between omic profiles, treatment response and disease progression over time. The focus of this research is to analyze the longitudinal transcriptomic profile of chronic myeloid leukemia patients (CML) in context of tyrosine kinase inhibitor (TKI) treatment and clinical status. The main objectives were to compare a series of post-TKI treatment transcriptome profiles to their baseline levels, and characterize the impact of TKI treatment and CML disease status on the individual's transcriptome over time. Our ultimate goal is to develop TKI response predictors using the longitudinal expression data collected over the treatment course.

Peripheral blood samples, buccal swabs and detailed clinical data were collected from each study participant (screened for BCR-ABL1 translocation) for a period of 6 months, in addition to pre-therapy baseline. RNA was extracted from granulocytes isolated from peripheral blood samples, and profiled using RNA sequencing. RNAseq profiles over TKI treatment course were compared to baseline, as well as against hematologic response (complete blood count), cytogenetic response (FISH), and clinical disease progression.

We investigated dynamic trends in RNAseq profiles associated TKI response, as well as with the clinical status of the patient over time. We identified genetic features that were either 1) Differentially expressed between baseline and post-TKI time points; 2) Showed non-random spikes in expression levels at specific time points; 3) Associated with hematological and clinical phenotypes, including white blood cell count, percentage granulocytes and percentage cells with BCR-ABL1 translocation; 4) Demonstrated highly correlated patterns of expression over time. Through clustering and enrichment analysis of the selected transcripts, we identified several pathways and molecular features associated with TKI-response, and altered disease state. Of note, we found mTOR signaling, and pro-apoptotic pathways to be significantly altered between baseline and TKI-responding individuals. In addition, we observed significant changes in transcription regulatory network of several transcription factors, notably AP-1, over the treatment time course.

To our knowledge, this is the first study to establish the utility of comprehensive longitudinal multiple transcriptome profile analysis of TKI-response in CML. We believe this study will pave way for future large-scale longitudinal omic profiling of CML and other cancer-types.

#2040

Influence of MDR1 and CYP3A5 genetic polymorphisms on trough levels and therapeutic response of imatinib in patients with chronic myeloid leukemia.

Natarajan Harivenkatesh, Lalit Kumar, Sameer Bakhshi, Atul Sharma, Madhulika Kabra, Thirumurthy Velpandian, Ajay Gogia, Shivaram Shastri, Yogendra Kumar Gupta. _All India Institute of Medical Sciences (AIIMS), New Delhi, India_.

Introduction:

Genetic polymorphisms in the genes coding for imatinib transporters & metabolizing enzymes might be responsible for marked inter-individual pharmacokinetic variability seen with imatinib & might result in altered therapeutic response.

Methodology:

Newly diagnosed patients with chronic phase CML started on imatinib therapy were enrolled & followed up for 24 months. The following single nucleotide polymorphisms (SNPs) were genotyped - C1236T, C3435T, G2677T & G2677A in MDR1 gene and A6986G in CYP3A5 gene. Genotyping was done using PCR-RFLP method & validated by direct gene sequencing. Plasma trough levels of imatinib were measured using LC-MS/MS. Patients who attained complete cytogenetic response by 12 months after imatinib therapy were called responders while those who failed to do so were called non-responders. Molecular response was assessed by quantitative RTPCR using international scale.

Results:

A total of 173 chronic phase CML patients were included in this study, out of which 71 were responders. Mean age at diagnosis was 35.9±14.3 years. All the alleles, except those for MDR1-C1236T, followed Hardy-Weinberg equilibrium. Marked variability in trough levels of imatinib was seen in patients with different genotypes (table1). The frequency of GG genotype for CYP3A5-A6986G and TT genotype for MDR1-C1236T, C3435T & G2677T/A was higher in responders; while the frequency of AA genotype for CYP3A5-A6986G, CC genotype for MDR1-C1236T & C3435T and GG genotype for MDR1-G2677T/A was higher in non-responders (table1).

Conclusions:

Genetic polymorphisms in MDR1 & CYP3A5 genes have a significant role in determining imatinib levels & therapeutic response in patients with CML. Genotyping of CPY3A5 & MDR1 genes may be considered in patients with CML to individualize the therapy & optimize the outcomes.

Table 1: Trough levels of Imatinib in patients with different genotypes and frequency of different genotypes in imatinib responders and non-responders  | |  | |  | |

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

SNP | Genotype | Trough levels (mean±SD) of Imatinib (ng/mL) | P value | Imatinib response | P value

Responders

(n=71) | Non-responders (n=102)

|

CYP3A5-A6986G | AA | 1415.1 ± 1036.3 | 0.016 | 4 (19%) | 17 (81%) | < 0.001

AG | 1610 ± 1161.1 | 20 (28%) | 51 (72%)

GG | 2229 ± 1524.6 | 47 (58%) | 34 (42%)

MDR1-C1236T | CC | 1559.8 ± 990.4 | 0.201 | 4 (21%) | 15 (79%) | 0.005

CT | 1819.6 ± 1450.2 | 42 (37%) | 71 (63%)

TT | 2178.4 ± 1246.7 | 25 (61%) | 16 (39%)

MDR1-C3435T | CC | 1418 ± 865.9 | 0.013 | 3 (16%) | 16 (84%) | < 0.001

CT | 1695 ± 1318.9 | 28 (32%) | 60 (68%)

TT | 2249.6 ± 1470.1 | 40 (61%) | 26 (39%)

MDR1-G2677T/A | GG | 1570.8 ± 1181.2 | 0.297 | 4 (22%) | 14 (78%) | 0.019

GT | 1726.5 ± 1489.3 | 23 (31%) | 51 (69%)

TT | 2133.7 ± 1338.4 | 37 (54%) | 32 (46%)

TA | 1840.5 ± 754.2 | 6 (60%) | 4 (40%)

GA | 1450.2 ± 743.5 | 1 (50%) | 1 (50%)

#2041

Pharmacogenomic effects of catechol-O-methyltransferase on aspirin and vitamin E cancer preventive treatment.

Kathryn T. Hall,1 Elisabeth Battinelli,1 Dani Passow,2 Ted J. Kaptchuk,3 Julie DSc Buring,1 Paul Ridker,1 Kenneth J. Mukamal,3 Daniel I. Chasman1. 1 _Brigham and Womens Hospital, Harvard Medical School, Boston, MA;_ 2 _Harvard University, Cambridge, MA;_ 3 _Beth Israel Deaconess Medical Center, Boston, MA_.

Despite demonstrated effects of catechol-O-methyltransferase (COMT) on estrogen metabolism and cancer cell proliferation, epidemiological studies of the effects of its genetic locus, COMT, on cancer incidence give conflicting results. These inconsistencies may in part be attributed to analyses that fail to account for pharmacogenomic interactions. We therefore examined the association between the common functional met/val substitution in COMT (rs4680) and incident cancer over 10 years among 23,294 initially cancer-free women of European ancestry from the Women's Genome Health Study (WGHS), a 2x2 factorial trial of randomly-allocated aspirin or vitamin E treatment in cancer prevention. We previously demonstrated interaction between rs4680 and random allocation to aspirin or vitamin E compared with placebo for association with incident cardiovascular disease in the WGHS and hypothesized that such interactions may be relevant to cancer. The association of rs4680 with total cancer in WGHS was non-significant overall. However, among placebo-allocated participants, the rs4680 val-allele was associated with lower total cancer incidence compared to the met-allele (per allele HR=0.82[0.72-0.95], p=0.006). In contrast, the val-allele was not associated with cancer rates among those allocated to aspirin (HR=1.02[0.90-1.16], p=0.8), and with higher rates among those assigned to vitamin E (1.14[1.01-1.30], p= 0.04) or both drugs (HR=1.15 [1.00-1.32], p=0.05). The interactions of genotype with treatment assignment were significant for both aspirin (p=0.03) and vitamin E (p=0.0006). Results were similar when controlling for hormone replacement therapy, BMI, smoking and alcohol use, for breast and colorectal but not lung cancer subtypes, and for a second COMT variant, rs4818, in linkage disequilibrium with rs4680. Together, these data support a role for COMT in cancer incidence and modification of its effects by commonly used drugs aspirin and vitamin E.

Age-adjusted Cox models of COMT rs4680 val-allele and rs4818 G-allele and total invasive cancer

---

|

Drug assignment arm* | rs4680** | rs4818**

Events/N | Aspirin: | Vitamin E: | HR[95% CI], p | Gene-drug interaction p | HR[95% CI], p | Gene-drug interaction p

415/5809 | Placebo | Placebo | 0.82 [0.72-0.95], p=0.006 | \-- | 0.82 [0.71-0.95], p=0.007 | \--

456/5805 | Aspirin | Placebo | 1.02 [0.90-1.16], p=0.8 | 0.026 | 0.97 [0.85-1.12], p=0.7 | 0.0009

455/5852 | Placebo | Vitamin E | 1.14 [1.01-1.30], p= 0.04 | 0.0006 | 1.19 [1.05-1.36], p=0.008 | 0.00015

406/5791 | Aspirin | Vitamin E | 1.15 [1.00-1.32], p=0.048 | 0.12*** | 1.13 [0.98-1.30], p=0.09 | 0.098***

*WGHS 2x2 treatment assignments

**rs4680 coded allele = G(val), reference allele = A(met); rs4818 coded allele = G, reference allele = C

***Gene-drug interaction p-value for 3-way COMT*aspirin*vitamin E interaction

#2042

A next-generation sequencing-based sample-to-result pharmacogenomics research solution enables both SNV and CNV detection at once.

Melvin S. Wei, Zhoutao Chen, Shann-Ching Chen, Manimozhi Manivannan, Emily Zeringer, Sunali Patel, Toinette Hartshorne, Guoying Liu, Fiona Hyland, Mark Andersen. _Thermo Fisher Scientific, Carlsbad, CA_.

Pharmacogenomics (PGx) is the study of genetic variations in terms of their response to drugs. Variations in gene sequence or copy number may result in complete loss of function, partial decrease or increase in enzyme activity, or an altered affinity for substrates, which may in turn significantly impact a drug's efficacy. PGx studies are increasing in significance as precision medicine is becoming a reality in standard practice. Different technologies have been developed to measure the sequence variation and copy number variation (CNV) in the PGx genes. Among them, a complete sample-to-result PGx workflow solution using the QuantStudio™ 12k Flex Real-Time PCR System is the most notable high throughput solution and has broad adoption by advanced PGx laboratories. Both PGx SNP/INDEL genotyping assays on OpenArray™ plates and copy number analysis on 384-well plates can be performed on the QuantStudio™ 12k Flex System. Integrated analysis software translates genotyping and copy number assay results into star allele genotypes for ease of interpretation. Recently we have developed a next generation sequencing (NGS) based PGx research solution with increased flexibility on the assay targets and combined detection of SNP/INDEL genotyping and CNV using Ion AmpliSeq™ technology for low to medium throughput laboratories. With a highly multiplexed PGx research panel, we can profile a set of 136 genetic markers in 40 known PGx related genes and CYP2D6 copy number variation in a single reaction using Ion Torrent™ semiconductor sequencing. The number of genetic markers can be customized easily based on the user need. To systematically compare these two end-to-end PGx workflows, we collected buccal swab samples from 20 individuals and performed both QuantStudio™ based assays and PGM™ based Ion AmpliSeq™ PGx research assay on them. Both systems generated high quality results. Compared with OpenArray™ plate genotyping results and 384-plate CYP2D6 copy number assay results from the QuantStudio™ system, the Ion AmpliSeq™ PGx research solution demonstrated >99.9% genotyping concordance, 100% CYP2D6 gene CNV concordance, >99.7% reproducibility, <0.2% no-call rate. The Ion AmpliSeq™ PGx solution enables flexible and integrated SNV & CNV detection for both standard genotyping practice and sophisticated exploratory research needs.

#2043

Effects of 24-h carboplatin pretreatment on olaparib clearance in women's cancers using noncompartmental and population pharmacokinetic analyses.

Andrew K.L. Goey, Cody J. Peer, Tristan M. Sissung, Jeffrey Roth, Shandiz Shahbazi, Jeffers Nguyen, Christina M. Annunziata, Nicole Houston, Elise C. Kohn, Jung-Min Lee, William D. Figg. _National Cancer Institute, Bethesda, MD_.

Background: Olaparib (OLA) is a PARP inhibitor approved for use in deleterious germline BRCA mutated recurrent or refractory ovarian cancer. Combining OLA with carboplatin (CARBO) could have additive effects based on platinum-DNA adducts requiring PARP for DNA repair. Preclinical data suggest greater cytotoxicity when CARBO is given prior to OLA. However, the optimal treatment sequence of these agents has not been studied previously in patients. We therefore investigated: 1) the effects of CARBO treatment on the pharmacokinetics (PK) and pharmacodynamics (PD) of OLA; 2) in vitro mechanisms of the interaction between CARBO and OLA.

Methods: Clinical PK and PD data of OLA were obtained from 58 patients with confirmed recurrent or refractory women's cancers participating in a two arm, parallel design, phase 1 trial (NCT01237067). In cycle 1 OLA tablets (200 mg BID) were given for 7 days either followed by CARBO (AUC 4) on day 8 (arm A) or after CARBO on day 1 (Arm B). In cycle 2 the arms received the reversed scheme. PK of OLA were assessed in both cycles by noncompartmental (NCA) and population pharmacokinetic (PPK) analyses. For PK/PD analyses, PAR levels were measured at baseline and 24 h after the first OLA dose. In vitro mechanistic studies were carried out by incubating whole human blood and avian DT40 PARP-1 KO cells with 10 μM CARBO for 24 h, followed by 1h-treatment of isolated PBMCs and PARP-1 KO cells with 10 μM OLA. Intracellular OLA concentrations were determined using UPLC-MS/MS.

Results: Both NCA and PPK analyses showed a ~50% increase in OLA clearance when CARBO was administered 24-h prior (P<0.02). The PPK model included a lag time parameter (P=1.1E-18), a second absorption compartment (P=7.7E-27), a single elimination compartment, and accounted for covariance among the clearance and volume parameters (P=6.7E-7). Presence of CARBO was the only significant covariate affecting OLA clearance (P=1.9E-13). Final estimates for clearance and volume of distribution were 6.8 L/h and 33 L, respectively, which were comparable with related reports. There were no trends between PK data and PAR levels, nor did the presence of CARBO affect PAR levels (P=0.89). PBMC experiments showed that 24-h pretreatment with CARBO significantly increased intracellular OLA concentrations by more than 30% compared with control samples (P=0.013). PARP-1 KO cells confirmed that intracellular PARP expression was not related to the increased OLA uptake. Possibly, CARBO affects other intracellular targets or transporters leading to increased intracellular uptake of OLA from the bloodstream.

Conclusion: This is the first known PK analysis showing a significant increase in OLA clearance after pretreatment with CARBO, possibly leading to subtherapeutic plasma concentrations of OLA. Preclinical experiments are ongoing to reveal the exact pharmacological mechanisms of this interaction.

#2044

Pharmacokinetics (PK) of nivolumab in patients with relapsed or refractory lymphoid malignancies.

Xiaoli Wang, Matthew Hruska, M. Brigid Bradley-Garelik, Bradly Boone, Akintunde Bello. _Bristol-Myers Squibb, Princeton, NJ_.

Objectives: Nivolumab, a fully human anti-programmed death-1 immunoglobulin G4 antibody, has demonstrated clinically meaningful responses and overall survival benefits in patients with solid tumors following administration of 3 mg/kg every 2 weeks (Q2W)—the currently approved dosing regimen for the treatment of advanced melanoma and non-small-cell lung cancer (NSCLC). Nivolumab has also demonstrated strong antitumor activity in lymphoid malignancies with an objective response rate of 87% in relapsed or refractory classical Hodgkin lymphoma (cHL) (Ansell SM, et al. N Engl J Med. 2015). The present analysis aimed to characterize the PK of nivolumab in 4 different lymphoid malignancies, including multiple myeloma (MM), T-cell non-Hodgkin lymphoma (NHL), B-cell NHL, and cHL, and to compare the PK to that seen in solid tumors.

Methods: This analysis was conducted as part of an open-label, dose-escalation phase 1 study (NCT01592370) investigating the safety, PK, and antitumor activity of nivolumab in patients with relapsed or refractory lymphoid malignancies. Patients were treated with nivolumab 1 mg/kg Q2W or 3 mg/kg Q2W in the dose-escalation phase, and with nivolumab 3 mg/kg Q2W during the dose-expansion phase. Serial blood samples were collected and analyzed for PK using a validated ligand-binding enzyme-linked immunosorbent assay. PK parameters of nivolumab, including area under the curve over the 2-week (336 hours) interval following the first dose (AUC336) and maximum plasma concentration (Cmax) after the first dose were characterized using noncompartmental analysis.

Results: The geometric mean (coefficient of variation [CV]) values of dose-normalized AUC336 of nivolumab in the MM (n = 22), B-cell NHL (n = 30), T-cell NHL (n =19), and HL (n =18) groups were 3759 µg•h/mL (23%), 3298 µg•h/mL (24%), 2800 µg•h/mL (29%), and 2977 µg•h/mL (27%), respectively. The geometric mean (CV) values of dose-normalized Cmax of nivolumab in the MM, B-cell NHL, T-cell NHL, and HL groups were 24 µg/mL (30%), 20 µg/mL (26%), 18 µg/mL (31%), and 18 µg/mL (25%), respectively. Dose-normalized exposures were similar at the 1- and 3-mg/kg dose levels.

Conclusions: The PK of nivolumab is similar among patients with different lymphoid malignancies and is similar to what has previously been observed for patients with solid tumors, including melanoma, renal cell carcinoma, and NSCLC, following administration of nivolumab 3 mg/kg Q2W. Based on the observed tolerable safety profile and strong antitumor activity, nivolumab 3 mg/kg Q2W is an appropriate dosing regimen for study in patients with lymphoid malignancies.

#2045

Relationship of venetoclax dose and exposure to response in relapsed or refractory chronic lymphocytic leukemia and non-Hodgkin's lymphoma subjects.

Kevin J. Freise,1 Aksana K. Jones,1 Doerthe Eckert,2 Sven Mensing,2 Shekman Wong,3 Rod Humerickhouse,1 Walid Awni,1 Ahmed H. Salem1. 1 _Abbvie, North Chicago, IL;_ 2 _Abbvie, Ludwigshafen, Germany;_ 3 _Abbvie, Redwood City, CA_.

Venetoclax is a selective, potent, orally bioavailable, small molecule, B-cell lymphoma-2 (Bcl-2) inhibitor that restores apoptosis in cancer cells. Clinical efficacy of venetoclax has been observed in a variety of hematological malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkins lymphoma (NHL). The objective of this research was to characterize the relationship between venetoclax exposures and efficacy in relapsed or refractory (R/R) CLL/small lymphocytic lymphoma (SLL) subjects, as well as safety in R/R CLL/SLL and NHL subjects. A total of 272 and 444 subjects from 4 clinical studies were included in the exposure-efficacy and exposure-safety analyses, respectively. Characteristics evaluated for their impact on efficacy and safety included: demographics (bodyweight, sex, etc.), baseline disease characteristics (e.g. lymphocyte count, tumor size, 17p del somatic mutation), and select co-medications. Model based simulations indicated that a venetoclax dosage of 400 mg daily maximized the probability of a typical subject with R/R CLL/SLL achieving an objective response (OR) by 6 months of venetoclax treatment with estimated OR rates of 80.9% (95% confidence interval [CI]: [77.5% - 84.0%]), 84.8% (95% CI: [81.5% - 88.0%]), and 85.5% (95% CI: [82.6% - 88.4%]) at the 200, 400, and 600 mg dosage regimens, respectively. The 17p deletion was not identified in any of the exposure-efficacy analyses to be related to the responsiveness of R/R CLL/SLL subjects to venetoclax. Therefore, subjects with the 17p deletion chromosomal aberration appear to be as sensitive to the effects of venetoclax as subjects who do not have the 17p deletion, consistent with its Bcl-2 inhibitor mechanism of action that bypasses the signaling events associated with the 17p deletion. The safety analyses of the adverse events (Grade ≥ 3) of neutropenia and infection in R/R CLL/SLL and NHL subjects both indicated that higher average venetoclax concentrations were associated with a decrease in adverse events, indicating that these evaluated safety endpoints are not dose-limiting. Sensitivity analyses supported that increasing venetoclax concentration did not increase these adverse events. It is hypothesized that the association is driven by disease improvement with venetoclax treatment and not by directly affecting neutrophils or granulocyte progenitor cells. Overall, the exposure-response-safety analyses indicated that a venetoclax dosage regimen of 400 mg daily maximizes (>80%) the probability of achieving objective response in R/R CLL/SLL subjects with minimal probability of neutropenia or infection.

#2046

Exposure - response (overall survival [OS]) analyses of patients with unresectable pancreatic cancer (PC) treated with galunisertib+gemcitabine (GG) or gemcitabine+placebo (GP) in a randomized phase 2, double-blind study.

Ivelina Gueorguieva,1 Josep Tabernero,2 Davide Melisi,3 Timothy H. Waterhouse,4 Colin Miles,1 Durisala Desaiah,4 Michael M. Lahn,4 Ann Cleverly,1 Karim A. Benhadji5. 1 _Eli Lilly and Company, Erl Wood, ELCL, United Kingdom;_ 2 _Vall d'Hebron University Hospital and Institute of Oncology (VHIO), Barcelona, Spain;_ 3 _Digestive Molecular Clinical Oncology, University of Verona, Verona, Italy;_ 4 _Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN;_ 5 _Eli Lilly and Company, Lilly France, Paris, France_.

Background: Transforming growth factor-beta (TGF-β) signaling pathway is active in PC. A pharmacokinetic/pharmacodynamic (PK/PD) approach integrated preclinical biomarkers and toxicity and allowed for prospective definition of therapeutic window (Gueorguieva et al. Br J Clin Pharmacol 2014;77:796-807). Here, we evaluate the observed exposure-OS relationship in patients with unresectable PC treated with either GG or GP with median (95% CI) OS (months) of 9.10 (7.43, 12.2) and 7.59 (4.04, 9.92), respectively.

Methods: A therapeutic exposure window for galunisertib between 3730 and 8380 ng*hr/mL was targeted in patients as described in Gueorguieva et al. 2014. Galunisertib 150 mg BID was given orally as intermittent dosing (14 days on/14 days off per cycle) and gemcitabine was administered as approved. Using nonlinear mixed effects modeling, data from six studies (n=297, 3097 observations) were combined; galunisertib population PK model was developed. Galunisertib exposure metrics were calculated for each patient in the phase 2 PC study (n=100 GG arm). Parametric survival models were used to identify influential covariates on OS such as dose, plasma exposure at steady state (AUC0-24,ss), Cmax,ss, ECOG, pre-treatment with gemcitabine, age, biomarkers (CA19-9 and TGFβ) and liver metastasis.

Results: PK profile of galunisertib was not altered when combined with gemcitabine. The population PK dataset included data from 297 patients/healthy subjects whose ages ranged from 22 to 84 years and who weighed between 39 and 126 kg. The PK of galunisertib was best described by a 2-compartment model with first order absorption and elimination. Galunisertib was rapidly absorbed with peak concentrations within 1-3 h and elimination half-life of 8 h. The mean (% SEE) population apparent clearance and volume of distribution of galunisertib was 35 (3%) L/hr and the steady state was 190 (11%) L. Between-patient variance was estimated to be 47% on the population apparent clearance. There was a small, but statistically significant effect of age on apparent clearance. None of the other investigated demographic patient characteristics were found to be significant. Galunisertib median (25th-75th percentile) AUC0-24,ss for PC patients was 5.56 (3.82-7.91) mg*h/L with Cmax and tmax of 904 (668-1194) ng/mL and 1.5 (1-2.5) h, respectively. A Weibull parametric survival model provided best fit to the OS data. There was a flat exposure-OS relationship within the observed exposure range, once all significant covariates (namely ECOG, pre-treatment with gemcitabine, age, CA19-9, and liver metastasis) were included.

Conclusion: This investigation confirms that 300 mg galunisertib administered as 150 mg BID for 14 days followed by 14 days off treatment is an appropriate dosing regimen for patients with PC.

#2047

Pharmacokinetics (PK) of CRLX301, a novel nanoparticle-drug conjugate (NDC) containing the payload docetaxel, in patients with refractory solid tumors.

William C. Zamboni,1 Ben Markman,2 Paul de Souza,3 E. Claire Dees,1 Tara Gangadhar,4 Scott Eliasof,5 Curran Murphy,5 Adrian Senderowicz,5 Hongwei Wang5. 1 _UNC Lineberger Comp. Cancer Center, Chapel Hill, NC;_ 2 _Monash Cancer Centre, Melbourne, Australia;_ 3 _Western Sydney University School of Medicine, Sydney, Australia;_ 4 _Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA;_ 5 _Cerulean Pharma, Inc., Cambridge, MA_.

Introduction. Nanoparticles (NPs) provide many advantages over small molecule drugs, such as greater solubility, increased duration of exposure, and enhanced tumor delivery, which may lead to improved antitumor activity and reduced toxicity. PK of NPs is complex as the PK is dependent upon the carrier and not the therapeutic entity until the active-drug payload is released. It is important to evaluate the PK of the inactive carrier-associated drug and active released drug. CRLX301 is an investigational NDC containing the payload docetaxel covalently conjugated to a cyclodextrin-polyethylene glycol (CD-PEG) co-polymer that is 10-30 nm in diameter. This study evaluated CRLX301 PK in a phase 1 study in patients with refractory solid tumors.

Methods. CRLX301 was administered at 7.5, 15, 30, 60 and 75 mg/m2 IV over 1 h every 21 days. The doses of CRLX301 represent mg of docetaxel. PK studies were performed on days 1-21 of cycles 1, 3 and 6. Plasma samples were processed to measure total (conjugated + released) and released docetaxel. PK parameters area under conc-time curve (AUC0-inf) and volume of distribution (Vd) were estimated. Results were compared to a prior PK study of Taxotere® at 75 mg/m2 IV x 1 every 21 days (Zamboni, et al. CCP 2011).

Results. Plasma PK of total and released docetaxel were linear after CRLX301 from 7.5 to 75 mg/m2. CRLX301 plasma PK was similar on cycles 1, 3 and 6 suggesting no accumulation after multiple doses. For CRLX301 at 75 mg/m2 on cycle 1, mean ± SD plasma AUC0-inf of total and released docetaxel were 321,355 ± 9,831 and 3,015 ± 904 ng/mL•h, respectively. Mean ± SD % of docetaxel released from CRLX301 into the plasma was 1.0 ± 0.2%.

Exposures of total and released docetaxel in plasma after administration of CRLX301 (7 days) were prolonged compared with Taxotere (24 h). Total docetaxel plasma AUC after CRLX301 at 75 mg/m2 was ~63-fold greater than for Taxotere. The Vd of CRLX301 total docetaxel and Taxotere were 2.1 ± 0.1 and 74-99 L/m2, respectively. The overall plasma AUC of released docetaxel after CRLX301 (3,015 ± 904 ng/mL•h) was similar to Taxotere (2,000-10,000 ng/mL•h); however, most of the released docetaxel AUC after CRLX301 is associated with exposures at 24-168 h. Early exposures of released docetaxel in plasma after CRLX301 (Cmax = 242 ± 44 ng/mL) were ~25-fold lower than Taxotere (Cmax = 5,000-8,000 ng/mL).

Conclusions. CRLX301 exhibits PK advantages over Taxotere such as higher retention of drug in plasma, slower clearance and controlled slow release of docetaxel from the carrier. CRLX301 exhibits low inter- and intra-patient PK variability. It is anticipated that this unique PK profile of CRLX301 may improve efficacy as well as the tolerability profile when compared to Taxotere. The study continues with dose escalation to further explore the safety, efficacy and PK of CRLX301.

#2048

Pharmacodynamic biomarker studies of TRC105 anti-endoglin (CD105) antibody revealed anti-angiogenic activity associated with CD105 depletion.

Liang Cao, Fatima Karzai, Andrea Apolo, Ravi Madan, Yunkai Yu, James Gulley, William Figg, William Dahut. _National Cancer Institute, Bethesda, MD_.

Introduction: CD105 is involved in normal vascular development. It is over expressed on the surface of proliferating vascular endothelial cells and is implicated in tumor angiogenesis. In hypoxic conditions, CD105 is upregulated through induction of hypoxia-inducible factor 1-α. TRC105 is a chimeric IgG1 antibody specific for CD105 and the agent for this phase I trial.

Methods: 20 patients with metastatic prostate cancer were treated with TRC105 at six dose levels in a phase I trial. Blood samples were analyzed for CD105 antigen depletion, VEGF as a marker for systemic hypoxia, and PSA.

Results: Maximum tolerated dose of 20 mg/kg every two weeks was reached. Significant plasma CD105 reduction was observed at high dose levels. The reduction of CD105 was associated with induction of plasma VEGF. Ten patients had stable disease, and the reduction of CD105 is associated with PSA stabilization.

Conclusion: A significant induction of VEGF was associated with CD105 reduction at three high dose levels, suggesting the anti-angiogenic activity of TRC105. Exploratory analysis showed a tentative correlation between the reduced CD105 and a decreased PSA velocity, suggestive of potential antitumor activity of TRC105 in metastatic prostate cancer.

#2049

Pharmacokinetic drug-drug interaction: a phase Ib dose escalation study of LY2780301 in combination with weekly paclitaxel.

Keyvan Rezai,1 Samuel Huguet,1 Olivier Madar,1 Jean-Marc Extra,2 Magali Provansal,3 Carole Tarpin,2 Nicolas Isambert,4 Jihane Pakradouni,2 Anthony Goncalves,2 François Lokiec1. 1 _Institut Curie, Saint Cloud, France;_ 2 _Institut Paoli Calmettes, Marseille, France;_ 3 _Institut Paoli Calmettes, Saint Cloud, France;_ 4 _Centre Gorges-François Leclerc, Dijon, France_.

Introduction: LY2780301 is an orally available small molecule dual inhibitor of p70 S6 kinase and AKT. LY2780301 is an inhibitor of CYP3A4. By the one hand, Paclitaxel (PXL) undergoes extensive metabolism via CYP450 2C8 and CYP450 3A4 pathways. By the other hand, PXL induces weakly CYP3A4 activity. In this study we investigated for the first time, the potential pharmacokinetic (PK) drug-drug interaction (DDI) related to the combination of both drugs LY2780301 and PXL.

Methods: Women with HER2- locally advanced or metastatic breast cancer, with and without PI3/AKT pathway activation, were treated with weekly administration of PXL on day1 (D1), D8 and D15. LY2780301 was administered by a daily flat dosing regimen starting at D3. Dose levels [DL, LY2780301 (mg/day)/PXL (mg/m2/week)] ranged from 400/70 to 500/80. For PK analysis, 7 time point samples were collected on D1 and D3 of cycle 1 for PXL and LY2780301 respectively. 7 samples were collected on D8 for both drugs. LY2780301 plasma concentrations were measured using Ultra Performance Liquid Chromatography (UPLC) coupled with tandem mass spectrometry validated method. PXL plasma concentrations were measured using UPLC coupled with UV validated method. A joint population PK (PK-POP) model has been developed for LY2780301 and

its main metabolite. PK-POP modelling has been performed with a non linear mixed effect model program (Monolix version 4.3.2).

Results: 12 patients, 35 to 67 years old, were treated. A total of 160 and 157 concentrations for LY2780301 and its metabolite were used respectively for PK-POP modeling. A one compartment open model adequately described LY2780301 and its metabolite concentration versus time courses respectively with the estimation of the fraction of absorbed dose (fm) of LY2780301 metabolised in that metabolite. The interindividual variabilities (ISV) could be well estimated for all structural parameters (clearance: CL, volume of distribution: V, L and the absorption constant: Ka). The population PK parameters obtained for the structural model were: Ka=0.536 h-1, CL/F=3.57 L/h, V/F=85.2 L, CL/(F*fm)=13.2 L/h, V2/(F*fm)= 13.2 L, fm=0.747 for LY2780301 and its metabolite respectively. PXL increases LY2780301 CL from 3.57 to 4.4 L/h (p =0.016).

Conclusions: We have demonstrated a PK DDI between LY2780301 and PXL. PXL increases LY2780301 CL and decreases LY2780301 AUC. PXL PK modeling is ongoing in order to verify the effect of LY2780301 on PXL PK.

#2050

**Porcine drug metabolism and toxicity** in vitro **model utilizing transformed hepatocyte cell lines (HepCre).**

Arun K. De, Kwame A. Darfour-Oduro, Laurie Rund, Kyle M. Schachtschneider, Kuldeep Singh, Lawrence B. Schook. _University of Illinois, Urbana, IL_.

To date, in vitro cytotoxicity assays are not highly predictive of in vivo toxicity. The adverse effects of new drugs are often not discovered until preclinical animal safety studies or even clinical trials; 40% of drugs drop out in preclinical animal studies and 89% of those that reach clinical trials fail. There is a critical need for more predictive and reliable in vitro testing methods. Due to its physiological similarities with humans, pigs have emerged as a suitable and reliable animal model for pharmacological and toxicological studies. To further the pigs' suitability, we have developed and characterized a transformed porcine hepatocyte cell line to support drug toxicity and metabolism assessments. Primary porcine hepatocytes isolated by a modified procedure of Panda's method (Panda et al., 2015) express phase I and II drug metabolism, phase-III transport, and nuclear receptors involved in regulation of drug metabolism transcripts. However, primary hepatocytes have a limited life span in culture and usually within 8 days post culture more than 50% of cells undergo apoptosis. Moreover, normal gene expression declines from day 5 in culture. To overcome these limitations, we have generated and characterized transformed hepatocyte cell lines (HepCre) derived from the transgenic Oncopig. Hepatocytes were isolated from Oncopigs (Schook et at., 2015) and transformed by treatment with Cre recombinase. When cultured in the presence of a differentiating agent (DMSO), the HepCre cell lines maintained normal hepatic functions, as well as the potential to express all the main drug metabolism and regulation genes comparable to that of cultured primary hepatocytes. The effect of model CYP (cytochrome P450) inducers (3- methylcholanthrene, rifampicin and phenobarbital) on the expression of CYP enzymes in primary hepatocytes and HepCre were studied. Treatment of 3-methylcholanthrene caused a significant upregulation of CYPA1 and CYPA2 transcripts, but the transcript of other CYPs remain unchanged. Both rifampicin and phenobarbital exposure resulted in the upregulation of several CYP transcripts, including CYP2A19, CYP2B22, and CYP3A. The gene transcription patterns in HepCre were similar to those of primary hepatocytes and in vitro human models. Toxicity responses of HepCre cells to hepatotoxic drugs (i.e. aflatoxin B1, chlorpromazine, amiodarone, and acetaminophen) was evaluated. After 72 hr of exposure, cell viability decreased in a concentration-dependent manner with IC50 of 5 µm, 50 µm, 20 µm, and 70 µm for aflatoxin B1, amiodarone, chlorpromazine, and acetaminophen, respectively. These findings indicate that this porcine transformed HepCre cell line represents a useful and predictive model for high throughput screening of new drugs as well as studies on metabolism and hepatotoxicity of chemicals.

References:

Panda S et al., (2015) PLoS ONE 10 (3): e0118841.

Schook, L.B et al (2015). PLoS One 10: e0128864.

#2051

Tumor-treating fields (TTFields) intensity in the gross tumor volume and peritumoral brain zone: implications for local recurrence in glioblastoma.

Aafia Chaudhry,1 Zeev Bomzon,2 Hadas Sara Hershkovich,2 Dario Garcia-Carracedo,1 Cornelia Wenger,3 Uri Weinberg,2 Yoram Palti2. 1 _Novocure, New York, NY;_ 2 _Novocure, Haifa, Israel;_ 3 _Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal_.

The purpose of this study was to simulate the intensity of TTFields delivered to the brain during the course of glioblastoma (GBM) treatment and to determine whether therapeutic intensities are delivered to the proximal peri-tumoral brain zone (PBZ). Background: TTFields are low-intensity (1-3 V/cm), intermediate frequency (200kHz), alternating electric fields delivered orthogonally in a localized manner during the course of GBM therapy. A recent phase 3 randomized, controlled trial conducted in patients newly diagnosed with GBM was stopped early for efficacy when the end points for progression-free survival (PFS) and overall survival (OS) were met at the interim analysis. Patients receiving TTFields in combination with temozolomide (TMZ) had a significantly longer PFS and OS compared with patients receiving TMZ alone. More than 90% of GBM recur at the margin of a resection cavity or within the PBZ where the presence of infiltrating tumor cells, inflammatory cells and tumorigenic stromal cells are thought to promote recurrence. Phantom model simulation studies suggest that field intensities >1V/cm are delivered to the brain in a non-uniform distribution, however the field distribution to the gross tumor volume (GTV) and PBZ have not been previously evaluated. Methods: Two MRI cases (frontal and posterior-parietal tumors) were used to generate TTFields treatment array layout maps using NovoTAL(TM) System planning software, targeting areas of contrast enhancement on T1 sequences. Simulations for the respective array layouts were created for solid tumors, resection cavities and for tumors with a necrotic core (modified Colin27 model, meshed and solved using the Sim4Life software solver package). Two orthogonal fields (left-right and antero-posterior) at a field frequency of 200 kHz were employed for all simulations. Field intensity was determined in the GTV, tumor margin(TM) and proximal PBZ (20mm) for all models. Results: Transducer array layout maps generated by the NovoTAL software deliver therapeutic intensities of TTFields in both L-R and A-P directions. Bi-directional intensities exceed therapeutic levels (>1 V/cm) in the GTV (median 1.84 V/cm), TM (median 1.9 V/cm) and PBZ (median 2.09 V/cm) in all solid tumors and in the PBZ (median 1.83 V/cm) surrounding a gross total resection (GTR) cavity. The highest areas of field intensity are observed directly adjacent to resection cavities and the ventricles. Conclusions: The delivery of therapeutic intensities of TTFields to patients who have undergone a GTR, subtotal resection or who have inoperable GBM, targets therapy to the area of active disease and importantly, to the PBZ. TTFields target residual tumor cells in the GTV and may also disrupt infiltrating tumor cells in the PBZ. Clinically, this may decrease local GBM recurrence rates and prospective clinical studies are warranted to explore this further.

### Drug Delivery

#2052

Efficient thermoregulation of heat-induced enzyme-prodrug therapy using recombinant E. coli containing a modified λpR promoter.

Venkata K. Nemani, Barjor Gimi. _Geisel School of Medicine at Dartmouth, Lebanon, NH_.

Introduction: The λpR-cI857 transcriptional promoter cassette drives heat-induced protein expression in recombinant E. coli by inhibiting transcription below 37 °C (1). We have earlier reported on the development of magnetic nanoparticle hyperthermia (MNP) induced enzyme-prodrug therapy based on recombinant E. coli cells. The E. coli are designed to express cytosine deaminase (CD) under the transcriptional control of the λpR-cI857 promoter (2). CD converts non-toxic 5-fluorocytosine (5-FC) to the toxic 5-fluorouracil (5-FU). E. coli cells were co-encapsulated with MNP in immunoisolative alginate microcapsules and heated to 43 °C using an external alternating magnetic field (AMF). Using in vitro MTT assays, we demonstrated that the cytotoxicity of the heat activated E. coli in the presence of 5-FC, against 9L, MCF-7 and PC-3 tumor cells, was comparable to direct 5-FU treatment (2). However, we observed significant cytotoxicity at 37 °C resulting from basal CD expression. We now report on an improved strain of E. coli cells using a modified λpR promoter to minimize CD expression at 37 °C.

Methods: A single base mutation was introduced in the λpR promoter during PCR amplification (1) from the pLDR20 vector. The modified promoter was ligated to the linearized pNEB206A vector and transformed into NEB-α cells using the USER™ cloning protocol (www.neb.com). NM522 cells containing the modified λpR-cI857 cassette and the CD gene were constructed and characterized following procedures established in our laboratory (2). The E. coli were then encapsulated in alginate microcapsules and heated at 43 °C to trigger CD expression. Cytotoxicity against CT26 colon cancer cells was evaluated using a MTT assay following the incubation of the cancer cells with the heated microcapsules and 5-FC (0.1 mM, 72 h, 37 °C).

Results summary: We observed thermoselective CD expression and catalytic activity in E. coli heated at 43 °C. MTT cell viability assay shows that the cytotoxicity of the encapsulated, heat-activated E. coli cells against CT26 colon cancer cells was comparable to direct treatment with 5-FU. Importantly, the basal cytotoxicity at 37 °C was significantly lower than the E. coli cells activated at 43 ºC. We are extending our work to express CD periplasmically in endotoxin free hosts for improved catalytic efficiency and to eliminate cytotoxicity arising from bacterial lipopolysaccharides. We expect that the improved E. coli cells should facilitate de novo synthesis of 5-FU, on-demand and locally in the tumor, thereby reducing systemic toxicity and increasing therapeutic gain.

Acknowledgement: This work is supported by a pilot grant from the Hitchcock Foundation.

References:

(1) Jechlinger et al, FEMS Microbiol Lett. 1999;173(2):347-52

(2) Nemani et al, J Biotechnol. 2015, 203:32-40.

#2053

Effects of thymoquinone encapsulation on its uptake, delivery and anticancer activity in breast cancer cells.

Isabelle Fakhoury,1 Walid Saad,1 Kamal Bou Hadir,1 Regine Schneider-Stock,2 Hala Gali-Muhtasib1. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _University of Erlangen, Erlangen, Germany_.

Introduction: Thymoquinone (TQ) is a promising anticancer molecule which suffers from limited bioavailability. Drug encapsulation is commonly used to overcome low drug solubility, bioavailability as well as nonspecific targeting. For this project, we synthesized different TQ nanoparticles (TQ-NPs), characterized their properties, uptake and delivery mechanisms, and assessed their anticancer potential in a panel of breast cancer cells.

Methods: The different TQ-NPs were prepared using Flash nanoprecipitation. Dynamic light scattering and scanning electron microscope were used for the characterization of the size, morphology and stability of the NPs. The anticancer effect was assessed by MTT. We subsequently formulated fluorescent TQ-NPs and evaluated their uptake and subcellular intake mechanism by both fluorometry and confocal microscopy.

Results: We were successful at formulating four different stable TQ-NPs that had an average diameter size between 45-130 nm. All TQ-NPs had also high entrapment efficiency and loading content. In vitro, TQ-NPs enhanced the antitumor activity of the drug in both MCF-7 and MDA-MB-231 cells, when compared to free TQ with no significant cytotoxicity of the blank NPs. Fluorescent TQ-NPs uptake was further found to be both time and concentration dependent. Finally, using inhibitors of endocytosis we determined that the endocytosis of TQ-NPs is caveolin mediated. This was also confirmed by examining the subcellular localization of TQ-NPs. The nanoparticle formulations colocalized with both caveolin and transferrin but not with lamp-1 and EEA-1 proteins.

Conclusion: Altogether, our results describe a new approach for the enhancement of TQ anticancer activity and provide insights on the uptake dynamics specific to our formulation.

#2054

Improved delivery of paclitaxel to prostate tumors: a membrane-type matrix metalloproteinase (MT-MMP)-targeted approach.

Paul M. Loadman, Javier Giménez-Warren, Andrew Mitchell, Amanda D. Race, Jade A. Spencer, Steve D. Shnyder, Jason H. Gill, Robert A. Falconer. _Univ. of Bradford, Bradford, United Kingdom_.

Introduction : Membrane-type matrix metalloproteinases (MT-MMPs) are highly active in prostate tumours, but absent or inactive in normal tissues. MT-MMPs are also known to be elevated in the majority of solid human tumours and to be central to tumor invasion and angiogenesis. Our objective has been to design inactive prodrugs of paclitaxel that are converted to the active drug by selected MMPs within the prostate tumor microenvironment.

Methods and Results : We report the synthesis and biological evaluation of a new series of peptide-based conjugates of paclitaxel designed to be selectively cleaved by MT-MMPs in the tumour microenvironment. Paclitaxel is conjugated to the peptide C-terminus via a self-immolative linker, while the N-terminus is protected from non-specific exopeptidase cleavage through the use of a masking group.

The relative importance of individual amino acids within the MT-MMP peptide recognition sequence has been investigated following extensive ex vivo metabolic studies. These studies employed tissue homogenates to assess activation of prodrug conjugates in tumour (PC-3) tissues and stability in normal tissues (liver kidney lung). ICTTax5 emerged as our lead agent, demonstrating in vitro stability in normal tissue with differential release of free paclitaxel in tumour tissue.

Further in vivo murine pharmacokinetic studies monitoring paclitaxel release in liver, lung, kidney heart and plasma revealed a substantial increase in tumour exposure to paclitaxel following ICTTax5 prodrug administration (20mg/kg) compared to the molar equivalent dose of paclitaxel alone (7mg/kg). AUC (uM.h) ratios of paclitaxel released from ICTTax5 compared to paclitaxel administered alone were 16.2 for tumour and in the range of 0.05-0.99 for normal tissues. Similar highly significant changes in Cmax were demonstrated with a 10-fold (9.77) increase in tumour concentrations and a substantially decreased Cmax ratio (0.02 - 0.19) in other tissues when administered as ICTTax5.

Anti-tumour efficacy studies (PC3 xenografts) resulted in a highly significant growth delay following single dose administration of ICTTax5 [20mg/kg] with paclitaxel alone (7mg/kg; the molar equivalent dose to the paclitaxel released from ICTTax5) having no significant anti-tumour effect.

Conclusion : A series of peptide-based prodrug conjugates of paclitaxel were synthesized. ICTTax5 was identified as the lead molecule, enabling selective delivery of active paclitaxel to PC3 prostate tumours resulting in superior pharmacokinetics and efficacy when compared to delivery of paclitaxel alone.

#2055

A first-in-class hemoglobin-floxuridine conjugate for the treatment of advanced stage liver cancer.

Steve Brookes, Murray J. Cutler, Jin Seog Seo, Gord Adamson, David Bell. _Therapure Biopharma Inc., Mississauga, Ontario, Canada_.

Hepatocellular carcinoma (HCC) is the second most common cause of death from cancer worldwide; few treatment options are available, especially for advanced stage disease. Therapure has developed a novel drug delivery platform based upon the attachment of drugs to hemoglobin (Hb) as a means of targeting the liver. TBI 302 is a hemoglobin-drug conjugate (HDC) designed to deliver the anticancer drug floxuridine to the liver to improve treatment for HCC. Systemic toxicity associated with free floxuridine restricts its use to locoregional administration (0.2-0.5 mg/kg/d) via continuous hepatic arterial infusion. Although hepatic arterial infusion of floxuridine can reduce hepatic tumor burden, toxicity from floxuridine and complications associated with direct hepatic infusion pumps can be significant. HDC technology exploits the natural pathway for hemoglobin clearance through the liver to provide selective drug targeting while preserving floxuridine activity following standard intravenous (IV) infusion. In a preclinical efficacy study of TBI 302, mice bearing human liver cancer cell line-derived orthotopic liver tumors received twice-weekly tail vein dosing of saline, 3.7 mg/kg floxuridine, or TBI 302. Following 6 weeks of treatment, 70% of the orthotopically implanted mice treated with 170.5 mg/kg TBI 302 (3.7 mg/kg floxuridine) had no measurable liver tumors, indicating suppression of tumor growth. In contrast, only 30% of mice treated with an equivalent dose of unconjugated floxuridine and 20% of mice treated with saline experienced liver tumor growth suppression. These results demonstrate the capability of the HDC platform to enhance the efficacy of cytotoxics through liver targeting. To establish a safe starting dose for a first-in-human Phase 1 trial, a GLP-compliant repeat dose preclinical safety study of TBI 302 in cynomolgus monkeys was conducted. TBI 302 administered by 1-hour IV infusion once per week for 8 weeks at doses of 2, 5, and 10.5 mg/kg (0.08, 0.19, and 0.4 mg/kg floxuridine, respectively) was well tolerated at all dose levels. Increasing doses of TBI 302 resulted in proportional increases in area under the concentration-time curve (AUC) and maximum concentration (Cmax) of total plasma floxuridine. No clinical signs or biochemical toxicity was associated with IV infusion of TBI 302. The no-observed-adverse effect level (NOAEL) of TBI 302 was determined to be the highest dose level of 10.5 mg/kg (0.4 mg/kg floxuridine). A Phase I safety study of TBI 302 as second-line therapy in HCC has been approved by the FDA. The primary objective is to determine safety and tolerability of TBI 302. Secondary objectives are to determine TBI 302 pharmacokinetics and effects on tumor burden. Therapure's HDC platform represents a new class of therapy that offers liver-specific targeting, while potentially reducing extra-hepatic toxicity of drugs.

#2056

Development of quaternary amine linkers for ADCs: Application to auristatin E and tubulysin.

Patrick J. Burke, Joseph Z. Hamilton, Thomas A. Pires, Jocelyn R. Setter, Joshua H. Hunter, Julia H. Cochran, Brian E. Toki, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. _Seattle Genetics, Inc., Bothell, WA_.

The recent clinical success of antibody-drug conjugates (ADCs) has spawned an increased effort to identify new technologies, and the development of new drug-linker chemistry is vital to expand the scope of conjugatable payloads. The tertiary amine functional group is a common structural motif present in many bioactive compounds, including antimitotics of the auristatin and tubulysin classes. Traditionally, conjugation of tertiary amines required drug derivatization or modification to remove an N-alkyl group, thus creating a readily conjugatable secondary amine. However, identifying appropriate modifications that do not compromise the activity of the drug is frequently time consuming and often unsuccessful. To eliminate the need for such structural modifications, we sought a method for stable conjugation and facile release through the tertiary amine functional group by creating linkers with a quaternary amine point of attachment. To validate the linker strategy, quaternary amine-based cleavable linkers bearing auristatin E were synthesized and evaluated as ADCs. The conjugates were stable in rodent plasma, and were potent and immunologically specific both in vitro and in vivo in a Hodgkin lymphoma xenograft model. A second application of this technology has been demonstrated with tubulysins, another class of potent antimitotics containing a tertiary amine at the N-terminus. A cleavable quaternary amine linker containing a tubulysin analog was synthesized and ADCs were prepared and evaluated. The tubulysin conjugates were potent and immunologically specific across a panel of cancer cell lines, including multiple MDR-positive lines. Furthermore, the tubulysin conjugate displayed 'bystander activity' in an in vitro co-culture assay. The quaternary amine linkers represent an advance in linker technology and will enable the evaluation of drug classes previously inaccessible as ADCs.

#2057

Dual peptide-mediated targeted delivery of siRNAs for the treatment of oral cancer.

Angela A. Alexander-Bryant,1 Haiwen Zhang,2 William Pugh,2 Lu Dinh,2 Christopher Attaway,2 Laurence Eggart,2 Robert Sansevere,2 Liliana Cantini,2 Andrew Jakymiw2. 1 _Clemson University, Clemson, SC;_ 2 _Medical University of South Carolina, Charleston, SC_.

Two major hurdles for small interfering RNA (siRNA)-mediated therapies are cell/tissue-type targeted delivery and endosomal entrapment of siRNAs, resulting in inefficient gene silencing. As a result, the objective of this study was to examine the feasibility of utilizing two peptides, one with cancer cell-targeting specificity and the second with endosome-disruptive properties, to co-deliver bioactive siRNAs into oral cancer cells overexpressing the epidermal growth factor receptor (EGFR) and induce silencing of the targeted oncoprotein, CIP2A. Previously, we designed a novel endosome-disruptive peptide, termed 599, that was demonstrated to mediate intracellular delivery of bioactive siRNAs targeting CIP2A (siCIP2A) in vitro. Furthermore, we recently demonstrated that 599 peptide-mediated delivery of siCIP2A into tumor tissues via intratumoral injections reduced the expression of CIP2A and significantly inhibited tumor growth. In order to induce targeted uptake via systemic administration, we designed the EGFR targeting peptide, GE11R9, which was found to deliver siRNAs specifically to EGFR-overexpressing oral cancer cells; however, it was incapable of mediating the delivery of bioactive siRNAs due to endosomal entrapment. Consequently, we developed a novel dual peptide delivery strategy, combining the 599 peptide, with the GE11R9 peptide and examined their ability to co-deliver siRNAs. Our results demonstrated that the dual peptide complex exhibited effective binding and specific uptake of siRNAs into EGFR-overexpressing cells compared to low-EGFR-expressing cells. The co-addition of the 599 peptide with the GE11R9 peptide also restored siRNA functionality. Furthermore, when administered systemically to mice bearing xenograft oral tumors, the dual peptide complex mediated increased targeted delivery of siRNAs into tumor tissues in comparison to the 599 peptide alone. Together, these data demonstrate the clinical potential of the dual peptide strategy for RNAi-based therapeutics by synergistically mediating the effective targeting and delivery of bioactive siRNAs into EGFR-overexpressing cancer cells.

#2058

Identification of a specific RNA aptamer targeting non-small cell lung cancer by in vivo SELEX.

Hanlu Wang,1 Yibang Zhang,2 Chang Chu,1 Wei Dai,3 Rihe Liu,4 Yongping Jiang1. 1 _Biopharmaceutical R &D Center, Peking Union Medical College of Tsinghua University, Suzhou, China; _2 _Biopharmagen. corp., Suzhou, China;_ 3 _NYU Langone Medical Center, Tuxedo, NY;_ 4 _Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, NC_.

In vivo selection of the RNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX) has demonstrated a great targeting tool for diagnostics and treatments in the clinic. In this study, in vivo selection of specific RNA aptamer for cells was established through the use of tumor-bearing xenograft mice injected with human non-small cell lung cancer NCI-H460 cells. Briefly, a synthetic DNA pool was first transcribed in vitro using mutant T7 RNA polymerase (Y639F), which is capable of recognizing 2'-fluoropyrimidines to produce nuclease-resistant RNA. PEGylated RNA library was then constructed to produce stable PEG-RNA molecules. A cDNA library was produced and was further transcribed for an RNA pool. The enriched nuclease-resistant PEG-RNA pool was re-injected into NCI-H460 tumor-bearing mice again. After multiple cycle selections in vivo, the cDNA pool was cloned into T-vector for sequencing analysis for enriched RNA motifs.

On round 8 of SELEX selection, an enriched RNA sequence (RA16) was identified at a frequency of 21.2%. The RA16 preparation was further enriched up to 94.7% on round 11. To characterize the specificity in vitro, NCI-H460 cells, along with additional tumor cells or normal control cells, were incubated with Cy3-labeled RA16 or the initial RNA library. The specific fluorescent binding was observed for HCI-H460 and H1299 cells, but not for HEK293T, Hela, and H226 cells. Binding affinity (Kd 9±2 nM) for RA16 was determined by flow cytometry using FITC-labeled RA16 to HCI-H460 cells. Meanwhile, cryosections from various organs of tumor-bearing mice also revealed strong binding of RA16 with HCI-H460 tumors but only slightly with lung tissue. No any significant binding was detected in other tissues of tumor-bearing mice or in any normal tissues of control mice.

Furthermore, a quantitative-RT-PCR was performed to trap specific RA16 sequence from various tissues of control or tumor-bearing mice. The selected RNA aptamer RA16 or the initial RNA library was injected into tumor-bearing(n=4) and normal control mice (n=4). Total RNAs were extracted from the mouse tumor or from heart, liver, spleen, lung and kidney 3.5 hrs post injection. There was an enrichment of 44.3±3.6-folds for the injected RA16 in mouse tumors treated by RA16 preparation comparing with mouse tumors treated by the initial RNA library. No significant enrichment of RA16 was detected in any other tissues of tumor-bearing mice or tissues from normal control mice. Significantly, in vivo tumor imaging study with Cy5.5-labeled RA16 showed that aptamer RA16 was gradually migrated toward tumor site and enriched with time at tumor area in mice. Our studies thus strongly suggests that RNA aptamer RA16 was specifically selected for targeting human NCI-H460 cells, as well as xenograft tumor tissues with high affinity both in vitro and in vivo.

#2059

Identification of novel paclitaxel sensitivity genes via lentiviral CRISPR/Cas9 screening in haploid human cells.

Jessica R. Hunt, Steffen Lawo, David Walter, Isabelle Nett, Joanne Yarker, Benedict C S Cross, Jonathan D. Moore. _Horizon Discovery, Cambridge, United Kingdom_.

Some cancers lack clearly druggable driver mutations and might also have too few neo-antigen generating mutations to provoke a tumor-specific immune response, even in the presence of antibodies targeting immune checkpoint mechanisms. Synthetic lethal/non-oncogene addiction represents an alternative approach to the therapy of such cancers, as exemplified by the recent approval of olaparib.

RNA interference has identified several putative synthetic lethal targets, but few, if any, of these have have withstood vigorous validation. The recent application of RNA-guided gene editing to mammalian cells offers new prospects for target identification. The complete ablation of gene expression, which this technology permits allows the identification of targets that have no phenotype when partially depleted by RNA interference.

Horizon has recently established a capability for CRISPR-Cas9 KO screening. Screens are performed by transducing a pool of cells with a complex lentiviral library. Next generation sequencing is then used to identify which sgRNAs are accumulating or being depleted from the population. As there are multiple sgRNAs per gene, we can rank genes for their likelihood of having a phenotype under the conditions used in the screen. Our proof-of-principle small molecule resistance and sensitivity screens using both whole-genome libraries and custom subset libraries have successfully identified previously published hits and also yielded novel hits.

Horizon has also developed a fully haploid cell line (eHAP), which has the potential to provide additional clarity in genetic screening. To evaluate the power of CRISPR-Cas9 KO screening in haploid cells, we have used our screening platform to identify genes required for survival in low doses of paclitaxel. Whole-genome screening using the GeCKOv2 library identified three kinesins amongst the most prominent of the hits whose depletion sensitised cells to this microtubule stabilising agent. Intriguingly, two of these kinesins, KIF2A and KIF2C are thought to destabilise mitotic microtubules, providing an attractive explanation for the synthetic lethality.

We will also present results from a second genome-wide CRISPR KO screen in haploid cells to identify genes required for survival under glucose starvation. Prominent hits included components of the electron transport chain and the tuberous sclerosis complex.

#2060

Polymer mediated RNAi-based combination therapy for treating prostate cancer.

Michael Danquah. _Chicago State University, Chicago, IL_.

Purpose. To determine whether RNAi-based combination therapy targeting androgen receptor (AR) and XIAP signaling pathways using polymeric micelles can treat prostate cancer.

Methods. Potency of siRNAs targeting AR and XIAP genes was assessed using real time RT-PCR, Western Blot and ELISA. Cell viability was determined by MTT assay. A library of polymers designed to degrade via hydrolysis and enhance siRNA delivery was synthesized by conjugating hexylamine, pentaethylenehexamine and 3-morpholinopropylamine to poly(ethylene glycol)-block-poly(5-methyl-5-carboxyl-propylene carbonate) backbone using EDC/HOBT coupling chemistry. Polymer structure and molecular weight were verified with proton NMR and Gel Permeation Chromatography, respectively.

Results. Three siRNA sequences targeting different regions of the AR and XIAP genes were designed, blasted against the human genome database to eliminate cross-silencing of nontarget genes and their potency screened in LNCaP and C4-2 cells following transfection. All siRNAs targeting AR (start site: 1172, 1648 and 1768) used in this study silenced AR mRNA and protein expression in both cell lines. Furthermore, 1172 was identified as the most potent sequence resulting in approximately 75% knockdown regardless of cell type. Also, the three siRNAs targeting XIAP (start site: 264, 297 and 458) down-regulated XIAP mRNA and protein levels in both cells and were generally more potent in C4-2 cells compared to LNCaP cells. C4-2 cells transfected with siRNA targeting AR (start site: 1172) and XIAP (start site: 264) alone or in combination and assayed after 96 hours revealed XIAP and AR down-regulation resulted in approximately 20% and 50% growth inhibition in C4-2 cells, respectively, compared to control. However, combined silencing of XIAP and AR genes decreased cell growth by 62% and was more potent in inhibiting cell proliferation compared to monotherapy. Additionally, combination of siRNA targeting AR (start site: 1172) and XIAP (start site: 264) significantly increased percentage of cells undergoing apoptosis (85%) compared to control or monotherapy. Poly(ethylene glycol)-block-poly(5-methyl-5-carboxyl-propylene carbonate) copolymer was synthesized by ring opening polymerization and conjugated with various ratios of hexylamine, pentaethylenehexamine and 3-morpholinopropylamine moieties. The resulting copolymer (PEG-b-P(CC-g-PHEXYL-g-PMORPH-g-PPHEA) were non-toxic even at concentration of 1500 µg/mL and successfully condensed siRNA at N/P of 25 with average hydrodynamic diameter of approximately 175 nm.

Conclusions. Combination based therapy using siRNA targeting AR and XIAP can potentially treat prostate cancer.

#2061

**DK049, a novel acid-sensitive prodrug of gemcitabine: design,** in vitro **properties and** in vivo **efficacy.**

David Koester, Javier Garcia, Olga Fuchs, Felix Kratz. _CytRx Corporation, Freiburg, Germany_.

Gemcitabine (Gemzar®) is an anticancer nucleoside that is widely used to treat solid tumors. Unfortunately, at its recommended dose of ~1000 mg/m2 approximately 90 % are deactivated by cytidine deaminase to the inactive uridine metabolite and excreted in the urine. A further disadvantage resulting in chemo-resistance is the low expression level of the human equilibrative nucleoside transporter 1 (hENT1) on the cell surface of cancer cells thus preventing substantial uptake of gemcitabine. In order to circumvent these disadvantages, we designed an innovative gemcitabine prodrug DK049 that a.) is capped at the amino group of the cytosine moiety with acetyl benzoic acid preventing deamination, and b.) incorporates a proprietary maleimide-bearing LADR™ linker forming a stable, but acid-sensitive hydrazone bond. At its 5´-OH position a phosphate group was introduced rendering water-solubility. Following intravenous administration, DK049 binds rapidly and selectively to circulating albumin and utilizes the advantages of albumin-mediated tumor uptake as well as cellular uptake by endocytosis (1).

After cleavage of the acid-sensitive benzoyl hydrazone bond, we could show that the acetyl benzoic acid is decapped by carboxylesterase 2. In a head-to-head comparison with gemcitabine (4 x 240 mg/kg on a weekly basis) in four subcutaneous human patient-derived tumor xenografts (NSCLC: LXFE397, LXFE937, ovarian cancer: OVXF899, and pancreatic cancer: Panc11159; 8-10 nude mice per group), DK049 (4 x 24 mg/kg gemcitabine equivalents or 8 x 18 mg/kg gemcitabine equivalents over 4 weeks) demonstrated distinctly superior antitumor efficacy versus gemcitabine (p < 0.05). In the LXFE937 and OVXF899 model, DK049 induced long-term significant regressions for several weeks after end of therapy without any loss in body weight or bone marrow toxicity. In summary, these results show a superior long-term antitumor efficacy of DK049 compared to gemcitabine at a significantly reduced dose.

(1) Felix Kratz, Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release 8;132:171-83, 2008.

#2062

Nanoemulsion formulations for anticancer agent piplartine characterization, toxicological, pharmacokinetics and efficacy studies.

Neel Fofaria, Alok Ranjan, Hussaini Syed Sha Qhattal, Xinli Liu, Sanjay K. Srivastava. _Texas Tech University Health Sciences Center, Amarillo, TX_.

Piplartine (PL) is an alkaloid found in black-pepper and known for its anticancer activity, however, due to poor solubility and lack of proper formulation, its use for oral

administration is a challenge. The objective of this study was to formulate PL into nanoemulsion drug delivery system for oral delivery and thereafter evaluate toxicity, pharmacokinetics and therapeutic efficacy. Optimized nanoemulsions were formulated by self-emulsification as well as by homogenization-sonication method. Two nanoemulsions enhanced the solubility of PL with low polydispersity index and high stability. Both PL loaded nanoemulsions exhibited enhanced dissolution, cellular permeability and cytotoxic effects as compared to pure PL. Formulation of PL into nanoemulsions did not obstruct its cellular uptake in cancer cells. Blank or PL loaded nanoemulsions did not exhibited toxicity in mice upon daily oral administration for 60 days. Pharmacokinetics of PL followed a two-compartment model after intravenous administration. PL loaded nanoemulsions showed 1.5-fold increase in oral bioavailability as compared to free PL. Finally, PL loaded nanoemulsions showed marked anti-tumor activity at a dose of 10 mg/kg in melanoma tumor bearing mice. In conclusion, for the first time we have developed a stable nanoemulsion delivery system for oral administration of PL, which enhanced its solubility, oral bioavailability and anti-tumor efficacy.

#2063

Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells.

Rohan A. Lalani, Priyanka Bhatt, Mohan Rathi, Ambikanandan Misra. _The Maharaja Sayajirao University of Baroda, Vadodara, India_.

Aim of the study was to prepare and characterize RGD grafted PEGylated liposomes of gemcitabine (PLGs) and to evaluate its cellular uptake, in vitro anti-proliferative activity and apoptotic effect in siRNA pretreated lung cancer cells.

PLGs were prepared by thin film hydration method and optimized for particle size, zeta potential and entrapment efficiency. Functionalization of liposomes was done by coupling reaction between DSPE mPEG and cRGD peptide by maleimide based reaction. Similarly, RGD grafted RRM1 siRNA liposomes were also formulated and evaluated. MTT assay was done to determine IC50 values of RGD grafted PLGs in A549 and H1299 cancer cells which were pre-treated with RGD grafted RRM1 siRNA loaded liposomes at a concentration of 50 pM of siRNA for gene silencing. DNA content analysis was done by flow cytometry using rhodamine in A549. The mode of cell death at different time and concentration was determined by FITC-AnnexineV assay in A549 cells and confocal microscopy was performed to assess the potential of RGD grafting on cellular uptake.

RGD conjugated and unconjugated PLGs were found Nano sized and had negative zeta potential with entrapment of 65%. H1299 cell line showed more amount of viable cells after 48 hr as compared to A549 cells in RGD conjugated PLGs. siRNA pre-treated PLGs exposed cells showed significantly less IC50 values as compared to cells without siRNA pretreatment and non-grafted liposomes. The results showed that the RGD conjugated liposomes at the concentration of 7nM showed 46% G1 phase arrest in siRNA pretreated cells as compared to 22% G1 phase arrest without prior siRNA treatment at 16hr. Two types of mode of cell death were found during the FITC-Annexine V assay. At 24 hr, the treatment with RGD grafted PLGs resulted in 17% & 4.4 % necrotic & apoptotic cell death respectively. While at equivalent drug concentration, the PLGs and drug solution showed 5.3% & 32.2% and 4.2% & 29.6% necrotic & apoptotic cell death respectively. Furthermore, the apoptosis was found to be time and concentration dependent. Results substantiate the sensitization effect by pre-exposure of siRNA in liposomal forms at Pico molar concentration along-with phagocytosis as mechanism of uptake of RGD-grafted liposomes.

To conclude, prior silencing of the resistance imparting gene can manifest the effect of therapy by conferring improved sensitivity in cancer cell lines. The effect can further be augmented by employing receptor targeting peptides such as RGD. Hence, Nano-constructs of chemotherapeutic drugs conjugated with RGD can effectively target lung cancer cells and pretreatment of RRM1 siRNA can probably reduce the limitation of drug resistance associated with lung cancer chemotherapy.

#2064

Novel HIF-2α targeted RNAi therapeutic for renal cell carcinoma.

So Wong, Weijun Cheng, Darren Wakefield, Aaron Almeida, Andrei Blokhin, Holly Hamilton, Vladimir Subbotin, Julia Hegge, Zane Neal, Guofeng Zhang, David Rozema, David Lewis, Steven Kanner. _Arrowhead Research Corp., Madison, WI_.

Background: Targeted therapy including VEGF and mTOR pathway inhibitors has dramatically transformed treatment options and outcomes for patients with metastatic clear cell renal cell carcinoma (ccRCC). However, alternate treatments are needed as resistance to these initially promising agents occurs frequently. RNAi interference (RNAi), an innate gene silencing mechanism, has been explored as a new class of therapeutics where conventional treatments are lacking or have failed. The challenge in leveraging this promising approach has been efficient delivery of an RNAi trigger (siRNA) to target tissue. Over 90% of ccRCC tumors express a mutant inactive form of the von Hippel-Landau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong evidence supports the observation that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF-2α), a tumorigenic driver of ccRCC.

Methods: We have developed a targeted delivery platform called Dynamic Polyconjugte™ (DPC) as an RNAi-based therapeutic targeting HIF-2α for advanced ccRCC. The ccRCC-specific DPC (ITG-DPC) comprises a membrane active polymer to promote RNAi trigger endosomal release, a ligand that binds to αV-containing integrin receptors expressed on tumor cells, reversible masking to prevent polymer activity before reaching the endosomal compartment, and a potent and specific RNAi trigger to HIF-2α. The modular nature of this delivery platform allows for flexibility to optimize each functional component independently. The ligand-dependent delivery of ITG-DPC was first evaluated in cultured tumor cells and then confirmed in ccRCC tumors established in nude mice using fluorescently-labeled ITG-DPC and confocal microscopy. To validate silencing of HIF-2α as an effective therapeutic approach, an inducible shRNA to HIF-2α was expressed in ccRCC tumors established in mice that significantly silenced HIF-2α gene expression and induced tumor regression.

Results: Bi-weekly injection of ITG-DPC into nude mice with established orthotopic A498 ccRCC tumors resulted in >80% knockdown of HIF-2α mRNA and significant tumor growth inhibition. Histological examination of tumor sections showed substantial cell killing and destruction of tumor architecture. Down-regulation of HIF-2α regulated pathways genes including VEGF-A, and the corresponding reduction in tumor-associated CD31 positive neovascularization, corroborated on-target effects of HIF-2α gene silencing.

Conclusion: Targeted delivery of a HIF-2α specific RNAi-based-therapeutic has the potential to radically impact the late-stage ccRCC treatment paradigm.

#2065

**Anti-FSHR antibody Fab' fragment conjugated immunoliposomes loaded with cyclodextrin-paclitaxel complex for improved** in vitro **efficacy on ovarian cancer cells.**

Priyanka Bhatt, Rohan Lalani, Rajashree Mashru, Ambikanandan Misra. _Department of Pharmacy, The Maharaja Sayajirao University of Baroda, Vadodara, India_.

Purpose of the study was to prepare and characterize cyclodextrin-paclitaxel (CD-PTX) complex encapsulated immunoliposomes (ILs) conjugated with Fab' fragment of anti-FSHR antibody and to determine their in vitro efficacy for cellular uptake, cytotoxicity, anti-metastatic activity and cell apoptosis on ovarian cancer (OC) cells.

CD-PTX complex was formulated using Heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) to improve aqueous solubility and liposomal loading of PTX. The complex was encapsulated in PEGylated liposomes (PLs) using reverse phase evaporation technique. ILs were prepared by covalent conjugation of Fab' fragments of anti-FSHR antibody to functionalized PLs via thioether linkage which was confirmed by SDS-PAGE analysis. The ILs were characterized for size, zeta potential, entrapment, loading efficiency and in vitro drug release profile. ILs were also evaluated for uptake efficiency and in vitro cytotoxicity in FSHR-expressing Caov-3 OC cells. To establish anti-metastatic effect, the ILs, PLs and equal amounts of PTX solution were studied on Caov-3 cells by in vitro wound scratch assay. The effect of PTX solution, PLs and ILs on Caov-3 cell apoptosis and cell cycle was examined using FACS technique.

Increased aqueous solubility of 11 mg/ml was achieved for PTX using DMBCD at molar ratio of 20 and the cytotoxic properties of CD-PTX complex was also retained similar to PTX as studied in Caov-3 cells. The PLs and ILs were found to be nanosized with optimum entrapment and improved loading efficiency of PTX. The in vitro release profiles exhibited controlled release of PTX from PLs (21.235±0.423%) and ILs (18.219±0.603%) after completion of 48 h. The IC50 values from MTT assay suggested that ILs was 3.7 and 8.1 times more cytotoxic than PLs and PTX solution respectively for Caov-3 cells after 24 h period. The ILs showed more anti-migration effect and less covered wound (39.66±5.2%) at a concentration of 3nM as compared with PTX solution (70.45±7.12%) and PLs (61.38±4.41%). After 24 h treatment no major distinction in % apoptosis was observed between PTX solution, PLs and ILs. However, after 48 h treatment, PLs and ILs respectively showed about 5.36% and 9.12% higher apoptosis when compared to PTX solution. In Caov-3 cells, after 48 h the ILs at 3nM concentration showed increased G2 phase cell arrest as compared to PLs which indicates the superiority of ILs.

To conclude, ILs displayed improved intracellular uptake and enhanced cytotoxic as well as anti-metastatic effects compared to PTX solution and PLs in OC cells. The results clearly highlight the importance of FSHR as one of the prominent targets for OC therapy and also the potential of anti-FSHR Ab Fab' conjugated nanocarriers to reduce the limitations associated with OC chemotherapy.

#2066

Albumin-binding doxorubicin prodrug selectively activated by radiation-induced apoptotic tumor cells.

Seung Woo Chung,1 Kwangmeyung Kim,2 Seong Who Kim,3 In-San Kim,2 Sang Yoon Kim,3 Youngro Byun1. 1 _College of Pharmacy, Seoul National University, Seoul, Republic of Korea;_ 2 _Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea;_ 3 _Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Republic of Korea_.

Despite the well-recognized potent anticancer effect of doxorubicin, the clinical use of doxorubicin is limited due to its dose-dependent toxicity. In this study, a novel drug delivery technology fundamentally different from the conventional tumor-targeting approaches was employed to improve the therapeutic index of doxorubicin. The prepared doxorubicin prodrug, EMC-DEVD-S-DOX, has two distinct features for effective tumor targeting: EPR effect-mediated passive targeting of tumor and extended plasma half-life by in situ albumin binding, and radiation-induced apoptosis targeting. The prodrug comprises of two important functional molecules: first, a maleimide group that allows in situ binding of the prodrug to the circulating albumin after intravenous administration; second, a DEVD motif that plays a crucial role in the activation of the prodrug through caspase-3-mediated cleavage in the apoptotic tumor cells that are exposed to radiation. As a result, EMC-DEVD-S-DOX showed a prolonged plasma half-life with selective accumulation within the tumor tissue, and was only activated and released free doxorubicin when combined with radiotherapy, thereby showing excellent synergistic effect. Our suggested drug delivery strategy completely relies on the anatomical feature of tumor vasculatures and the upregulated caspase-3 after exposure to radiation. In this reason, the genomic variations of tumor cells are unlikely to influence the efficacy of the prodrug. Considering that the conventional active tumor targeting approach is seriously challenged by the intratumor heterogeneity, EMC-DEVD-S-DOX could be an outstanding alternative for an effective cancer treatment.

#2067

Attacking prostate cancer with a prodrug-doped cellular Trojan horse.

Nathaniel Brennen,1 Oren Levy,2 Edward Han,2 David Marc Rosen,1 Juliet Musabeyezu,2 Helia Safaee,2 Sudhir Ranganath,2 Jessica Ngai,2 Martina Heinelt,2 Sandrine Billett,3 Neil Bhowmick,3 Samuel Denmeade,1 Jeffrey Karp,2 John Isaacs1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Prostate cancer is the second leading cause of cancer-related deaths in American men. Despite considerable advances in prostate cancer research, there is a major need for a systemic delivery platform that efficiently targets anti-cancer drugs to sites of disseminated prostate cancer while minimizing host toxicity. Human mesenchymal stem cells (MSCs) are excellent candidates for cell-based drug delivery since they display tropism towards cancer sites and clinical studies have demonstrated that hundreds of millions of allogeneic human MSCs can be safely administered intravenously without adverse effects in a variety of pathological settings. Furthermore, we have previously documented that MSCs can be detected in radical prostatectomy tissue from men with prostate cancer. In this proof-of-concept study, human MSCs were loaded with poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) encapsulating the macromolecule G114, a thapsigargin-based prostate specific antigen (PSA)-activated prodrug. G114-loaded MPs were successfully internalized by MSCs without impacting MSC viability, followed by sustained release of G114 as an intact prodrug from loaded cells. Moreover, G114 released from G114 MP-loaded MSCs is selectively toxic to the PSA-secreting prostate cancer cell line, LNCaP. Finally, G114 MP-loaded MSCs inhibited tumor growth when co-inoculated with CWR22 prostate cancer xenografts, suggesting that cell-based delivery of G114 does not compromise potency of the prodrug in vitro or in vivo. We envision that this MSC-based platform may be developed into an effective, systemic 'Trojan Horse' therapy for the targeted delivery of therapeutic agents to sites of metastatic prostate cancer.

#2068

Engineering of hairpin loop enhances the loading of endogenously expressed pre-miRNA into extracellular vesicles.

Dhruvitkumar S. Sutaria,1 Jinmai Jiang,1 Ola A. Elgamal,2 Ana-Clara P. Azevedo-Pouly,3 Ryan E. Pavlovicz,2 Chenglong Li,2 Mitch A. Phelps,2 Thomas D. Schmittgen1. 1 _University of Florida, Gainesville, FL;_ 2 _Ohio State University, Columbus, OH;_ 3 _University of Texas Southwestern Medical Center, Dallas, TX_.

Extracellular vesicles (EVs) hold tremendous potential as drug delivery carriers for therapeutic nucleic acids such as microRNA (miRNA) due to their natural composition and ability to be engineered to contain targeting peptides on their surface. We have engineered HEK293T cells to secrete EVs by overexpressing Lamp-2A protein containing liver cancer targeting peptide PC94 and therapeutic pre-miR-199. The novel feature of this system is that both the cargo and the microvesicles are synthesized by the same cells, thus abrogating the need for loading synthetic oligonucleotides into EVs. The pre-miR-199a loop region was modified such that it resembled the HIV-1 transactivation response (TAR) RNA which was engineered into the Lamp2A intron. Correct splicing of the intron portion and processing of the mature miRNA was evaluated after its transfection into HEK293T cells. Computational modeling was used to study the interaction between the modified pre-miR-199a loop (TAR RNA) and the TAT peptide. This study exhibited a stable interaction between the two and also showed that the peptide is not bound to the stem portion of the RNA loop, permitting the insertion of any pre-miRNA sequence. EMSA gel shift, rDICER processing and luciferase assays were performed to study the correct binding, processing and functionality of the modified sequence. These results show that the modified loop is actively involved in binding with the TAT peptide and it enables the modified miRNA to be loaded in the microvesicles. In an effort to assess the loading of the therapeutic miR-199a-3p relative to other endogenous miRNAs contained within the EVs, we performed small RNA sequencing on RNA isolated from both the producing cells and purified EVs. Triplicate samples of RNA isolated from 3 different HEK293T producing cells were sequenced: wild type HEK293T and those stably transfected with the empty vector (empty) or the TAT/TAR pre-miR-199a (full). The expression of the miRNAs were ranked from the RNA sequenced in both the cells and EVs. Producing cells engineered to express miR-199a-3p containing the TAT/TAR loading motif ranked third when comparing the ratio of miRNAs loaded in the EVs from the full to empty cells. Interestingly, certain mature miRNAs were preferentially loaded into the EVs, including miR-451a, miR-122 and miR-1246. As reported by Villarroya-Beltri, et al., (Nature Comm., 2013) in human peripheral blood mononuclear cells, all three mature miRNAs contained 4 nucleotide "exo motifs" in their 3' end that likely caused preferential loading into HEK293T EVs. Our data demonstrate that stably transfecting HEK293T cells with vectors expressing therapeutic miRNAs dramatically increases their loading into EVs. These mature miRNAs may be further engineered to contain exo motifs that could conceivably enhance their loading into EV drug carrier systems.

#2069

Activity of an EphA2-targeted docetaxel nanoliposome in pancreatic patient-derived models as monotherapy and in combination with gemcitabine.

Daryl C. Drummond,1 Ninfa L. Straubinger,2 Tista Roy Chaudhuri,2 Michael Moser,3 Walid S. Kamoun,1 Lia Luus,1 Zhaohua Richard Huang,1 Suresh Tipparaju,1 Bryan Gillard,3 Carl Morrison,3 Elizabeth Repasky,3 Dmitri B. Kirpotin,1 Robert M. Straubinger2. 1 _Merrimack, Cambridge, MA;_ 2 _U. Buffalo, Buffalo, NY;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Pancreatic cancer remains one of the deadliest cancers with survival described in number of months and weeks. Recent advances in the treatment of pancreatic cancer led to the recent approval of a liposomal irinotecan (ONIVYDETM (irinotecan liposome injection), previously MM-398). Given the activity of taxanes in pancreatic cancer and the ability of nanoliposomes to deliver drugs, we developed a novel EphA2-targeted nanoliposomal docetaxel (MM-310) and evaluated its activity in patient derived xenograft (PDX) models of pancreatic cancer as a monotherapy, as well as in combination with gemcitabine. Additionally, we aimed to test the predictive potential of key biomarkers that are linked to the MM-310 mechanism of action.

Several PDX models developed at Roswell Park Cancer Institute were screened for the expression of EphA2 (MM-310 target), CD31 (blood vessels), Massons Trichrome (fibrosis), CA XI (hypoxia), and E-Cadherin (adhesion molecule that can potentially inhibit target engagement). Eight EphA2\+ PDX models were used to evaluate the activity of MM-310 and compare it to clinically relevant agents including nab-paclitaxel, liposomal irinotecan, oxaliplatin, and gemcitabine. We also tested the combination potential of MM-310 and gemcitabine.

MM-310 was able to statistically significantly control tumor growth in all tested models with tumor regression in more than 85% of the models. When compared with standard of care agents in tumor models, at equitoxic dosing, MM-310 demonstrated greater activity to nab-paclitaxel in 80% (4/5), gemcitabine in 100% (5/5), and oxaliplatin in100% (5/5), and liposomal irinotecan in 80% (4/5). Gemcitabine is currently considered a standard of care in pancreatic cancer in combination with nab-paclitaxel, thus we conducted a study to evaluate the potential combination benefits of gemcitabine with MM-310. The combination of suboptimal doses of MM-310 and gemcitabine led to significant tumor growth control which was greater to either arm alone. Additionally, at equitoxic dosing of 50% maximum tolerated dose, MM310 + gemcitabine showed greater effect than ABRAXANE (paclitaxel protein-bound particles for injectable suspension) + gemcitabine. Although we have excluded EphA2 negative models from these studies, biomarker analysis showed that MM-310 effects are not correlated with the EphA2 expression level, suggesting that a low level EphA2 might be sufficient to mediate activity and that liposome delivery might be the rate limiting step. Additional biomarker analysis will be conducted.

In conclusion, we found that MM-310 is highly active in several patient derived models of pancreatic cancer and that it was equal or greater to most standard of care agents. Future studies will aim at identifying markers for differentiating response to MM-310 (EphA2 targeted nanoliposomal docetaxel) and ONIVYDE (irinotecan liposome injection).

#2070

Gemcitabine-loaded microparticles promote cancer cell death in subcutaneous pancreatic cancer xenografts.

Maria Munoz-Sagastibelza,1 Vadim Kurbatov,1 Sophia Dynes,1 Jennifer Caceres,1 Michael Chen,1 Raavi Gupta,1 Catherine Burkhart,2 Laura Martello-Rooney1. 1 _SUNY Downstate Medical Center, Brooklyn, NY;_ 2 _Buffalo BioLabs, Buffalo, NY_.

Pancreatic cancer is the fourth leading cause of cancer death in the United States with only 7% of diagnosed patients surviving 5 years. Most pancreatic cancer patients are not surgical candidates due to advanced stage at diagnosis. Current systemic chemotherapies, while exposing patients to the adverse side effects of treatment, have not been very effective at decreasing tumor burden primarily due to poor systemic drug uptake resulting from the dense stromal nature of pancreatic tumors. Poly(lactic-co-glycolic acid)-based (PLGA) microparticles (MPs) are a promising tool for localized drug delivery within the tumor due to their high biocompatibility, flexibility in the encapsulation of different drugs and extended drug release inside the tumor. The present study investigated whether gemcitabine-loaded microparticles (GMPs) in the range of 10-50 microns, in comparison with blank (no drug) MPs (BMPs), saline intraperitoneal injection (SIP) and gemcitabine intraperitoneal injection (GIP) as controls, are able to promote cancer cell killing effects in vivo.

In vitro studies with PANC-1 and MIAPaCa-2 human pancreatic adenocarcinoma cell lines treated with different PLGA co-polymer ratios used to encapsulate gemcitabine showed enhanced cell killing and decreased colony formation in the longer release co-polymer ratio after 2 weeks of treatment. Subsequently, the in vivo efficacy of GMPs was tested by direct injection of GMPs into established subcutaneous MIAPaCa-2 tumors in nude mice. Treatment commenced when tumor volume was approximately 250 mm3. Following two weeks of treatment, there was a trending decrease in tumor volume in the GMPs-injected MIAPaCa-2 tumors compared to the BMPs-injected tumors. When comparing the SIP to GIP groups, there was no difference in final tumor volume emphasizing the lack of effective penetration of systemic gemcitabine into the tumor. In addition, we observed less tumor progression in the GMPs group compared to the others. At the endpoint, the tumors were excised, frozen in OCT compound and sectioned to visualize fluorescent MPs and to detect apoptosis by immunofluorescence. Interestingly, we observed a significant increase in apoptosis in the tumors treated with GMPs compared to the BMP tumors (p<0.05) as well as the SIP (p<0.05) and GIP tumors (p<0.05).

In conclusion, our data suggest that gemcitabine-loaded MPs could decrease tumor volume and increase local pancreatic tumor cell death. Further studies are needed to optimize the MPs loading and injection to confirm its efficacy. The described drug delivery method has the potential to be a more efficient local treatment modality than systemic gemcitabine against pancreatic cancer.

#2071

**SW43-DOX - a potential new targeted drug for the treatment of liver malignancies - An** in vitro **evaluation of receptor affinity and tumor internalization.**

Johannes M. Ludwig,1 Yongkang Gai,2 Sun Lingyi,2 Dexing Zeng,2 Hyun S. Kim3. 1 _Division of Interventional Radiology, Department of Radiology and Biomedical Imaging, Yale University, New Haven, CT;_ 2 _Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, PA;_ 3 _Division of Interventional Radiology, Department of Radiology and Biomedical Imaging, Yale University, New Haven, CT; Interventional Oncology Translational Laboratory, University of Pittsburgh School of Medicine, PA; Yale Cancer Center, Yale, New Haven, CT_.

Purpose: The σ2-receptor is known to be overexpressed on many rapidly growing cancer cells, thus represents a tumor biomarker and is potentially attractive for targeted tumor therapy. We have synthesized the conjugate SW43-DOX consisting of the σ2-receptor agonist SW43 and the anticancer agent Doxorubicin (DOX). The purpose of this study was to compare the half maximal effective concentration (EC50) of SW43-DOX and DOX and to evaluate the receptor affinity and internalization of SW43-DOX in hepatocellular carcinoma and other cancer cell lines in vitro.

Material & Methods: SW43 was synthesized and conjugated with Doxorubicin (A Chemtek Inc.) For saturation binding & internalization assays, the chelator L-NETA was attached & loaded with Lu177. Hep G2, Hep 3B, Panc-1 & HT-29 cell lines were evaluated. EC50 was assessed by treatment with various concentrations ranging from 0.5-700 µM of SW43-DOX/Doxorubicin (CellTiter-Glo®; Promega). Saturation binding assay: σ2-receptor blocking was achieved with 10-100 µM SW43 for 15 min. at RT prior to adding 1-300 µM SW43-DOX-Lu177 in HBSS buffer (+0.1% BSA) incubating for 3.5h on ice. Internalization assay: After receptor blocking (7.5 µM SW43; 15 min.), cells were incubated with 75 nM SW43-DOX-Lu177 for .5, 1 & 2h at 37°C/5% CO2. The cell surface bound fraction from the internalization assay was scavenged by incubation with HBSS (+20 mM NaOAc; pH4.0; 10 min; 37°C/5% CO2). Cells were lysed with 0.5% SDS, radioactivity measured (Cobra -counter, Packard Bell) & normalized to whole cell protein. Statistics: EC50: Dose response non-linear fitting model; Internalization: ANOVA + Bonferroni post-hoc test, Saturation binding: One site saturation non-linear regression fit model. Prism V6.0.

Results: EC50 showed lower needed concentration (µM) for SW43-DOX compared to Doxorubicin: Hep G2 (12.0 (95%CI: 10.3-13.9) vs. 81.5 (95%CI: 66.7-99.5); p<0.001), Hep 3B (8.0 (95%CI: 7.2-9) vs. 17.6 (95%CI: 14.1-22); p<0.001), Panc-1 (21.59 (95%CI: 15.44-30.2) vs. 64.5 (95%CI: 50.3-82.6); p=0.31), and HT-29 (20.7 (95%CI: 18.3-23.3) vs. 179.8 (95%CI: 65.9-593.5); p<0.001). Maximal specific cell surface binding capacity (Bmax; pmol/mg): Hep G2 (21.5; 95%CI: 14.3-28.6), Hep 3B (35.2; 95%CI: 27.9-42.5), Panc-1 (66.5; 95%CI: 51.6-78.2) & HT-29 (33.4; 95%CI: 8.43-58.5). Specific binding affinity (KD) in nM: Hep G2 (49.2; 95%CI: 2.2-96.3), Hep 3B (40.8; 95%CI: 13.4-68.1), Panc-1 (96.4; 95%CI: 51.4-141.5), HT-29 (31.3; 95%CI: -40.5-84.4). Specific internalization was evident at all time points for all cell lines (p<0.05). Specific internalization (pmol/mg ±SD) after 2h was 29.5 ±6.6 (Panc-1), 10.3 ±1.6 (HT-29), 18.2 ±2.36 (Hep G2) & 30.7 ±8.2 (Hep 3B).

Conclusion: SW43-DOX exerts a higher antitumoral effect than Doxorubicin & binds specifically onto the cell surface with high affinity & subsequently specific uptake via the σ2-receptor.

#2072

Inhibition of breast cancer stem cells by Hedgehog-inhibitory peptide conjugated with Elastin-like biopolymers.

Jung Su Ryu, Drazen Raucher. _University of Mississippi Medical Center, Jackson, MS_.

The Hedgehog(Hh) signaling pathway has been reported to play a pivotal role in cancer. Studies have shown that suppression of this aberrantly activated Hh pathway may be effective in controlling the development and proliferation of many cancers. However, to date, there is no drug in clinical application that can be used to inhibit the Hh signaling pathway and suppress cancer growth.

In attempt to address this problem, we developed a thermally responsive polypeptide inhibitor of the Hh pathway. This polypeptide is derived from a mammalian tropo-elastin protein which will aggregate and accumulate at the tumor site where local hyperthermia is applied. For this study, ELP was fused to a peptide that inhibits Hh signaling by blocking interaction of Sonic Hh ligand (Shh) with the Patch-1 docking site. To improve bioavailability, ELP was further modified by adding the cell penetrating peptide, Tat.

The anti-proliferative activity of Tat-Hhi-ELP was examined in three breast cancer cell lines: MCF7, MDA-MB-231, and SKBR-3. Treatment of the cells with 20 uM of peptide for 2 days resulted in maximally 40% inhibition of cell proliferation. Observed cytotoxicity was further increased two fold by application of hyperthermia.

To validate that Tat-Hhi-ELP is targeting the Hh pathway, we determined the levels of GLI-1, which is a downstream target in the Hh pathway. Treatment of the breast cancer cells with Tat-Hhi-ELP resulted in reduction of GLI-1 levels, indicating that the cytotoxicity is based on hedgehog pathway inhibition. Furthermore, the formation of mammospheres was significantly reduced by treatment with Tat-Hhi-ELP in SKBR-3 cells. These results suggest that thermal targeting of ELP-based Hedgehog inhibitory peptides to breast cancer cells may be an effective and promising treatment strategy against breast cancer stem cells.

#2073

Focused ultrasound-mediated transport of poly(alkyl) cyanoacrylate nanoparticles across the blood-brain barrier in a melanoma brain metastasis model.

Habib Baghirov,1 Andreas Åslund,1 Sofie Snipstad,1 Sigrid Berg,2 Rune Hansen,2 Frits Thorsen,3 Yrr Mørch,4 Catharina de Lange Davies1. 1 _Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway;_ 2 _Department of Circulation and Medical Imaging, Norwegian University of Science and Technology; SINTEF Medical Technology, Trondheim, Norway;_ 3 _Molecular Imaging Center, Department of Biomedicine, University of Bergen; Kristian Gerhard Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen, Bergen, Norway;_ 4 _SINTEF Materials and Chemistry, Trondheim, Norway_.

Drug delivery into the brain is impeded by the blood-brain barrier (BBB) - a dynamic interface that protects brain homeostasis, but also screens the brain from the vast majority of large and/or hydrophilic drug molecules. Focused ultrasound (FUS) has emerged as one of the promising methods to open the BBB safely and reversibly. We have previously reported FUS-mediated BBB opening using a novel platform consisting of microbubbles surrounded by poly(alkyl cyanoacrylate) nanoparticles that could prospectively be used for FUS- and nanoparticle-mediated drug delivery into the brain (1,2).

Here, we have been investigating FUS-mediated BBB opening using a novel ultrasound system capable of generating FUS at two frequencies, 1.1 MHz and 7.8 MHz during the same experiment. This system allows a very precise selection of the exposure area. We used FUS exposure at 1 MHz to open the BBB by cavitation, while exposure at 7.8 MHz was employed to enable the action of acoustic radiation force. This force is caused by a transfer of momentum from acoustic waves to the tissues in which they propagate, and can facilitate nanoparticle transport in the extracellular matrix. FUS-mediated BBB opening was performed in NOD/SCID mice with melanoma brain metastases developed after intracardiac injection of human melanoma brain metastasis cells (3). The cells were fluorescently labeled, which enabled their detection in the brain. Poly(isohexyl) cyanoacrylate (PIHCA) nanoparticle-microbubbles similar to those reported in our earlier works (1,2) were injected immediately before the FUS exposure.

Successful opening of the BBB was verified by MRI using a gadolinium-based contrast agent. An optimal window of exposure intensities that allow BBB disruption was found to be close a mechanical index of 0.3. Location of fluorescently labeled nanoparticles relative to blood vessels and tumor cells was determined in frozen sections using confocal microscopy. Tissue damage and FUS-induced changes at the cellular and molecular level were studied using histological and molecular techniques.

In conclusion, the dual-frequency ultrasound transducer setup and our novel nanoparticle-microbubble platform showed promising results which will be used to develop a novel treatment of brain metastasis combining dual-frequency FUS with drug delivery using microbubbles.

1.

Nanoparticle-stabilized microbubbles for multimodal imaging and drug delivery. Mørch Ý et al. Contrast Media Mol Imaging. 2015 Sep;10(5):356-66

2.

Nanoparticle delivery to the brain - By focused ultrasound and self-assembled nanoparticle-stabilized microbubbles. Åslund AK et al. J Control Release. 2015 Oct 28;220(Pt A):287-294

3.

Automated tracking of nanoparticle-labeled melanoma cells improves the predicted power of a brain metastasis model. Sundstrøm T et al. Cancer Res. 2013 Feb;73(8):2445-2456.

#2074

Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic cancer stem cell characteristics by suppressing sonic hedgehog pathway.

Wei Yu, Yiming Ma, Sharmila Shankar, Rakesh K. Srivastava. _Kansas City VA Medical Center, Kansas City, MO_.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in US, with a 5-year survival rate of approximately 6%. Patients develop resistance to currently used anticancer drugs which are mainly toxic and also demonstrate severe side effects. Cancer stem cells (CSCs) / tumor initiating cells (TICs) have been proposed for tumor initiation, promotion, metastasis, and chemotherapy failure. Thus, there is an urgent need to develop novel strategies for the management of pancreatic cancer. Sonic hedgehog (Shh) pathway has been implicated in pancreatic carcinogenesis and is constitutively active in CSCs. The α-Mangostin is derived from the plant mangosteen (Garcinia mangostana) which is a tropical evergreen tree. Mangosteen contains Mangnoids, such as α-Mangostin, and other phytochemicals. The main objective of the study was to demonstrate the biological activities of α-Mangostin-encapsulated PLGA nanoparticles (Mang-NPs) in human pancreatic CSCs and cells lines. We have also performed docking of α-Mangostin on to the DNA binding sites of Gli to assess whether α-Mangostin disrupts Gli-DNA binding and acts as a Gli inhibitor. Mang-NPs inhibited cell proliferation, colony formation, and induced apoptosis in pancreatic cancer AsPC-1, PANC-1 and Mia-Paca-2 cell lines, and CSCs. However, Mang-NPs had no effect on human pancreatic normal ductal epithelial cells. In addition, Mang-NPs inhibited epithelial to mesenchymal transition. α-Mangostin disrupted Gli-DNA binding activity. Western blot analysis has demonstrated that Mang-NPs inhibited pluripotency maintaining factors Nanog and c-Myc. Furthermore, Mang-NPs inhibited the expression of Gli1, Gli2, Patched1 and Patched2, and Gli transcriptional activity in pancreatic CSCs. Our data demonstrate that Mang-NPs can inhibit pancreatic CSC characteristics by disrupting Gli-DNA binding activity and may act as a Gli transcription inhibitor.

#2075

Differential tissue clearance results in improved therapeutic index for irinotecan liposome injection (ONIVYDE) when combined with the PARP inhibitor veliparib in preclinical cervical tumors.

Alexander Koshkaryev,1 Jonathan Fitzgerald,1 Jaeyeon Kim,2 Ashish Kalra,2 Walid Kamoun,2 Sarah Blanchette,1 Lia Luus,2 Stephan Klinz,2 Ozan Alkan,2 Tad Kornaga,2 Susan Bates,3 Yves Pommier,4 Daryl C. Drummond1. 1 _Merrimack Pharmaceuticals, Cambridge, MA;_ 2 _Merrimack, Cambridge, MA;_ 3 _Columbia University, New York, NY;_ 4 _National Cancer Institute, Bathesda, MD_.

PARPs (poly ADP ribose polymerases) are a family of enzymes involved in DNA repair via two mechanisms: catalytic inhibition and trapping of PARP-DNA complexes. Inhibition of this repair pathway can result in cell death following DNA damage. Irinotecan liposome injection has shown promising preclinical and clinical activity in a range of cancer types, and was recently approved in the United States in combination with 5-FU/LV for patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. Irinotecan liposome injection is a highly stabilized liposomal formulation of irinotecan. It is hypothesized that combining PARP inhibitors with Top I inhibitors will result in increased efficacy compared to either agent alone. However, the preclinical promising activity has also given rise to unacceptable toxicity in the clinic for the combinations.

In preclinical study, compared with free irinotecan, irinotecan liposome injection has an extended PK profile with prolonged local tumor exposure of irinotecan and SN-38. Since SN-38 is cleared more quickly from normal tissues than from tumor, it is hypothesized that delayed dosing of the PARP inhibitor, veliparib, relative to irinotecan liposome injection will allow for the expected window of maximum irinotecan-induced toxicity to pass in the absence of concurrent veliparib toxicity. However, the tumor levels of SN-38 are predicted to be sustained by the time veliparib is given, therefore maintaining the ability of both drugs to act on tumor tissue simultaneously to maintain synergy.

To test the hypothesis that delayed dosing of veliparib relative to irinotecan liposome can alleviate systemic toxicity, a pre-clinical dose tolerability study was performed. Mice were dosed chronically with irinotecan liposome once weekly at various doses on Day 1, while veliparib was dosed once daily at a fixed dose for 3 consecutive days each week (either on Days 2-4, Days 3-5, or Days 4-6) and body weight was followed as a gross measure of toxicity. Toxicity of the combination was seen at the highest doses of irinotecan liposome when given in close proximity to the veliparib doses. However, this toxicity could be alleviated either by dose reducing irinotecan liposome or delaying the start of veliparib dosing. This dosing schedule was followed in studies in two cervical cancer tumor xenograft models, one in which veliparib alone was not efficacious, and a second in which neither irinotecan liposome or veliparib were efficacious as single agents, however the combination demonstrated tumor growth inhibition.

This promising preclinical regimen will be examined further in a clinical novel phase I study design led by the NCI/CTEP where both the dose and schedule will be optimized in parallel. The study is currently planned to be initiated in 2016.

#2076

Albumin-linked proaerolysin based molecular grenades: A systemic therapeutic for disseminated castration resistant prostate cancer.

Freddie L. Pruitt, Nathaniel Brennen, Lizamma Antony, David M. Rosen, Samuel Denmeade, John Isaacs. _Johns Hopkins School of Medicine, Baltimore, MD_.

The progression of prostate cancer (PCa) to metastatic castration resistant prostate cancer (CRPC) is an ominous clinical finding that is a significant therapeutic challenge for clinicians. Current treatments for men with CRPC are not curative and only extend survival for a little more than 12 months. Conventional anti-cancer therapies induce death of cancer cells by exploiting the relatively fast rate of growth cancer cells display in relation to normal cells. Radiation and chemotherapeutics are designed to arrest replication of the tumor DNA. The dose limiting toxicities associated with various anti-cancer therapies, is thought to play a role in tumors acquiring resistance to conventional therapeutics (chemoresistance). To overcome the adverse side effects of conventional anti-cancer therapies, the Isaacs laboratory has advocated the use of chemical engineering principles to modify potent killing toxins to produce "molecular grenades." These molecular grenades are selectively "detonated" by the prostate tumor; thereby, liberating their killing toxin efficiently only within the immediate environment surrounding cancer sites. The objective for this project is to use a bio-engineering approach to produce recombinant pro-toxins designed for specific cleavage by a defined protease whose high expression is restricted to the tumor microenvironment at sites of metastatic CRPC. In contrast to benign tissue, where the vasculature is only permissive to the diffusion of micromolecules across the endothelial barrier, the leaky nature of the tumor vasculature allows for macromolecules to pass this barrier as well. Human serum albumin (HSA) is a large macromolecule, which will aid in the targeted delivery of the toxin, proaerolysin (PA), to malignant sites via the enhanced permeability and retention (EPR) effect. We are able to produce a recombinant HSA linked to the N-terminus of Proaerolysin (HSA/PA) via a peptide linker specific for the protease prostate specific antigen (PSA). This recombinant HSA/PA shows selective activation in the presence of PSA expressing PCa cell lines with toxicity at low nanomolar concentrations. The PA toxin is extremely toxic when intravenously (IV) injected in mice, displaying an LD100 of 100ng. Restricting the potent killing ability of PA to sites of PCa is key, so that it can be delivered as a systemic therapeutic. Linking HSA with PA to produce the recombinant HSA/PA has neutralized the extreme toxicity observed when PA was injected directly in mice. With the recombinant HSA/PA we can safely dose mice up to 12ug. The characteristics of the prostate tumor microenvironment show promising potential for preventing chemoresistance and death from metastatic disease by lending itself to therapeutic targeting with our molecular grenade Proaerolysin.

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#2077

Carbonate prodrugs derived from triterpenoids with high cytotoxic activity.

Petr Dzubak,1 Renata Burianova,1 Martina Michalova,1 Barbora Liskova,1 Milan Urban,1 Jan Sarek,1 Adela Galandakova,2 Jitka Ulrichova,2 Marian Hajduch1. 1 _Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic;_ 2 _Dpt. of Medicinal Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic_.

Number of active anticancer compounds failed in the drug development due to unfavorable pharmacological properties. Design of pro-drugs is plausible approach to improve drug-like properties of biologically active compounds. Betulinic acid is a well-known representative of triterpenes, which possess a moderate cytotoxic activity and high selectivity towards the tumor cells. However, its pharmacological properties, particularly solubility and bioavailability, are poor. In order to improve pharmacology of betulinic acid derivatives, we have synthesized and tested activities of above 40 carbonate based prodrugs. We have developed methods for activation of "pro-drug" derivatives of betulinic acid by primary rat hepatocytes. Cytotoxicity of pro-drugs and activated metabolites was measured on the sensitive tumor cell line CCRF-CEM (human T-lymphoblastic leukemia). We have identified 21 prodrugs >10 times more cytotoxic to CCRF-CEM cells after pre-incubation with rat hepatocytes. The best candidate pro-drug showed >100 fold increase of cytotoxicity due to metabolic activation and it was cytotoxic to cancer cells in sub-micromolar concentrations. Our concept and predicted mechanisms of pro-drug activation were verified by HPLC-MS/MS where the pro-drugs and corresponding active compounds were identified and quantified. For the most active derivatives we have tested ADME characteristics and in vivo pharmacokinetics which showed significantly improved drug-like properties. Analysis of anti-cancer activities of the pro-drugs is currently being performed. This work was supported by grants LF_2015_031 and by the National Sustainability Program (LO1304).

#2078

Interleukin-12 gene therapy in combination with bevacizumab and pegylated liposomal doxorubicin for treatment of disseminated ovarian cancer.

Jason G. Fewell, Majed M. Matar, Jennifer Rice, Diane McClure, Elaine Brunhoeber, Jeff Sparks, Stefanie Greenleaf, Kelley Smith, Khursheed Anwer. _Celsion, Inc, Huntsville, AL_.

Despite recent improvements in treatment options for ovarian cancer patients, notably, the approval of using bevacizumab in combination with chemotherapies including pegylated liposomal doxorubicin (PLD), this disease is still the most deadly of all gynecological malignancies requiring new and novel therapeutics. Interleukin-12 (IL-12) is a highly active cytokine that can induce a potent anti-cancer immunity mediated through activation of cytotoxic T-lymphocytes, natural killer cell proliferation, and secretion of interferon-γ. We are developing an IL-12 based gene therapy for the treatment of gynecological malignancies that have spread into the peritoneal cavity. Our approach utilizes IL-12 plasmid (pIL-12) formulated with the PPC delivery system, which is comprised of a low molecular weight polyethylenimine covalently linked to polyethyleneglycol and cholesterol.

Previously we have shown in a mouse model of disseminated ovarian cancer efficacy of a treatment regimen of pIL-12/PPC used in combination with paclitaxel and carboplatin. The combination treatment significantly improved survival compared to either pIL-12/PPC alone or chemotherapy alone and demonstrated the feasibility of using an IL-12 immunotherapy in combination with cytotoxic chemotherapies to achieve additive therapeutic effects. Results from a Phase I clinical trial in platinum resistant patients have shown that intraperitoneal delivery of pIL-12/PPC in combination with PLD produced an overall clinical benefit of 57.1% (PR=21.4%; SD=35.7%) in patients with measurable disease. The highest percentage of PRs were found at the highest dose level (28.6%) along with highest percentage of patients achieving SD (57.1%).

Here we describe studies evaluating the combination of pIL-12/PPC with bevacizumab and PLD. For these studies 5,000,000 human SKOV3 cells were implanted into the peritoneal cavity of immunocompromised Hsd:Athymic Nude-Foxn1nu mice. Treatment with pIL-12/PPC alone and bevacizumab alone resulted in a 50% and a 39% reduction of animals with visible tumors at the end of the study. Combining pIL-12/PPC + bevacizumab improved the response to 78% of animals with no visible tumors. Further, combining pIL-12/PPC + bevacizumab + PLD resulted in a >98% decrease in tumor burden in animals compared to controls and ~92% decrease in tumor burden compared to animals treated only with bevacizumab + PLD. All treatments were well tolerated and analysis of serum chemistries and hematology showed normal ranges of all parameters examined for all groups. There were no significant differences in animal weights between groups during the experiment. Together these results suggest synergistic efficacies can be achieved by combining a novel pIL-12/PPC immunotherapy with anti-angiogenesis therapies and cytotoxic chemotherapies in disseminated ovarian cancer.

#2079

IONP-LPrA2 and PEG-LPrA2 therapies for triple negative breast cancer.

Tia L. Harmon,1 Adriana Harbuzariu,1 Antonio Rampoldi,1 Courtney Dill,1 Viola Lanier,1 Danielle Daley-Brown,1 Crystal Lipsey,1 Cynthia Tchio,1 Lily Yang,2 Ruben Rene Gonzalez-Perez1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

Background: Breast cancer (BC) is an epidemic in the US. It is estimated that there will be over 230,000 new BC diagnoses in 2016. Triple Negative Breast Cancer (TNBC) comprises ~15 % of BC cases and lacks targeted therapeutic options. Obesity and high leptin levels are associated with higher TNBC incidence and poorer patient outcomes. Overexpression of leptin and its receptor, Ob-R, induce BC cell growth and angiogenesis; therefore leptin/Ob-R may serve as a TNBC therapeutic target. We have developed a Leptin Peptide Receptor Antagonist, LPrA2, which effectively inhibits leptin signaling. To increase its efficacy LPrA2

was coupled to iron oxide nanoparticles (IONPs) and polyethylene glycol (PEG), and subsequently tested in vitro and in vivo in obese mice hosting syngeneic TNBC-like mammary tumors.

Methods: The conjugation of LPrA2 to IONPs and PEG was confirmed by immunoblotting analysis. E0771, mouse BC cells, which are progesterone receptor and HER2 negative were made insensitive to estrogen stimuli by long-term treatment with Tamoxifen (TAM). Cell cycle and MTT assays were performed to determine the effectiveness of leptin signaling inhibition by conjugated LPrA2 in vitro. C57BL/6 female mice were fed a 60% fat diet

to induce obesity. The obese mice were injected with E0771-TAM cells in the mammary fat pad and treated with IONP-LPrA2 and PEG-LPrA2 after tumor development. Obese mice treated with IONP-LPrA2 Scramble (Sc) and PEG-LPrA2 Sc served as negative controls.

Results and Conclusion: IONP-LPrA2 and PEG-LPrA2 attenuated leptin signaling in vitro and decreased tumor growth and progression in vivo in comparison to the controls. These findings indicate that conjugated LPrA2

may serve as a targeted therapy for TNBC. IONP-LPrA2 may be especially useful in treating this more aggressive form of BC due to its ability to capture multiple LPrA2 peptides as well as its small and uniform particle size. 

### Experimental Therapeutics

#2080

Demonstration of casual relationship between blood-tumor barrier permeability changes and chemotherapeutic uptake and effect in brain micrometastases of breast cancer.

Afroz Shareef Mohammad, Chris E. Adkins, Emma L. Dolan, Tori B. Terrell-Hall, Paul R. Lockman. _West Virginia University, Morgantown, WV_.

Background: 10-16% of women with advanced breast cancer will develop symptomatic brain metastases, the survival rate of which is less than 2 years. When metastasis develop within the brain, the BBB anatomy changes due to increased angiogenesis with increase in permeability of BBB in the tumor region (> 1mm diameter). We have also demonstrated in our previous study that, once the metastases are established in the brain (>1 mm in diameter) chemotherapy fails to induce cytotoxicity in most of them. The efficacy of a chemotherapeutic agent in the metastatic brain tumors significantly correlates to its uptake in the micro metastases (< 0.5 mm in diameter). Therefore, it is important to characterize the permeability changes in micrometastases using permeability markers 14C-AIB (104 Da) and Texas Red dextran (3kDa) in five novel brain seeking cell lines (4T1, MDA-MB-231BR HER2+, MDA-MB-231BR, JIMT-1Br and Sum190) which preferentially develop brain metastases of breast cancer.

Methods: Female nude mice were intracardially injected with different human brain seeking breast cancer cell and allowed it to metastasize. Metastases were allowed to develop until neurologic symptoms appeared and animals were injected with IV bolus dose of permeability markers 14C-AIB (104 Da) and Texas Red dextran (3kDa) 10 minutes prior to euthanasia. After euthanasia rain sample were harvested, sectioned and analyzed by autoradiography and fluorescent microscopy.

Results: Passive permeability changes in 4T1 metastatic lesions was 3.171 + 1.52 (SD) for 14C-AIB, and 2.529 + 0.84 (SD) for the passive diffusion marker 3kDa Texas Red Dextran(TRD). MDA-MB-231BR HER2 metastatic lesions has 14C-AIB permeability 4.936 + 3.6 (SD) and for TRD 1.301 + 0.49 (SD). For MDA-MB-231Br cell line permeability changes with 14C-AIB was found to be 3.001 ± 0.1514 (SD) and the BTB permeability to TRD ranged was found to be 1.159 ± 0.3137 (SD). In JIMT-1Br, 14C-AIB permeability was found to be 2.212 + 0.72 (SD) and TRD was 1.502 + 0.344 (SD). Sum190 micrometastases showed maximum 14C-AIB permeability in the range of 1 to 28.64 fold (Mean was 10.11 ± 5.68; SD) and TRD was found to be in the range of 1 to 2.10 (Mean was 1.287 ± 0.293; SD).

Conclusions: We have demonstrated that there are differences in passive permeability between each cell line and there are differences in overall survival and the size of lesions at the time neurological symptoms develop. The increased permeability of the vasculature of CNS micrometastases is critically important for both diagnosis and treatment.

#2082

Fc-FcγR interaction impacts the clearance and antitumor activity of antibody-drug conjugates in NSG mice.

Fu Li, Michelle Ulrich, Joshua Hunter, Lori Westendorf, Devra Olson, Cassie Baker Lee, Dennis Benjamin, Che-Leung Law. _Seattle Genetics, Inc, Bothell, WA_.

Majority of preclinical efficacy studies for cancer therapeutics are performed on tumor models implanted in immune-deficient mouse strains, including athymic nude, severe combined immunodeficiency (SCID), or NOD-SCID/IL2Rgamma null (NSG) mice. To better understand the correlation between drug exposure and preclinical antitumor activity of biologics, we evaluated the pharmacokinetic (PK) profiles of antibodies and antibody-drug conjugates (ADC) in four strains of mice commonly used in cancer research, including BALB/c, athymic nude, SCID, and NSG. To our surprise, antibodies and ADCs had an abnormally short serum half-life in NSG mice, compared to other strains of mice.

We hypothesized that the fast clearance from plasma in NSG mice may reduce the overall exposure of xenografts to the ADCs, resulting in underestimation of antitumor activity. This was confirmed by comparing cAC10-vcMMAE in CD30+ Karpas-299 tumors implanted in either SCID mice or NSG mice. A single dose of 1 mg/kg cAC10-vcMMAE ADC resulted in complete remission of Karpas-299 tumors implanted in SCID mice (5/5). In contrast, the same treatment regimen had no effect on Karpas-299 tumors implanted in NSG mice (0/5). This observation suggests indeed the shortened exposure penalizes ADC efficacy in NSG models.

We then hypothesized that the fast clearance of ADCs from plasma in NSG mice could be mediated by Fc-FcγR interaction; compromising this interaction may improve the serum half-lives of antibodies and ADCs and thus enhance ADC-mediated antitumor effects in NSG mice. To test this, we utilized a mutant antibody with the amino acid substitutions of E233P:L234V:L235 in its Fc domain that result in significantly reduced FcγR binding (G1V1). These mutations restored the serum exposure in NSG mice to that level comparable to other strains of mice. As a result, treatment of 1 mg/kg cAC10G1V1-vcMMAE led to tumor remission in Karpas-299 tumors grown in NSG mice, correlating with extended exposure. In parallel, pretreatment with human IV immunoglobulin also enhanced ADC exposure and antitumor activity in the NSG mice.

In summary, these results suggest preclinical PK/PD assessment should be performed in the same strains of mice, especially when NSG xenograft models are used to evaluate activities. Moreover, reduced FcγR binding may enhance ADC exposure and allow a better assessment of therapeutic potential of biologics in NSG-based xenograft models.

#2083

Toxicity, bioavailability, pharmacokinetics, tissue distribution and metabolism of a novel small molecule inhibitor of IL-6-induced STAT3 activation.

Brian F. Kiesel,1 Robert A. Parise,1 Jianxia Guo,1 Donna M. Huryn,2 Paul A. Johnston,2 Rafaelle Colombo,2 Malabika Sen,2 Jennifer Grandis,3 Julie L. Eiseman,1 Jan H. Beumer1. 1 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 2 _University of Pittsburgh, Pittsburgh, PA;_ 3 _University of California, San Francisco, CA_.

Introduction: The oncogenic transcription factor STAT3 is frequently hyper-activated in head and neck cancer and promotes gene transcription involved in cancer development, maintenance and progression. Several selective small molecule inhibitors of IL-6-induced STAT3 activation were identified in a screening campaign, and four analogs from a lead optimization series were analyzed. Compound UPCDC10205 was prioritized for in vivo testing to evaluate its toxicity, pharmacokinetics (PK) and metabolism in mice.

Methods: The four inhibitors of IL-6-induced STAT3 activation were incubated with liver microsomes from Foxn1 +/nu mice up to 90 min. An LC-MS/MS assay was developed to quantify substrate depletion. Single IV dose toxicity was determined in male and female Foxn1 +/nu mice at the maximum soluble dose of 4 mg/kg of compound UPCDC10205 in 10% Solutol. In the multiple IV dose study in female mice UPCDC10205 was dosed QDx5 at 4, 2.7, and 1.3 mg/kg/day. During toxicity studies clinical health status was observed daily and body weight was recorded twice weekly for 14 days after treatment, followed by necropsy. To evaluate PK, single doses of UPCDC10205 IV 4 mg/kg, PO IV 4 mg/kg, or PO 30 mg/kg UPCDC10205 suspension in 1% CMC, were administered to groups of female mice. Mice were euthanized from 5 min to 24 h after dosing (n=3). RBCs, plasma and tissues were collected and stored at -80 °C. UPCDC10205 concentrations were quantified by LC-MS/MS. Non-compartmental PK were evaluated. LC-MS/MS was used to screen for metabolites in plasma and urine.

Results: Approximately 80% of compound UPCDC10205 remained after a 90 min microsomal incubation compared to <50% for the other analogs. Mice exhibited no signs of toxicity after single or multiple doses of 4 mg/kg IV. Exposure in liver, lungs, kidney, skeletal muscle and brain were 1.6-3.2-fold that of plasma. Plasma AUC after IV 4 mg/kg (1022 ng/mL*h) compared to PO 4 mg/kg dosing (53 ng/mL*h) yielding a bioavailability of ~5%. Compound UPCDC10205 was not detected beyond 6 h in any tissue. The plasma half-life was 0.6 h, clearance 3.9 L/h/kg and distribution volume 3.4 L/kg. The major metabolite identified in both plasma and urine was UPCDC10205 N-glucuronide

Conclusion: In vitro, UPCDC10205 was metabolically stable. No gross toxicity was observed in mice administered the maximum soluble dose. UPCDC10205 was widely distributed into tissues and cleared rapidly. Bioavailability was ~5%. In vivo metabolism of UPCDC10205 was by direct glucuronidation, explaining why microsomal stability (reflective of phase I metabolism) did not translate to in vivo metabolic stability.

Support: P30CA047904; P50CA097190

#2084

Pharmacokinetic and pharmacodynamic assessment of autophagy inhibition following hydroxychloroquine in mice.

Kristen M. Jackson, Ryan J. Hansen, Daniel L. Gustafson. _Colorado State University, Fort Collins, CO_.

Hydroxychloroquine (HCQ) is being tested in a number of human clinical trials to determine the role of autophagy in response to standard anticancer therapies. However, preclinical studies in mouse models are equivocal with regard to HCQ exposure (pharmacokinetics) and inhibition of autophagy (pharmacodynamics) in various tissues. Understanding HCQ pharmacokinetics (PK) and pharmacodynamics (PD) in mice allows for a comparison to human PK as reported in recent trials and as such the subsequent PD associated with autophagy inhibition in tissues. In this study, female BALB/c mice were treated with a single intraperitoneal dose of 20, 40, or 80 mg/kg HCQ and tissues and whole blood collected at 3, 6, 12, 24, 48 and 72 hours. Levels of HCQ and desethylhydroxychloroquine (dHCQ), an active metabolite, in whole blood and tissues were determined via a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Non-compartmental analysis (NCA) measured exposure, as determined by area under the drug concentration versus time curve (AUC0-inf), to both HCQ and dHCQ was dose proportional in whole blood, liver and brain. The maximum concentration (Cmax) of HCQ was 1.3, 3.1 and 4.7 µg/mL and 20.6, 41.7 and 114.7 µg/mL in whole blood and liver, respectively, following 20, 40 and 80 mg/kg. Brain exposure was far less with Cmax achieving 272, 717 and 2220 ng/mL. In general, the time of maximal concentration (Tmax) of HCQ and dHCQ was observed at 3 hr or 6 hr, regardless of dose and location measured. PD assessment for autophagy inhibition was determined by western blotting. The LC3 protein, which is involved in membrane dynamics during autophagy, was visualized and the ratio of LC3-II to tubulin was measured. In the liver, autophagy was inhibited approximately two-fold following all doses at 24 and 48 hr compared to controls. Autophagy was inhibited in both the gut and kidney at 24 and 48 hr at all doses. In the brain, autophagy inhibition was not dose related and may be a result of low HCQ levels. These results demonstrate that a dose of 20 mg/kg is sufficient to inhibit autophagy in most tissues investigated; however, while PK parameters appear to be dose proportional, increased tissue drug levels does not necessarily equate to increased PD effects/autophagy inhibition. In addition, the data establishes a dose of 40 mg/kg in mice results in Cmax and AUC0-inf of HCQ equivalent to that observed in human clinical trials targeting autophagy modulation following a dose of 600 mg daily. Studies are ongoing to assess this relationship in solid tumors.

#2085

Development of a mechanism-based pharmacokinetic/pharmacodynamic model to characterize tumor killing effect of an anti-VISTA monoclonal antibody in tumor bearing mice.

Xiling Jiang, Jocelyn Leu, Indrajeet Singh, Linda A. Snyder, Weirong Wang. _Janssen Research & Development LLC., Spring House, PA_.

The V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a recently discovered co-inhibitory molecule that shares limited sequence homology with the IgV domain of immune regulators of the B7 family. A first-in-class monoclonal antibody (mAb) that targets VISTA is currently in development for evaluation in patients with non-small cell lung cancer and other types of cancer. Data from a preclinical mouse model (in human VISTA knock-in mice) showed that treatment with its surrogate antibody demonstrated tumor growth inhibition, which is believed to be associated with modulation of the myelomonocytic and T cell compartments. A mechanistic pharmacokinetic/ pharmacodynamic (PK/PD) modeling approach was applied that integrated the in vitro and in vivo information (PK, target occupancy (TO) and tumor growth), and incorporated key physiological processes. A one-compartment pharmacokinetic model with Michaelis-Menten elimination kinetics was used to characterize the nonlinear clearance of the surrogate antibody. The growth and killing of tumor cells was described with a cell distribution tumor growth/ killing model. Tumor killing effects can be described well by a linear relationship to VISTA TO. The developed PK/PD model captured the observed data in mice. This work also presents the potential of how mechanistic modeling and simulation can support first-in-human dose selection and dosing regimen optimization for immunomodulatory mAbs for anticancer immunotherapy.

#2086

Noscapine chemosensitization enhances docetaxel anticancer activity and tumor stroma disruption against triple negative breast cancer.

Ravi Doddapaneni, Ketan Patel, Nusrat Chowdhury, Mandip Sachdeva. _Florida A &M University, Tallahassee, FL_.

Purpose:

The use of Noscapine (Nos) as a chemosensitizer followed by docetaxel (DTX) treatment therapy could be a novel approach for the treatment for breast cancer and possibly reduce the adverse side effects associated with DTX based chemotherapy. The goal of this study was to examine the chemo-sensitizing effect of Nos to DTX and also tumor stromal disruption effect of Nos in mice bearing xenograft TNBC tumors.

Methods:

Effect of Nos chemosensitization on DTX cytotoxicity was evaluated in MDA-MB-231 cells by trypan blue dye method. Apoptosis was measured by AnnexinV/FITC method using flow cytometer. Expression of different proteins like phospho-p38, pJNK, bcl-2, α-tubulin, Akt, pAkt, survivin was evaluated by immunoblot. Alpha-tubulin binding assay was done by fluorescent microscopy. In vivo antifibrotic efficacy of Nos and uptake of coumarin-6 loaded fluorescent liposomes was evaluated by picro-sirius red staining and fluorescent microscopy respectively in xenograft breast tumors.

Results:

MDA-MB-231 TNBC cells were exposed at sub-therapeutic dose of Nos (4 µM) which increased the cytotoxicity of DTX by 3.0-fold. Flow-cytometric analysis showed significant increase (30 percent) of late apoptotic cells in Nos chemosensitized, DTX-treated MDA-MB-231 cells compared with DTX alone treatment. Further, chemosensitization of TNBC cells with Nos at different time intervals (6 h, 12 h and 24 h), the effect on stress transducer p38 stress activator protein kinase was significantly activated (p<0.01). Also, Nos (4 µM) chemosensitized cells followed by DTX treatment showed downregulation of bcl2 (2.3-fold), survivin (1.3-fold) and pAKT (1.9-fold) expression further illustrating the role of Nos in inducing apoptosis. Interestingly, at this dose of Nos (4 µM), there was no impact on alpha-tubulin. Also, in vivo studies with 100 mg/kg Nos given orally to MDA-MB-231 tumor bearing athymic nude mice revealed significant reduction in tumor collagen-1 levels in Nos treated group compared to control as determined by picro-sirius staining. In Nos treated tumors, uptake of coumarin-6 loaded fluorescent liposomes was 7-fold higher compared to controls suggesting antifibrotic role of Nos.

Conclusion:

In conclusion, chemosensitization with sub-therapeutic dose of oral noscapine could be a promising approach to increase anticancer activity of DTX and can further enhance uptake of liposomes suggesting that this approach may have potential in TNBC treatment.

#2087

The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) appears as an efficient targeted therapy when used in an adjuvant setting in patient-derived xenografts of uveal melanoma.

Béré Diallo,1 Gerald Massonnet,1 Rania El-Botty,1 Chloé Raymondie,1 Guillaume Carita,1 Sergio Roman-Roman,1 Paul Smith,2 Emma Davies,2 Didier Decaudin,1 Fariba Némati1. 1 _Inst. Curie, Paris, France;_ 2 _Astra Zeneca, Macclesfield, United Kingdom_.

Uveal melanomas (UM) constitute the most common primary intraocular tumors in adults and are characterized by a constitutive activation of the MAPK pathway due to mutations of the GTPase genes GNAQ or GNA11 in almost 80% of cases. The most commonly used treatments for UM are alkylating agents such as dacarbazine (DTIC) and temozolomide (TMZ). The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) has shown clinical activity compared to DTIC/TMZ in a recent Phase II clinical trial and has recently completed a Phase III clinical trial in combination with DTIC (NCT01974752). In parallel with this trial we sought to evaluate the efficacy of DTIC + selumetinib in UM patient-derived xenografts (PDXs).

Three models were included in the study (MP34, MP55, and MM26), all bearing a GNAQ or GNA11 mutation. Selumetinib was administered orally at 25 mg/kg/day, 5 days a week, and DTIC at a dose of 40 mg/kg/day on Days 1 to 5 every 4 weeks.

A significant tumor growth inhibition (TGI) of 54% was observed in the MP34 model but not in the two remaining PDXs. In one model, MM26, DTIC induced a strong TGI of about 99% with 6/9 complete remissions (CRs). The combination of selumetinib + DTIC did not significantly increase efficacy compared to monotherapy in any of the models; in the MM26 PDX, the combination induced a similar TGI (99%) and CR rate (5/9) as DTIC alone. In this experiment, after two courses of DTIC + selumetinib, selumetinib was continued alone, showing a significant increased growth delay (p<10-3 at Day 113), compared with DTIC alone. Pharmacokinetics, pharmacodynamics, and molecular studies on the three UM PDXs are ongoing.

In conclusion, we have observed that response of UM PDX models to DTIC was not increased when combined with selumetinib; these results are similar to those seen in the Phase III study of this drug combination. The observation that MEK inhibition was effective in delaying progression in the DTIC-sensitive PDX and published clinical studies demonstrating MEK inhibitor monotherapy activity indicate that MEK inhibition may have value as a treatment for UM; perhaps in the adjuvant setting in two specific clinical situations, i.e. patients with irradiated or enucleated high-risk primary intraocular or surgically resected metastatic UM.

#2089

Therapeutic index prediction of the agonistic aglycosylated TWEAK receptor binding antibody BAY-356.

Anders Viberg,1 Eva Hanze,1 Lisa Dietz,2 Ruprecht Zierz,3 Sandra Berndt,3 Sabine Wittemer-Rump3. 1 _qPharmetra, Stockholm, Sweden;_ 2 _Bayer AG, Wuppertal, Germany;_ 3 _Bayer AG, Berlin, Germany_.

BAY-356 is a novel aglycosylated anti-TWEAK receptor antibody with potent agonistic activity evaluated for cancer therapy. In order to predict an efficacious therapeutic dose of BAY-356 in man, we sought to determine a therapeutic index where the exposure related to therapeutic efficacy was compared with the exposure obtained following doses tested in toxicology studies.

BAY-356 (1 mg/kg) was administered intravenously to healthy Cynomolgus monkeys and plasma concentrations measured in order to determine pharmacokinetic (PK) parameters. Using allometric scaling, the PK in humans was predicted. Efficacy data of BAY-356 in WiDr, HN10321, A253 and SCaBER xenograft models representing colorectal, head and neck squamous cell carcinoma and bladder cancer, were used to derive exposure-response models for each tumor model where the plasma exposure of BAY-356 is assumed to have an effect on the tumor size in a tumor growth model. NONMEM 7.3 was used in the estimations.

The tolerability of BAY-356 was tested in monkeys at doses of 10, 20 and 40 mg/kg by weekly intravenous injections over 4 weeks. Treatment resulted in slight to moderate toxicity in liver, kidneys and pancreas and 10 mg/kg was set to be an exposure dose well tolerated and not to be exceeded by therapeutic exposure. A dosing strategy in humans predicted to result in the same exposure as 10 mg/kg weekly in monkey is 18 mg/kg every third weeks (Q3W).

Using the estimated exposure-response in xenograft models, human tumor doubling times of 8 weeks (A253 and HN10321), 24 weeks (WiDr) and 12 and 24 weeks (SCaBER) in combination with human predicted PK parameters, tumor reduction over time in humans was predicted based on a human dosing strategy of 18 mg/kg BAY-356 Q3W. For the A253 tumor model, stable disease in humans was predicted while the results from HN10321 and SCaBER models predicted a 13-60% decrease in tumor size in humans following 12 weeks treatment. For WiDr, this dosing strategy was predicted to not be efficacious in humans.

The results based on modelling of xenograft data indicate that 18 mg/kg BAY-356 dosed Q3W is predicted to be efficacious in humans for certain tumor cells.

#2090

Evaluation of the cytotoxic profile of Metformin and Y15 in platinum resistant ovarian cancer cells.

Arkene S. Levy,1 Zara Khan,2 Samuel Batko,2 Keerthi Thallapureddy,2 Robert Smith,2 Thanigaivelan Kanagasabai,2 Julie Torruellas Garcia,2 Appu Rathinavelu2. 1 _University of the West Indies, Mona Campus, Kingston, Jamaica;_ 2 _Nova Southeastern University, Fort Lauderdale, FL_.

Platinum resistance remains a major challenge in the chemotherapeutic management of ovarian cancer. The anti-diabetic drug metformin has been previously shown to induce cytotoxicity in platinum resistant ovarian cancer cells and overexpression and increased phosphorylation of a tyrosine kinase called Focal Adhesion Kinase (FAK) has been implicated in the development of this platinum resistance. Therefore, in the present study we evaluated the combined cytotoxic efficacy of Metformin and the focal adhesion kinase inhibitor 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) in platinum resistant OVCAR3 ovarian cancer cells. Cells were initially treated with concentrations of Y15 ranging from 10-100 μM, and metformin from 10-100mM to determine 1C50 values. Subsequently, cells were treated with Y15 (80 μM) and metformin (26mM) alone and in combination. All treatments were triplicated with duration of 24hrs and control cells exposed to media only. The cytotoxic profile of each treatment was assessed using the automated trypan blue assay. DNA fragmentation and poly ADP ribose polymerase (PARP) cleavage assays were performed to evaluate the mechanism of cell death and we further evaluated the expression of phosphorylated FAK, p53 and p21 in response to treatments using western blot.

Y15 alone produced 48% cell death. In combination, Y15 significantly increased the cytotoxic efficacy of metformin by 22%, when compared to the metformin only treatment. Cell death by apoptosis was confirmed by PARP cleavage and the presence of DNA fragments in Y15, metformin, and metformin +Y15 treatment groups. The Metformin +Y15 combination significantly downregulated the expression of phosphorylated FAK when compared to the individual treatments and control and this confirmed reduced FAK activity. Reduced FAK auto phosphorylation also correlated with increased expression of p53 AND p21 in metformin and Y15 treatment groups.

Our findings show that Y15 significantly enhances the cytotoxic profile of metformin in platinum resistant OVCAR-3 cells. Furthermore, a FAK dependent apoptotic mechanism appears to underlie the cytotoxic effect of metformin as well as Y15 as both drugs significantly reduced the phosphorylation of FAK alone, and in combination. Reduced FAK activity also correlated with increased p53 and p21 expression. This study is the first to report a FAK dependent cytotoxic mechanism of metformin in ovarian cancer and in further work we will evaluate the mechanisms why which metformin cooperates with Y15 to inhibit FAK activity in platinum resistant ovarian cancer.

#2091

Atypical relationship between cytochrome P450 1B1 (CYP1B1) polymorphism and cell proliferation and invasiveness in head-and-neck carcinoma.

Valérie Le Morvan, Elodie Richard, Maud Cadars, Alban Pasquies, Amélie Lansiaux, Jacques Robert. _Inst. Bergonié, Bordeaux, France_.

We have previously shown a close association between a single nucleotide polymorphism (SNP) of cytochrome P450 1B1 (CYP1B1) (V432L, rs1056836) and in vitro sensitivity to several anticancer agents. From squamous cell carcinoma (HNSCC) cell lines not expressing this cytochrome, CAL27 and CAL33, we generated by lentiviral infection isogenic derivatives expressing to similar levels the two variant forms of CYP1B1.

Cell lines re-expressing CYP1B1 had higher proliferation and invasiveness than the original ones, and this was further increased when they expressed the variant form, which were significantly resistant to cisplatin and camptothecin. This was confirmed by in vivo studies in SCID mice: tumors re-expressing CYP1B1 had growth acceleration, especially those expressing the variant form. Treatment by cisplatin, irinotecan and doxorubicin induced regression of tumors obtained from vector-only infected cells, while those obtained from cells re-expressing CYP1B1, especially the variant form, were markedly resistant to all three drugs.

Four proteins representative of epithelial and mesenchymal phenotypes were determined by western blots. In the CAL33 cell line, the expression of E-cadherin and claudin-1 (epithelial markers) was decreased upon re-expression of CYP1B1, whereas the expression of N-cadherin and vimentin (mesenchymal markers) was increased. In the CAL27 cell line, this was the same only for mesenchymal markers. Using the Aldefluor assay kit, a stem cell phenotype was exhibited by the CAL27 line re-expressing the CYP1B1 variant genotype.

In silico studies on the collections of cancer cell lines (NCI-60 and CCLE) confirmed the significant association between CYP1B1 polymorphic status and resistance to DNA-damaging drugs. In addition, the variant genotype was associated to a mesenchymal phenotype defined by significant overexpression of series of 8-12 genes including vimentin and moesin, whereas the common and heterozygous genotypes were associated to an epithelial phenotype defined by significant overexpression of a series of 20-30 genes, including claudins 3, 4 and 7, E-cadherin and EPCAM.

The clinical consequences of the CYP1B1 L432V gene polymorphism were studied in a prospective population of 125 head-and-neck cancer patients (pts) treated in the palliative setting: 48 pts with common homozygous genotype (CH), 40 heterozygotes (HT) and 35 variant homozygous pts (VH). No differences in response rates as a function of the polymorphism were noticed. Overall median survival was significantly worse in VH pts (6.6 months) than in CH (13.2 months), HT falling in between (9.8 months) (p = 0.017).

As a conclusion, CYP1B1 appears to be involved in cell proliferation and invasiveness, these properties being magnified when expressed under its homozygous variant form, which is suggested to be associated to the epithelial-to-mesenchymal transition.

#2092

Characterizing NIR dye-IL13RA2 antibody conjugates for biodistribution studies in xenograft tumor models by fluorescence molecular tomography (FMT).

Parul Gupta,1 Dangshe Ma,2 Rachel Roach,1 Mary Spilker,1 Mauricio Leal,2 Cedo Bagi3. 1 _Pfizer Inc., San Diego, CA;_ 2 _Pfizer Inc., Pearl River, NY;_ 3 _Pfizer Inc., Groton, CT_.

Background: The antibody based therapies are promising anti-cancer therapeutic modalities with minimal toxicity and maximum efficacy. The efficacy of these agents is regulated by their biodistribution and targeting. The contemporary methods of testing the biodistriution of large molecule drugs are expensive and tedious. The development of simple and rapid methodology, such as optical imaging, can enable effective screening of a larger number of compounds. Here we have used Fluorescence Molecular Tomography (FMT), an optical imaging technique to study biodistribution and tumor targeting of IL3RA2 antibody (Ab). Methods: Different near infrared (NIR) fluorophores (λmax:650-800nm) were conjugated to the Ab. The fluorophore conjugation protocol was optimized to achieve a degree of labeling (DOL) of 1-3 for all the conjugates. The properties of Ab-fluorophore conjugate (Ab-F) were compared to unlabeled Ab using SEC-HPLC and cell binding assays in three cell lines with varying expression of IL13RA2: A375(+++), U87MG(+) and H460(-). For in vivo evaluation, Ab-F conjugates were administered intravenously at a dose of 2 nmol fluorophore to nu/nu mice bearing A375. Similar studies were also conducted in U87MG and H460 xenografts with selected Ab-F. The mice were imaged longitudinally (6 time-points) for up to 96 hrs using FMT4000 and the data were analyzed using TrueQuant software. Results: The SEC-HPLC and flow cytometry studies demonstrated that conjugation of Ab with most of the fluorophores did not change its stability or functionality. The in vivo fluorescence data from all the NIR dyes showed a peak tumor accumulation of the Ab-F at 6h and was maintained until 96 hrs. Quantitation of various Ab-F conjugated revealed that 2-6% of the injected dose was accumulated in the A375 tumors. In contrast to tumor profile, there was a steep decline in heart signal (a surrogate for blood/ plasma concentration), suggesting fast clearance from blood. The in vivo and ex vivo data suggested that there was 15-80pmol of Ab-F conjugate accumulated in the tumors at 96h. In addition, Alexa Flour® (AF)680 and AF750 showed minimal non-specific accumulation in other organs, whereas VivoTag® (VT)680 and BODIPY®630 showed a significantly higher non-specific accumulation in liver. Conclusions: These results show that the biological properties of Ab were not changed by conjugation with various NIR fluorophores at DOL <3. Optical imaging using fluorescent tags can effectively track and quantitate the tumor targeting/ biodistribution of large molecule drugs.

#2093

Imaging single cell drug target engagement in vivo reveals heterogeneous fractional occupancy.

Matt Dubach, Ralph Weissleder. _MGH, Boston, MA_.

To produce effective therapeutic treatment, drugs need to reach and engage the cognate target in the cells of interest. Incomplete target occupancy may lead to cell survival and disease progression. Therefore, for maximum efficacy, it is critical to achieve high target occupancy within each cell of a tumor. However, the tumor microenvironment likely creates a dynamic, heterogenous distribution of drug that limits target occupancy in some cells. Unfortunately, we lack the tools to determine target engagement at the cellular level, which prevents studies on heterogeneous target occupancy and contributes to the poor understanding of drug failure mechanisms. Here, we developed an approach to measure target engagement of small molecule inhibitors within single cells in mouse xenograft in vivo tumor models. Our method uses fluorescently labeled, target specific companion imaging drugs, fluorescence polarization imaging, and intravital microscopy, to measure target occupancy of unlabeled, clinical drugs in single cells. We calculated the equivalent in vitro exposure of ibrutinib, a covalent BTK inhibitor, in vivo, but found increased cellular heterogeneity, highlighting differences between in vivo activity and in vitro measurements. Additionally, following systemic delivery of the reversible PARP inhibitor olaparib, we found cells with substantial uninhibited target when average measurements indicated all target was occupied, illustrating drug activity heterogeneity that may lead to tumor survival. These results demonstrate that our technique, which is the first to measure unlabeled drug engagement with target in single cells, can quantitate cellular fractional occupancy heterogeneity during drug treatment. We expect this approach to serve as a valuable tool when measuring cellular pharmacology in vivo, providing insight on dosing, drug activity in the tumor and sources of heterogeneous drug distribution.

#2094

Translating EPHARNA (DOPC nanoliposomal EphA2-targeted siRNA) to the clinic.

Michael J. Wagner, Rahul Mitra, Wallace Baze, Kirstin Barnhart, Robert L. Coleman, Gabriel Lopez-Berestein, Anil K. Sood. _M.D. Anderson Cancer Center, Houston, TX_.

Introduction: To address the need for efficient and biocompatible delivery systems for systemic siRNA delivery, we have developed a 1,2-Dioleoyl-sn-Glycero-3-Phosphatidylcholine (DOPC) nanoliposomal EphA2-targeted therapeutic (EPHARNA). We have previously demonstrated the efficacy of EPHARNA in multiple tumor models. Here, following FDA guidance, we performed safety studies of EPHARNA in murine and primate models.

Methods: Single dosing of EPHARNA was tested at 5 concentrations in mice (N=15 per group) and groups of animals were sacrificed on days 1, 14, and 28 for evaluation of clinical pathology and organ toxicity. Multiple dosing of EPHARNA was tested in mice twice weekly for 1 month (N=10 per group) and in Rhesus macaques weekly (N= 4 per group) at two dose levels in each model. Possible effects on hematologic parameters, serum chemistry, coagulation, and organ toxicity were assessed in all animals.

Results: Following single dose EPHARNA administration to mice, no gross pathological or dose-related microscopic findings were observed in either the acute (24 hrs) or recovery (14 and 28 days) phases of this study. The no-observed-adverse-effect level (NOAEL) for EPHARNA is considered > 225 μg/kg when administered as a single injection intravenously in CD-1 male and female mice. With twice weekly injection, EPHARNA appeared to stimulate a mild to moderate inflammatory response in a dose-related fashion. In addition, there appeared to be a mild hemolytic reaction in the female mice. Systemic toxicity related to both the DOPC formulation as well as EphA2 silencing was assessed in Rhesus macaques due to the near 100% homology between Rhesus and human EphA2. One animal developed signs consistent with an infusion reaction on its first administration, and was dosed with a slow infusion over 15-30 minutes with subsequent infusions. Minimal to moderate infiltration of mononuclear cells was found in some organs including the GI tract, heart, and kidney in all Rhesus macaques in all groups. The significance of these infiltrates to the general health of the animals is unknown. One female in the control group and one in the high dose group had ovarian mineralization, which was considered incidental in this study. No differences attributed to EPHARNA were observed in the hematologic or anatomic parameters assessed.

Conclusion: These results demonstrate that EPHARNA is well tolerated at all doses tested without significant complications. These data, combined with previously published validation studies demonstrating siRNA as an effective therapeutic in vivo, led to the development of a first in human Phase I clinical trial that is currently underway (NCT01591356).

#2095

Toxicity of selective allosteric FGFR2 inhibition in preclinical studies.

Nataliya V. Lapina, Marina V. Melikhova, Olga A. Vakunenkova, Vadim A. Kashuro. _Institute of Toxicology of Federal Medical-Biological Agency, Saint Petersburg, Russian Federation_.

Alofanib (RPT835, Ruspharmtech LLC) is a novel selective allosteric inhibitor binding to the extracellular domain of FGFR2. Alofanib had no direct effect on activation of FGFR1 and FGFR3 in vitro. Selective effect of the compound may reduce toxicity compared to multi-targeted VEGFR/FGFR or pan-FGFR inhibitors. This study characterizes the organ toxicity of alofanib in Sprague-Dawley rats and Chinchilla rabbits, and the reversibility of any treatment-induced effects.

240 rats and 30 rabbits received alofanib (0-40.5 and 0-21.6 mg/kg/day, respectively) intravenously on a consecutive daily dosing schedule for thirteen weeks. Clinical observations and laboratory parameters were recorded. Necropsy was conducted following treatment/recovery periods, and histologic examinations were performed.

There was no treatment-related mortality, severe toxicity and significant changes in organs in the thirteen-week study. Alofanib was well-tolerated in vivo. Clinical signs of first injection (8.1 mg/kg) included moderate local irritative reaction and decreased activity during hour in rats. Clinical symptoms of toxicity were not observed over study. Animals were active. Body weight remained stable and it was comparable to vehicle groups. Abnormal electrocardiogram findings and blood pressure changes were not detected. It was notable that in the study, none of the hematologic changes was present at the end of the recovery period. Minimal significant decrease in ALT/AST, slight increase in alkaline phosphatase in male rats and mild increase in urea, decrease in chlorine in female rats were found throughout treatment period. Minimal significant decrease in albumin and chlorine was identified in rabbits. Rate of hyperphosphatemia, an on-target side-effect of pan-FGFR inhibitors, will be presented at the AACR Meeting.

We conclude that toxicity of alofanib was mild in the thirteen-week study. Slight decrease of chlorine in blood was most common side effect. The final conclusion will be made after six-month study. This study was supported by a grant from the Skolkovo Foundation.

#2096

Mechanistic insights into the pathogenesis of anti-DLL4-related hepatic sinusoidal dilatation.

Monika A. Jarzabek,1 Rupal Desai,2 Yuda Zhu,3 Christina Z. de Zafra,4 Joe Beyer,4 Gary Cain,4 Rajiv Raja,2 Annette T. Byrne,1 Priti Hegde,2 Jacqueline M. Tarrant2. 1 _Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _Oncology Biomarker Development, Genentech Inc., South San Francisco, CA;_ 3 _Nonclinical Biostatistics, Genentech, Inc., South San Francisco, CA;_ 4 _Safety Assessment, Genentech, Inc., South San Francisco, CA_.

Background: The Delta-like 4 (DLL4)-mediated NOTCH signaling pathway is an attractive therapeutic target in cancer. However, chronic blockade of DLL4 signalling has been observed to result in vascular toxicities, such as hepatic sinusoidal dilatation. As the underlying pathogenesis is unclear, the current study was undertaken to interrogate gene expression changes and potential safety biomarkers associated with anti-DLL4 related hepatic sinusoidal dilatation.

Methods: Formalin-fixed paraffin-embedded (FFPE) liver sections were derived from male and female cynomolgus monkeys administered FDLL8566 (recombinant humanized anti-DLL4 F(ab')2 antibody) by intravenous injection once weekly for 8 weeks at dose levels of 0, 5, 15 and 50 mg/kg/week (n=3/sex/group). RNA was extracted from whole liver sections and laser capture-microdissected (LCM) hepatic regions with or without sinusoidal dilatation. mRNA expression was quantified using a high-throughput RT-qPCR approach, with 96 pre-validated, species-specific TaqMan gene expression assays. A non-parametric statistical test was used to assess differential gene expression between sinusoidal dilatation-affected and non-affected liver tissues. The Benjamini-Hochberg multiple testing comparison error-based False-Discovery-Rate (FDR) method was applied to calculate the adjusted p-values for each probe set.

Results: Fourteen candidate genes were differentially expressed between sinusoidal dilatation-affected and non-affected cynomolgus monkey livers with an FDR adjusted p value <0.05. The presence of sinusoidal dilatation could be discriminated based on hierarchical clustering and principal component analysis (PCA). Modulation of expression in genes associated with the VEGF/NOTCH pathway, vascular remodeling, and inflammation were related to anti-DLL4-induced sinusoidal dilatation.

Conclusions: This work highlights the involvement of the NOTCH pathway in the maintenance of hepatic sinusoidal homeostasis in the nonhuman primate (NHP). As preclinical toxicities in NHPs have translated to patients for other anti-DLL4 inhibitors, the changes in candidate genes and potential mechanism of toxicity identified in this study are likely to be relevant to humans. The phenotypic characteristics of hepatic sinusoidal dilatation associated with anti-DLL4 resemble the microscopic and molecular features observed in the liver following oxaliplatin treatment of patients (including involvement of angiogenesis), suggesting that similar molecular mechanisms of hepatic toxicity may exist between anti-DLL4 and oxaliplatin.

This work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528.

#2097

Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models.

Xin Yu,1 Ashley T. Tsang,2 Tracy Withers,1 John Gelleran,3 David Augeri,3 S. David Kimball,3 Darren R. Carpizo1. 1 _Rutgers The Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ;_ 3 _Rutgers Ernest Mario School of Pharmacy, Piscataway, NJ_.

Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to KRAS as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. We identified ZMC1 as an allele specific mutant p53 reactivator and lead compound for mutant p53 targeted drug development. ZMC1 restores wildtype structure and function by functioning as a zinc metallochaperone to restore zinc binding to mutant p53 proteins with impaired zinc binding. Our aim was to translate this novel mechanism in vivo using murine pancreatic cancer models. We investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of ZMC1 and performed efficacy studies for allele specificity in nude mice with subcutaneous tumors from murine pancreatic cancer cell lines derived from a genetically engineered mouse model (KPC) expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). Tumor growth inhibition became apparent only in KPCp53-R172H xenografts but not in KPCp53-R270H. We then performed efficacy studies in the autochthonous KPC model and found that, ZMC1 extended the median survival from 15 to 26 days in the KPCp53-R172H mice (p=0.05) but not in the KPCp53-R270H mice. We sought to improve the efficacy of ZMC1 by synthesizing it complexed with zinc (Zn-1) in a 2:1 molar ratio. Zn-1 significanlty increased the median survival of KPCp53-R172H mice from 15 to 35 days (p=0.0042). The apoptosis rate in the tumors (by Immunohistochemistry staining with Cleaved Caspase 3) was also increased by treatment of ZMC1 and Zn-1. These studies indicate that ZMC1 can function in vivo as a mutant p53 targeted anti-cancer drug at doses that are well tolerated. Furthermore, ZMC1 can be optimized by synthesizing it complexed with zinc.

#2098

Understanding the synergy between temozolomide and PARP inhibition in Ewing sarcoma.

Ruth Carlson, Raushan Kurmasheva. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Ewing sarcoma is the fourth most common highly malignant childhood cancer; it is defined by a tumor-specific chromosomal translocation. In approximately 85% of all tumors, the EWSR1 gene on chromosome 22 is fused to a member of E26 transformation-specific sequence family of transcription factors, the FLI1 gene on chromosome 11. DNA damage induced by expression of EWSR1-FLI1 fusion gene is potentiated by PARP1 inhibition in Ewing cells, where EWSR1-FLI1 genes act in a positive feedback loop to maintain the expression of PARP1. As single agents, PARP inhibitors have shown promising activity in vitro, but only modest activity in in vivo models. In our previous work (in PPTP), we showed that low level damage to DNA by temozolomide (TMZ) can be potentiated up to 40-fold through inhibition of PARP by BMN-673, leading to dramatic tumor regressions in 5 of the 10 Ewing sarcoma xenograft models. Therefore, we were aimed to determine the differences between the tumors that respond to treatment with BMN-673 and those intrinsically resistant to it, and to understand the underlying mechanisms of such resistance, with the ultimate goal to develop more effective therapy for Ewing sarcoma.

We have used the same cell lines to investigate biochemical differences between those Ewing sarcoma lines where there is synergy in xenograft models in mice, and those where the combination is inactive. Our studies using cell lines show that, 1) in vitro BMN-673 and TMZ are synergistic in all cell lines, irrespective of their response as xenografts; 2) In vivo synergy correlates with intrinsic sensitivity to BMN-673 and TMZ in vitro. For Ewing lines, where there is no in vivo synergy when they are grown as xenografts, they are ~9-fold resistant to BMN-673, and ~15-fold resistant to TMZ relative to lines where there is synergy as xenografts.

The resistance of the Ewing sarcoma xenografts to the synergy of the combination of BMN-673 and TMZ in mice is a consequence of intrinsic resistance to either or both of the drugs.

The combination of BMN-673 and TMZ in Ewing sarcoma xenografts is the most dramatic synergy between two drugs to be reported. The synergy in vivo is restricted to Ewing sarcoma, yet only half of the models respond to combination treatment with the other half completely resistant. Studies proposed in this application will potentially identify biomarkers that will allow identification of patients likely to benefit from such treatment, and spare toxicity for those unlikely to respond.

#2099

Model-riven optimization of anti-angiogenics combined with chemotherapy: application to bevacizumab + pemetrexed/cisplatin doublet in NSCLC-bearing mice.

Joseph Ciccolini,1 Sebastien Benzekry,2 Sarah Giacometti,1 Fabrice Barlesi,3 Dominique Barbolosi1. 1 _Aix Marseille University, Marseille, France;_ 2 _INRIa, Bordeaux, France;_ 3 _APHM, Marseille, France_.

Bevacizumab-containing protocols are all based upon the concomitant administration of the drugs given in a row. Bevacizumab is expected to induce a transient normalization of the tumor neo-vasculature prior to exert its anti-angiogenic properties. The resulting increase in blood flow could lead to higher drug delivery to the tumors and therefore to higher efficacy, provided that cytotoxics are administered after bevacizumab. Determining this time-window cannot be performed by empirical practice, but mathematical modeling can help to achieve this goal. To this end, we have developed an original mathematical PK/PD model that enables the description of the effect of bevacizumab on the quality of the vasculature and the resulting effects of drugs sequential administration on tumor growth. The model was able to simulate a variety of scheduling and suggested that a 5-days lag between bevacizumab and cytotoxics should achieve higher antiproliferative efficacy as compared with standard administration. To test the predictivity of the model, a comparative study was undertaken in tumor-bearing mice. Human H460 NSCLC cells stably transfected with luciferase were ectotopically implanted in swiss nude mice. Treatment consisted in the combo bevacizumab (20 mg/kg) plus pemetrexed (100 mg/kg) and cisplatin (3 mg/kg) given as 3 cycles administered every 2 weeks following different scheduling. Mice were randomly allocated into 4 groups (n=12 mice per group): control, concomitant (bevacizumab and chemo given the same day), sequence-1 (chemo followed by bevacizumab 5 days later) and sequence-2 (bevacizumab followed by chemo 5 days later). Tumor growth was monitored twice a week by bioluminescence imaging after i.p. injection of 150 mg/kg luciferine. As predicted by our mathematical model, results showed that the sequential administration of bevacizumab followed 5 days later by the pemetrexed/cisplatin doublet led to a better efficacy (-42% reduction in tumor growth at treatment completion and -35% at study conclusion, p<0.05, One-Way Anova) whereas standard dosing or reverse sequence did not significantly reduce tumor growth. Additionally, this sequential administration of bevacizumab first and cytotoxics next led to a median survival of 75 days, whereas other treatment groups could only achieve 52 days survival and control mice 32 days, respectively. Altogether, our experimental data demonstrate that delaying the administration of the chemotherapy after that of bevacizumab leads to higher efficacy and longer survival as compared with standard dosing. Although preliminary, this study suggests that current administration of bevacizumab could be an underpowered strategy. Besides, this preclinical study confirms the accuracy of our mathematical model to identify the optimal sequencing between anti-angiogenics and cytotoxics.

#2100

Imputation of drug sensitivity levels in The Cancer Genome Atlas using machine learning identifies novel predictors of chemotherapeutic response.

Paul Geeleher, R. Stephanie Huang, Fan Wang, Gladys Morrison. _University of Chicago, Chicago, IL_.

The Cancer Genome Atlas (TCGA) represents a landmark undertaking that has collected genomic data from tumors of almost 15,000 cancer patients. Most tumors have been assayed using many high-throughput genomics techniques, such as whole-genome DNA copy number, gene expression, mutation status and DNA methylation. The project has also compiled a large set of matching clinical data; however, the utility of TCGA for pharmacogenomic discovery has been severely limited by the lack of cleanly measured drug response data. The absence of such data owes to the difficulty in obtaining this information from cancer patients, because individuals are typically treated with complex multi-drug regimes, which renders the precise measurement of a drug specific response phenotype intractable. However, precisely measured drug response data can be readily obtained in pre-clinical models, such as cell lines. Thus, in this study, we have implemented a machine learning based approach, where gene expression based predictive models of drug response are constructed on approximately 700 cancer cell lines; these models are then applied to nearly 15,000 TCGA tumor samples, for which gene expression data is also available, yielding a predicted drug sensitivity value in each TCGA sample. We used this approach to impute a drug sensitivity estimate for 138 drugs that were treated against the cell lines.

Our approach allowed us to transform TCGA into a vast pharmacogenomics dataset, on an unprecedented scale (and thus power), which could be mined for novel associations relevant to chemotherapeutic response. As a proof-of-concept, we first investigated whether the existing small number of known clinically relevant associations could be recapitulated in these imputed data. As an example, we identified the predicted difference in sensitivity to Lapatinib, which interrupts ERBB2, as significantly greater in ERBB2 amplified tumors (P = 6 × 10-12), an association that is drug specific. In ERBB2 amplified tumors, large chunks of chromosome 17, containing many genes, are typically amplified; this recurring phenomenon often renders it impossible to identify the causative genes in genome wide copy number data using conventional approaches. Strikingly however, by interrogating the observed effect size of genes in this amplicon, ERBB2 could be identified as the precise causative drug target in our data, demonstrating the impressive increase in power obtained from this approach. Additionally, the second most significant association for Lapatinib is for EGFR, which is the known secondary target of this drug (P = 5 × 10-4).

We also identified many novel associations; e.g. that a copy number amplification in ERLIN2, a gene shown to play a role in stabilizing microtubules, is associated with resistance to a class of chemotherapeutics that interact with the microtubules. This was subsequently validated with follow-up experiments.

#2101

A gene-expression fingerprint predicting sensitivity to all-trans-retinoic acid in breast cancer cells is tumor-context independent.

Enrico Garattini, Maurizio Gianni', Marco Bolis, Maddalena Fratelli, Gabriela Paroni, Mineko Terao. _Mario Negri Inst. for Pharmacol. Research, Milan, Italy_.

All-trans retinoic acid (ATRA) and derived natural as well as synthetic retinoids are promising agents in the treatment and chemoprevention of various types of neoplasia, including mammary tumors. ATRA is an important component of the therapeutic schemes used for the treatment of a rare form of Acute Myelogenous Leukemia known as Acute Promyelocytic Leukemia (APL). A rational use of the paradigmatic retinoid, ATRA, in a heterogeneous disease, like breast cancer, requires the definition of the cellular and molecular determinants of sensitivity to the agent. The major aim of the study was the definition of a predictive gene expression fingerprint that can be used for the selection of patients who may benefit from treatment protocols containing ATRA. To this purpose, we selected 53 breast cancer cell lines characterized for the constitutive whole genome gene expression profiles. The sensitivity of 30 cell lines (training set) to the anti-proliferative action of ATRA was defined after challenge with increasing concentrations of the retinoid for 3, 6 and 9 days. This analysis established that Luminal and ER+ cell lines are enriched within the ATRA sensitive group. In contrast, cell lines characterized by a Basal-like phenotype, according to the PAM50 gene expression signature, are generally refractory to the growth inhibitory action of ATRA. The sensitivity of Luminal-A and Luminal-B and the general refractoriness of Basal-like tumors to ATRA was validated in short-term tissue slice cultures of surgical breast cancer specimens. The training set was used to define a gene-expression fingerprint consisting of morethan 100 genes significantly associated with ATRA sensitivity. The fingerprint was generated by reprocessing the RNA sequencing data contained in the CCLE (Cancer Cell line Encyclopedia) of the Broad Institute and it was built from approximately 60,000 coding and non-coding loci. The approach involved the use of general linear models (machine learning algorithm). The identified gene-expression fingerprint was subsequently used to successfully predict ATRA sensitivity in a test set consisting of the remaining 23 cell lines. As a first step towards the use of the fingerprint for the stratification of patients, we evaluated the proportion of predicted ATRA sensitive breast tumors in the TCGA dataset. In accordance with the cell line and primary tumor data, approximately 30% of the Luminal tumors present with a high similarity score to the identified gene expression fingerprint associated with ATRA sensitivity. In contrast, only 5% of the Basal-like or Triple-negative mammary tumors are characterized by the same high similarity score. Interestingly, the ATRA sensitivity signature seems to be tumor context independent, as it correctly identifies the Acute Promyelocytic Leukemia patients present in the Acute Myelogenous Leukemia patients present in two publicly available datasets.

#2102

Targeting mTOR downstream of LIN28 in atypical teratoid rhabdoid tumors promotes apoptosis and suppresses tumorigenicity.

Jeffrey Rubens,1 Antoinette Price,1 Brent Orr,2 Charles Eberhart,1 Eric Raabe1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _St Jude Research Hospital, Memphis, TN_.

LIN28 is a somatic cell reprogramming and stem cell factor that binds to and regulates RNA involved in growth, invasion and metabolic genes. We have previously shown that LIN28 is upregulated in atypical teratoid rhabdoid tumors (AT/RT) and contributes to the aggressive nature of these tumors. One of the canonical downstream targets of LIN28 is the mTOR pathway. We hypothesized that AT/RT tumorgenicity is dependent on mTOR signaling downstream of LIN28 and inhibition of this pathway would disrupt tumor growth. We found that primary human AT/RT samples have high expression of the mTOR pathway as determined by immunohistochemistry staining for P-S6 and P-AKT Ser473 (21% of tumors with 2+ P-S6 staining; 87% with 2+ p-AKT staining). The dual TORC1/2 inhibitor MLN0128 has good brain penetration and is currently being tested in phase I clinical trials. Treatment of AT/RT cell lines with MLN0128 inhibits TORC1/2 targets in vitro and suppresses cell proliferation at 100nM concentration in multiple AT/RT cell lines (MTS assay for BT12 p<0.005 vs DMSO control; BT37 p=0.006 vs DMSO control; and CHLA-06 p<0.005 vs DMSO control by t-test). MLN0128 also slows cell proliferation via BrdU assay (BT12 p<0.005 vs DMSO control; BT37 p<0.005 100nM vs DMSO control; CHLA-06 p<0.005 100nM vs DMSO control by t-test) and induces apoptosis measured by Western Blot for cleaved PARP and cleaved caspase 3 assay (BT12 p<0.005 100nM vs DMSO; BT37 p<0.005 100nM vs DMSO; CHLA-06 p=0.007 100nM vs DMSO by t-test). MLN0128 induces apoptosis synergistically with cisplatin, which is the backbone of conventional AT/RT therapy (CC3 assay BT37 p<0.005 vs DMSO by t-test) and slows cell growth (MTS assay BT37 p<0.005 vs DMSO control by t-test). MLN0128 treatment of AT/RT xenograft mouse models extends overall survival from a median of 23 days to 39 days (Log-rank test p=0.002). Targeting the mTOR pathway with MLN0128 leads to potent in vitro and in vivo activity against AT/RT and can be combined with conventional chemotherapy to provide an important survival benefit. MLN0128 may be a candidate for future clinical trials to treat this deadly tumor.

#2102A

Simultaneous targeting of EGFR and ALK in EML4-ALK positive lung cancer to inhibit tumor cell proliferation and migration.

Chunrong Li, Joseph G. Kern, Shyhmin Huang, Eric A. Armstrong, Lauryn R. Werner, Paul M. Harari. _University of Wisconsin School of Medicine, Madison, WI_.

Approximately 4-7% of non-small cell lung cancers (NSCLC) harbor the EML4-ALK chromosomal rearrangement. ALK has been identified as a molecular driver of cancer and ALK inhibitors have been developed for clinical use. We have confirmed high expression of EGFR in EML4-ALK-positive cell lines (H3122 and H2228). Recognizing the capacity of both EGFR and ALK signaling to regulate tumor cell growth, we investigated the effects of co-inhibition of ALK and EGFR on cell proliferation and migration in EML4-ALK-positive cells in vitro. We also examined the anti-tumor effect of ALK and EGFR inhibitors in xenograft mouse models. Cell proliferation was determined by CCK8 and cell migration was examined by wound healing assay and transwell migration assay. ALK inhibitors (crizotinb, ceritifinib and alectinib) and EGFR inhibitors (erlotinib and gefitinib) were utilized to block ALK and EGFR signaling activity.

We confirmed that H3122 and H2228 cells were resistant to EGFR inhibitors in comparison to EML4-ALK negative cell lines H226 and A549. Simultaneous knockdown of ALK and EGFR by siRNA significantly inhibited tumor cell proliferation. Combined inhibition of ALK and EGFR inhibited cell proliferation more potently than single inhibitor treatment alone. In addition, NIH3T3 cells expressing EML4-ALK variants 1, 2 or 3a displayed enhanced ability for cell migration, suggesting a role of EML4-ALK in cell migration. Individual ALK inhibitors and EGFR inhibitors inhibited cell migration. Further, combined ALK and EGFR blockade resulted in a stronger cell migration inhibition than single agent therapy. The combination of ALK inhibitor with EGFR inhibitor was more potent than single target treatment in tumor xenograft growth inhibition. Mechanistic analysis of the co-regulation of cell migration by ALK and EGFR signaling in EML4-ALK positive cells is under investigation.

These results identify enhanced inhibitory effects on tumor cell proliferation and migration with simultaneous blockade of ALK and EGFR signaling suggesting that dual targeting may prove more effective than single agent approaches. These findings indicate the potential for therapeutic benefit of co-targeting ALK and EGFR in patients with EML4-ALK positive NSCLC.

### Mechanisms of Drug Resistance 2

#2103

Activating alternative receptor tyrosine kinases induced alectinib-resistance in ALK rearranged non-small cell lung cancer cells.

Hideko Isozaki,1 Eiki Ichihara,1 Masayuki Yasugi,2 Nagio Takigawa,3 Kadoaki Ohashi,4 Toshio Kubo,4 Takashi Ninomiya,4 Nobuaki Ochi,3 Daisuke Minami,4 Kenichiro Kudo,1 Yuka Kato,1 Hiroe Kayatani,1 Tomoki Tamura,1 Kiichiro Ninomiya,1 Toshio Higo,1 Tsuyoshi Makimoto,1 Akiko Sato,4 Katsuyuki Hotta,4 Kunio Matsumoto,5 Toshiaki Sendo,1 Mitsune Tanimoto,1 Katsuyuki Kiura4. 1 _Okayama University, Okayama city, Japan;_ 2 _Chugoku Central Hospital, Fukuyama city, Japan;_ 3 _Kawasaki Medical School, Okayama city, Japan;_ 4 _Okayama University Hospital, Okayama city, Japan;_ 5 _Kanazawa University, Kanazawa city, Japan_.

Background

The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), alectinib, demonstrated high response rate, long response duration and a favorable toxic profile in patients with ALK-rearranged advanced non-small cell lung cancer in a phase II study (Lancet Oncol 14:590-8, 2013). However, even this promising drug is predicted to develop acquired resistance. Therefore, we investigated the mechanisms of resistance using two alectinib-resistant cell lines.

Methods

We established alectinib-resistant cell lines, H2228/CHR and ABC-11/CHR, from H2228 (EML4-ALK fusion genes variant 3a/b E6) and ABC-11 (EML4-ALK fusion genes variant 3b E6) respectively, by continuous exposure to alectinib. They were characterized using MTT assay, Western blotting, receptor tyrosine kinase array, ELISA, FISH, RT-PCR, and xenograft models.

Results

H2228/CHR and ABC-11/CHR cells were 117- and 40-fold more resistant than the parental lines, respectively, and maintained downstream AKT and ERK phosphorylation even in the presence of 10 μM alectinib. There were no ALK secondary mutations in those resistant cell lines. H2228/CHR lost the EML4-ALK fusion gene, and exhibited increased activation of insulin-like growth factor-1 receptor (IGF-1R) and human epidermal growth factor receptor 3 (HER3) with overexpression of the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF-1R and HER3 signaling overcame the resistance. In ABC-11/CHR, MET was activated by stimulated hepatocyte growth factor (HGF) autocrine signaling. We found HGF gene translocation underlying the HGF autocrine system. Anti-HGF antibody suppressed the MET activation and combined treatment with alectinib and anti-HGF antibody or a MET inhibitor suppressed downstream signaling in ABC-11/CHR cells. Finally, crizotinib, which targets both ALK and MET, most effectively inhibited the growth of ABC-11/CHR both in vitro and in vivo.

Conclusions

We identified novel alectinib resistance mechanisms caused by the activation of alternative tyrosine kinase receptors. Our findings provide new insights into constructing a therapeutic strategy for ALK-positive lung cancer.

#2104

ERK-dependent IL-6 autocrine signaling mediates adaptive resistance to pan-PI3K inhibitor in head and neck squamous cell carcinoma.

Mi Ran Yun,1 Han Na Kang,1 Byoung Chul Cho2. 1 _Je-uk Laboratory of Molecular Cancer Therapeutics, Yonsei Cancer Research Institute, University College of Medicine, Seoul, Republic of Korea;_ 2 _Division of Medical Oncology, Yonsei Cancer Center, Yonsei Unversity College of Medicine, Seoul, Republic of Korea_.

Purpose: To investigate resistant mechanisms to NVP-BKM120, a pan-PI3K inhibitor, and potential combination therapies in head and neck squamous cell carcinoma (HNSCC).

Experimental Design: A panel of 10 human HNSCC cell lines including NVP-BKM120-resistant HNSCC patient-derived cell lines were used to evaluate the antitumor activity of NVP-BKM120. Patient-derived tumor xenograft (PDTX) models were established from recurrent/metastatic HNSCC patients treated with and resistant to NVP-BKM120.

Results: Treatment of NVP-BKM120 led to upregulation of interleukin 6 (IL-6) and subsequent activation of either extracellular signal-regulated kinase (ERK) or signal transducers and activators of transcription 3 (STAT3), resulting in a modest antitumor effects on HNSCC cells. In a subset of cell lines, blockade of IL-6 autocrine signaling with IL-6 receptor (IL-6R) siRNA or IL-6R neutralizing antibody completely abolished constitutive or NVP-BKM120-induced activation of ERK and STAT3 as well as expression of oncogenic factors including Bcl2 and cMYC, resulting in the enhanced sensitivity to NVP-BKM120 with a marked reduction of S phase and significant increment of G0/G1 arrest. Interestingly, trametinib, a MEK inhibitor, significantly reduced NVP-BKM120-induced IL-6 and cMYC upregulation, whereas it had no effect on STAT3 inhibition. Moreover, combination of NVP-BKM120 with trametinib yielded more potent anti-tumor effects than combination with a pharmacologic inhibitor of STAT3 or siSTAT3. Furthermore, combination of NVP-BKM120 with trametinib or tocilizumab, a humanised anti-IL-6 receptor antibody, synergistically suppressed the growth of NVP-BKM120-resistant PDTX models as compared with either agent alone, which was confirmed in two PDTX-derived cell lines.

Conclusions: Collectively, these results suggested that IL-6/ERK signaling contributes to intrinsic and adaptive resistance to NVP-BKM120 in HNSCC, providing a rationale for a novel therapeutic strategy to overcome resistance to pan-PI3K inhibitors.

#2105

c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors.

Puyu Shi,1 You-Take Oh,1 Guojing Zhang,1 Weilong Yao,1 Ping Yue,1 Rajani Kanteti,2 Jacob Riehm,2 Ravi Salgia,2 Taofeek Owonikoko,1 Suresh S. Ramalingam,1 Mingwei Chen,3 Shi-Yong Sun1. 1 _Emory University Winship Cancer Institute, Atlanta, GA;_ 2 _Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL;_ 3 _Department of Respiration, First Affiliated Hospital of Medical College of Xi'an Jiaotong University,, Xi'an, Shaanxi, China_.

c-Met amplification and acquisition of a second T790M mutation are key mechanisms accounting for majority of resistant cases to first generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs; i.e., erlotinib). The third generation EGFR-TKIs (e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants while sparing wild-type EGFR, represent very promising therapeutic options for NSCLC patients who have become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. We show that c-Met amplification and hyperactivation is an universal mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed elevated levels of c-Met (due to gene amplification) and p-c-Met and were cross-resistant to AZD9291 or erlotinib. Both chemical and genetic inhibition of c-Met overcame the resistance of these cell lines to AZD9291 including enhancement of apoptosis or G1 cell cycle arrest. Consistently the combination of AZD9291 and c-Met inhibition effectively inhibited the growth of both erlotinib- and AZD9291-resistant HCC827 xenografts in nude mice. Hence, we suggest that inhibition of c-Met is also an effective strategy to overcome resistance of EGFR-mutated NSCLCs with c-Met amplification or hyperactivation to AZD9291, providing the rationale for clinical development of this novel combination strategy. (SSR, TKO and SYS are Georgia Research Alliance Distinguished Cancer Scientists)

#2106

Transcriptome analysis of patient-derived bladder cancer xenografts identifies genes associated with chemoresistance.

Aimy Sebastian,1 Kelly A. Martin,1 Chong-xian Pan,2 Ai-hong Ma,2 Ralph W. deVere White,2 Gabriela G. Loots1. 1 _Lawrence Livermore National Laboratories, Livermore, CA;_ 2 _UC Davis Comprehensive Cancer Center, Sacramento, CA_.

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

Methods: We employed a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms contributing to resistance to gemcitabine-cisplatin combination therapy in advanced bladder cancer and to identify novel candidates that can be targeted to treat chemotherapy resistant bladder cancer. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis.

Results: PDXs retained the morphology fidelity and shared 92-97% of genetic alterations of parental cancer cells. The RNA-seq data suggested the presence of significant differences between the transcription profiles of drug-sensitive and drug-resistant tumors. We identified 333 genes >2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Genes down-regulated in drug resistant tumors include tight junction protein CLDN3 and regulators of G-protein signaling RGS2 and RGS3. Significantly up-regulated genes include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane glycoprotein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment.

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

#2107

PDK1 blockade overcomes intrinsic resistance to PI3Kα inhibition.

Pau Castel,1 Haley Ellis,1 Ruzica Bago,2 Eneda Toska,1 Kannan Srinivasaraghavan,3 F Javier Carmona,1 Pedram Razavi,1 Chandra Verma,3 Maura Dickler,1 Sarat Chandarlapaty,1 Edi Brogi,1 Dario Alessi,2 José Baselga,1 Maurizio Scaltriti1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _University of Dundee, Dundee, United Kingdom;_ 3 _Bioinformatics Institute (A*STAR), Singapore, Singapore_.

Somatic mutations in PIK3CA, the gene encoding the α isoform of PI3K (p110α), are frequent in breast cancer. These mutations lead to constitutive activation of the PI3K/AKT/mTORC1 pathway and are a potential therapeutic target. Despite the encouraging activity shown by selective PI3Kα inhibitors in early clinical studies, many breast cancer patients with PIK3CA-mutated tumors remain insensitive to these agents.

We have previously shown both in preclinical models and in patients undergoing treatment with PI3Kα inhibitors that intrinsic resistance to PI3Kα inhibition is associated with sustained mTORC1 activation despite full AKT blockade. To elucidate how tumor cells can activate mTORC1 bypassing the AKT node, we performed a synthetic lethal RNAi screening using a library against the human kinome and phosphatome. We found that knockdown of phosphoinositide-dependent kinase 1 (PDK1) sensitized resistant cell lines to PI3Kα blockade, preventing mTORC1 signaling. These results were also validated by either shRNA-mediated PDK1 genetic knockdown or using the selective PDK1 kinase inhibitor, GSK2334470. The combination with PI3Kα inhibition abolished mTORC1 activity and resulted in superior antitumor activity in vitro and in vivo.

Mechanistically, the combination of GSK2334470 with PI3Kα inhibition decreased the phosphorylation of the transcription factor FOXO3 at its T32 residue, causing its nuclear translocation, occupancy at the promoters of its target genes, and increased transcription. Given that full suppression of AKT activity was already achieved with PI3Kα inhibition alone, we hypothesized that another kinase downstream of PDK1 is responsible for FOXO3 phosphorylation and is inhibited upon PDK1 blockade. We demonstrated that SGK1, an AGC kinase upstream of FOXO3 and downstream of PDK1, is responsible for mediating resistance to PI3Kα inhibitors by activating mTORC1 through the direct phosphorylation of the tumor suppressor TSC2. Consistently, we found that SGK1 mRNA and protein levels are higher in PIK3CA-mutant cells resistant to PI3Kα.

Our results were further corroborated through the characterization of the biochemical properties of a first-in-class SGK1 kinase inhibitor. This compound, in combination with a PI3Kα inhibitor, phenocopied the effects of PDK1 inhibition in all the models tested.

Our findings uncover a parallel signaling cascade triggered by PI3Kα pharmacological inhibition that reconciles the sustained mTORC1 activation with lack of AKT enzymatic activity. This offers a novel therapeutic approach for PIK3CA-mutant breast tumors intrinsically resistant to PI3Kα inhibition.

#2108

Mechanism underlying acquisition of dual resistance to EGFR and MET inhibitors by EGFR-mutant lung adenocarcinoma cells.

Toshimitsu Yamaoka,1 Tohru Ohmori,1 Motoi Ohba,1 Yasunori Murata,2 Yasunari Kishino,2 Sojiro Kusumoto,2 Hiroo Ishida,3 Tsukasa Ohnishi,2 Yasutsuna Sasakii3. 1 _Institute of Molecular Oncology, Showa University, Tokyo, Japan;_ 2 _Allergology & Respiratory Medicine, Showa University School of Medicine, Tokyo, Japan; _3 _Medical Oncology, Showa University School of Medicine, Tokyo, Japan_.

BACKGROUND. We established a cell line with acquired resistance to gefitinib by continuously exposing lung adenocarcinoma PC-9 cells to gefitinib. MET amplification was confirmed in this clone, designated as PC-9MET. In PC-9MET, no T790M mutation was detected in EGFR, and combined treatment with EGFR/MET inhibitors suppressed cell proliferation. To investigate further bypass pathways, we established a clone with dual-resistance to EGFR/MET inhibitors by exposing PC-9MET cells to increasing concentrations of MET-TKI (PHA665752) in the presence of 1 μM gefitinib. This clone was designated as PC-9DR2.

EXPERIMENTAL PROCEDURES. To investigate new bypass signals, we used a human phospho-receptor tyrosine kinase (phospho-RTK) array. Cell proliferation and signal transduction were determined by MTT assay and Western-blot analysis, respectively. mRNA expression was quantified by real-time PCR. Mouse embryonic fibroblast cell lines derived from IGF-1R-deficient mice and engineered to overexpress human IGF-1R were used to measure IGF bioactivity in cell culture media.

RESULTS. In PC-9DR2 cells, up-regulation of IGF-1R activity was confirmed on phospho-RTK array, whereas MET expression was partially attenuated. Exposure of PC-9DR2 cells to the IGF-1R-TKI OSI906 plus the MET-TKI PHA665752 suppressed cell proliferation and induced apoptosis. Interestingly, IGF-1R activation leads to PI-3K/AKT via IRS-1, and MET activation separately leads to ERK1/2 via Gab2. Furthermore, combined treatment with the MET-TKI PHA665752 and the IGF-1R inhibitor BI 836845, a humanized monoclonal antibody against IGF-1 and IGF-2, also suppressed cell proliferation, suggesting that increased ligand activation might lead to the phosphorylation of IGF-1R as a bypass signal. In a cell-based human IGF-1R phosphorylation assay, the culture medium of PC-9DR2 more potently phosphorylated IGF-1R than did the medium of PC-9MET cells. However, PC-9DR2 cells were not associated with increased expression of either IGF-1 or IGF-2, the ligands of IGF-1R. The insulin-like growth factor binding proteins (IGFBP) are a family of six proteins that function as transport proteins for IGF-1 and IGF-2 in the circulation and regulate their access to the potentially oncogenic IGF-1R. The expression levels of IGFBP2 and IGFBP4 were significantly attenuated in PC-9DR2 cells. Moreover, exposure of PC-9DR2 cells to human recombinant IGFBP2 or IGFBP4 inhibited IGF-1R phosphorylation, and concurrent treatment with PHA665752 suppressed cell proliferation.

CONCLUSION. Our findings suggest that combined treatment with IGF-1R/MET inhibitors might be of value clinically.

#2109

Clonal heterogeneity of EGFR TKI-resistant NSCLC revealed through single-cell dilutional cloning and high-throughput functional screens.

Dawn Pingxi Lau,1 Shivaji Rikka,2 Hui Sun Leong,1 Gek San Tan,3 Eleni G Christodoulou,1 Sai Sakktee Krisna,1 Shen Yon Toh,1 Xue Lin Kwang,1 Fui Teen Chong,1 Audrey Ann Liew,1 Tony Kiat Hon Lim,3 DasGupta Ramanuj,2 N Gopalakrishna Iyer,4 Daniel Shao-Weng Tan5. 1 _Cancer Therapeutics Research Lab, National Cancer Centre Singapore, Singapore;_ 2 _Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, Singapore;_ 3 _Department of Pathology, Singapore General Hospital, Singapore;_ 4 _Singhealth/Duke-NUS Department of Head and Neck Surgery, Singapore General Hospital, Singapore;_ 5 _Department of Medical Oncology, National Cancer Centre Singapore, Singapore_.

While EGFR tyrosine kinase inhibitors (TKIs) can elicit significant tumor shrinkage in the clinic, drug resistance inevitably ensues, likely due to a persistent subpopulation. T790M mutation is a common resistance mechanism found in 60% of tumors progressing on TKI. The aim of this study is to uncover novel mechanisms of gefitnib-resistant (GR) NSCLC by selecting TKI naïve PC9 single cell clones (SCC) differing in stemness and vulnerabilities to gefitinib (G), and inducing resistance through sublethal drug exposures. A high throughput drug screen was performed in 4 SCC to discover alternate therapies to target T790M negative GR clones.

231 SCC of PC9 were isolated and confirmed to be T790M negative (0.3% sensitivity) using Agena SABER assay. All clones were characterized by their vulnerability to G (viable after 48 hour exposure to 0.1uM) and ALDH1 activity using the ALDEFLUOR assay and flow cytometry. Four "diverse" clones (stratified according to ALDH1 and sensitivity to TKI) were selected to generate GR cell lines. Agglomerative hierarchical clustering of differentially expressed genes revealed that the clone exhibiting extreme low survival and ALDH1 signature clusters significantly apart from the rest. Subsequent iterations of this experiment (in total 5 per clone) revealed unique (and mostly stochastic) patterns of acquiring T790M across all clones.

GR clones were then subjected to mutational analysis using the Ion AmpliSeq™ Colon and Lung Cancer Panel v1, and showed that 11/20 (55%) PC9 SCC GR lines have acquired the T790M mutation. T790M positive (AF range: 2.4 - 41.0%) GR lines were sensitive to Afatinib and WZ4002, a 2nd and 3rd generation EGFR TKI, with EGFR1086 phosphorylation being completely absent in all non-responding lines. T790M negative GR lines further acquired NRAS Q61K, G12D mutation and KRAS Q22K mutation. A high throughput drug screen with kinase inhibitor library from Selleck Chemicals (n=273 kinases, n=349 anticancer library) confirmed the selectivity of MEK inhibitors, as well as identified a subset of T790M-ve lines that are sensitive to Aurora kinase inhibitors.

Our study highlights the potential for pEGFR to complement T790M status as a selection biomarker, particularly where low tumor cell concentration may yield false negative results. Taken together, we have uncovered potential therapeutically tractable subsets of T790M negative EGFR TKI resistant cell lines. pEGFR may identify a subset of T790M negative tumors that remain driven by and may be an alternate selection biomarker to 2nd/3rd gen TKI. Combinational approaches with MEK and aurora kinase inhibitors could be further explored in T790M negative/ pEGFR negative tumors.

#2110

The role of VAV-interacting Kruppel-like factor (VIK) in resistance to palbociclib in estrogen receptor-positive breast cancer.

Catherine Lenihan, Francesca Cavicchioli, Karen O'Leary, Alice Shia, Peter Schmid. _Barts Cancer Institute, Queen Mary University of London, London, United Kingdom_.

Background: The cyclin D-CDK4/6-Rb pathway, regulating the G1/S phase transition of the cell cycle, is frequently dysregulated in estrogen receptor (ER) positive breast cancer, and has been connected with endocrine resistance. Combined treatment of palbociclib, a highly selective CDK4/6 inhibitor, with endocrine therapy has led to a substantial improvement in outcome of patients with ER positive metastatic breast cancer. Increasing clinical use means acquired resistance to palbociclib is emerging as a new major clinical challenge, understanding of resistance mechanisms will be crucial. VAV interacting Kruppel-like factor (VIK) is a potential novel transcription factor known to be involved in cell cycle regulation, interacting with CDK4. Previously our lab reported a series of breast cancer patient tumour samples where VIK methylation was associated with an increased risk of recurrence and decreased survival in tamoxifen-treated patients, indicating a role for VIK in ER positive breast cancer.

Results: Methylation analysis of the VIK promoter region by pyrosequencing revealed dense methylation of the CpG island located in the 5' regulatory sequences of the gene, in 8 out of 20 breast cancer cells lines. Methylation above 30% correlated with absent mRNA expression, confirming methylation transcriptionally silences VIK. Subsequently, VIK was knocked down in the ER positive, VIK unmethylated breast cancer cell lines T47D and SUM44. Knockdown resulted in significant cell death due to induction of apoptosis as measured by Annexin V staining and increased levels of cleaved PARP. The knockdown of VIK altered expression of regulatory cell cycle proteins decreasing levels of Rb, pRb, cyclin D and CDK4, whilst upregulating cyclin E. Treatment with the CDK4/6 inhibitor, palbociclib, in cells with reduced VIK expression resulted in decreased sensitivity to the drug. There was a shift in IC50 value towards resistance from 100nM to 500nM in T47D cells and 80nM to 1.2µM in SUM44 cells. In a model of acquired resistance, T47D cells were cultured long-term with palbociclib generating resistant clones with increased IC50 value 10 fold higher than sensitive cells. VIK was significantly down regulated in all resistant clones to barely detectable mRNA levels, indicating an important role for VIK in resistance to CDK4/6 inhibition.

Conclusions: VIK is a novel epigenetically regulated gene in breast cancer, playing a key role in the regulation of the G1/S phase transition in the cell cycle. VIK plays an important role in development of resistance to CDK4/6 inhibitors in estrogen receptor positive breast cancer.

#2111

Mitogen-activated protein kinase-driven Sprouty2 expression mediates resistance to receptor tyrosine kinase-targeted therapeutics in glioblastoma cells.

Evan K. Day, Matthew J. Lazzara. _University of Pennsylvania, Philadelphia, PA_.

The therapeutic efficacy of inhibitors of oncogenic kinases in glioblastoma multiforme (GBM) has been disappointing to date. In glioblastoma and other malignancies, resistance to targeted therapeutics can arise through dynamic rewiring of cell signaling pathways responsible for tumorigenesis and survival. Here, we identify one such signaling rewiring process in glioblastoma cells that promotes expression of Sprouty2 (SPRY2). SPRY2 expression is of interest because we recently identified SPRY2 as a driver of glioblastoma cell and tumor proliferation and resistance to kinase inhibitors. Specifically, we found that SPRY2 depletion reduced the ability of glioblastoma cells to form colonies in soft agar, to form subcutaneous tumors in mice, and to resist co-inhibition of the EGFR and MET receptor tyrosine kinases. In the present study we found that, in a panel of glioblastoma cell lines, SPRY2 expression was initially reduced in response to inhibition of EGFR and MET. However, at later time points (24-48 hr after receptor inhibition) resurgent SPRY2 expression was observed even as EGFR and MET phosphorylation remained suppressed. To identify regulators responsible for resurgent SPRY2 expression, we undertook a quantitative systems biology approach based on partial least squares regression (PLSR) modeling wherein the phosphorylation states of multiple signaling pathways known to be regulated by EGFR and MET were measured at six time points during the 48 hrs after treatment of cells with EGFR and MET inhibitors. The PLSR model identified late stage (48 hr) phosphorylation of extracellular signal-regulated kinase (ERK) as having a role in cellular resistance to the inhibitors distinct from ERK phosphorylation at all other times. Inspection of the experimental data revealed that phosphorylated ERK, which was initially suppressed in response to EGFR and MET inhibition, also displayed a resurgence that paralleled the trend in SPRY2 expression. This correlation between ERK phosphorylation and SPRY2 expression is consistent with the documented ability of ERK to regulate SPRY2 expression transcriptionally. The importance of resurgent ERK phosphorylation was confirmed in experiments showing that its suppression through the use of a MEK inhibitor prevented resurgent SPRY2 expression and augmented cell death in response to EGFR and MET inhibitors. We hypothesize that resurgent ERK phosphorylation and resultant SPRY2 expression occur due to the ability of glioblastoma cells to activate receptor tyrosine kinases other than EGFR and MET in response to inhibition of those receptors. Antibody microarray experiments have provided some initial clues about the identity of those receptors, and experiments are ongoing to validate those results.

#2112

ABCC11/MRP8 and ABCB1/MDR1 confer eribulin resistance to breast cancer cells.

Takaaki Oba,1 Ken-ichi Ito,1 Shun'ichiro Taniguchi2. 1 _Breast, endocrine, and thoracic surgery, Shinshu University, Matsumoto, Japan;_ 2 _Institute for Biomedical Sciences, Shinshu Univ, Matsumoto, Japan_.

Background: Eribulin is a key drug for the patients with metastatic breast cancer; however, the mechanisms of how breast cancer cells become resistant to eribulin have not been fully elucidated. The purpose of this study is to analyze the mechanisms of eribulin resistance in breast cancer cells.

Cell lines:

Eribulin resistant cell lines were established for three estrogen receptor (ER) positive cell lines (MCF7/E, BT474/E,ZR75-1/E), three triple negative cell lines (MDA-MB-231/E, Hs578T/E, MDA-MB-157/E), and one HER2 positive cell line (SKBR3/E) by exposing to stepwise increased doses of eribulin.

Results: The mRNA and protein expressions of both ABCC11 and ABCB1 were increased in all eribulin resistant cell lines regardless of their subtype. Inhibition of either ABCC11 or ABCB1 by small interfering RNA (siRNA) partially restored the sensitivity to eribulin in MCF7/E, BT474/E, and ZR75-1/E cells. Moreover, dual inhibition of ABCC11 and ABCB1 additively restored the sensitivity to eribulin in MCF7/E cells. On the other hand, overexpression of exogenous ABCC11 caused eribulin resistance in MCF7 and MDA-MB-231 cells (resistance ratio; MCF7; 13.8±0.85, MDA-MB-231; 11.4±0.74). Interestingly, inhibition of ER expression by siRNA or down regulation of ER by fluvestrant decreased ABCC11 expression and increased the sensitivity to eribulin in MCF7/E cells.

Discussion: Our data demonstrates that ABCC11/MRP8 and ABCB1/MDR1 confer eribulin resistance to breast cancer cells regardless of their subtype; moreover, our data suggests the possibility of involvement of ER pathway in the regulation of ABCC11 expression in the ER positive breast cancer cells. Although further studies should be required, the expression of ABCC11/ABCB1 may be used as biomarkers for prediction of response to eribulin in breast cancer. In addition, the inhibition of ABCC11/ ABCB1 might be of service in a novel strategy to enhance the antitumor effect of eribulin.

#2113

Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC.

Matthew S. Sung,1 Xingzhi Tan,1 Christine Hosselet,1 Michael Cinque,1 Erik Upeslacis,1 Jonathon Golas,1 Fang Wang,1 Bingwen Lu,1 Laurie Tylaska,2 Lindsay King,2 Jeremy Myers,1 Edward Rosfjord,1 Judy Lucas,1 Hans-Peter Gerber,1 Frank Loganzo1. 1 _Pfizer Inc., Pearl River, NY;_ 2 _Pfizer Inc., Groton, CT_.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprised of the HER2-targeting antibody trastuzumab and DM1, a microtubule depolymerizing agent covalently attached via a non-cleavable thioether linker. T-DM1 has demonstrated clinical benefit for patients with metastatic breast cancer, however activity may be limited by inherent or acquired resistance during prolonged treatment periods. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 positive tumors, are not well understood. We used the HER2+ cell line N87 to develop a model of T-DM1 resistance utilizing a cyclical dosing schema in which cells received T-DM1 in an "on-off" routine until a T-DM1 resistant population of N87 cells (N87-TM) was generated. N87-TM cells displayed 103-fold resistance toward T-DM1 treatment compared to the parental N87 cells. The N87-TM cells were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug efflux pump (e.g. MDR1) protein expression compared to parental N87 cells. Comparative proteomic profiling suggested an enrichment in proteins (e.g. caveolin-1, CAV1) that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize ADCs into intracellular CAV1+ puncta and alter their trafficking to the lysosome compared to N87 cells. Intriguingly, T-ADCs utilizing auristatin payloads attached via an enzymatically cleavable linker (i.e. ValCit linker) overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice in vivo formed T-DM1 refractory tumors which remain sensitive to T-ADCs with cleavable-linked auristatin payloads. When comparing antigen positive patient-derived xenograft models that were refractory to T-DM1 yet responded to T-ADCs with ValCit linker-payloads, CAV1 was found to be a predictive protein biomarker identifying T-DM1 refractory tumors. These data implicate caveolae-mediated endocytosis in ADC biology and suggest that alterations in this pathway may impact a tumor's response profile to ADCs with non-cleavable linkers. We also propose CAV1 as a novel protein biomarker whose high tumoral expression predicts a refractory response to the T-DM1 ADC.

#2114

Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells.

Asmi Chakraborty, Matthew Schultz, Hoa Q. Trummell, James A. Bonner, Susan Bellis. _University of Alabama at Birmingham, Birmingham, AL_.

ST6Gal-I is a sialyltransferase that adds α2-6 linked sialic acids to cell surface proteins as they pass through the trans-Golgi. Sialic acids, being negatively charged, are able to alter the function of selected cell surface receptors, which leads to dysregulation of various downstream cellular pathways. Overexpression of ST6Gal-I has been observed in various cancers including ovarian and pancreatic cancer. We have previously shown that knockdown of ST6Gal-I expression increases ovarian cancer cell susceptibility to the chemotherapeutic drug cisplatin. In the present study we further investigate whether resistance to additional drugs such as gemcitabine, the front line treatment for pancreatic cancer, is affected by ST6Gal-I activity. MiaPaCa-2 and BxPC3 pancreatic cancer cell lines, having high ST6Gal-I expression, were employed for our studies. To elucidate the mechanistic role of ST6Gal-I in gemcitabine resistance we created stable ST6Gal-I knockdown lines of MiaPaCa-2 and BxPC3 cells. Gemcitabine induced cell death was more pronounced in the knockdown cell lines, indicated by heightened activation of caspase-3. Gemcitabine is metabolized to a nucleoside analogue, gemcitabine triphosphate, which induces apoptosis by promoting DNA damage. Increased single stand DNA damage in the knockdown cells was confirmed using alkaline comet assay. Gemcitabine treatment led to greater activation of DNA damage markers and response elements (ɣH2AX, phospho CHK1, phospho CHK2), along with increased levels of cleaved caspase-3 in the knockdown cells as compared to empty vector control cells. We next developed a stable gemcitabine resistant MiaPaCa-2 cell line by growing parental cells in gemcitabine. We selected for the population of cells that survived and were able to replicate in gemcitabine containing media. ST6Gal-I levels were found to be increased in the stable gemcitabine resistant lines relative to parental cell lines, suggesting that cells with high ST6Gal-I expression selectively survive gemcitabine treatment. By measuring the levels of genes involved in activation over those responsible for inactivation of gemcitabine, we were able to obtain a gemcitabine sensitivity predictive ratio for MiaPaCa-2 and BxPC3 ST6Gal-I knockdown, empty vector, parental and stable gemcitabine resistant cell lines. This ratio has been described previously in literature as a metric for gauging gemcitabine resistance. According to the ratios obtained, ST6Gal-I knockdown enhanced drug sensitivity whereas high expression of ST6Gal-I offered protection against gemcitabine induced apoptosis. Collectively these data indicate that upregulation of ST6Gal-I imparts tumor cell survival through prevention of gemcitabine induced DNA damage.

#2115

MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation.

Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. _Mayo Clinic, Rochester, MN_.

Background. Oncogenic BRAFV600E mutations in colorectal cancer (CRC) are associated with poor prognosis and treatment resistance. BRAF mutations activate MAP kinase signaling and may confer apoptosis resistance by upregulating anti-apoptotic BCL-2/BCL-XL/MCL-1 proteins.

Results. Ectopic expression of mutant BRAFV600E was shown to potently activate MEK/ ERK that mediated the phosphorylation (T163) and upregulation of MCL-1, but not BCL-2 or BCL-XL. MEK/ERK-mediated phosphorylation of MCL-1 (T163) increased its protein stability, shown by an MCL-1 phospho-mimic mutant. In human colon cancers, mutant vs wild-type BRAF was associated with increased MCL-1 expression by IHC. MEK/ERK inhibition by cobimetinib induced pro-apoptotic BIM in BRAF mutant CRC cell lines (HT-29, RKO), and suppressed phospho-MCL-1/MCL-1 proteins as did ERK siRNA. Suppression of MCL-1 by shRNA or using a small molecule MCL-1 inhibitor (A-1210477) synergistically enhanced cobimetinib-induced apoptosis (Table) that was associated with BAK/BAX activation. A-1210477 antagonized MCL-1 by disrupting its interaction with BAK and BIM proteins, shown by immunoprecipitation.

Conclusion. BRAF/MEK/ERK induces upregulation of MCL-1 via phosphorylation/stabilization to confer apoptosis resistance that can be overcome by combined ERK and MCL-1 inhibition, suggesting a novel therapeutic strategy against BRAF mutant CRCs.

Drug-induced apoptosis (annexin V) in CRC cell lines

---

Drugs | HT-29

% apoptosis | RKO

% apoptosis

DMSO | 16.84 | 2.11

A-1210477 (2.5 µM) | 29.52 | 22.88

Cobimetinib(5 µM) | 28.04 | 21.05

Combination | 53.22 | 50.93

#2116

Chemotherapy-induced exosomal secretion promotes chemoresistance in pancreatic cancer cells.

Girijesh K. Patel, Arun Bhardwaj, Sanjeev K. Srivastava, Mohammad Aslam Khan, Seema Singh, Moh'd Khushman, Ajay P. Singh. _Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death from cancer in the United States, with a dismal median survival rate of 2-8 months after diagnosis and a 5-year overall survival rate of ~7%. It carries a notoriously poor prognosis due to advanced stage at presentation for most patients, lack of very effective therapy and development of resistance to available chemotherapy. Intercellular communication between tumor-tumor and tumor-nontumor cells is critical for tumor growth and progression. Extracellular vesicles (EVs) comprising of exosomes, microvesicles and apoptotic bodies are considered important mediators of intercellular communication and play pivotal roles in different physiological and pathological processes. However, role of EVs in the development of acquired resistance to chemotherapy in PDAC cells is largely unexplored. Here, we examined the role of EVs in chemotherapeutic resistance of PDAC cells.

Methods: Conditioned media from vehicle-treated and (V-CM) gemcitabine-treated PDAC cells (Gem-CM) were collected and fractionated into soluble (Gem-Sol) and vesicular (GEM-EV) factions. PDAC cells were pre-treated with Gem-CM, Gem-Sol and Gem-EV followed by treatment with various doses (0-20 μM) of gemcitabine for different time intervals (48 and 72 h). EVs were then separated by differential ultracentrifugation, size was determined using Dynamic Light Scattering (DLS) and assessed for their chemoresistance conferring activity in PDAC cells. Immunoblotting was performed to examine the specific markers associated with EVs.

Results: Both Gem-CM and Gem-EV provided significant protection to gemcitabine-treated cells, while Gem-Sol had only negligible effect. DLS based-size distribution analysis identified EVs of three different size viz. large (> 1500 nm), medium (500-1500 nm) and small (100-300 nm). Treatment of large and medium sized Gem-EVs did not show any chemoprotective effect in PDAC cells, while smaller Gem-EVs provided remarkable survival benefits to PDAC cells against gemcitabine treatment. Immunoblot data revealed that these active EVs are enriched with CD9 and CD63, specific markers of exosomes. Interestingly, we observed that exosomes of gemcitabine-treated PDAC cells were larger than those secreted by vehicle treated cells. Moreover, the amount of exosomes in Gem-CM was significantly higher as comparison to the CM of vehicle-treated cells. Studies are being conducted to identify the cargo encapsulated by exosomes responsible for exosomes-mediated chemoresistance in PDAC cells.

Conclusion: Our findings suggest that chemotherapy induces secretion of exosomes in PDAC cells that enhances chemoresistance by spreading the molecular signals.

#2117

The novel and comprehensive screening of genes resistant to an anticancer drug and radiation in esophageal squamous cell carcinoma.

Hirofumi Kawakubo, Kazumasa Fukuda, Yu Kigasawa, Tomoko Takesue, Mai Tsutsui, Tsunehiro Takahashi, Rieko Nakamura, Norihito Wada, Hiroya Takeuchi, Yuko Kitagawa. _Keio University, School of Medicine, Tokyo, Japan_.

Background: Esophageal cancer has a poor prognosis due to early metastasis and direct invasion. Multimodal therapies including surgery, chemotherapy, and radiotherapy are necessary for advanced esophageal cancer. Although chemo or radiosensitivity is closely related to prognosis, it is currently impossible to predict the therapeutic effect of therapies. Patients with resistance to chemo or radiotherapy cannot derive benefit from the therapy but suffer side effects. Therefore, detecting resistant genes and mechanisms is essential for tailoring treatment to improve the prognosis.

Drug and radiation resistance may be mediated by genetic changes in the tumor; therefore, the identification of gene mutations may lead to better therapeutic outcomes. We used a novel method involving transposons to screen and identify drug-resistant genes and radio-resistant genes.

Methods: A modified piggyBac transposon was designed as an insertion mutagen, and a cytomegalovirus (CMV) promoter sequence was added to induce strong transcription. After establishing a transposon-tagged cell library, we treated cell lines derived from esophageal squamous cell carcinomas (ESCC) [Tohoku Esophagus (TE)] with cisplatin (CDDP) or radiation. We performed splinkerette PCR and TOPO cloning on the resistant colonies. Bacterial colonies were sequenced, and Next-Generation sequencing was used to identify the over-expressed/down-regulated sequences as candidate genes for CDDP resistance or radiation resistance,

Results: We established 4 cell lines of transposon-tagged cells, TE 4, 5, 9, and 15. We treated the 2 relatively viable cell lines, TE4 and TE15, with CDDP or irradiation. We identified 37 candidate genes from 8 resistant colonies for CDDP. Eight genes were over-expressed whilst 29 were down-regulated. Eleven genes were detected as candidate radioresistant genes.

Conclusions: The new gene screening technique was useful for detecting candidate of drug-resistant genes and radio-resistant genes in esophageal squamous cell carcinoma. We identified 37 candidate genes responsible for CDDP resistance in the two cell lines derived from ESCC cells. Eleven genes were detected as candidate radioresistant genes in the two cell lines derived from ESCC cells.The method is inexpensive, relatively simple, and capable of introducing activating and de-activating mutations in the genome, allowing for drug-resistant genes and radio-resistant genes to be identified.

#2118

Aurora kinase A (AURKA) dependence underlies the emergence of acquired resistance to 3rd generation EGFR inhibitors in NSCLC.

Khyati Niral Shah,1 Henry J. Haringsma,2 Andrew D. Simmons,2 Sourav Bandyopadhyay1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Clovis Oncology, San Francisco, CA_.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have been used to treat non-small cell lung carcinoma (NSCLC) patients that have EGFR-activating mutations. Despite the initial favorable response, lung cancer cells eventually develop resistance to 1st-generation EGFR-TKIs driven in approximately 60% of cases by a secondary mutation (T790M) in EGFR. Third-generation EGFR-TKIs, including osimertinib (AZD9291) and rociletinib (CO-1686), target mutant forms of EGFR (including T790M) and have shown evidence of clinical activity in patients with T790M-positive NSCLC. However, like previous EGFR-TKIs, acquired resistance to these agents eventually occurs through largely unknown mechanisms. To better understand mechanisms of resistance, we generated in vitro acquired resistant cell line models, including one from the NCI-H1975 cell line harboring EGFR T790M. We utilized a pathway-oriented chemical screen to identify compounds with potent synergy when used in combination with rociletinib in acquired resistance models. We reveal that NSCLC cells escape from EGFR blockade by activation of Aurora kinase signaling and that co-treatment with EGFR and Aurora kinase (AURKi) inhibitors can cause apoptosis in these cells. In addition, up-front co-treatment of parental NSCLC cells blocks the emergence of acquired resistance in multiple models. In PC-9 2A10 resistant xenograft model, the combination of rociletinib and the AURKi MLN-8237 prevents tumor growth with minimal toxicity. Molecular studies reveal that co-treatment with rociletinib and AURKi cooperates to suppress EGFR and AURKA signaling, whereas treatment with either compound alone incompletely inhibits signaling. Consistently, EGFR-TKI/AURKi cotreatment cooperates to trigger NFkB inactivation, loss of the pro-survival factor survivin, loss of mitochondrial membrane potential, and induction of apoptosis. The identification of a synthetic lethal interaction between EGFR and AURKA inhibitors has important implications for the development of novel treatment strategies in T790M positive NSCLC tumors.

#2119

Acquired nintedanib resistance in FGFR1-driven small cell but not non-small cell lung cancer is mediated by ABCB1.

Bernhard Englinger,1 Daniela Lötsch,1 Christine Pirker,1 Sushilla van Schoonhoven,1 Bernd Boidol,2 Charles Lardeau,2 Stefan Kubicek,2 Pal Szabó,3 Gergely Szakacs,4 Walter Berger1. 1 _Institute of Cancer Research, Vienna, Austria;_ 2 _Research Center for Molecular Medicine, Vienna, Austria;_ 3 _Institute of Organic Chemistry, Hungarian Academy of Sciences, Budapest, Hungary;_ 4 _Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary_.

Lung cancer accounts for the highest number of cancer-related deaths worldwide. Patient outcome is dismal and approval of targeted compounds is yet restricted to non-small cell lung cancer (NSCLC) histology. In defined subgroups of NSCLC, but also of small cell lung cancer (SCLC), genetically amplified fibroblast growth factor receptor 1 (FGFR1) is an oncogenic driver and inhibition of the FGFR1 signaling axis exerts potent antitumor effects. The FGFR/PDGFR/VEGFR inhibitor (TKI) nintedanib has recently been approved for second-line treatment of lung adenocarcinoma (AC) and is also being investigated in clinical trials for the treatment of SCLC. Despite initial treatment success, tumor recurrence due to acquired drug resistance is anticipated. We therefore aimed at characterizing the molecular mechanisms underlying resistance development of FGFR1-driven lung cancer against nintedanib.

One SCLC line (DMS114) and two NSCLC squamous cell carcinoma (SCC) lines (NCI-H1703, NCI-H520) driven by FGFR1 based on gene amplification were selected for nintedanib resistance in vitro. Cytotoxicity was evaluated by MTT and FACS. Intracellular responses to drug exposure were analyzed by qPCR and Western Blot. To compare transcription levels of parental cells with their selected sublines, whole-genome gene expression array was performed. Cross-cytotoxicity profiles were determined by a large-scale anticancer compound screen. ATP-binding cassette (ABC) transporter activity was analyzed by calcein AM efflux- and ATPase assay, intracellular Nintedanib levels were determined by liquid chromatograpy-mass spectrometry (LC-MS).

Chronic exposure to the FGFR/PDGFR/VEGFR inhibitor nintedanib led to expression of the ABCB1 multidrug-resistance (MDR) efflux pump in the SCLC cell line DMS114 but not in the NSCLC SCC cell lines NCI-H1703 and NCI-H520. In the nintedanib-selected subline of DMS114 (DMS114/NIN), the FGFR1 signaling axis remained active. Insensitivity of DMS1114/NIN towards Nintedanib was mediated by efflux via ABCB1, revealing Nintedanib as a substrate for this efflux pump. Ponatinib, another non-ABCB1-substrate FGFR inhibitor, retained strong cytotoxic activity. Additionally, DMS114/NIN cells exerted profound and ABCB1-dependent collateral sensitivity against the MDR-selective lanthanum compound KP772.

ABCB1 needs to be considered as a factor underlying intrinsic and acquired nintedanib resistance. On one hand, in second-line therapy, chemotherapy-induced MDR might render tumors resistant to Nintedanib. On the other hand, in first-line treatment, high ABCB1 expression in cancer cells or the microvasculature e.g. in clear-cell renal carcinoma or CNS tumors might account for intrinsic Nintedanib resistance. Switching to MDR-selective anticancer compounds or to non-ABCB1-substrate small molecule TKIs in FGFR/PDGFR/VEGFR-driven tumors might be an option to circumvent nintedanib resistance.

#2120

Molecular characterization of cancer stem cells derived from drug-resistant melanoma.

Elyse J. Berlinberg, William Crosson, Arbis Rojas, Ramin Nazarian. _UCLA David Geffen School of Medicine, Los Angeles, CA_.

BRAF kinase is a key intermediate in the MAP kinase pathway, which is associated with cell growth and proliferation. BRAFV600E activating mutation is found in 50-60% of all melanomas and 7% of all cancer malignancies. Despite an unprecedented initial response, patients treated with a highly effective BRAF inhibitor, vemurafenib (FDA approved 2011), can acquire drug resistance in as little as 6-9 months. Epigenetically driven epithelial-to-mesenchymal transition (EMT), a hallmark of cancer stem-like cells (CSCs), is a potential mechanism that melanomas employ to evade targeted therapy. We are investigating molecular alterations due to EMT in parental and vemurafenib-resistant melanoma sublines. Characterization of differential protein expression in parental and resistant cell lines through immunocytochemical, flow cytometric, and immunoblot analysis has indicated that EMT could confer drug resistance that prolongs survival of malignant melanomas. Furthermore, comparison of parental to isogenic resistant melanoma cell lines showed upregulated expression of EMT-related biomarkers that may be suggestive of the development of CSCs. These CSCs may serve as progenitor cells for therapy-resistant sublines in a heterogeneous melanoma population. Our research has identified notable EMT biomarkers in CSCs that can be used as novel targets for diagnosis and treatment of tumor resistance in melanoma patients.

#2121

Src mediates acquired resistance to ALK inhibitor in ALK-rearranged non-small cell lung cancers.

Ryohei Yoshida, Shunsuke Okumura, Takaaki Sasaki, Yoshinobu Osaki. _Asahikawa Medical University, Asahikawa city, Hokkaido, Japan_.

ALK tyrosine kinase inhibitors (TKIs) play a significant role in the treatment of ALK-rearranged non-small cell lung cancer patients.After the significant initial response to ALK-TKIs, the emergence of acquired resistance ultimately occur for all patients.

Understanding the origin of resistance mechanisms within a patient is crucial to guide treatment and developing drugs/combinations to circumvent resistance.

There are reported that some of the tyrosine kinases are involving by bypassing ALK signaling, but comprehensive identification of proteins that are significant for acquired resistance in ALK-TKIs is still required.

Quantitative proteomic analysis provides a powerful approach in screening for alterations in protein levels. An analysis of the differential proteins expression between original cancer cell and acquired resistance cell would be useful for identification of proteins drug resistance.

To investigate the changes of chaperon holding proteins, ALK rearranged-cell line H3122 were used. ALK-TKI Alectinib resistant cell lines were established by exposing increased dose of alectinib to ALK rearranged H3122 cell line (AFR). Luminespib (AUY922, Novartis Pharm.) was used as HSP90 inhibitor and Saracatinib (Selleck Chemicals.) was used as Src inhibitor in this study.

We used iTRAQ (isobaric tags for relative and absolute quantitation) technology using nano-LC-MS/MS analysis, to identify alterations in H3122 protein levels of pre/post HSP90 inhibitor treatment. For further analysis, protein expression was measured by western blot. To assess the cancer cell growth inhibition for ALK and/or Src inhibitors, we used colony formation assay.

Our results revealed that Crk-Src related proteins were overexpressed in ALK TKI resistance cells and had a significant protein expression changes after HSP90 inhibitor treatment. To address the impact of overcoming resistance by blocking Crk-Src pathway, Drugs/combinations with ALK inhibitor and/or Src inhibitors showed that significant treatment effect in vitro and in vivo study using tumor-bearing mouse model.

Conclusion

Src is one of the significant signaling pathways in the ALK-rearranged NSCLCs resistant to ALK-TKIs including alectinib, and inhibiting Src signaling could be a potential target for ALK-rearranged NSCLC after ALK-TKI resistance.

#2122

Downregulation of RGS10 induces PI3K/mTOR inhibitor resistance by Ras activation in glioblastoma.

Xiaolong Li, Shaofang Wu, Dimpy Koul, W.K. Alfred Yung. _MD Anderson Cancer Center, Houston, TX_.

Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults. The prognosis of affected patients is poor and median survival is only one year. The essential role of PI3K signaling in GBM proliferation, metabolism and differentiation has been highlighted. The major mechanism for the oncogenic activity of PI3K is by preventing apoptosis through the downstream activation of Akt and mTOR. The importance of mTOR in the regulation of cell survival downstream of PI3K signaling provides a strong rationale for the use of dual PI3K/mTOR inhibitor. However, the limited effect of dual PI3K/mTOR inhibitor used as single agent is most likely due to activation of collateral pathways, suggesting that additional refinement of this overall approach is necessary. We investigated potential bypass mechanisms to PI3K/mTOR inhibition as a single agent and generated three resistant cell lines to PI3K/mTOR inhibitor with 3 to 5-fold increase in IC50 value and with an aim to identify the potential bypass of survival to PI3K/mTOR inhibitor. We show that the IC50 of the resistant cell lines and parental lines is correlated with s6 phosphorylation and the three resistant cell lines express increased level of phosphorylated ERK1/2, p70S6K and p90RSK protein levels. Further investigation show that RGS10, a regulator of G protein signaling, is down regulated in the resistant cell lines. Down regulation of RGS10 increases the GTP-bound Ras, implying that Ras activation defines the acquired resistance by activating the ERK/p90RSK pathway. Knockdown RGS10 in the parental cells induces ERK1/2, p70S6K and p90RSK phosphorylation and the resistance to PI3K/mTOR inhibitor. Raf1 or p90RSK knockdown can re-sensitize resistant cell lines to PI3K/mTOR inhibition. Combination of PI3K/mTOR inhibitor with Raf inhibitor Sorafinib efficiently inhibited the growth of the resistant cell lines showing Ras/Raf activation serves as an escape mechanism to PI3K/mTOR inhibition.

These observations suggest that Ras/Raf signaling serves as an survival signal upon PI3K/mTOR inhibition and provides a strong rationale for the combined use of PI3K/mTOR and Raf1 inhibition to induce lethal growth inhibition in human GBM.

#2123

Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines.

Tanuka Biswas,1 Rebecca Fritzemeier,2 Adam Mark,3 Tobias Meißner,3 Brandon M. Young,3 Mark D. Pegram1. 1 _Stanford University, Stanford, CA;_ 2 _University of Washington, Seattle, WA;_ 3 _Avera Cancer Institute, La Jolla, CA_.

Resistance to monoclonal antibody (mAb) therapy limits clinical utility. ADCC is a significant mechanism of action of mAb-elicited tumor response. Postulated mechanisms of mAb resistance include, alteration in cell signaling pathways (PI3K/Akt/Ras-MAPK signaling), over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes. However, most studies on mAb resistance have utilized cancer cells conditioned with mAbs alone, in absence of immune effector cells. We developed an in vitro selection model wherein human HER2 positive breast cancer cell lines (BT474, SKBR3) and EGFR-expressing colorectal cancer cell lines (HT29, DLD1) were subjected to acute ADCC (>90% cell death) with high effector-target (100:1) ratio repeatedly with trastuzumab and cetuximab, respectively, using human peripheral blood mononuclear cells (PBMCs) as effectors. Surviving cells were allowed to recover to confluence over 8-10 weeks for 10 total rounds of ADCC selection. Internal controls included mock-treated parental, isotype-IgG1, effector cells only, and mAb alone. In vitro ADCC assays based on calcein fluorescent labeling of live target cells, demonstrated significant reduction (20%, p<0.005) in cell lysis in immune-selected cell lines compared to parental controls. ADCC-conditioned HT29 cells demonstrated 5-fold reduction in EGFR gene copy number by fluorescence in situ hybridization (FISH, p<0.0001), 2-fold reduction in mRNA level (qPCR, p< 0.01) and 1.8-fold reduction in protein level (immunoblot densitometry, p<0.01). In BT474 breast cells, ADCC-conditioned cells acquired elongated fibroblast-like morphology. Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 x 150 bp paired-end, with over 100 million paired-end reads/sample), coupled with bioinformatic analyses (Reactome pathway database) revealed gene expression changes in immune-selected cells. These involve changes in key immune signaling pathways such as NK-cell and T-cell maturation, class I MHC antigen processing, and immune regulatory pathways, among others. Further validation through q-PCR analyses and loss/ gain of expression is ongoing. Future investigation will also determine whether mAb combination with agonist CD137 mAb (co-stimulatory NK receptor) or Fc-engineered mAbs (e.g. afucosylated) will re-sensitize resistant cells. Our data indicate immune-selection by effector cells contributes to ADCC resistance in vitro. Such investigation may help to elucidate potentially targetable pathways/ markers that emerge from immune-selection with mAbs and effector cells.

#2124

Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ breast cancer.

Andrew T. Baker, Kinnari Pandya, Clodia Osipo. _Loyola University Chicago, Maywood, IL_.

ErbB-2 (HER2) is over expressed in 15 - 25% of breast cancer. First line therapy for HER2+ breast cancer is a humanized, monoclonal antibody, trastuzumab. Resistance is a major clinical problem. Previous work has suggested that Notch-1 is critical for resistance. PTEN, a breast tumor suppressor and potent inhibitor of the PI3-K/AKT is decreased in HER2+ breast cancer and loss of PTEN predicts for worst outcome. Notch-1 downregulates PTEN expression and we sought to determine the mechanism by which Notch-1 attenuates PTEN and whether this decrease is necessary for trastuzumab resistance. Our hypothesis is that Notch-1 decreases PTEN to maintain trastuzumab resistance. Human HER2+ breast cancer cell lines: sensitive (BT474HS) or resistant (BT474HR and HCC1954) to trastuzumab were used to measure Notch-1 modulation of PTEN expression when treated with trastuzumab. Protein and transcript expression of PTEN were measured by Western blot and RT-PCR, respectively. ChIP assays were preformed to identify whether Notch-1 regulated PTEN expression was mediated by direct recruitment of Notch-1 to the PTEN promoter. Growth and mammosphere forming assays were performed to determine if trastuzumab resistance could be rescued in trastuzumab treated cells with simultaneous Notch-1 and PTEN knockdown, or increased downstream PI3K pathway activity using a constitutive active form of AKT1 (Myr-AKT1). Western blot and RT-PCR results showed that Notch-1 protein levels are increased while PTEN mRNA and protein levels are decreased in trastuzumab resistant cells (BT-474HR) compared to sensitive (BT-474HS) cells. Results revealed that Notch-1 knockdown increased PTEN transcript and protein expression. Notch-1 ChIP assays demonstrated that Notch-1 was enriched on the PTEN promoter and a Notch target gene promoter, HEY1 in resistant cells. Notch1 knockdown significantly decreased proliferation and mammosphere forming efficiency (MFE) of resistant cells regardless of trastuzumab treatment. PTEN knockdown rescued the decrease in proliferation and MFE mediated by Notch-1 knockdown. As PTEN is an attenuator of PI3-K/AKT signaling, we asked whether constitutive activation of AKT1 was sufficient to rescue proliferation upon Notch-1 knockdown. The results showed that myr-AKT1 increased AKT1 signaling and rescued the decrease in proliferation mediated by Notch-1 knockdown. These results demonstrate, to our knowledge, for the first time that Notch-1 is a direct transcriptional repressor of PTEN in HER2+ breast cancer cells. Increased activation of the PI3K pathway through Notch-1 mediated inhibition of PTEN is necessary to maintain proliferation and possibly survival of the cancer stem cell population of trastuzumab resistant cells. Inhibiting the expression and/or activity of Notch-1 in HER2+ breast cancer could prove to be an effective therapy to prevent expansion of cancer stem cells and/or re-sensitize HER2+ breast cancer to ant-HER2 therapy.

#2125

ERG and AR-v7 involvement in taxane resistance of metastatic castration-resistant prostate cancers.

Adeline Berger, Seaho Kim, Ada Gjyrezi, Giuseppe Galletti, Andrea Sboner, Mark A. Rubin, Scott Tagawa, Paraskevi Giannakakou, David S. rickman. _Weill Cornell Medicine, New York, NY_.

While taxane-class drugs remains the only chemotherapy agents shown to improve survival of patients with metastatic castration-resistant prostate cancer (mCRPC), most patients with mCRPC ultimately become refractory due to the development of drug resistance. The molecular mechanisms involved in this resistance remain an unmet need. We previously showed that ERG, an oncogenic transcription factor, directly interacts with soluble tubulin dimers impairing the ability of taxanes to bind to microtubules in vitro and in circulating tumor cells (CTCs) from CRPC patients. We also demonstrated that expression of AR-v7 confers taxane resistance in vitro and in animal models of CRPC, owing to the lack of the microtubule-binding hinge domain which renders AR-v7 insensitive to microtubule stabilization by the taxanes. The purpose of the present study is to evaluate the impact of ERG and AR-v7 co-expression in prostate cells and to characterize their cooperative role in mediating taxane resistance. We generated multiple isogenic cell lines over-expressing ERG alone or in combination with AR-v7, and engineered VCaP cells (harboring endogenous ERG rearrangement and expressing AR-v7) to express a TetOn-shRNA targeting ERG mRNA. We also developed and optimized a Digital Droplet PCR (DD-PCR) assay to quantify the expression of ERG derived from the TMPRSS2-ERG gene fusion and of AR-v7. We tested the sensitivity and specificity of the assays using VCaP cells spiked into healthy donor blood samples. We detected ERG and AR-v7 co-expression in single VCaP cells. We also found that ERG and AR-v7 form a protein complex and we are testing their potential co-binding at common target genes using ChIP-seq. To determine the clinical relevance of our findings we queried available RNAseq data from benign prostate, locally advanced hormone naïve prostate cancer and a subset of the Stand-up-to-Cancer cohort of CRPC patients. We found high levels of AR-v7 expression in CRPC samples and in none of the benign or PCa samples and a co-overexpression of AR-v7 and ERG with 75% of ERG positive CRPC also expressing AR-v7. We are currently using our assays to characterize ERG and AR-v7 co-expression in CTCs of mCRPC patients samples collected at baseline, on treatment and relapse- as part of a fully-enrolled clinical trial (TAXYNERGY). We expect an enrichment overtime of CTCs that are positive for both ERG and AR-v7. In conclusion, determining ERG and AR-v7 status in mCRPC patients and their involvement in taxane resistance mechanism will aid refer ERG and AR-v7 positive mCRPC patients to an effective therapy.

#2126

Androgen receptor expression is associated with sunitinib resistance in renal cell carcinoma models.

Remi M. Adelaiye-Ogala,1 Sreenivasulu Chintala,1 Ashley Orillion,1 Piergiorgio Pettazzoni,2 May Elbanna,1 Bennett Elzey,3 Kiersten Marie Miles,4 Chinghai Kao,1 Giulio F. Draetta,2 Roberto Pili1. 1 _Indiana University-Purdue University Indianapolis Indiana, Indianapolis, IN;_ 2 _The University of Texas, MD Anderson Cancer Center, Houston, TX;_ 3 _Purdue University, Lafayette, IN;_ 4 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Androgen receptor (AR) expression has been reported in renal cell carcinoma but its biological role remains elusive. Sunitinib is a potent anti-angiogenic drug approved for the treatment of advanced renal cell carcinoma (RCC). However, eventually RCC tumors develop drug resistance. We have hypothesized that tumor cells resistance to sunitinib is associated with kinome reprograming and anti-apoptotic genes upregulation. To date, there is no report on the association between AR expression and resistance to TKI such as sunitinib in RCC. Our study was designed to investigate the role of AR in sunitinib resistance in RCC. We used our previously reported sunitinib resistant ccRCC cells and patient derived xenograft models. Methods: Human RCC cell lines; 786-0, 786-0R (sunitinib resistant), C2, C2R (sunitinib resistant), ACHN and Caki 2 were utilized to determine sensitivity or resistance to sunitinib. Patient derived xenograft (PDX) models of advance and metastatic RCC and RCC cell lines, sensitive and resistant tumors were used to detect AR expression by qRT-PCR, immunohistochemistry and Western blot analysis. Reverse phase protein array (RPPA) was used to assess 249 protein including AR in sunitinib sensitive and resistant tumors. Results: Our qRT-PCR data showed an increase by 1000 folds in mRNA levels of AR in our sunitinib resistant cell lines. Similarly, RPPA data revealed AR to be increased in sunitinib resistant RCC PDX tumors. This observation was confirmed by Western blot analysis. Conclusion: Overall our data suggest the potential role of AR and its association with resistance to sunitinib. Ongoing studies are testing the in vitro and in vivo combination treatment of RCC models with sunitinib and AR antagonists.

#2127

Identification of a novel NAMPT inhibition resistance mechanism utilizing the de novo NAD+ biosynthesis pathway.

jun guo, Chris Tse, William N. Pappano. _AbbVie, North Chicago, IL_.

Targeting NAD(H) metabolism is being interrogated as a novel anti-cancer strategy. Nicotinamide phoshophoribosyltransferase (NAMPT) was found to be overexpressed in tumor cells, and plays a key role in the replenishment of the NAD(H) pool in cells. In this study we have used a commercially available NAMPT inhibitor, GMX1778, which exerts a cytotoxic effect by decreasing the cellular level of NAD(H) . Through serial selection with increasing sub-lethal concentrations of GMX1778, we derived a GMX1778-resistant HT1080 human fibrosarcoma cell line (HT1080-GMX). The IC50 value for GMX1778-induced cell killing of the resistant cell line was 100-fold greater than that determined for the parental cell line. No significant change in the level of expression of NAMPT was observed in HT1080 vs. HT1080-GMX cells. However, a key enzyme in the de novo NAD(H) synthesis pathway known as quinolinate phosphoribosyl transferase (QPRT) was significantly upregulated as validated by Western blot and bDNA analysis. Knockdown of QPRT partially restored the sensitivity to GMX1778 exposure in HT1080-GMX cells, but did not affect the parental cells. The cytotoxicity of GMX1778 can be partially rescued with exogenous quinolinic acid, which permits NAD(H) repletion via QPRT in the de novo pathway, in HT1080-GMX cells but not HT1080 parental cells. Overexpression of QPRT in HT1080 cells did not fully rescue GMX-induced cytotoxicity and strongly suggests that there are additional mechanisms of resistance in the HT1080-GMX cell line beyond QPRT overexpression. Exome sequencing of the NAMPT gene in the HT1080-GMX resistant lines identified a single mutation of amino acid residue 18 from tyrosine to cysteine [NAMPT(Y18C)] located in the first exon of one allele. Tyrosine 18 is located near the active site of NAMPT and associates with NAMPT small molecule inhibitors through a pi stacking interaction, as determined by studies of the enzyme's crystal structure. Overexpression of the NAMPT mutant Y18C in HT1080 cells significantly reduced the sensitivity to GMX1778 exposure, but overexpression of a NAMPT Y18F mutant had no effects compared to the parental line. Based on our results, activation of an alternate NAD(H) biosynthesis pathway via QPRT or the Y18C mutation within the NAMPT coding sequence can both be contributors to resistance in this line. The characterization of resistance inducing up-regulation of other sources of NAD(H) such as de novo synthesis or mutations in NAMPT may be useful in developing next-generation NAMPT inhibitors that are less affected by known resistance mechanisms.

Disclosures:

All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

#2128

Influence of estrogen receptor status on acquisition of doxorubicin resistance in breast cancer cells.

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

Breast cancer is the most commonly diagnosed invasive cancer in women. Doxorubicin is being the mainstay treatment for solid cancers including breast cancer. However, chemotherapy resistance, either innate or acquired, is a major limitation in breast cancer treatment. The mechanism of resistance as well as the role of estrogen receptor (ER) status in doxorubicin resistance in breast cancer is not clear. Therefore, objective of this study was to determine whether ER status influence the doxorubicin resistance, and to further identify the molecular mechanism in this process. To address this question, ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cell lines were given continuous treatment of clinically relevant concentration of doxorubicin and the pattern of resistance was monitored. Resistance was evaluated using various parameters such as cell count, cell growth and cytotoxicity by MTT assay, cell cycle analysis using flow cytometer. Expression of genes and protein related to cell cycle and cell survival, drug transport, DNA damage repair, metastasis/invasion and epigenetic regulatory complex were measured by qRT- PCR and western blot respectively. Soft agar assay and wound healing assay were performed to determine the anchorage-independent growth and migration potential of resistance cells. Results of MTT assay, and cell cycle revealed that ER-positive MCF-7 cells developed relatively earlier and high level of resistance when compared to MDA-MB-231 cells. This was further confirmed by additional features such as, cancer stem cell markers, epithelial to mesenchymal transition, and increased tumorigenicity in MCF-7 cells than in MDA-MB-231 cells during resistance development. These changes were associated with alteration in drug transporters, cell cycle genes and epigenetic regulatory proteins such as HDAC1 and DNMT1. Pretreatment with demethylating agent 5-aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly resensitized resistance MCF-7 and MDA-MB-231 cells to doxorubicin in comparable to sensitive parental cells. However, this resenstitivty was higher in MCF-7 cells when compared to MDA-MB-231 cells. In summary, result of this study suggests that acquisition of doxorubicin resistance depends on status of estrogen receptor in breast cancer cell lines. Additionally, this differential resistance pattern could be due to differences in epigenetic changes in these two types of breast cancers, thus the acquired resistance could be potentially resensitized using epigenetic therapy

#2129

Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer.

Adam D. Swick, Tan Xiaojun, Tyler Fowler, Kwangok Nickel, Richard A. Anderson, Randall J. Kimple. _University of Wisconsin, Madison, WI_.

Background and purpose:

Despite decades of research into improved treatments for head and neck cancer, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Among the plethora of intrinsic and acquired mechanisms of resistance, recent evidence has suggested the cellular process of autophagy may be an additional contributor. This stress response, which normally functions during periods of nutrient deprivation to maintain cellular homeostasis, may have the unintended consequence of protecting tumor cells from the cytotoxic effects of chemotherapy and radiotherapy. We show that autophagy mediates resistance to chemotherapies targeting the EGFR and mTORC pathways in addition to radiotherapy in several head and neck cancer models, begin to elucidate the mechanism of this resistance, and demonstrate that the addition of a specific autophagy inhibitor can block cytoprotective autophagy.

Methods and results:

Building on well established evidence that mTORC inhibition can directly induce autophagy, recent work has established a novel mechanism by which kinase inactive EGFR resulting from either growth factor starvation or anti-EGFR antibody (cetuximab) can upregulate this response via an interaction with LAMPT4B. Treatment of a panel of HNC cell lines with either serum starvation or cetuximab produced increased formation of LC3-B puncta, a marker for autophagasome formation, by IF staining, and was confirmed by Western blot for increased LC3-B flux and decreased p62. SiRNA knockdown of either EGFR or LAMPT4B in HNC cells abrogated the induction of autophagy in response to either serum starvation or cetuximab treatment. We have also shown that radiation induces autophagy in a time and dose dependent manner. The mechanism of radiation-induced autophagy remains a subject of study; preliminary data suggest that both EGFR and LAMPT4B play a role as siRNA knockdown of those mRNAs reduces the autophagic response. To determine whether blockade of cytoprotective autophagy can help overcome therapeutic resistance, we have tested specific autophagy inhibitors in combination with either chemotherapy or radiation. The novel ULK1 inhibitor SBI-0206965 successfully blocks the induction of autophagy in a panel of HNC lines. More compellingly, while neither SBI-0206965 nor the NEDD4 inhibitor Heclin, impacted cell growth in untreated cells, both were effective in reducing cell survival following treatment with radiation, cetuximab, or mTORC inhibitor (AZD8055).

Summary:

These pre-clinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anti-cancer treatments including EGFR or mTORC inhibition and radiotherapy. Further we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance.

#2130

The novel G-quadruplex with hairpin loop formed immediately upstream of the human BCL-2 P1 promoter represses BCL-2 transcription.

Buket Onel, Guanhui Wu, Megan Carver, Daria Timonina, Marti Larriva, Danzhou Yang. _The University of Arizona, Tucson, AZ_.

The abnormal overexpression of the BCL-2 protein is linked to a large number of cancers. Elevated levels of BCL-2 are found to promote resistance to chemotherapy and radiation. Therefore, BCL-2 is considered to be an attractive target for cancer therapeutics. We have previously identified a 39-base-pair GC-rich sequence located in the major P1 promoter region of the human BCL-2 gene, whose G-rich strand (Pu39) can form two interchangeable G-quadruplex (G4) structures, a hybrid-type G-quadruplex and a parallel G-quadruplex with a 13-nt middle loop. However, in our functional study of the BCL-2 P1 promoter activity using a luciferase reporter system containing this region in tumor cells, we found that the construct with a complete G-quadruplex-knock-out Pu39 mutant is still affected by G4-interactive compounds. In this study we report a new 29-mer G-quadruplex-forming sequence, P1G4, just downstream of Pu39 and immediately upstream of the human BCL-2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. Using NMR spectroscopy, the P1G4 G-quadruplex is shown to be a novel dynamic equilibrium of two parallel structures, one regular G4 with two 1-nt loops and a 12-nt middle loop and another broken-strand G4 with three 1-nt loops and a 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) structure in the long loop. Using dimethyl sulfate (DMS) footprinting assays, we show that G-quadruplexes can readily form in the P1G4 sequence under physiological salt conditions, and that P1G4 and previously identified Pu39 G-quadruplexes appear to form independently in adjacent regions. To further understand the respective function of Pu39 and P1G4, we created luciferase-constructs driven by the BCL-2 promoter sequence including both Pu39 and P1G4, and examined their functions by knocking out either or both of them. We found P1G4 independently and predominantly represses BCL-2 transcription. Unlike the 1245 parallel G-quadruplex formed in Pu39, P1G4 structure is easily compromised by modifications in the hairpin loop, indicating the loop is crucial for the maintenance of this dynamic structure. The dynamic equilibrium of two closely related structures and the unique hairpin loop conformation are specific to the P1G4 sequence and distinguish the P1G4 quadruplex from other parallel structures, and may provide a unique target for small molecules to modulate BCL-2 gene transcription. The presence of P1G4 and Pu39 G-quadruplexes in adjacent regions suggests a mechanism for precise regulation of BCL-2 gene transcription.

#2130A

Epigenetic silencing of TGFβ1, BCL6, KILLIN and CTSZ is related to trastuzumab resistance in HER2-positive breast cancer models.

Sonia Palomeras,1 Angel Diaz-Lagares,2 Sarrats Ariadna,1 Crujeiras Ana Belen,2 Giro-Perafita Ariadna,1 Juan Sandoval,2 Marc Rabionet,1 Manel Esteller,2 Teresa Puig1. 1 _TargetsLab (Oncology Unit); Medical Sciences Dept.; Faculty of Medicine, University of Girona, Girona, Spain;_ 2 _Epigenetics and Cancer Biology Program (PEBC); IDIBELL; Hospital Duran i Reynals, Barcelona, Spain_.

Introduction: The major clinical problem for HER2 breast cancer target therapies is the acquisition of resistance. Trastuzumab has been used for the treatment of HER2-positive early stage and metastatic breast cancers. Notoriously, up to 62% patients that respond to the initial treatment develop resistance within a year. Different cellular and molecular alterations may contribute to the development of Trastuzumab resistance, including epigenetic changes. The aim of our study has been the evaluation of epigenetic differences between Trastuzumab-sensitive and -resistant breast cancer cell lines to determine whether they might be used as potential biomarkers to help in the treatment assessment and monitoring.

Experimental procedures: To understand the epigenetic changes that may be associated with Trastuzumab resistance we compared the DNA methylation status of ~450000 CpG sites from a DNA methylation microarray performed in Trastuzumab-sensitive and resistant cells. To identify the differentially methylated CpGs we considered differences between methylation groups of ≥60%. The functions associated to genes were analysed using the Gene Ontology and were used to select a small number of candidate genes. Their differences in promoter methylation status were validated by expression techniques (qPCR) and subsequently confirmed by demethylation agent (5-aza-DC) treatment followed by qPCR. The Trastuzumab resistant HER2-positive cell line (SKTR) had been previously developed in our laboratory by exposing SKBr3 cells to Trastuzumab, starting with 1µM for three months of exposure and increasing up to 2 µM for a 12 months period.

Summary of the new, unpublished data: The DNA methylation microarray data showed that different 5'-CpG island sites had altered methylation profile when comparing Trastuzumab-resistant (SKTR) and -sensible SKBr3 cells. We selected 4 candidate genes with a hypermethylation profile in -resistant vs. -sensible cells: TGFβ1 (Transforming Growth Factor Beta 1), BCL6 (B-Cell CLL/Lymphoma 6), KILLIN (P53-Regulated DNA Replication Inhibitor) and CTSZ (Cathepsin Z). The hypermethylation of these four candidate genes in SKTR was in agreement with their expression downregulation observed by PCR analysis. SKTR treatment with the demethylation agent 5-aza-2'-deoxycytidine restored the expression levels of all four genes to the levels of Trastuzumab-sensitive cells. Although previously related to breast cancer, TGFβ1, BCL6, KILLIN and CTSZ have not earlier been associated with the development of Trastuzumab resistance in breast cancer or in other cancer types and may be further studied as potential biomarkers.

Conclusions: These results provide a basis for future studies to validate the hypermethylation status of these genes as a predictive or monitoring biomarker of Trastuzumab resistance in HER2+ breast cancer patients.

### New Drugs, Therapeutic Targets, and Treatment Approaches

#2132

The novel, rationally-designed, ALK/SRC inhibitor TPX-0005 overcomes multiple acquired resistance mechanisms to current ALK inhibitors.

Dayong Zhai, Wei Deng, Zhongdong Huang, Evan Rogers, J. Jean Cui. _TP Therapeutics, Inc., San Diego, CA_.

Non-small cell lung cancers harboring rearrangements involving the ALK gene are sensitive to treatment with the ALK inhibitor crizotinib. However, the emergence of drug resistance is universal and limits clinical applicability. The mechanisms of resistance include ALK gene amplification, acquired ALK missense mutations, bypass activation, and epithelial-mesenchymal transition (EMT). More than ten acquired ALK mutations have been identified from patients and preclinical cell lines resistant to crizotinib. Although the second generation of ALK inhibitors (ceritinib and alectinib) overcome some of the crizotinib resistant mutations, they are plagued by a different spectrum of ALK resistant mutations. For example, the ALK G1202R mutation confers resistance to crizotinib, ceritinib, and alectinib. None of the current ALK inhibitors approved or in clinical trials can overcome bypass and EMT resistance mechanisms. SRC kinase has been identified to contribute broadly to primary intrinsic resistance to ALK inhibitor treatment, the development of bypass resistance and EMT during ALK inhibitor treatment. Saracatinib, a selective SRC inhibitor, can re-sensitize ALK inhibitor-resistant cell lines, suggesting a therapeutic role. There are currently no available ALK/SRC dual inhibitors, and we designed a novel ALK and SRC inhibitor TPX-0005 to address the need to overcome resistance. TPX-0005 is a novel three-dimensional macrocycle with a much smaller size (MW <370) than current ALK inhibitors designed to efficiently target the center of the ATP binding site and circumvent the steric interference from mutations outside the ATP binding boundary. As expected, TPX-0005 potently inhibited WT ALK (IC50 1.01 nM) and mutant ALKs including ALK G1202R (1.26 nM) and ALK L1196M (1.08 nM). Moreover, TPX-0005 effectively inhibited tumor growth in vivo in ALK WT and ALK G1202R xenografts. TPX-0005 is also a potent SRC inhibitor (IC50 5.3 nM). The elevated SRC kinase activity in H2228 lung cancer cell line confers resistance to crizotinib (IC50 1200 nM) and ceritinib (IC50 1000 nM) in cell proliferation assays. TPX-0005 effectively overcame this primary resistance (IC50 100 nM in cell proliferation assay) with strong inhibition of the phosphorylation of EML4-ALK (IC50 13 nM) and the SRC substrate paxillin (IC50 107 nM), along with other downstream signaling targets. TPX-0005 inhibited H2228 cell migration in a wound healing assay with similar activity to saracatinib. Taken together, TPX-0005 was able to not only inhibit the wild-type and a broad spectrum of mutant ALKs, but also overcome primary resistance and suppress metastatic features by inhibiting SRC. Overall, TPX-0005 has a highly favorable profile with ability to overcome multiple ALK resistance mechanisms including secondary mutations, bypass signaling activation, EMT, and warrants clinical investigation.

#2133

Ending the endless acquired tyrosine kinase resistance mutations — Design of TPX-0005, a multi-target ALK/ROS1/TRK inhibitor with broad spectrum activity against wild-type and mutants including ALK G1202R, ROS1 G2032R and TRKA G595R.

J. Jean Cui, Evan Rogers, Dayong Zhai, Wei Deng, Zhongdong Huang, Jeffrey Whitten, Yishan Li. _TP Therapeutics. Inc., San Diego, CA_.

The enormous success of kinase inhibitors in the treatment of cancer is being rapidly overshadowed by the emergence of drug resistance. Drug-resistant mutations increase the kinases' binding affinity with ATP, shifting them to the active conformation, and/or introduce new steric hindrance that interferes with the inhibitor structural motifs outside the highly conserved ATP binding boundary. Most kinase inhibitors are oversized, and often have motifs close to or across the gatekeeper to the hydrophobic back pocket to gain high potency, rendering them vulnerable to the clinical gatekeeper mutations, such as ALK L1196M, EGFR T790M, and ABL T315I. Another common vulnerability of kinase inhibitors is interaction with the conserved glycine residue at the hinge C-terminal that forms a hydrophobic sandwich with the kinase β1 sheet. Kinase inhibitors often use an aromatic ring or a flat motif to go through this narrow sandwich to the solvent exposure area. Alterations at the conserved glycine commonly referred to as solvent-front mutations clash with the inhibitor motif inside the sandwich, underlying clinical resistance. For example, ALK G1202R confers a common resistance to crizotinib and other ALK inhibitors, ROS1 G2032R and TRKA G595R render resistances to crizotinib and entrectinib, respectively. Overall, the traditionally designed, oversized kinase inhibitors are destined to develop many different resistance profiles in the clinic. Continuously introducing new inhibitors to overcome new evolving mutations is becoming increasingly hard to sustain. Here, we propose targeting resistance-driven kinase active conformation with a compact molecule that is completely located inside the ATP binding boundary as a novel strategy to tackle the ever evolving mutation resistances. We designed TPX-0005, a novel three-dimensional macrocycle with a much smaller size (MW <370) than current ALK, ROS1, and TRK inhibitors in the clinic. Efficiently targeting the center of the ATP binding site avoids the steric interference with mutations outside the ATP binding boundary. As expected, TPX-0005 potently inhibited WT ALK (IC50 1.01 nM) and mutant ALKs including ALKG1202R (1.26 nM) and ALKL1196M (1.08 nM). TPX-0005 demonstrated even stronger potency against the closely related kinases ROS1 (ROS1 WT 0.07 nM and ROS1 G2032R 0.456 nM), and TRK family (TRKA 0.826 nM, TRKB 0.052 nM, and TRKC 0.096 nM). TPX-0005 potently inhibited the phosphorylation of LMNA-TRKA G595R (IC50 < 1 nM) in NIH3T3 LMNA-TRKA G595R cells. Taken together, TPX-0005 is a novel ALK/ROS1/TRK inhibitor and can effectively overcome a broad spectrum of acquired resistance mutations, especially the solvent front mutants ALK G1202R, ROS1 G2032R and TRKA G595R.

#2134

Rational design of selective MNK 1 and 2 kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia patients.

Kassoum Nacro,1 Haiyan Yang,1 Melvyn Wai Tuck Ho,1 Yoon Sheng Yeap,1 Lohitha Rao Chennamaneni,2 Shi Hua Ang,1 Eldwin Sum Wai Tan,1 Athisayamani Jeyaraj Duraiswamy,1 Sharon Lim,3 Boping Liu,1 Esther Hongqian Ong,1 Meng Ling Choong,1 Shi Jing Tai,1 Vithya Manoharan,1 Vishal Pendharkar,1 Lijun Ding,1 Yun Shan Chew,1 Joma Kanikadu Joy,1 John LW Kuan,1 Perlyn Z. Kwek,1 Anders Poulsen,1 May Ann Lee,1 Kanda Sangthongpitag,1 Charles Chuah,3 Tiong S. Ong,3 Jeffrey Hill,1 Thomas H. Keller,1 Alex Matter1. 1 _Experimental Therapeutics Centre, Singapore, Singapore;_ 2 _Institute of Chemical & Engineering Sciences, Singapore, Singapore; _3 _Duke-NUS Graduate Medical School, Singapore, Singapore_.

The marketed BCR-ABL tyrosine kinase inhibitor (TKI), imatinib (Gleevec™) is a very successful targeted anti-cancer therapy. It has revolutionized the treatment of early stage or chronic phase (CP) chronic myeloid leukemia (CML). Unfortunately, a proportion of CP patients experience suboptimal responses to BCR-ABL TKIs, and progress to blast crisis (BC) stage of CML with poor survival rate. A potential cause of the resistance to TKI is the elevated level of phosphorylated eukaryotic initiation factor 4E (eIF4E), which has been found to be a consistent feature in patient-derived BC-CML samples. Importantly, both in vivo and in vitro studies have demonstrated that the MAP kinase-interacting serine/threonine-protein kinases 1 and 2 (MNK1/2) phosphorylate eIF4E on Ser209, and that the overexpression of eIF4E drives oncogenesis in a variety of cancers including BC-CML. Furthermore, several reports have indicated that eIF4E phosphorylation at Ser209, as well as eIF4E overexpression, is critical to tumor progression.

We found that a BC-CML cell line, K562, that expresses a serine to alanine phospho-mutant at position 209 of eIF4E, shows reduced ability to form tumors in mice compared to wildtype eIF4E. In addition, our recent work has demonstrated the importance of the MNK-eIF4E axis in activating BC leukemia stem cell (LSC) function (Lim et al., PNAS18; 110(25):E2298-307, 2013). These data highlight the critical importance of MNK1/2-dependent eIF4E phosphorylation in cancer progression and maintenance, and suggests that inhibition of MNK1/2 is an attractive therapeutic approach to treat BC-CML. Consequently, we set out to identify selective inhibitors of the MNK1/2 kinases to treat BC-CML patients.

Here, we report our hit finding strategy, as well as our hit to lead optimization process. Results describing structure activity relationships, pharmacokinetics properties, and biochemical characteristics of a highly specific MNK1/2 inhibitor, are presented. Our data demonstrate that drug-like molecules can be developed to potently and specifically inhibit the MNK kinases. We also show that simultaneous inhibition of MNK and BCR-ABL is effective at inhibiting BCR-ABL-driven growth and proliferation, as well as inhibiting the MNK-eIF4E-dependent self-renewal function of BC-LSCs. A combination of selective MNK and BCR-ABL inhibitors may provide clinical benefit to BC-CML patients.

#2135

Estrogen receptor-beta and mutant p53 signaling crosstalk as a novel molecular target to overcome therapeutic resistance in high-grade serous ovarian cancer.

Chetan C. Oturkar, Gokul M. Das. _Roswell Park Cancer Institute, Buffalo, NY_.

The high-grade serous ovarian carcinoma (HGSOC), the most aggressive ovarian cancer subtype, constitutes 90% of serous carcinomas and it accounts for two-thirds of ovarian cancer deaths. It is a poorly understood disease with high therapeutic resistance and extremely poor prognosis. The current study is an attempt to analyze molecular mechanisms underlying HGSOC and to identify new therapeutic targets. The Cancer Genome Atlas (TCGA) analyses of ovarian carcinoma demonstrated that p53 mutations are near universal (90%) in HGSOC and forkhead box M1 protein (FOXM1) network is significantly altered in 87% of HGSOC leading to transcriptional upregulation of proliferation-related target genes. Moreover, Mutant p53 and FOXM1 have important roles in resistance to chemotherapy. In the present study, we have used various experimental procedures such as RNAi technology, quantitative real-time PCR, immunoblot analysis, proximity ligation assay (PLA) (to determine protein-protein interaction in situ) and assays for cell cycle, apoptosis and proliferation to analyze signaling crosstalk in OVCAR3 (HGSOC) cells. Our experiments have identified estrogen receptor-beta (ER-beta) as a novel member of the mutant p53-FOXM1 axis. We show that not only mut-p53 and ER-beta physically interact, but also they mutually regulate each other's expression at the gene transcriptional level leading to changes in expression of downstream signaling targets such as FOXM1. Importantly, the effect of knocking down ER-beta on transcription of FOXM1 was similar to that of knocking down endogenous mut-p53, suggesting that both mutant p53 and ER-beta are transcriptional activators of FOXM1. Knocking down ER-beta results in decreased mut-p53 transcript levels suggesting transcription of mut-p53 is activated by ER-beta in OVCAR cells. On the other hand, mut-p53 represses transcription of ER-beta gene. Thus, our experiments have revealed a feedback loop between mut-p53 and ER--beta at the transcriptional level. . We show that when ER-beta was knocked down, FOXM1 levels were decreased and apoptosis was increased. Of note, knocking down ER-beta decreased resistance to cisplatin and carboplatin, major therapeutic agents currently in use for ovarian cancer. Our data provides new insight into the mechanisms underlying HGSOC by illustrating how signaling crosstalk between ER-beta and mut-p53 leads to increased expression of pro-proliferative and pro-tumorigenic downstream targets such as FOXM1, overexpression of which has been associated with resistance to therapeutic agents.in HGSOC patients. Our study will be the first, to the best of our knowledge, to investigate whether the crosstalk between ER-beta and mut-p53 play an important role in the onset and progression of HGSOC. Thus, we have identified mutant p53-ER beta signaling axis as a novel therapeutic target in HGSOC.

#2136

Entrectinib is effective against the gatekeeper and other emerging resistance mutations in NTRK-, ROS1- and ALK- rearranged cancers.

Ge Wei,1 Elena Ardini,2 Roopal Patel,1 Nicholas Cam,1 Jason Harris,1 Jean-Michel Vernier,1 Nanqun Zhu,1 Litain Yeh,1 Robert Shoemaker,1 Pratik Multani,1 Zachary Hornby,1 Robert Wild,1 Gary G. Li1. 1 _Ignyta, Inc, San Diego, CA;_ 2 _Nerviano Medical Sciences, Milan, Italy_.

Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK result in oncogenic fusion proteins that have been identified in many types of cancer, including lung, colorectal, salivary gland, sarcoma, papillary thyroid, glioblastoma, melanoma and other histologies. Entrectinib (RXDX-101) is an orally available, highly potent and selective ATP-competitive pan-Trk, ROS1 and ALK inhibitor. In preclinical studies, entrectinib effectively inhibits target kinase activity and cancer cell proliferation and in vivo tumor growth across various fusion partners and cancer types. More importantly, entrectinib's activity has been validated clinically in patients across multiple fusion partners and tissue histologies.

Trk inhibitors, including entrectinib, have shown promising clinical activity in molecularly selected patients. Predictably, potential resistance mechanisms have also begun to emerge. For example, mutations in the Trk kinase domain were identified as one of the in vitro induced resistance mechanisms to the Trk inhibitor, Loxo-101. The three reported resistance mutations in the Ba/F3-MPRIP-NTRK1 cell line model treated with Loxo-101 were F589, G667 and V573. The F589 location on TrkA is equivalent to the gatekeeper mutations, L1196 location on ALK and L2026 location on ROS1. These gatekeeper mutations often arise as resistance mechanisms in patients treated with ALK and ROS1 inhibitors. To test the activity of entrectinib against these three reported NTRK1 mutations, we introduced mutated Trk proteins into Ba/F3 and cancer cell lines and performed dose-dependent proliferation studies. Entrectinib was able to inhibit proliferation of cells harboring each of these three mutations that confer resistance to other Trk inhibitors. Particularly, the IC50 values of entrectinib against kinase domain wildtype and gatekeeper mutated (F589) are essentially unchanged (low single-digit nM), which is consistent with the observation that entrectinib is also able to inhibit the gatekeeper mutation in ALK (L1196) in both cell based assays and in vivo tumor growth inhibition studies.

In conclusion, our preclinical data suggest that entrectinib is an effective treatment for patients with NTRK-rearranged tumors, including cancers that harbor certain resistance mutations to other Trk inhibitors.

#2137

Discovery of dual MNK 1 and 2 and BCR-ABL kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia.

Joseph Cherian,1 Kassoum Nacro,1 Zhi Ying Poh,1 Samantha Guo,1 Melvyn Wai Tuck Ho,1 Haiyan Yang,1 Sharon Lim,2 Meng Ling Choong,1 Joma Kanikadu Joy,1 Perlyn Z. Zekui,1 Boping Liu,1 Esther Hongqian Ong,1 Vishal Pendharkar,1 Lijun Ding,1 Anders Poulsen,1 May Ann Lee,1 Kanda Sangthongpitag,1 Charles Chuah,2 Tiong S. Ong,2 Jeffrey Hill,1 Thomas H. Keller,1 Alex Matter1. 1 _Experimental Therapeutics Centre, Singapore, Singapore;_ 2 _Duke-NUS Graduate Medical School, Singapore, Singapore_.

Chronic Phase Chronic Myelogenous Leukemia (CP-CML) is well treated with tyrosine kinase inhibitors (TKI) through BCR-ABL kinase modulation. However, TKI are ineffective for late-stage or blast crisis (BC) CML in which phase, granulocyte macrophage progenitors (GMPs) have acquired the ability to function as leukemic stem cells (LSCs), and are thought to act as a reservoir for TKI resistance with poor prognosis. An effective BC-CML therapy will likely have to target the BC-LSC population to ensure long-term disease control.

Recent studies have shown the importance of the MAP kinase interacting serine/threonine kinase (MNK1/2)-eukaryotic translation initiation factor 4E (eIF4E) axis in sustaining BC-LSC self-renewal. Thus, a single agent which inhibits both BCR-ABL and MNK1/2 kinase simultaneously represents a rational approach to target BC-LSCs. Such an agent should be able to distinguish between normal hematopoietic stem cells (HSC) and LSCs.

Here, we report the discovery of a potential BC-CML therapy that relies on the inhibition of MNK1/2 and BCR-ABL kinases using a dual specific MNK/ABL inhibitor. We will also report the in vitro and in vivo biological data, pharmacokinetic properties, and biochemical characteristics of such compounds. These compounds are able to prevent eIF4E phosphorylation, thus selectively inhibiting the MNK-eIF4E-dependent self-renewal function of BC-LSCs as a single agent while leaving normal HSCs unscathed.

#2138

Functional evaluation of interferon/STAT1 pathway activation in response to genotoxic treatment.

Julie Gaston,1 Laura Cheradame,1 Marie-Emmanuelle Legrier,2 Olivier Deas,1 Jean Gabriel Judde,1 Vincent Goffin,3 Stefano Cairo1. 1 _Xentech, Evry, France;_ 2 _Curie, Paris, France;_ 3 _Inserm U1151, Necker Hospital, Paris, France_.

Triple negative breast cancer (TNBC) accounts for approximately 15-25% of breast cancers at diagnosis, and is one of the most aggressive subtypes, with around 70% of patients that live free of disease 5 years post-diagnosis. One of the most reliable predictive markers of patient outcome is the pathological complete response (pCR), with no tumor cells detectable at histopathological level after neoadjuvant chemotherapy.

As pCR is observed in only about 20-30% of TNBC, it is mandatory to identify new therapeutic options to improve pCR rate upon neoadjuvant chemotherapy. The mechanisms of tumor resistance remain an unmet need in oncology. In a recent study, we used our breast cancer PDXs collection to tackle this question, and address the mechanisms of tumor response to treatment vs tumor recurrence. To do this we analyzed the gene expression profile of laser-microdissected residual tumor nodules interspersed in the murine stroma upon very efficient tumor response to Adriamycin/Cyclophosphamide (AC). When doing so, we identified several genes of the IFN/STAT1 pathway that were strongly over-expressed when compared to pre-treatment tumors. In this study, we investigated the mechanisms leading to IFN/STAT1 pathway activation following chemotherapy in tumor cells and we evaluated the functional involvement of the IFN/STAT1 signature as a whole or at single target gene level in residual tumor cell resistance to chemotherapy after genotoxic treatment.

To this aim, we set up an ex-vivo/in vitro assay to mimic induction of the IFN/STAT1 signature in tumor cells after genotoxic treatment. We identified cell lines and primary cultures from a patient-derived xenograft (PDX) that mimicked the in vivo situation, as only the cell lines that respond to genotoxic stress in vivo activated IFN/STAT1 pathway in response to genotoxic treatment in vitro. When exposed to conditioned medium collected from mafosfamide-treated cancer cells, these cells activated luciferase reporter genes harboring ISRE (interferon stimulated response elements) and GAS (gamma interferon activated sequence) response elements, suggesting active ligands of the IFN/STAT1 pathways were secreted. STAT1 or IFNAR1 gene silencing (siRNA) resulted in markedly attenuated gene signature expression after mafosfamide treatment. The addition of conditioned medium significantly reduced mafosfamide-induced cancer cell death suggesting that IFN/STAT1-related genes over-expression may ultimately have protective effect on cancer cell viability. Accordingly, the inhibition of several genes of the IFN/STAT pathway, or DNA sensors acting upstream, increased cell mortality when combined with genotoxic treatment and delayed colony formation.

Overall, this study supports the functional implication of IFN/STAT1-related genes in tumor resistance to genotoxic treatment.

#2139

RNA nanoparticles as a two-in-one siRNA delivery carrier synergistically suppress drug-resistance cancer via combination therapy with doxorubicin.

Hyung Jun Ahn. _Korea Institute of Science and Technology, Seoul, Republic of Korea_.

To overcome multi-drug resistance (MDR) caused by malfunction of genes in chemotherapy, we synthesize RNA nanoparticles (RNA NPs) as a "two-in-one" siRNA delivery system for MDR1 siRNA and BCL2 siRNA by using rolling circle transcription and base-pairing with folate-DNA-cholesterol fragments. The resulting RNA nanoparticles (Dsi RNPs), which contain multiple tandem copies of two siRNA sequences, form the self-assembled structure with about 100 nm of diameter, and simultaneously suppress pump and non-pump phenotypes in the same cancer cells. Using Dsi RNPs, we show that there exists an optimal time point within the window of time at which the synchronous silencing of MDR1 and BCL2 gene markedly enhances the chemosensitivity of MDR cancer cells to doxorubicin. Accordingly, a two-step sequential treatment of MDR cancer cells with Dsi RNPs and doxorubicin leads to the optimal synergistic cancer suppression. Finally, we demonstrate that such a synergism is attributed to the synchronous silencing of both genes, as well as to the precisely controlled timings of drugs and Dsi RNPs treatment. RNA NPs exhibit the folate receptor-specific cellular uptake, lowering the risk of severe adverse side effects. Also, RNA NPs do not induce innate immunostimulatory response and their highly condensed structure reveals the enhanced stability against nuclease attack in bloodstream. Therefore, RNA NPs delivery system provides an alternative tool as a two-in-one siRNA carrier that is suitable for the spatial-temporal synchronization of double gene silencing, and also, Dsi RNPs have great potential as combination chemotherapeutics with traditional anticancer drugs for suppression of MDR cancer.

#2140

**Cabazitaxel is more active than first generation taxanes in** ABCB1 **(+) cell lines due to its higher intracellular accumulation and reduction in ATPase stimulation.**

George E. Duran,1 Dietmar Weitz,2 Dorothée Sémiond,3 Diego Gianolio,3 Sandrine Macé,4 Branimir I. Sikic1. 1 _Stanford University School of Medicine, Stanford, CA;_ 2 _Sanofi-Aventis, Frankfurt, Germany;_ 3 _Sanofi Oncology, Cambridge, MA;_ 4 _Sanofi Oncology, Vitry-sur-Seine, France_.

The taxanes paclitaxel (Taxol) and docetaxel (Taxotere) have substantial clinical activity in breast, ovarian, lung, and other cancers, but their clinical efficacy is limited by preexisting or acquired drug resistance, such as the expression of the ATP-dependent multidrug resistance (MDR) transporter P-glycoprotein (P-gp). Cabazitaxel (Jevtana), the dimethoxy derivative of docetaxel, was identified because of its activity in taxane-resistant models both in vitro and in vivo. Our studies confirm that cabazitaxel is more active in cell variants that express P-gp, and the objective of this study was to investigate cabazitaxel's affinity for the transporter. Taxane accumulation patterns were studied in ABCB1(+) variants using [14C]-radiolabeled docetaxel and cabazitaxel over a time course up to 1 h. Time points were collected and spun (10,000 x g, 1 min) through Nyosil M20 oil thereby terminating uptake, cell pellets lysed with 2% (w/v) SDS, and counts determined by liquid scintillation, with all measurements normalized to protein content. The kinetics of drug accumulation between the two taxanes is different, with the maximum intracellular drug concentration achieved faster with cabazitaxel (5 min) than docetaxel (15-30 min) in all cell lines. ABCB1(+) variants accumulated less taxane, and these levels could be restored to parental levels in the presence of known P-gp inhibitors (2 µM PSC-833, valspodar). However, the MDR cell models tested accumulated twice as much cabazitaxel than docetaxel under identical experimental conditions. Efflux in drug-free medium confirmed that ABCB1(+) variants retained 2.2x more cabazitaxel than docetaxel. We observed a strong association (r2 = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. Several low P-gp-expressing cell models were not cross-resistant to cabazitaxel while demonstrating resistance to docetaxel. Furthermore, we determined ATPase stimulation in membranes isolated from our MDR cell models as an indirect measure of P-gp activity following taxane treatment. We observed a 1.9x reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel than with docetaxel (1 µM for 30 min). Future experiments will include direct photoaffinity labeling of P-gp with [3H]-azido-cabazitaxel and docetaxel, and competition assays with known P-gp substrates. Our studies indicate that the improved activity of cabazitaxel in MDR models might be due to its reduced affinity for P-gp compared to docetaxel.

#2141

TAS-115, a novel and highly potent VEGFR/MET inhibitor, shows prominent antitumor efficacy by restoring HGF-mediated hypoxia-resistant phenotype in clear cell sarcoma.

Hidenori Fujita, Yukari Yamada, Miki Terasaka, Yayoi Fujioka, Naomoto Harada, Akihiro Hashimoto, Kazutaka Miyadera, Kenichi Matsuo, Kazuhiko Yonekura. _Taiho Pharmaceutical Co., Ltd., Tokyo, Japan_.

<Background>

Hepatocyte growth factor (HGF) / HGF receptor (MET) signaling is considered to be involved in chemoresistance to cancer treatment. Notably, many reports have described reduced sensitivity to vascular endothelial growth factor receptor (VEGFR)-targeted inhibitors in patients with high serum HGF levels compared to those with low serum levels. This study investigated whether HGF directly influenced sensitivity to VEGFR-targeted inhibitors in human clear cell sarcoma (CCS) xenograft models using human HGF knock-in (hHGF KI) mice, and evaluated the potency of TAS-115.

<Material and methods>

MET signaling pathway in SU-CCS-1, a human CCS cell line, were analyzed using Western blotting analysis. To study the effects of hypoxia in SU-CCS-1 cells, culture plates were placed in airtight jars under anaerobic conditions and cell viability was assessed by ATP-based assay. In the in vivo study, SU-CCS-1 cells were subcutaneously implanted into hHGF KI mice, and compounds were orally administered once daily for 14 consecutive days. Tumor vessel density (TVD) was determined by immunohistochemistry using anti-CD31 antibody.

<Results>

SU-CCS-1 cells expressed MET, and exogenous HGF phosphorylated MET and its downstream factors in SU-CCS-1 cells. Exogenous HGF also significantly enhanced SU-CCS-1 cell proliferation. TAS-115 inhibited HGF-induced MET phosphorylation and SU-CCS-1 cell proliferation in a dose-dependent manner at concentrations higher than 10 nM. In contrast, pazopanib, a VEGFR-targeted multi-kinase inhibitor, could not block HGF-induced phenotypes in SU-CCS-1 cells, even at 1 µM. To evaluate the effects of HGF on the antitumor activities of TAS-115 and pazopanib against SU-CCS-1 cells, these agents were administered in hHGF KI and wild-type (WT) mice bearing subcutaneous SU-CCS-1 tumors. TAS-115 completely suppressed tumor growth and reduced TVD in SU-CCS-1 tumor tissue in both models at a dose of 200 mg/kg/d. In contrast, pazopanib suppressed tumor growth at a dose of 100 mg/kg/d in WT mice but not in hHGF KI mice. However, pazopanib significantly reduced TVD in SU-CCS-1 tumor tissue in both models. HGF is therefore suggested to impact SU-CCS-1 cells rather than mouse endothelial cells. We considered that HGF modulated cell proliferation or survival in SU-CCS-1 cells under hypoxia. Actually, hypoxic conditions induced apoptosis in SU-CCS-1 cells, and endogenous HGF dose-dependently attenuated hypoxia-related apoptosis. TAS-115 clearly restored HGF-mediated cytoprotection under hypoxia but pazopanib did not.

<Conclusion>

These results suggest that effects of VEGFR-targeted inhibitors are attenuated by HGF-mediated hypoxia resistance, and simultaneous inhibitors of the MET and VEGFR axes, such as TAS-115, are more effective in sarcoma patients with high serum HGF than inhibition of the VEGFR axis alone.

#2142

Plumbagin, a medicinal plant-derived naphthoquinone, inhibits the growth of docetaxel resistant prostate cancer cells via inhibition of the expression of P-glycoprotein and NF-kB activation.

Ashok Singh, Anupama Singh, Satyamedha Bathula, Saivenkateshkomal Bathula, Ajit K. Verma. _University of Wisconsin-Madison, Madison, WI_.

Prostate cancer (PCa) is one of the leading causes of death in men across the world. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy manifests by progression to castration resistant prostate cancer (CRPC), which is the end stage that accounts for the majority of PCa patient deaths. Docetaxel (DTX) is an approved drug for the treatment of CRPC. However, development of DTX resistance and toxicity are the major limitations in the use of DTX in the treatment of CRPC. Now we investigated to determine whether these limitations may be overcome by combining DTX with plumbagin (PL). PL is a quinoid constituent isolated from the root of Plumbago zeylanica L. The roots of Plumbago zeylanica L. have been used in Indian medicine for more than 2500 years for treatment of various ailments. We were the first to report that PL inhibits the growth and PCa metastasis. Now we present that PL: 1) sensitizes CRPC cells to DTX and thus reduces its effective therapeutic dose to prevent DTX resistance and toxicity, and 2) inhibits the viability and growth of DTX resistant cells (22Rv1R). In combination therapeutic experiments, the effects of different concentrations of PL (0, 1.0, 2.0, and 5.0 μM) alone or in combination with DTX (0.25 nM and 0.5 nM) on colony formation in 22Rv1 and C4-2 prostate cancer cells, was determined. Combined treatment of PL and DTX has more than additive inhibitory effects on colony formation. This synergism of PL and DTX on colony formation can be explained on the basis of unique mechanism of action of PL and DTX. PL-induced inhibition of PCa growth and metastasis involves inhibition of the expression of multiple targets including PKCε, which correlates with the aggressiveness of PCa and plays roles in both androgen independent androgen receptor (AR) activation and promotion of PCa cell survival. DTX targets microtubules, impairs AR signaling, and inhibits cell division. To determine the effects of PL on DTX resistant PCa cells, we established DTX-resistant 22Rv1 cell line (22Rv1R). 22Rv1R overexpresses multidrug resistant protein 1 (MDR1, also known as P-glycoprotein). MDR1 is overexpressed in many tumors and thus causing intrinsic drug resistant. Transcription factor NF-ΚB, which is linked to cell proliferation and survival, is also activated in 22Rv1R. PL inhibited the colony forming efficiency of 22Rv1R cells which accompanied inhibition of MDR1 expression and NF-ΚB activation. In summary, the results presented here and the ones reported by us previously with preclinical mouse models (TRAMP and PTEN knockout mice) of PCa indicate that PL has the potential to prevent as well as treat PCa. PL is a potential natural agent worthy of investigation for prevention and treatment of human PCa. (Support: NIH grant CA102431).

#2143

Mechanistic study of the relative cytotoxicity of doxorubicin loaded nanoparticle formulation compared to free doxorubicin in hepatocellular carcinoma (HCC) cell lines.

Véronique Trochon-joseph,1 Caroline Lemarchand,1 Vincent Hayes,1 Yamina Rayah,1 Jean-Louis Labernardière,1 Graham Dixon2. 1 _onxeo, paris, France;_ 2 _Onxeo, Paris, France_.

Background and Aims

Efficacy of doxorubicin encapsulated into a biodegradable network (NP) is currently studied in phase III compared to Best Standard of Care in patients with advanced HCC (Relive study). Preclinical studies demonstrated that NP allowed the reversion of chemo-resistance and enhanced cytotoxicity compared to free doxorubicin on resistant human hepatoma and mestastatic liver cells. In this study the mechanism of action of NP in overcoming HCC cellular resistance as well as the mechanisms of cellular uptake were investigated in representative cell lines.

Methods

Determination of IC20 of NP in the presence of influx drug inhibitors targeting endocytosis (clathrin or caveolae-dependent; sucrose and Genistein respectively), phagocytosis

(LY-294002) or macropinocytosis (Cytochalasin D) were performed on two HCC cell lines (HuH7 and HepG2) using a proliferation assay. Each uptake inhibitor was added 30 min before incubation with NP on cultured cells for measurement of proliferation followed by cellular quantification of free doxorubicin by HPLC. The same assays were also performed using Verapamil as an inhibitor of the major multidrug resistance protein P-glycoprotein (P-gp). The P-gp ATPase activity in plasma membrane preparations from vehicle, doxorubicin and NP-treated cells was determined using the Pgp-Glo assay system.

Results

NP showed lower IC20 than free doxorubicin in HCC proliferation assays. None of the influx inhibitors used in combination with NP impaired the inhibitory effect of NP on the rate of proliferation of HCC cell lines. Similarly, Verapamil did not modulate the cellular potency of NP on the rate of proliferation in these cell lines. Doxorubicin quantification in cellular extracts and supernatant of NP-treated cells confirmed these results. Furthermore, NP had no effect on P-gp ATPase activity in isolated plasma membrane samples.

Conclusions

All these results demonstrated that NP did not enter into the HCC cell via an active transport mechanism, but by passive diffusion as occurs with free Doxorubicin. The absence of inhibition of the P-gp ATPase by NP implies that the Doxorubicin-nanoparticle conjugate (ion pair) probably 'hinders' the doxorubicin from being a substrate for the pump rather than by a direct interaction. The implication of these data on the role of NP in the treatment of HCC will be discussed.

#2144

Next generation inhibitors to reverse ABCB1 transport mediated multidrug resistance in cancer.

Anna Maria Barbuti, Bhargav A. Patel, Tanaji T. Talele, Zhe-Sheng Chen. _St. John's University, Queens, NY_.

The therapeutic efficacy of anticancer drugs is often limited by chemoresistance. Cancer can present intrinsic or acquired resistance to chemotherapy drugs through different mechanisms of multidrug resistance (MDR). The accelerated drug efflux facilitated by overexpression of ATP-binding cassette (ABC) transporters is one of the key mechanisms of MDR. Specifically, overexpression of ABCB1 (MDR1, P-gp) is known to be a major cause of resistance to structurally dissimilar anticancer drugs such as paclitaxel and doxorubicin. To circumvent ABC transport mediated MDR, many small molecules and kinase inhibitors have been researched and employed to retain the efficacy of the anticancer drugs. Though many small molecule inhibitors demonstrate to be useful reversal agents, understanding the mechanisms at a molecular level is important in order to design candidate drug inhibitors which are safe, selective and effective. To investigate the functional groups and molecular structures which can efficiently reverse ABCB1-mediated MDR, multiple series of thiazole-valine peptide compounds were designed, synthesized and tested in vitro. The results indicated BTT-10A was most effective in sensitizing ABCB1-mediated resistance to anticancer drugs. Both transfected and drug-selected cell lines overexpressing the ABCB1 transporter were utilized in this study. We identified BTT-10A, at nontoxic concentrations (10 µM), to have optimal MDR reversal activity through cell viability MTT cytotoxicity and reversal bioassays. BTT-10A sensitized resistant cells to the anticancer drugs in a concentration dependent manner, without downregulating the transporter expression. BTT-10A significantly increased the accumulation of ABCB1 anticancer substrate drugs by blocking the efflux function of ABCB1 transporter, as demonstrated by significant increased [³H]-paclitaxel accumulation, and increased intracellular doxorubicin accumulation through immunofluorescent detection in ABCB1 overexpressing cells. Alongside these findings, docking studies showed site-1 to be the preferable binding site for BTT-10A within the drug-binding pocket of human ABCB1 homology model. Therefore, we report that BTT-10A antagonizes MDR by inhibiting the efflux activity of the overexpressed ABCB1 transporter. These findings reveal more evidence of specific molecular structure and function of ABCB1-mediated MDR reversal agents. The therapeutic application of BTT-10A co-administered with ABCB1 substrate anticancer drugs holds promise for cancer patients resistant to specific chemotherapies, overexpressing the ABCB1 transporter. Further in vivo studies will be conducted using drug resistant xenograft models. This research will help inspire further investigations of ABC transport-mediated MDR in cancer, to find the next generation of targeted and safe reversal agents to combat chemoresistance.

#2145

Elimination of multidrug-resistant glioma cells using unique sequence of high-intensity focused ultrasound and low-dose generic chemotherapeutic.

Howard Q. Vo. _Houston Methodist Research Institute, Houston, TX_.

BACKGROUND

Despite medical advances, multidrug-resistant (MDR) gliomas continue to challenge the patients. In this study, I evaluate a new strategy in which the unique sequence of High-Intensity Focused Ultrasound (HIFU) and low-dose generic chemotherapeutic is utilized to attack MDR glioma cells.

METHODS

This strategy, motivated by recent clinical trials in which HIFU was used to non-invasively disrupt the blood-brain barrier and to treat brain tumors, led to the in vitro study during which human glioma cell line U87 (ATCC), modified for overexpression of epidermal growth factor receptor (EGFR), was paradoxically treated with low-dose Etoposide (VP-16).

Day 1: Each data sample consisted of ~10K cells grown inside a well of 8-well glass slides (Lab-Tek). Each slide was then filled with growth media, sealed, submerged in a 37ºC degassed water bath and secured onto a fixture. The targets for HIFU therapy are the center points of the 4 quadrants of each well's base. The constant HIFU parameters for each slide were acoustic pressure 4.5 MPa, repetition rate 1 Hz, focal-zone depth and 30 sec/sonication/center point while HIFU duty cycle (DC) ranged 0-50% among slides. After HIFU treatment, the slides were incubated for 24h.

Day 2: Cell media for each slide was replaced with fresh media containing [VP-16] 0-100 uM prior to repeating the HIFU procedure from day 1.

Day 4: After 48h of exposure to VP-16, cell survivability study was performed using cell-count technique to determine the contribution of HIFU-mediated necrosis as well as VP-16-mediated apoptosis.

RESULTS

Cell survivability decreased by increasing [VP-16] or HIFU DC. In VP-16-only group (HIFU DC 0%), average survivability was 85% (n=4) at [VP-16] 1 uM. In HIFU-only group ([VP-16] 0 uM), average survivability was 34% (n=4) at DC 50%. In contrast, average survivability was 12% (n=4), using [VP-16] 1 uM and DC 50%.

CONCLUSION

The unique sequence of HIFU and low-dose Etoposide virtually eliminated MDR glioma cells on day 4.

in vitro Treatment of Multidrug-Resistant Glioma Cell Line U87 Using HIFU-Etoposide Sequence

---

[Etoposide] = [VP-16], (uM) | 0 | 0.5 | 1 | 5 | 10 | 50 | 100

Average cell survivability (%) ± SD on day 4

(Etoposide only; DC 0%; n=4) | 100 ± 47

(control) | 62 ± 23 | 85 ± 13 | 41 ± 5 | 37 ± 7 | 25 ± 4 | 21 ± 6

Average cell survivability (%) ± SD on day 4

(DC 50%; n=4) | 34 ± 5

(HIFU only) | 14 ± 7 | 12 ± 6 | 6 ± 2 | 4 ± 2 | 3 ± 1  | 4 ± 1

Note: The above data suggests that, in principle, drug resistance may be reversible and that the unique sequence of HIFU and a low-dose generic drug may be a promising alternative to current treatments (multidrug regimen, surgery or radiotherapy) against other MDR solid cancers. In addition to high efficacy within a relatively short time, the low cost and the potential absence of drug side effects from using low-dose generic chemotherapeutic are exciting motivations to further investigate this new cancer treatment in animal studies.

#2146

YAP is an important pathway downstream of EGFR that is a potential target for EGFR-TKI-resistance lung adenocarcinomas.

Ting-Fang Lee,1 Yu-Chi Tseng,1 Ying-Pu Liu,1 Yu-Rung Kao,2 Cheng-Wen Wu1. 1 _National Yang-Ming University, Taipei, Taiwan;_ 2 _Academia Sinica, Taipei, Taiwan_.

Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. Using lung adenocarcinoma cell lines, we detected enhanced YAP expression and activity mediated by EGFR signaling due to enhanced protein stability. The Src family protein YES was involved in EGFR-regulated YAP expression. The YAP/YES pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP levels by mechanisms bypassing EGFR were effective in killing EGFR-dependent cells including EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP in EGFR mutant lung adenocarcinomas and identified YAP as an promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance.

#2147

Overcoming mTOR resistance mutations with a new generation mTOR inhibitor.

Vanessa S. Rodrik-Outmezguine,1 Masanori Okaniwa,2 Zhan Yao,1 Chris Novotny,2 Claire McWhirter,3 Arpitha Banaji,1 Helen Won,1 Wai Wong,1 Mike Berger,1 Elisa de Stanchina,1 Derek G. Barratt,3 Sabina Cosulich,3 Teresa Klinowska,3 Neal Rosen,1 Kevan M. Shokat4. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA;_ 3 _AstraZeneca, Macclesfield, United Kingdom;_ 4 _University of California San Francisco, San Francisco, CA_.

First generation mTOR inhibitors (rapalogs) have modest activity in some tumors. We sought to delineate the likely resistance mechanisms to existing mTOR inhibitors as a guide for next generation therapies. We identified FRB domain and kinase domain mutations as causing acquired-resistance to rapamycin and mTOR kinase inhibitor respectively.

Resistance to rapamycin was due to loss of binding of the FKBP12-rapamycin complex to the FRB domain of mTOR mutant protein. Resistance to the mTOR kinase inhibitor was not due to a gatekeeper mutation but rather to an increase in the intrinsic kinase activity of the mTOR kinase domain mutant. These and similar mutations have also been identified in tumors from untreated patients. These findings suggest that these mutations are gain of function mutants but also that tumors with these activating mutations will be intrinsically resistant to second generation mTOR kinase inhibitors.

We have designed a new class of mTOR inhibitor, RapaLink-1, that takes advantage of the juxtaposition of two drug binding pockets to create a bivalent interaction. This compound potently inhibits mTOR activation in cells with mTOR wild-type and overcomes resistance to existing first and second generation inhibitors. Moreover, intermittent dosing of RapaLink-1 effectively suppresses mTOR-dependent tumor growth in vivo. Thus, such third generation mTOR inhibitors could be useful for treating tumors with activating mutations of mTOR and for tumors with acquired-resistance to current mTOR inhibitors.[N.R. and K.S. contributed equally to this work.]

#2148

MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines.

Hongfang Wang,1 Shawn P. Carey,1 Olga Burenkova,1 Jian Tang,1 Nastaran Gerami-Moayed,1 Sabrina Arena,2 Alberto Bardelli,3 Rachel Nering,1 Jeffrey D. Kearns1. 1 _Merrimack Pharmaceuticals, Cambridge, MA;_ 2 _Candiolo Cancer Institute – FPO, IRCCS and FIRC Institute of Molecular Oncology (IFOM), Candiolo,Torino, Italy;_ 3 _Candiolo Cancer Institute – FPO, IRCCS, Candiolo and University of Torino, Candiolo, Torino, Italy_.

Mutations in the extracellular domain (ECD) of EGFR have been reported in colorectal (CRC) patients as a mechanism of acquired resistance to the EGFR-directed antibodies cetuximab and panitumumab. Similar to EGFR intracellular domain mutations observed in patients treated with EGFR kinase inhibitors, these EGFR-ECD mutations inhibit the binding of the antibodies while maintaining EGFR signaling activity. MM-151, an investigational agent consisting of three anti-EGFR IgG1 antibodies, has been recently reported to overcome these mutations in over-expression and patient-derived cell line models. Here, we extend these studies to characterize the mechanisms of action that enable MM-151 to provide robust inhibition of EGFR-ECD mutations beyond that achievable by other monoclonal and oligoclonal EGFR antibodies. First, utilizing a cell-free binding assay with recombinant EGFR-ECD mutants, we found that MM-151, but not the Sym004 combination of two antibodies, maintained oligoclonal binding to all mutants. Second, we performed a panel of 2D and 3D in vitro experiments with over-expression and genome-edited (CRISPR-Cas9) cell lines harboring EGFR-ECD mutations to characterize target engagement, cell signaling, proliferation, and the impact of hetero- versus homozygous expression of EGFR wild type or mutant alleles. We observed that the three mechanisms of action for MM-151- antagonism of both low- and high-affinity EGFR ligands, EGFR down-regulation, and immune-effector activity— were preserved in these models. Third, we performed xenograft experiments to evaluate the behavior of MM-151 and found that MM-151 maintained inhibitory activity in an in vivo context. The results of our multiscale experiments identified the impact of combining three antibodies to overcome the high plasticity of the EGFR extracellular domain and thus reveal the potential for clinical evaluation of MM-151 in both EGFR-naïve and EGFR-refractory CRC populations.

#2149

The anti-EGFR antibody mixture Sym004 overcomes acquired resistance to cetuximab in colorectal cancer.

Francisco J. Sanchez-Martin,1 Alba Dalmases,2 Beatriz Bellosillo,2 Guillem Argiles,3 Mariona Gelabert,1 Israel Cañadas,1 Joana Vidal,4 Giulia Siravegna,5 Sabrina Arena,1 Klaus Koefoed,6 Laura Visa,4 Oriol Arpí,1 Ivan D. Horak,6 Mar Iglesias,2 Christopher Stroh,7 Michael Kragh,6 Ana Rovira,1 Joan Albanell,4 Alberto Bardelli,5 Josep Tabernero,3 Clara Montagut4. 1 _Cancer Research Program, IMIM. Hospital del Mar, Barcelona, Spain;_ 2 _Pathology Department, Hospital del Mar, Barcelona, Spain;_ 3 _Medical Oncology Department, Vall d´Hebron Institute of Oncology (VHIO), Barcelona, Spain;_ 4 _Medical Oncology Department, Hospital del Mar, Barcelona, Spain;_ 5 _Candiolo Cancer Institute – FPO, IRCCS, Candiolo, Torino, Italy;_ 6 _Symphogen A/S, 2750, Ballerup, Denmark;_ 7 _Merck-Serono, Germany_.

The anti-EGFR monoclonal antibodies (MoAbs) cetuximab and panitumumab are used to treat 'RAS' wild type metastatic colorectal cancer (MCRC), providing significant clinical benefit in patients. However, all patients ultimately develop disease progression, driven by acquisition of mutations in downstream effectors and in the extracellular domain (ECD) of EGFR, in approximately 25% of the cases. Sym004 is a novel 1:1 mixture of two non-overlapping anti-EGFR MoAbs that has recently shown promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored an EGFR mutation in the post-cetuximab tumor sample. Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring EGFR ECD mutations maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As a proof of principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. These data suggest that Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. Overall, EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials.

#2150

Alternative treatment strategies to overcome MAPK inhibitor resistance in melanoma.

Eva Goetz, Bokang Rabasha, Levi Garraway. _Dana-Farber Cancer Institute, Boston, MA_.

Inhibitors of the BRAF/MEK/ERK pathway have promise in the treatment of BRAF mutant melanoma and other MAPK activated cancers. Treatment of metastatic melanoma patients with the combination of RAF + MEK inhibitor has extended progression free survival over single agent therapy. However, in a large subset of patients, resistance invariably develops. Most clinical models show reactivation of the MAPK pathway in resistant samples, suggesting further downregulation of MAPK signaling might be beneficial. Therefore, we took BRAF mutant melanoma grown to resistance by continuous culture in dabrafenib plus trametinib (RAF + MEK inhibitor) or SCH772984 (ERK inhibitor), and treated them with a triple combination of RAF+MEK+ERK inhibitors. We found cells resistant to RAF+MEK inhibitors were cross-resistant to ERK inhibitor, but sensitive to RAF+MEK+ERK inhibitors. Conversely, cells resistant to the ERK inhibitor were cross-resistant to RAF+MEK inhibitor, but sensitive to the triple combination. However, given the possible deleterious side effects of a triple combination in patients, we explored whether alternative dosing and timing strategies could delay development of resistance. We compared chronic dosing of RAF+MEK or ERK inhibitors to weekly exposures alternating between RAF+MEK and ERK inhibitors. This alternative dosing strategy delayed development of resistance in vitro over chronic dosing, while also resulting in a slower proliferation rate in resistant cells. In addition, cells exposed to a higher dose of inhibitor for a shorter period of time was equivalent to chronic exposure. These studies suggest that alternative dosing strategies (other than chronic exposure) may prevent development of resistance, and allow for treatment of patients with higher order combinations and less side effects.

#2151

Gingerol, paradol and shogaol overcome colorectal cancer cell resistance to sorafenib via enhancing its cellular uptake, entrapment and intracellular metabolism.

Mohamed G. Mehanna,1 Fahad A. Al-Abbasi,1 Tarek Elawady,1 Ali M. El-Halawany,2 Ahmed M. Al-Abd3. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Cairo University, Cairo, Egypt;_ 3 _National Research Center, Giza, Egypt_.

Sorafenib is tyrosine kinase inhibitor used for the treatment of hepatocellular and renal carcinomas but suffers from resistance in some tumors such as colorectal cancer. Colorectal cancer multi-drug resistance might be attributed to over-expression/over-activation of P-glycoprotein (P-gp) efflux pump and/or intracellular metabolism. Gingerol, paradol and shogaol are naturally occurring major compounds within the family Zingiberaceae. Gingerol and its related hydroxyphenylalkanones are known for their potential interference with P-gp function as well as intracellular drug metabolism. In the present study, we investigated the influence of gingerol, paradol and shogaol on the cytotoxic profile of sorafenib in colorectal cancer cell lines (HT-29, HCT-116, LS-174T and CaCo-2) via their influence on sorafenib cellular accumulation and intracellular metabolism. Sorafenib alone showed considerable cytotoxic activity against all testes colorectal cell lines with IC50 ranging from 2.0±0.3 to 11.10±0.8 µM. Gingerol, paradol and shogaol significantly enhanced the cytotoxicity of sorafenib against HT-29 cells and decreased its IC50 from 9.2±0.4 µM to 6.6±0.7, 3.8±0.4 and 4.7± 0.4 µM, respectively. Similarly in HCT-116, gingerol, paradol and shogaol significantly decreased sorafenib IC50 from 11.3±0.8 µM to 5.7±0.7, 5.9±0.4 and 7.7± 0.5 µM, respectively. Using DNA cytometric analysis, combination of gingerol, paradol or shogaol with sorafenib significantly decreased the proliferating cell fractions from 16.9±1.1% to 11.6±1.1%, 14.7±1.1% and 9.16±1.8%, respectively. In parallel, combination of gingerol, paradol or shogaol with sorafenib significantly increased the c-PARP concentration from 60.0±20.0 pg/cell to 256.5±25.8, 244±61.4, and 202.9±37.2 pg/cell, respectively. Gingerol and paradol, but not shogaol increased the intracellular accumulation of the P-gp probe, rhodamine at concentration range (3-100 µM). However, using recombinant ATPase attached p-gp molecules, shogaol and paradol significantly inhibited the ATPase activity. Gingerol, paradol and shogaol increased sorafenib uptake and decreased its extracellular concentration from 1491.7±132.0 ng/ml to 511.0±16.0, 216.0±39.0 and 256.4±15.6 ng/ml, respectively. Only gingerol and paradol increased sorafenib intracellular accumulation and increased its level from 239.0±16.0 pg/cell to 297.0±32.6 and 327.0±12.0 pg/cell, respectively. Both paradol and shogaol enhanced the intracellular metabolism of sorafenib to its N-oxide active metabolite and entrapped it within cellular compartment. In conclusion, gingerol, paradol and shogaol significantly enhanced the anti-cancer profile of sorafenib via influencing its cellular pharmacokinetics.

#2152

PJ34-mediated PARP-1 enzymatic activity inhibition in cervical cancer modulates the cellular response to cisplatin.

Minakshi Mann, S. S. Chauhan, Sunesh Kumar, Neerja Bhatla, Sameer Bakhshi, Ritu Gupta, Sobuhi Iqbal, Lalit Kumar. _All India Institute of Medical Sciences, New Delhi, India_.

BACKGROUND: Manifestation of chemotherapy (Cisplatin) resistance is an important cause of treatment failure in patients of locally advanced (FIGO stage, IIB to IVA) cervix cancer. We hypothesised that Cisplatin (CDDP) resistance might be associated with PARP-1 hyper-activation and inhibition of PARP-1 enzyme through PJ34 could sensitise cells to cisplatin mediated cytotoxicity.

METHODS: We studied combined effect of "PJ34 (PARP-1 inhibitor) and CDDP" in vitro in SiHa and HeLa cell line. Cytotoxicity and cell cycle progression was determined with MTT assay and FACS analysis, respectively. Poly(ADP-ribosyl)ation was assessed by western blotting to determine PARP-1 enzymatic activity inhibition by PJ34. Statistical analysis was done by two-tailed Student t tests using GraphPad software and P-values were considered significant at <0.05.

RESULTS: In both cell lines, poly(ADP-ribosyl)ation was inhibited by PJ34 when used at very low concentration (≤ 3μM) and negligible band was observed at concentrations selected for further experiments. In SiHa cells, PJ34 significantly potentiated CDDP mediated cytotoxicity as assessed by MTT assay (p<0.05). Compared to CDDP alone, treatment with combination of PJ34 and CDDP lead to decrease in CDDP IC50 value from 10.8μM to 3.6μM; 7.7μM to 3.2μM; 5.3μM to 2.2μM; 3.9μM to 1.3μM at 48 h, 72 h, 96 h and 120 h treatment, respectively (p <0.01). In HeLa cells, treatment with low concentration of PJ34 (for 24 h) increased survival as well as CDDP IC50 value compared to CDDP alone. However, increase in incubation time decreased both cell survival and CDDP IC50 value. At its higher concentration, PJ34 significantly decreased cell survival (p<0.01), also decrease in CDDP IC50 value was observed from 10μM to 3.4μM; 8.9μM to 3.75μM; 6.1uM to 2μM and 5.2μM to 1.1μM at 24 h, 48 h, 72 h and 96 h treatment respectively (p <0.005). It is reported that CDDP causes cell cycle arrest at late S, early G2/M phase and death at even higher dose, cell cycle analysis revealed that PJ34 increased this late S, early G2/M block and an increased cell death as evaluated by sub G0 peak, in both the cell lines. PJ34, itself had minimum cytotoxic effect at concentrations used in the study; IC50 value was determined to be 24.8μM (24 h, p = 0.0068) for HeLa and 60μM (48 h, p = 0.00067) for SiHa.

CONCLUSION: Suppression of PARP-1 enzymatic activity makes cervix cancer cells more sensitive toward CDDP induced toxicity. It seems to be a plausible mechanism for CDDP-resistant cancer cells to develop a dependency to PARP-1. Thus, PARP-1 enzyme inhibition could be explored as a potential therapy for treating CDDP resistance in cancer cervix.

#2153

ATP7B is a promising therapeutic target for uterine leiomyosarcoma.

Mamoru Kakuda. _Osaka University, Suita-City, Japan_.

Objective

Resistance to platinum drugs remains a significant problem in uterine leiomyosarcoma (LMS). We investigated the role of ATP7B in the resistance to platinum drugs in LMS using both in vitro and in vivo models.

Methods

The expression of the typical platinum transporters (MDR1, MRP2, ATP7A, and ATP7B) was examined in LMS cell lines using Western blotting analysis. ATP7A expression was investigated by immunohistochemistry (IHC) using clinical samples of LMS. IC50 values to cisplatin were measured in SK-LMS cells, SK-LMS-ATP7B-suppressed cell line (SK-LMS-7B cells), which permanently transfected PRS ATP7B shRNA vector. We established xenografted mice by inoculating SK-LMS cells and SK-LMS-7B cells, and examined in vivo platinum sensitivity with cisplatin for both tumors. This study was approved by the Institutional Review Board and the Ethics Committee of the Osaka University Hospital (approval #10302, approved on March 11, 2011).

Results

The expression of ATP7B was identified in the SK-LMS cells. ATP7B expression was identified in 70% (14/20) of the LMS clinical samples using IHC. The IC50-values to cisplatin improved from 17 mM to 4.3 mM after the suppression of ATP7B in SK-LMS-7B cells. A significant anti-tumor effect of cisplatin was observed in SK-LMS-7B xenografted mice than in SK-LMS xenografted mice. We also identified CuSO4 as an preferentially inhibitor of ATP7B in vitro.

Conclusion

ATP7B is associated with platinum resistance. CuSO4 acting as an inhibitor of ATP7B can be a therapeutic target for LMS.

#2154

TX1111: a peptide homologue of Topoisomerase-1 sensitizes pancreatic cancer cells to gemcitabine.

Manu Gnanamony,1 Victoria Stepanova,2 Lily Criscione,1 Jerusha Boyineni,1 Stephen J. Marshall,1 AiXuan Holterman,1 Christopher S. Gondi1. 1 _University of Illinois College of Med. at Peoria, Peoria, IL;_ 2 _University of Pennsylvania School of Medicine, Philadelphia, PA_.

Pancreatic Ductal Adeno Carcinoma (PDAC) is the fourth most common cause of cancer deaths in the United States and accounts for over 95% of all pancreatic cancers. The combined 1- and 5-year survival rates for PDAC are very poor, at 25% and 6% respectively. A major hallmark of pancreatic cancer is tumor recurrence and extremely poor response to chemotherapy. The current standard of care for patients with advanced pancreatic cancer is a chemotherapy regimen that includes gemcitabine. This treatment results in a modest benefit i.e., an increase in survival of only 5 weeks. The high mortality in patients diagnosed with this pancreatic cancer is primarily due to its drug-resistant nature and the lack of effective therapeutic strategies to overcome drug resistance. This disappointing situation strongly suggests that an improved and increased understanding of drug resistance mechanisms could lead to the development of novel therapeutic strategies for the successful treatment of patients with pancreatic cancer.

Our studies have shown that one of the contributing factors that lead to chemotherapy resistance is the protease activator urokinase plasminogen activator (uPA). To validate the contribution of uPA in chemoresistance, we overexpressed uPA in MIAPaCa-2 and PANC-1 cells and determined chemoresistance. We observed that uPA overexpressed cells showed increased chemoresistance in both Mia PaCa-2 (>25 fold) and PANC-1 (>6 fold). Mass-Spec analysis of nuclear uPA-IP from pancreatic cancer cells revealed that uPA interacts with Topoisomerase-1 (TOPO-1). This was further validated by western blot analysis of nuclear co-IP in pancreatic cancer cells. Further the binding domains of TOPO-1 to uPA were identified using overlapping a peptides array (PepSpot-JPT) followed by synthesis of these peptides. We observed that a TOPO-1 homologous peptide designated TX1111 was capable of significantly suppressing gemcitabine resistance in pancreatic cancer cells. Our observations show that the specific peptide TX1111 suppresses chemoresistance by 45% in Mia Paca-2 cells (p<0.001) and by 40% in PANC cells (p=0.002).

These results demonstrate that:

1. Overexpression of uPA promotes gemcitabine resistance in pancreatic cancer cells

2. uPA and TOPO-1 interact in the nucleus of pancreatic cancer cells

3. The peptide homologue of TOPO-1, TX1111 sensitizes gemcitabine resistant pancreatic cancer cells.

#2155

Acquired tamoxifen resistance sensitises breast cancer cells to bisphosphonates.

Dionysia Lymperatou, Christopher Smith, Wen Guo Jiang, Bronwen Evans, Stephen Hiscox. _Cardiff University, Cardiff, United Kingdom_.

Acquisition of resistance to endocrine therapies is a major limiting factor to their clinical effectiveness, resulting in disease relapse and poor prognosis. This may be due in part to the observations that acquired resistance to agents such as tamoxifen is accompanied by a gain in aggressive cellular features likely to promote disease spread and survival at metastatic sites. Bisphosphonates (BPs) are potent antiresorptive drugs used to treat osteoporosis and bone metastasis. Recent evidence also suggests that these agents may possess anti-tumour properties independently of their action on osteoclasts in a range of different tumours including breast cancers resulting in suppression of proliferation and migration and an induction of apoptosis. Here we have explored the potential of the bisphosphonate, zoledronic acid (ZOL) to suppress the aggressive phenotype of acquired endocrine-resistant breast cancer models versus their endocrine-sensitive counterparts.

ER+, endocrine-sensitive MCF7 cells and their highly-proliferative, ER+, tamoxifen-resistant ('TamR') counterparts were exposed to increasing concentrations of ZOL (1-160µM) for 3 days and changes in growth determined by MTT assay and cell counting. Immunohistochemical analysis of Ki67 confirmed cell growth effects whilst cell cycle analysis was performed using FACS and propidium iodide. Changes in signalling pathways were investigated by Western blotting.

The proliferative capacity of TamR cells was greatly suppressed by ZOL in a dose dependent manner in contrast to MCF7, where the effect was minimal (IC50 ± SD: 30 ± 4 µM (TamR) versus 150 ± 15 µM (MCF7); p<0.05). This was confirmed with Ki67 analysis which showed a reduction in Ki67 positivity of 40% (TamR) versus 25% (MCF7); p<0.05. ZOL treatment of TamR cells resulted in a significant loss of cells in G1 phase and an accumulation of the cells in S phase. ZOL treatment of TamR cells also resulted in loss of AKT activity, whilst AKT was unaffected in MCF7 cells.

Our data suggests that acquisition of resistance to tamoxifen confers a sensitivity to BPs where treatment of resistant cells with agents such as zoledronic acid results in suppression of proliferation and modulation of AKT signalling. These data suggest that BPs may exert direct anti-tumour effects on breast cancers which have relapsed on endocrine treatment.

#2156

Targeting estrogen receptor mutations for treatment of endocrine therapy resistance in breast cancer.

Prasanna G. Alluri, Jose Larios, Rohit Malik, James Rae, Arul M. Chiinaiyan. _University of Michigan, Ann Arbor, MI_.

Approximately 70% of all breast cancers express the Estrogen Receptor (ER) and inhibition of the ER signaling remains the mainstay of systemic therapy in such cancers. The advent of endocrine therapies (ET) have contributed significantly to reduction in the incidence, recurrence and mortality associated with breast cancer. However, approximately one-third of patients with ER-positive breast cancer do not benefit from adjuvant ET, and nearly all patients with ER-positive metastatic breast cancer (MBC) ultimately become refractory to all known ETs. While several mechanisms of resistance to ET have been proposed, there are currently no clinically effective treatments for ET resistance. Thus, ET resistance remains a major obstacle to the effective treatment of a significant subset of ER-positive breast cancers. We have recently reported recurrent somatic mutations in the ligand binding domain (LBD) of the Estrogen Receptor gene (ESR1) in metastatic breast cancer patients with a history of prior treatment with ET. The ESR1 mutations result in constitutive activation of the ER and confer ET resistance when ectopically expressed in ER-positive cell lines. In an attempt to develop novel therapeutic strategies to treat ET-resistant breast cancer, we have evaluated OTX015, a clinical bromodomain and extraterminal (BET) domain inhibitor, in pre-clinical models of ET resistance due to ESR1 mutations. Our studies suggest that BET inhibition may serve as an attractive strategy for treatment of ET resistance due to ESR1 mutations.

#2157

**Enhanced** in vitro **activity of dianhydrogalactitol (VAL-083) in combination with platinum drugs: Impact of p53 and platinum-resistance.**

Anne Steino,1 Guanghan He,2 Michelle Martinez-Rivera,2 Jeffrey A. Bacha,1 Dennis M. Brown,3 Zahid H. Siddik2. 1 _DelMar Pharmaceuticals, Vancouver, British Columbia, Canada;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _DelMar Pharmaceuticals, San Francisco, CA_.

Cisplatin is an important frontline drug for ovarian carcinoma and non-small cell lung cancer (NSCLC). However, the initial response rates of up to 70% are usually followed by relapse due to the onset of drug resistance. Mechanistically, platinum resistance is multifactorial, with loss of p53 function (by mutation or inhibitory protein binding to wild-type (wt) p53) playing a central role. The appearance of drug resistance is a major clinical barrier and, therefore, new agents are needed to overcome this limitation. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent that induces DNA interstrand cross-links at N7-guanine, a mechanism that is distinct from the intrastrand cross-links by platinum-based drugs. VAL-083 is clinically approved in China for lung cancer, and is undergoing clinical trial in the US for glioma. In the present study, we have examined the in vitro cytotoxicity of VAL-083 as a single agent and in combination with cisplatin or oxaliplatin using the 5-day MTT assay. In the isogenic HCT-116p53+/+ and HCT-116p53-/- colorectal models, loss of p53 increased IC50 of (or resistance to) cisplatin and oxaliplatin by 3- to 6-fold, whereas resistance to VAL-083 was increased only 1.7-fold by loss of p53. These results indicate that the cytotoxicity of VAL-083 is less impacted than the platinum drugs by loss of p53. When tested in cisplatin-sensitive (A2780) vs. cisplatin-resistant wt p53 ovarian tumor models (2780CP-16, OVCAR-10, Hey and OVCA-433) there was a 10- to 40-fold increase in IC50 for cisplatin, while the corresponding increase in IC50 for VAL-083 was only 4- to 7-fold. This indicates only partially cross-resistance between VAL-083 and cisplatin and thus suggests a distinct mode of action for VAL-083 as compared to cisplatin. To further investigate, immunoblots were developed after a 24-h exposure of isogenic A2780 or 2780CP-16 models to cisplatin or VAL-083. The two drugs were equally effective at stabilizing and activating p53 in A2780 cells. However, in cisplatin-resistant 2780CP-16 cells, VAL-083 was more effective than cisplatin at increasing p53 and p21 levels, and at inducing Ser-15 and Ser-20 phosphorylation of p53. This is consistent with the ability of VAL-083 to circumvent cisplatin resistance and demonstrated that this alkylating agent also has the capacity to partially restore wt p53 function in ovarian tumor cells. The independent mode of actions of these drugs suggested the potential for combining VAL-083 with cisplatin or oxaliplatin. These combinations in wt (H460 and A549) and mutant (H1975 and H157) p53 NSCLC models demonstrated significant super-additivity (p<0.05) and/or synergy (CI < 1). Taken together, these results demonstrate the antitumor activity of VAL-083 against both wt and mutant p53 cancers and raise the clinical potential for treatment in a combination setting with platinum drugs.

#2158

Pharmacologic, pharmacodynamic action of MELK kinase inhibitor OTS167 in cancer cells.

Suyoun Chung,1 Kyoko Kijima,1 Yosuke Harada,1 Naofumi Takamatsu,1 Takashi Miyamoto,1 Yo Matsuo,1 Yusuke Nakamura2. 1 _OncoTherapy Science, Inc., Kawasaki, Japan;_ 2 _The University of Chicago, Chicago, IL_.

Cancer is the second most common cause of death in US in 2015 and has been the leading cause of death in Japan since 1981. Despite of improved therapeutic strategies in cancer, only limited treatment options can still be available to a subset of patients. Many efforts have been made to develop molecular targeted drugs, but the success rate did not reach to patients' expectation. Unlike the conventional chemotherapy, the use of maximum tolerated dose (MTD) and the measurement of bulk tumor volume are not always appropriate for the clinical evaluation of molecular targeted drugs. To optimize and personalize the dosing for targeted agents, detailed molecular pathway and biomarkers that are affected by the drug should be elucidated. OTS167 was developed as a novel and potent MELK kinase inhibitor that we previously reported. The expression of MELK is elevated in various human cancers both in solid and hematological tumor and MELK is known to be associated with cancer progression and poor prognosis. Because MELK is indicated its critical roles in cancer stem cell proliferation as well, targeting MELK is an attractive and promising therapeutic strategy for cancer patient.

Here, we report the molecular mechanism of action of OTS167 in preclinical model. OTS167 suppressed MELK activity and promoted MELK protein degradation by inhibition of autophosphorylation. OTS167-treated cells showed drastic morphological transformation with the induction of p53 and p21 expression. We also investigated the expression of stem cell markers to elucidate whether OTS167 suppresses cancer stem-like properties through inhibition of MELK. Furthermore, we evaluated antitumor activity of OTS167 using human xenograft model and molecular changes in tumor tissue. The expression of MELK and downstream molecules were decreased in OTS167-treated xenograft tumor tissues by IHC. The change of expression and pharmacological effect were well correlated. Our data provide the evidence for the concept that OTS167 suppresses tumor growth through the inhibition of MELK signaling pathway and suggest the possibility of biomarkers for the assessment of clinical efficacy.

#2159

In vitro **characterization of PTAU and FU interactions in colon cancer cells.**

Esther A. Suswam, Gaurav Kumar, Hyung-Gyoon Kim, Mohamed Osmar, Mahmoud H. el Kouni, Upender Manne. _University of Alabama at Birmingham, Birmingham, AL_.

Background: Fluorouracil (FU) remains a main anticancer therapy for colorectal cancer (CRC) and other solid tumors. The mechanism of action of FU is associated with inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA. High toxicity and wide-spread chemoresistance, however, have limited the utilization of FU-based therapies. Five intracellular enzymes, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), thymidine phosphorylase (TP), and uridine phosphorylase (UP) are key determinants of FU sensitivity or resistance. Combination therapies aimed at improving the bioavailability of FU and reducing host toxicity has been evaluated. Recently, our group developed a new uridine phosphorylase inhibitor, 5-phenylthio-acyclouridine (PTAU) that has shown promise in reducing host toxicity and increasing FU efficacy in vivo. The current studies were to characterize interactions of PTAU and FU in vitro and to assess its effects on key metabolic enzymes in human colon cancer cells. Hypothesis: We hypothesized that inhibition of uridine phosphorylase by PTAU would ameliorate the toxic effects of FU and enhance its efficacy by permitting increased doses of FU. Methods: Three colon cancer cell lines with differing p53 status, HCT116-p53wt (wild-type), HCT116-p53null, and HT-29 (p53-mutant); and a normal colonic epithelial cell line, CRL-1790, were used. Doubling times were determined, and IC50 values for FU were derived from kill curves using MTT assay. Cells were treated for 72 hours with IC50 doses of FU, PTAU (100 μM) or uridine (25 μM), alone or in combination, or with the solvent, DMSO. mRNA levels for DPD, TS, TP, and UP were quantitated by qRT-PCR; for intracellular protein expression, cells were fixed and stained with monoclonal antibodies and analyzed by flow cytometry. Results: FU sensitivity correlated with cell doubling times. Basal expression of FU metabolic enzymes differed between CRC and normal cells. mRNAs for DPD and UP were higher in normal relative to cancer cells, but TS mRNA was higher in cancer cells. FU treatment upregulated mRNA expression of DPD, UP, and TS in cancer cells but not in normal cells. Only in cancer cells, DPD induction by FU was potentiated by PTAU. In contrast, treatment with FU decreased protein levels of DPD, TP, and TS in cancer cells. PTAU induced TS protein expression in cancer cells, an effect that was reduced by co-treatment with FU. Conclusions: FU induces mRNA levels of DPD, UP, and TS in cancer cell lines, but protein levels are reduced; the discrepancy between mRNA and protein levels following FU treatment underscores the need for evaluation of colon cancer samples to assess response to FU therapy. This work was supported by a Supplement of the MSM/TU/UABCCC U54 Partnership grant (3U54-CA118948-09S1) from NIH.

#2160

Targeting SMC1 in combination therapy for triple negative breast cancer.

Sushma Yadav, David Berz, George Somlo, Kimlin Ashing, Yate-Ching Yuan, Robert J. Hickey, Sailee Yadav, Arthur D. Riggs. _City of Hope National Medical Center, Duarte, CA_.

Triple negative breast cancer (TNBC) defined primarily by lack of expression of estrogen and progesterone hormone receptors (HR) and lacking HER2 overexpression and/or gene amplification (HER2) is an aggressive subtype with poor prognosis. Presentation is frequently at an advanced stage, and TNBC is particularly prevalent in younger women, African Americans, and/or in women with BRCA1 and less frequently with BRCA2 gene mutations. Over the last several years, although great progress has been made in the treatment of early HR positive and HER2+ (as well as advanced stage) breast cancer, relapse/progression-free and overall survival of patients with TNBC remains a serious problem. Molecular subtyping identified subsets of TNBC, several of which are thought to be DNA-repair deficient (either due to germ-line mutations such as BRCA1 or 2, or somatic mutations affecting DNA-repair), may be amenable to treatment with DNA-damaging agents such as platinum compounds and PARP (poly(ADP-ribose) polymerase-1) inhibitors. This could be particularly useful in BRCA-mutated cancers. As for the other TNBC subtypes, development of novel therapeutic agents, represents a high priority.

In response to this clinical need, we tested the efficacy of a novel combinatorial strategy which targets differentiated cells and TNBC cancer stem cell like population (CSCs). Structural maintenance of chromosome-1 (SMC1) was selected as a tumor antigen based on our published studies showing its surface localization and the role in cell proliferation, survival and metastasis in TNBC cells. Bioinformatics analyses using public available patient cohorts with clinicopathological information such as TCGA, NCBI and BRAVO showed statistically higher mRNA overexpression of SMC1 in TNBC compared to HR+ and HER2+ breast cancer. To target SMC1, we developed several monoclonal antibodies against the epitope of SMC1 which showed similarity to cell-adhesion peptides in order to facilitate biochemical and cellular assays. The impact of SMC1 inhibition in combination with veliparib, an inhibitor of PARP-1 was tested in BRCA1-wild-type (MDA-MB-231) vs. mutant (MDA-MB-436) TNBC cells and in CSCs (CD44+/CD24low-) sorted from these cells by MTT and colony forming assays. TUNEL and Annexin-V flipping assays were used to determine cell apoptosis. The role of intrinsic vs. extrinsic pathways of apoptosis was examined by measuring activation of Caspase 8 and 9 as well as measurements of cytochrome-c release from mitochondria.

Our results showed that Mab-S15 recognizes SMC1 which is selectively expressed on the surface of cancer cells (including CSC's) and differentiated TNBC's, but has a minimal distribution in normal cells. The antibody distinctly inhibits the survival of TNBC in culture, including CSC's, sorted from TNBC's. The Mab-S15 and veliparib combination was more effective than the individual agents in inhibiting the growth and survival of TNBC cells including those with a functional BRCA1. 

## CANCER CHEMISTRY:

### High Throughput Screening and Natural Products

#2161

Development of radiolabelled drug carrier systems for molecularly targeted radiotherapy.

Jyothi U. Menon, Joshua Owen, Robert Carlisle, Katherine A. Vallis. _University of Oxford, Headington, Oxford, United Kingdom_.

Introduction

Tagging of radioisotopes to targeting moieties such as peptides, antibodies and nucleotides aids in more precise delivery of these isotopes to cancer cells for treatment. However some of the obstacles to successful delivery of these radiolabelled molecules include poor circulation half-life and inefficient tumour extravasation and penetration. Liposomal drug carrier systems have extended circulation profiles but would benefit from enhanced binding and entry into target cancer cells. To address some of these challenges, liposomes encapsulating a chemotherapeutic agent (doxorubicin) and tagged with radiolabelled targeting moieties were designed, formulated and then tested for specific binding to and killing of cancer cell lines.

Experimental Procedures

Distearoylphosphatidylethanolamine-poly ethylene glycol (DSPE-PEG) based liposomes were developed and surface modified with epidermal growth factor-diethylenetriamine pentaacetate (EGF-DTPA) followed by 111In radiolabelling. The EGF-tagged liposomes were characterised using zetasizer and transmission electron microscopy (TEM). Direct particle cytotoxicity was assessed using the MTT assay and uptake kinetics was determined by treating EGF+ve MDA-MB-468 (1.3 × 10^6 EGFR/cell) and EGFR-ve MCF7 cells (1.5 X 10^4 EGFR/cell) with Nile red-containing liposomes and quantifying Nile red fluorescence in the cell lysates. Payload delivery capabilities were studied by exposing EGFR+ve luciferase-expressing MDA-MB-231 or EGFR-ve luciferase expressing MCF7 breast cancer cells to EGF-tagged or non-tagged particles containing the model drug luciferin. Therapeutic efficacy of Doxorubicin-containing EGF-tagged liposomes to MDA-MB-468 and MCF7 cells was then assessed by the MTT assay.

Results

The particles were spherical in morphology with a diameter of 140-160 nm. More than 95% of the 111In added was tagged to the particles. The liposomes did not show intrinsic cytotoxicity and were taken up by cells in a dose-dependent manner with greater uptake observed in MDA-MB-468 than in MCF7s. Greater delivery (2-fold increase, p<0.05) of EGF-tagged luciferin-containing particles was observed in MDA-MB-231 cells than in MCF7 cells, this was not the case for non-tagged particles. EGF-tagged particles also gave better delivery (3.5-fold increase, p<0.05) than non-tagged particles in MDA-MB-231 cells, this was not the case in MCF7 cells. Doxorubicin-loaded EGF-tagged liposomes had an IC50 of 0.02 mg/ml with MDA-MB-468 and 0.04 mg/ml with MCF7s.

Conclusions

This work, using 111In- and EGF-tagged liposomes has shown promising in vitro targeted cellular uptake and drug delivery. Receptor binding kinetics and the feasibility of using these systems in vivo for ultrasound-assisted radiopharmaceutical delivery will follow.

#2162

Novel anti-gastrin nanoparticle inhibits growth of pancreatic cancer.

Julian Burks,1 Sandeep Nadella,1 Abdullah Mahmud Mahmud,2 Scott McNeil,2 Jong-IN Hahm,1 Stephan Stern,2 Jill Smith1. 1 _Georgetown University, Washington, DC;_ 2 _NIH Nanotechnology Characterization Lab, Frederick, MD_.

Background: Receptor-targeted therapies or inhibition of growth factors have improved cancer survival. Pancreatic ductal adenocarcinoma (PDAC) markedly over-expresses the cholecystokinin-B (CCK-B) receptor and re-expression gastrin stimulates growth of PDAC by an autocrine mechanism through the CCK-B receptor. When gastrin mRNA is down regulated by RNAi techniques, PDAC growth and metastases are inhibited in animal models. However, anti-gastrin gene therapy cannot be readily used in humans unless nontoxic gene delivery strategies are implemented. The purpose of this project was to develop a unique drug delivery nanoparticle (NP) that selectively binds to the CCK-B receptor and safely delivers siRNA to down-regulate gastrin expression and inhibit growth of PDAC.

Methods: In order to develop the targeted NP, a thiol functionalized polyethylene glycol-block-poly(L-lysine) (SH-PEG-PLL) polymer was synthesized. To render the NP target-specific for the CCK-B receptor we used a maleimide link to conjugate Gastrin-10 to the PEG via Michael addition reaction. The resulting Ga-PEG-PLL was extensively purified using a PD-10 column and by dialysis. The polyplex micelle was prepared by mixing 1mg/mL of the Ga-PEG-b-PLL with a gastrin siRNA (si286 GUGCUGAGGAUGAGAACUA), which decreases gastrin mRNA 90%. The PEG protects the siRNA from degradation in solution or blood and the lysine polymer forms a micelle shielding the positive charge and eliminating toxicity. The NP was analyzed by dynamic light scattering (DLS) and zeta potential. Efficacy of the NPs to inhibit growth was tested on PANC-1 human PDAC cells that have a high number of CCK-B receptors. Cells were plated into 6-well plates overnight and then treated for 72h with PBS, 100x NP (10nM siRNA) or 50x NPs (5nM siRNA) in serum-free DMEM media. Viable cell counts were performed by trypan blue exclusion.

Results: Characterization of the functionalized polyplex NP confirmed a molecular weight of 9700 Da. Trityl deprotection and conjugation of Ga-10 to the SH-PEG-PLL polymer were confirmed by NMR which demonstrated complete removal of the trityl group and greater than 70% conjugation of the peptide to target the receptor. The polyplex NP complex was confirmed by DLS measurement, which demonstrated size distributions of 44.3 ± 0.3 and 48.2 ± 0.3 nm for receptor-targeted and untargeted polyplex respectively. Treatment of PANC-1 cancer cells with the anti-gastrin NPs significantly inhibited growth by 98% compared to untreated controls (p=0.005).

Conclusion: A novel NP that targets the over-expressed CCK-B receptor on PDAC was successfully synthesized and characterized. The NP can deliver anti-gastrin gene therapy in the form of siRNA into human pancreatic cancer cells to significantly inhibit cell growth by downregulation of gastrin expression. This technique may provide a safe and novel gene therapy delivery method to treat those with advanced PDAC.

Supported by: Otto J Ruesch Center for the Cure of GI Cancers

#2163

Targeting brain tumor and brain metastases using brain penetrating peptide and long circulating nanoemulsions.

Xinli Liu, Tanvirul Hye. _Texas Tech University Health Sciences Center, Amarillo, TX_.

Purpose: Glioblastoma brain tumor and brain metastases from primary breast or lung cancer have limited treatment options, primarily due to the extremely invasive and aggressive nature of tumors; the largely impermeable blood-brain barrier (BBB); and presence of efflux transporters at BBB. We aimed to improve brain delivery of drugs by intravenous injection of a mixture of brain penetrating peptide K16ApoE and drug encapsulated long circulating nanoemulsions. We also explored the potential mechanisms that account for its brain delivery.

Methods: Oil-in-water nanoemulsions (NE) containing medium-chain triglycerides oil and co-surfactants were prepared by a spontaneous self-emulsification method (SNNE) and conventional high speed homogenization and sonication method (CNE). The nanoemulsions were mixed and injected with 20 nmol of K16ApoE peptide that comprising 16 lysine residues and 20 amino acids corresponding to the low-density lipoprotein receptor binding sequence from apolipoprotein E in healthy and tumor bearing mice.

Results: Nanoemulsions had mean particle size less than 200 nm (CNE: 190 nm,+35 mV; SNNE: 120 nm, -10 mV). In vitro, both NE was taken up by brain capillary endothelial cells bEnd.3 and glioblastoma cell U87-luc in a serum- dependent manner (10% FBS> 5%FBS> 2.5%FBS> serum free). Both the bEnd3 and U87-luc cells express high levels of an endocytic low density lipoprotein receptor-related protein 1 (LRP1) receptor. Using LRP1 antagonist receptor-associated protein (RAP) to block a ligand binding to LRP1 led to significant decrease of cellular uptake of NE. The addition of one of LRP1 ligand apolipoprotein E (12.5-25 µg/mL) in the serum free medium resulted in significantly higher cellular uptake of NE in bEnd3 cells and U87-luc cells, suggesting the NE can be imported into the brain and brain tumor cells by binding to apolipoprotein E via LRP1 receptor mediated transcytosis. In vivo, intravenous injection of near-infrared labeled NE significantly improved its brain accumulation in comparison to that of free dye. When 20 nmol of K16ApoE peptide and NE mixture was inject intravenously, the peptide transiently opened the BBB and allowed even higher amount of NE accumulation at brain tumor.

Conclusions: Our study demonstrates that nanoemulsion is a promising nanocarrier to deliver imaging or therapeutic agents for the diagnosis or systemic treatment of brain tumor or brain metastasis. K16ApoE peptide is a brain penetrating peptide and can help transport nanoparticles to the brain tissue without the need for chemical modification of surface of nanoparticles. Binding of nanoemulsion to apolipoprotein E and involvement of LRP1 receptor-mediated transcytosis could be important steps in mediating the uptake of nanoemulsion in the brain endothelial cells and brain tumor cells.

#2164

Tenascin-C binding peptides for cancer targeting.

Prakash Lingasamy,1 Allan Tobi,1 Hedi Hunt,1 Pille Säälik,1 Markko Salumäe,1 Tambet Teesalu,1 Tambet Teesalu2. 1 _University of Tartu, Tartu, Estonia;_ 2 _Sanford-Burnham Medical Research Institute, La Jolla, CA_.

Extracellular matrix protein tenascin C (TNC) is strongly overexpressed in the stroma of many solid tumors. TNC expression is upregulated at the invasive front of tumors while being nearly undetectable in normal adult tissues. TNC-targeted humanized antibodies are undergoing clinical development for targeting compounds to solid tumors. Compared to antibodies, homing peptides may have more favorable extravasation and tumor penetration properties.

We used in vitro T7 peptide display to identify peptides that bind to the alternatively spliced domain C of the large isoform of Tenascin-C (TNC FnIIIC). The selected phage pool showed 500-fold increase in binding to immobilized TNC FnIIIC. Candidate TNC FnIIIC-binding peptides (designated PL1- 3) were found to bind to TNC FnIII C protein, to the extracellular matrix extracted from PC3 prostate tumors and to cultured tumor cells known to overexpress TNC. To evaluate candidate peptides in vivo, we used mice bearing patient-derived xenografts of P3 glioma (R. Bjerkvig, Bergen, Norway), 4T1 breast tumors, and PC-3prostate carcinoma. A pool of the phages displaying TNC FnIIIC binding peptides, and a panel of phages displaying published tumor homing and control peptides was injected intravenously in tumor-bearing mice, and relative tumor homing and background in control tissues was determined for each phage using high-throughput sequencing (HTS) of phage genomic DNA. These studies demonstrated that among TNC FnIIIC targeting peptides, the PL3 peptide showed the best tumor homing and specificity profile. To evaluate the potential of the synthetic PL3 peptide for tumor targeting of nanoparticles, we coupled PL3 peptide to iron oxide nanoworms (IONW) - paramagnetic nanoparticles that are PEGylated to extend blood half-life, and have because of their elongated shape more effective targeting properties than spherical nanoparticles. 5 hours after intravenous administration of 7.5mg/kg of IONW in mice bearing PC3 prostate cancer xenografts, PL3-IONW but not control IONW showed a robust accumulation in tumor vasculature, and some extravasation into extravascular tumor tissue. Our data suggests that the newly identified TNC FnIIIC targeting peptide, PL3, may be used for payload delivery into tumor stroma. Currently, we are evaluating PL3-targeted IONWs as a MRI contrast agent and as carriers for cytotoxic compounds to TNC expressing tumors.

#2165

Enhanced uptake and accumulation of temozolomide and irinotecan in orthotopically-implanted gliomas by vascular priming with RRx-001.

Pedro Cabrales,1 Bryan Oronsky,2 Jan Scicinski2. 1 _Department of Bioengineering, University of California and San Diego, La Jolla, CA;_ 2 _EpicentRx, Inc, Mountain View, CA_.

Background and Objective

RRx-001 is a radio-, immune- and chemosensitizing pan-epigenetic inhibitor with vascular normalizing properties under investigation in multiple Phase II clinical trials. Vascular normalization refers to the remodeling of the immature, crowded and dysfunctional tumor capillary network that is pruned typically through the action of anti-VEGF therapy and results in improved tumor blood flow and oxygenation. One benefit of vascular normalization is the improved penetration of cytotoxic and other therapies into the tumor leading to better responses and potentially improved survival.

MRI studies in a Phase 1/2 clinical trial of RRx-001 with radiation in brain metasteses showed alterations in tumor blood flow suggesting vascular normalization. The purpose of these experiments was to determine whether vascular normalization after administration of RRx-001 that had been previously observed both clinically and preclinically in heterotopically implanted syngeneic tumors, would increase the delivery and accumulation of systemically administered irinotecan (CPT-11) or temozolomide (TMZ) in orthotopically implanted gliomas and whether an increase in survival would be observed.

Methods

Human glioblastoma cells (GBM43), maintained as serially passaged subcutaneous xenografts in athymic mice were implanted intracranially in six-week-old athymic mice. In three separate experiments, 12 days after implantation, mice were treated with (1) RRx-001, (10 mg/kg IV); (2) TMZ, (20 mg/kg, IV or 25 mg/kg, PO) or irinotecan (0.4 mg); (3) TMZ (20 mg/kg or 25 mg/kg, PO) or irinotecan (0.4 mg) 4 hours after RRx-001 (10 mg/kg) and (4) saline control. Dosing was every 6 days in 4 cycles. Animals were euthanized as the symptoms from the increasing tumor burden prevented access to food or water. Tumor tissue was collected from a subset of animals 24 h after last dose of chemotherapy. Analysis of irinotecan content in tumor tissue was carried out by HPLC and temozolomide by LCMS.

Results

In all three experiments, pretreatment with RRx-001 increased the concentration of irinotecan and IV and PO temozolomide in the tumor tissue. The combination groups also had statistically significant greater survival compared to the cytotoxic drug and RRx-001 alone and against control.

Conclusion

Vascular normalization-enhanced delivery of standard cytotoxics mediated by RRx-001 may improve the delivery of cytotoxic therapy into tumor tissue, particularly in difficult to treat brain cancers, leading to enhanced responses and improved survival and warrants further preclinical and clinical investigation.

#2166

Targeted delivery of chemotherapeutic agents to solid tumors via systemic Nano-diamino-tetrac.

Thangirala Sudha, Dhruba J. Bharali, Noureldien HE Darwish, Melis Debreli-Coskun, Qishan Lin, Paul J. Davis, Shaker A. Mousa. _Pharmaceutical Research Institute, Albany College of Pharmacy & Health Sciences, Rensselaer, NY_.

Covalently bound to a poly (lactic-co-glycolic acid) (PLGA) polymer via a linker, tetraiodothyroacetic acid (tetrac) as Nano-diamino-tetrac (NDAT) targets a specific cell surface receptor on the extracellular domain of integrin αvβ3. The integrin is primarily expressed by cancer cells and rapidly dividing endothelial cells. The conjugation of tetrac and linker to a 120 nm PLGA nanoparticles prohibits cell entry of NDAT and offers an opportunity to load the nanoparticle with a generic cancer chemotherapeutic agent for cancer-targeted delivery. Even without a payload, NDAT has anticancer and anti-angiogenesis properties. For the current studies, xenografts of human urinary bladder cancer (263JBV) cells and of human pancreatic cancer (SUIT-2) cells were established in the nude mouse. Tumor growth and tumor content of cisplatin (263JBV xenografts) and of paclitaxel (SUIT-2 xenografts) were compared in the following animal cohorts: 1) untreated controls; 2) controls treated with void PLGA nanoparticles (NPs), 3) daily treatment with chemotherapeutic agent (cisplatin, 1 mg/kg, or paclitaxel, 0.3 mg/kg), alone, 4) daily treatment with sub-maximal NDAT, alone (0.3 mg tetrac equivalent/kg), 5) daily treatment with chemotherapeutic agent encapsulated into PLGA NPs (no tetrac), and 6) daily treatment with chemotherapeutic agent loaded into NDAT. NDAT dose in group 5 was 0.3 mg tetrac/kg. Daily drug treatments were continued for 3 weeks after establishment of xenografts. Urinary bladder tumor weight at animal sacrifice decreased insignificantly vs. controls in animals that received cisplatin or cisplatin + PLGA. Submaximal dosing of NDAT, alone, induced a small, but significant, decreased in tumor weight. Tumors in animals treated with NDAT bearing a payload of cisplatin decreased in weight by 50% (P <0.01) and mean cisplatin content (LC/MS/MS) of tumors (ng/gm of tissue) was 5-fold more than in animals treated with cisplatin, alone, and 3-fold more than in animals treated with PLGA NPs with encapsulated cisplatin. Animals receiving cisplatin, alone, developed neurotoxicity (inability to use the hind limbs). In pancreatic cancer xenografts, paclitaxel encapsulated into PLGA NPs, NDAT, alone, and NDAT bearing a payload of paclitaxel caused significant decreases in tumor weight. The greatest reduction in tumor weight (60%, P<0.01) was in animals exposed to NDAT bearing a drug payload and mean concentration of paclitaxel in these tumors (ng/gm tissue) exposed to an NDAT/paclitaxel payload was 5-fold more than in xenografts exposed to paclitaxel, alone, or paclitaxel encapsulated into PLGA NPs. Thus, the payload capacity of NDAT for cancer chemotherapeutic agents and tumor-specific delivery of cancer chemotherapeutic agents significantly increased bladder and pancreatic cancer content of, respectively, cisplatin and paclitaxel. Enhanced tumor size reduction and deceased systemic toxicity were achieved with this targeted approach.

#2167

Efficient and rapid cellular delivery of bioactive proteins using EXPLOR: exosomes engineered for protein loading via optically reversible protein-protein interaction.

Nambin Yim, Seung-Wook Ryu, Kyungsun Choi, Kwang Ryeol Lee, Seunghee Lee, Hojun Choi, Jiho Park, Daesoo Kim, Wondo Heo, Chulhee Choi. _Korea Advanced Inst. of Science & Tech., Daejeon, Republic of Korea_.

Despite the long list of therapeutic proteins available for treating various human diseases, the vast majority of commercial protein-based drugs, such as cytokines, hormones, and monoclonal antibodies, have been limited to extracellular mechanisms of action. Many intracellular proteins with great potential as biopharmaceutical drugs have been identified; however, many of the challenges associated with intracellular protein delivery have yet to be solved. Although protein transduction and lipid nanoparticle-mediated protein delivery methods have been proposed for direct protein delivery into target cells and tissues, many obstacles remain before these methods can be successfully employed in vivo, including low purification efficiency, failure to separate from nanoparticles in recipient cells, and induction of immune responses against host immune cells. To address these limitations, we developed an opto-genetically engineered exosome system, named 'exosomes for protein loading via optically reversible protein-protein interaction" (EXPLOR) that can deliver soluble proteins into the cytosol via controlled, reversible protein-protein interactions (PPI). Among nanoparticles, cell-derived exosomes have recently been highlighted as new therapeutic strategies for the in vivo delivery of nucleotides and chemical drugs. Exosomes are natural cell-derived extracellular vesicles that originate from internal endocytic compartments and multi-vesicular bodies and participate in intercellular communication. Recent studies have sought to use exosomes as a new method for the in vivo delivery of siRNA or miRNA to specific target tissues by systemic injection. These methods were based on the passive loading of siRNAs or miRNAs into isolated exosomes by electrophoresis, a method poorly suited for the intracellular delivery of cellular proteins. By integrating a reversible PPI module controlled by blue light with the endogenous process of exosome biogenesis, we were able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs was shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in both a time- and dose-dependent manner. In the present study, we have demonstrated the intracellular delivery of mCherry, Cre enzyme, Bax, and Super repressor IκB proteins as functional proteins in the target cells and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based drugs into recipient cells and tissues both in vitro and in vivo.

#2168

Optimizing doxorubicin derivatives delivery using temperature sensitive biopolymers in multidrug resistant breast cancer cells.

Sonja Dragojevic,1 Jung Su Ryu,1 Felix Kratz,2 Drazen Raucher1. 1 _University of Mississippi Medical Center, Jackson, MS;_ 2 _CytRx Inc., Germany_.

The anticancer agent doxorubicin is an anthracycline compound that shows high potency in treating cancer, and it is one of the most widely used chemotherapeutics. However, efficiency of doxorubicin treatment is limited by low blood plasma solubility, poor blood pharmacokinetics, and non-selective cell killing that results in serious toxicity to healthy tissues. Additionally, cancer cells often develop drug resistance, which is a significant limiting factor to the drug's effectiveness. Motivated by these problems, we have designed a drug delivery system that can specifically deliver drug to the tumor site while overcoming doxorubicin resistance in breast cancer cell lines. This drug delivery system consists of: ELP - Elastin like polypeptide, CPP - Cell penetrating peptide, a cleavable linker -- to enable doxorubicin release in the targeted low pH environment, and a derivative of the anticancer agent Doxorubicin (modified by a 6-maleimidocaproyl moiety for conjugation to a terminal cysteine residue on ELP). ELP is thermally responsive and improves the complex's pharmacokinetics by prolonging its clearance rate while the CPP mediates cellular uptake of large macromolecules. The linker is an acid sensitive amino acid sequence (Gly-Phe-Leu-Gly) that serves as a substrate for lysosomal enzymes. In this study, we compared cytotoxicity of a cleavable (cDox) and non-cleavable (ncDox) doxorubicin derivative delivered by ELP biopolymer in the breast cancer cell lines MCF-7 and MCF7/ADR (doxorubicin resistant cell line). We showed that cDox had two fold higher cytotoxicity than ncDox in both cell lines. When ncDox, however, was conjugated to the ELP biopolymer containing a lysosomally degradable GFLG spacer, the drug delivery construct was equally cytotoxic to both sensitive and resistant cell lines, indicating that the construct delivers doxorubicin into cells by a mechanism that bypasses doxorubicin resistance. Confocal fluorescence microscopy showed that after two hours of exposure to the doxorubicin derivatives, cDox was predominantly localized in the nucleus in both cell lines. However, ncDox was localized in the plasma membrane and cytosol. Intracellular doxorubicin accumulation examined by flow cytometry indicated 3 fold higher uptake of ELP-cDox in sensitive MCF 7 cells compared to resistant MCF7 ADR cells. Cellular uptake of ELP-cDox was further enhanced two fold when conjugated with CPP. In conclusion, our current results indicate that ELP-doxorubicin conjugates may successfully overcome drug resistance in breast cancer cells, providing a promising approach for the use of chemotherapeutic agents such as doxorubicin in patients with drug resistant breast cancers.

#2169

High content screening in MCF7 and MCF10A cells show differential responses depending on oxygen levels and mechanistic readout for viability.

Michelle Yan, Michael O'Grady, Anderson April, Scott Clarke, Quentin Low, Carolyn DeMarco, Veronica Calderon, Kathy Kihn, Leticia Montoya, Carmen Finnessy, Marcy Wickett. _ThermoFisher Scientific, Eugene, OR_.

Oxygen levels in typical cell culture conditions do not accurately reflect the oxygen levels cells are exposed to within the body. Furthermore, oxygen levels can vary within the tumor microenvironment. These variances can affect how cells respond to a variety of drugs and small molecules often used during cancer treatment. Previous studies have shown altered drug sensitivities in MCF7 cells depending on the oxygen levels the cells are grown in. To further understand how oxygen levels affect drug sensitivity, the response of tumorigenic MCF7 cells were compared to non-malignant MCF10A cells, cultured under low and high oxygen.

The goal of this study was to examine the differences in the sensitivity of MCF7 and MCF10A cells to drugs from the Tocriscreen Total compound library when cultured under low and high oxygen levels and for different drug exposure time periods. Cells were grown using standard cell culture conditions (19% oxygen) or low oxygen (5%). Using high-content imaging and high-throughput analysis methods, viability was assessed using two different mechanistic readouts; cellular metabolic activity and membrane permeability. Post-screening analysis was performed to confirm positive hits by performing dose-responses and determining IC50 concentrations using the same reagents as in the screen, and reagents to assess oxidative stress and cellular proliferation.

Results showed that viability differed depending on mechanistic readout and platform method used to determine hits as well as duration of exposure to compound. In addition, the response of MCF10A cells was not always identical to that of the MCF7 cells. Post-screening analysis of "hits" indicated different potencies of compounds tested depending on oxygen level and cell type. These data suggest that some drugs may affect MCF7 and MCF10A cells differently depending on environmental oxygen levels and mechanistic readout used to determine cellular health.

#2170

Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of prostate cancer.

Ahmad Salam,1 Vincent Hembrick,1 Jesse Jaynes,1 Timothy Turner,2 Mohamed Abdalla1. 1 _Tuskegee University, Tuskegee, AL;_ 2 _Jackson State University, Jackson, MS_.

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Due to the harsh side effects of conventional chemo- and radiotherapies, targeted drug delivery is now gaining the focus of cancer researchers. The key for success of any newly developed targeted drug delivery systems is the novel design of targeting peptides which have high binding affinities to differentially over-expressed receptors on the surface of PCa cells. Two over-expressed such receptors, which can be targeted to the cell surface of PCa cells are: Gonadotropin Releasing Hormone Receptor (GnRH-R), and Prostate Specific Membrane Antigen (PSMA). The analysis done from molecular dynamics simulation data gave an important information about each interaction; such as RMSD, The number of contacts between receptor and peptide within a cutoff distance of 5 Å and the binding free energies, ∆GGBSA of the interactions. In all systems, the back bone RMSD deviation of the receptor was calculated using the last 10 ns of the sampling process and averaged over all trajectories. The RMSD data deviated between 1.22 - 2.51 Å. The free binding energies or ∆GGBSA was calculated from the last 10 ns of the sampling process. The QH1 system has the more negative ∆GGBSA -36.15 kcal mol-1 and the QH4 system has the least negative ∆GGBSA of -18.4 kcal mol-1 among the eight systems of GnRH-R. QH2 system exhibits a highly interactive hydrogen bond (bond distance is 2.65 Å) between 134th position of the receptor amino acid sequence GLU and 5th position of the qh2 peptide's TYR . The ∆GGBSA is for the qh2 system is – 34.92 kcal mol-1. In the PSMA and its targeting peptide interactions, T2IA has the least negative ∆GGBSA of -47.37 kcal mol-1. A salt bridge was observed between the 10th position of the T2IA peptide sequence ARG and the 328 position of the 3RBU receptor's GLU. Based on our molecular modeling studies, we have determined the best targeting peptides among all the newly designed peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated surface plasmon resonance (SPR) in near future.

#2171

Beta-hairpin peptide hydrogels as scaffolds for 3D high-throughput cancer drug discovery.

Peter Worthington,1 Katherine M. Drake,2 Andrew D. Napper,2 Darrin J. Pochan,1 Sigrid A. Langhans2. 1 _University of Delaware, Newark, DE;_ 2 _Nemours/AI duPont Hospital for Children, Wilmington, DE_.

High-throughput screening (HTS) remains a promising initial step in discovering novel lead compounds for cancer therapy, but its value is limited by the poor predictability of clinical effectiveness of candidate compounds. One compelling reason for this lack of reliability to predict in vivo efficacy is the fact that most HTS is done using traditional two-dimensional (2D) cell cultures. While 2D cultures are convenient and can easily be automated, compelling evidence suggests that cells cultured in these non-physiological conditions differ from cells grown in the more in vivo like three-dimensional (3D) systems that better mimic microenvironments. Thus, a 3D culture model is expected to be a better platform for cancer drug discovery. Here we demonstrate the feasibility of a beta-hairpin hydrogel as a novel platform for 3D HTS. MAX8 forms a bonded fibrillar hydrogel network under physiological conditions and moreover, due to its unique shear-thinning abilities can be incorporated into standard HTS equipment. MAX8 is biocompatible with a variety of cancer cell lines, can be tuned for porosity, permeability and stiffness, and can be functionalized with common and cell-type specific ligands to more closely resemble an in vivo-like tumor environment. Using medulloblastoma cells that are known to display more immature, tumor-like features in 3D and that were automatically dispensed onto 384-well plates we have developed a robust assay to measure cell viability on a HTS platform that will have broad applicability for other cancers for which 3D HTS is desirable.

#2172

Circle Akt in: Epitope catalyzed assembly of macrocyclic therapeutics against phosphorylated Akt.

Arundhati Nag. _California Institute of Technology, Pasadena, CA_.

An approach to drugging 'undruggable' target proteins using macrocyclic, epitope targeted peptide based affinity reagents is demonstrated. An unbiased comprehensive Cu-catalyzed Azide Alkyne Cycloaddition cyclized peptide macrocyclic library is synthesized and screened against the phoshoS474 containing Hydrophobic Motif (HM) peptide epitope of Akt2. The best macrocyclic ligand, which exhibits specificity at the peptide and protein levels, is further extended through an in situ click screen to yield bivalent macrocyclic reagent with high affinity and specificity. The bivalent peptide, targeted against the phospho-S474 region of Akt, inhibits the Akt kinase in in vitro kinase assays. This ligand is being optimized, through systematic changes in its composition, to increase its cell permeability characteristics, so that its effects can be studied in Akt overexpressing carcinoma cell lines. The optimized macrocyclic peptide can eventually be used as an in vivo imaging probe and as a peptide inhibitor drug.

#2173

Sulforaphane suppresses the growth of triple-negative breast cancer stem cells in vitro and in vivo.

Nadia P. Castro,1 Maria Cristina Rangel,1 Anand S. Merchant,2 Karen Saylor,1 David Salomon,1 Young Kim3. 1 _NCI-CCR, Frederick, MD;_ 2 _NCI, Bethesda, MD;_ 3 _NCI-Division of Cancer Prevention, Rockville, MD_.

Triple-negative breast cancer (TNBC) represents the poorest prognosis among the breast cancer subtypes and no current standard therapy. TNBC tumors are enriched with stem-like cells that are resistant to standard targeted drugs. Thus, it is urgent to develop new agents that are non-toxic and specifically efficacious against these cancer stem cells (CSCs) that are suggested to be responsible for the initiation and maintenance of tumors. Here, we show that sulforaphane (SFN), which is a dietary component abundant in broccoli and its sprouts, inhibits cell proliferation and sphere formation of the CSC population in TNBC cells and also reduces mammary tumor growth.

To evaluate the effects of SFN in TNBC we isolated CSC population from MDA-MB-231 Luci D3H1 human cells using fluorescently activated cell sorting (FACS) analysis. The population containing CD49f+, CD24-/CD44high showed the ability to form mammospheres, which is a characteristic of stemness. When these cells were treated with natural dietary constituent SFN at the concentration of 7.5 μM, they lost the property to form mammospheres in vitro. In vivo, when BalbC/nude mice were supplemented with SFN before and after cell inoculation (daily i.p. injection of 50mg SFN/kg for 5 and 3 weeks respectively), they exhibited reduced tumor volume. The suppressive effect of SFN on the tumor volume was higher when the treatment started before tumor implantation with a thirty percent reduction for pre- (n=20, 5 weeks) and fourteen for post-treated (n=20, 3 weeks) group compared to respective controls (saline treated group, n=20). These results suggest that SFN could be used as a chemopreventive agent considering its nontoxicity and efficacy. Next, we examined the effects of SFN in CSC during tumor growth using a panel of 192 stem cell markers by Nanostring mRNA analysis. The results showed that 95% of the genes were down- regulated in the pre-treatment group (n=10) compared with the control group (n=10). Twenty-seven genes presented p value <0.02 and fold change >1.5. We selected 8 genes for further real time validation confirming reduced gene expression of FOXD3, FDZ10, GAS1, WNT3, and NOTCH4 in pre-treatment group (n=10) compared with control (n=10). By real time only, we also found the same gene expression pattern for two CSC markers of our interest, CRIPTO-1 and NFkB.

Our results indicated that SFN suppresses the formation of triple negative mammospheres and tumor development both in vitro (7.5uM) and in vivo (50mg/kg) possibly by targeting a CSC population. Further analysis on gene expression revealed that SFN particularly decreased the expression of stem cell markers. These results suggest that the use of SFN for chemoprevention of TNBC is plausible and warrant further clinical evaluation.

#2174

A high-throughput, high-content assay for the discovery of new inhibitors of DNA double-strand break repair.

Yulia Surovtseva,1 Vikram Jairam,2 Ranjini Sundaram,2 Ranjit Bindra,2 Seth Herzon2. 1 _Yale University, West Haven, CT;_ 2 _Yale University, New Haven, CT_.

DNA repair pathways are being intensively investigated as targets for chemotherapeutic interventions. Despite the immense interest in this area, few whole-cell screens for the discovery of DNA repair inhibitors have been described. We have developed an automated, whole-cell, high-throughput assay for the unbiased discovery of inhibitors of the non-homologous end joining (NHEJ) and homologous recombination HR) repair pathways, the two primary pathways that ameliorate DNA double-strand breaks. Our assay enables indirect measurement of DNA repair activity by monitoring the kinetics of γH2AX and 53BP1 foci resolution after treatment with ionizing radiation (IR) and candidate DNA damage response (DDR) inhibitor. We also have developed a unique platform of secondary assays to rank-order the potency of lead compounds and gain insights into their mechanism of action. In addition to hit validation and rigorous exclusion of false-positive hits, our follow-up platform includes orthogonal assays for measuring mutagenic NHEJ and HR repair activity, as well as high-throughput assays for quantitative analysis of cell viability and cell cycle. Notably, both our primary and secondary assays are fully-automated and may be applied to screen large libraries of compounds.

We validated our high-throughput assay using known DDR and DNA damage checkpoint inhibitors, and applied this screen toward the evaluation of 2,366 structurally-diverse small molecules with known bioactivity. Vorinostat and quercetin, compounds known to possess DDR inhibitory activity, were identified in the screen, supporting the sensitivity of our approach. Notably, the screen also lead to the identification of cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, as potent inhibitors of DNA double-strand break repair. Many studies have noted the anticancer properties of the cardiac glycosides, but the basis of this activity has remained a "black box". Our data suggest an explanation for this long-standing unsolved problem, and argue that this activity should be taken into consideration when evaluating other clinical applications of these agents. Because these compounds are already approved for use in humans, we believe they are excellent candidates for repurposing as chemo and radiosensitizers.

#2175

Anemarrhena rhizome and Schisandra chinensis inhibit viability and promote apoptosis in pancreatic cancer cell lines.

Catherine MarElia, Brant Burkhardt. _University of South Florida, Tampa, FL_.

Pancreatic cancer has one of the highest mortality rates of any cancer, with just a 6% 5-year survival rate. The current standard chemotherapy is gemcitabine, alone or in combination with 5-fluorouracil. However, the prognosis still remains poor due to apoptotic resistance, presenting a need for an adjuvant therapy option that will induce apoptosis within pancreatic tumor cells. Emerging evidence has supported the use of herbal extracts as alternative or adjuvant treatments for pancreatic cancer. In this study, we evaluated the effects on pancreatic cancer cells of a mix of 8 herbal extracts, selected for their high clinical usage and documented ability to alleviate pancreas-specific ailments such as type 2 diabetes, yet not examined for application in pancreatic cancer.

The objective of this study was to determine the inhibitory effects of Astragalus membranaceus, Pseudostellaria heterophylla, Gypsum fibrosum, Anemarrhena rhizome, Solomon's seal rhizome, Rehmannia, Schisandra chinensis, and Cornus fruit extracts on pancreatic cancer cell viability in combination (referred to as 8-mix) and individually on pancreatic cancer cells. We also examined extracts previously demonstrated to inhibit pancreatic cancer cells, Cordyceps chinensis and American ginseng, as a control.

The viability of pancreatic adenocarcinoma cell lines (BxPC-3 and Panc-1) and the human pancreatic islet: Panc-1 hybrid cell line (1.1B4) was evaluated in response to the 8-mix, C. chinensis, and A. membranaceus via MTT assay. To further characterize which herbs in the 8-mix were responsible for the reduction in cell viability, BxPC-3 and Panc-1 cells were assessed upon treatment with each individual herbal extract via MTT assay following a 12 hour exposure. To examine the impact of these herbs on a non-cancerous cell line, the T80 ovarian epithelial cell line was also evaluated. We measured the apoptotic activity of the 8-mix and two most potent individual herbs within the mix using a commercially available caspase 3/7 assay on BxPC-3 cells.

Initial testing showed significant inhibitory effects in all pancreatic cell lines induced by the 8-mix in a dose-dependent manner. A. rhizome and S. chinensis were the most potent inhibitors of pancreatic cancer cell viability among the individual extracts. A. rhizome had the greatest apoptotic activity in the BxPC-3 cells compared to S. chinensis, the 8-mix, and vehicle-treated controls. Within the non-cancerous ovarian T80 cell line, A. rhizome and S. chinensis significantly inhibited cell viability.

Taken together, our data suggests that A. rhizome induces apoptosis in pancreatic cancer cells and both A. rhizome and S. chinensis significantly inhibit viability in numerous pancreatic cancer cell lines. These herbs may therefore have potential as an adjuvant therapy in combination with traditional chemotherapeutic agents for the treatment of advanced pancreatic cancer.

#2176

Targeting eEF-2Kinase by thymoquinone in triple negative breast cancer.

Nashwa N. Kabil, Recep Bayraktar, Nermin Kahraman, Bulent Ozpolat. _MD Anderson Cancer Center, Houston, TX_.

Triple-negative breast cancers (TNBCs) constitute a heterogeneous subtype of breast cancers that present a significant challenge for the treatment and management, and have a poor clinical outcome. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical calcium/calmodulin (Ca2++/CaM)-dependent Ser/Thr-kinase that regulates peptide chain elongation by phosphorylating its substrate elongation factor 2 (eEF2), resulting in decreased protein translation. Recently, we have reported that eEF-2K expression is significantly upregulated in TNBC cell lines and is associated with poor survival and worse prognosis, indicating that eEF-2K is a potential therapeutic target in TNBC. Moreover, we demonstrated that eEF-2K promotes TNBC cell proliferation, invasion/migration, and tumorigenesis. Its inhibition by systemically administered liposomal siRNA resulted in marked inhibition of tumor growth and enhancement in the efficacy of chemotherapy in orthotopic TNBC mouse models (Tekedereli 2012). Currently, there are no reported inhibitors of eEF-2K. Natural dietary poly phenolic compounds have been shown to inhibit various cancers by regulating various kinases and molecular targets, with both in-vitro and in-vivo activity. Thymoquinone (TQ) is an active ingredient isolated from Nigella sativa and has been investigated for its antioxidant, anti-inflammatory, and anticancer activities in several in-vitro and in-vivo models. However, the mechanism by which TQ mediates its effects not well understood. Here, we investigated if TQ inhibits eEF-2K in TNBC cells. We demonstrated, for the first time, that treatment with TQ decreases the expression of eEF-2K in a dose dependent manner as wells as its downstream targets, resulting in decreased proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, eEF-2K knockdown by siRNA recapitulates the effects of TQ, including cell proliferation, migration/invasion, and correlated events. Additionally, TQ treatment inhibits Src activity and other molecular targets, and induces significant apoptosis in TNBC cells in a caspase independent manner. Currently, we are investigating the downstream effects of TQ by RPPA analysis and its in-vivo efficacy in orthotopic xenograft mouse models of TNBC. Overall, our studies suggest that TQ could present a novel therapeutic strategy in targeting eEF-2K and inhibition of TNBCs.

#2177

Rutin enhances the antiproliferative effect of 5-FU and oxaliplatin in colon cancer cells.

Farnoud Nasiri,1 Gorkem Kismali,1 Merve Alpay,2 Funda Kosova,3 Dilek Ulker Cakir,4 Tevhide Sel1. 1 _Ankara University, Ankara, Turkey;_ 2 _Duzce University, Duzce, Turkey;_ 3 _Celal Bayar University, Manisa, Turkey;_ 4 _Canakkale Onsekiz Mart University, Canakkale, Turkey_.

Background: Rutin is a strong antioxidant molecule and it has advantageous over other flavonoids due to it is a nontoxic and nonoxidizable molecule. The concept of dual therapy of anti-cancer drugs with natural compounds has become a very promising approach in new strategy to treatment. The aim of this study was to evaluate the antiproliferative and apoptotic effects of rutin flavonoids and its efficacy in enhancing the anticancer effects of 5-FU (5-fluorouracil) and oxaliplatin against colon cancer cells.

Material and Methods: Caco-2 human colon cancer cells were treated with rutin and/or anticancer drugs (5-FU and oxaliplatin), cell viability and apoptotic parameters were examined. Cell viability was determined by MTT assay. Apoptotic markers (cleaved caspase 3, cleaved PARP and phosphor-Bad) were determined using specific enzyme-linked immunosorbent assay kits. Caspase-8 and caspase-9 were analyzed by colorimetric activity assay kit. DNA fragmentation was analyzed by agarose gel electrophoresis.

Results: The exposure Caco-2 human colon cancer cells to rutin, 5-FU and oxaliplatin resulted growth inhibition in a dose- and time-dependent manner. Cell viability assay shows that rutin inhibiting growth of Caco-2 cells at high concentrations (over 1000 µM). Combination with rutin markedly enhanced 5-FU and oxaliplatin growth inhibiting effects on Caco-2 cells. Results suggested that rutin had significant anticancer abilities; such rutin were capable of causing multifold decreases in the half maximal inhibitory concentration IC50 value of 5-FU and oxaliplatin. This flavonoid increased the levels of proteins associated with apoptotic cell death (phospho-Bad, cleaved caspase 3 and cleaved PARP) alone and combination. The activities of caspase 8 and caspase 9 were stimulated by the combination treatment of rutin and oxaliplatin then alone. DNA fragmentation was observed only on combination treatment.

Conclusions: Combined treatment with rutin and anticancer drugs (5-FU and/or oxaliplatin) is more effective than the individual treatments of drugs at inhibiting growth of Caco-2 cells. The use of lower 5-FU and oxaliplatin doses, with similar effects, could be also useful to reduce possible adverse effects of these drugs. However, further studies at molecular level are required to elucidate chemopreventive and chemotherapeutic effects of rutin on colon cancer.

#2178

A novel injectable formulation of quercetin reliant on the ability of the drug to complex copper.

Kent T. Chen, Malathi Anantha, Ada W.Y. Leung, Mohamed Wehbe, Brent Sutherland, Marcel B. Bally. _BC Cancer Research Centre, Vancouver, British Columbia, Canada_.

Low levels of fruit and vegetable consumptions have been linked to increased risk of cancer. Quercetin is one of the most ubiquitous flavanoids found in fruits and vegetables and there is increasing evidence that points to the anti-cancer potential of quercetin. This flavanoid acts as a promoter of apoptosis by modulating apoptosis through Bcl-2, Akt-1, and/or NF-κB. In addition, quercetin also demonstrated promise when used in combination with approved chemotherapeutic agents such as cisplatin and irinotecan. However, quercetin has remained largely clinically irrelevant despite all the potential anti-cancer benefits noted in in vitro assays. This is due in part to the low bioavailability of the compound and its poor aqueous solubility (solubility <100µg/mL). In order to realize the full therapeutic potential of quercetin, we have developed an injectable quercetin formulation prepared using a method that relies on its ability to bind copper. Quercetin was added to pre-formed copper-containing liposomes (,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio)); a strategy that involved addition of quercetin powder directly to the liposomes at 60⁰C. In vivo pharmacokinetics of the liposomal quercetin formulation was assessed by injecting the product intravenously at a dose of 50mg/kg into RAG2m mice. Plasma samples were taken at 0.5, 1, 4, 8, and 24 hours post-injection. Quercetin concentration in plasma was analyzed via HPLC and lipid concentrations were determined using liquid scintillation counting. The plasma copper concentration was determined using atomic absorbance spectrometry. All data were plotted in Prism 6.0 (mean ± SEM). Quercetin encapsulation into copper liposomes resulted in a final drug-to-lipid ratio (mol:mol) of 0.2. The encapsulation process was copper-dependent. The formulation is stable at 4⁰C for at least three weeks, maintaining a particle size of approximately 135nm and >98% encapsulation efficiency. Compared to previous data on free quercetin, the liposomal formulation of quercetin shows a markedly improved pharmacokinetic profile. The formulation demonstrated a 10-fold increase in elimination half life and a 7-fold increase in AUC when compared to previously published studies with free quercetin. A novel liposomal formulation of quercetin has been generated and the resulting formulation resolved the solubility issues faced when considering the use of quercetin for studies attempting to define its therapeutic activity in various cancer models.

#2179

Macrolide toxin Mycalolide B is a potent inhibitor of HER2 cancer cell invasion and is the basis of actin targeted therapy for metastatic cancers.

Rodette N. Williams, Andrew W. Craig, John S. Allingham. _Queen's University, Kingston, Ontario, Canada_.

Breast and ovarian cancers are among the leading causes of cancer related deaths in women worldwide. Human epidermal growth factor receptor 2 (HER2) is highly expressed in a subset of these cancers that show high rates of progression to metastatic disease. Metastasis is driven by filamentous actin (F-actin) based protrusions that penetrate and degrade extracellular matrix (ECM) to facilitate tumor invasion. In this study, we tested an actin-depolymerizing macrolide toxin Mycalolide B, as a potential suppressor of highly metastatic HER2+ breast and ovarian cancer cell models. Dose responses with Mycalolide B in SKBR3 breast cancer and SKOV3 ovarian cancer cells resulted in similar cytotoxicity (~65 nM IC50) profiles. In addition, Mycalolide B doses well below the IC50 showed potent suppression of leading edge protrusions, and motility in SKBR3 and SKOV3 cancer cells. This also correlated with reduced ECM degradation and Transwell invasion at low nanomolar doses of Mycalolide B. In contrast, other F-actin based processes such as endocytosis of HER2 and EGFR, were less sensitive to Mycalolide B treatment. However, Mycalolide B treatment did skew the size of endocytic vesicles, which may reflect defects in F-actin based vesicle motility or maturation. Given that HER2 cancers have been effectively targeted by Trastuzumab and Trastuzumab-based antibody-drug conjugates, we are currently testing the compatibility of combined treatments with Mycalolide B and Trastuzumab, for their effects on F-actin based invasion of HER2+ cancers. This strategy may yield a novel class of antibody-drug conjugate, and lead to new HER2 targeted therapies that suppress tumor metastasis by disrupting early steps that are dependent on efficient F-actin polymerization.

#2180

Drug-like inhibitors of SGK1: Discovery and optimization of low molecular weight fragment leads.

James Zapf,1 Todd Meyer,2 Warren Wade,1 Laura Lingardo,1 Ayse Batova,1 Gordon Alton,1 Peter Pallai2. 1 _Visionary Pharmaceuticals, San Diego, CA;_ 2 _BioBlocks, San Diego, CA_.

Triple negative (ER, PR and HER2/neu negative) breast cancers (TNBC) constitute 15-24% of breast cancers, but account for a disproportionate share of mortality. TNBC tumors are more aggressive, lack a targeted therapy, and are prone to relapse and metastasis after cytotoxic drug treatments, the only drug therapy currently available. Serum and glucocorticoid-regulated kinase 1 (SGK1) promotes the growth, invasiveness and chemo-resistance of cancer cells and is overexpressed in 48% of breast cancers, yet is not detected in normal breast tissue. In particular, SGK1 is overexpressed in TNBC cells and knockdown of SGK1 blocks the proliferation and invasiveness of TNBC cells. Thus, SGK1 is an attractive targeted therapy for TNBC. In the SGK1 inhibitors that have been reported previously, poor physicochemical properties have prevented these inhibitors from being active in vivo and advancing to the clinic. To discover drug-like inhibitors of SGK1, we used our technology platform (Leap-To-Lead™) and a fragment-based screening approach to find SGK1 inhibitor scaffolds. Low molecular weight inhibitors like ours are more readily optimized for potency while retaining drug-like properties. From this screen, we identified 11 scaffolds as novel inhibitors of SGK1. Four scaffold series had clear structure activity relationships (SAR). To illustrate the potential of these scaffolds, we carried out a limited synthetic expansion around one scaffold and improved potency more than 600-fold (SGK1 IC50 = 1 µM; LE = 0.4). Several compounds dose-dependently inhibited the proliferation of a TNBC cell line (MDA-MB-231) and inhibited the phosphorylation of the biomarker protein N-Myc downstream regulated 1 (NDRG-1). Unlike previously reported SGK1 inhibitors, our fragment lead compounds show excellent cellular penetration in Caco-2 assays.

In summary, a fragment-based screening approach was used to identify four novel scaffolds with clear SAR trends. A single scaffold series was further optimized and shows anti-proliferative activity and biomarker modulation in a TNBC cell line. These SGK1 inhibitor series represent excellent starting points for further SAR studies and development of a novel preclinical candidate.

#2181

Chemotherapeutic, chemomodulatory and vascular protective effects of naturally occurring hydroxyphenylalkanes and diarylheptanoids.

Mohammad A. Baghdadi,1 Eman A. Al-Ghamdi,2 Abdulmohsin J. Alamoudi,2 Fahad A. Al-Abbasi,2 Hany M. El-Bassossy,2 Ali M. El-Halawany,3 Ahmed M. Al-Abd4. 1 _King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia;_ 2 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 3 _Cairo University, Cairo, Egypt;_ 4 _National Research Center, Giza, Egypt_.

Hydroxyphenylalkanes and diarylheptanoids possess potential therapeutic values in different patho-physiological conditions such as inflammation, oxidative stress, cardiovascular disorders and malignancy. Doxorubicin is widely used chemotherapeutic agent against several types of neoplastic disorders. In the current study, naturally isolated hydroxyphenylalkane and diarylheptanoid compounds were investigated for their potential chemo-modulatory effects on the top of their potential vascular protective role with doxorubicin. Shogaol and 4-methoxygingerol showed considerable cytotoxic effects against HCT116, HeLa, HepG2 and MCF7 cells with IC50 ranging from 3.1 to 18.7 µM and from 9.1 to 19.4 µM, respectively. Using DPPH free radical scavenging assay, diarylheptanoids were stronger antioxidant than hydroxyphenylalkanes with EC50 ranging from 0.5 to 5.9 µM and from 4.6 to 9.5 µM, respectively. In addition, all tested diarylheptanoids significantly ameliorated CCl4 induced disturbed intracellular GSH/GSSG balance. Gingerol moderately enhanced the cytotoxic profile of doxorubicin against HepG2 and Huh7 cells decreasing their IC50's from 0.52 to 0.37 µM and from 50 to 30 nM, respectively. Combination index of gingerol with doxorubicin (combination at equitoxic ratio of 1/100) was indicative of additive interaction (CI-value were 1.1 and 1.0, respectively). To further investigate the interactive characteristics between doxorubicin and gingerol, cell cycle distribution of HepG2 and Huh7 cells was studied using DNA cytometry after exposure to their single and equitoxic combination. Doxorubicin induced cell accumulation at S-phase and G2/M-phase while, gingerol combination with doxorubicin significantly induced cell cycle arrest at G2/M-phase. To study the potential vascular protective effect of gingerol against doxorubicin induced vascular toxicity, doxorubicin (10 µM for 1h) was incubated with or without different concentrations of 6-gingerol (3, 10 and 30 µM for 1h) on isolated aortic rings and their response to phenylephrine (PE) and acetylcholine (ACh) was examined. Co-incubation of 6-gingerol (30 µM) completely blocked the exaggerated vasoconstriction and impaired vascular relaxation induced by doxorubicin

#2182

Flavokawain A and B from kava extract exhibits low toxicity and up-regulates tumor suppressor miRNAs in human osteosarcoma cells.

Wendong Zhang,1 Jonathan Morris,1 Sajida Piperdi,1 Yi Guo,2 Tao Ji,2 Rui Yang,1 Michael Roth,1 Jonathan Gill,1 David S. Geller,1 Richard Gorlick,1 Kevin Jones,3 Xiaolin Zi,4 Bang H. Hoang1. 1 _Montefiore Medical Center, New York, NY;_ 2 _People's Hospital Peking University, Beijing, China;_ 3 _University of Utah Medical Center, Salt Lake City, UT;_ 4 _University of California - Irvine, Irvine, CA_.

Objective: Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Flavokawain A and B (FKA and FKB) from kava extract have been reported to have significant anti-tumoral effects on human cancer cells. The consumption of kava-containing beverage has been associated with a low cancer incidence. The objective of this study is to investigate the anti-tumoral effects of flavokawains in OS. In a previous report, mice treated with high-dose FKA did not demonstrate any significant major organ toxicity. MicroRNAs (miRNA), endogenous, non-coding RNA, are associated with cell-cycle regulation, differentiation, proliferation, apoptosis, and migration. There remains a paucity of literature on the effects of flavokawains on the expression of miRNA in OS. As such, we hypothesize that flavokawains will up-regulate tumor-suppressive miRNA in OS cell lines.

Methods: Human OS cell lines from American Type Culture Collection (ATCC), patient-derived OS cell lines, human mesenchymal stem cells, human intestinal epithelial cells, and mouse bone marrow cells were cultured. Cell lines were divided into control and treatment groups with increasing doses of FKA or FKB. Cell viability was determined by MTT proliferation assays and cytotoxicity to bone marrow cells was examined by colony formation. Effects of flavokawain on intestinal cells were examined by fluorescent cytotoxicity and flow cytometry. Apoptotic assays were performed following flavokawain treatments. The expression of miRNA after FKA and FKB treatment was examined by qRT-PCR. SCID mice were injected with OS 143B cells in the tibia and treated with either vehicle control or FKA (200 mg/kg) by oral gavage.

Results: By flow cytometry, FKB increases apoptosis of OS 143B cells while sparing intestinal FHs 74 intestinal cells. Mouse bone marrow cells treated with FKB showed no significant difference in colony forming units compared to control cells. Exposure of OS cell lines to FKA or FKB resulted in a loss of cell viability in a dose-dependent manner. In all cell lines tested a dose-dependent expression of miRNA was observed after exposure to FKA or FKB. Flavokawain B induced miR-26a while FKA induced miR-26a, miR-34, and miR-203 expression in OS cell lines. Mice bearing 143B xenograft tumors treated with FKA did not exhibit weight loss compared to control animals. Animals treated with FKA sustained less lung metastasis than animals treated with vehicle control.

Conclusions: Taken together, the evidence suggests flavokawains up-regulate tumor suppressive miRNAs in OS cells. In vivo experiments suggest that FKA exerts anti-metastatic effects in OS. Given their low toxicity profile, flavokawains may be useful as a chemopreventive strategy for OS and thus warrant further investigations.

#2183

Baicalein and meformin decrease small cell lung cancer growth by inhibiting the mTOR pathway in itro.

Emily F. Marcinkowski, Dan Raz, Bing Shen, Quanhua Xing, Jin Yan, Wei Wen, Ernest Han, John Yim. _City of Hope, Duarte, CA_.

Background: Metformin use has been associated with decreased lung cancer risk in several observational studies and is currently in clinical trial in conjunction with standard chemotherapeutics. One possible antitumor mechanism is the negative regulation of the mTOR pathway. We previously reported that the natural product Baicalein has antitumor effects through targeting mTOR pathway. We hypothesized that Baicalein to have antitumor effects in small cell lung cancer. Methods: H1417 small cell lung cancer cells were cultured and treated with increasing doses of Baicalein or Metformin. The cells were harvested at 24 hours and subjected to Western blotting staining for the downstream products of the mTOR pathway. Cell proliferation was determined by MTT assay at 24, 48, and 72 hours. MTT conversion to formazan dye correlates with the number of living cells. Results: We found a dose dependent decrease in the downstream mTOR1 targets pS6K1 and pS6 using both Baicalein and Metformin. There was also an increase in the expression of the mTOR inhibitors DDIT4 and IRF-1. Using the MTT assay, we were able to demonstrate a marked dose dependent decrease in cell proliferation that was sustained over 72 hours from treatment. Interestingly, Baicalein has a markedly higher potency working at micromolar doses verses Metformin which required millimolar doses. Conclusions: Both Baicalein and Metformin effectively decrease cell proliferation in small cell lung cancer cells in vitro. We have shown these drugs to target the mTOR pathway. Cell proliferation is inhibited at a markedly smaller dose by Baicalein compared with Metformin. Baicalein may be useful in cancer chemotherapy and chemoprevention.

#2184

The effects of specific heterocyclic compounds on angiogenesis and apoptosis factors in cancer cells.

Funda Kosova,1 Özlem Temiz-Arpacı,2 İbrahim TUĞLU,3 Ercüment ÖLMEZ,4 Feyzan ÖZDAL KURT,5 Gorkem KISMALI,6 Zeki Ari,7 Mustafa ARISOY8. 1 _Medical Biochemistry of Celal Bayar University, Manisa, Turkey;_ 2 _Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Tandogan, Ankara, Turkey;_ 3 _Department of Histology and Embryology of Celal Bayar University Medical Faculty, Manisa, Turkey;_ 4 _Department of Pharmacology of Celal Bayar University Medical Faculty, Manisa, Turkey;_ 5 _Celal Bayar University Science and Art Faculty, Manisa, Turkey;_ 6 _Department of Biochemistry of Ankara University Veterinary Faculty, Manisa, Turkey;_ 7 _Department of Biochemistry of Celal Bayar University Medical Faculty, Manisa, Turkey;_ 8 _Drogsan Pharmaceuticals, R &D Department, Ankara,, Turkey_.

Introduction: Benzoxazole have received considerable attention during last few decades as they are endowed with variety of biological activities and have wide range of therapeutic properties. The present study aimed to evaluate cytotoxic and antiapoptotic activities of new synthesize benzoxazole derivative in breast cancer cell lines.

Material and Method: In this study, the breast cancer cell lines MDA-MB and MCF-7 were used. Cytotoxic activities of novel benzoxazole-derived compound were analyzed with MTT assay. Additionally, the level of its effects on NF-κB and apoptosis-related proteins were examined by the western blot. Immunohistochemical stainings were done for VEGF, eNOS proteins and Tunnel assay was performed to show DNA damage.

Results: The structure of the compound synthesized in our study 5-amino-2-(p-bromophenyl)-benzoxazole was proved by elemental analysis, IR, 1H NMR and mass

spectroscopy analysis methods. Novel benzoxazole analogue detected cytotoxic on breast cancer cells dose dependent manner, MCF-7 cells were found more sensitive by MTT assay. When the protein was examined in immunohistochemistry with regard to VEGF, eNOS and TUNEL, it was observed that it caused a reduction in VEGF and an increase in eNOS and TUNEL. We observed that it wasn't between groups in Apaf-1 and BCL-2 levels, but down regulation was observed in caspase 3 and Nfkβ levels compared to control group. Novel benzoxazole compound increased Cytochrome C level in treated cells compared to the control group in MDA-MB cells but not in the MCF-7 cells.

Conclusion: In summary, It is felt that this newly synthesized heterocyclic compound increases apoptozis by reducing the activation of Nfkβ, and in this way has shown an effect of inhibiting tumor growth in cancer treatment.

Key Words: Heterocyclic Compounds, NF-κB, APAF-1, Cytochrome C, Caspase-3, bcl-2

#2185

Sann-Joong-Kuey-Jian-Tang can inhibit human breast cancer cells through dural- blocking both the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways.

CHIN CHENG SU. _CHANGHUA CHRISTIAN HOSPITAL, CHANGHUA, Taiwan_.

Sann-Joong-Kuey-Jian-Tang (SJKJT) consists of 17 species of medicinal herbs, is a traditional Chinese medicine prescription, and has been used as complementary medication for a number of types of solid cancer in Taiwan. Transmembrane tyrosine kinase has been strongly implicated in the growth, survival, and metastasis of a wide variety of human tumors. The phosphoinositide-3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) and RAS/RAF/MEK/ERK pathways are two of the most frequently dysregulated kinase cascades in human cancer were well documented. Both pathways represent important signal transduction mechanisms that facilitate the proliferation and survival of cancers driven by growth factor receptors, such as vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor-I receptor (IGF-IR), human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR).The individual downstream components of these signaling cascades either through somatic mutation or epigenetic modification, are also known to be frequently altered in cancer, thus contributing to tumorigenesis and resistance to anticancer therapies. Targeting both the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR Pathways for suppressing Inhibitor Resistant Cells is necessary. In the present study, the human breast cancer BT-20 and MCF-7 cells were treated with SJKJT in vitro. The cytotoxicity of SJKJT were evaluated by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SJKJT on the protein expressions of VEGFR, IGF-IR, PI3K, AKT, mTOR, Ras, Raf, MEK and ERK and β-actin in the BT-20 and MCF-7 cells were examined by western blot analysis. The results showed that SJKJT can induce the proliferation inhibition with time and dose dependent. As a potential mechanism, it was noted that SJKJT treatment significantly inhibited the activity of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways, which played a protective role against the cytotoxicity. In addition, it was demonstrated that SJKJT treatment inhibited the protein expressions of VEGFR, EGFR and IGF-IR significantly. These results suggest that one of the molecular mechanisms for SJKJT to inhibit breast cancer BT-20 and MCF-7 cells maybe through inhibiting the protein expressions of VEGFR, EGFR, IGF-IR and both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways. The use of traditional chinese medicine prescription SJKJT may become a feasible novel therapy option. Further studies are warranted to fully elucidate its mechanisms of action.

#2186

Incorporation of natural compounds into biomaterials to prevent breast cancer recurrence.

Lauren Jordan, Bailey-Jean Walker, Christopher Moody, Brian W. Booth. _Clemson University, Clemson, SC_.

Numerous studies have demonstrated that naturally occurring phytochemicals such as Tannic acid (TA) possess anti-cancer properties. Apoptotic activity is increased in breast cancer and prostate cancer cells in response to exposure to tannin extracts. Collagen type I is a biomaterial routinely used for cosmetic and reconstructive surgeries, including breast reconstruction following breast cancer mandated surgeries. TA functions as a collagen crosslinking agent through both hydrogen bonding and hydrophobic effects. Thus, if TA-crosslinked collagen type I is used for breast reconstruction procedures, the crosslinked collagen will be remodeled by growing breast epithelial cells and adipocytes where the incorporated TA will be released killing nearby residual cancer cells preventing recurrence. When adipocytes are grown on TA-crosslinked collagen beads the measureable released TA induces caspase-mediated apoptosis in ER+ and HER2+ breast cancer cells but has no effect on triple negative breast cancer cells and reduced apoptotic-inducing activity in normal breast epithelial cells. We are developing an injectable matrix comprised of TA-crosslinked collagen beads and adipocytes to serve as a tissue regeneration platform in patients post-lumpectomy. Variables such as collagen temperature, syringe pump rate, and drop distance have been studied to determine ideal method for producing beads of the desired size and shape. These promising results will lead to the construction of a matrix designed to not only promote tissue regeneration but also simultaneously help to prevent tumor recurrence. This matrix provides protection in breast cancer patients and holds promise for patients suffering from other soft tissue cancers.

#2187

Garcinone inhibits nasopharyngeal carcinoma cell growth and arrests cell cycle via modulating the expression of ATR/Stat3/4E-BP1.

Yudui Xia, Xia Liu, Chunllin Zou, Hongwei Guo, Yeguo Yang, Yong Lei, Jian Zhang, Yi Lu. _Key Laboratory of Longevity and Ageing-related Diseases, Ministry of Education, China & Center for Translational Medicine, Guangxi Medical Univ., Nanning, China_.

Nasopharyngeal carcinoma (NPC) is one of the common head and neck malignancies. Radiotherapy and chemotherapy are the standard treatments for NPC. Garcinone, a natural compound isolated from Garcinia oblongifolia Champ., is a xanthone derivative and has been shown to have potential cytotoxic effects on certain cancer types. However, there have been limited studies performed on its effects on NPC cells and the mechanisms of these effects remain unknown. In this study, we firstly found that garcinone significantly inhibited cell viability of human NPC cell lines CNE1, CNE2, HK1, and HONE1. This inhibition was in a time- and dose-dependent manner. Then, flow cytometry analysis was performed on cell cycle analysis. We observed that garcinone arrested cell cycle at S phase. With 10μM of high-dose garcinone treatment, the cells exhibited necrotic morphology changes including cell swelling, rough endoplasmic reticulum (ER) degranulation, ER dilatation, mitochondria swelling, and vacuolar degeneration. Finally, we found that garcinone stimulated the expression levels of ATR and 4E-BP1, while efficiently inhibited the expression levels of Cyclin B1, Cyclin D1, Cyclin E2, cdc2, CDK7, and Stat3. Collectively, the ability of garcinone to inhibit NPC in vitro suggested that garcinone might be a novel agent for the management of NPC. Supported by NSFC Key Project 81130046; NSFC Project 81171993 and NSFC81272415; Guangxi Key Projects 2013GXNSFEA053004 and 2012GXNSFCB053004; Guangxi Ministry of Education 201202ZD022 and 201201ZD004.

#2188

Isolation and anticancer properties of some naturally occurring sesquiterpene lactones from Pulicaria undulate.

Mohammed M. Arab,1 Fahad A. Al-Abbasi,1 Mohamed-Elamir F. Hegazy,2 Ali M. El-Halawany,3 Ahmed M. Al-Abd2. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _National Research Center, Cairo, Egypt;_ 3 _Cairo University, Cairo, Egypt_.

Sesquiterpene lactones are natural compounds abundant in several plant families with a variety of biological activities such as antitumor effects. Camptothecin is famous topoisomerase interactive agent with anticancer properties against several types of malignancies. The purpose of this study is to investigate the potential chemotherapeutic effects of some naturally occurring sesquiterpene lactones isolated from Pulicaria undulate. Compounds with promising anticancer profile has been further investigated for potential chemomodulatory effects to camptothecin in different tumor cell types. Cytotoxicity of the isolated terpenoids were assessed against human breast adenocarcinoma (MCF7), human colorectal cancer (HCT116 and LS174T) and human hepatocellular carcinoma (HepG2) cells using sulpharhodamine-B assay after cell exposure for 72 h. Cytotoxic parameters (IC50 and R-fraction) were calculated from cell viability after fitting using Emax model. Two sesquiterpene lactones, 2α-hydroxy alantolactone and tomentosin, showed considerable cytotoxic profile against cell lines under investigation with IC50's ranging from 5 to 22 µM and from 14 to 39 µM, respectively. Treatment with camptothecin alone showed significant cytotoxicity with IC50's range from 0.13 to 1.5 µM in the same panel of cell lines. Equitoxic combination of tomentosin with camptothecin (combination ratio was 1:100) resulted in 60% reduction for the IC50 of camptothecin alone in HCT116 cells. On the other hand, equitoxic combination for hydroxy alantolactone with camptothecin did not significantly enhance the cytotoxicity of camptothecin against HCT116 cells. To explain the interactive characteristics of hydroxy alantolactone and tomentosin with camptothecin, annexin-V/PI staining coupled with flow cytometry analysis for apoptosis and necrosis was assessed after treatment with camptothecin alone or in combination with hydroxy alantolactone or tomentosin. Again, tomentosin combination with camptothecin significantly increased apoptotic as well as necrotic cell populations compared to camptothecin treatment alone by 2-3 folds. On the other hand, combination of camptothecin with hydroxy alantolactone significantly increased apoptotic cell population with reciprocal decrease in necrotic cell population. In conclusion, hydroxy alantolactone and tomentosin might be promising anticancer agents. However, further molecular investigation is required to understand the nature of interaction between the isolated sesquiterpene lactones and cornerstone chemotherapeutics such as camptothecin.

#2190

Isolated cannabidiol from Cannabis sativa plant extracts inhibit growth and induce apoptosis in cervical cancer cells.

Lesetja Raymond Motadi, sindiswa lukhelo. _Univ. of the Witwatersrand, Johannesburg, South Africa_.

Cervical cancer remains a global health related issue among females of Sub-Saharan Africa, with over half a million new cases reported each year. Different therapeutic regimens have been suggested in various regions of Africa, however, over a quarter of a million women die of cervical cancer, annually. Therefore, it is important to search for new drugs through effective screening of medicinal plant extracts to identify lead anti-cervical cancer drugs. The aim of this study was to evaluate for the anti-growth effects of Cannabis sativa extracts and its isolate, cannabidiol on cervical cancer cell lines HeLa, SiHa, and ME-180. To determine for the presence of important constituents and evaluate for the anti-growth effects, phytochemical screening, MTT assay, cell growth analysis, flow cytometry, morphology analysis, Western blot, caspase 3/7 assay, and ATP measurement assay were conducted were conducted. Results obtained indicate that both plant extracts induced cell death at an IC50 of 50 - 100µg/ml and the Inhibition of cell growth was cell line dependent. Flow cytometry confirmed that, with or without cell cycle arrest, the type of induced cell death was apoptosis. Cannabis sativa extracts led to the up-regulation of apoptosis proteins (p53, Bax, caspase-3, and caspase-9) and the down regulation of anti-apoptosis proteins (Bcl-2 and RBBP6), signalling the execution of apoptosis. Apoptosis induction was further confirmed by morphological changes, an increase in Caspase 3/7 and a decrease in the ATP levels. In conclusion, this data implies Cannabis sativa crude extracts has the potential to inhibit growth and induce apoptosis in cervical cancer cell lines, which may be due to the presence of cannabidiol.

### Nanotechnology and Drug Delivery

#2191

Imaging the kinetics of targeted nanoparticles to pancreatic cancer.

Jonathan Moreno, Qin qin Fei, Maria P. Lambros. _Western Univ. of Health Sciences, Pomona, CA_.

Purpose: The purpose of this work is to image the kinetics of targeted lipid nanoparticles to an SCL transporter of pancreatic cancer using a near infrared (NIR) fluorescent dye DyLight® 747 and a mouse orthotopic model of fluorescent pancreatic cancer.

Methods: Initially and before the in vivo experiments, the uptake of nanoparticles by human pancreatic cancer cells was evaluated in vitro using fluorescent microscopy and flow cytometry. A quantity of 1.5 x105 PANC-1, MIA PaCa, or BxPC-3 cells/ml was placed in each well of a six-well plate and incubated for 5 hours with different formulations of fluorescent lipid targeted and non-targeted nanoparticles encapsulation daunorubicin which has natural fluorescence. After incubation, the growth media were removed and the cells were washed with PBS. The uptake of the fluorescent lipid nanoparticles by the cancer cells was evaluated an EVOS® microscope. All the in vitro experiments were done in triplicate. For the in vivo targeting studies, nude mice (Balb/c Ola Hsd-Fox1 nu) were implanted orthotopically (in the tail of the pancreas) with 3 x106 PANC-1 cells expressing GFP. The injection volume for the implantation was 50 microliters and contained 3 x106 PANC-1-GFP cells mixed with serum free media and HC Matrigel (1:1). One month after the orthotopic implantation, the implanted pancreatic tumor was visualized in live, anesthetized animals using UVP Scientia (UVP, Upland, CA). The animals were separated into three groups (three animals in each group) and injected via the tail vein as follows: Group A, was injected with only the fluorescent dye, Dylight 747; group B was injected with fluorescent non-targeted nanoparticles; and group C was injected with fluorescent targeted nanoparticles.

Results: Both the flow cytometry and fluorescence microscopy data show a significantly higher uptake of the targeted lipid nanoparticles compared to the non-targeted ones. The imaging of the fluorescent lipid nanoparticles in live mice shows that both targeted and nontargeted nanoparticles reach the pancreatic tumor. The nontargeted nanoparticles reside less than 48 hours on the tumor, whereas the targeted nanoparticles can reside for 96 hours on the tumor. The non-encapsulated fluorescent dye did not reach the tumor and was excreted in the urine.

Conclusion: The results show that Dylight® 747 can be used to image the kinetics of a drug delivery system in a live animal. Furthermore while both lipid nanoparticles reach the tumor, the targeted lipid nanoparticles reside significantly longer on the tumor and this may translate to a better therapeutic outcome.

#2192

Fusogenic targeted liposomes as next-generation nanomedicine for prostate cancer.

Jihane M. Mriouah,1 Rae Lynn Nesbitt,2 Deborah Sosnowski,1 Desmond Pink,1 Roy Duncan,3 Andries Ziljstra,4 John D. Lewis1. 1 _University of Alberta, Edmonton, Alberta, Canada;_ 2 _Entos Pharmaceutical, Edmonton, Alberta, Canada;_ 3 _University of Dalhousie, Halifax, Nova Scotia, Canada;_ 4 _Vanderbilt University, Nashville, TN_.

Metastatic or castrate-resistant is the second-leading cause of cancer mortality in males. In the past 3 years, chemotherapies have extended survival, but efficacy is limited by dose-limiting toxicities due to suboptimal biodistribution. We address the lack of specificity and accumulation in Castrate Resistant Prostate Cancer (CRPC) by developing a unique nano-carrier for drug delivery: targeted fusogenic liposomes.

We have developed a platform whereby liposomes are formulated with fusion associated small transmembrane protein p14 displaying targeting ligands. The p14 protein catalyzes mixing of the liposomal bilayer with cell membranes to deliver the cargo directly into the cytoplasm, while the targeting ligand bombesin, allows for targeting to the Gastrin-releasing Peptide (GRPR) that is overexpressed in prostate cancer.

We hypothesized that this novel targeted fusogenic liposome formulation would significantly improve the biodistribution and efficacy of chemotherapy.

A clinical liposomal doxorubicin formulation (DOXIL) was modified to incorporate targeted fusogenic p14 protein.

We then evaluated the efficacy and biodistribution of these new formulations using in vitro and in vivo models of CRPC. In PC3 cells, compared to conventional liposomes, intracellular levels of doxorubicin are increased by 15 and 25 times when p14 or p14-bombesin liposomes are used. Additionally, the IC50 is reduced from 85mM to 2mM. In mice bearing PC3 tumors treated with targeted fusogenic liposomal doxorubicin, we observed tumor growth inhibition of 57% (vs control).

This establishes a proof of concept for an innovative targeted drug delivery system that may improve the outcome of patients with CRPC by enhancing the effect of approved drugs.

#2193

Stabilityof injectable phospholipid nanoparticles loaded with paclitaxel: influence oflipid composition, drug concentration, storage temperature, lyophilization, andadditives.

Shrada Prabhulkar,1 Gary Williams,1 Steve Miller,1 Zachary Yim,2 Walter McConathy,2 Tapas De2. 1 _Autotelic Inc, Fountain Valley, CA;_ 2 _LipoMedics Inc, Fort Worth, TX_.

Paclitaxel is one of the most effective chemotherapeutic drugs for solid tumors including breast, lung and ovarian cancers. It has been formulated as a nanoparticle formulation, Abraxane, to improve its solublity (0.35-0.7 µg/mL) and to avoid the use of harmful solvents like cremophor EL. We previously reported the next-generation Abraxane - Cynviloq - a polymeric micelle paclitaxel formulation which uses a chemical polymer instead of biological polymer to stabilize the nanoparticle [1]. Here we investigate and report the use of phospholipids instead of a chemical polymer for the creation of the next-generation Abraxane. The effect of lipid composition on drug loading and physical stability of paclitaxel-loaded lipid-coated nanoparticle formulation was evaluated before and after lyophilization. The nanoparticles were prepared by microfluidization-solvent evaporation method. The formulation parameters included type of phospholipids, phospholipid fatty acid chain length, ratio of phospholipid and lysophospholipid combination, and drug-phospholipid ratio. The process parameters such as microfluidization pressure and number of microfluidization cycles were studied and their impact on drug loading, particle size and physical stability were evaluated. The short-term stability evaluation of nanoparticles prepared with different phospholipid ratios demonstrated that 4:1 as the optimum phospholipid-lysophospholipid ratio to achieve a loading of more than 60% paclitaxel with particle size of approximately ~200nm. The nanoparticle size increased with an increase of carbon chain length of the phospholipid fatty acid, but no significant trends were observed for drug loading with changes in microfluidization pressure or number of cycles. The stability of the formulation was evaluated at different temperatures before and after lyophilization. The optimization of phospholipid composition, drug-lipid ratio, process parameters and additives for stability on lyophilization led to a physically stable paclitaxel-loaded phopholipid-coated nanoparticulate formulation that maintains size, charge and particulate integrity during storage. Phospholipid bound paclitaxel nanoparticle was successfully manufactured in lab scale with the desired properties. Scaling up and additional work-up for clinical evaluation is being evaluated.

Reference:

1. Motamed K, Goodman Y, Hwang L, Hsiao C, Trieu V. (2014). IG-001 - A non-biologic nanoparticle paclitaxel for the treatment of solid tumors. J Nanomater Mol Nanotechnol 3:1. doi:10.4172/2324-8777.1000136.

#2194

New oxazaphosphorine prodrugs for immunotherapy and nanomedicine against cancer.

Charles Skarbek,1 Henri Gonde,2 Mélanie Desbois,3 Julia Delahousse,1 Nathalie Chaput-Gras,3 Jean-Pierre Benoit,2 Emilie Roger,2 Angelo Paci1. 1 _CNRS, Univ. Paris-Sud, Gustave Roussy, Université Paris-Saclay, Villejuif, France;_ 2 _INSERM, Angers, France;_ 3 _INSERM / CNRS, Villejuif, France_.

Oxazaphosphorines (Oxaza) are widely used in the treatment of numerous cancers, activity against various tumor types, from soft tissue sarcomas to genito-urinary cancers for ifosfamide (IFO), from lymphoma to breast cancer for cyclophosphamide (CPA) and are still the corner stone of several polychemotherapy protocols. Limiting their toxicity and increasing their efficacy and safety through a better specificity could be of major interest.

By consequence, we have designed new pre-activated oxazaphosphorines (X-Oxaza, Skarbek et al J Med Chem. 2015 Jan 22;58(2):705-17) to improve antitumoral response with reduced toxicity or side effects, as metabolization is not needed to liberate the active metabolite. We also aimed to design new drug delivery systems (DDS) based on these X-Oxaza to take advantage of passive and active targeting in order to improve the response selectivity. Indeed, nanocarriers formulations take advantage of the so-called enhanced permeability and retention effect (EPR effect) and increase the circulation time in the bloodstream. Active targeting is planed by grafting targeting ligands on the surface of DDS such as monoclonal antibody-mAbs and amino acids...

The different steps leading to the design of the X-Oxaza has been developed within our laboratory and the proof of concept has been validated by their in vitro evaluation on a panel of tumor cell lines (Skarbek et al J Med Chem. 2015 Jan 22;58(2):705-17; Paci et al Bioorg Med Chem Lett. 2001 May 21;11(10):1347-9). These pre-activated Oxaza have been formulated as DDS either using the physicochemical properties of the terpene derivatives, which can self-assembly into nano-assemblies (NAs), or by encapsulating the short-chain X-Oxaza into Lipid nanocapsules (LNCs). Both DDS were tested on rhabdomyosarcoma and Ewing sarcoma cell lines and showed an increased activity compared to the free form.

Regarding the immunological field, CPA was shown to enhance the immune defenses particularly promoting differentiation of CD4+ cell toward Th1. The isomeric form of CPA, IFO, leads us to believe that IFO could have an influence on the immune system. Indeed, we have studied the immunomodulatory activity of IFO and the results show that IFO participates in the modulation of the secretion of cytokines from immune cells with a dose / effect relationship.

In the meantime, we investigate the influence of IFO and these X-Oxaza on the immune system using immunocompetent and immunodepressed mouse models. With the link of immune checkpoint antibodies, this strategy will benefit of the immunomodulatory effects of X-Oxaza combined to the antiproliferative properties of these antibodies.

These new functionalized DDS may provide a useful strategy to give specificity to active drugs used for many years in clinical practice. Both DDS could be grafted with mAbs which could lead to a new family of DDS aiming to combine antiproliferative and immunomodulatory properties for a dual antitumoral action

#2195

Small molecule drug conjugates targeted to cholecystokinin 2 receptors.

Jyoti Roy, Charity Wayua, Philip S. Low. _Purdue University, Lafayette, IN_.

Cholecystokinin 2 receptor (CCK2R) is over expressed on many cancers of lungs, pancreas, liver and GI tract (esophagus, colon and GIST), whereas its expression in normal tissues is limited to epithelial cells of the GI tract and brain. This restricted expression pattern renders CCK2R an excellent candidate for use in tumor-targeted drug delivery. Here we used a high affinity, low molecular weight CCK2R antagonist (CRL) to deliver imaging agents and cytotoxic drugs specifically to CCK2R positive tumors. We demonstrated that in vitro CRL conjugated to either NIR dye or radioactive imaging agent binds selectively to CCK2R positive cancer cells and exhibits low nano molar affinity for the receptor. We further showed that In vivo NIR dye and radiolabeled conjugate localizes primarily to the CCK2R positive murine tumor xenografts. Both of these conjugates also exhibited low serum protein binding and cleared rapidly from blood. Thus CRL-NIR dye conjugate have a potential application in fluorescence guided surgery where as CRL-radioactive conjugate seems promising for radioimaging in the clinic. Encouraged by the results of the imaging agents, CRL was conjugated to two potent microtubule inhibitors (tubulysin B hydrazide and desacetyl vinblastine). In vitro these two conjugates demonstrated high affinity and specificity for CCK2R. In vivo CRL conjugated to tubulysin B hydrazide was found to be more effective in eliminating tumor compared to desacetyl vinblastine conjugate. Neither of the CRL-cytotoxic drug conjugates showed any toxicity associated with the base drug nor any weight loss was observed in the mice treated with these conjugates. Results suggest that CRL has the proficiency to delivery drug specifically to tumors over expressing CCK2R receptor and lower the toxicities associated with conventional ways of treating cancer.

#2196

Improving cytotoxicity against cancer cells by chemo-photodynamics combined modalities using silver/graphene quantum dots/doxorubicin nanoconjugates.

Joel Encarnación-Rosado, Khaled Habiba, Kenny García-Pabón, Brad R. Weiner, Gerardo Morell. _University of Puerto Rico, Rio Piedras Campus, San Juan, PR_.

Tumor microenvironment complexity render chemotherapy and radiotherapy ineffective to eradicate highly aggressive tumors. For this reason, we are interested to find new avenues that can improve clinical outcomes. Recently, different nanomaterials such as Graphene Quantum Dots (GQDs) have been explored in treatments for a broad range of diseases, including cancer therapeutics. GQDs are nanometer-sized fragments (2-20 nm) of graphene, which have shown unique electrical, optical and mechanical properties. We hypothesize that the unique physical and chemical properties of the Graphene Quantum Dots (GQDs) along with the anticancer properties of silver (Ag) can provide an alternative to the challenges presently encountered in traditional cancer therapy regimens. In this study functionalized silver decorated graphene quantum dots (Ag-GQDs) nanocomposites were synthesized by a bottom-up approach by the pulsed laser irradiation of a liquid hydrocarbon precursor containing silver ions. The Ag-GQDs were characterized by UV-Vis spectroscopy, transmission electron microscopy, and Fourier transform IR spectroscopy. The Ag-GQD nanocomposites were tested in the treatment of cervical cancer HeLa cells and prostate cancer cells DU-145. The Ag-GQD nanocomposites demonstrated high potential in the delivery of Doxorubicin to cancer cells and an anticancer activity boost due to their intrinsic properties. Interestingly, we observed an increase in the activity of caspase-3/7 in DU145 and HeLa when treated with such nanoparticles. The photo-activation of Ag-GQDs with 425 nm radiation increased the levels of Reactive Oxygen Species (ROS) in both cell lines, inducing cell death by DNA damage. The combination of the chemo-photodynamic therapies using Ag-GQDs conjugated with DOX remarkably enhanced the treatment efficacy of HeLa and DU145, as compared to treatment by using each modality alone. HeLa. Fluorescence imaging results showed that Ag-GQDs deliver DOX to the nucleus of cancer cells, which suggests they may deliver other cargos. Also, to confirm whether the decrease in the cells viability upon treatment with Ag-GQDs was caused by toxicity, we tested a non-transformed cell line. The data suggest that the effect is due to the intrinsic effect of Ag-GQDs and not by toxicity. The Ag-GQDs thus offer a general platform for incorporating multiple therapeutic modalities for treating different types of cancer and they represent a significant breakthrough in nanomedicine for potential translation to the clinic. Further studies are necessary to elucidate the exact mechanism(s) of Ag-GQDs in releasing their cargo, and to test them in vivo. In summary, we developed a novel, multi-functional, and biocompatible PEGylated Ag-GQDs nanocomposites that combines two therapeutic modalities, chemotherapy and photodynamic therapy, into one platform for treatment of different type of cancers.

#2197

Mucopenetrating magnetic nanoparticles for drug delivery.

Viajayakumar N. Boya, Renn Lovett, Saini Satua, Vaibhav Gandhi, Prashanth K.B. Nagesh, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. _University of Tennessee Health Science Center, Memphis, TN_.

Objectives: Cervical cancer (CxCa) is one of the most common cancers among women worldwide. Current standards of care for cervical cancer includes surgery, radiation, and chemotherapy. However, systemic chemotherapy fails to elicit therapeutic responses and causes severe systemic toxicity due to limited concentration of drug reaching to mucosal tissue and blocking of drug penetration through the epithelial mucus surface of the cervix. Mucus in general functions as a protective barrier to for viruses and bacteria, but in cervical cancer condition it poses a serious issue for drug delivery modalities. Therefore, our study was aimed to develop a nanoparticle formulation that can effectively bind and penetrate the mucin barrier of the tissues for effective drug delivery applications.

Methods: Iron oxide based magnetic nanoparticles (MNPs) were prepared by precipitation of iron salts in the presence of ammonia with subsequent coating with β-cyclodextrin (β-CD) and/or pluronic polymer (F127). Four different compositions of MNP formulations, plain MNPs, MNPs with β-CD (MNP+βCD), MNPs with F127 (MNP+F127), and MNPs with β-CD and F127 (MNP+βCD-F127) were formulated for this study. Particle size, distribution, and zeta potential of MNPs and MNP-mucin were measured using the Zetasizer based on dynamic light scattering technique, mucin binding ability of MNPs was measured using SpectraMax M2e plate reader, migration of MNPs in the presence of mucin was measured using Boyden chamber assay, and tissue uptake/internalization was measured by Prussian blue staining and fluorescence techniques.

Results: Among four different formulations (MNPs, MNPs+βCD, MNP+F127, and MNP+βCD-F127), MNP+βCD-F127 formulation is unique due to its triple layered composition. This MNP formulation was prevented from aggregation in particle size anaysis in solution, indicating its dispersive nature along with penetration compatibility in mucus network. Instantaneous and long term mucin binding experiments suggest that MNP+βCD-F127 nanoparticles strongly interacted with mucin layer. Further, our mucin incubated nanoparticles effectively pass through membrane in Boyden chamber migration assay. Moreover, tissue uptake/internalization studies demonstrated an efficient penetration of MNP+βCD-F127 nanoparticles through mucin layer.

Conclusion: All above biophysical properties of MNP+βCD-F127 nanoparticles demonstrate that our unique formulation can bind and penetrate the mucus barrier. This study suggest that this unique nanoparticle formulation could a good candidate for local drug delivery applications for overcoing problems associated with cervical cancer treatment.

#2198

Integrin αvβ3-targeted lipase-labile docetaxel-prodrug micelles preferentially treat breast cancer bone metastases.

Michael H. Ross,1 Alison K. Esser,1 Anne H. Schmieder,1 Grace Cui,1 Xiaoxia Yang,1 Xinming Su,1 Dipanjan Pan,2 Gregory M. Lanza,1 Katherine N. Weilbaecher1. 1 _Washington University School of Medicine, St. Louis, MO;_ 2 _University of Illinois, Urbana, IL_.

Background: Bone metastases occur in 70% of metastatic breast cancer patients and are a leading cause of morbidity. Current therapies are often palliative, in part due to a lack of specificity for tumor targets within the bone. Integrin αvβ3 is overexpressed on neo-angiogenic blood vessels, tumor-promoting macrophages, osteoclasts, and more aggressive breast cancer cells, making it an attractive therapeutic target.

Objective: The goal of this study was to use Sn2 lipase-labile docetaxel-prodrug nanoparticle, targeted against activated integrin αvβ3, to attenuate breast cancer metastases.

Methods: A novel phospholipid-based micelle (∼12.5 nm) was functionalized with a peptidomimetic for activated integrin αvβ3, and designed to carry either rhodamine for fluorescent labeling or Sn2 lipase-labile prodrug of docetaxel (DTX-PD) for drug delivery. For microscopic localization studies, fluorescently labeled micelles were prepared with or without integrin αvβ3-targeting. C57BL/6 female mice received MMTV-PyMT breast cancer cell line (luciferase-labeled) via intracardiac (IC) injection to achieve tumor metastasis in all major organs. On day 8 post-IC injection, micelle preparations were administered i.v. and circulated within C57BL/6 mice for 3 hours prior to sacrifice and tissue collection. For drug efficacy studies, Sn2 lipase-labile docetaxel-prodrug was incorporated into αvβ3-micelles (αvβ3-DTX-PD). C57BL/6 female mice IC injected with MMTV-PyMT cells (luciferase-labeled) were treated with αvβ3-DTX-PD, or molar equivalent dose of free-DTX, or saline. Beginning on day 4 post-IC injection, mice were treated 3 times, once every 3 days (1.85mg/kg DTX per treatment). On day 12 post-IC injection, metastatic burden in the major organs was analyzed via ex vivo bioluminescent imaging.

Results: Fluorescent histological analysis of the tibiofemoral bone region showed significant colocalization of αvβ3-micelles with breast cancer bone metastases, as compared with non-targeted micelles (6.5-fold increase, p<0.001). αvβ3-micelles did not localize within tumor-free bones. In drug efficacy analysis, αvβ3-DTX-PD micelles significantly attenuated bone metastases (3-fold, p=0.007) and significantly reduced osteolytic bone destruction as assessed by x-ray analysis (3.5-fold, p=0.021). Serum chemistry analysis of enzymes indicative of liver damage were elevated in the DTX treated group, but not in αvβ3-DTX-PD or saline treated groups. Interestingly, αvβ3-DTX-PD micelles did not attenuate breast cancer metastases within the liver, lungs, or kidneys. Histological examination of metastatic tumor tissue demonstrated elevated integrin β3 expression in bone metastases, as compared to the other sites.

Conclusion: These findings suggest that the unique elevated expression of integrin αvβ3 within breast cancer bone metastases could be exploited with αvβ3-DTX-PD micelles for effective therapy.

#2199

TLR3-induced immune response using PLGA nanoparticles for dendritic cell-based cancer immunotherapy.

HEEDONG HAN. _Konkuk University, Seoul, Republic of Korea_.

Dendritic cells (DCs) are potent professional antigen presenting cells (APCs) capable of initiating a primary immune response and activating T cells, and they play a pivotal role in host immune responses to cancer. Prior to antigen presentation, efficient antigen and adjuvant uptake by DCs is necessary to induce their maturation and cytokine generation. Nanoparticles (NPs) are capable of intracellular delivery of both antigen and adjuvant to DCs. Here, we developed an advanced poly(lactic-co-glycolic acid) (PLGA)-NP encapsulating both ovalbumin (OVA) as model antigen and poly I:C as adjuvant to increase intracellular delivery, and promote DC maturation. These NPs were taken up by mouse primary DCs and their uptake greatly facilitated MHC class I antigen presentation in vitro. Moreover, vaccination with PLGA-NP treated DCs led to the generation of OVA-specific CD8+ T cells, and the resulting antitumor efficacy was significantly increased in EG.7 tumor-bearing mice compared to control (p < 0.01). Taken together, we demonstrate that this PLGA-NP platform may be an effective method for delivering tumor-specific antigens or adjuvant to DCs.

#2200

Omega-3 fatty acid conjugated paclitaxel dendrimers exhibit enhanced anticancer activity in various preclinical models of gastrointestinal cancers.

Tanmay Dichwalkar,1 Samhita Bapat,1 Priya Pancholi,1 V. K. Yellepeddi,2 Vikas Sehdev1. 1 _Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY;_ 2 _College of Pharmacy, Roseman University of Health Sciences, South Jordan, UT_.

Background: Paclitaxel (PTX) is frequently used for the treatment of advanced gastrointestinal (GI) cancers. However, development of drug resistance, dose-dependent toxicity, and lack of oral bioavailability are some of the major limiting factors that impede the therapeutic use of PTX for treatment of GI cancers. Drug conjugates of dendrimers, such as poly (amidoamine) (PAMAM), have been reported to effectively overcome bioavailability and dose-dependent toxicity related limitations. Therefore, in this study we synthesized and investigated the anticancer potential of omega-3 fatty acid [docosahexanoic acid (DHA)]-poly (amido)amine (PAMAM)-paclitaxel conjugates in various in vitro models of gastrointestinal cancers. Methods: DHA was conjugated to amine-terminated PAMAMG4 dendrimers using coupling agents HOBt and HBTU. PAMAMG4-DHA was then conjugated to PTX using PTX-NHS-ester. The conjugates were purified by size exclusion chromatography and characterized using 1H-NMR, MALDI-TOF-MS and high-resolution ESI-MS. The percentage payload of PTX conjugated to PAMAMG4-DHA and in vitro stability of PAMAMG4-DHA-PTX were evaluated using HPLC. Cell viability, clonogenic cell survival, and western blot analyses were done to evaluate the cell viability, survival, and induction of apoptotic proteins (P53 and P21), respectively. The in vitro cytotoxicity and induction of apoptosis was investigated in AGS, FLO-1, and MIA PaCa-2 GI cancer cell lines after treatment with PTX or PAMAMG4-DHA-PTX. Results: The 1H-NMR, MALDI-TOF and high-resolution ESI mass spectra confirmed the conjugation of DHA to PAMAM and PTX to PAMAM-DHA. The in vitro stability data showed that PAMAM-DHA-PTX conjugate was stable in human plasma for 24 hours. The cell viability data indicated that treatment with PAMAMG4-DHA-PTX is significantly more potent than PTX at inhibiting proliferation of esophageal (FLO-1: PTX IC50: 3.8±0.68 nM, PAMAMG4-DHA-PTX IC50: 1.1±0.17 nM), gastric (AGS: PTX IC50: 5.1±0.58 nM, PAMAMG4-DHA-PTX IC50: 1.4±0.23 nM), and pancreatic (MIA PaCa: PTX IC50: 5.1±0.75 nM, PAMAMG4-DHA-PTX IC50: 1.8±0.35 nM) cancer cell lines. The clonogenic cell survival data shows a similar activity where treatment with PAMAMG4-DHA-PTX exhibited enhanced inhibition of long term survival of FLO-1, AGS, and MIA PaCa cells when compared to PTX. The protein expression data further showed that compared to PTX treatment with PAMAMG4-DHA-PTX induced higher expression of P53 and P21 pro-apoptotic protein in AGS gastric cancer cell line. Conclusions: Overall our findings indicate that paclitaxel was successfully conjugated with PAMAMG4-DHA resulting in the synthesis of a novel PAMAMG4-DHA-PTX conjugate that exhibits enhanced anticancer activity than PTX alone in various in vitro models of GI cancers.

#2201

Tea nanoparticle, a safe and biocompatible nanocarrier, greatly potentiates the anticancer activity of doxorubicin.

Yi-Jun Wang,1 Yujian Huang,2 Nagaraju Anreddy,1 Guan-Nan Zhang,1 Yun-Kai Zhang,1 Meina Xie,1 Derrick Lin,2 Dong-Hua Yang,1 Mingjun Zhang,2 Zhe-Sheng Chen1. 1 _St. John's University, Queens, NY;_ 2 _The Ohio State University, Columbus, OH_.

An infusion-dialysis based procedure has been developed as an approach to isolate organic nanoparticles from green tea. Tea nanoparticle (TNP) can effectively load doxorubicin (DOX) via electrostatic and hydrophobic interactions. We established an ABCB1 overexpressing tumor xenograft mouse model to investigate whether TNP can effectively deliver DOX into tumors and bypass the efflux function of the ABCB1 transporter, thereby increasing the intratumoral accumulation of DOX and potentiating the anticancer activity of DOX. MTT assays suggested that DOX-TNP showed higher cytotoxicity toward CCD-18Co, SW620 and SW620/Ad300 cells, as compared to DOX. Animal study revealed that DOX-TNP resulted in a greater inhibitory effect on the growth of SW620 and SW620/Ad300 tumors than DOX alone. The cTnI levels in mice showed that TNP had no cardiotoxicity and DOX-TNP had moderate cardiotoxicity. Blood Smear tests demonstrated that TNP and DOX-TNP did not cause neutropenia or thrombocytopenia in mice. Therefore, TNP is a safe nanocarrier with excellent biocompatibility and minimal toxicity. In pharmacokinetics study, DOX-TNP greatly increased the SW620 and SW620/Ad300 intratumoral concentrations of DOX, as compared to DOX alone. But DOX-TNP had no effect on the plasma concentrations of DOX up to 240 min after administration. Furthermore, ex vivo IHC analysis of SW620 and SW620/Ad300 tumor sections revealed evidence of prominent antitumor activity of DOX-TNP. In conclusion, our findings suggested that natural nanomaterials could be useful in combating multidrug resistance (MDR) in cancer cells and potentiating the anticancer activity of chemotherapeutic agents in cancer treatment.

#2203

Targeting monoclonal antibodies to the tumor microenvironment for cancer immunotherapy.

Frederick L. Hall,1 Sant P. Chawla,2 Neal S. Chawla,2 Erlinda M. Gordon2. 1 _Counterpoint Biomedica LLC, Santa Monica, CA;_ 2 _Sarcoma Oncology Center/Cancer Center of Southern California, Santa Monica, CA_.

Therapeutic antibodies and immune checkpoint inhibitors can be delivered more efficiently to the tumor microenvironment (TME) by targeting the exposed collagenous (XC) proteins at the site/s of tumor invasion, stroma formation, and neoangiogenesis.Purpose: To assess the ability of bifunctional fusion polypepetides (mAb-Tropins) to selectively deliver monoclonal antibodies (mAbs) to the TME in tumor-bearing nude mice.Materials and Methods: Synthetic peptide probes and polypeptide aptamers (25-45 aa), fluorescein-labeled IgGs, and an anti-VEGF human mAb were tested in vitro in exposed collagen (XC)-agarose binding assays, in HUVEC cultures, and in vivo in a human xenograft model of pancreatic cancer in nude mice.Results: The XC-targeted mAb-Tropins, bound non-covalently to FITC-labeled IgGs [but not to truncated F(Ab')2 fragments], exhibited selectivity for XC-agarose over control agarose matrices. Importantly, the biological activity of the XC/mAb-Tropin-bound anti-VEGF mAbs was fully preserved, as demonstrated by inhibition of endothelial cell proliferation in HUVEC cultures. In vivo, intense fluorescence was observed in tumors of mice injected with mAb-Tropin targeted IgGs at 15 and 60 minutes after intravenous injection, but not in non-targeted IgG-treated mice (see Figure below).Conclusions: Tumor XC-targeted mAb-Tropins are an effective method of delivering therapeutic mAbs precisely to the tumor compartments, with meaningful implications for cancer immunotherapy.

Figure

Figure Legend: The performance of mAb-Tropin 277 is evaluated in vitro in XC-binding assays and in vivo in a murine xenograft model of metastatic cancer. XC-agarose chromatography using mAb-Tropin 277 and FITC-IgGs, followed by stringent washing and analysis of column eluates and retentates (A,B), demonstrates aptamer-dependent binding of IgGs to XC-proteins (retentates), as well as depletion of the fluorescent IgGs from solution (column pass-through). The mAb-Tropin 277 dependent targeting of fluorescent IgGs (via the systemic circulation) to tumors is demonstrated in a murine metastatic cancer model (C). Taken together with the in vitro binding data—which showed that the bifunctional mAb-targeting onco-aptamers efficiently sequester IgGs (A) and concentrate the bound IgGs on collagen matrices (B)—this in vivo data indicates that mAb-Tropins concentrate and compartmentalize IgGs/mAbs within the TME (C).

#2204

Targeted, controlled anticancer drug delivery and release with magnetoelectric nanoparticles.

Alexandra Rodzinski,1 Rakesh Guduru,1 Emmanuel Stimphil,1 Tiffanie Stewart,1 Ping Liang,2 Carolyn Runowicz,1 Sakhrat Khizroev1. 1 _Florida International University, Miami, FL;_ 2 _Cellular Nanomed, Weston, FL_.

Most cancer therapy is nonspecific, aimed at all dividing cells, resulting in untoward side effects. Our study demonstrates the effectiveness of magnetoelectric nanoparticles (MENs), which have been developed to address the critical issue of normal cell off-targeting in cancer treatment, in both in-vitro and in-vivo studies, as well as characterizes their biodistribution and clearance.

Exploiting the difference in electric properties between normal and cancer cell membranes, MENs are able to enter cancerous cells carrying a therapeutic payload and release the payload intracellularly with the application of an external magnetic field, while not affecting normal cells. SKOV-3 human ovarian carcinoma cells were used as a model to showcase the unique cancer targeting capabilities of these CoFe2O4@BaTiO3 nanostructures coated with the mitotic inhibitor Paclitaxel (PTX). The MENs-PTX bond was characterized in the lysate of treated cells using spectroscopic analysis and scanning probe microscopy. SKOV-3 xenografted athymic nude mice were treated via subcutaneous or IV injection on a weekly basis with a MEN, conventional ferromagnetic nanoparticle (MN), or polymer nanoparticle (PLGA) formulation. Biodistribution and clearance of MENs is one of the most important open questions addressed in this study. Our approach is to investigate the key parameters that affect the therapeutic index, i.e. the maximum tolerated dose, blood circulation half-life and biodistribution due organ accumulation. The approach is to study factors such as the size and shape of MENs, chemical composition, targeting ligand functionalization, MENs' biodegradability, and microenvironment and other biological barriers. Besides using conventional fluorescent markers, a novel nanoparticle distribution approach based on energy-dispersion spectroscopy (EDS) is exploited.

In-vitro studies on the cell lysate of MENs treated SKOV-3 cells determined reliable entry into the cells by MENs with the application of a small magnetic field (~100 Oe) and reliable payload release with the application of an a.c. magnetic field (~50 Oe, 100 Hz). In-vivo studies demonstrated that the MENs-PTX formulation in combination with an externally applied magnetic field reduces tumor growth rate when injected subcutaneously, and fully cures the cancer when delivered via IV-injection. The MENs formulation was more successful in treating the tumor than both MN and PLGA formulations. EDS confirmed the presence of MENs in tumor tissues.

MENs provide a novel mechanism by which cancer cells are targeted (using the difference in the cancer electric cell membrane properties compared to normal cells) and a drug payload is released (externally triggered with the application of an a.c. magnetic field) reliably. The underlying physics of the electric field interactions involved in the MENs drug delivery system was demonstrated here using ovarian cancer, but can be applied to virtually any cancer.

#2205

Exosomal encapsulation of doxorubicin reduces the cardiac toxicity of mice.

Flavio Rizzolio,1 Mohamad Hadla,1 Giuseppe Corona,1 Isabella Caligiuri,1 Stefano Palazzolo,1 Sabrina Semeraro,2 Amelia Gamini,2 Vincenzo Canzonieri,1 Giuseppe Toffoli1. 1 _CRO- National Cancer Institute, Aviano, Italy;_ 2 _Trieste University, Trieste, Italy_.

Exosomes are nanovesicles produced by cells embedded of cellular information and represent a formidable natural cargo for communication. The name "FedExosomes" denotes the general idea that exosomes could deliver cargo that conveniently manipulated could be of help for patient therapy. Up to now, a few papers demonstrated that modified exosomes could deliver drugs specifically to cancer cells and many laboratories are now studying their properties and compositions. Differently from previously published papers that focused on the efficacy of the doxorubicin encapsulated in exosomes (exoDOX), for the first time, we demonstrated that unmodified exosomes loaded with DOX are less toxic than free DOX by altering the biodistribution of the drug. The heart tissue of mice treated with a toxic dosage of DOX exhibited scattered cytoplasmic paranuclear vacuoles and focal intense eosinophilic cytoplasm, devoid of their transversal striations with moderate disarrangement of cardiomyocytes. The heart tissue of exoDOX treated mice at the same dosage appeared normal. Mass spectrometry studies confirmed that although the quantity of exoDOX in the blood was the same compared to DOX, accumulation in the heart was reduced of about forty percent. Maximum tolerated dose of exoDOX treated mice is double than free DOX. Mice treated at double concentration of exoDOX vs DOX have reduced tumor burden compared to free DOX without observable toxicity.

We demonstrated that exoDOX increased the therapeutic index of DOX by reducing toxic side effects and envisage a similar mechanism for others chemotherapeutic drugs. Since the exosomes could be prepared directly from the patients, our preclinical approach could have a possible immediately application in the clinic.

#2206

Development and characterization of an injectable copper clioquinol formulation: repurposing of a topical antifungal for treatment of cancer.

Moe Wehbe,1 Malathi Anantha,1 Ian Backstrom,1 Armaan Malhotra,1 Katarina Edwards,2 Marcel Bally1. 1 _BC Cancer, Vancouver, British Columbia, Canada;_ 2 _University of Uppsala, Uppsala, Sweden_.

Clioquinol is an FDA approved antifungal and antiprotozoal drug that has recently garnered attention for its anti-cancer activities. In vitro studies with CQ activity suggest that its mechanism of activity may be dependent its ability to bind and transport copper. CQ binds copper to form a stable dimeric complex (2 mol CQ:1 mol Cu ((CuCQ2)). Although therapeutically interesting, CuCQ2 is virtually insoluble in aqueous solutions and therefore it has been difficult to develop formulations suitable for administration. To address this, a liposomal CuCQ2 formulation was developed and characterized for maximum tolerated dose, pharmacokinetics and efficacy in xenograft models. Liposomes (~100nm) composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio) were prepared to contain 300 mM CuSO4. CQ is added to the outside of these pre-formed liposomes; at the appropriate incubation temperature CQ crosses the liposomal membrane where it can interact with the encapsulated Cu to form the insoluble CuCQ2 complex which becomes trapped. The maximum tolerated dose of the resultant formulation was determined in CD-1 mice. The pharmacokinetics of the formulation (liposomal lipid, CQ and Cu) were measured following intravenous administration at a well tolerated dose. Efficacy was measured in two cancer models (sc U251 glioblastoma and i.p A2780 ovarian). The maximum tolerated dose of liposomal CuCQ2 was 30 mg/kg when administered i.v (q2d for two weeks) and 15 mg/kg when administered i.p (QD for two weeks). Pharmacokinetics studies with liposomalCuCQ2 administered iv at 30 mg/kg indicated an terminal elimination half-life (t1/2) of 5.4 hr and an AUC of 994 ug•hr/mL. Results demonstrate that the CuCQ2 complex remains intact upon injection. When used to treat animals bearing established sc U251 glioblastoma tumors showed 30% smaller tumours than those treated with vehicle alone. The activity of liposomal CuCQ2 in the A2780 intraperitoneal cancer model will be presented. This work describes, for the first time, a novel injectable formulation of CuCQ2; this formulation is active against established xenograft models. The activity of this formulation will now be assessed in the context of copper-dependent inhibition of NFκB signalling.

#2207

**Using AFM to predict the specificity of tumor targeting nanomedicine** in vivo **.**

Peng Guo,1 Biran Wang,2 Daxing Liu,2 Jiang Yang,1 Marsha Moses,1 Debra Auguste2. 1 _Boston Children's Hospital, Boston, MA;_ 2 _The City College of New York, New York, NY_.

Most cancer target and biomarker studies focus on the identification of overexpressed genes or proteins of cancer cells. However, their potential feasibility as tumor-specific antigens targeted by antibody-guided nanomedicine has not consistently been based on quantitative assessments of tumor specificity and binding strength. Cancer cell membrane surfaces possess hundreds of different functionally versatile proteins (receptors and ligands) which are heterogeneously upregulated or downregulated to promote cancer development and progression. Selection of appropriate drug targets is pivotal to the successful development of tumor-targeted therapeutics because tumor-specific affinity of potential cancer targets is a key factor modulating cellular binding and uptake, and has the potential to translate to therapeutic bioactivity. To date, the quantitative measurements of cancer target antibody-antigen interactions are often limited by dynamic biophysical and biomechanical methods for probing such interactions.

To address this challenge, we used flow cytometric analyses to compare 40 potential cancer cell surface target candidates for triple negative breast cancer (TNBC), an aggressive malignancy with limited treatment options. We selected intercellular adhesion molecule-1 (ICAM-1) as the "optimized" TNBC target for nanomedicine. Reasoning that adhesion forces between the ICAM-1 antibody and live human breast cancer cells could be used to guide nanomedicine delivery to TNBC tumors, we used atomic force microscopy (AFM) to evaluate the binding strength between the ICAM-1 antibody and antigen on live human TNBC cells and non-neoplastic cells. Adhesion forces between the ICAM-1 antibody and live human TNBC MDA-MB-231 cells (523 ± 113 pN) were significantly higher than those of non-neoplastic MCF10A cells (336 ± 33 pN). We next validated these findings by engineering ICAM-1-targeted immunoliposomes and then tested their TNBC-specific binding affinity and cytotoxicity on three TNBC cell lines. In vivo imaging experiments further confirmed that the ICAM-1-targeted immunoliposomes preferentially accumulated in orthotopic TNBC tumors, in comparison with nonspecific, IgG-conjugated liposomes. These data support the conclusion that ICAM-1 antibody-conjugated immunoliposomes containing chemotherapeutic drugs have the potential to preferentially target and destroy TNBC cells, and may also be applicable to a range of metastatic cancers that demonstrate an overexpression of ICAM-1. These findings also support the use of AFM as a valuable tool in the characterization of surface antigen-antibody interactions that predict in vitro and in vivo binding affinity.

Acknowledgements: D. Auguste acknowledges the support of NIH 1DP2CA174495. M. Moses acknowledges the support of NIH R01CA185530 and the Breast Cancer Research Foundation.

#2208

Novel nanoparticle formulation of Plumbagin for pancreatic cancer treatment.

Bilal B. Hafeez, Vivek K. Kashyap, Vijayakumar N. Boya, Aditya Ganju, Mohammad Sikander, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. _University of Tennessee Health Science Center, Memphis, TN_.

Pancreatic cancer (PanCa) is one of the most fatal of all cancers and is ranked as the fourth most common cause of cancer related deaths among both men and women in the US. The management of PanCa, is exceptionally difficult due to the extremely poor response to available therapeutic modalities. Highly desmoplastic microenvironment in pancreatic tumor causes suboptimal drug delivery and increases chemo-resistance. Plumbagin (PL), a naturally occurring napthoquinone derived from the root of Plumbago zeylanica L., has showed potent cancer preventive and therapeutic activity against variety of cancers. However, the clinical translation of PL has been significantly hampered due to its toxicity and suboptimal bioavailability. To address these clinically relevant issues, we have developed and characterized a novel PL-loaded magnetic nanoparticle (MNP-PL) formulation. This MNP-PL formulation was prepared using Magnetic nanoparticles (MNPs) composed of an iron oxide core which has distinct advantages in i) bio/hemo-compatibility, ii) biodegradation, iii) higher drug loading capacity and iv) improved bioavailability. Our novel MNP-PL formulation provided average size of 125 nm in dynamic light scattering (DLS) and exhibited -9.42 to -10.79 mV zeta potential with an outstanding PL loading efficiency. We have evaluated anti-cancer potential of MNP-PL formulation in human PanCa cells (HPAF-II, AsPc1 and Panc-1). We first performed MTS and colony formation assays to determine the effects of free PL and MNP-PL formulation on growth of PanCa cells. In this experiment, cells were treated with various concentrations of free PL (1-15 µM) or MNP-PL (1-15 µM) for 24 hrs. Results exhibited efficient internalization of the MNP-PL formulation in a dose-dependent manner. As a result, the MNP-PL formulation showed four fold dose advantage over free PL. IC50 of free PL was recorded 10 µM which was significantly reduced to 2.5 µM in MNP-PL. MNP-PL also showed four fold inhibition in colony formation compared to free PL. MNP-PL treatment more efficiently inhibited oncogenic CXCL12/CXCR4 signaling pathway in both PanCa and patient derived stromal fibroblast cells. MNP-PL treatment also showed decreased expression of CXCR4 protein levels in PanCa cells. Moreover, MNP-PL treatment inhibited stromal derived factor 1 (SDF-1)/CXCL12 expression in stromal fibroblasts. These results suggest that our novel MNP-PL formulation has more anti-cancer potential than free PL against PanCa. In conclusion, MNP-PL formulation may reduce the toxicity and improve the bioavailability of free PL and could be used for the treatment of PanCa. 

## CLINICAL RESEARCH:

### Adoptive Cell Therapy, Immune Checkpoints, and Vaccines

#2209

Costimulation of T cells by CD81 enhances CAR transduction of naive T cells.

Liora M. Schultz, Debra Czerwinski, Chiung-Chi Kuo, Shoshana Levy, Ronald Levy. _Stanford, Palo Alto, CA_.

T cells genetically modified to express chimeric antigen receptors (CARs) mediate the eradication of antigen expressing tumor cells. The process of ex vivo T cell transduction to express CARs influences the phenotype, function and ultimate fate of the final CAR+ T cell product infused into patients. Pre-clinical data supports that naive-derived effector T cells have superior anti-tumor effect and enhanced persistence upon adoptive transfer as compared to memory-derived effector T cells. CD81 is a member of the tetraspanin family that functions as a T cell costimulator with activity independent of and additive to CD28. The addition of CD81 to CD3 and CD28 costimulation specifically induces increased activation of naive T cells. Retroviral genetic engineering methods require cells to be activated and in cell cycle to permit optimal gene integration. Here we demonstrate that introducing CD81 costimulation to the process of CAR transduction results in enhanced activation of naive T cells and resultant improved CAR transduction of the naive T cell subset.

Human naive T cells were isolated from healthy donors using magnetic bead negative selection. The purity of isolation was confirmed using flow cytometry showing CD62L+, 45RO- surface expression of the selected T cells. Naive T cells were differentially activated using CD3 antibody alone or in combination with CD28 or dual CD28 and CD81 costimulation. Using CD69 as a measure of activation, we demonstrate that stimulation with anti-CD3 alone or CD3 and CD28 antibodies does not activate isolated naive T cells. The addition of a CD81 antibody induces activation of both CD4+ and CD8+ naive T cells at 24 and 48 hours. We then transduced these activated naive T cells with a retrovirus encoding1928z, a CD19 specific second generation CAR. We used flow cytometry to confirm CAR transduction and compared the transduction efficiencies of the differentially costimulated groups.

We demonstrate that adding CD81 costimulation to CD3 and CD28 T cell activation permits retroviral-mediated CAR transduction of naive T cells. In contrast, stimulating CD3 alone or CD3 and CD28 does not induce activation, nor CAR transduction of isolated naive T cells. Transduction efficiency correlated with the degree of activation pre-transduction. Although the starting population was comprised of purified naive T cells, the activation and retroviral transduction process induced a change in surface markers. Surface expression of CD45RO increased and CD62L decreased, generating a final population of both naive T cells and naive-derived memory and effector T cells. This confirms that phenotyping T cells using surface markers following transduction is not reflective of the original T cell parent population.

We conclude that inclusion of CD81 co-stimulation into the process of CAR transduction improves transduction of naive T cells resulting in a final CAR+ T cell pool enriched for more potent naive derived CAR+ T cells.

#2210

Multiple antigens stimulating cellular therapy increases immune responses in patients with hepatocellular carcinoma.

Yanyan Han,1 Jing Huang,2 Ran Tao,1 Yabing Guo,2 Li Liu,2 Dongyun Wu,1 Jin Li,1 Xiangjun Zhou,1 Jinlin Hou2. 1 _SYZ Cell Therapy Co., Shenzhen, China;_ 2 _State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Guangzhou, China_.

Background & Aims: Hepatocellular carcinoma (HCC) is one of the most common tumors in China, and frequently occurs in the patients with chronic hepatitis B virus (HBV) infection. To investigate whether adoptive cell therapy induces tumor specific immune responses in HCC patients and further brings clinical benefits, Multiple Antigens Stimulating Cellular Therapy (MASCT) was applied in addition to conventional therapies.

Methods: Activated T cells that were prepared from the autologous peripheral blood mononuclear cells (PBMCs). Cells were stimulated and amplified with dendritic cells (DCs) pulsed by multiple tumor antigen peptides, including tumor associated antigen peptides and HBV antigen peptides. All patients received infusions of 5-10x107cells/kg of body weight of the resulting T cells every 1-3 months . The immune responses in the patients` PBMCs were examined before and after the treatment.

Results: The resulting amplified cells were almost exclusively CD3+ (95% ± 1%), with a major percentage being CD3+CD8+ cells (70% ± 5%) and a minor proportion being CD3+CD4+(19% ± 3%) and CD3+CD56+ (14% ± 1%) cells, while there were insignificant amounts of CD4+CD25+Foxp3+ regulatory T cells (0.4% ± 0.1%). These T cells have restored immune functions and HLA-restricted specific cytotoxicity against HCC cells. After repeating treatment of MASCT, the frequency of regulatory T cells in the patients' PBMCs was significantly decreased, and the proliferation and function of tumor antigen specific T cells were enhanced. The specific immune responses against each kind of tumor antigens were also detected in patients. And these immune responses were gradually increased during the treatment of MASCT. Moreover, in retrospective study, the disease control rate (DCR) in one year was found to be raised to 80% in stage B HCC patients receiving MASCT combined with conventional therapies.

Conclusions: Our study has demonstrated that MASCT is a well tolerate immunotherapy in patients with HCC. Specific T cell responses against tumor antigens can be strongly induced in vivo. Our study also indicated that repeated MASCT treatment improves both the immunologic function and disease control of patients with HCC.

#2211

**Enhanced** in vitro **and** in vivo **targeting of rituximab-sensitive and -resistant Burkitt lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR)-modified expanded natural killer cells in combination with a histone deacetylase inhibitor, romidepsin.**

Yaya Chu,1 Ashlin Yahr,1 Bokun Cheng,1 Bian Hang,1 Janet Ayello,1 Matthew Bath,2 Mitchell S. Cairo1. 1 _New York Medical College, Valhalla, NY;_ 2 _State University of New York at Buffalo, Buffalo, NY_.

Background: For BL patients who relapse, the prognosis is dismal due to chemo-immunotherapy resistance (Cairo et al, JCO, 2012, Goldman/Cairo et al, Leukemia, 2013). Our group has successfully modified expanded peripheral blood Natural killer cells (exPBNK) with an anti-CD20 CAR to target rituximab sensitive/resistant CD20+ BL cells in vitro and in NSG mice (Chu/Cairo, et al, Can Imm Res 2015). Romidepsin is a histone deacetylase (HDAC) inhibitor that increases the expression of NKG2D ligands in BL (Chu/Cairo, et al, Cytotherapy, 2014).

Objective: We investigated the effect of romidepsin alone and in combination with anti-CD20 CAR modified exPBNK cells against CD20+ BL cells in vitro and in humanized BL NSG mice.

Methods: Anti-CD20 CAR modified exPBNK cells were produced as we have described (Chu/Cairo, et al, Can Imm Res 2015). HDACs levels in BL cells were examined by western blot. Raji, Raji-2R and Raji-4RH cells were treated with 10ng/ml romidepsin, generously provided by Celgene. Raji-Luc engrafted mice were injected i.p. with romidepsin (2.2mg/kg) or PBS once a week for 3 consecutive weeks followed by anti-CD20 CAR exPBNK cell or mock exPBNK cell injections 24hrs later. Tumor regression and/or progression were monitored weekly by tumor volume measurement and by in vivo bioluminescent imaging (Chu/Cairo, et al, Can Imm Res 2015).

Results: HDAC 1, 3, 6 levels were significantly increased in Raji, Raji-2R, Raji-4RH, Ramos, and Daudi compared to white blood cells. Total H3K9 acetylation was enhanced in Raji, Raji-2R, and Raji-4RH following romidepsin. More importantly, romidepsin significantly inhibited BL cell proliferation (p<0.001), induced apoptosis only in rituximab sensitive BL cells with increased active caspase 3, and induced cell cycle arrest in resistant BL cells (p<0.001). Romidepsin significantly inhibited in vivo Raji and Raji-2R cells growth in xenografted NSG mice with reduced tumor size (p<0.05) and bioluminescence (p<0.05).

In vitro cytotoxicity of anti-CD20 CAR exPBNK cells was significantly enhanced against romidepsin treated BL cells compared to the untreated at E:T=3:1 (P<0.001; n=4), or compared to the mock exPBNK (P<0.05; n=4).

In humanized Raji xenograft NSG mice, survival time in romidepsin+CAR exPBNK treated mice was significantly extended compared to the untreated mice (median 28 days, P<0.001), the mock exPBNK treated mice (median 29 days, P<0.001), the CAR exPBNK treated mice (median 42.5 days, P<0.05), the romidepsin treated mice (median 30 days, P<0.001), and the romidepsin+mock exPBNK treated mice (median 34.5 days, P<0.05).

Conclusion: These results suggest the therapeutic potential of the combination of anti-CD20 CAR modified exPBNK cells and romidepsin against chemo-immunotherapy resistant BL.

#2212

Induction of α5β1 integrin expression rescues geriatric CAR-T cell function.

Prajna Guha,1 Marissa Cunetta,1 Ponnandai Somasundar,1 N J. Espat,1 Richard P. Junghans,2 Steven C. Katz1. 1 _Roger Williams Medical Center, Providence, RI;_ 2 _Tufts Medical Center, Boston, MA_.

Chimeric antigen receptors expressing T cells (CAR-Ts) are a promising form of immunotherapy for solid tumors. The majority of cancer patients in the United States are over 65 years old. The impact of age-related physiologic changes on cancer therapy and immunotherapy in particular require further study. We tested our hypothesis that CAR-T from geriatric donors (gCAR-T, n=8, >65 years) are functionally impaired relative to CAR-T from younger donors (yCAR-T, n=8, 18-45 years). T cells were genetically modified to express CAR specific for carcino-embryonic antigen (CEA), presently in use in our phase I trials. CAR-T cells were expanded ex vivo with IL2 (3000IU/ml) or IL15 (5ng/ml) for 20 days. Higher transduction efficiencies (36.9%+4.3 yCAR-T vs 21.3%+2.2 gCAR-T, p=0.002) and improved cell expansion (2.5 fold in IL2, p=0.01, two-tailed t-test) were observed in yCAR-T cells compared to gCAR-T. To define the underlying signaling mechanisms that led to increased proliferation in yCAR-T cells, we examined pERK, pAKT, pSTAT3 and pSTAT5. Following activation with anti-CD3, yCAR-T demonstrated significantly increased levels of pERK (13.4 fold in IL2, p=0.03 and 2.3 fold in IL15, p=0.04), pAKT (4.2 fold in IL2, p=0.04 and 3.0 fold in IL15, p=0.04), pSTAT3 (1.5 fold in IL2, p=0.02 and 2.9 fold in IL15, p=0.01) and pSTAT5 (5.7 fold in IL2, p=0.02 and 2.1 fold in IL15, p=0.03) compared to gCAR-T. Furthermore, yCAR-T contained higher proportions of CD4 effector cells (EC) (p=0.003), CD4 effector memory (EM) cells (p=0.002) and CD8 effector memory cells (EM) (p=0.002). In accordance with higher numbers of CD4 and CD8 EM, yCAR-T demonstrated higher levels of CEA specific cytotoxicity compared to gCAR-T (1.3-fold over gCAR-T, p=0.03), with maximum cytotoxicity observed in IL15 treated yCAR-T cells. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin (1.3+0.1 ng/ml, p=0.01) and granzyme B (0.4+0.03 ng/ml, p=0.02). Unmodified geriatric T cells (gT) had decreased α5β1 integrin expression as compared to unmodified young T cells (yT) (yT cells 44.4 +4.2 vs gT cells 22.3 +2.0, p=0.0003), a known mediator of retroviral transduction. We found that M-CSF or TGF-β1 rescued CAR expression by gCAR-T (yCAR-T 36.9+4.3, gCAR-T 29.9+1.0, gT+M-CSF 41.4+1.0, gT+TGF-β1 40.1+2.0, p<0.002 vs gCAR-T) through restoring α5β1 integrin expression (gT cells +M-CSF 41.0+1.0, gT cells + TGF- β1 31.8+1.4, yT cells 44.41 +4.2, p<0.0005). We performed cytotoxicity assays and demonstrated that M-CSF and TGF-β1 (up to 1.5 fold over untreated gCAR-T, p<0.05) rescued g-CAR-T tumor killing function. Our study suggests that functional impairment of gCAR-T can be mitigated through M-CSF of TGF-β1 treatment to restore α5β1 integrin expression.

#2213

Comparison of infusion hepatic transarterial devices for hepatic artery delivery of CAR-T cells.

Josephine K. Darpolor, Marissa Cunetta, Matthew Lima, Jillian Gardell, Gary Point, Zhi Liu, N J. Espat, Prajna Guha, Steven Katz. _Roger Williams Medical Center, Providence, RI_.

We previously tested the efficacy of chimeric antigen receptor T cell (CAR-T) treatment via hepatic artery infusion in a phase I clinical trial. Though these results were promising, our current protocol for hepatic immunotherapy for metastasis (HITM) includes transcatheter arterial embolization in order to reduce off-target toxicity from CAR-T delivery to gastric, enteric, or pancreatic tissues. Surefire Infusion System devices employ a soft fabric one way valve that prevents back-flow during hepatic artery infusion and generates a pressure gradient, which is clinically shown to increase delivery of therapeutic agents into tumor. We performed in vitro and in vivo testing in preparation for a phase Ib trial to ensure that the SIS does not adversely affect CAR-T performance. Viability, phenotype and activation status of CAR-T with no catheter (NC) and following passage through a standard microcatheter (STD), Surefire Infusion System (SIS), or Surefire Precision (SP) infusion system were tested via flow cytometry. CAR-T viability was preserved as measured by Zombie NIR. We did not detect a significant phenotype shift when examining CD4, CD8, CD25, and CD69. A CFSE assay and an LDH were done to track proliferation and measure the cytotoxicity of the CAR-T when stimulated by tumor. NC CAR-T had significantly more proliferation than each of the catheter groups. Specific Lysis was not significantly different between the NC, STD, SIS, and SP groups. We also performed in vivo testing in mice with intraperitoneal (IP) tumor with IP NC or SIS CAR-T. By Day 10, there was significantly less tumor present in the NC (5.7- fold reduction, p=0.02) and the SIS (44- fold reduction, p=0.04) groups than in the tumor only control group. The SIS CAR-T group and the NC CAR-T group were not significantly different (p=0.38). The SIS and SP devices did not adversely impact CAR-T viability, phenotype, or performance implying that these catheters could be interchangeably used for more efficient and targeted CAR-T dose delivery.

In Vitro Experimental Data  | |  | |

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

Experiment | NC

(Control) | STD

(Control) | SIS | PS

Viability

(Zombie NIR) | 6% | 7.9%

p= 0.7 vs NC | 7.4%

p=0.9 vs. STD | 9.9%

p=0.7 vs STD

Phenotype

(CD3+CD4+) | 33.1% | 30.6%

p= 0.9 vs NC | 33.3%

p=0.9 vs STD | 21.3%

p= 0.8 vs STD

Phenotype

(CD3+CD8+) | 68.8% | 69.5%

p= 0.9 vs NC | 69.1%

p=0.9 vs STD | 66.7%

p= 0.7 vs STD

Activation

(CD69+) | 34.93% | 32.3%

p= 0.9 vs NC | 33%

p= 0.9 vs STD | 25.6%

p= 0.7 vs STD

Treg

(CD25+) | 35.2% | 36.2%

p= 0.9 vs NC | 36.8%

p= 0.6 vs STD | 40.5%

p= 0.6 vs STD

Proliferation

(CFSE) | 41.7% | 26.7%

p= 0.005 vs NC | 27.3%

p= 0.8 vs STD | 24.2%

p= 0.4 vs STD

Cytotoxicity

(LDH Assay) | 51.2% | 42.1%

p= 0.1 vs NC | 44%

p= 0.7 vs STD | 41.1%

p= 0.9 vs STD

#2214

GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory.

Minu K. Srivastava, Rui Yun, Erin Mayes, Hyun_Bae Jie, Fumiko Axelrod, Jorge Monteon, Ming-Hong Xie, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. _Oncomed, RedWood City, CA_.

GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF superfamily and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. GITR is found primarily on activated T effector (Teff) cells and regulatory T (Treg) cells. Co-stimulation of GITR by agonist agents is hypothesized to promote anti-tumor immunity by enhancing Teff cell activity and inhibiting Treg suppression. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc). GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in multiple syngeneic mouse tumor models. GITRL-Fc enhanced tumor specific T-cell responses, particularly of the Th1 type, and also led to reduction in Treg-mediated immunesuppressive activity. GITRL-Fc displayed single agent activity in inhibiting tumor growth and promoting complete tumor rejection in the murine CT26 colon carcinoma model and combination activity with anti-PDL1 as compared to anti-PDL1 and control IgG2a alone. Mice "cured" with GITRL or GITRL/anti-PDL1 combination treatments were protected from re-challenge with tumor cells, suggesting the existence of immunologic memory. More mice were protected from tumor re-challenge with the combination of GITRL-Fc and anti-PDL1, as compared to GITRL-Fc alone. Our results demonstrate that agonist GITRL-Fc induces potent T cell responses, overcomes Treg inhibition, and promotes anti-tumor activity in preclinical models as a single agent or in combination with anti PDL1. The mechanism of tumor eradication and induction of long-term immune memory response by the combination is under investigation and will be discussed at the presentation.

#2215

Pembrolizumab and ipilimumab reduce human RKO colon carcinoma tumor growth in HLA matched CD34-NSG humanized mice.

Marcio Lasaro, Jason M. Davis, Mark Ellison, Martin R. Graf, Vivek Mahajan, Aidan Synnott, Robert J. Mullin, Paula L. Miliani De Marval. _Charles River Discovery Research Services, Morrisville, NC_.

Over the past decade there has been an increasing demand for preclinical models useful for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. The recently developed humanized mouse models composed of various types of human immune cells has the potential to recapitulate the human anti-tumor response modulated by checkpoint inhibitors. Tumor cells may develop multiple mechanisms to evade immune surveillance, including PD-L1 upregulation which suppresses T-cell effector responses towards the tumor. In the present study, we evaluated the efficacy of the checkpoint inhibitors pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) in the human RKO colon carcinoma xenograft model in CD34-NSG humanized mice. These mice were engrafted with human hematopoietic stem cells (HSC) that matched the HLA haplotype of the RKO cells (HLA-A*01). Flow cytometry analysis confirmed high level of expression of PD-L1 in RKO colon carcinoma cells. The results from this study revealed that pembrolizumab and ipilimumab monotherapies significantly inhibited tumor growth. Surprisingly, combination therapy did not provide additive or synergistic effects and resulted in the same level of efficacy as the individual regimens. In order to understand the impact of these therapies on distinct immune cell populations, we analyzed the distribution of CD4 and CD8 T lymphocytes, Treg cells, NK cells and B cells in peripheral blood, spleen and tumor samples from tumor bearing humanized mice, via flow cytometry. We found that these checkpoint inhibitors can enhance effector functions of CD4+ and CD8+ T cells associated with increased expression of IFN-Γ. A parallel study with these checkpoint inhibitors carried out with humanized mice engrafted with HSC of the same haplotype as the RKO cells but from a different donor resulted in identical anti-tumor efficacy. In summary, these studies validate the use of humanized mouse models to test the tumor response to immune-checkpoint based therapies as it shows significant anti-tumor growth as a result of pembrolizumab and ipilimumab monotherapies associated with activation of T lymphocytes.

#2216

Study of PDL-1 regulation and expression in glioblastoma and its role in cancer resistance.

Michele Russo,1 Paolo D'Arrigo,1 Felix Hausch,2 Anna Rea,1 Martina Tufano,1 Maria Fiammetta Romano,1 Simona Romano1. 1 _University of Naples Federico II, Naples, Italy;_ 2 _Max Planck Institute of Psychiatry, Munich, Germany_.

Among the high-grade malignant gliomas (III-IV), grade IV glioma, or glioblastoma (GBM), is the most common and destructive form of brain cancer. Despite rigorous molecular, preclinical, and clinical research these tumors remain a challenge to treat and 70% of patients diagnosed with GBM will succumb to the disease within 2 years. New immune-based cancer treatments are currently in development, among which, anti-PD-1 is in a phase II clinical trial for recurrent glioblastoma. Tumor cells exploit PDL-1/PD1 pathway to induce T cell exhaustion and evade host antitumour immunity. PDL-1 expression on glioblastoma has been shown to correlate with a bad prognosis. There is strong demand for knowledge about the mechanisms regulating PDL-1 expression in glioblastoma. Our research group, has previously identified a relevant role for FKBP51, and its spliced isoform FKBP51s, in PDL-1 expression and melanoma resistance to the therapy. FKBP51 is an immunophilin capable of immunosuppression, originally cloned in lymphocytes. Because FKBP51 was identified as highly expressed in glioblastoma, correlating with tumor aggressiveness, we investigated whether this protein and/or its spliced isoform FKBP51s could be involved in regulation of PDL-1 expression in glioblastoma. In addition, we looked at the role of these protein-isoforms in glioblastoma resistance to chemotherapy. To this aim, we used two different glioblastoma cell lines termed D54 and U251. Our results suggest that, differently from melanoma, in which PDL-1 is inducible and associated with FKBP51 splicing, glioblastoma cell lines express high basal levels of PDL-1. Interestingly, both the canonical and the spliced FKBP51 isoforms were highly expressed in the glioblastoma cell lines. Expression of PDL-1 was regulated by FKBP51s knockin and knockout and the levels changed in accordance with FKBP51s levels. Moreover, in glioblastoma cells silenced for FKBP51s, the decrease in PDL-1 levels was associated with a significant increase in apoptosis, either spontaneous apoptosis and the one stimulated by etoposide or temozolomide. Two recently developed compounds, selective inhibitor of FKBP51, termed SAFit1 and SAFit2 were able to reduce expression of PDL-1 on glioblastoma cell lines. Particularly, SAFit 2 was found to be more effective, in accordance with the notion that it is more brain-permeable than SAFit1. SaFit 2 was also shown to increase sensitivity to etoposide of glioblastoma cell, although to a lesser extent when compared to FKBP51siRNAs. In conclusion, our preliminary results suggest that FKBP51s plays a relevant role in glioblastoma resistance and PDL-1 expression. Future studies are needed to shed light on the mechanisms underlying FKBP51s-induced PDL-1 expression and signaling pathway downstream to FKBP51s/PDL-1 that sustain glioblastoma resistance.

#2217

Combining CEA-IL2v and FAP-IL2v immunocytokines with PD-L1 checkpoint blockade.

Valeria Nicolini, Inja Waldhauer, Anne Freimoser-Grundschober, Stefan Evers, Jose Saro, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. _Roche Pharma Research and Early Development, Schlieren, Switzerland_.

Introduction: CEA-IL2v (RG7813, RO6895882) and FAP-IL2v (RG7461, RO6874281) are novel CEA-/FAP-targeted immunocytokines based on a novel IL2 variant (IL2v) with abolished CD25 binding that are currently in clinical phase 1 trials. PD-L1 is found on the surface of cells in various tumors and is induced by interferon gamma (IFNg). It prevents the immune system from destroying cancer cells by interacting with the inhibitory receptors PD-1 and B7.1 on T cells. Atezolizumab blocks the interaction of PD- L1 with its receptors and is currently being investigated in pivotal clinical trials for various cancers.

Materials and Methods: PD-L1 expression on A549 tumor cells was analyzed by flow cytometry after treatment with CEA-IL2v alone or in the presence of PBMCs at varying E:T ratios. The combination of muCEA-IL2v, a murinized version of CEA-IL2v, with a muPD-L1 specific surrogate antibody was investigated in syngeneic stably CEA expressing PancO2 and MC38 cell lines in human CEA transgenic C57BL/6 mice. As control an untargeted DP47-muIL2v control immunocytokine was applied in the PancO2-CEA model. The combination of muFAP-IL2v, a murinized version of FAP-IL2v, with a muPD-L1 specific surrogate antibody was investigated in a syngeneic stably FAP expressing MC38 cell line in C57BL/6 mice.

Results: In vitro, co-cultures of human PBMCs and the human A549 lung cancer cell line showed that CEA-IL2v induces the up-regulation of PD-L1 on A549 tumor cells in a concentration-dependent manner mediated through release of IFNg. In the syngeneic s.c. MC38-CEA model in CEA/hCD16 transgenic C57BL/6 mice, muCEA-muIL2v (0.5 mg/kg, q1wx2) and the muPD-L1 antibody (10 mg/kg, q1wx2) resulted in superior combined efficacy compared to the respective single agent therapies with respect to tumor growth inhibition. In the syngeneic orthotopic Panc02-CEA model in CEA transgenic C57BL/6 mice, muCEA-muIL2v (0.25 mg/kg, q1wx4) and the muPD-L1 antibody (10 mg/kg, q1wx4) resulted in superior median and overall survival model compared to the respective single agent therapies. Notably, the combination of muCEA-muIL2v was superior to the combination of the untargeted DP47-muIL2v control immunocytokine (exposure matched dose: 0.2 mg/kg, q1wx4) with the muPD-L1 antibody. Similarly, in the syngeneic s.c. MC38-FAP model the combination of muFAP-muIL2v (2 mg/kg, q1w4) and the muPD-L1 antibody (10 mg/kg, q1w4) resulted in superior combined efficacy compared to the respective single agent therapies in terms of tumor growth inhibition as well as median and overall survival.

Conclusions: Taken together these nonclinical combination data support the clinical testing of combinations of CEA-IL2v and FAP-IL2v with atezolizumab. A clinical Phase 1b study combining CEA-IL2v and atezolizumab is currently ongoing (NCT02350673).

#2218

Distribution and prognostic relevance of PD-1/PD-L1 immune checkpoints and tumor-infiltrating lymphocytes in early-stage pulmonary basaloid squamous cell carcinoma.

Marius Ilie,1 Catherine Butori,2 Véronique Hofman,1 Christelle Bonnetaud,3 Sandra Lassalle,2 Elodie Long,2 Salomé Lalvée,2 Nicolas Vénissac,4 Jérôme Mouroux,4 Charles Hugo Marquette,5 Paul Hofman1. 1 _CHU Nice, Laboratory of Clinical and Experimental Pathology and Hospital-Integrated Biobank BB-0033-00025, Nice, France;_ 2 _CHU de Nice, Laboratory of Clinical and Experimental Pathology, Nice, France;_ 3 _CHU Nice, Hospital-Integrated Biobank BB-0033-00025, Nice, France;_ 4 _CHU de Nice, Thoracic Surgery Department, Nice, France;_ 5 _CHU de Nice, Pneumology Department, Nice, France_.

Background: The activation of immune cells by immune checkpoint inhibitors showed promising results with increased patient survival in several types of cancer. Since no data exist for pulmonary basaloid squamous cell carcinoma (BSCC), we aimed to characterize the expression of PD-L1 and PD-1 as well as tumor infiltrating lymphocytes (TILs) in operable BSCC.

Materials and Methods: We retrospectively included 57 patients with operable stages I-III BSCC. The expression of PD-L1 (clone SP142, Spring Bioscience), PD-1 (clone NAT105, Ventana) and CD8 (clone SP57, Ventana) was evaluated by automated immunohistochemical analysis in FFPE specimens of BSCC. PD-L1 expression was scored on tumor cells (TC3≥50% positive tumor cells; TC2≥5% but <50% positive tumor cells; TC1≥1% but <5% of positive tumor cells; and TC0<1% positive tumor cells) and tumor-infiltrating immune cells (IC3≥10% of positive immune cells; IC2≥5% but <10% of positive immune cells; IC1≥ 1% but <5% of positive immune cells; and IC0< 1% of positive immune cells). Survival analysis was determined by Kaplan-Meier analysis and Cox proportional hazard models.

Results: PD-L1 expression on tumor cells (TC1-3) was observed in 30% of patients (17/57), with high-PD-L1 expressing TC (TC2/3) in 14% of cases (8/57). PD-L1 expression on tumor-infiltrating immune cells was noticed in 36% of patients (20/57), with high-PD-L1 expressing IC (IC2/3) in 18% of cases (10/57). PD-L1 expression strongly correlated with CD8+ TILs and PD-1+ expression. CD8+ TILs infiltrated the tumors in three different patterns (stromal, peritumoral, diffuse). High PD-L1 expression either on TC or IC was significantly associated with better progression-free survival (PFS) (TC0, P=0.024; TC0/IC0, P=0.03) and overall survival (OS) (TC0, P=0.01; TC0/IC0, P=0.007) in BSCC patients. High amounts of TILs and PD-1+ cells were significantly correlated with improved PFS (TILs, P=0.035; PD-1+, P=0.007) and OS (TILs, P=0.016; PD-1+, P=0.03).

Conclusion: Increased PD-L1 TC and IC levels, TILs density and PD-1+ infiltrates are associated with better outcome in early-stage BSCC. Assessment of PD-L1 expression along with PD-1+ and CD8+ TILs could be useful to predict response to immune checkpoint inhibitors in BSCC. [M.I. and C.B. contributed equally to this work.]

#2219

Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows synergistic anti-tumor activity when combined with PD-1 blockade in a mouse model of colon cancer.

Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. _Karyopharm Therapeutics Inc, Newton, MA_.

Introduction: Selinexor is an oral, first-in-class SINE compound that specifically binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports over 200 cargos, including major tumor suppressor proteins (TSPs), leading to their functional inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. Interestingly, XPO1 also mediates the export of NFAT1c, STAT1 and STAT3, which have been implicated in regulation of the inhibitory T-cell receptor PD-1 (NFAT1c, STAT3) and its ligand, PD-L1 (STAT1). Therefore, we hypothesized that selinexor treatment will result in up-regulation of PD-1 and PD-L1, making tumor cells more amenable to immunotherapy.

Methods: The effect of selinexor on PD-1 and PD-L1 gene expression was tested in human normal donor leukocytes and in tumor cells [HCT-116(colon), PC-3 (prostate) and MDA-MB-468 (breast)], respectively by quantitative PCR. A Colon26 syngeneic mouse model of CRC was generated and animals were assigned to the following treatment groups: (i) vehicle, (ii) selinexor at sub-therapeutic dose of 5 mg/kg (M/W/F), (iii) Anti PD-1 (BioXCell), 100ug biwk and (iv) selinexor + anti-PD-1 combination. Tumor growth, weight loss and signs of toxicity were monitored for 45 days. Xenografts were harvested, RNA and DNA were collected and tumors were analyzed microscopically and by immunohistochemestry (IHC).

Results: In-vitro studies showed that selinexor induces PD-1 gene expression by ~2-fold in normal donor leukocytes as early as 4 hrs after treatment initiation and that this change is sustained for at least 24 hrs. A similar effect was measured on PD-L1 gene expression induction in HCT-116, PC-3 and MDA-MB-468 tumor cells. In-vivo, the combination was well tolerated and no weight loss or signs of toxicity were evident. Selinexor-anti-PD-1 combination therapy exhibited a synergistic effect on percent Tumor Growth Inhibition (%TGI) were the selinexor-treated group and the anti-PD-1-treated group alone were very similar with mean %TGI measuring 27% and 24% respectively, and the combination group measuring a mean %TGI of 67%. Importantly, 4-out-of-10 mice in the combination group did not have detectable tumors at day 22 and these mice were also tumor free at the end of the study, at day 45. IHC analysis showed overall enhanced effect of the combination treatment on induction of apoptosis and reduced proliferation as well as effect on specific T-cell checkpoint regulatory molecules.

Conclusion: Selinexor induces PD-1 and PD-L1 gene expression, sensitizing tumor cells to immunotherapy. Selinexor synergizes with anti-PD-1 to inhibit tumor cell proliferation and to induce apoptosis in-vivo. These data provide rational support for study of selinexor/ anti-PD-1 combination in clinical trials.

#2220

PD-L1 expression in primary tumor and circulating tumor cells in patients with small cell lung carcinomas.

Marius Ilie,1 Véronique Hofman,1 Elodie Long,1 Catherine Butori,2 Salomé Lalvée,2 Eric Selva,3 Nicolas Vénissac,4 Jérôme Mouroux,4 Charles Hugo Marquette,5 Paul Hofman1. 1 _CHU Nice, Laboratory of Clinical and Experimental Pathology and Institut for Research on Cancer and Aging; Nice (IRCAN); CNRS UMR7284/INSERM U1081; Faculty of Medicine; Nice University, Nice, France;_ 2 _CHU Nice, Laboratory of Clinical and Experimental Pathology, Nice, France;_ 3 _CHU Nice, Hospital Integrated Biobank BB-0033-00025, Nice, France;_ 4 _CHU Nice, Thoracic Surgery Department, Nice, France;_ 5 _CHU Nice, Pneumology Department and Institut for Research on Cancer and Aging; Nice (IRCAN); CNRS UMR7284/INSERM U1081; Faculty of Medicine; Nice University, Nice, France_.

Background: Although small cell lung carcinomas (SCLC) show an initial response to treatment, patients rapidly suffer disease recurrence and become refractory to chemotherapy. Despite intensive research, the prognosis of SCLC remains poor, and therefore new strategies to improve outcome are urgently needed. Blockade of immune checkpoints PD-1/PD-L1 with monoclonal antibodies has recently emerged as a new therapeutic tool in oncology. Due to the difficulty in obtaining adequate tissue in diseases such as SCLC for PD-L1 expression assessment, we investigated the usefulness of circulating tumor cells (CTCs) detection as a non-invasive method to evaluate PD-L1 status in SCLC patients.

Materials and Methods: CTC capture and PD-L1 expression was evaluated in a cohort of 18 SCLC patients for whom resected tissue specimens were available and were positive for CTCs, as detected by using the filtration-based ISET platform (Rarecells Diagnostics, Paris, France). PD-L1 expression was evaluated in ISET-captured CTCs and FFPE tissue sections by immunohistochemistry (IHC) using the SP142 rabbit monoclonal antibody (Spring Bioscience, USA). PD-L1 expression was scored on tumor cells (TC3≥50% positive tumor cells; TC2≥5% but <50% positive tumor cells; TC1≥1% but <5% of positive tumor cells; and TC0<1% positive tumor cells) and tumor-infiltrating immune cells (IC3≥10% of positive immune cells; IC2≥5% but <10% of positive immune cells; IC1≥ 1% but <5% of positive immune cells; and IC0< 1% of positive immune cells).

Results: None of the SCLC cases showed PD-L1 protein expression in tumor cells on tissue specimens. PD-L1 expression was observed in the stroma. Using IHC assay, 67% of cases (8/12) were scored IC1-3 based on PD-L1 expression in tumor-infiltrating immune cells and 42% (5/12) were scored IC2/3. On the ISET platform, CTCs enumeration ranged from 3 to 637 CTCs/6 ml, with median 11 CTCs/6 ml, and clusters of CTCs in 83% of patients. No PD-L1 expression was observed in CTCs. However, 42% of cases showed PD-L1 expression in circulating immune cells, with PD-L1 status on filters being concordant with the IC2/3 status in patient-matched tumor tissue.

Conclusion: PD-L1 seems expressed in only a fraction of SCLC, with carcinoma cells being negative in all cases, and PD-L1 expressed in tumor-infiltrating immune cells. Patients with stromal PD-L1 expression may potentially respond to anti-PD-1 treatment. Besides conventional IHC, ISET platform seems suitable for non-invasive real-time detection of circulating PD-L1 expression to determine PD-L1 status of SCLC patients entering clinical trials.

#2221

HHLA2, a novel member of the B7 family of inhibitory ligands, is highly expressed in osteosarcoma.

Pratistha Koirala,1 Michael Roth,2 Jonathan Gill,2 Jordan Chinai,1 Michelle Ewart,3 Sajida Piperdi,1 David Geller,3 Bang Hoang,3 Yekaterina Fatakhova,4 Maya Ghorpade,2 Xingxing Zang,1 Richard Gorlick1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Children's Hospital at Montefiore, Bronx, NY;_ 3 _Montefiore Medical Center, Bronx, NY;_ 4 _Sophie Davis School of Biomedical Education, Manhattan, NY_.

Purpose: Over the past four decades there have been minimal improvements in outcomes for patients with osteosarcoma (OS). New targets and novel therapies are needed to improve outcomes for these patients. In this study, we sought to evaluate the prevalence and prognostic utility of the immune checkpoint inhibitor HHLA2 in OS.

Experimental Design: HHLA2 expression was evaluated in two cohorts of OS patients using a tumor microarray (TMA) (n=62) and whole slides (n=48). HHLA2 expression was assessed in primary tumor specimens and metastatic disease, and correlated with the presence of tumor infiltrating lymphocytes (TILs), and event free survival.

Results: HHLA2 was expressed in 68% of OS tumors in the TMA and in 54% in the second cohort. HHLA2 was expressed in almost all metastatic disease specimens and was more prevalent than in primary specimens without known metastases (93% vs 53%, p=0.02). TILs were present in 75% of all osteosarcoma specimens. Patients whose tumors were ≥25% or ≥50% HHLA2 positive had significantly worse five-year event-free-survival (33% vs 64%, p=0.03 and 14% vs 59%, p=0.02).

Conclusions: HHLA2 is expressed in the majority of OS tumors, is associated with metastatic disease, and is associated with poorer survival. Further studies are needed assessing the effect of HHLA2 expression on the function of the immune microenvironment, as well as assessing the feasibility and utility of targeting HHLA2 in OS.

#2222

Dual inhibition of cancer stemness and immune checkpoint genes by targeting Stat3.

Yuan Gao, Sarah Keates, Eric Hsu, Janet Huang, Emily Brooks, Matt Hitron, Youzhi Li, Harry A. Rogoff, Chiang Li. _Boston Biomedical, Inc., Cambridge, MA_.

Highly malignant stemness-high cancer cells, also termed cancer stem cells, have been found to hijack stemness genes important for embryonic stem cells to acquire a state of high-stemness. Cancer stemness has been associated with enhanced tumorigenicity and resistance to chemotherapies. Moreover, stemness-high cancer cells have been isolated from a variety of human cancer types. Therefore, targeting cancer stemness holds significant promise for advancing cancer treatment.

Recent clinical success with antibodies targeting PD1 and PDL1 has validated that cancer cells can hijack immune checkpoint genes to subvert endogenous anticancer surveillance by the immune system. It is, therefore, highly desirable to develop therapeutics that simultaneously target both cancer cell stemness and immune checkpoints.

The transcription factor Signal Transducer and Activator of Transcription 3 (Stat3) is activated by a multitude of upstream oncogenic pathways and cytokine receptors. Aberrant and constitutive activation of Stat3 has been found in a wide variety of human cancers, and has also been implicated in cancer cell proliferation, survival, and immune evasion.

By using aiRNA (asymmetric RNA duplexes) we have achieved potent Stat3 silencing with precision in stemness-high cancer cells. We observed that Stat3 silencing leads to simultaneous down regulation of cancer cell stemness and immune checkpoint gene expression. Treatment of stemness-high cancer cells with BB608, a small molecule inhibitor of Stat3, led to simultaneous inhibition of stemness-high cancer cell survival and self-renewal, as well as downregulation of immune checkpoint genes, including IDO1 in vitro and in vivo. Furthermore, while expression of stemness genes and immune checkpoint genes in tumor tissues are known to predict poor patient survival, patients with higher expression levels of stemness genes and immune checkpoint genes showed prolonged overall survival after treatment with BB608 in clinical trials. Targeting Stat3 is, therefore, a promising strategy to achieve dual inhibition of cancer cell stemness and immune evasion.

#2223

Membranous expression of programmed cell death-ligand 1 (PDL1) on cancer cells is induced by cisplatin in an ATR dependent manner.

Lea Schaaf,1 Meng Dong,1 Simon Heine,1 Heiko van der Kuip,1 Walter E. Aulitzky2. 1 _Dr. Margarete Fischer-Bosch Institute and University of Tuebingen, Stuttgart, Germany;_ 2 _Robert Bosch Hospital, Stuttgart, Germany_.

Cancer cells frequently develop strategies allowing to escape potential immune attacks by the host. One central component of this defense mechanism is the PDL1-PD1 axis with PDL1 expression on the surface of antigen presenting cells. DNA damaging chemotherapeutic are known to have suppressive effect on the immune system. Possible effects on immune checkpoints, however, are poorly understood. We investigated if Cisplatin, a widely used chemotherapeutic for treatment of ovarian and lung carcinomas, might influence expression of PDL1 on cancer cells of these pathologies. Treatment of cell lines as well as precision-cut tissue slices from patient tumors with clinically relevant dosages of cisplatin led to a significant induction of PDL1 protein in cancer cells as revealed by Western Blot and IHC analyses. This effect was independent on ATR or ATM activity since pre-treatment with ATR or ATM inhibitors had no significant effect on cisplatin induced total PDL1 protein levels. Interestingly, however, we observed an almost complete inhibition of cisplatin-induced membranous PDL1 upon ATR but not ATM inhibition as revealed by FACS analysis in unfixed cells. The role of the ATR axis was further corroborated by the finding that presentation of PDL1 at the cell surface was paralleled by phosphorylation of CHK1.

Together our data indicate an important role of DNA damage induced ATR/CHK1 activity on the localization of PDL1 at the surface of cancer cells. These data implicate that combination of cisplatin with either anti-PD1 or anti-PDL1 antibodies or ATR/CHK1 inhibitors should reduce immunosuppressive mechanisms and enhance chemotherapy efficacy.

#2224

Discovery, development and characterization of a new class of therapeutic anti-PD-1 antibody.

Lei Wang, Najmia Amirina, Fellix Scheuplein, Vikki Spaulding, Yanyan Wang, Sri Vadde, M. Isabel Chiu, Daniel Doty, Thomas McQuade, Bin Feng, Sheila Ranganath, Anhco "Cokey" Nguyen. _Enumeral Biomedical, Cambridge, MA_.

Blockade of immune checkpoint (IC), such as PD-1, confers durable clinical benefit to a minority of melanoma and lung cancer patients. Challengingly, the 5-year survival rate is still below 30% in those patients, and most other solid tumors are not particularly responsive to immunotherapy. Therefore, more IC blocking mAbs are under development to meet clinical needs. Using Enumeral's unique single cell immunoprofiling platform, we developed a novel class of anti-PD-1 antibodies, named 244C8, with distinct epitope binding site than currently marketed anti-PD-1 antibodies. To evaluate the function of 244C8 on anti-tumor immunity, we employed ex vivo methods to determine responsiveness of tumor infiltrating lymphocytes (TILs), isolated from human lung cancer biopsies, to various anti-PD-1 antibodies, including our lead anti-PD1 therapeutic candidate 244C8. The results show that all anti-PD-1 antibodies tested reversed T cell exhaustion in tumor microenvironment. Further, 244C8-treated cells produced increased IFN-y compared to nivolumab, indicating a stronger anti-tumor immunity. In the settings of mixed lymphocyte reaction (MLR) and CMV antigen-recall assays, 244C8 treatment also led to higher levels of T cell activation compared to conventional anti-PD-1 antibodies. Interestingly, in both ex vivo and in vitro models, we observed a pattern of cytokine production in 244C8-treated cells that is consistently distinct from that produced by conventional/marketed anti-PD-1 antibodies, suggesting a differentiated mechanism of action.

#2225

Image analysis-based PD-L1 companion and complementary diagnostics.

Joseph S. Krueger, Roberto Gianani, Brooke Hirsch, Stefan Pieterse, Famke Aeffner, David Young. _Flagship Biosciences, Westminster, CO_.

The PD-1 pathway, comprised of the immune cell co-receptor Programmed Death 1 (PD-1) and its ligands PD-L1 and PD-L2, mediates local immunosuppression in the tumor microenvironment. Immune checkpoint modulators are designed to block the local immunosuppression caused by this pathway. The FDA approved anti-PD-1 antibody therapies Opdivo® (nivolumab; Bristol-Myers Squibb) and Keytruda® (prembrolizumab; Merck & Co) rely on PD-L1 immunohistochemistry (IHC) in vitro diagnostic (IVD) tests to determine the PD-L1 status in patients in non-small cell lung cancer (NSCLC), in order to predict response to these drugs. The current complementary diagnostic for Opdivo® (Dako 28-8 PharmDx®) relies on a pathologist scoring paradigm which considers any patient with ≥1% positive tumor cells an optimal candidate for Opdivo® treatment. However, overall survival (OS) is further increased when patients have ≥5% or ≥10% PD-L1 positive tumor cells. This scoring approach is vastly different than the PD-L1 scoring approach used in the Keytruda® companion diagnostic (Dako 22C3 PharmDx®), which utilizes a ≥50% positive tumor cells value to predict a positive Overall Response Rate (ORR; OS not yet determined). Thus, the 28-8 test for Opdivo® utilizes a more precise approach than the 22C3 test for Keytruda®, and requires a more calibrated scoring approach. This calibrated approach for Opdivo® requires the difficult challenge of pathologists reliably distinguishing membrane staining to define the fine gradations of 1%, 5% and 10% PD-L1 positive neoplastic cells. To best meet this challenge, we developed a digital Tissue Image Analysis (TIA) solution which enabled accurate, unbiased quantification of PD-L1 on a cell-by-cell basis to classify the percentage positive tumor cells in patients with high granularity. Using Flagship's proprietary CellMapTM algorithm, we evaluated 40 formalin-fixed paraffin-embedded (FFPE) NS-NSCLC samples which were stained using the Dako 28-8 PharmDx® PD-L1 IHC test. The TIA strategy digitally separated tumor cells from other cell types, and quantified membrane staining intensity according to a consistent threshold. The performance of the resulting IHC-TIA assay was evaluated in the context of a CLIA validation study performed by Flagship. The results demonstrated equivalency to the manually scored IVD reference standard; however, the TIA scoring of this assay provided consistent, unbiased, and more detailed scoring of PD-L1 stained tissues for determining the patients with ≥1, ≥5, and ≥10% PD-L1 positive tumor cells with greater confidence than a manual scoring approach. Moving forward, these TIA tools can be utilized to assess PD-L1 positive cell frequencies with greater reliability and granularity to identify optimal treatment cutpoints for these and other PD-L1 IHC tests used to predict response to PD-L1/PD-1 inhibitors.

#2226

Anti-human PD-1 antibody BGB-A317 exhibits potent immune cell activation.

Tong Zhang, Jing Song, Yucheng Li, Jie Ma, Yingdi Shi, Xiaoran Wu, Lanlan Xu, Qi Liu, Yanping Cao, Yali Wang, Hao Peng, Hongjia Hou, Xu Zhao, Xiaomin Song, Lai Wang, Min Wei, Lusong Luo, Kang Li. _BeiGene, Beijing, China_.

Background: PD-1 is a "check point" inhibitory receptor, which is primarily expressed in activated and/or exhausted T cells. Engagement of PD-1 receptor by its ligand PD-L1 or PD-L2 (expressed on APCs and some tumor cells), leads to negative regulatory signaling in T-cells, promoting functional exhaustion of tumor-infiltrating T-lymphocytes. BGB-A317 is a novel humanized IgG4 anti-PD-1 antibody under clinical development. The immunomodulatory activity of BGB-A317 was evaluated in in vitro assays and reported here.

Materials and methods: BGB-A317 was generated through hybridoma fusion, humanized by CDR grafting and structural simulation. The Fc and hinge regions of BGB-A317 were engineered to remove Fc gamma receptor (FcγR) binding and to stabilize the hinge region. The binding affinity and specificity were studied by ELISA, FACS and SPR (Biacore). The immunomodulatory functions of BGB-A317 were evaluated using both T-cell lines and primary immune cells.

Results: BGB-A317 binds to the extracellular domain of human PD-1 with high affinity (KD = 0.15nM) and specificity. In competition assays, BGB-A317 efficiently blocks the interactions between PD-1 and its ligands. In vitro cell-based assays, BGB-A317 significantly enhances cytokine (IL-2 and IFN-γ) production using T-cell line or primary T cells cocultured with signal-sending cells in a dose-dependent manner. BGB-A317 also potently increases NK cell-mediated IFN-γ production and cytotoxicity against PD-L1+ tumor cells. As an important functional attribute, BGB-A317 exhibits no binding to any of FcγRs, including FcγRI, FcγRIIA, FcγRIIB and FcγRIIIA. Therefore, no antibody-dependent cell-mediated (ADCC) or complement-dependent cytotoxicity (CDC) has been observed in activated T cells treated with high concentration of BGB-A317 up to 100 μg/ml. BGB-A317 is stable as bivalent antibody unlike the wild type human IgG4 going through dynamic conformational change, forming bispecific antibody by Fab-arm exchange.

Conclusions: BGB-A317 demonstrated potent immune cell activation in in vitro assays, supporting its clinical development for the treatment of human cancers.

Keywords: Anti-PD-1, immunotherapy, functional assays

#2227

Positive correlation between the gamma-H2AX and PD-L1 expressions in lung squamous cell carcinoma.

Atsushi Osoegawa, Takafumi Hashimoto, Yohei Takumi, Miyuki Abe, Hitomi Hiraishi, Shuji Suehiro, Michiyo Miyawaki, Kenji Sugio. _Oita University Faculty of Medicine, Yufu, Japan_.

Lung cancer is a leading cause of cancer death worldwide. Recently, molecular targeting therapy has been developed and it has shown promising results against advanced lung cancer. Among them, lung squamous cell carcinoma has fewer treatment options because it is not driven by oncogenic mutations, but by alterations in tumor suppressor genes and subsequent chromosomal instability. Programmed death-ligand 1 (PD-L1) is one of the immune checkpoint molecules that is expressed on the surface of tumor cells, where it inhibits activities of cytotoxic T cells. Recent studies revealed that the PD-1/PD-L1 blockade could improve the overall survival in lung squamous cell carcinoma, potentially because it carries high mutation burdens and neoantigens which could be targeted by cytotoxic T cells. We hypothesized that DNA damage could accumulate in tumors with high mutation burdens, thereby inducing the PD-L1 expression on tumor cells, sensitizing them to anti-PD-1 therapy. An immunohistochemical analysis of 41 consecutive lung squamous cell carcinoma cases, which received surgery at our institution between April 2013 and March 2014, revealed that a high PD-L1 expression was observed in 15 patients (37%) that was associated related with a poor recurrence-free survival (p=0.028). The PD-L1 expression level was also positively associated with the γH2AX expression (a direct marker for DNA damage) (p=0.02). The γH2AX expression in tumor cell nuclei was confirmed by immunofluorescent staining. It forms foci in tumor cell nuclei, indicating the incidence of DNA double strand breaks. Our findings demonstrate for the first time that the nuclear γH2AX expression in lung squamous cell carcinoma is positively associated with the PD-L1 expression. Further studies are warranted to investigate whether γH2AX could be a biomarker for PD-1 targeting therapy and whether combination therapy with PD-1 targeting therapy and a DNA-damaging agent has synergistic effects.

#2228

Cbl-b inhibitors as novel intra-cellular checkpoint inhibitors for cancer immunotherapy.

Saket Agarwal,1 Jian Wu,1 Chris Riling,1 Matthew Kodrasov,1 Joseph Weinstock,1 Ivan Sokirniy,1 Michael Mattern,1 Taku Kambayashi,2 Suresh Kumar1. 1 _Progenra Inc, Malvern, PA;_ 2 _University of Pennsylvania, PA_.

The approval by the FDA and clinical use of immune checkpoint inhibitors, such as anti-PD1 and anti-CTLA4 antibodies, that activate cytotoxic T lymphocyte (CTL) mediated anti-tumor immune responses against advanced solid tumors has resulted in a paradigm shift in cancer therapy. Antitumor activity of CTLs and NK cells is tightly regulated by intracellular signaling events orchestrated by the ubiquitin signaling system. In particular, Cbl-b (Casitas B-lineage lymphoma proto-oncogene b), a RING finger E3-ubiquitin ligase primarily expressed in immune cells, acts as an intracellular checkpoint and a master negative regulator of both CTLs and NK cells. Accordingly, Cbl-b-/- mice have been observed to reject a variety of implanted metastatic and non-metastatic tumors and to delay significantly the outgrowth of spontaneous tumors; these observed antitumor effects were seen to be mediated by activated CD8+ T cells and NK cells. Based on the overwhelming evidence supporting the role of Cbl-b in immune suppression, this ubiquitin conjugating enzyme is considered a novel target for developing small molecule cancer immunotherapy agents. Using proprietary high throughput screening technologies, Progenra identified novel, selective Cbl-b inhibitors that were shown to decrease ubiquitination of substrates such as TAM receptor kinases (Tyro3) and to activate T cells (as judged by increased IL-2 production) and NK cells (as judged by increased IFNγ production and degranulation) in ex vivo functional assays. ADME/DMPK evaluation and in vivo efficacy of selected Cbl-b inhibitors will be discussed in relation to the therapeutic utility of this class of small molecule agent, which is expected to potentiate the anticancer effects of approved immunotherapy agents.

#2229

PD-L1 is expressed on CD11bhighCD14highHLA-DR+ myeloid cells in the tumor microenvironment of non-small cell lung cancer (NSCLC).

Chung-Hee Sonn, Yunji Oh, Tae Eun Kim, Jung Sun Ha, Hong Kwan Kim, Jong Ho Cho, Sumin Shin, Jhingook Kim. _Samsung Medical Center, Seoul, Republic of Korea_.

Understanding the immune system of cancer patients is essential for the development of optimal treatment. We analyzed tumor cells and infiltrating immune cells in tumor tissues of NSCLC to determine the type of cells which expressed immune checkpoint molecules and/or their ligands, and compared them to clinicopathological characteristics. To obtain a single cell suspension from the tumor tissues of lung cancer patients, each fresh tumor sample was chopped and treated with collagenase and DNase. The dissociated tumor tissue cells (DTCs) were stained with fluorochrome-labeled mAbs and the distribution of immune cell components and expression of surface markers in DTCs were determined by FACSuite analysis. The yield of DTCs was 5.87±4.73x10^7/gram. PD-L1, which is a ligand for immune checkpoint molecule PD-1, is expressed on CD11bhigh myeloid cells in the blood, regardless of HLA-DR expression. On the other hand, PD-L1 is expressed on CD11highCD14highSSClow myeloid cells in the tumor site. All PD-L1 expressing cells were positive for HLA-DR, a ligand for a T cell receptor of CD4 T cells. Furthermore, these cells also expressed both CD86 and CD124. Interestingly, most tumor cells expressed very low levels of the PD-L1 molecule. And we found that CD11highCD14+HLA-DR- myeloid cells did not express PD-L1. The proportion of CD11highCD14+HLA-DR-PD-L1- myeloid cells was higher in advanced stage patients than early stage patients. In conclusion, PD-L1 is expressed on CD11bhighCD14highHLA-DR+SSClow myeloid cells, but not CD11bhighCD14+HLA-DR- myeloid cells. We propose that the PD-L1+ myeloid cells are likely to play an important role in tumor immunity. Further functional studies will be necessary to determine how the interaction between the cells will affect immunity.

code no | age | gender | smoking | stage | cell type | PD-L1+HLA-DR+CD14highCD11bhigh cells (%) | PD-L1-HLA-DR-CD14+CD11bhi cells (%)

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

L-032 | 57 | male | yes | IA | mucinous adenocarcinoma | 39.8 | 1.2

L-052 | 81 | male | yes | IA | adenocarcinoma | 19.4 | 4.1

L-048 | 49 | male | yes | IIIA | squamous cell carcinoma | 4.0 | 9.0

L-024 | 71 | male | yes | IIIA | adenocarcinoma | 11.2 | 0.4

L-036 | 65 | male | yes | IV | adenocarcinoma | 11.4 | 5.3

L-042 | 73 | male | yes | IV | adenocarcinoma | 21.6 | 5.5

L-046 | 74 | male | no | IA | adenocarcinoma | 6.1 | 1.6

L-021 | 67 | female | no | IB | large cell, adenocarcinoma | 14.7 | 2.3

L-019 | 76 | female | no | IB | adenocarcinoma | 15.8 | 16.7

L-271 | 45 | female | no | IV | adenocarcinoma | 11.2 | 17.2

L-272 | 45 | female | no | IV | adenocarcinoma | 9.9 | 24.8

#2230

Evaluation of PD-L1 expression in ovarian carcinosarcoma by immunohistochemistry and RNAscope in-situ hybridization.

Li Liang,1 Jun-Song Chen,1 Yi Jiang,1 Na Niu,1 Connie Zhang,2 Emily Park,2 Xiao-Jun Ma,2 Jinsong Liu1. 1 _The University of Texas MD Anderson Cancer Center, HOUSTON, TX;_ 2 _Advanced Cell Diagnostics, Inc., Heyward, CA_.

Ovarian carcinosarcoma, also called malignant Müllerian mixed tumor, is a rare, but very aggressive biphasic tumor with both malignant epithelial and stromal components. PD-L1 is one of the ligands of immune inhibitory receptor PD-1. PD-L1/PD-1 pathway‎ suppresses T cell immune response, increases programmed cell death of activated T cells and plays an important role in tumor immune escape. Anti-PD-L1 immunotherapy has shown promising results in certain malignancies, such as melanoma and lung cancer. Therefore, we performed PD-L1 immunohistochemical staining on 30 specimens of ovarian carcinosarcoma collected from patients that underwent surgeries at our institution between 2002 and 2015. In addition, we performed RNAscope in-situ hybridization to examine PD-L1 mRNA expression in 16 specimens that were collected within past 5 years (2010-2015). Our result demonstrated focally membranous expression of PD-L1 protein in 23 of 30 cases (77%). PD-L1 mRNA expression correlated with membranous, but not cytoplasmic expression of PD-L1 protein. In conclusion, PD-L1 protein and mRNA are frequently expressed in ovarian carcinosarcoma, suggesting anti-PD-L1 immunotherapy may be helpful in these patients.

#2231

IO125 is a potent inducer of tumor immunogenicity.

Sanghamitra Mylavarapu,1 Shelly Kaushik,1 Yashpal Yadav,1 Monideepa Roy,1 Aniruddha Sengupta,1 Shiladitya Sengupta2. 1 _Invictus Oncology, New Delhi, India;_ 2 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA_.

Combinatorial treatments with chemotherapeutics and immunotherapies are currently being investigated in several cancers. Chemotherapeutics can increase the immunogenicity of tumors besides modulating the immune system. Here we describe the development of a novel platinum-based therapeutic, IO-125, which assembles into stable nanoscale supramolecular structures. IO125 demonstrates enhanced activity compared with standard Pt drug across cancer lines. IO-125 preferentially targets tumors in vivo, and demonstrated improved efficacy in triple negative breast cancer (4T1), Lewis lung carcinoma (LLC) and melanoma (B16/F10) tumor models. Treatment with IO-125 elevates expression of immune-markers (including IGKC, PD-L1, PD-1, CD8A), which are associated with good prognosis. In addition, combinatorial treatment with IO-125 and antibody against immune checkpoint inhibitor(s) resulted in superior antitumor efficacy compared to treatment with antibody alone. This study shows that supramolecular therapeutics, especially IO125, can emerge as a novel approach to focally modulate the tumor immune contexture.

#2232

Immune-inflammatory markers predict the outcome of metastatic colorectal cancer patients treated with the thymidylate synthase poly-epitope peptide (TSPP) vaccine: results from a multi-arm TSPP/VAC phase Ib program.

Pierpaolo Correale,1 Cirino Botta,2 Elodia Claudia Martino,1 Valerio Nardone,1 Cristina Ulivieri,3 Claudia Gandolfo,4 Tatiana Cosima Baldari,3 Stefano Lazzi,5 Alessandro Ginori,5 Antonella Fioravanti,6 Giacomo Maria Guidelli,6 Luigi Pirtoli,1 Antonio Giordano,7 Pierfrancesco Tassone,2 Pierosandro Tagliaferri,2 Maria Grazia Cusi4. 1 _Unit of Radiotherapy and Unit of Pathology, Department of Oncology, Azienda Ospedaliera Universitaria Senese, Siena, Italy;_ 2 _Department of Experimental and Clinical Medicine, "Magna Graecia" University and Medical Oncology Unit, and Medical Oncology Unit, AUO "Materdomini", Catanzaro, Siena, Italy;_ 3 _Life Sciences Department, University of Siena, Siena, Italy;_ 4 _Microbiology and Virology Unit, Department of Medical Biotechnology, Azienda Ospedaliera Universitaria Senese, Siena, Italy;_ 5 _Unit of Pathology, Department of Oncology, Azienda Ospedaliera Universitaria Senese, Siena, Italy;_ 6 _Unit of Rheumatology, Department of Clinical Medicine and Immunologic Sciences, Siena, Italy;_ 7 _Department of Biotechnology, Temple University, "Sbarro" Foundation, Philadelphia, PA_.

Thymidylate synthase (TS) is a tumor-associated enzyme critical for DNA replication. TSPP is a poly-epitope-peptide vaccine to TS, which elicits a multi-epitopic CTL response with antitumor activity in preclinical models. TSPP has been tested in 50 advanced cancer patients enrolled in a multi-arm dose-finding TSPP/VAC1 phase Ib trial program (Eudract:# 2009-016897-33)(Table1) between May 2011 and January 2013. The trial results revealed that TSPP is safe and immune-biologically active, and provided preliminary evidence of a antitumor activity in pretreated mCRC patients (Cusi MG et al 2015 and Correale P et al 2015). This population of 41 patients presented a progression-free-survival and an overall-survival (OS) of 6.9 and 11.3 months, respectively, with a one-year survival rate of 32% (13 patients). We focused on mCRC patients as they represented the most relevant and homogeneous population in the trial. We performed Kaplan-Meier curves, log-rank tests and regression curves to correlate immunobiological findings and patients' outcome. We found that their OS correlated with lower (under the median) ECOG scores (p:0.039), CEA levels (p:0.021), and serum levels of inflammatory markers (NLR, CRP, ESR, and LDH/LDHNR and ENA; p inferior 0.04) at baseline. Patients' OS also correlated with greater (over the median) baseline levels of IL4 (p:0.028) and 17 (P:0.049), and with a post-treatment increase in anti-neutrophil-cytoplasm-antibodies/anti-proteinase-3 (Fold change to baseline values grater than 1; p:0.039). Activating K-ras mutations did not correlate with survival, however, inflammatory markers, cytokines, and autoantibodies lost their correlation with the survival in patients bearing these mutations. These results suggest that TSPP-antitumor activity in mCRC patients is affected by their baseline inflammatory/immunological status at baseline.

TSPPVAC1 Phase Ib program; patients' distribution

---

ARM A

1. ADJ + TSPP 100 µg (n=3)

2. ADJ + TSPP 200 µg (n=3)

3. ADJ + TSPP 300 µg (n=6)

ARM B

1. ADJ + TSPP 100 µg + IG-1 (n=3)

2. ADJ + TSPP 200 µg + IG-1 (n=3)

3. ADJ + TSPP 300 µg + IG-1 (n=3)

ARM C (DL1-3)

1. GOLFIG + ADJ+TSPP 100 µg + IG-1 (n=3)

2. GOLFIG + ADJ+TSPP 200 µg + IG-1 (n=3)

3. GOLFIG + ADJ+TSPP 300 µg + IG-1 (n=11)

ARM C (DL0)

1. GOLFIG (12 courses) -> ADJ + TSPP 300 µg + IG-1 (n=10)

Figure legend: ADJ = montanide;

n= number of patients;

IG1 = sc. GM-CSF and sc. Aldesleukine;

GOLFIG = gemcitabine + FOLFOX4 polychemotherapy + IG1 regimen

#2233

PI3K/Akt/mTOR signaling and plasma membrane proteins are implicated in responsiveness to adjuvant dendritic cell vaccination for metastatic colorectal cancer.

David Qian,1 Jinyoung Byun,1 Xiangjun Xiao,1 Stephanie Her,2 Arief Suriawinata,3 Christopher Amos,1 Richard Barth3. 1 _Dartmouth Geisel School of Medicine, Hanover, NH;_ 2 _Dartmouth College, Hanover, NH;_ 3 _Dartmouth-Hitchcock Medical Center, Lebanon, NH_.

Purpose: We have previously demonstrated that metastatic colorectal cancer patients who exhibit immune responses to a dendritic cell (DC) vaccine have superior recurrence-free survival following surgery, compared to patients in whom immune responses do not occur. We sought to characterize the patterns of T lymphocyte infiltration and somatic mutations in metastases that are associated with and predictive of response to the DC vaccine.

Methods: Cytotoxic, memory, and regulatory T cells in resected metastases and in surrounding normal liver tissue from 22 patients (11 responders and 11 non-responders) were enumerated by immunohistochemistry prior to vaccine administration. In conjunction with tumor sequencing, the Combined Multivariate and Collapsing method was used to identify gene mutations that are associated with vaccine response. We also derived a response prediction score for each patient using his/her tumor genotype data and variant association effect sizes computed from the other 21 patients; greater weighting was placed on genes that encode plasma membrane proteins.

Results: There was no relationship between vaccine response and intra-tumor, peri-tumor, or hepatic densities of T cell subpopulations. Associated genes were found to be statistically enriched in PI3K, Akt, and mTOR signaling pathways (P < 0.001). With respect to prediction, applying a consistent score cutoff over the 22 rounds of leave-one-out cross-validation correctly inferred vaccine response in 21 of 22 patients (95%).

Conclusions: Adjuvant dendritic cell vaccination has shown promise as a form of immunotherapy for patients with metastatic colorectal cancer. Its efficacy may be influenced by somatic mutations that affect pathways involving PI3K, Akt, and mTOR, as well as neoantigens on tumor cell surfaces.

#2234

Exploring the oncolytic potential of Newcastle Disease Virus.

James A. Harper,1 Jon Travers,1 Ruth Franks,1 Shannon Burke,1 Christel Navarro,1 Cheng Xing,2 Weijia Wang,2 Qi Xu,2 Robert W. Wilkinson,1 Hong Jin,2 Danielle Carroll1. 1 _Medmmune Ltd, Cambridge, United Kingdom;_ 2 _Medmmune LLC, Mountain View, CA_.

Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell death occurs as a natural consequence of the viral life cycle and newly produced virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of potentially producing novel tumour antigens that can induce an immune-mediated therapeutic response.

Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of mammalian cancers in PhI clinical studies. Using reverse genetics, we generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. We have additionally inserted the GM-CSF gene in order to maximize anti-tumour immune response and provide an in situ, patient/tumour- specific vaccine, combined with potent oncolysis. We have evaluated the biological characteristics of recNDVGM-CSF in vivo and in vitro. In vitro recNDVGM-CSF selectively replicates in and kills a wide variety of tumour cell types. In vivo, using a fibrosarcoma model that supports viral replication, a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. Additionally, following IV administration recNDVGM-CSF localises to and replicates within the tumour leading to a localised GM-CSF production within the tumour. The inherent properties of NDV (self-propagation, tumour-selective replication, immune activation) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients.

#2235

Clinical results of a Phase I/II trial of adjuvant therapeutic vaccination in high risk resected prostate cancer patients using autologous dendritic cells loaded with mRNA from primary prostate cancer tissue, hTERT and survivin.

Anne Merete Aa. Tryggestad,1 Karol Axcrona,1 Iris Bigalke,1 Else M. Inderberg-Suso,1 Gjertrud Skorstad,1 Ulrika Axcrona,1 Steinar Aamdal,1 Gustav Gaudernack,2 Dolores Schendel,3 Wolfgang Lilleby,1 Svein Dueland,1 Gunnar Kvalheim1. 1 _Oslo University Hospital, Norwegian Radium Hospital, OSLO, Norway;_ 2 _Ultimovacs AS, Norway;_ 3 _Medigene Immunotherapies GmbH, Germany_.

Prostate cancer patients diagnosed with high Gleason score (≥ 8) and large tumors (≥T2c) are considered high-risk patients and >50% will develop an early biochemical relapse. Presently, there is no curative therapy available for patients with biochemical relapse. Based on these findings we initiated in January 2011 a Phase I/II dendritic cell (DC) vaccine study. Patients included have pathological stage pT2 - pT3b, Gleason score 7b-10, pN0, pN+ or pNx and postoperative PSA < 0.2 µg/L. Following surgery autologous tumor cell lines were established from each patient using an in-house culturing method. mRNA from the tumor cell line was produced and used for DC vaccination in combination with mRNA hTERT and mRNA Survivin. DCs were differentiated from enriched monocytes, cultured for 2 days with IL4 and GM-CSF and matured with Jonuleit-maturation cocktail for 24 hours. The matured DCs were transfected separately with the 3 different mRNAs and then frozen and stored until use. The vaccination regimen includes one vaccine per week for four weeks, followed by monthly "vaccine boost" during the first year, then every 3 months the second and third year. Recently, a novel 3 days DC protocol using a TLR7/8-agonist maturation cocktail has been implemented at our department. Based on encouraging clinical results with this type of DCs in compassionate use patients with different types of tumors, we decided to change our DCs protocol to the new generation DCs. Of the 20 patients included in this trial 15 patients have been given the standard fast DCs and 5 have been vaccinated with the new type of DCs. 8 Patients given standard DC has completed 3 years of vaccination and 4 has completed 2 years of vaccination. 3 of 15 patient given standard DC has experienced PSA relapse during vaccination. None of the patients given the new type of DC has so far experienced raise in PSA levels. To our knowledge this is the first adjuvant DC vaccine study in high risk prostate cancer and we conclude that the study is feasible, safe and utmost promising. Extensive immune monitoring is ongoing taking advantage of the established autologous tumor cell lines from all patients.

#2236

Long lasting CR status of stage IV gall bladder cancer and colon cancer after combined treatment including autologous formalin-fixed tumor vaccine (AFTV).

Fumito Kuranishi,1 Yusuke Sumi,1 yoij Uemae,2 Tadao Ohno2. 1 _Innoshima Ishikai Hospital, Onomichi, Japan;_ 2 _Cell-medicine Inc.,, Tsukuba, Japan_.

(INTRODUCTION) The prognosis of advanced (stage IV) cancer is very poor. However, we have already reported two cases of eradication of advanced breast cancer with bone metastasis (AACR 2013 and AACR 2015) after combined treatments including AFTV. We here additionally report successful cases of gall bladder cancer (stage IV, liver metastasis) and colon cancer (stage IV, lung metastasis).

(CASE 1) A 61-year-old female with stage IV gall bladder cancer (T3N1M1: liver metastasis) was operated in May 2011 including partial resection of liver. She was treated with, as the first-line adjuvant therapy, AFTV followed with conventional chemotherapy. This patient is still alive without any recurrence confirmed by CT etc.

(CASE 2) A 60 year-old male with stage IV (N3, H0, P1, M1: lung, lt. adrenal gland, abdominal wall) was operated in August 2009. Then conventional chemotherapy, 2 courses of AFTV and radiation were applied sequentially. No recurrence was observed for more than 5 years.

(DISCUSSION) We diagnosed both patients staying at long lasting complete remission. The prognoses of stage IV gall bladder cancer and abdominal wall-metastasized colon cancer are very poor despite of conventional treatments. Adverse effects of AFTV were less than grade 2, therefore combination therapies including AFTV are worth to consider, although larger scale clinical trial is essential.

#2237

Gene expression measurement of immuno-oncology targets in a single FFPE section using a novel targeted sequencing assay.

Monica M. Reinholz, Debrah Thompson, James Cooley, Xiao-Bo Chen, Iris Howlett, John Luecke, Patrick Roche, Bonnie LaFleur. _HTG Molecular, Tucson, AZ_.

Background and Purpose: The field of immuno-oncology (IO) covers a broad set of research disciplines and presents a highly diverse set of experimental requirements. The challenges include sample types of varying quality and quantity of material (including both small fixed samples and blood products) as well as an expanding multiplicity of targets to assay for immunological response. The HTG EdgeSeq system combines HTG's proprietary quantitative nuclease protection assay chemistry with a Next Generation Sequencing (NGS) platform to enable semiquantitative analysis of hundreds of targeted genes in a single assay. The novel HTG EdgeSeq Immuno-Oncology Assay measures 549 IO-related genes and can be used with most clinically relevant samples.

Methods: An internal verification study was performed to evaluate a five-point, two-fold titration series (6 concentrations) in a variety of sample types with as few as a 250 cells, 32 µl of PAXgene, or 1.56 mm² of a 5 µm FFPE section. FFPE sample types included: melanoma; DLBCL; and tissue from lung, breast, colon and renal carcinomas. Additionally, examples of the biological relevance of this data are provided by examination of expression from several pairs of samples, each profiled using a single 5 µm FFPE section.

Results: Equivalent expression across the dynamic range was obtained within each of the sample types (Pearson correlations ranging between 0.84 to 0.98). For DLBCL, while most canonical markers of immune cells showed similar patterns of expression, different patterns of expression of T cell and NK cell genes were obtained, likely indicating a different composition of the immune cell infiltrates within these tumors. Specific to the two different melanoma tumors the lymphocyte infiltrates appear to be similar in these tumors. Interestingly, a markedly different immune response appears to be occurring in the melanoma tumors. One tumor appears to be mounting a significant type I interferon response, which is not as apparent in the second tumor. Differential expression of other well-known metastasis-associated genes are also observed between the two tumors. These observations suggest that patterns of differential expression could be used to assist in directing patients to targeted therapies.

Conclusion: The HTG EdgeSeq Immuno-Oncology Assay provides a valuable tool for researchers exploring the immune response to tumors across a wide variety of tissues. Combining highly reproducible results with very small sample input amounts allows the assay to be utilized for the very small and precious samples available to researchers in this field.

#2238

Understanding immune phenotypes and their spatial relationships to breast adenocarcinoma in FFPE tissues.

Yi Zheng, Pallavi Thuse, Linying Liu, Edward C. Stack, Michael Campisano, Kent Johnson, Darryn Unfricht, Nara Narayanan, Clifford Hoyt, Milind Rajopadhye. _PerkinElmer, Hopkinton, MA_.

The relationship between elements of the immune system and breast tumors in situ requires an approach that leverages multiplexed immunohistochemistry (mIHC) with multispectral imaging to facilitate precise image analyses. To achieve this, we developed a novel 7-color mIHC assay, based on tyramide signal amplification, that allowed us to reliably interrogate CD3, CD4, CD8, FoxP3, CD68, and cytokeratin, in formalin-fixed, paraffin-embedded (FFPE) samples of human breast cancer. Imaging was performed using the multispectral Mantra system and inForm image analysis software. Using this specific mIHC panel and the cell segmenting and phenotyping tools in inForm, we were able to reliably identify cytotoxic T cells (CD3+ CD8+), helper T cells (CD3+ CD4+), regulatory T cells (CD3+ CD4+ FoxP3+), tumor associated macrophages (CD68+) and breast tumor cells (CK+). With cell phenotypes within the tumor microenvironment determined based on specific colocalized staining combinations, we then employed spatial point pattern analyses to examine spatial relationships between specific phenotypes. With this analysis, we were able to describe distances between cytotoxic T cells and regulatory T cells, cytotoxic T cells and tumor associated macrophages, as well as cytotoxic T cells and breast tumor cells. With this combined mIHC, multispectral imaging and advanced image analysis, we demonstrate a novel method that allows for unique tumor microenvironment assessments within in breast cancer. Through the preservation of tumor architecture available in archival FFPE tissues, these methods can advance our understanding of unique tumor microenvironment interactions, and could provide the ability to stratify responses to immunotherapies. 

### Biomarkers for Lung Cancer

#2239

Isolation and characterization of circulating tumor cells (CTCs) in non small cell lung carcinoma.

Marianna Scrima,1 Donatella Malanga,2 Carmela De Marco,2 Giuseppe Viglietto2. 1 _Biogem, Ariano Irpino (AV), Italy;_ 2 _Università Magna Graecia Catanzaro, Italy_.

Purpose: Lung cancer is the most common cause of cancer-related deaths that frequently metastasizes prior to disease diagnosis. Circulating tumor cells (CTCs) are of great clinical interest in terms of prognosis and therapy intervention and may provide important prognostic and predictive information in non-small cell lung cancer (NSCLC). The purpose of this study was to evaluate diagnostic performance of a non-invasive method based on filtration-based technology (ISET) using cytomorphologic criteria in patients undergoing surgery comparing with histologic features of corresponding primary lesions.

Materials and Methods: The CTCs analysis was performed through the following steps: isolation by ISET filtration method (ScreenCell®) from 5 ml of peripheral blood of 36 patients with NSCLC and 23 with benign lung lesions; classification of CTCs with haematoxylin and eosin (H&E) staining; identification of cell marker with co-immunostaining for epithelial and mesenchymal markers (Vimentin and Cytocheratin). CTCs were classified as tumor cells if: (i) they have a large (≥12 μm), hyperchromatic, irregular-shaped nuclei; (ii) high nuclear-cytoplasmic ratio (>0.5).

Diagnostic performance was evaluated against pathologist reported diagnoses of cancer from histological or cytological analysis.

Results: CTCs were detected in 27 of 36 (75%) NSCLC patients and in 2 of 23 (9%) negative control patients (p=0.001). No significant differences were found for the percentage of CTCs for primary and metastatic cancer as well as for cancer stages. A test sensitivity and specificity for diagnosing lung cancer was 75% and 91% respectively. After co-immunostaining (Cytocheratin, Vimentin, CD45) CTCs showed positivity (14/15, 93%) only to vimentin (mesenchymal marker) and completely absence of cytocheratin signal (epithelial marker).

Conclusions: The performance of the tested filter-based antibody-independent technology to capture CTCs using standard cytomorphologic criteria provides the potential of a diagnostic blood test for lung cancer showed by high test sensitivity and specificity. The vimentin high expression in CTCs showed both that is a possible marker to CTCs identification and isolation and the importance of EMT mechanism transition in CTCs.

#2240

A case series of ERBB2 indel driver mutations in non-small cell lung cancer identified by cell-free circulating tumor DNA NGS.

Nir Peled,1 Anna Belilovski-Rozenblum,1 Lior Soussan-Gutman,2 Christine Lee,3 AmirAli Talasaz,3 Richard B. Lanman3. 1 _Tel Aviv University, Tel Hashomer, Israel;_ 2 _Oncotest Teva, Teva Pharmaceuticals, Shoham, Israel;_ 3 _Guardant Health, Inc., Redwood City, CA_.

Background: In-frame insertions between codons 775 and 881 in exon 20 of the ERBB2 gene, of which a 12 base pair YVMA insertion is the most common, are activating mutations in 2%-4% of non-small cell lung cancers (NSCLC), and have also been reported in exon 19 and 20 in breast cancer. These driver mutations are not captured with IHC or FISH staining because in the majority of cases, the ERBB2 gene is not amplified and HER2 protein is not overexpressed. Next generation sequencing (NGS) of circulating cell-free DNA (cfDNA) provides a non-invasive means of identifying these potential driver mutations.

Method: Guardant360TM is a targeted cfDNA NGS panel using hybrid capture and complete exon sequencing for single nucleotide variant detection in 70 genes, copy number amplifications (CNA) in 16 genes, and fusions in six genes and indels in EGFR, ERBB2 and MET exon 14 skipping. De-identified pathology and genotyping reports were reviewed for consecutive patients in which ERBB2 indels were identified in clinical practice.

Results: Guardant360 identified ERBB2 indels in 27 of 2,093 (1.3%) of non-squamous NSCLC cases, with a single concomitant ERBB2 gene amplification. For this ERBB2 indel series, pathology reports revealed no patients with HER2 amplification via IHC or FISH, nor ERBB2 point mutation via NGS, but were only available in 25% of cases. Eight of the ERBB2 indels were confirmed by tissue NGS reports with zero false positives (100% PPV). 80% of ERBB2 indels were the common p.Tyr772_Ala775dup (YVMA insertion) in exon 20 followed by other insertions in codons 772 through 814. A single ERBB2 exon 20 p.Leu755_Glu757delinsProLys net deletion at 3.9% mutant allele fration (MAF) was noted in one patient, for whom outcome data was available. Initial tissue was IHC negative for HER2 overexpression at the referring hospital where the archival tissue biopsy was exhausted and so could not be sequenced. Based on the cfDNA finding of ERBB2 indel, the patient was switched from cytotoxic chemotherapy to trastuzumab with objective response on PET/CT and a repeat Guardant360 showed the ERBB2 indel MAF had dropped below the test limit of detection. After four months the patient progressed and it was decided to switch to ado-emtansine trastuzumab (T-DM1) in late November.

Results: ERBB2 indels can be identified without tissue in NSCLC patients with 100% PPV in this modest cfDNA series. In a patient whose tissue was not available for sequencing, an objective response with trastuzumab was obtained.

#2241

Serum and plasma IL-17A concentrations in lung cancer patients and controls.

Animesh Shukla, Sheldon Grove, Simone Barbero, Galina N. Nikolenko, Anu Mathew, Martin K. Stengelin, Eli N. Glezer, Jacob N. Wohlstadter. _Meso Scale Diagnostics, LLC, Rockville, MD_.

Purpose: IL-17A, a cytokine produced by Th17 cells, is reportedly a central regulator of lung tumor growth (Reppert et al., OncoImmunology 1, 2012) and a potential serum biomarker for lung cancer (Xu et al., Biomarkers, 2014). Serum concentrations of IL-17A are too low to measure in most samples (on the order of 0.1 pg/mL) using commercially available ELISAs. In order to overcome this limitation, we developed the S-PLEX™ Human IL-17A ultrasensitive assay which is capable of accurately measuring IL-17A in serum and plasma. We used this assay to measure IL-17A concentrations in serum samples from lung cancer patients and controls.

Methods: Serum samples from 59 lung cancer patients and 44 apparently healthy controls (including 14 heavy smokers) were analyzed using S-PLEX technology to determine IL-17A concentrations. In addition, IL-17A concentrations were measured in matched plasma samples from 24 of the controls. S-PLEX is a new ultrasensitive immunoassay format based on MSD's MULTI-ARRAY® electrochemiluminescence technology. The IL-17A assay requires only 25 µL of serum or plasma per measurement and can be run on the MESO® SECTOR S 600 and MESO QuickPlex® SQ 120 instruments.

Results: The S-PLEX Human IL-17A assay was capable of accurately measuring IL-17A concentrations in 125 of the 127 samples tested. The lower limit of detection was 6 fg/mL and the assay range (LLOQ to ULOQ) was from 20 fg/mL to 200,000 fg/mL. Typical intra-plate coefficients of variation (CVs) ranged between 5% and 15%. Control samples run at 3 levels, in multiple replicates over multiple days, produced CVs <20%. Spike recovery and dilution linearity were between 80% and 120%, and IL-17A levels in matched serum and plasma samples were similar. Specificity of the assay was demonstrated by analyte depletion using several anti-IL-17A specific antibodies, indicating the assay is specific for the IL-17A homodimer. There was no detectable cross-reactivity to the IL-17F homodimer, and the IL-17A/F heterodimer cross-reactivity was less than 1%. In this study, IL-17A serum concentrations were not found to be significantly different between lung cancer patients and controls, with a median concentration of 183 fg/mL and interquartile range (IQR) of 88-369 fg/mL (n=59) for lung cancer patients, compared to 128 fg/mL with an IQR of 75-198 fg/mL (n=44) for controls.

Conclusion: We have developed a highly specific and sensitive IL-17A assay that is 100 times more sensitive than the current limits of ELISA technology. This assay enables accurate determination of IL-17A concentrations in serum and plasma samples from lung cancer patients and healthy controls. The results from this study do not support the use of IL-17A as an effective serum biomarker for the presence of lung cancer.

#2242

cMET in non-small cell lung cancer: pieces of the puzzle.

Nele Van Der Steen,1 Vanessa Deschoolmeester,1 Filip Lardon,1 Christian Rolfo,2 Elisa Giovannetti,3 Godefridus J. Peters,3 Patrick Pauwels2. 1 _University of Antwerp, Wilrijk, Belgium;_ 2 _Antwerp University Hospital, Edegem, Belgium;_ 3 _VU Medical Center, Amsterdam, Netherlands_.

A rapidly growing domain in present-day oncology is the development of targeted therapies, including several cMET inhibitors against non-small cell lung cancer (NSCLC). Nowadays, cMET amplification is used as a standard biomarker for patient selection, however there is no clear cut-off value to determine amplification, nor is there a real consensus about the way to test this. More recently, splicing variants of cMET, which skip exon 14, are gaining importance since it has been shown that patients harboring this mutation can respond to cMET directed targeted therapy. In this study, we explored the occurrence of cMET aberrations and their correlation with cMET expression in a population of 155 primary EGFR-TKI naive NSCLC tumors. Since cMET is considered as important in metastatic behavior, we also investigated the correlation of cMET expression and amplification between the primary tumor and metastases. Finally, given the link between the EGFR and cMET pathways, the EGFR status was also studied.

Samples were collected at the Antwerp University Hospital and the Onze-Lieve-Vrouw Hospital Aalst. The expression of EGFR and cMET was determined by immunohistochemistry (Ventana, clones 3C6 and SP66 respectively), while cMET amplification was examined by in situ hybridization (Ventana, MET DNP and CEN7 DIG probes). EGFR mutations and cMET splice site mutations were detected by Sanger sequencing. Significant correlations were tested using the Chi² and kappa test (SPSS 23).

In 146/155 tumors cMET expression could be determined; 73/146 samples showed high (2+ or 3+ score) cMET expression and 4/118 samples showed amplification (ratio ≥ 2). No significant correlations could be determined between cMET expression and histological subtype of NSCLC (p=0.065), differentiation degree (p=0.468) and cMET amplification (p=0.214). However, a significant correlation was found between cMET expression, EGFR expression (p=0.015) and EGFR mutations (p=0.029). Splice site mutation regions were sequenced in 87/155 samples, all of which were wild-type. When comparing cMET amplification between the primary tumor and the corresponding metastases (n=40), only one sample showed amplification in the metastases and not in the primary tumor. Hence, cMET expression showed a significant correlation between primary tumors and their metastases (kappa=0.003).

The overall results of our study are in agreement with earlier data. Moreover, we showed that overall expression levels of cMET in primary tumors and metastases are very similar despite large intratumor heterogeneity. In conclusion, this study shows that high cMET expression in most NSCLC samples does not originate from presently known genetic cMET aberrations. High cMET expression is likely to be caused by temporary changes in transcription and translation, influenced by EGFR-signaling, miRNAs or other regulatory mechanisms.

#2243

Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts).

Zai Ahmad,1 Jen Fraser-Fish,1 Rajiv Kumar,2 Bernadette Ebbs,1 Gemma Fowler,1 Penny Flohr,1 Mateus Crespo,1 Sanjay Popat,2 Jaishree Bhosle,2 Udai Banerji,1 Mary O'Brien,2 Johann S. de Bono,1 Timothy A. Yap1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _The Royal Marsden NHS Foundation Trust, London, United Kingdom_.

Background: NSCLC exhibits intratumor heterogeneity, with subpopulations of cells undergoing epithelial-mesenchymal transition. Such CTCs from NSCLC pts may be missed by the EpCAM-based CELLSEARCH® system (CS). The label-free ClearCell® FX inertial microfluidic system (FX) isolates CTCs based on size and inertia and may lead to more accurate CTC capture. PD-1/PD-L1 inhibitors have shown benefit in PD-L1+ NSCLC pts, but responses are still seen in PD-L1- pts, suggesting limitations in tumor PD-L1 testing. Also, PD-L1 testing on CTCs may be more practical than tumor rebiopsies and may provide insights into cancer heterogeneity.

Methods: FX was validated with EpCAM-high and EpCAM-low cancer cell lines labeled with CellTracker dyes spiked into healthy volunteer (HV) blood for repeatability and reliability. Enriched cells were detected with the automated Bioview Duet imaging system for recovery (%). Identical spiking studies were conducted on CS for comparison. Antibodies for 5-color immunofluorescence (IF) (CK, CD45, DAPI, TTF1 [to detect lung adenocarcinoma cells], PD-L1) were optimized on EpCAM-high and EpCAM-low cell lines. 8ml of blood from NSCLC pts were enriched with FX for 5-color IF characterization. 7.5ml of blood from the same pts taken at identical timepoints were enumerated with CS for comparison. Blood was obtained from HV for CTC enumeration on FX and CS as controls.

Results: Cell recovery of EpCAM-high cancer cell lines using FX produced similar counts to CS, e.g. lung NCI-H2066 cells: FX 64.9%±4.5 vs CS 74.4%±9.2 (p=0.05); lung H1975 cells: FX 74.1%±11.9 vs CS 74.7%±10.7 (p=0.91). In contrast, for EpCAM-low cells, a significant difference in cell recovery between FX and CS was seen, e.g. lung A549 cells: FX 59.3%±5.8 vs CS 26.1%±7.6 (p<0.0001); and prostate PC3 cells: FX 68.6%±7.4 vs CS 35.5%±6.9 (p<0.0001). CTCs were detected in 19/21 pts with progressing stage IV NSCLC (adenocarcinoma) (mean 57; range 4-304) using FX, vs 7/21 pts (mean 7; range 1-38) using CS. CTC counts were higher with FX vs CS in 19/21 (90%) NSCLC pts, p=0.012. No CTCs were detected in HV with FX (n=10) and CS (n=5). After FX enrichment, CTCs from 17/21 NSCLC pts were available for 5-color IF testing. Of these 17 pts, 15 had TTF1+ CTCs. All 15 pts with TTF1+ CTCs had ≥1 PD-L1+ CTC. 6/15 (40%) pts only had PD-L1+ TTF1+ CTCs, with no PD-L1- TTF1+ CTCs. PD-L1 CTC heterogeneity was seen in the other 9/15 pts, with co-existing PD-L1+ and PD-L1- TTF1+ CTCs. 6 of these 9 pts had more PD-L1+ TTF1+ CTCs than PD-L1- TTF1+ CTCs.

Conclusions: FX resulted in consistently high cell recovery rates regardless of EpCAM status. Higher CTC counts were isolated with FX vs CS in 90% of NSCLC pts. 40% of NSCLC adenocarcinoma pts had PD-L1+ TTF1+ CTCs, but PD-L1 CTC heterogeneity was seen in other pts, which may in part explain differences in responses to PD-1/PD-L1 inhibitors in NSCLC.

#2244

Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients.

Satomi Yagi,1 Yasuhiro Koh,2 Hiroaki Akamatsu,2 Woong Kim,2 Ayaka Tanaka,2 Kuninobu Kanai,2 Atsushi Hayata,2 Ryota Shibaki,2 Masayuki Higuchi,1 Hisashige Kanbara,1 Takashi Kikuchi,2 Keiichiro Akamatsu,2 Masanori Nakanishi,2 Hiroki Ueda,2 Nobuyuki Yamamoto2. 1 _Hitachi Chemical Co., Ltd., Tokyo, Japan;_ 2 _Third Department of Internal Medicine, Wakayama Medical University, Japan_.

Background and Purpose:

Circulating tumor cells (CTCs) are relatively rare cells defined as tumor cells circulating in the peripheral blood of patients with solid tumors. Diagnosis utilizing CTCs is expected to help guide decision-making for precision cancer medicine. We developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Here we evaluated its performance using preclinical spike-in model and blood samples from non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients.

Material and method:

The automated MCA system consists of components such as chambered cartridge containing micro metal filter, reagent and waste reservoirs, and peristaltic pump. To evaluate the recovery of CTCs, preclinical experiments using NSCLC cells, NCI-H820, A549, NCI-H441 and NCI-H23 spiked into peripheral whole blood from healthy volunteers were performed. For clinical evaluation, 6 mL of peripheral whole blood was collected from 50 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Samples were collected in an EDTA-containing tube and were processed within 3 hours of blood draw. Recovered cells on the filter were then fixed, permeabilized, and stained automatically and high-resolution fluorescent images were obtained using fluorescence microscope. We defined CTC as DAPI-positive, cytokeratin-positive and CD45-negative cell.

Results:

Results of the preclinical study showed that up to 90% of spiked-in tumor cells were recovered, confirming that the detection sensitivity by this automated device is on par with that by previous manual detection procedure. Demographics of 50 lung cancer patients enrolled in clinical study were as follows: median age 72 (range, 48 to 85); male 66%; stage III/IV 12/88%; NSCLC/SCLC 78/22%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 / 6 mL, respectively. Three or more CTCs were detected in 71% of patients with advanced lung cancer (39 out of 50) and five or more CTCs were detected in 52% of patients (26 out of 50) (median CTC count 13.5). Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those with intrathoracic metasitasis (median CTC count, 8 versus 4, p=0.058). A head-to-head comparison between CellSearch system and our system was conducted in NSCLC patients, showing the superiority of our system (median CTC count, 0 versus 11.25, p=0.0001, n = 17).

Conclusions

Our results suggest that the automated MCA device has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This device also enables higher throughput owing to its automated procedure. Further clinical evaluation including the detection of PD-L1 expression will be performed in an expansion cohort.

#2245

FDG PET quantification for prediction of early recurrence in patients with stage I and II non small cell lung cancer.

Irene A. Burger, Michael Arvanitakis, Seraina Steiger, Walter Weder, Sven Hillinger. _Univ. Hospital Zürich, Zürich, Switzerland_.

Background: Although surgical resection remains the optimal treatment for early-stage NSCLC, up to 50% of patients with stage I and II relapse and die within 5 years after curative resection. Therefore prognostic markers are important as these patients might benefit from adjuvant therapy. The goal of this study was to evaluate established PET quantification metrics including: maximal standard uptake volume (SUVmax), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) as prognostic markers for early recurrence and overall survival in resected early stage lung cancer.

Methods: Between January 2003 and December 2010 182 surgically resected patients with stage I-II NSCLC who underwent 18 F FDG PET/CT less than one month prior to surgery have been evaluated. All patients had at least 5 years of follow-up. Cox proportional hazard model was used to determine the association between variables and survival respectively time to recurrence. For the multivariate analysis the following variables have been included: tumor size on CT, age tumor stage, histology, SUVmax, TLG (for TLG42% (threshold at 42% SUVmax) and TLG2.5 (cut-off at SUV 2.5) and MTV42% and MTV2.5).

Results: 133 patients were included, 71 with adeno carcinoma, 62 with squamous cell carcinoma. TLG2.5 and MTV2.5 values have been a significant prognostic factor for recurrence (P<0.0001). Patients with a MTV2.5 above 42 cm3 had a mean recurrence time of 0.8±0.9 years, while patients with MTV2.5 ≤ 42 cm3 recurred within 2.8±1.3 years. TLG2.5 and MTV2.5 PET volume metrics have not been predictable for overall survival in adenocarcinoma patients, recurrence or overall survival in squamous cell cancer. SUVmax, TLG42% and MTV42 were not predictable for early recurrence or survival for both histologies.

Conclusions: TLG2.5 and MTV2.5 may be useful prognostic variables in stage I-II NSCLC depending on the tumor type. Using a cut-off at 42 cm3 for early stage adenocarcinoma patients a high risk of recurrence within one year might be identified and adjuvant therapy following surgical resection could improve outcome for those patients.

#2246

Androgen receptor expression in non-small cell lung cancer circulating tumor cells.

Jennifer L. Schehr, Zachery Schultz, Anwaar Saeed, Jamie M. Sperger, Ticiana Leal, Kara Mattox, Anne Traynor, Joshua M. Lang. _University of Wisconsin - Madison, Madison, WI_.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a leading cause of death worldwide. There is a critical need to develop new therapeutic options for patients with advanced NSCLC. Androgen Receptor (AR) expression has been observed in lung cancer, along with castration associated inhibition of tumor growth in mouse models. The AR is a high value therapeutic target in prostate cancer and many targeted therapies are clinically available. However, significant genomic and functional alterations occur in NSCLC over the course of targeted therapies that may predict response and resistance to AR targeted therapies. The development of predictive and pharmacodynamic biomarkers of the AR signaling pathway in NSCLC may be critical to developing appropriate therapeutic strategies in this context. To evaluate AR expression and the AR signaling pathway, we developed a circulating tumor cell (CTC) assay to evaluate orthogonal endpoints across protein and gene expression analytics in patients with advanced NSCLC.

METHODS: CTCs were isolated from patients with metastatic NSCLC using an integrated CTC capture and analysis technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. This microscale platform integrates tumor cell capture, for any target of interest, with enumeration and nucleic acid extraction for analysis of orthogonal endpoints from a blood sample. CTCs were captured with multiple antibodies, including EpCAM, MUC1, and Vimentin. mRNA was extracted from CTCs for gene expression analysis of the AR, AR splice variants, downstream targets in the AR signaling pathway and activation markers in NSCLC.

RESULTS: Enumeration of CTCs from patients with metastatic NSCLC shows significant tumor burden, ranging from 10-150 CTCs per 7.5mL of blood. Gene expression analysis of CTCs identified expression of proliferative markers KRAS, BRAF, and RET. In our initial cohort of patients, full length AR expression was identified in 13/13 patients with NSCLC. The AR V7 splice variant was detected at low levels in 2/13 patients. PSMA, but not TMPRSS2, was identified in a subset of these patients suggesting activity of the canonical AR signaling pathway

CONCLUSIONS: The VERSA effectively captures CTCs with high sensitivity to detect the AR and AR variants in NSCLC CTCs. The AR is detectable in patients with NSCLC CTCs at high frequency. A subset of patient samples show activity in the AR signaling pathway that could potentially drive disease progression. The identification of the AR V7 splice variant suggests shared mechanisms of resistance in NSCLC and prostate cancer. These assays may serve as predictive and pharmacodynamic biomarkers for AR targeted therapies in NSCLC.

#2247

A multicenter clinical trial of lung cancer circulating tumor cell assay with the largest sample size (1210 cases) in China.

Jiatao Lou,1 Caicun Zhou,2 Jing Wu,1 Lihua Qiao,1 Xiaohui Liang,1 Xiaoqian Wang,1 Xiaoxia Chen,2 Xuefei Li,3 Chao Zhao2. 1 _Shanghai Chest Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China;_ 2 _Shanghai Pulmonary Hospital, Tongji University of Medicine, Shanghai, China;_ 3 _Shanghai Pulmonary Hospital, Tongji University, Tongji University Medical School Cancer Institute, Shanghai, China_.

Background: As a novel biomarker of primary tumors, circulating tumor cells (CTCs) are well known to play an important role in cancer diagnosis, recurrence and metastasis, and disease monitoring. This study investigated the application of CTCs in lung cancer therapy by quantitative determination of folate receptor-positive CTCs.

Methods: This study enrolled 1210 subjects (including 560 lung cancer patients, 350 patients with benign lung diseases, 150 healthy subjects and 150 non-lung cancer malignant tumor patients) and quantified the tiny amounts of CTCs in peripheral blood by negative enrichment using immunomagnetic beads in combination with folate receptor-directed polymerase chain reaction (PCR).

Results: Following ROC curve analyses of CTC levels in the patients with benign lung diseases or benign lung diseases and the healthy subjects, the cut-off of CTCs was 8.70 CTC Units/3mL with a specificity of 88.2% (441/500) and a sensitivity of 79.6% (446/560) as determined by the method mentioned above. The ROC area under

curve of more than 0.8 (AUC=0.8797, P<0.0001) suggested the excellence of ligand-directed PCR to diagnose lung cancer. For stage II-IV lung cancer patients, the sensitivities were 70.8%(34/48), 79.6% (125/157), and 90.2% (185/205) respectively, and the sensitivity in stage I patients can reach up to 68.0% (102/150) as well. CTC levels appeared to have greater accuracy to diagnose lung cancer (AUC: 0.8833, P<0.0001) than the combination of the other four lung cancer-specific serum tumor markers (CEA, NSE, CYFRA21-1 and SCC)(AUC: 0.8557, P<0.0001). CTCs were related to tumor stage and tumor size other than pathological type. By quantitative determination of folate receptor-positive cells in 150 different types of cancers, the CTC level in lung cancer was found to be higher than those in gastric, breast, colorectal, liver, and esophageal cancers.

Conclusions: LT-PCR achieves quantitative determination of folate receptor-positive CTCs to diagnose lung cancer with high sensitivity and specificity, and significantly greater diagnosis accuracy than the model of combined lung cancer-specific serum tumor markers.

#2248

Plasma epidermal growth factor receptor mutation (EGFR) testing in advanced non-small-cell lung cancer patients harboring EGFR mutations by chip-based digital PCR system.

Norimitsu Kasahara,1 Hirotsugu Kenmotsu,1 Masakuni Serizawa,1 Rina Umehara,1 Akira Ono,1 Kazushige Wakuda,1 Shota Omori,1 Kazuhisa Nakashima,1 Tetsuhiko Taira,2 Tateaki Naito,1 Haruyasu Murakami,1 Noriaki Sunaga,3 Yasuhiro Koh,4 Keita Mori,1 Masahiro Endo,1 Takashi Nakajima,1 Masanobu Yamada,3 Masatoshi Kusuhara,1 Toshiaki Takahashi1. 1 _Shizuoka Cancer Center, Shizuoka, Japan;_ 2 _Minami Kyushu National Hospital, Kagoshima, Japan;_ 3 _Gunma University Hospital, Gunma, Japan;_ 4 _Wakayama Medical University, Wakayama, Japan_.

Background: EGFR mutation testing is important in the treatment decision for advanced non-small cell lung cancer (NSCLC). Because T790M mutation in exon 20 is associated with approximately 50% of cases resistant to treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), the importance of re-biopsy for detecting T790M mutation during EGFR-TKI therapy is increasing. Circulating tumor DNA (ctDNA) is recognized as a promising target of minimally invasive diagnosis. Digital polymerase chain reaction (dPCR) is a new PCR technology for accurate quantification of the copy number of target molecules from low-input DNA. This study aimed to develop a blood-based EGFR mutation analysis in patients with advanced NSCLC by using a chip-based dPCR system that enables easy handling at a moderate costs.

Methods: From October 2013 to March 2014, 49 patients with NSCLC harboring EGFR-activating mutations detected with the Scorpion ARMS method were enrolled in this study. ctDNA samples were extracted from plasma samples of 21 and 28 patients before treatment and after progression with EGFR-TKI, respectively. dPCR was performed on the QuantStudio 3D (QS3D) Digital PCR System (Life Technologies) by using 20 ng ctDNA, TaqMan probes for three EGFR mutations (exon 19 deletion, L858R in exon 21, and T790M in exon 20), and QS3D Digital PCR 20K Chips (2 chips per assay), according to the manufacturer's instructions. The performance of the dPCR assay was assessed by using HDx Reference Standards (Horizon) containing each mutant sequence serially diluted with that containing wild-type EGFR sequence.

Results: Mutation of 0.1% was successfully detected in the dPCR assay for the three EGFR mutations. The median copy numbers of the EGFR mutation-positive samples were 2.9 copies (range, 0.3-84.2) for exon 19 deletion, 5.1 copies (0.6-742.1) for L858R in exon 21, and 1.4 copies (0.6-36.9) for T790M in exon 20. The sensitivity and specificity of each dPCR assay calculated by comparison with corresponding tumor samples were 61.8% (21/34) and 93.3% (14/15) for exon 19 deletion, and 66.7% (10/15) and 100% (34/34) for L858R, respectively. The T790M mutations were detected in 43% (12/28) of the ctDNA samples after progression with EGFR-TKIs therapy but in none of the samples before treatment with EGFR-TKIs. Among the 12 patients with NSCLC harboring T790M mutation in ctDNA, T790M mutations were detected in 5 of the 11 patients who underwent re-biopsy.

Conclusions: This system showed similar performance to other dPCR assays used in previous studies (sensitivity: 66.6-92.0% and specificity: 95.7-100%). These results indicate the possibility that EGFR mutation testing with ctDNA using the chip-based dPCR for easy handling at a moderate costs is useful as a minimally invasive monitoring method in clinical settings.

#2249

Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing.

Ken Suzawa, Shuta Tomida, Takahiro Matsubara, Kadoaki Ohashi, Yuho Maki, Hiromasa Yamamoto, Mizuki Morita, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Katuyuki Kiura, Shinichiro Miyoshi, Shinichi Toyooka. _Okayama University, Okayama, Japan_.

[Background] Mutational analysis is essential for recent personalized therapeutic approaches of non-small cell lung cancer (NSCLC) patients. Circulating cell-free DNA (cfDNA) from plasma is an emerging topic as a tool for liquid biopsy and possibly enable repetitive genetic diagnosis or monitoring of cancer status. However, detection of cfDNA from the circulation still remains challenging because of its highly fragmented and low concentration nature. In this study, we analyzed cfDNA samples by targeted deep sequencing at high coverage.

[Materials and Methods] Twenty matched DNA samples were collected from metastatic NSCLC patients with EGFR mutation, which was detected by a clinical EGFR mutant enriched PCR assay using PNA-LNA PCR clamp method. These samples were extracted from tissue samples of fresh tumor tissue except one formalin fixed paraffin embedded (FFPE) sample (tDNA), and plasma samples (cfDNA). Mutation hotspots were enriched by multiplex PCR using the GeneRead DNAseq Targeted Panels V2 Human Tumor Actionable Mutations panel (QIAGEN, Hilden, Germany), followed by library construction, and sequenced on the MiSeq sequencer. Resulting data was analyzed using CLC Genomics Workbench and Genomics Server and mapped to the full human genome.

[Results and discussion] The mean read depth in target region was 59,000 reads. Matched EGFR driver mutations between cfDNA and tDNA were detected with an overall concordance of 25% (5 of 20 samples). The concordance rate of exon21 L858R point mutation, exon19 deletions and exon20 T790M point mutation were 32.5% (3 of 8 samples), 16.7% (2 of 12 samples), and 66.7% (2 of 3 samples), respectively. The mutant allele frequencies in cfDNA ranged from 0.1% to 44.5% (median, 0.3%). While further improvement is mandatory, these results suggest that targeted deep sequencing of cfDNA at high coverage may have a potential utility as a non-invasive mutational analysis. In addition, there seemed to be a tendency of better detection rate for point mutations (45.5%) than deletions (16.7%), while the difference didn't reach statistical significance (p= 0.19), which were corresponding to previous reports.

#2250

STK11 mutations in non-small cell lung cancer (NSCLC): descriptive analysis and prognostic significance.

Francesco Facchinetti, Maria Virginia Bluthgen, Gabrielle Tergemina Clain, Laura Faivre, Jean-Pierre Pignon, David Planchard, Jean-Charles Soria, Benjamin Besse, Ludovic Lacroix. _Gustave Roussy Cancer Campus, Villejuif, France_.

Background: STK11 (also called LKB1) is among the 3 most mutated genes in lung adenocarcinoma, but its prognostic and predictive significance in advanced NSCLC patients is still not clear. Recent preclinical and translational evidence suggest its role in conditioning KRAS mutant lung adenocarcinomas aggressiveness and response to targeted treatment.

Materials and methods: This retrospective analysis, in a single center, included consecutive NSCLC patients who had undergone a platinum-containing regimen, for which standard or next generation (NGS) sequencing analyses of STK11 gene (exons 1-9) had been successfully performed. STK11 gene was considered mutated if effect on protein was predicted as deleterious. Clinical features along with EGFR (exons 18-21), KRAS (exons 2-4) and TP53 (exons 1-11) mutational status were correlated to STK11 status. Kaplan-Meier methods, log-rank test, and Cox proportional hazards models were used for survival analysis, adjusting for stage (advanced yes/no) and histology for progression-free survival (PFS), and smoking status, stage, EGFR/KRAS status and number of lines of chemotherapy for overall survival (OS).

Results: Among the 302 patients included between November 2007 and January 2015, 63% were male, 16% non-smoker and median age was 60 year; 75% had an adenocarcinoma, 88% a stage IIIB or IV and 80% no previous treatment for advanced disease; 25 (8%) patients had a STK11 mutation, 30 (10%) an EGFR mutation and 81 (27%) a KRAS mutation. Deletions/insertions, missense and false-sense mutations mainly involved exon 4-6 of STK11. No statistical differences were observed between STK11 status and sex, smoking status, age, stage at diagnosis or histology (adenocarcinoma versus others). Among the 25 patients with STK11 mutation, 13 patients (52%) had a KRAS mutation, compared with 67 (24%) among those without STK11 mutation (p=0.008). No correlation between STK11 and EGFR or TP53 mutational status was observed. Median follow-up time was 34.8 months (95% confidence interval [CI95]: 30.7-42.3). In univariate analysis, OS was shorter for STK11 mutated patients compared to wild type ones (p=0.085; HR: 1.53; CI95: 0.94-2.49) with a median OS of 10.4 (CI95: 6.1-15.7) and 17.3 (CI95: 14.0-21.1) months for mutated and wild-type STK11 patients respectively. In multivariate analysis, the HR was 1.21 (CI95: 0.74-1.99; p=0.45). Similar non-significant results were observed for PFS.

Conclusion: We confirm the reported data of a positive correlation between STK11 and KRAS mutations. No correlation between STK11 status and PFS or OS in patients receiving platinum-based treatment was observed when taking into account other prognostic covariates.

#2251

Intra-tumor heterogeneity in non-small-cell lung cancer (NSCLC) carrying EGFR mutations.

Anna Maria Rachiglio,1 Matilde Lambiase,1 Francesca Fenizia,1 Alessandro Morabito,1 Gaetano Rocco,1 Domenico Galetta,2 Vienna Ludovini,3 Bruno Vincenzi,4 Emiddio Barletta,5 Gerardo Botti,1 Nicola Normanno1. 1 _Istituto Nazionale Tumori "Fondazione G. Pascale"-IRCCS, Naples, Italy;_ 2 _National Cancer Research Center "Giovanni Paolo II", Bari, Italy;_ 3 _S. Maria della Misericordia Hospital, Perugia, Italy;_ 4 _University Campus Bio-Medico, Rome, Italy;_ 5 _Oncology Unit, "G. Rummo" Hospital, Benevento, Italy_.

Introduction. Driver mutations of the epidermal growth factor receptor (EGFR) are usually detected in 10% to 15% of Caucasian non-small cell lung cancer (NSCLC) patients. Evidence suggests that a fraction of EGFR mutant NSCLC might have intra-tumor heterogeneity which could potentially affect response to EGFR tyrosine kinase inhibitors (TKIs). We performed next generation sequencing (NGS)-based analysis of a large cohort of Italian EGFR mutant NSCLC to assess the level of intra-tumor heterogeneity.

Materials and methods. Genomic DNA from EGFR mutant NSCLC samples as assessed with routine diagnostic methods (N. 181; 141 tissue specimens and 40 cytology samples), was retrospectively analyzed with the Ion AmpliSeq Colon and Lung Cancer Panel using Ion Torrent semiconductor sequencing. The panel assesses over 500 somatic mutations in 22 genes at a sensitivity of 2%. Variants with allelic frequency >2% and <5% were confirmed by droplet digital PCR (ddPCR), if material was available.

Results. Analysis of EGFR mutant samples with NGS revealed the presence of two different hotspot EGFR mutations in 17/181 cases (9.4%). In 11 cases a EGFR sensitizing mutation and the p.T790M resistance mutation were detected; 6 tumors carried two different EGFR activating mutations at different allelic frequency. In addition, the presence of at least one variant in genes other than the EGFR was observed in 84/181 cases (46.4%), with TP53 being the most frequent mutant gene in EGFR mutant NSCLC (32/181; 17.7%). In 42 samples (23.2%) hotspot mutations were found in genes other than the EGFR, such as KRAS, NRAS, BRAF, ERBB2, PIK3CA or MET, which might cause primary resistance to EGFR targeting drugs. In 29 samples the additional mutations were at an allelic frequency >5%, and in 13 at a frequency >2% and <5%. All mutations at low allelic frequency for which material was available were confirmed by ddPCR (N.5). The allele frequency of the driver EGFR mutation and of the additional mutations were usually different, suggesting the sub-clonal origin of some driver mutations. In particular, in 15/42 cases the EGFR mutant allele frequency was lower as compared with the additional driver mutation.

Conclusion. These preliminary data suggest that a subgroup of EGFR mutant tumors have intra-tumor heterogeneity and are likely to carry tumor clones with different molecular profile. Follow-up data are being collected to assess whether this phenomenon might affect the activity of first line EGFR TKIs.

#2252

Correlation of baseline value of folate receptor-positive circulating tumor cells and efficacy of pemetrexed and dynamic monitoring study in non-small cell lung cancer patients receiving first-line platinum-based chemotherapy.

Xiaoxia Chen,1 Caicun Zhou,1 Xuefei Li2. 1 _Shanghai Pulmonary Hospital, Tongji University of Medicine, Shanghai, China;_ 2 _Shanghai Pulmonary Hospital, Tongji University, Tongji University Medical School Cancer Institute, Shanghai, China_.

Background: The efficacy of pemetrexed associates with the expression of folate enzymes, however, few serological marker is available to predict its efficacy. Moreover, dynamic monitoring of circulating tumor cell (CTC) count can predict the outcome of tumor therapy, the progress of tumor and the overall survival of the patient. By quantitative determination of folate receptor-positive CTCs, this study investigated the correlation of its baseline values to the efficacy of pemetrexed and performed a dynamic monitoring study in non-small cell lung cancer (NSCLC) patients who underwent first-line platinum-based chemotherapy.

Methods: This study enrolled 70 NSCLC patients who received first-line platinum-based chemotherapy. The CTCs were quantified by negative enrichment using immunomagnetic beads in combination with folate receptor-directed polymerase chain reaction(PCR) that allows secondary amplification of tiny amounts of CTCs in peripheral blood, thereby enhancing the sensitivity of detection. In this study, the CTC levels were examined by collecting 3mL of anti-ENTA whole blood samples before the treatment and after every chemotherapy, and followed up until progressive disease (PD) or completion of first-line chemotherapy. Based on the changes in CTC levels, we evaluated the correlation of the changes in CTC levels and the progress of the disease as well as the correlation of the CTC levels and the therapeutic outcomes.

Results: The CTC level of seventy patients was 13.68 CU/3mL. Of twenty-two patients who received pemetrexed disodium/platinum combined therapy (AP/AC), the patients harboring high levels of folate receptor showed greater efficacy than those with low expression levels (8.7<CTC level<16, n=7; PFS: 448 vs.94days, P=0.0199; ORR: 75% vs.11%). In the dynamic monitoring study, the CTC level (AUC=0.8026,P=0.0033), the CTC ratio (AUC=0.8422,P=0.0003), the rate of CTC changes (OR=102.005,P=0.0012) after the second chemotherapy and the CTC level (AUC=0.9487,P<0.0001), the CTC ratio (AUC=0.8889,P<0.0001), the changing rate of CTCs (OR=51.662,P=0.0031) after the fourth chemotherapy positively correlated to the disease progression.

Conclusions: The patients with high expression of folate receptor-positive CTCs appear to have superior response to pemetrexed than those with low expression. Additionally, the changes of CTC count can be used as a dynamic monitoring indicator in the treatment process to evaluate tumor burden and therapeutic outcomes.

#2253

Digital sorting and single-cell genomic profile comparison of lung adenocarcinoma CTCs between EpCAM and size-based enrichment methods.

Francesca Fontana,1 Francesco Gelsomino,2 Claudio Forcato,1 Mario Terracciano,1 Chiara Bolognesi,1 Annalisa Altimari,3 Genny Buson,1 Chiara Mangano,1 Paola Tononi,1 Francesco Bacchi,1 Gianni Medoro,1 Michelangelo Fiorentino,3 Nicolò Manaresi,1 Michele Tognetto,2 Andrea Ardizzoni2. 1 _Silicon Biosystems S.p.A., Bologna, Italy;_ 2 _Unità Operativa di Oncologia Medica, Policlinico Sant'Orsola – Malpighi, Bologna, Italy;_ 3 _SSD Diagnostica Istopatologica Molecolare degli organi solidi e del relativo trapianto, Policlinico Sant'Orsola-Malpighi, Bologna, Italy_.

Introduction

Several methods are currently available for Circulating Tumor Cells (CTCs) enrichment. Many studies compared CTC counts between FDA-approved CellSearch® system (based on EpCAM enrichment) and size-based selection methods. However, little is known about genomic differences across CTCs obtained from different platforms. For the first time, we report here a genomic characterization of copy-number and sequence variants in single CTCs enriched with different methods from the same patient.

Methods

Peripheral blood (PB) and FFPE tissue from a pelvis bone metastasis were collected from an advanced lung adenocarcinoma patient, treated with cisplatin-pemetrexed-necitumumab as first line therapy and with carboplatin-gentamicin as second-line. Two PB samples were collected at the same time: one was enriched with CellSearch® System, and one with Parsortix PR1 (size-based selection), followed by Cytokeratin-APC, CD45-PerCp and DAPI staining.

Both samples were analyzed with DEPArray™ system and single CTCs along with single WBCs as controls, were isolated.

Collected cells were whole genome amplified with Ampli1™ WGA kit and their Genome Integrity Index (GII) was assessed. On IonTorrent™ PGM Platform, we profiled Genome-wide copy-number alterations (CNA) by low-pass whole-genome sequencing with Ampli1™ LowPass Kit, and analyzed cancer-gene sequence variants with Ampli1™ CHP Custom Beta targeted panel.

FFPE tissue was disaggregated down to single-cell suspension and labelled with Keratin and Vimentin to distinguish tumor from stromal cells. Aliquots of pure cells were recovered with DEPArray™ system; the analysis of CNVs and mutations on tissue-derived samples is in progress.

Results

Single CTCs (CK+, CD45-,DAPI), showing GII≥3, either isolated from CellSearch®-enriched or PR1-enriched blood, along with WBC, were selected for NGS.

Ampli1™ LowPass Kit data showed several aberrations confirming tumor origin of all CTCs, while WBCs (n=6) produced balanced profiles. Unsupervised hierarchical clustering segregated PR1-derived (n=6) from CellSearch®-derived (n=4) single CTCs in two separate branches. PR1-derived CTCs were very similar to each other, whereas CellSearch®-derived CTCs were more heterogeneous, although certain aberrations were common to all CTCs across platforms. At the sequence level, the targeted panel revealed somatic mutations shared by all CTCs (FLT3), a PTPN11 subclonal mutation (in 3/6 PR1 and 1/4 CellSearch), and other private mutations in single CTCs.

Discussion

The combination of low-pass whole-genome sequencing and targeted sequencing on single-CTCs sorted from PB enriched with different platforms reveals genetic similarities and diversities.

Integration of results with genetic analysis of pure tumor cells isolated from the tumor tissue, will provide further insight of tumor diversity in different compartments.

#2254

Variations in the epithelial-mesenchymal transition (EMT) program by non-small cell lung cancer (NSCLC) circulating tumor cells (CTCs) do not influence survival.

Colin R. Lindsay, Vincent Faugeroux, Emma Pailler, Maria-Virginia Bluthgen, Chloé Pannet, Maud Ngo-Camus, Guillaume Bescher, Fanny Billiot, Jordi Remon, Philippe Vielh, David Planchard, Jean-Charles Soria, Benjamin Besse, Françoise Farace. _Institut Gustave-Roussy, Villejuif, France_.

Background

By definition, CTCs must undergo the EMT to enter the bloodstream where they can be isolated from cancer patients for translational and biological study. Here we examined survival patterns in relation to CTC EMT expression in different molecular subgroups of NSCLC.

Methods

125 patients (pts) with advanced treatment-naïve stage IIb-IV NSCLC were prospectively included for CellSearch CTC analysis as part of the Gustave-Roussy MSN study. Patients signed an informed consent for one CellSave tube prior to chemotherapy. Anti-vimentin (vim) antibody was added to the free channel in the CellSearch system for examination of EMT. Association of CTC number with clinical characteristics were assessed using Fisher's exact, Mann-Whitney and chi-squared tests. Kaplan-Meier method and log-rank tests were used to analyse progression-free survival (PFS) and overall survival (OS) of vimentin-expression in molecular subgroups of NSCLC.

Results

51/125 pts (40.8%) were CTC-positive by CellSearch (≥2 CTC), and 29/125 (23.2%) patient samples contained at least 1 vimentin-positive (+) CTC. 19/76 (25%) adenocarcinomas were KRAS mutated. In the KRAS subgroup, 0/19 patient samples (0%) from pts with mutated KRAS contained vim+ CTC, compared to 17/48 pts (35.4%) with wild-type (WT) KRAS (p=0.0027). There was also a significantly higher overall number of vim+ CTCs in pts with KRAS WT cancer compared to KRAS mutated cancer (mean 0 vs 1.63, respectively, p=0.0035). For KRAS WT pts, no survival difference was evident between vim+ and vim- subgroups in terms of PFS or OS. 21/89 adenocarcinomas were EGFR mutated (23.6%). In this subgroup, statistically higher numbers of EGFR mutated pts with both vim+ and total CTCs were observed compared to EGFR WT pts (vim+ CTC: 9/21 EGFR mutated vs 9/56 EGFR WT, p=0.0134; total CTC: 12/21 EGFR mutated vs 18/56 EGFR WT, p=0.0451). Similarly, there was a significantly higher overall number of vim+ CTCs in pts with EGFR mutated cancer compared to pts with EGFR WT cancer (mean 1.24 vs 0.91, respectively, p=0.0189), but no PFS or OS difference was evident between vim+ and vim- subgroups in EGFR mutated pts. 14/71 (19.7%) adenocarcinomas were ALK rearranged, with further results pending.

Conclusions

At baseline stage IIIb-IV disease, there are statistically fewer vim+ CTCs (and pts with vim+ CTCs) in KRAS mutated NSCLC, while vim+ CTC (and vim+ CTC pts) are statistically higher in EGFR mutated NSCLC. Despite this differential CTC vimentin expression between molecular subgroups, no PFS or OS difference is evident between vim+ and vim- patients. This biological variation coupled to a lack of overall clinical impact favours the hypothesis that each individual CTC is a highly plastic cell that can cover a range of EMT expression.

#2255

Evaluation of the diagnostic value of serum anti-cyclin B1 autoantibody as a biomarker in lung cancer.

Mengtao Xing, Pei Li, Jianxiang Shi, Liping Dai, Jitian Li, Jianying Zhang. _University of Texas at El Paso, El Paso, TX_.

To detect the diagnostic value of cancer-associated autoantibodies in lung cancer, a panel of 9 tumor-associated antigens (TAAs) were used in this study. The TAAs were composed of p62, p16, Koc, p53, Cyclin B1, Cyclin E, Survivin, HCC1, and Rol-A full-length recombinant proteins. Enzyme-linked immunosorbent assay and immunoblotting were used to detect antibodies in 50 lung cancer sera with clinicopathological characteristics and 42 sera from normal individuals. Following, antibodies to Cyclin B1, survivin, p53 and HCC1 were selected and confirmed in another 62 sera from patients with lung cancer but without clinical date and evaluate the diagnostic value of these TAAs. The results showed that the positive rate of autoantibodies against nine selected TAAs (cyclin B1, survivin, HCC1,p53, KOC,p16,p62, RalA and cyclin E) reached 36%, 36%, 26%, 12%, 8%, 6%, 6%, 2%, 0% in 50 lung cancer sera and 4.8%, 2.4%, 0%, 0%, 2.4%, 4.8%, 2.4%, 2.4%, 4.8% in 42 normal sera, respectively. The AUC values for distinguishing lung cancers from normal sera were 0.767, 0.400, 0.567, 0.395, 0.230, 0.517, 0.635, 0.194, 0.411, respectively. These data suggest that cyclin B1, survivin, HCC1 and p53 has higher clinical diagnostic quality and value than other TAAs in lung cancer. In the confirmation group with 62 lung cancer sear , autoantibodies against these four TAAs were also significantly higher in patients with lung cancer (AUC values of 0.764,0.653,0.623 and 0.622, p<0.05). Evaluation of the diagnostic value of these four TAAs in 50 sera from lung cancer patients with clinical characteristics, and results revealed that serum anti-cyclin B1, anti-survivin and anti-p53 autoantibodies significantly increased in patients with stage I but only serum anti-cyclin B1 increased in stage II and III lung cancer (p <0.05, respectively), ROC curve analysis has shown that anti-cyclin B1 and anti-p53 autoantibodies can discriminate stage I lung cancer from normal controls with AUC value of 0.757,0.697, respectively (p<0.01), and anti-survivin and anti-HCC1 autoantibodies failed to discriminate stage I lung cancer from normal controls . Our results suggest that these four TAAs has higher clinical diagnostic quality and value than other TAAs in lung cancer and serum cyclin-B1 may have the potential to serve as novel non-invasive diagnostic biomarkers in patients with lung cancer, especially in early stage lung cancer.

#2256

Establishment and characterization of circulating tumor cell-derived xenografts in non-small cell lung cancer.

Vincent Faugeroux,1 Olivier Deas,2 Judith Michels,1 Jean Gabriel Judde,2 Stefano Cairo,2 Philippe Vielh,1 Virginie Marty,1 Fanny Billiot,1 Maud Ngocamus,1 Benjamin Besse,1 Patricia Kannouche,1 Françoise Farace1. 1 _Institut Gustave Roussy, Villejuif, France;_ 2 _XenTech, Evry, France_.

Low numbers of circulating tumor cells (CTCs) have so far limited the establishment of CTC-derived xenografts (CDXs) to improve our understanding of tumor progression, drug resistance mechanisms, and their biological properties. We report the establishment and the phenotypic and molecular characterization of one NSCLC CDX.

Blood samples (30 ml) were drawn from 49 NSCLC patients with advanced metastatic disease. CTCs were enriched by RosetteSep, embedded in matrigel, and implanted in the interscapular aspect of NSG mouse (Nod/Scid-IL2Rγ-/-). Mice were followed-up for one year according to ethical regulations. CDX tumours and CDX derived cell lines were phenotypically and molecularly characterized by immunofluorescence, immunohistochemistry, CGHarray, exome sequencing and transcriptome gene expression.

CTCs from one NSCLC patient with 750 CTCs detected by CellSearch gave rise to a tumor 5 months after initial murine injection. Histological analysis confirmed the human origin of the tumor and the presence of a poorly differentiated adenocarcinoma consistent with the patient's biopsy. Tumor was positive for EpCAM, EMA, CK8;18, and Ki67, and negative for vimentin. A fraction of cells (25%) from freshly dissociated tumors exhibited ALDH activity. CGH from CDX tumors at passage 1 and 2 shows multiple gene rearrangement, revealing a high degree of genomic instability. Transcriptome analysis of ALDH positive and negative cells is ongoing and should help of identifying a cancer stem cell gene expression signature. Whole-exome sequencing of CDX tumor is ongoing and will be compared to data obtained from single CTCs from the patient.

A cell line established in vitro from the CDX model grows in 3D clusters and is tumorigenic in mice. Interestingly, this cell line is positive for cytokeratins, EpCAM, E-cadherin, N-cadherin, vimentin, and expresses multiple cancer stem cell markers including CD166, CD24, CD133 and, ALDH activity. The cell line is hypotetraploid (about 70 chromosomes) and its CGH profile was similar to that of the CDX tumour, revealing a high level of genome instability. By investigating DNA replication process in this cell line, we found that it exhibits a spontaneous enhanced DNA damage signaling associated to an accumulation of DNA double strand breaks mainly in S phase strongly suggesting that the CDX-derived cell line displays hallmarks on replication stress that could explain, at least partially, the genomic instability in the cells.

We report a low success rate in the establishment of NSCLC CDX (2%). However one NSCLC CDX model harboring cancer stem cell properties and deficiency of DNA replication maintenance was established. Ongoing work to identify a cancer stem cell signature and characteristics replication stress markers in this CDX model will be presented. This NSCLC CDX model will be useful to test drugs targeting these alterations in vivo and improve our knowledge of drug resistance.

#2257

Differential expression of PD-L1 on circulating tumor cells among patients with advanced lung cancer.

Woong Kim,1 Yasuhiro Koh,1 Hiroaki Akamatsu,1 Satomi Yagi,2 Ayaka Tanaka,1 Kuninobu Kanai,1 Atsushi Hayata,1 Ryota Shibaki,1 Masayuki Higuchi,2 Hisashige Kanbara,2 Takashi Kikuchi,1 Keiichiro Akamatsu,1 Masanori Nakanishi,1 Hiroki Ueda,1 Nobuyuki Yamamoto1. 1 _Wakayama Medical University, Wakayama, Japan;_ 2 _Hitachi Chemical Co., Ltd, Chikusei, Japan_.

Background and purpose:

Immune-checkpoint blockade with anti-programmed death-1 (PD-1) antibodies is rapidly emerging for the treatment of human malignancies including lung cancer. Although programmed death-ligand 1 (PD-L1) has been studied as a predictive biomarker, detection and evaluation of PD-L1 expression level on tissue samples remain challenging due to its dynamic and unstable expression. Thus the diagnostic tool for real-time monitoring of PD-L1 expression is critically needed. Here, we assessed the expression pattern of PD-L1 on circulating tumor cells (CTCs) by using microcavity array (MCA) system in patients with advanced non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC).

Experimental procedure:

PD-L1 staining on CTCs was established using NSCLC cell lines H820, H441, A549 and H23 expressing varying levels of PD-L1 spiked in the peripheral blood obtained from healthy donors. For clinical evaluation, 3 ml of peripheral whole blood was collected from 20 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Cells were captured and immuno-stained by using the automated MCA system (Hitachi Chemical Co., Ltd). CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. PD-L1 expression level on CTCs was visualized by addition of PD-L1 immunocytochemistry procedure. High-resolution fluorescent images were obtained using fluorescence microscope (Carl Zeiss Microscopy Co., Ltd).

Results:

Characteristics of 20 lung cancer patients enrolled in clinical study were as follows: median age 74 (range, 48 to 84); male 60 %; stage III/IV, 10/90 %; NSCLC/SCLC, 70/30 %. More than 2 CTCs were identified in 14 patients (median 22.5; range, 4 to 71), and PD-L1 positive CTCs were detected in 12 patients (median 5; range, 2 to 15). No correlation was detected between the number of total CTCs and that of PD-L1 positive CTCs in each patient (R2 = 0.05). We found a total of 25 CTC clusters from 20 patients, of which PD-L1 expression was both homogenous and heterogeneous. It is noteworthy that clustered CTCs have larger proportion of PD-L1 positive CTCs per whole clustered CTCs than that of non-clustered CTCs (24/54, 44% versus 51/347, 15%, respectively). We further focused on CTC-interacting white blood cells, which intensively bound with aggregated CTCs rather than single CTC (12/54, 22% versus 43/337, 13%, respectively). Our data implicate that PD-L1 expression on CTC correlates with aggregation of CTCs (p < 0.05).

Conclusions:

Our results showed that PD-L1 expression on CTCs was detectable and there is intrapatient heterogeneity of its expression in patients with advanced lung cancer. Further investigation is warranted to better understand the biological importance of the correlation between PD-1 expression and CTC aggregation and CTC bound to white blood cells.

#2258

Digital whole slide quantitative image analysis of TOPO II and P53 in sarcomas evaluating both primary and lung metastatic tumors.

Margaret Strack,1 George Sandusky,1 Daniel Rushing2. 1 _IU School of Medicine, Indianapolis, IN;_ 2 _IU Simon Cancer Center, Indianapolis, IN_.

Sarcomas are a heterogeneous group of tumors that account for about 200,000 cancers worldwide each year (~1% of all human malignant tumors) and represent 15% of all pediatric malignant tumors. They comprise over fifty subtypes that can broadly be classified into bone and soft-tissue sarcomas. The vast majority of sarcomas fall into the soft-tissue group, primarily connective tissues such as muscle (smooth and skeletal), fat, and blood vessels. Bone sarcomas represent only about 20% of all diagnosed sarcomas. It is estimated that over 50% of human tumors have a TP53 mutation and over 80% of tumors have dysfunctional p53 signaling. Topoisomerase II (TOPO II) is the target for many chemotherapeutic agents. TOPO II increases at the end of the S to G2 phase and pushes the cancer cell through cell division.

In this study, 84 clinical sarcomas were evaluated from the IU Medical Center clinical case files. Some were metastatic lung lesions while others were from the primary tumor. These were Liposarcoma, Leiomyosarcoma, Fibrosarcoma, Malignant Fibrous Histiosarcoma(MFH), Synovial Sarcoma, Osteosarcoma, and Ewings. Paraffin-embedded tissue blocks were obtain from Indiana University Simon Cancer Center Tissue Procurement and Indiana University Health Pathology Laboratory under approved IRB protocols. The tumors were fixed in 10% neutral buffered formalin (NBF), processed into a paraffin block, microtomed, and stained with H&E. Immunostains with both TOPOII and P53 were performed using used the DAKO platform with the LSAB2 system. Both primary antibodies were from DAKO. The slides were scanned using Aperio Imaging System was performed using the positive-pixel algorithm in Image Scope.

The results showed that the primary tumors expressed a lower level of TOPO II and P53 in the primary tumor and a higher expression in the metastatic lung lesions in the majority of the sarcomas. However, in the subgroups MFH and Fibrosarcomas were higher in the primary tumor compared to metastatic lesions. The Aperio imaging results showed that the primary tumors were 5.238% positive for TOPO II and 2.4818% positive for P53 on average compared to the metastatic lung lesions with 9.64% positive for TOPO II and 4.5692% positive for P53. In conclusion the metastatic tumors of both biomarkers were increased in the lung. The conclusion suggests that TOPOII is overexpressed and driving cell division while a mutant P53 is dysfunctional and does not prevent tumor cell from going into the cell cycle. The results are consistent with previous studies showing that the metastatic lesions are in a state of cell cycling due to increased drug resistance.

#2259

Comprehensive evaluation of promising biomarkers for lung cancer risk prediction.

Mattias A. Johansson,1 Ayumu Taguchi,2 David Muller,3 Samir Hanash,2 Paul Brennan1. 1 _International Agency for Res. on Cancer, Lyon, France;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Imperial College, London, United Kingdom_.

Introductory sentence indicating the purposes of the study:

Lung cancer will remain the number one cancer killer worldwide for the foreseeable future (Globocan 2012). Although low-dose CT screening has been shown to reduce mortality, there is an urgent need to improve risk prediction models to identify those subjects at high risk and most likely to benefit from screening in order its improve the screening efficacy (PMID: 23697514, 25372087).

Brief description of pertinent experimental procedures:

We have assembled a compendium of promising blood-based risk biomarkers for lung cancer that are currently being validated in order to identify a panel of validated risk markers for use in lung cancer risk assessment. Using pre-diagnostic serum samples from CARET cohort participants (current or former heavy smokers) we have recently validated pro-surfactant protein B (Pro-SFTPB) and Diacetyl spermine (DAS) as risk markers for non-small cell lung cancer (PMID: 26282655). Validation of additional markers from the compendium using Pro-SFTPB and DAS as anchor markers with further refinement of the risk prediction models is currently being performed in two large independent cohort studies (EPIC and NSHDS). Biospecimens from these cohorts include pre-diagnostic plasma from 550 former and current smoking lung cancer cases that were diagnosed within 5 years of blood draw, along with 1,100 matched controls.

Summary of the new, unpublished data and conclusion:

Preliminary data indicate that blood-based biomarkers of lung cancer risk have a strong potential to improve on questionnaire-based risk prediction models for lung cancer (PMID: 26282655). During the meeting we will present detailed results of the individual risk biomarkers and their associations with lung cancer risk up to 2 years prior to diagnosis, as well as an assessment of the extent to which they can improve traditional risk prediction models.

#2260

Classification of patients with lung cancer and benign lung disease via assessment of DNA methylation of SHOX2 and PTGER4 in plasma.

Anne Schlegel, Oliver Hasinger, Selina Esche, Melanie Martini, Thomas König, Gunter Weiss. _Epigenomics AG, Berlin, Germany_.

Introduction:

Annual screening for lung cancer (LC) with low-dose computed tomography (LDCT) is recommended in the US for specific high risk patients. Despite this, LC patients are frequently symptomatic at time of presentation and are often diagnosed with advanced stage disease associated with poor prognosis. At the same time the substantial rate of false positive findings of LDCT raised controversy about the risk profile of the groups eligible for LDCT screening. Non-invasive diagnostic tools may contribute to further risk stratification for screening candidates or patients with findings in LDCT. Aberrant DNA methylation detected in cell free-floating DNA has been shown to be clinically useful. Specifically, a DNA methylation panel of the genes SHOX2 and PTGER4 has been evaluated in three independent case-control studies comprising a total of 330 plasma specimens from LC patients and healthy individuals with promising results (AUC = 91 to 95%). Here, we report on further evaluation of the methylation marker panel in patients with malignant (LC) or benign lung disease (BLD).

Material and Methods:

For this case-control study 116 plasma specimen were collected from patients in Bulgaria and the US. The majority of patients were either diagnosed with LC (30 patients) or a BLD (50 patients). The remaining 36 subjects were healthy controls. A triplex real-time PCR assay for methylated DNA of SHOX2 and PTGER4 and the methylation independent ACTB control assay was developed. Total DNA was extracted from 3.5ml plasma samples and bisulfite converted utilizing a commercially available kit. After purification DNA was assayed in PCR triplicates. Cycle threshold values were aggregated utilizing a predefined algorithm. Receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) were analysed.

Results:

The marker panel showed significant discriminatory power to distinguish LC cases from the remaining subjects (AUC = 0.84), including BLD patients (AUC = 0.83) and healthy subjects (AUC = 0.86). At a fixed specificity level of 95% the sensitivity for LC was 50%, while for a fixed 90% sensitivity the observed specificity was 53%. BLD and healthy subjects were similar with respect to the methylation markers (AUC = 0.55, p-value = 0.45). The clinically important group of COPD patients (18 cases) was significantly different from the LC group (AUC = 0.76, p-value < 0.01).

Conclusions:

Combination of DNA methylation markers SHOX2 and PTGER4 can be used to distinguish lung cancer patients from healthy subjects as well as patients with non-malignant diseases with high sensitivity at a reasonable false positive rate. The marker panel will be developed into a blood-based lung cancer diagnostic tool that may ultimately show clinical utility in combination with current imaging techniques to improve lung cancer risk stratification.

#2261

PKR and Jagged1 associated with lymph node metastasis in lung cancer.

Apar Pataer, Chuncheng Hao, Arlene M. Correa, Annikka Weissferdt, Carmen Behrens, Ignacio I. Wistuba, Stephen G. Swisher. _UT MD Anderson Cancer Center, Houston, TX_.

In the current study, we analyzed the association between lymph node metastasis and over 50 biomarkers in 319 NSCLC patients. We observed that tumor location and the protein expressions of Jagged1 and RNA-dependent protein kinase (PKR) have significantly association with the lymph node metastasis. We further investigated the mechanism of interaction between Jagged1 and PKR, and demonstrated that induction of PKR promotes Jagged1 protein degradation through proteasome system in lung cancer cells. We also found that combination of Jagged1 with PKR improved prognostic value in all stages NSCLC patients. However, combination of Jagged1 with PKR did not improve lymph node metastatic value. Taken together, our data suggest that PKR and Jagged1 are independent markers for predicting lymph node metastasis, and PKR may reduce lymph node metastasis partly by promote Jagged1 proteins degradation in NSCLC patients.

#2262

Thioredoxin reductase 1: the key member in metabolism affects PFS of 1st line erlotinib to advanced non-small cell lung cancer harboring EGFR mutation-positive.

Yongchang Zhang. _Hunan Cancer Hospital, Changsha, China_.

Background:

Deregulating cellular energetics is one promising hallmark of cancer and Thioredoxin reductases 1 (TrxR1) plays a key role in cell metabolism [1-4]. It was found in our previous study that the activity of TrxR1 in serum is significantly higher in cancer patients than volunteers (based on 1122 cancer patients VS 84 volunteers), and over expression of TrxR1 correlates with poor prognosis in metastatic EGFR wild-type non-small-cell lung cancer patients treated with standard 1st line platinum doublets [5]. Yet the role of TrxR1 remained unclear in metastatic non-small-cell lung cancer harboring EGFR sensitive mutation treated with standard 1st line erlotinib.

Aim:

to explore the prognostic role of TrxR1 in metastatic non-small-cell lung cancer harboring EGFR sensitive mutation treated with 1st line erlotinib.

Methods:

We have built several cohorts to explore the clinical significance of TrxR1. In this prospective observational study, 37 metastatic non-small-cell patients harboring EGFR 19 deletion or L858 mutation were recruited from cohorts of NCT01980212, NCT01991418 and NCT02098954. EGFR mutation status was test by ARMS. The activity of serum TrxR1 was measured by ELISA assay after receiving one month first line standard EGFR-TKI (erlotinib). The cut off value was set as 12. Survival data were collected and analyzed to TrxR1 levels.

Results:

Until Nov.2015, all of the 37 patients are alive. Progression free survival (PFS) was not mature yet. Follow-up data and dynamic changes of TrxR1 were in process. Among the 17 patients who progressed, survival data analysis showed that the lower TrxR1 activity group was with significantly longer PFS compared to the higher group (15.2m vs 7.0m, p=0.029)to both mutation types, also to the exon19del subtype(17.0m vs 13.0m, p=0.19) and to the exon 21L858R subtype(11.5m vs 4.5m, p=0.096).

Conclusions:

High activity of TrxR1 may suggest an independent role of poor prognosis in EGFR mutation-positive advanced NSCLC treated with 1st line erlotinib, including both exon19del and L858R mutation subtypes, which helps to illustrate the different results of erlotinib treatment. Possibly, those with high TrxR1 should be given a complex of EGFR-TKI plus TrxR1 inhibition. Several inhibitors are under developed in various tumors and hopefully our study could contribute to improve the current standard treatment of lung cancer.

[1]Douglas Hanahan et al; Cell. 2011 Mar 4;144(5):646-74.

[2]Lincoln DT et al; Anti-cancer Res. 2003, 23(3B):2425-33.

[3]Turunen N, et al; APM IS. 2004, 112(2):123-32.

[4]Fan C, et al;. Cell Death Dis. 2014 Apr 24;5:e1191.

[5]Yongchang Zhang et al; Thirteenth Annual International Conference on Frontiers in Cancer Prevention Research, Poster Session B

#2263

Prognostic significance of REV3like and TYMS gene expression in blood samples from non-small cell lung patients treated with platinum-based chemotherapy plus pemetrexed.

Mª Teresa Agullo-Ortuño,1 C.Vanessa Díaz-García,1 José A. López-Martín,2 Elena Prieto-García,1 Santiago Ponce,2 Jon Zugazagoitia,1 Lara Iglesias-Docampo,2 Ana B. Enguita,2 Luis Paz-Ares,2 Juan A. Nuñez-Sobrino2. 1 _Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain;_ 2 _Hospital Universitario 12 de Octubre, Madrid, Spain_.

BACKGROUND: Pemetrexed is an effective antineoplastic agent as monotherapy (second line and maintenance setting) or in platinum-based combination regimes (first-line). Pemetrexed inhibits three key enzymes in the folate metabolic pathway: thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). As a consequence, pemetrexed interferes with the synthesis of both pyrimidine and purine, thereby effectively inhibiting both DNA and RNA synthesis. The aim of this study was to investigate the impact of the PBMC (peripheral blood mononuclear cells) expression levels of AEG1, BRCA1, REV3like, ant TYMS mRNA in the clinical outcome in NSCLC patients treated with Pemetrexed-based therapy.

MATERIAL AND METHODS: This is a prospective, observational cohort study, of consecutively selected patients. We included patients with non-squamous NSCLC, stage IV, treated with platinum-based chemotherapy plus Pemetrexed. Blood samples from each patient were collected at the beginning of treatment. Quantitative reverse transcription (qRT)-PCR was performed with the use of the TaqMan gene expression assay (Applied Biosystems) according to the manufacturer's instructions. Amplifications were carried out on the Applied Biosystem 7500 RT-PCR system, and relative gene expression values were calculated by the ΔΔCt method (Sequence Detection System 2.0.5). Results from analysis, done in triplicate, are expressed relative to expression levels of GAPDH.

RESULTS: We here present preliminary results of 16 patients included in the study: median age was 63.4 (range 52-82); 12 were males. Histological subtypes were 13 adenocarcinomas, and 3 large cell carcinomas. Pemetrexed was combined with Carboplatin in 9 patients and Cisplatin in 7 patients. Best response obtained was partial response in 7 patients, stable disease in 3 patients, and progression in 5 patients. We found significant differences in the REV3like expression levels between patients with PR&SD and patients with PD (0.69 ± 0.14 vs 0.44 ± 0.07; P= 0.003). In addition there were statistically significant differences in disease free survival (3 ± 0.74 vs 13 ± 1.89, P= 0.035; 12 ± 2.32 vs 3 ± 2.19, P= 0.003, months), and overall survival (10 ± 5.21 vs not reached, P= 0.026; not reached vs 12 ± 2.38, P= 0.007, months) between patients with mRNA levels of REV3like and TYMS under mean value of the cohort and patients with levels over mean value of the our cohort.

CONCLUSIONS: This study supports the value of the PBMC expression profiling of the REV3like and TYMS genes in the prognosis of NSCLC treated with platinum-based chemotherapy plus Pemetrexed.

#2264

Tumor fragmentation is a poor prognostic histologic biomarker for lung squamous cell carcinoma.

Ruben Casanova, Alex Soltermann. _University Hospital Zurich, Zürich, Switzerland_.

Introduction: Lung squamous cell carcinoma (SCC) is the second most frequent histologic type of non-small cell lung carcinoma (NSCLC). Next to pTNM staging, grading is also important for histomorphologic classification of tumors into relevant prognostic groups. SCC forms cohesive tumor sheets of various sizes, ranging from large pushing boundaries to small detached clusters. The presence of small clusters such as single cells, tumor buds 15 cells) could also contain prognostic information and hence be pertinent for tumor grading. Herein we propose a computer-based morphometric analysis for the global assessment of lung SCC fragmentation.

Methods: Surgically resected lung SCC tumors (discovery tissue microarray cohort n=208, 2 cores per patient; validation whole section cohort n=99, 2 blocks per patient) were immuno-histochemically stained with pan-cytokeratin. Color-based segmentation (tumor epithelia brown, stroma blue-grey) allowed to separate tumor from stroma and to compute the number of tumor fragments. Associations with disease-free (DFS) and overall survival (OS) were assessed by univariate Cox regressions.

Results: Tumor fragments were defined as tumor clusters >3000µm2 (circa >15 cells) entirely surrounded by stroma. Criteria for high fragmentation were: > 5 fragments summed over 2 TMA cores (range: 2-18, median = 5) and > 700 fragments summed over 2 whole sections (range: 94-3988, median = 570). Respectively, 44% and 42% of the patients had highly fragmenting tumors in the discovery (D) and validation cohort (V), correlating with blood vessel infiltration (cc=0.181, p<0.001 (D); cc=0.220, p=0.029 (V)). High fragmentation was also associated with decreased OS (HR=1.77, p=0.001) and DFS (HR=1.83, p=0.027) in (D). This was confirmed in (V): OS (HR=2.09, p=0.004), DFS (HR=2.60, p=0.005).

Conclusions: We used an automated morphometric approach for unbiased scoring of tumor fragmentation on digitalized histological sections. This versatile method could potentially be applied to virtually any solid human carcinoma. Our analysis revealed that tumor fragmentation is a poor prognosticator for lung squamous cell carcinoma, e.g. due to increased likelihood of vessel infiltration.

#2265

An integrated analysis of gene mutations and gene sets for predicting paclitaxel response in lung adenocarcinoma.

Chia-Yu Huang,1 Yu-Chiao Chiu,1 Tzu-Pin Lu,2 Liang-Chuan Lai,3 Mong-Hsun Tsai,4 Tzu-Hung Hsiao,5 Eric Y. Chuang1. 1 _Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan;_ 2 _Department of Public Health, Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan;_ 3 _Graduate Institute of Physiology, National Taiwan University, Taipei, Taiwan;_ 4 _Institute of Biotechnology, National Taiwan University, Taipei, Taiwan;_ 5 _Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan_.

Lung cancer is the leading cause of cancer death worldwide. A prevalent histological subtype of lung cancer is adenocarcinoma. Chemotherapies and targeted therapies have been developed to treat such malignancy. However, due to the heterogeneity of cancer genomes, drug responses vary considerably among patients and the average survival rate remains quite unsatisfactory. Therefore, integrated biomarkers for predicting drug responses are greatly needed. Addressing this, in the present study we aimed to develop a prediction model based on an integrated analysis of gene mutations and gene sets. Briefly, the two-tailed Student's t-test was performed to identify the gene mutations and gene sets of which activities were associated with drug sensitivity, and classification trees were derived from these genomic features. We applied the analysis to genomic datasets and drug sensitivity data from the Cancer Cell Line Encyclopedia (CCLE) and gene sets defined in the Molecular Signatures Database (MSigDB), and constructed a prediction model for response to paclitaxel, a widely used drug for cancers, in lung adenocarcinoma. Taking KRAS mutation as an example, we identified 20 and 15 drug response-associated gene sets in KRAS-mutant and KRAS-wild type cell lines, respectively. The two lists of gene sets were mutually exclusive, suggesting the need of building individual prediction models for groups of cancer subtypes. We then built a classification tree for each of the two groups and tested their prediction performance by leave one out cross-validation tests; ~64% and ~81% accuracy was achieved for KRAS-mutant and KRAS-wild type cell lines, respectively. Gene sets of "SIG_CHEMOTAXIS" and "PID_ERB_GENOMIC_PATHWAY" served as crucial nodes for the trees of KRAS-mutant and KRAS-wild type cells, respectively. In conclusion, we developed a novel method that integrates gene mutations and gene sets for predicting drug responses and demonstrated its high performance in lung adenocarcinoma. Our model is widely applicable to identify potent biomarkers for anticancer drugs in cancers and accelerate the realization of precision medicine.

#2266

Molecular and genomic characterization of SCLC.

Stephen V. Liu,1 Edward S. Kim,2 Rebecca A. Feldman,3 Zoran Gatalica,3 Jeffrey Swensen,3 Hossein Borghaei,4 Alexander I. Spira,5 Gerold Bepler,6 Sandeep Reddy,3 Afshin Dowlati7. 1 _Georgetown University - Lombardi Comprehensive Cancer Center, Washington, DC;_ 2 _Levine Cancer Institute Carolinas HealthCare System, Charlotte, NC;_ 3 _Caris Life Sciences, Phoenix, AZ;_ 4 _Fox Chase Cancer Center, Philadelphia, PA;_ 5 _Virginia Cancer Specialists, Fairfax, VA;_ 6 _Karmanos Cancer Institute, Detroit, MI;_ 7 _University Hospitals Case Medical Center, Cleveland, OH_.

Introduction: Small cell lung cancer (SCLC), strongly tobacco-associated, has been described to have a heavy mutation burden, harboring high rates of TP53 and RB1 alterations. While initially responsive to radiation and chemotherapy, SCLC is characterized by eventual progression and resistance to traditional therapy. We retrospectively analyzed a molecular profiling (MP) database to identify potentially actionable alterations using a multi-platform approach which includes massively parallel sequencing. Experimental Procedures: SCLC patient samples were referred to a central CLIA laboratory (Caris Life Sciences, AZ) for MP (immunohistochemistry [IHC] and next generation sequencing [NGS]). Expression of PD1 (MRQ-22, ≥1+) on tumor infiltrating lymphocytes (TILs) and PDL1 (130021, SP142, ≥2+≥5%) in tumor cells was performed by IHC. Additional IHC (ERCC1, TOPO1) and NGS on 591 genes was performed on FFPE samples using the Illumina NextSeq platform in a subset of patient samples. All variants were detected with > 99 % confidence. Variants are described as follows: pathogenic, presumed pathogenic, variants of unknown significance and unclassified variants (excluding SNPs). Results: 203 SCLC samples were identified, 48% were females (97) and 52% were males (106). Median age was 65 [range: 29-88]. Cancer cells expressed PDL1 in 2.5% of cases (5/203) and PD1+ TILs were detected in 38% (75/197). For comparison, internal PDL1+ in non-small cell lung carcinomas (NSCLC) was 31% (339/1098). Notable findings from IHC included ERCC1 negative status in 93% (14/15) and TOPO1 + in 70% (14/20). CNV and mutational analysis (NGS) was available for 10 and 22 patients, respectively. Amplifications were found in the following genes: CCND3, CRKL, FGF4, FGFR1 and NFKB1A (n=1, respectively), and CCND1, CCNE1, CDKN2A and FGF3 (n=2, respectively). As previously reported, the most frequently altered genes were TP53 (73%) and RB1 (68%). Clinically relevant pathogenic or presumed pathogenic variants included: EGFR (exon 19 deletion), BRAF (G469A), APC (T1556fs), NF1 (A1610fs, D699fs), NOTCH1 (E473fs, C332Y, G546X) and PTCH1 (N1351fs). It was thought the patient with EGFR mutation is a case of NSCLC transformation to SCLC. Variants with unknown significance or unclassified variants detected in genes with clinical relevance and of potential interest for targeted therapy in SCLC include: DDR2, cMET, RET, FGFR1/3, BRCA2, IGF1R, RICTOR and NTRK1. Conclusion: Genomic and molecular characterization of SCLC samples reveals a heterogeneous population. Several potentially actionable targets are identified by NGS. Early reported trial data suggests susceptibility of SCLC to immune checkpoint inhibitors. We observed higher levels of PD1+ TILs, however differences in antibody clones, thresholds or staining localization (tumor cells vs. stromal lymphocytes), may account for the observed overall low PD-L1 expression. Further efforts are needed to identify and validate new therapeutic targets in SCLC.

#2267

Efficacy of pemetrexed-based chemotherapy in patients with NSCLC harboring KRAS mutations.

Shunsuke Okumura, Takaaki Sasaki, Shin-ichi Chiba, Masatoshi Sado, Naoyuki Miyokawa, Hiroshi Funakoshi, Yoshinobu Ohsaki. _Asahikawa Medical University, Asahikawa, Japan_.

[Background]

Biomarkers like epidermal growth factor receptor (EGFR) gene mutations or anaplastic lymphoma kinase (ALK) rearrangement have been discovered in lung cancer, leading to targeted therapies developed in non-small cell lung cancer (NSCLC). Several oncogenes have been reported to be negative biomarkers for response to tyrosine kinase inhibitors (TKIs), for example, NSCLC with KRAS mutations are resistant to EGFR-TKIs. A preclinical study showed that KRAS mutant lung cancer cells were sensitive to pemetrexed, while a retrospective study reported that pemetrexed-based chemotherapy didn't show a favorable outcomes in patients with KRAS mutant NSCLC. We, therefore, studied efficacy of pemetrexed-based chemotherapy in patients with NSCLC, and compared the efficacy between KRAS and wild-type NSCLC.

[Methods]

We retrospectively studied the medical records of patients diagnosed with NSCLC between 2013 to 2015. Genomic DNA (gDNA) was extracted from FFPE tissue samples (QIAamp DNA FFPE Tissue Kit, QIAGEN). KRAS mutations were evaluated using Ion Torrent NGS systems (Ion PGM, Thermo Fischer Scientific) or PCR-rSSO. AmpliSeq Colon and Lung Cancer Research Panel v2 was used in the present study. We used Ion Reporter software for next-generation sequencing data analysis.

[Results]

We analyzed the data of 226 patients with NSCLC and successfully extracted gDNA from 94 tissue samples. Among them, mutation analysis by Ion PGM system was performed in 48 samples and PCR-rSSO in 46 samples. We will report response rate and progression free survival in the patients treated with pemetrexed-based chemotherapy.

#2268

Detection of T790M mutation in EGFR gene, an EGFR-TKI resistant mutation, in tumor samples unexposed to EGFR TKIs.

Hayato Koba, Hideharu Kimura, Shingo Nishikawa, Taro Yoneda, Takashi Sone, Kazuo Kasahara. _Kanazawa University Hospital, Kanazawa, Japan_.

Background: T790M mutation in EGFR gene is the commonest mechanism of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients with EGFR mutation. The third-generation EGFR-TKIs are expected to overcome the resistance caused by T790M existence. On the other hand, several reports using highly sensitive detection assays showed a minute amount of T790M mutation alleles was detected in tumor tissues obtained before EGFR-TKI treatment.

Aim: To clear whether T790M dominancy in tumor cells before EGFR-TKI treatment reflects between about the EGFR-mutated lung cancer which was not until exposed EGFR TKI therapy and actual resistance mechanism. Methods: We assessed T790M dominancy in tumor cells from 29 NSCLC patients with EGFR mutations, who had not received EGFR-TKIs. T790M mutation and common mutations, such as deletional mutations in exon 19 and L858R, were detected by droplet digital PCR (ddPCR) separately. T790M dominancy was calculated from each level of mutation-alleles about common mutation and T790M mutation.

Results: We can detect the T790M mutation in initial biopsy tissues from 21 cases (72%). The mean value of T790M mutation dominancy ratio before EGFR-TKI treatment was 0.48% (0.00-69.09%) and it is not difference between in patients with T790M-positive at resistance and in that with negative (p=0.87). Conclusions: T790M mutation was detected in most of the tumor tissues unexposed to EGFR TKIs. T790M mutation dominancy calculated by our assays in tumor tissues before EGFR-TKI treatment may not be related to induce T790M resistance to EGFR-TKIs.

### Mechanisms of Response to Targeted Agents and Potential New Targets

#2269

Comparative effectiveness of everolimus + endocrine therapy vs endocrine monotherapy among postmenopausal women with HR+/HER2- advanced breast cancer: a multicountry retrospective chart review.

Fernando Petracci,1 Jose Zarba,2 Andrea Michelotti,3 Lorenzo Livi,4 Cristian Villanueva,5 Roberto Bordonaro,6 Viktor Sherstnev,7 Rubén Kowalyszyn,8 Nina Marinsek,9 Zhou Zhou,10 Alexander Macalalad,10 Valerie Koo,10 Erich Trieschman,10 Jipan Xie,10 James Signorovitch,10 Barbara Ratto,11 Keiko Higuchi,11 Mahasti Saghatchian12. 1 _Instituto Fleming, Buenos Aires, Argentina;_ 2 _Centro Médico San Roque Tucuman, San Miguel de Tucuman, Argentina;_ 3 _Oncologia Medica I, Ospedale S.Chiara, Azienda Ospedaliera Universitaria Pisana, Pisa, Italy;_ 4 _Azienda Ospedaliero Universitaria Careggi Firenze, Firenze, Italy;_ 5 _Centre Hospitalier Régional Universitaire Besançon, Besançon, France;_ 6 _UOC Oncologia Medica ARNAS Garibaldi Nesima-Catania, Nesima-Catania, Italy;_ 7 _Oncology Clinical Oncology Dispensary #5, Moscow, Russian Federation;_ 8 _Clinica Viedma, Viedma, Argentina;_ 9 _Navigant Consulting, Inc, London, United Kingdom;_ 10 _Analysis Group, Boston, MA;_ 11 _Novartis Pharmaceuticals Corporation, East Hanover, NJ;_ 12 _Institut Gustave Roussy, Paris, France_.

Background: This study aims to compare patient characteristics and outcomes of everolimus (EVE) + endocrine therapy (ET) vs ET monotherapy among postmenopausal women with hormone receptor-positive (HR+) human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (aBC) across multiple countries.

Methods: This retrospective chart review included postmenopausal patients with HR+/HER2- aBC who were previously treated with a nonsteroidal aromatase inhibitor (NSAI) and later received EVE + ET, ET monotherapy, or chemotherapy (the index treatment) from 17 sites in 6 countries (Canada, the Netherlands, France, Italy, Russia, and Argentina). This is a subset analysis comparing patients who received EVE + ET vs ET monotherapy. Stratified sampling by index treatment and line of therapy was used to ensure the sample groups included different treatments and lines of therapy. Patient characteristics were compared using Wilcoxon rank-sum tests for continuous variables and Fisher exact tests for categorical variables. Progression-free survival (PFS) was estimated using Kaplan-Meier analysis and was compared between the 2 groups using Cox proportional hazards models adjusting for age, country, line of therapy, metastatic sites, symptoms, comorbidities, prior chemotherapy, recurrent vs de novo cancer, and time from the last adjuvant ET to aBC diagnosis.

Results: A total of 119 patients in the EVE + ET group (77 in 1st/2nd, and 42 in 3rd/4th lines) and 102 patients in the ET monotherapy group (76 in 1st /2nd, and 26 in 3rd/4th lines) were included in the study. All but 2 patients in the EVE + ET group received EVE + exemestane; ET monotherapy included fulvestrant (46.1%), exemestane (21.6%), NSAIs (16.7%), and tamoxifen (14.7%). Patients in the EVE + ET group were numerically younger (median 63.0 vs 66.0 years; P = .10); appeared to have fewer comorbidities, particularly hypertension (11.8% vs 35.3%; P < .01); had a higher proportion of visceral metastasis (43.7% vs 32.4%; P = .10) and a higher

number of metastatic sites (median 2.0 vs 1.0; P = .14); had a lower proportion of bone/joint symptoms (30.3% vs 42.2%; P = .07); and had a shorter time from initiation of the last adjuvant ET to aBC diagnosis (median 43.1 vs 55.6 months; P = .05). In the unadjusted analysis, patients in the EVE + ET group had similar PFS as those in the ET monotherapy group (median 9.1 vs 9.2 months; unadjusted HR = 0.98; P = .90), but after adjustment for baseline characteristics, the hazard of progression or death of any cause was lower (adjusted HR = 0.67; P = .05).

Conclusions: Patients who received EVE + ET tended to be younger with fewer comorbidities yet have disease with faster progression and a higher burden of metastases. After adjusting for these characteristics, EVE + ET was associated with longer PFS compared with ET monotherapy.

#2270

A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer.

Niamh E. Buckley, Paula Haddock, Ricardo De Matos Simoes, Eileen Parkes, Gareth Irwin, Frank Emmert-Streib, Stephen McQuaid, Richard D. Kennedy, Paul B. Mullan. _Queen's University Belfast, Belfast, United Kingdom_.

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or 'BRCAness', is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies.

We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways.

#2271

bcl-2 expression in HER2-positive breast carcinoma subtypes.

Kalliopi P. Siziopikou,1 Alexandra Larson,1 Luis Z. Blanco,1 Virginia Kaklamani2. 1 _Northwestern University Feinberg School of Medicine, Breast Pathology Section and The Robert H. Lurie Comprehensive Cancer Center, Chicago, IL;_ 2 _Breast Oncology Program CTRC, University of Texas Health Science Center, San Antonio, TX_.

Background: HER2-positive breast cancers comprise 20-30% of all breast cancers; amplification of HER2 confers a poorer prognosis. While treatment with anti-HER2 targeted therapies has dramatically improved outcomes in this group, a significant subset of patients develops resistance, highlighting the need for novel therapeutic options. In addition, like triple negative breast carcinomas, HER2-positive invasive carcinomas also consist of different intrinsic subtypes with different responses to therapy and different overall prognosis. Finally, recent reports suggest that bcl-2 expression contributes to resistance to anti-HER2 therapies. In this study, we evaluated expression of bcl-2 in HER2-positive breast tumors and correlated the expression of bcl-2 with HER2 subtype, patient demographics and histologic and molecular characteristics.

Design: The study population consisted of 114 patients with HER2-positive invasive breast carcinoma treated with surgical excision or mastectomy at Northwestern Memorial Hospital (2009-14) (mean age 53, range 18-80). Electronic medical records were reviewed for patient demographics. Pathologic tumor characteristics (histologic type, size, grade, lymph node status) and tumor marker profile (ER, PR, p53 and Ki-67) were evaluated. Tissue microarrays were constructed (3 cores from each case to account for tumor heterogeneity) for immunohistochemical evaluation of bcl-2 (Dako, clone 124).

Results: Overall, these HER2-pos breast carcinomas were almost exclusively of ductal histologic type (101/114, 88.6%). Almost ¾ of the cases were poorly differentiated, grade 3 carcinomas (83/114, 73%), while the other 31 were grade 2 tumors. No grade 1 tumors were seen. bcl-2 was expressed in 64/114 (56%) of the HER2-pos carcinomas. No association of the bcl-2 expression with age, race, tumor size, lymph node status, p53 expression, or Ki-67 proliferation index was seen. Of interest, this sample of HER2-pos carcinomas was almost equally balanced between ER-neg/HER2-pos (HER2 subtype) and ER-pos/HER2-pos (Luminal B subtype), 54 vs 60 cases respectively. bcl-2 was differentially expressed in these two HER2-pos tumor subtypes: only 9/54 (14%) of the HER2 subtype expressed bcl-2 compared to 55/64 (85.9%) of the Luminal B subtype (p<0.0001).

Conclusions: 1. bcl-2 is expressed in just over half of the HER2-pos carcinomas. 2. bcl-2 expression is far more common in Luminal B compared to the HER2-pos subtype of breast carcinomas. 3. bcl-2 expression does not appear to have a differential expression between age or racial groups and does not correlate with tumor size or lymph node status. Our findings add to the understanding of the molecular mechanisms that drive the different subtypes of HER2-pos breast carcinomas and raise the possibility that bcl-2 targeted therapies may lead to novel therapeutic approaches for the management of subgroups of patients with HER2-pos breast tumors.

#2272

Association of G-protein estrogen receptor (GPER) in primary breast cancer in Caucasian and Black women by tumor immunophenotype and menopausal status.

Sandra L. Deming-Halverson,1 Carl Graeber,2 Jason Machan,2 Edmond Sabo,3 Wei Zheng,4 Edward J. Filardo2. 1 _Social & Scientific Systems, Inc., Durham, NC; _2 _Brown University School of Medicine, RI;_ 3 _Technion Institute and Rambam Health Care Campus, Israel;_ 4 _Vanderbilt University Medical Center, TN_.

Background: Assignment of breast cancer therapies is largely defined by immunohistochemical analyses that evaluate steroid hormone and growth factor receptor status in primary tumor biopsies. Patients whose tumors are devoid of these receptors have fewer treatment options, and poor survival. This outcome is more commonly measured among Black versus Caucasian patients, and in patients with intact ovarian function. The G-protein coupled estrogen receptor, GPER, holds significance for breast cancer biology and treatment as 17Β-estradiol and ER antagonists trigger the release of epidermal growth factor (EGF)-ligands and GPER associates directly with clinicopathological factors that predict advanced disease.

Experimental design: GPER was measured by immunohistochemical analysis and a semi-quantitative scoring method in 521 primary breast tumors obtained from the NCI Cooperative Breast Cancer Tissue Resource and the Nashville Breast Health Study, including tumor specimens derived from 389 Caucasian and 108 Black patients. GPER was correlated with disease progression variables and evaluated by race, menopausal status and tumor immunophenotype.

Results: GPER is expressed in more than two-thirds of invasive breast carcinomas in this study with no difference observed between Caucasian and Black women (p=0.69). Coexpression of GPER and ER was measured in less than half of all tumors (48%). Additionally, GPER is commonly expressed in ER-negative tumors (107/171; 62%) and in triple negative breast cancer (TNBC) (42/56; 75%) and did not differ by race for this newly defined measure of estrogen responsiveness (p=0.42). Moreover, while ER-negative tumors are more common in pre- (79/153; 52%) versus postmenopausal women (92/336; 27%), GPER expression is not influenced by menopausal status. GPER is positively associated with tumor size (particularly in premenopausal women) in both studies, and with frank metastases at first diagnosis in the NCI population. No significant association was seen with nodal invasion. Survival data in NBHS patients found that women who died of breast cancer are more likely to have GPER-positive tumors than survivors.

Conclusion: GPER is expressed in the majority of invasive breast tumors (> 60%) at diagnosis and is commonly retained in patients with ER-negative or triple-negative disease regardless of race or menopause. Unlike ER, which varies inversely with variables that predict advanced disease, GPER is positively associated with these same prognostic parameters. These data support GPER as an independent marker of estrogen responsiveness and breast cancer progression. Most importantly, our results suggest that GPER represents a promising therapeutic target since its expression is constant across multiple types of invasive breast cancer, including patients with TNBC and in tumors from pre- and postmenopausal women.

#2273

Targeting the PI3K/AKT/mTOR pathway for the treatment of metaplastic breast cancer: Does location of PIK3CA mutation or histology affect response.

Reva K. Basho,1 Michael Gilcrease,1 Rashmi K. Murthy,1 Thorunn Helgason,1 Daniel J. Booser,1 Daniel D. Karp,1 Funda Meric-Bernstam,1 Kenneth R. Hess,1 Shelley M. Herbrich,1 Vicente Valero,1 Constance Albarracin,1 Jennifer Litton,1 Mariana Chavez-MacGregor,1 Nuhad K. Ibrahim,1 James L. Murray,1 Kimberly B. Koenig,1 David Hong,1 Vivek Subbiah,1 Razelle Kurzrock,2 Filip Janku,1 Stacy Moulder1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of California San Diego Moores Cancer Center, San Diego, CA_.

Background: Metaplastic breast cancers (MpBCs) are a chemo-refractory group of tumors that contain a component of squamous and/or mesenchymal differentiation identifiable by light microscopy. MpBCs contain a high frequency of aberrations in the PI3K/AKT/mTOR pathway, making this pathway a potential target for therapy.

Methods: Patients with advanced MpBC (N=52) were treated with liposomal doxorubicin (D), bevacizumab (A) and the mTOR inhibitors temsirolimus (T) or everolimus (E). D and A were administered IV on day 1 with T (IV on days 1, 8 and 15) or E (continuous daily oral administration) using 21 day cycles. All tumors were evaluated to assess histology of metaplasia (spindle, mixed spindle vs non-spindle cell). Response was assessed every 6 weeks using RECIST. When available, archived tissue was evaluated for aberrations in the PI3K pathway using standard assays.

Results: Fifty-two MpBC patients were treated with DAT (N=39) or DAE (N=13). Median age was 58 (range 37-79); median number of prior regimens for metastatic disease was 1 (range 0-5). The objective response rate (ORR) was 21% [complete response (CR)=4 (8%); partial response (PR)=7 (13%)] and 10 (19%) pts had stable disease (SD)≥6 months for a clinical benefit rate (CBR) of 40%. Tissue was available in 43 pts and 32 (74%) had a PI3K pathway activating aberrations. PI3K pathway aberration was associated with a significant improvement in ORR (31 vs 0%; P=0.04) but not CBR (44 vs 45%; P=1.00) or progression-free survival (median 5 vs 3 months; P=0.35). The most frequent PI3K pathway aberration was mutation in PIK3CA, occurring in 19 patients. Outcomes were similar if mutations of PIK3CA were located in the helical or kinase domain (ORR 25% vs 27%; P=1.00 and CBR 38% vs 47%; P=1.00, respectively). Spindle cell was the most frequent metaplastic histology seen, occurring in 18 tumors and mixed with other metaplastic histologies including squamous, chondroid and osseous in 14 additional tumors, while 20 tumors had non-spindle cell morphologies. The incidence of PI3K pathway aberration was similar across histologies (61% in spindle vs 67% in mixed spindle vs 60% in non-spindle cell). Tumors with mixed histology had lower ORR, CBR and PFS, but this was not statistically significant, likely due to small numbers in each cohort: ORR 22% in spindle vs 7% in mixed spindle vs 30% in non-spindle cell, P=0.27; CBR 50% in spindle vs 21% in mixed spindle vs 40% in non-spindle cell, P=0.25; and PFS median 4 months in spindle vs 2 months in mixed spindle vs 5 months in non-spindle cell, P=0.68.

Conclusions: Response to mTOR inhibition is enhanced in MpBCs with PI3K pathway aberrations. However, specific aberrations in PIK3CA do not lead to differential response to mTOR inhibition. PI3K pathway aberrations and response to mTOR inhibition are seen across all histologies of MpBC, and the response is not enhanced in particular histologies.

#2274

Deregulated Hippo pathway is a potential therapeutic target for advanced gallbladder cancer.

Carolina Bizama,1 Patricia García,1 Jaime A Espinoza,1 Nicole Elgueta,1 Ismael Riquelme,2 Francisca Alfaro,1 Diego Romero,1 Helga Weber,2 Pamela Leal,2 Javier Retamal,1 Lorena Rosa,1 Juan Carlos Roa1. 1 _Pontificia Universidad Católica de Chile, Santiago, Chile;_ 2 _Universidad de La Frontera, Temuco, Chile_.

Gallbladder cancer (GBC) is lethal and aggressive disease characterized by late diagnosis, poor prognosis and lack of effective therapeutic options. An excellent candidate for targeting therapy in cancer is the Hippo signaling pathway that has been reported to be deregulated in different types of cancer and recently has emerged as a master regulator that plays a critical role in multiple processes such as tumorigenesis, apoptosis, tumor stem cell phenotype, drug cell resistance and metastatic potential. However, little is known about the status of the Hippo kinase pathway in human GBC. The aim of this study is determinate the Hippo pathway activity in GBC and evaluate the potential therapeutic role of YAP1 siRNA and the pharmacological inhibitor Verteporfin, a small molecule that inhibit TEAD-YAP interaction. Immunohistochemistry was used to analyze the expression of YAP1 and Survivin, a major downstream effector and transcriptional target gene of Hippo signaling pathway, in tissue microarray containing 198 advanced GBC samples. Then, we performed an exploratory analysis of Hippo pathway activity by qRT- PCR in advanced GBC and cholecystitis samples. The levels of expression of the Hippo core kinase proteins in GBC cell lines was studied by western blot and the inhibition of YAP1 was performed thought specific siRNA and Verteporfin. Immunohistochemistry showed high nuclear expression of YAP1 in vast majority of advanced GBC, which is correlated significantly with nuclear Survivin expression. The 5-year survival rates of patients with survivin-positive cancer were significantly lower compared with survivin-negative cancer patients (p<0.05, Log-rank test). The gene expression analysis showed a significant down-regulation of the Hippo kinase core suppressor genes STK3 (MST2), STK4 (MST1), SAV1, LATS2 and up-regulation of GPCR5A and BIRC5 in GBC tissues compared with cholecystitis. In addition, western blot analysis show high levels of nuclear YAP1 in 4 GBC cells and its expression were associated with a reduction in the phosphorylation status of LATS1 and MOB. Our findings indicate that targeting YAP1 with siRNA reduced mRNA and protein levels of this oncogene in almost 80% and inhibited the clonogenic capacity of GBC cell lines. Otherwise, the in vitro pharmacological inhibition of YAP1 with Verterporfin reduces YAP1 protein in a dose-dependent manner and was associated with down-regulation of survivin. Our results showed that the Hippo- pathway is deregulated in advanced GBC and mediates its oncogenic effects through of attenuation of their suppressor core components, nuclear translocation of YAP1 and up-regulation of survivin expression. Targeting YAP1 by siRNA and Verteporfin leads to inactivation of YAP1 and survivin affecting cancer cell survival capacity. Targeting the Hippo/YAP has the potential to provide a novel strategy for GBC therapy. Research supported by FONDECYT 3140426, 1130204, 11130515, 3140308.

#2275

Intestine-specific homeobox (ISX) upregulates E2F1 expression and related oncogenic activities in HCC.

Shen-Nien Wang, Li-Ting Wang, Shih-Hsien Hsu. _Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan_.

Intestine-specific homeobox (ISX), a newly identified proto-oncogene, is known to regulate cell proliferation and drive hepatocellular carcinoma (HCC) formation; however, the underlying mechanisms linking gene expression and tumor formation remain obscure. In this study, we showed that ISX transcriptionally activates E2F1 expression and related oncogenic activity by directly binding to its promoter region. Mechanistically, forced ISX expression upregulated the expression and nucleus translocation of the E2F1-DP1 complex phosphorylated by the cyclin D1-CDKs complex, which promoted oncogenic activities of ISX-E2F1 axis in hepatoma cells. In addition, co-expression of both ISX and E2F1 significantly promoted cell proliferation and anti-apoptotic signals instead of apoptosis and autophagy induced by the tumor suppressors p53 and RB1. In contrast, short hairpin RNA-mediated attenuation of ISX and E2F1, respectively, in hepatoma cells decreased cell proliferation and malignant transformation in vitro and in vivo. The mRNA expression was compared in 238 paired specimens of HCC and adjacent normal tissues, and E2F1, as an expression of ISX, pathologically exhibited a tumor-specific expression pattern and was highly correlated with ISX expression, patient survival time, progression stage, and poor prognosis. Taken together, our results highlight that E2F1 is an important downstream gene of ISX in hepatoma progression.

#2276

Sorafenib/Refametinib potently inhibits Wnt/β-catenin in vitro and patient-derived xenograft models of human hepatocellular carcinoma.

Huynh T. Hung,1 Richard Ong,1 Florian Puehler,2 Arne Scholz,2 Oliver Politz,2 Dieter Zopf,2 Dominik Mumberg,2 Karl Ziegelbauer2. 1 _National Cancer Ctr. Singapore, Singapore, Singapore;_ 2 _BAYER Pharma AG, Berlin, Germany_.

Background: Mutations affecting the Wnt/β-catenin pathway have been found in 26-40% of HCC samples. Aberrant constitutive activation of this pathway leads to uncontrolled cell proliferation growth and survival of tumor cells. Thus, identification of Wnt/β-catenin pathway inhibitors might lead to a new treatment option for a large subset of HCC patients. In the present study, we aim to investigate the effects of Sorafenib plus the allosteric MEK1/2 inhibitor Refametinib (Sorafenib/Refametinib) on Wnt/β-catenin signaling in vitro and in PDX models of HCC. Methods: We treated 15 patient-derived HCC xenograft models with i) 10 mg/kg Sorafenib QD, ii) 15 mg/kg Refametinib QD, and iii) Sorafenib/Refametinib. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant Wnt/β-catenin signaling. Apoptosis, cell proliferation and localization of β-catenin were analyzed by immunohistochemistry. In-vitro explant culture from 3 patient-derived HCC lines was also tested. Results: In both wild-type and activated-mutated β-catenin HCC primary cells, Refametinib and Sorafenib/Refametinib potently inhibited cell proliferation and caused cell death, which were correlated with downregulation of Dvl3 and β-catenin target genes (Axin2, c-myc, cyclin D1 and survivin). Inhibition of p-LRP6 at Ser1490 and p-β-catenin at Tyr142 and elevation of E-cadherin were also observed. These changes were not observed in Sorafenib-treated samples. In addition, the Sorafenib/Refametinib combination also suppressed Wnt3a-induced p-LRP6 at Ser1490 and β-catenin accumulation in cells. In vivo, significant reductions in p-LRP6 at Ser1490 and p-β-catenin at Tyr142 were observed in tumor samples from the Sorafenib/Refametinib treatment group and to a lesser extent, samples from the Refametinib. Although the overall protein levels of β-catenin were not significantly changed, non-phosphorylated (active) β-catenin Ser33/37/Thr41 and WNT/β-catenin target genes in Refametinib- and Sorafenib/Refametinib-treated tumors were significantly reduced. While vehicle-treated β-catenin-mutated tumors showed strong cytoplasmatic and nuclear β-catenin localization, most of β-catenin in Sorafenib/Refametinib- and, to a lesser extent, Refametinib-treated tumors was accumulated at the membrane. Conclusion: Sorafenib/Refametinib and, to a lesser extent, Refametinib inhibit the growth of patient derived explant cultures and PDX models. They also inactivate β-catenin signaling. Since activating mutations in the Wnt/β-catenin pathway are detected in a high proportion of HCC, combination of Sorafenib and Refametinib may represent an alternative treatment for β-catenin-dependent HCC.

#2277

Multiplexed gene expression profiling of resected intrahepatic cholangiocarcinoma: Implication of FGFR4-FGF19 as a novel treatment target.

Changhoon Yoo,1 Kyu-pyo Kim,1 Baek-Yeol Ryoo,1 Seung-Mo Hong,1 Jung Jin Hwang,1 Seong-Yun Jeong,1 Klaus Hoeflich,2 Oleg Schmidt-Kittler,2 Stephen Miller,2 Eun Kyung Choi1. 1 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Blueprint Medicine, Cambridge, MA_.

Background: The fibroblast growth factor receptor 4 (FGFR4) pathway is an essential regulatory component of bile acid synthesis and its relationship with hepatocellular carcinoma (HCC) has been reported. We investigated the gene expression and clinical significance of FGFR4 and related pathways in intrahepatic cholangiocarcinoma (IH-CCa).

Methods: We assessed the expression of 98 genes in formalin-fixed paraffin embedded tumor tissue specimens from 46 patients with IH-CCa surgically resected using the NanoString platform. This included 10 FGF pathway genes (e.g. FGFR1-4, KLB, FGF3, 4, 19, 21, 23), 19 distal marker genes (e.g. CYP7A1, CYP17A1) and 31 genes relevant to HCC and CCa (e.g. AFP, TS) and 18 CNV matched genes and 20 control genes. Log-transformation of the gene expression was done for normalization and statistical analysis. Overall survival of 44/46 patients was correlated with gene expression (< median vs ≥ median) using a log-rank test. Two patients were excluded from analysis, due to death from postoperative complications and metastasectomy respectively.

Results: The median age of our patient cohort was 56 years (range 30-78) and 34 patients (74%) were male. All but one tumor specimen (98%) were from the primary tumor resection and microscopic complete resection (R0) was achieved in 37 patients (80%). Six patients (13%) had hepatitis B virus infection with or without liver cirrhosis. FGFR4 was expressed 100 times over background in IH-CCa, the expression of FGF19, FGF21, CYP7A1, CYP17A1, AFP and KLB was lower. Overall survival was significantly stratified according to the expression of FGFR4 (p=0.004), FGF19 (p=0.046), FGF21 (p=0.04), KLB (p=0.03), CYP8B1 (p=0.02), ADH1B (p=0.045), CDKN1B (p=0.005), NCOR1 (p=0.03), NF1 (p=0.006), NROB2 (p=0.001), MAP2K3 (p=0.009), MYC (p=0.04), CYP2E1 (p<0.001), and LGR5 (p=0.03).

Conclusions: Our results indicate that FGFR4 might have a crucial role in IH-CCa, and its overexpression was associated with better prognosis, supporting that FGFR4 may define a subset of IH-CCa and be used as a promising target for therapeutic interventions.

#2278

Next-generation sequencing (NGS) analysis from a phase II single-agent gedatolisib study in patients with endometrial cancer.

Nuzhat Pathan, Patricia English, Keith Ching, Stephen Huang, Mehran Jalaie, Kristen Pierce, Jennifer Vermette. _Pfizer, San Diego, CA_.

Genetic alterations in the PI3K pathway are abundant in endometrial cancer. Hence it is hypothesized that PI3K/mTOR inhibitors will have utility for treatment of endometrial cancer. Gedatolisib, also known as PF-384, is a potent and selective dual PI3K-mTOR inhibitor with broad anti-tumor activity in preclinical studies. Gedatolisib delivered weekly intravenously was investigated in a Phase II single-agent study of advanced endometrial cancer. The cumulative clinical benefit rate from 38 response evaluable patients was 39.5%, comparable to historical rates with mTOR inhibitors. A retrospective NGS analysis of archival biopsies from patients in the study was conducted using the Foundation Medicine Inc (FMI) Foundation One Test to explore potential predictive biomarkers for clinical benefit. Of the 26 patient samples submitted for analysis, data was obtained from 19 samples of which 17 had evaluable response data. Of these 17 samples, 6 were from patients who exhibited progressive disease (PD), 6 were from patients with stable disease (SD) and 5 were from patients that showed a partial response (PR) to treatment. In general, the best responders had a low mutation load while the worst responders had a higher mutation load. Whether large mutation burden was due to mismatch-repair defects determined by MSI status of the tumors is currently under investigation. PTEN alterations were found in tumors from patients that exhibited PD and SD, but not PR. Multiple genetic alterations in ARID1A were observed in 3 of 6 tumors from patients with PD. While PIK3CA alterations were frequently present, 2 of 5 patients with PR and 1 with SD exhibited an activating PIK3CA mutation at H1047R. Among 175 genes that were observed to be mutated in the analysis of 17 independent tumor samples, the top ranking genes associated with a reduction in tumor size (% change from baseline by RECIST 1.0; Wilcoxon rank sum test) were MAP3K1 (p=0.027) and CTNNB1 (p=0.050). Activating mutations in β-catenin were observed in tumor from 3 of 5 patients with PR and were not present in tumor from patients with PD or SD. Tumor from a patient who exhibited an outlier clinical response (stayed on study after 2 years on treatment) had an activating mutation in Akt at E17K and also a novel mutation in mTOR at F2184L. Computational modeling analysis revealed that the mutation did not have a significant impact on kinase structure or ligand binding. KRAS mutations did not appear to correlate with clinical response. This study highlights both the wealth of information provided by NGS analysis, but also the complexity of NGS data analysis and interpretation.

#2280

Functional characterization of combining epigenetic modifiers azacitidine and AG-221 in the TF-1:IDH2R140Q AML model.

Vivek S. Chopra,1 Brian Avanzino,1 Konstantinos Mavrommatis,1 Adam Olshen,2 Jorge DiMartino,1 Kyle J. MacBeth1. 1 _Celgene Corp., San Francisco, CA;_ 2 _UCSF, San Francisco, CA_.

Approximately 15% of AML patients have an IDH2 mutation which leads to production of the oncometabolite 2-hydroxyglutarate (2HG). Accumulation of 2HG inhibits aKG-dependent DNA and histone demethylases, resulting in epigenetic disregulation, which in turn leads to a block in cellular differentiation, promoting AML. AG-221 is a selective inhibitor of IDH2 mutant enzyme and is in development for AML patients carrying IDH2 mutation. Clinical responses to AG-221 therapy have been observed, including decreases in blast percentages and evidence of differentiated functional blood cells; however, preliminary analysis indicated that the mutant allelic burden was not reduced in a majority of subjects treated with AG-221. Azacitidine (AZA) is another epigenetic modifying agent with clinical activity in AML. We hypothesized that combining AG-221 with AZA could synergize in releasing the differentiation block in IDH2-mutant AML cells and enhance cell killing. TF-1:IDH2R140Q are human erythroleukemia cells engineered to express R140Q-mutant IDH2 and model the differentiation block conferred by 2HG accumulation. TF-1:IDH2R140Q cells were treated with AG-221, AZA or the combination of AG-221 + AZA, and measures of cell differentiation (hemoglobinization, KLF1 and HBG qRT-PCR, CD34/CD38 flow cytometry) and death (IncuCyte Zoom caspase 3/7 ) were evaluated. Measures of cell differentiation were evaluated in TF-1:IDH2R140Q cells, using an in vitro erythropoietin differentiation assay. Single agent AG-221 and AZA increased heme production in a dose-dependent manner, as evidenced by increased red color of cells. With AZA + AG-221 combination, hemoglobinization was greater than with single agents. Dose-dependent increases in RNA expression of differentiation markers KLF1 and HBG were observed with single agents, and the combination resulted in additive, or greater than additive, increases. Quantification of hematopoietic stem /progenitor cell populations demonstrated that as single agents, both AG-221 and AZA reduced CD34+/CD38+ and CD34+/CD38- cell populations, and the combination resulted in additive or greater than additive decreases. Real-time quantification of cell death showed that single agent AG-221 had no effect, while single agent AZA increased apoptosis. Concurrent combination of AZA + AG-221 increased cell death beyond that of single agents. Whole genome expression data (RNA-seq) showed enrichment of differentiation and cell death gene signatures with the combination when compared to single agents alone. We have demonstrated the beneficial effects of combining AG-221 + AZA in the TF-1:IDH2R140Q AML cell line, including greater than additive increases in hemoglobinization and expression of differentiation markers, reduced stem /progenitor cell populations, and potentiation of death. Further exploration of the molecular mechanism of the combination is ongoing.

#2281

DNA methyl transferase inhibitors (DNMTis) upregulate NY-ESO expression in several human primary cells: implications for co-therapy with engineered adoptive T-cell therapies.

Bruno Laugel,1 Alexandra Sevko,1 Karen Howe,1 Zoltan Ferjentsik,1 Tiago Ferronha,1 Miguel Maroto,1 Joanna Brewer,1 Bent Jakobsen,1 Gwendolyn Binder-Scholl2. 1 _Adaptimmune, Oxford, United Kingdom;_ 2 _Adaptimmune, Philadelphia, PA_.

The DNA methyl transferase inhibitors 5-aza-2'-deoxycytidine (decitabine or DECI) and 5-azacytidine (azacitidine or AZA) have recently been approved for the treatment of a variety of hematologic malignancies. These cytidine analogues remove repressive epigenetic DNA methyl marks and induce the transcription of numerous genes normally silenced during the processes of cellular differentiation or transformation. DNMTis have a net anti-neoplastic activity through the up-regulation of tumor-suppressor genes involved in the induction of apoptosis, cellular differentiation and cell cycle arrest. In addition, DNMTis promote the expression of several cancer testis antigens (CTAs) by tumor cells, including NY-ESO and MAGE-A1/A3. These observations suggest that the combination of DNMTis with immunotherapeutic modalities may provide some clinical benefit.

Adoptive immunotherapies using autologous CD4+ and CD8+ T cells expressing an affinity optimized NY-ESO TCR are in clinical trials and show robust clinical responses in patients across several indications including synovial sarcoma, multiple myeloma or melanoma. DNMTis were previously shown to induce the expression of NY-ESO in antigen-low or -negative tumor cells and consequently enable the activation of NY-ESO-specific T-cells. While there is compelling evidence for the combination of DNMTis with T cells containing engineered affinity-enhanced TCRs recognizing NY-ESO-1, safety data supporting this approach are lacking. Initial tests into the safety of combining these treatments investigated the effects of DECI and AZA on NY-ESO and LAGE-1 expression in a panel of primary adult human cells of different tissue origins. Our data demonstrate that treatment with either compound resulted in a significant up-regulation of NY-ESO transcription in a subset of primary cells tested. The magnitude of increase in transcript expression varied between cell types and, in some cases, was sufficient to activate T cells expressing an affinity-optimized NY-ESO-specific TCR. These findings raise important safety concerns with regards to the potential combination of DNMTi compounds with NY-ESO T-cell therapy and suggest that such co-therapies may result in on-target off-tumor toxicities.

#2282

Decitabine response in FLT3-negative AML is associated with mitochondrial priming.

Elisha Dettman,1 Camille Doykan,1 Jo Ishizawa,2 Weiguo Zhang,2 Alison Walker,3 William Blum,3 Rebecca Klisovic,3 Michael Cardone,1 Michael Andreeff,2 Amy Johnson3. 1 _Eutropics Pharmaceuticals, Cambridge, MA;_ 2 _M.D. Anderson Cancer Center, Houston, TX;_ 3 _The Ohio State University, Columbus, OH_.

To date, acute myeloid leukemia (AML) remains a poor-prognosis disease with only approximately 25% of patients surviving five years after being diagnosed (SEER – 2005-2011). There are a wide variety of treatment options ranging from intense chemotherapy to targeted therapies. Beyond tolerability, identifying the ideal treatment strategy for each patient remains a difficult task. Mitochondrial profiling of AML patient samples using Bcl-2 homology domain (BH3) mimetics has proven to accurately identify patients that respond to several therapies in AML. In this method, cancer cells are exposed to BH3 mimetics designed to test for susceptibility to induction of apoptosis. The readout, called "BH3-Priming" indicates anti-apoptotic protein dependencies in cancer cells.

In this study, samples from AML patients that were treated with at least one cycle of the hypomethylating agent, decitabine, at The Ohio State University were collected and BH3 profiled (n = 64). The patients were assessed for a complete response to the drug and were characterized by several molecular biomarkers including FLT3 mutational status. We found that the FLT3 mutation negative patients (n = 32) who responded to decitabine had significantly higher mitochondrial responses to the BIM and HRK mimetics (p = 0.04) compared with non-responders. We also found that FLT3 mutant patients (n = 12) had significantly (p = 0.02) higher priming than those patients lacking those lesions. Surprisingly, despite being apparently more predisposed towards apoptosis, these patients are less likely to respond to decitabine (42% versus 20%) in vivo than those without these mutations. This could indicate that additional non-mitochondrial inhibitory mechanisms are present in those FLT3 mutant leukemias that prevent response to decitabine.

To further examine the interaction between FLT3 and mitochondrial priming, we profiled engineered cell lines with and without FLT3 mutations. We found that mitochondrial priming was significantly lower (p = 0.03) in BAF3 cells with unmutated FLT3 compared with five BAF3 cell lines containing FLT3 mutations (ITD and TKD mutations). Further, we examined the Broad Institute's Cancer Therapeutics Response database (CTRP v 2.0) for cancer cell line response to decitabine and found that wild type FLT3 cell lines that responded to decitabine had significantly higher mitochondrial priming than those that did not respond to the drug (p = 0.04), consistent with the patient data.

Taken together, we have discovered that response to decitabine in AML cell lines and AML patient samples depends on mitochondrial dependencies and FLT3 mutational status. Genetic lesions in FLT3; however, may be able to over-ride mitochondrial responsiveness to pro-apoptotic factors, illustrating the probable need to measure multiple attribute types including genetics and ex vivo functional testing to accurately determine the likelihood of response to AML therapies.

#2283

Molecular signaling in multiple myeloma: association of RAS/RAF mutation status and MAPK pathway activation in primary myeloma patient biopsies.

Jing Xu,1 Nicole Pfarr,2 Volker Endris,2 Elias K. Mai,3 Nur Hafzan Md Hanafiah,4 Nicola Lehners,5 Roland Penzel,2 Wilko Weichert,2 Anthony D. Ho,3 Peter Schirmacher,2 Hartmut Goldschmidt,6 Mindaugas Andrulis,2 Marc S. Raab5. 1 _Max Eder Group Experimental Therapies for Hematologic Malignancies, Heidelberg University Hospital and German Cancer Research Center (DKFZ); Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany;_ 2 _Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany;_ 3 _Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany;_ 4 _Max Eder Group Experimental Therapies for Hematologic Malignancies, Heidelberg University Hospital and German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 5 _Max Eder Group Experimental Therapies for Hematologic Malignancies, Heidelberg University Hospital and German Cancer Research Center (DKFZ); Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany;_ 6 _Department of Internal Medicine V, Heidelberg University Hospital and National Center for Tumor Diseases (NCT), Heidelberg, Germany_.

Multiple myeloma (MM) is an incurable plasma cell malignancy. Recent studies have identified a dominant mutation cluster in RAS/BRAF genes, highlighting the MEK/ERK signaling pathway as a potentially promising therapeutic target. However, treatment of RAS/RAF mutant MM with the MEK inhibitor, trametinib, resulted in moderate response rates. There were, however, durable remissions in those patients (pts) who responded. This suggests a variable degree of dependency on MEK/ERK signaling in RAS/RAF mutant MM. To address this issue, we correlated RAS/RAF mutations with the RAF/MEK/ERK cascade activation in primary MM patient biopsies.

Primary material consisted of bone marrow or soft tissue biopsies from 179 pts, including 102 pts with newly diagnosed myeloma (NDMM) and 77 pts with relapsed/refractory myeloma (rrMM). Activation of the RAF/MEK/ERK cascade was assessed using an immunohistochemical (IHC) assay for phospho-ERK. In parallel, KRAS, NRAS, HRAS and BRAF mutation status was assessed by targeted re-sequencing using the Ion Torrent NGS technology.

The mutation frequencies found were KRAS (25%), NRAS (24%), and BRAF (8%) while no HRAS was detected, consistent with previous studies. Overall, RAS/RAF mutations were significantly more frequent in rrMM (p = 0.010), mainly driven by an increase in NRAS mutations (p = 0.017), proving the importance of RAS/RAF-mediated signaling in MM and indicating a potential role for NRAS mutations in drug resistance development. The top nine recurrently detected individual mutations were KRAS Q61H (n = 11), NRAS Q61R (11), NRAS Q61K (10), KRAS G12D/G12V (6 each), BRAF V600E (6), NRAS G13D (5), NRAS G13R/Q61H (4 each).

When correlated with ERK activation status, KRAS but not NRAS mutations were associated with pathway activation compared to RAS/RAF-wild type (wt) (p = 0.003). However, these were not mutually exclusive, suggesting that ERK activation is dependent on the type of mutation rather than the fact that the RAS/RAF gene is mutated. Therefore, each recurrent RAS/RAF mutation was tested against all RAS/RAF-wt samples: Only KRAS G12D and BRAF V600E were consistently associated with ERK activation (p< 0.001 each).

In summary, we found that RAS/RAF mutations are not generally associated with RAF/MEK/ERK pathway activation in MM. However, we did identify specific amino acid changes in KRAS and BRAF which are more likely to be associated with pathway activation and two mutations which were consistently associated with MEK/ERK signaling. These findings indicate that confirmation of protein-level pathway activation is needed when considering targeted therapy. In this regard, IHC is a cost-effective and reliable method to assess pathway activation and should therefore be considered in future. The clinical relevance of RAS/RAF mutations in MM should be further elucidated in prospective cohort studies.

#2284

Synergistic anti-tumor effect of the combination of amrubicin and erlotinib in non-small cell lung cancer with wild type EGFR.

Sakiko Otani,1 Jiichiro Sasaki,2 Kuniko Masuda,3 Kazuya Okamoto,3 Akiko Namubu,4 Toshihide Matsumoto,5 Masumi Tanaka,1 Mikiko Ishihara,1 Yoshiro Nakahara,1 Tomoya Fukui,1 Satoshi Igawa,1 Noriyuki Masuda1. 1 _Dept. Respiratory Med., Kitasato University School of Medicine, sagamihara Knagawa, Japan;_ 2 _Dept. comprehensive Med.Resarch and Development Center for New Medical Frontiers, Kitasato University School of Medicine, sagamihara Knagawa, Japan;_ 3 _Pharmaceutical Research Laboratories, Nipponkayaku co., LTD., Tokyo, Japan;_ 4 _Dept. Tumor Genetics and Biology,Faculty of Medical Science Kumamoto University, Kumamoto, Japan;_ 5 _Dept. pathology, Kitasato University School of Medicine, sagamihara Knagawa, Japan_.

Background: Standard second-line chemotherapy against advanced non-small-cell lung cancer (NSCLC) is a single agent such as docetaxel, pemetrexed, or erlotinib (Erl). We recently reported that the combination of amrubicin (AMR) and Erl was clinically safe and effective for previously treated NSCLC patients. The purpose of this research is to confirm the experimental effectiveness of the combination and to investigate mechanism of the action.

Methods: We perform to evaluate the NSCLC cell lines proliferation inhibition by using XTT assay and to make two-dimension isobolograms for assessment of the combination effect. Flowcytemetry analysis (FACS) and Western blot analysis (WB) is also done in vitro. In vivo growth inhibition assay using subcutaneous model on SCID mice is performed. In addition, we measure intracellular concentration of amrubicinol(AMR-OH) which is converted as active metabolite from AMR in both cells treated with AMR alone and the combination.

Results: The combination of AMR and Erl had significant synergistic effect on EGFR wild type NSCLC cell line A549, while additive effect on EGFR mutant cell line PC-9. Sub G0-G1 population on FACS was significantly increased in A549 cells treated with combination. The combination activated apoptosis related proteins in WB. The strong growth inhibition of subcutaneous tumors on SCID mice was observed in the combination cohort. Intracellular concentration of AMR-OH was significantly increased in the combination than in AMR monotherapy.

Conclusion: The combination of AMR and Erl is synergistically induced apoptosis and growth inhibition on A549 cells via increasing intracellular concentration of AMR-OH. This combination may be a promising therapeutic option against advanced NSCLC with wild type EGFR.

#2285

Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression.

Sun-Hee Kim,1 Yuuri Hashimoto,1 Sung-Nam Cho,1 Jason Roszik,1 Denái R. Milton,1 Fulya Dal,1 Sangwon F. Kim,2 David G. Menter,1 Peiying Yang,1 Suhendan Ekmekcioglu,1 Elizabeth A. Grimm1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _The Perlman School of Medicine at University of Pennsylvania at University of Pennsylvania, Philadelphia, PA_.

COX-2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS-mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy.

#2286

Targeting the notch pathway: A potential therapeutic approach for desmoid tumors.

Danielle Braggio,1 Hui Shang,2 Ya-Jung Lee,3 Ghadah A. Al-Sannaa,4 Chad J. Creighton,5 Svetlana Bolshakov,3 Alexander JF Lazar,4 Dina Lev,6 Raphael E. Pollock1. 1 _The Ohio State University, Columbus, OH;_ 2 _Department of Orthopedics, Taihe Hospital, Hubei University of Medicine, Shiyan, China;_ 3 _Department of Surgical Oncology, University of Texas MD Anderson Cancer Center (MDACC), Houston, TX;_ 4 _Department of Pathology, University of Texas MD Anderson Cancer Center (MDACC), Houston, TX;_ 5 _Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX;_ 6 _Sheba Medical Center, Surgery B, Tel Aviv, Israel_.

Desmoid tumors (DTs) are rare mesenchymal lesions that can recur repeatedly. When it is feasible, DTs are surgically resected; however, this often results in high recurrence rates. Recently, treatment with PF-03084014, a potent γ-secretase inhibitor, has been shown to have antitumor activity in several tumor types by affecting the WNT/β-catenin pathway. Consequently, Notch pathway inhibition by PF-03084014 might be a promising approach for DT treatment. The expression of Notch pathway components was analyzed in DT tissues and cell strains with immunohistochemistry and Western blotting, respectively. A panel of DT cell strains was exposed to PF-03084014 and evaluated for cell proliferation. Antitumor effects were assessed via cell cycle, apoptosis, and migration and invasion analysis. Cells treated with PF-03084014 were characterized with a gene array analysis combined with Ingenuity Pathway Analysis. The results showed that Notch pathway components were expressed at different levels in DTs. Hes1 (Hes Family BHLH Transcription Factor 1) was overexpressed in DT tumors versus dermal scar tissue, and PF-03084014 caused significant decreases in Notch intracellular domain and Hes1 expression in DT cell strains. PF-03084014 decreased DT cell migration and invasion and also caused cell growth inhibition in DT cell strains, most likely through cell cycle arrest. Gene array analysis combined with Ingenuity Pathway Analysis showed that Wnt1-inducible signaling pathway protein 2 possibly regulated Notch and WNT pathways after treatment with PF-03084014 through integrin. Our findings suggest that the Notch pathway is an important DT therapeutic target. Furthermore, PF-03084014 has significant antitumor activity against DTs, and it may be an alternative strategy for DT treatment.

#2287

Final results of the pharmacodynamic (PD) data of PQR309-001, a first-in-human trial of a combined PI3K/mTOR inhibitor in advanced solid tumors.

Andreas Wicki,1 Vincent Prêtre,1 Reto Ritschard,1 Nicholas Brown,2 Vincent Bize,3 Thomas Fabbro,1 Natasa Cmiljanovic,4 Sasa Dimitrijevic,4 Deborah Schmitz,4 Michael Stumm,4 Rebecca Kristeleit5. 1 _University Hospital Basel, Basel, Switzerland;_ 2 _University College Hospital, London, United Kingdom;_ 3 _Swiss Group for Clinical Cancer Research, Bern, Switzerland;_ 4 _Piqur Therapeutics, Basel, Switzerland;_ 5 _University College London Cancer Institute, London, United Kingdom_.

Background: PQR309 is a novel, oral, balanced pan-class 1 PI3K, mTORC1 and mTORC2 inhibitor. PQR309-001 was a first-in-man dose escalation study evaluating PQR309 in patients with advanced solid tumours.

The primary objective of the study was to establish the maximum tolerated dose (MTD) of PQR309, its safety and tolerability. Secondary objectives included characterization of PQR309 pharmacokinetics (PK) and pharmacodynamics (PD).

Methods: We investigated PQR309 treatment-induced PD, including changes of 16 PI3K-mTOR and MAPK associated phospho-proteins, and 84 mRNAs in serial tumor biopsies obtained from patients enrolled in study PQR309-001. 13 patients were eligible for PD analysis after histologic evaluation of fresh-frozen tissue. A solid-phase assay with phospho-specific antibodies was used for the investigation of PI3K/mTOR and MAPK signalling. RNA analysis was performed with an RTK/PI3K-specific RNA array.

Results: Under treatment with PQR309, phospho-Akt (p_T308 and p_S473), phospho-mTOR and phospho-S6 were significantly downregulated compared to baseline (p<0.05, Wilcoxon signed-rank test). Moreover, patients with radiographic tumor shrinkage (n=7) had significantly stronger phospho-Akt (p_T308), phospho-mTOR and phospho-S6 reduction than patients whose tumor was growing (n=6) (p<0.05, Mann-Whitney test). Importantly, ERK1/2 phosphorylation, as the readout of MAPK activity, was decreased overall with no statistically significant difference between shrinking and growing tumors. The analysis of mRNAs showed a trend towards upregulation of PDGFRA (3.2 times, not statistically significant) after 21 days of therapy with PQR309. Intriguingly, no consistent changes of other PI3K-related RNAs was observed.

Conclusion: PD assessments in tumour tissue samples from patients treated with PQR309 demonstrated a reduction of phosphorylation of direct targets for PQR309 (PI3K and mTOR) as well as downstream effectors of mTOR. Furthermore, the data indicate that downregulation of phospho-Akt (p_T308) and mTORC1 activity may be predictive of tumor shrinkage while upregulation of PDGFRA may represent a mechanism of resistance against PI3K/mTOR inhibition.

#2288

Targeting mTOR pathway in rare and recalcitrant tumors.

Veronica Rojas,1 Shridar Ganesan,2 Kim M. Hirshfield,2 Robert S. Dipaola,2 Lorna Rodriguez-Rodriguez2. 1 _Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ;_ 2 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

INTRODUCTION: Genomic alterations affecting the PI3K/AKT/mTOR pathway are commonly seen among various cancer types [Kandoth 2013]. The objective of this study is to determine the clinical utility of using comprehensive genomic profiling (CGP) in the course of clinical care to identify genomic alterations affecting the PI3K/AKT/mTOR pathway in patients with either rare or refractory cancers.

METHODS: CGP was performed by Foundation Medicine, Inc. Genomic alterations were reviewed by members of an institutional molecular tumor board (MTB). Consensus recommendations on genomically targeted, clinically relevant FDA-approved, on- and off-label therapies and clinical trials were sent to the treating physician for their consideration as treatment alternatives. Ninety-seven tumors with alterations in the PI3K/AKT/mTOR pathway were analyzed.

RESULTS: Thirty-three patients with cancers that were either rare or refractory to standard therapy received drug targeting the PI3K/AKT/mTOR pathway. Age range was 18-78 years old. Thirteen patients were non-Hispanic White, three non-Hispanic Black, two Hispanic and one Asian. Tumors tested included 6 gynecologic, 5 renal, 2 sarcomas and one each of pancreatic, melanoma, T cell lymphoma, bladder, adrenal, and skin-not melanoma. Treatment outcomes were available for 19/33 patients (one patient died soon after presentation at the MTB and could not be treated). Seven (37%) patients derived clinical benefit from a drug targeting the PI3K/AKT/mTOR pathway; the duration of clinical benefit ranged from 90-245 days. The clinical benefit was not tumor type specific and included two endometrial tumors, 2 renal tumors, one sarcoma, one adrenal paraganglioma and one micropapillary urothelial carcinoma. Eight patients have not been started on PI3K/AKT/mTOR targeted therapy yet.

CONCLUSION: These data suggest that tumors with alterations in the PI3K/AKT/mTOR pathway may respond to targeted therapy regardless of tumor of origin. 

## IMMUNOLOGY:

### Adoptive Cell Therapy

#2289

Allogenic TCRa/CS1 double knockout T-cells bearing an anti-CS1 chimeric antigen receptor: An improved immunotherapy approach for the treatment of multiple myeloma.

Roman Galetto, Isabelle Chion-Sotinel, Agnes Gouble, Julianne Smith. _Cellectis, Paris, France_.

Multiple Myeloma (MM) is a B-cell neoplasia characterized by clonal expansion of malignant plasma cells in the bone marrow. Although currently available therapies can improve patient's overall survival, it still remains incurable in most of them cases. On the other hand, the use of autologous chimeric antigen receptor (CAR)-redirected T-cells have allowed to achieve long-term durable remissions in patients with B cell leukemia, indicating that CAR technology may become a new alternative in cancer treatment. Extensive research is therefore being made in immunotherapy against MM, in order to identify novel antigenic targets to be considered for this approach.

In the present work we have assessed the feasibility of CAR-mediated targeting of the CS1 antigen (SLAMF7), which is highly expressed on tumor cells from most patients with MM. However, expression of CS1 on normal CD8+ T-cells is potentially an obstacle for the development of CAR T-cells against this protein, since antigen-expressing T cells will be targeted, impacting both on the number and the phenotype of the final CAR T cell population.

To address this limitation, Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology was used to inactivate the CS1 gene in T-cells, prior to transduction with a lentiviral vector encoding an anti-CS1 CAR. Our results demonstrate that while non-gene-edited T-cells expressing the anti-CS1 CAR display cytotoxic activity against MM cell lines, CS1-gene-edited CAR cells display significantly increased cytotoxic activity. In addition, a progressive loss of CD8+ T-cells was observed in non-edited CAR+ T-cells, while this population was preserved in CS1 knockout T-cells.

Gene editing technology was also used here to inactivate the TCRa constant (TRAC) gene, minimizing the potential for engineered T-cells to mediate Graft versus Host Disease (GvHD). We have indeed previously shown that editing of the TRAC gene can be achieved at high frequencies, allowing efficient production of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, we were able to perform multiplex genome editing that led to the production of double KO (TRAC and CS1) T-cells

Finally, we evaluated the in vivo activity of double knockout CAR T-cells (UCARTCS1) by performing experiments in an orthotopic MM mouse model, showing that CS1 disrupted T-cells were able to mediate an in vivo anti-tumoral activity.

Our results show that multiplex genome editing is possible and can lead to the production of double KO TRAC/CS1 T-cells, allowing large scale manufacturing of allogeneic, non alloreactive CS1 specific T-cells with enhanced antitumor activity. Moreover, these allogenic T-cells could be easily available for administration to a large number of MM patients.

#2290

Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells.

Loretta Gammaitoni,1 Lidia Giraudo,1 Marco Macagno,2 Giulia Cattaneo,2 Valeria Leuci,2 Francesco Sassi,1 Alessandro Zaccagna,1 Alberto Pisacane,1 Valentina Coha,1 Fabrizio Carnevale-Schianca,1 Massimo Aglietta,2 Dario Sangiolo2. 1 _Candiolo Cancer Institute FPO-IRCCS, Candiolo, Italy;_ 2 _University of Torino, Department of Oncology, Candiolo Cancer Institute IRCCS, Candiolo, Italy_.

Purpose of the study was to explore the activity of cytokine-induced killer (CIK) cells against autologous chemo-surviving melanoma cancer stem cells (mCSCs) in vivo.

Elimination of mCSCs is an ambitious goal as considered responsible for chemo-resistance and relapses.

CIK cells are ex vivo expanded T-NK lymphocytes with MHC-independent antitumor activity. We provided proof of concept that CIK cells can kill mCSCs in vitro (Gammaitoni et al. Clin Cancer Res 2013) and demonstration of their activity in vivo, within an autologous model, would be relevant in clinical perspective.

Experimental procedures and results.

In vivo activity of CIK cells against autologous mCSCs was explored in NOD/SCID mice bearing tumors generated by subcutaneous implantation of primary melanoma cells or alternatively surgically resected tumor samples (patient-derived xenografts, PDX).

mCSCs were visualized by a lentiviral CSC-detector encoding eGFP under control of stem-gene promoter OCT4 (Gammaitoni et al. Clin Cancer Res 2013). Melanoma cells were engineered with the CSC-detector before implantation or alternatively, in PDX, after removal of residual tumors at the end of experimental treatments.

In vivo chemotherapy (CHT) consisted of intravenous (i.v.) Fotemustine (600µg days 1;15). Immunotherapy consisted of i.v. CIK cells (1x107) every 5 days. Antitumor activity was evaluated assessing tumor proliferative index by Ki67 expression in residual tumors. Activity against mCSCs was evaluated assessing the rate of residual GFP+-mCSCs in residual tumors after treatments.

In vivo infusion of CIK cells for 2 weeks determined significant tumor response (n=6, p < 0.0001) that fully involved autologous eGFP+-mCSCs. On the contrary, CHT spared mCSCs that resulted significantly increased in residual tumors compared with mice treated with CIK cells (p=0.001) or untreated controls (p=0.001). CIK cells infused after a previous CHT treatment (n=6) retained significant antitumor activity (p=0.001) and were capable to revert the CHT-induced enrichment of eGFP+-mCSCs, compared to mice treated with CHT alone (p=0.01).

Similarly, in the PDX model, the infusion (n=4) of autologous CIK cells exerted significant antitumor activity that involved eGFP+mCSCs (p=0.009), confirmed by the absence of their increment in residual tumors (p=0.3).

In vitro CHT treatment of melanoma from 8 different patients significantly enriched eGFP+ mCSCs (2.2 ±0.2 fold, p=0.0018) compared to untreated controls. Melanoma treated with CHT, enriched in eGFP+mCSCs, were efficiently killed by autologous CIK cells with mean killing values ranging from 94±2% (40:1 E/T) to 21%±4 (1:3 E/T).

Conclusions.

Our findings provide first demonstration that immunotherapy with CIK cells is active against autologous mCSCs, surviving chemotherapy, in vitro and in vivo. We provide an experimental platform to study mCSCs and rationale to design clinical studies with CIK cells against melanoma.

#2291

On- and off target toxicity profiling for adoptive cell therapy by mass spectrometry-based immunopeptidome analysis of primary human normal tissues.

Oliver Schoor,1 Jens Fritsche,1 Sarah Kutscher,1 Andrea Mahr,1 Lea Stevermann,1 Annika Sonntag,1 Franziska Hoffgaard,1 Dominik Vahrenhorst,1 Julia Leibold,1 Valentina Goldfinger,1 Leonie Alten,1 Sebastian Bunk,1 Dominik Maurer,1 Steffen Walter,2 Hans-Georg Rammensee,3 Harpreet Singh-Jasuja,1 Toni Weinschenk1. 1 _Immatics Biotechnologies GmbH, Tuebingen, Germany;_ 2 _Immatics US Inc, Houston, TX;_ 3 _University of Tuebingen, Tuebingen, Germany_.

A major constraint for the broad and safe application of Adoptive Cellular Therapy (ACT) is the limited number of validated tumor targets, especially for solid tumors. For T-cell receptor (TCR)-based approaches, presentation of targeted HLA-peptides on normal tissues can lead to on-target toxicity, such as severe inflammatory colitis reported upon re-directing T cells to an HLA-A*02 restricted carcinoembryonic antigen (CEA) epitope. Independently, off-target cross-reactivity of TCRs occurred in previous ACT trials, e.g. when a MAGEA3-directed TCR cross-recognized an HLA-A*01 restricted epitope from titin expressed on heart, which led to fatal cardiac toxicities. Here we present a novel approach allowing the prediction of severe on- and off-target side effects before entering into clinical trials.

We used a target discovery engine (XPRESIDENT) combining highly sensitive, quantitative mass spectrometry (LC-MS/MS), RNA-Seq-based differential transcriptomics, immunology and bioinformatics to characterize the human immunopeptidome directly on shock frozen primary human tissues. Over the last years we have built an according database for > 600 tumor samples from > 20 different tumor types and, importantly, > 300 samples from > 40 different normal tissue types, resulting in hundreds of thousands of unique HLA-peptide sequences. These data allow conclusions on which HLA peptides are actually presented on primary normal tissues in a quantitative manner, taking into account relative differences between normal tissues and tumors as well as absolute peptide copy numbers per cell. In order to assess the off-target risk for a TCR, we predict all theoretical HLA- and TCR-binding peptides in the proteome, ideally based on the binding motif of the TCR, and specifically search for actual peptide presentation by normal tissues.

When analyzing the above described CEA case, we were able to detect the CEA-derived peptide IMIGVLVGV on HLA-A*02 positive colorectal cancer samples, but importantly also on normal colorectal samples. In the original study describing the titin case tremendous experimental efforts and sophisticated cell culture models were required to retrospectively identify cross-recognition of the peptide on cardiomyocytes as the cause of toxicity. In contrast, with our approach we easily and directly identified the critical peptide ESDPIVAQY as one of the most abundantly presented peptides on an HLA-A*01 positive primary human heart sample. We show that this approach can lead to noteworthy results also for other pre-clinical and clinical stage TCR candidates.

In conclusion our data demonstrate that ultrasensitive LC-MS/MS of primary tissue may represent a fast, straightforward and meaningful complementary method to common in vitro or animal models for the prediction of on- and off-target toxicities in TCR-based immunotherapy approaches.

#2292

Reconstitution of myeloid derived suppressor cells after the induction of lymphopenia and TIL therapy.

Krithika N. Kodumudi, Doris Wiener, Amy Weber, Erica Royster, Linda Kelley, Amod Sarnaik, Shari Pilon-Thomas. _H.Lee Moffitt Cancer Center, Tampa, FL_.

Factors such as T regulatory (Treg) cells and myeloid derived suppressor cells (MDSC) limit the effectiveness of current immunotherapy protocols. Several reports have shown that administration of chemotherapeutic agents or total body irradiation (TBI) before adoptive transfer of tumor-specific T cells reduces or eliminates the immunosuppressive populations such as Tregs and MDSC. A previous study in our lab has shown that MDSC that reconstitute after lymphodepletion are highly suppressive compared to the endogenous MDSC in a B16 melanoma model. Blockade of MDSC expansion in combination with adoptive T cell transfer delayed tumor growth and improved survival rate. However, there is very little known about the MDSC reconstitution after the adoptive transfer of tumor infiltrating lymphocytes (TIL) in melanoma patients. Melanoma patients were treated with non- myeloablative therapy that included cyclophosphamide and fludarabine. At the time of TIL transfer, no circulating lymphoid or myeloid cells were detected in the PBMC. One day after completion of chemotherapy, up to 100 billion TIL were transferred followed by high dose bolus IL-2. At weeks 1-4 after TIL transfer, PBMC were collected and we investigated the reconstitution and the suppressive function of MDSC. MDSC were phenotyped by flow cytometry as HLA-DR- Lin- CD14+CD33+ or CD11b+CD14-CD33+. While 3-15% of MDSC was observed in the PBMC of melanoma patients as their baseline, after lymphodepletion and one week post TIL infusion, the percent of MDSC increased to 10-30% and gradually dropped to baseline by week 4. To examine suppressive function, MDSC were isolated from peripheral blood at 1-2 weeks after TIL infusion and co-cultured with healthy donor T cells or patient TIL. MDSC were highly suppressive and abrogated T cell proliferation and IFN-gamma secretion. Furthermore, in patients that responded to TIL therapy, the ratio of peripheral MDSC to total CD8+ T cells was lower. These studies demonstrate that MDSC that reconstitute post- lymphodepletion are suppressive and may potentially interfere with robust T cell function. Further studies are warranted to clarify the role of MDSC in the efficacy of TIL therapy. Blocking MDSC in combination with TIL therapies represent an improved strategy for the treatment of melanoma patients.

#2293

Vector-free engineering of immune cells for adoptive cell therapy.

Armon Sharei,1 Jonathan Gilbert,1 Darrell Irvine,2 Klavs Jensen,2 Robert Langer2. 1 _SQZ Biotechnologies, Boston, MA;_ 2 _Massachusetts Institute of Technology, Cambridge, MA_.

Ex vivo manipulation of the immune system for therapeutic purposes has shown incredible clinical promise with the advent of cell therapies such as Chimeric Antigen Receptor modified T-Cell Therapies (CAR-T). However expanding methods to manipulate cells beyond using plasmids and viruses requires a new delivery paradigm. Here we describe a microfluidic approach discovered at MIT where cells are mechanically deformed as they pass through a constriction smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer directly into the cytosol. The method was recognized as one of the World Changing Ideas of 2014 by Scientific American since it has demonstrated the ability to deliver a range of material, such as nanoparticles and proteins to primary cells including embryonic stem cells and immune cells. This is in contrast to existing vector-based and physical methods that have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or oncogenic off-target effects. Supporting the enabling potential of the new deformation based method, we previously reported that delivering protein transcription factors to primary fibroblasts produces a 10-fold improvement in induced pluripotent stem cell colony formation relative to electroporation and cell-penetrating peptides.

In this work we describe the use of the vector-free technology to deliver antigen protein directly to the cytoplasm of antigen presenting cells to drive a powerful antigen specific T-cell response. Current efforts to use antigen presenting cells to drive T-cell responses rely on an inefficient process called cross-presentation that relies on material escaping the endosome and entering the cytoplasm. We believe that by delivering antigen directly to the cytoplasm of antigen presenting cells we can overcome this long standing barrier and drive powerful and specific T-cell responses. Our results show that by adoptively transferring antigen presenting cells that have antigen delivered into them we can drive a significant T-cell response. Specifically, we found that this results in a ~50x increase in antigen specific T-cells in vivo when compared to endocytosis. This advance has the potential to dramatically enhance the therapeutic potential of therapeutic vaccination with antigenic material for the treatment of a wide variety of cancers. Indeed, the ability to deliver structurally diverse materials to difficult-to-transfect primary cells indicate that this method could potentially enable many novel clinical applications.

#2294

Reversible regulation of chimeric antigen receptor surface expression.

Sarah A. Richman,1 Liang-Chuan Wang,2 Edmund K. Moon,2 Steven M. Albelda,2 Michael C. Milone2. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

The purpose of this study is to develop and characterize a chimeric antigen receptor (CAR) T cell platform that can be rapidly and reversibly down-regulated. The rationale for this approach is to mitigate the risk of tissue toxicity as CAR T cell therapies are expanded from hematologic malignancies to solid tumors. Whereas off-tumor/on-target activity can be tolerable in the case of the B cell hypoplasia that occurs with CART therapy for ALL, targeting of other normal tissues predicted to occur in the solid tumor setting likely will be more clinically challenging. Currently implemented design approaches to arresting CAR-mediated toxicity have involved suicide domains. While effective in triggering CAR T cell apoptosis, these approaches are not reversible, and additional therapy would require repeated infusions, which may not be feasible under certain circumstances. A system of temporary down-regulation could overcome this obstacle and provide a useful alternative to current strategies. To this end, we have fused a mouse anti-human GD2 neuroblastoma antigen-specific CAR to a LID (ligand-induced degradation) domain. The LID domain, developed at Stanford University, targets the polypeptide for degradation upon the addition of rapamycin or a similar ligand, "shield." In the study presented here, bulk human T cells were transduced with lentivirus encoding either the control anti-GD2 CAR or the anti-GD2-CAR-LID fusion construct. Transduced T cells, after a standard procedure of stimulation and expansion, were then incubated in either standard media alone or media with shield ligand. CAR expression was measured at a range of shield ligand concentrations and at various time points up to 24 hours by flow cytometry using goat-anti mouse antibody. We observed a shield dose-response reduction in CAR expression and found that this shield-induced reduction in expression started at 8 hours and approached ~ 20% of baseline by 24 hours. As early as 24 hours following the removal of the shield-containing media and washing, CAR surface expression had returned to ~65% of baseline. Next, we assessed in vitro T cell effector functions including proliferation as measured by flow cytometric cell counting, IFN-gamma release as measured by ELISA, and cytotoxicity as measured by chromium release. We observed that in the presence of shield ligand, T cell effector functions were reduced to near background levels. The shield compound appeared to have no effect on the control anti-GD2 CAR that did not contain the LID domain. Our conclusion from these in vitro experiments is that the CAR-LID construct has the potential to allow for temporary and tunable down-regulation of CAR T cell activity in a clinical setting and warrants further characterization. In vivo experiments designed to test the CAR-LID T cells in a mouse model of xenografted disseminated neuroblastoma are ongoing, and we plan to expand this approach to other tumor systems.

#2295

GDNF family receptor alpha 4 (GFRa4)-targeted adoptive T-cell immunotherapy for medullary thyroid carcinoma.

Vijay G. Bhoj,1 Selene Nunez-Cruz,1 Kenneth Zhou,1 Dimitrios Arhontoulis,1 Michael Feldman,1 Keith Mansfield,1 Haiyong Peng,2 Christoph Rader,2 Don L. Siegel,1 Michael C. Milone1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _The Scripps Research Institute, Jupiter, FL_.

Metastatic medullary thyroid cancer (MTC) is a rare, but often aggressive, thyroid malignancy with a 5-year survival rate of 28% and few effective therapeutic options. Adoptive T-cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CARTs) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4), which associates with the RET receptor tyrosine kinase, as a putative antigenic target for CAR-based therapy of MTC. Using RNA in situ hybridization (RNAscope) and quantitative RT-PCR, we show that GFRα4 is highly expressed in MTC. Normal tissue expression of GFRα4 mRNA is restricted to parafollicular cells (C-cells) within the thyroid, the normal cell type from which MTC originates, and normal thymus.Based upon the highly restricted expression, we generated two high affinity single chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by selecting a naïve rabbit antibody library by phage display. Second generation CARs bearing the CD137 costimulatory domain were constructed using these GFRα4-specific scFvs. GFRα4-specific CARTs trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to control pre-established TT cell tumors in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CARTs, and support this molecule as a promising target for adoptive T cell immunotherapy and other antibody-based therapy of MTC.

#2296

**Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple** in vivo **tumor models.**

Shannon Grande, Molly R. Perkins, Amanda Hamel, Holly M. Horton, Fay Eng, Claire J. Rhodes, Tracy E. Garrett, Sara M. Miller, John W. Evans, Howard J. Latimer, Christopher Horvath, Michael Kuczewski, Kevin Friedman, Richard A. Morgan. _bluebird bio, Cambridge, MA_.

Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells for treatment requires ex vivo culture to facilitate CAR gene transfer and to achieve a therapeutic dose of the modified cells. Recent data suggests that specific T cell subtypes can provide enhanced anti-tumor efficacy, spurring efforts to optimize the production of therapeutic T cells via the cumbersome physical isolation of central memory T cells or culture in cytokines such as IL-7 and IL-15. Here we explored the potential for a simple culture modification to improve the therapeutic potential of CAR T cells without adding manufacturing complexity. To this end, we produced CAR T cells specific to B cell maturation antigen (BCMA) using standard IL-2 culture conditions supplemented with a PI3K inhibitor, or with IL-7 and IL-15 in place of IL-2. The in vivo activity of CAR T cells was studied in mouse models of human Burkitt's lymphoma (Daudi) and multiple myeloma (RPMI-8226), both of which express BCMA. In the Daudi model, NSG mice were injected intravenously with 2 x 106 tumor cells and allowed to accumulate a large tumor burden to model late stage disease observed in relapsed and refractory lymphoma. In this advanced disease model, anti-BCMA CAR T cells (4 x 106/mouse) cultured either in IL-2 or IL-7 and IL-15 had little or no effect on tumor growth (p = 0.22 and 0.23, respectively) and all mice succumbed to tumors within two weeks of treatment. In contrast, all animals treated with the same number of anti-BCMA CAR T cells cultured with PI3K inhibition survived and had complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were studied in a model of multiple myeloma. NSG mice were injected subcutaneously with 107 RPMI-8226 cells and 22 days later received a single administration of anti-BCMA CAR T cells (4 x 105/mouse) cultured under various conditions. In this model, tumor regression occurred regardless of in vitro culture conditions. To model tumor relapse and evaluate CAR T cell durability, surviving animals were re-challenged with RPMI-8226 cells on the opposite flank two weeks after initial tumor clearance. In contrast to other conditions, all animals treated with anti-BCMA CAR T cells cultured with PI3K inhibition were protected against subsequent tumor challenge (p=0.005). This improved therapeutic activity of anti-BCMA CAR T cells cultured with PI3K inhibition was associated with an increased frequency of CD62L+ CD8+ T cells in the drug product (p < 0.001) suggesting enrichment of this distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion

with IL-2 may generate an improved anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies.

#2297

Tandem anti-CD20 and -CD19 scFV-based chimeric antigen receptors (CARs) mitigate tumor escape in hematologic malignancies.

Dina Schneider, Ying Xiong, Darong Wu, Boro Dropulic, Rimas Orentas. _Lentigen Technology Inc., Gaithersburg, MD_.

In recent years, adoptive cell therapy using T cells engineered with chimeric antigen receptors (CAR T) specific for CD19 has shown marked clinical efficacy. Nevertheless, cases of tumor escape due to the loss of CD19 antigen have been reported. We hypothesized that tumor escape in B cell malignancies could be mitigated by CAR T approaches that target the CD19 and CD20 tumor antigens simultaneously.

Second generation CARs containing scFv specific for CD19 and CD20 were generated by lentiviral transduction of human primary T cells. The configurations were: a) single targeting domain, b) tandem targeting domains, or c) two full-length CAR receptors co-expressed bicistronically in the same T cell. CAR T expression for all constructs was confirmed by Western blotting and protein L flow cytometry. CAR T cytolytic activity and IFN gamma secretion were evaluated by killing assays and ELISA, respectively.

Single, tandem, and bicistronic CAR T cells exhibited CAR surface expression of 50-70%. All anti-CD19 and anti-CD20 CAR T cells demonstrated target-specific cells lysis and induction of IFN gamma. Interestingly, the bicistronic CAR19 CAR20 construct yielded high CAR T surface expression and IFN gamma production, but inferior in vitro cytolytic activity. To confirm CAR T specificity, K562 CD19+ and K562 CD20+ cell lines were generated. In flow-based 1h killing assays, CAR19 and CAR20 lysed their respective target lines only, whereas tandem CAR constructs lysed both K562 CD19+ and K562 CD20\+ cells, but not K562 control. In a subsequent series of co-culture experiments, Raji target cells were combined with very low ratios of CAR T cells, to allow for the evolution of escape variants. On day 5 of co-incubation in the presence of CAR19 T cells, the surviving Raji cells numbers were comparable to control (85,000 cells/well vs 77, 000 cells/well in sham control). In comparison, viable Raji numbers decreased drastically post co-incubation with CAR20-, CAR19_20- and CAR20_19-T cells (546, 1,355 and 1,543 cells/well, respectively). Among the surviving Raji population, CD19 surface expression was reduced to 1.6%, vs 93.29% for sham control, whereas the expression of CD20 and CD22 remained high (97% and 78%, respectively). By contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration.

Thus, targeting two tumor antigens simultaneously using tandem CAR T cells is a promising new strategy for mitigation of tumor escape via antigen down-regulation. Moreover, expression of two scFv in a single CAR format is superior to expression of two independent CARs from a bicitsronic vector. It also appears that the order of the targeting domains within the tandem CAR impacts anti-tumor activity. These findings are currently being explored further in animal model systems.

#2298

Cytokine-induced killer cells redirected with anti-CD44v6 chimeric antigen receptor against soft tissue sarcomas.

Valeria Leuci,1 Monica Casucci,2 Giovanni Grignani,3 Ramona Rotolo,1 Elisa Vigna,1 Loretta Gammaitoni,3 Giulia Mesiano,3 Lorenzo D'Ambrosio,1 Massimo Aglietta,1 Attilio Bondanza,2 Dario Sangiolo1. 1 _University of Torino, Department of Oncology, Candiolo Cancer Institute IRCCS, Candiolo, Italy;_ 2 _San Raffaele Hospital Scientific Institute, Innovative Immunotherapies Unit, Milan, Italy;_ 3 _Candiolo Cancer Institute FPO-IRCCS, Candiolo, Italy_.

Purpose of our study is to explore the anti-sarcoma activity of cytokine-induced killer (CIK) cells engineered with a chimeric antigen receptor (CAR) against the isoform variant 6 of adhesive receptor CD44 (CD44v6). CD44v6 may be an ideal target for immunotherapy as it is a tumor-promoting antigen, associated with the metastatic process and tumor initiating cells. Advanced and metastatic soft tissue sarcomas (STS) are currently incurable and in great need for new therapeutic strategies.

CIK cells are ex vivo expanded T lymphocytes endowed with MHC-independent antitumor activity.

CIK cells are active against STS (Sangiolo et al. Cancer Research 2014) but their function decreases at low effector/target (E/T) ratios with limitations in clinical perspective. We hypothesized that CIK cells may be effective candidates for CAR-based strategies, considering their intense ex vivo expansibility and innate antitumor activity.

Experimental procedures and results. CIK cells were expanded from 9 STS patients and engineered with a lentiviral vector encoding for anti-CD44v6 CAR containing a CD28 signaling domain (Casucci et al, Blood 2013) and the HSV-TK suicide switch. Tumor killing was assessed in vitro against 10 STS (undifferentiated pleomorphic n=5; Liposarcoma n=2; Fibrosarcoma n=1; GIST n=2), in 2 cases STS targets were autologous.

All 10 STS expressed CD44v6 (Relative Fluorescence Intensity=4, SE=1). Mean transduction efficiency was 60% (SE=6%). Expansion rates and phenotype of CAR-CIK were comparable with unmodified controls (CD3+CD56+=50%; CD8=66%; NKG2D=85%).

Anti-CD44v6 CAR-CIK cells efficiently killed STS in vitro. Mean tumor-specific killing, at low E/T ratios, was significantly higher compared with unmodified CIK cells: 98% vs 84% (E/T=10:1, p>0.05), 89% vs 41% (E/T=1:1, p=0.0001), 65% vs 26% (E/T=1:4 p=0.0001).

In vitro treatment with Ganciclovir (10 µM) significantly inhibited tumor killing activity of CAR-CIK from 70 % to 10 % ( E/T 1:1, n=3, p=0.008).

Blocking experiments (n=2) with anti-CD44v6 antibody against the same epitope recognized by the CAR (VFF-18) decreased tumor-specific killing from 96% to 43% (E/T=1:1) and 27% (E/T=1:2). Adoptive infusion of anti-CD44v6 CAR-CIK cells significantly delayed tumor growth (p=0.01) and reduced proliferative index (p=0.001) of established subcutaneous fibrosarcoma xenografts in NOD/SCID mice (n=3) compared to untreated controls (n=3) without any sign of toxicity.

Conclusions. Ours is the first report of CAR-engineered CIK cells against solid tumors. This approach significantly potentiates the innate tumor killing ability of CIK cells with a new redirected antitumor specificity. CIK cells may be appealing alternative candidates to conventional T cells for future CAR-based strategies against solid tumors. Our findings support CD44v6 as a valuable target for adoptive cell therapies against currently incurable sarcomas.

#2299

ET1402L1-CART, a T cell therapy targeting the intracellular tumor antigen AFP, demonstrates potent antitumor activity in hepatocellular carcinoma models.

Hong Liu, Yiyang Xu, Jingyi Xiang, Li Long, Shon Green, Zhiyuan Yang, Jingwei Lu, Neal Cheng, Lucas H. Horan, Bin Liu, Su Yan, Pei Wang, Juan Diaz, Lian-xing Liu, Vivien Chan, Cheng Liu. _Eureka Therapeutics, Emeryville, CA_.

Hepatocellular carcinoma (HCC) is the 5th most prevalent and 3rd most lethal cancer worldwide, due to limited treatment options. The lack of tumor-specific membrane targets is one of the key challenges currently facing immunotherapy for solid tumors, where on-target/off-tumor activity can lead to severe adverse responses. Most HCCs overexpress Alpha-Fetoprotein (AFP), a secreted fetal glycoprotein rarely expressed in adult tissues, making AFP an ideal target for immunotherapy. However, since AFP is only expressed intracellularly and secreted, it cannot be targeted with traditional antibodies against cell-surface antigens. To overcome this limitation, we exploited the fact that intracellular antigens, including AFP, are processed into peptides and presented by class I major histocompatibility complexes (MHCs) on the surface of tumor cells. Using an antibody discovery platform optimized for identifying candidates that bind peptide/MHC complexes, we developed ET1402L1, a fully-human scFv specifically targeting an immunogenic AFP peptide complexed with human leukocyte antigen (HLA)-A*02:01. ET1402L1 binds HLA-A*02:01/AFP with exquisite specificity and does not cross-react with naked HLA-A*02:01 molecules or with HLA-A*02:01 complexed with a panel of control peptides from endogenous human proteins. T cells engineered to express a second generation chimeric antigen receptor (CAR) constructed with ET1402L1 responded to HLA-A*02:01/AFP-expressing cells by degranulating and releasing IFN-γ, IL-2, IL-6, IL-8, IL-10, GM-CSF, and TNFα in an antigen-selective manner. In a cell killing assay, ET1402L1-CART selectively lysed HCC cells that expressed both HLA-A*02:01 and AFP, while sparing cells from multiple tissue types that were negative for either. In vivo, ET1402L1-CART demonstrated potent anti-tumor activity in multiple HCC xenograft models. While intravenous administration of ET1402L1-CART significantly inhibited the growth of established subcutaneous (s.c.) Hep G2 tumors (an HCC cell line positive for HLA-A*02:01 and AFP), intratumoral delivery resulted in rapid and prolonged tumor regression with complete regression observed in 75% of animals. These effects were recapitulated in a s.c. tumor model using another liver-derived HLA-A*02:01-positive cell line (SK-HEP-1) engineered to express the AFP peptide. Lastly, intraperitoneal injection of ET1402L1-CART led to regression of tumors in a disseminated abdominal HCC xenograft model. Our data demonstrates that CAR-T cell therapy targeting intracellular antigens via peptide/MHC complexes can effectively eradicate solid tumors in vivo. This approach expands the spectrum of antigens available for redirected T cell therapy against solid malignancies and provides a promising future therapy for HCC. A phase I clinical trial testing ET1402L1-CART in HCC patients is expected to begin in 2016.

#2300

Human CD133-specific chimeric antigen receptor (CAR) modified T cells target patient-derived glioblastoma brain tumors.

Parvez Vora,1 Chitra Venugopal,1 Sujeivan Mahendram,1 Chirayu Chokshi,1 Maleeha Qazi,1 Minomi Subapanditha,1 Mohini Singh,1 David Bakhshinyan,1 Ksenia Bezverbnaya,1 Jarrett Adams,2 Nicole McFarlane,1 Sachdev Sidhu,2 Jason Moffat,2 Jonathan Bramson,1 Sheila Singh1. 1 _McMaster University, Hamilton, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Glioblastoma (GBM), an aggressive primary brain tumor in adults, is feared for its near uniformly fatal prognosis and is characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. We developed patient-derived brain tumor initiating cell (BTIC) early passage lines that describe the extent of intertumoral heterogeneity, presenting a powerful preclinical model of GBM. Numerous studies have implicated CD133+ BTICs as drivers of chemo- and radio-resistance in GBM. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of chimeric antigen receptor (CAR) T-cell strategy that specifically targets CD133+ GBM cells.

From the previously generated CD133-specific humanized monoclonal antibody, we derived the single chain variable fragment (scFv) and cloned it into an antigen-specific second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR vector in two different orientations: Light chain-linker-Heavy chain (CD133 CAR-LH) and Heavy chain-linker-Light chain (CD133 CAR-HL). Following lentiviral preparation, the T cells isolated from PBMCs were transduced with CD133 CAR-LH and CD133 CAR-VH constructs. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. While expression of ΔNGFR was observed in all CAR T cells (including controls), we found expression of the myc tag in both variations of CARs, CD133 CAR-HL and CD133 CAR-LH. Furthermore, we used Presto Blue-based killing assays to test the ability of CD133 CARs to selectively bind and kill CD133+ GBM BTICs. Our data shows that CD133-specific CAR T cells not only recognized, but killed the CD133+ GBM cells selectively, validating this adoptive T-cell therapeutic strategy. CAR-expressing T cells were activated in presence of CD133high GBM cells showed increase surface expression of activation markers CD69 and CD25. Both, CD4+ and CD8+ CD133-specific CAR-T cells showed upregulation in surface expression levels of activation markers.

This rigorously obtained data offers compelling evidence that CAR-T induced cytotoxicity against treatment-resistant and evasive CD133+ GBM BTICs could provide a very potent, specific and novel therapeutic strategy for GBM patients.

#2301

Intraperitoneal CAR-T infusion results in durable protection against peritoneal metastases and control of systemic tumor growth.

Marissa Cunetta,1 Gary Point,1 Zhi Liu,1 Prajna Guha,1 Josephine Darpolor,1 Matthew Lima,1 N J. Espat,1 Nader Hanna,2 Cherif Boutros,2 Richard P. Junghans,3 Steven Katz1. 1 _Roger Williams Medical Center, Providence, RI;_ 2 _University of Maryland, MD;_ 3 _Tufts Medical Center, MA_.

Colorectal carcinomatosis due to peritoneal metastases (PM) is a significant cause of morbidity and mortality. Present treatments fail to cure the majority of patients with PM. We are developing a chimeric antigen receptor T cell (CAR-T) regional infusion immunotherapy platform for PM. We treated mice with established CEA+ MC38 PMs with anti-CEA CAR-Ts either via regional intraperitoneal (IP) or systemic tail vein (TV) infusion. When compared to TV infusion, IP delivery resulted in significantly improved responses (37-fold reduction in tumor growth, p<0.01). To measure the durability of protection from IP tumor growth, we re-challenged mice with IP tumor following successful treatment with IP CAR-Ts. Mice who had received initial IP CAR-T treatment were protected from tumor growth following re-challenge for 20 days compared to control animals (1390-fold reduction in tumor growth, p=0.016). Assessment of IP tumor 10 and 28 days following tumor re-challenge revealed a high proportion of intratumoral CAR-Ts (Day 10- 70.0%, Day 28- 46.6%; p=0.10) and the proportion of CAR-Ts with an effector memory phenotype (CD44+CD62L-CCR7-, Day 10- 19.3%, Day 28- 60.0%; p<0.01). In separate experiments, we studied the effect of IP CAR-T infusions on tumor growth at distant sites. We established both IP and subcutaneous (SQ) flank tumors in mice prior to CAR-T infusions. Following treatment, IP CAR-T infusion resulted in a significant decrease in SQ tumor growth on day 8 compared to control (5.7-fold, p=0.03), and a trend toward improvement compared to TV (4.9-fold, p=0.224). While CAR-Ts were not detected in whole blood, SQ tumor, or draining lymph nodes following IP CAR-T infusion, serum IFNγ levels were significantly elevated on day 4 compared to control mice (p=0.04), and returned to baseline by day 7. Our findings demonstrate that regional IP CAR-T delivery provides durable protection against IP tumor growth in association with CAR-T effector memory programming, in addition to a systemic anti-tumor effect.

#2302

An 'off the shelf,' GMP-grade, IL-2-independent NK cell line expressing the high-affinity Fc-receptor to augment antibody therapeutics.

Laurent Boissel,1 Hans Klingemann,1 Kerry Campbell,2 Karen Nichols,1 Frances Toneguzzo,1 Paula Marcus,3 Brent Williams,3 Armand Keating,3 Patrick Soon-Shiong4. 1 _NantKwest, Inc., Cambridge, MA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 4 _NantKwest, Inc., Culver City, CA_.

A number of approved IgG1 monoclonal antibodies (mAb) used in cancer therapy, including trastuzumab, cetuximab, and rituximab, depend on natural killer (NK) cell-mediated antibody dependent cell-mediated cytotoxicity (ADCC) to induce cancer cell death, mediated through the Fcγ receptor (FcγR). Multiple studies report that survival benefit is significantly improved in cancer patients receiving IgG1 antibody therapy who have NK cells that express the high-affinity FcγRIIIa receptor (CD16-158V). Only 12% of the normal human population, however, is homozygous for expression of this high-affinity variant CD16. We hypothesized that infusion of an NK-92 cell line modified to express CD16-158V (high-affinity NK cells [haNK]) in combination with an IgG1 mAb may result in superior antitumor effects compared with IgG1 mAb alone.

A GMP-grade plasmid-transfected variant of NK-92 expressing the high-affinity CD16 receptor was developed utilizing a novel transfection vector containing the erIL-2 gene, enabling the resulting haNK cells to grow independently of IL-2. Using limiting dilution, clones having identical surface molecule expression to the parent cell line were generated and shown to have long-term expression of CD16. The haNK cells displayed high ADCC in combination with rituximab, trastuzumab, and daratumumab against target cell lines that were not killed by the parental NK-92 cells. Of note, despite non-viral transfection, the plasmid is fully integrated into chromosome 5. The haNK cell line secretes about 200 pg/106 cells of IL-2, and like the parental NK-92 cell line, produces enough IFNγ to potentially stimulate an adaptive immune response. A GMP-grade master cell bank of haNK cells has been established; haNK cells can be cryopreserved with retention of ADCC activity.

In vivo studies demonstrated the efficacy of haNK cells. In a multiple myeloma model of CD38+ NCI-H929 cells xenografted into NOD/SCID gamma null (NSG) mice, treatment with haNK cells plus the anti-CD38 mAb daratumumab led to prolonged survival versus mice treated with haNK cells plus an isotype control (log rank p= 0.0169), demonstrating therapeutically relevant in vivo ADCC. haNK cells have also been transfected with various CARs using mRNA, thereby enabling this platform to make a bispecific effector cell product.

In summary, haNK cells are a potent and versatile platform for cellular immunotherapy, and clinical trials of these novel "off the shelf" high-affinity NK cells are scheduled for 2016.

#2303

The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients.

Yan Kong, Jiayi Yu, Lu Si, Zhihong Chi, Chuanliang Cui, Xinan Sheng, Jun Guo. _Peking University Cancer Hospital & Research, Beijing, China_.

Background: Chimeric antigen receptor (CAR)-engineered T cells have demonstrated potent clinical efficacy in patients with B cell lymphoma. However, the application of CAR-T cell therapy targeting other solid tumors has been limited. The GD2 gangliosides are sialic acid-containing glycosphingolipids that play a role in signal transduction and cell-cell recognition. 1st generation gangliosides GD2 chimeric antigen receptor T cells in some patients with refractory neuroblastoma already studied in a phase I clinical trials, indicate that T cells expressing 1st generation anti-GD2 chimeric antigen receptors were safe and mediated modest antitumor activity. In a phase I study, the murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, was administered intravenously to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma. Antitumor responses occurred in 7 of 17 patients. However, the expression of the gangliosides GD2 in Asian melanoma populations has not been reported.

Methods: This study involved tumor samples from 228 melanoma patients (129 acral melanomas, 65 mucosal melanomas, 13 melanomas on skin with chronic sun-induced damage, 21 melanomas on skin without chronic sun-induced damage) in Peking University Cancer Hospital & Institute. Clinical data were collected. Immunohistochemistry assays using antibodies against gangliosides GD2 were performed on formalin-fixed, paraffin-embedded specimens. The ability of gangliosides GD2 chimeric antigen receptor T cells to kill gangliosides GD2\+ melanoma cell was evaluated by MTT in vitro.

Results: Among the 228 samples,40.3% of the cases(92/228) demonstrated positive staining with gangliosides GD2. Expression of gangliosides GD2 were relatively more frequent in acral (48.1%) and mucosal (36.9%) melanomas than in CSD (15.4%) and Non-CSD (19.0%) melanomas. The median survival time for patients with expression of gangliosides GD2 was significantly shorter than patients with absence of gangliosides GD2 expression (21.4 vs 57.6 months, P<0.05). Statistical differences were found between primary melanomas with metastatic melanomas (34.9% vs 53.0%, P<0.05). In addition, the gangliosides GD2 chimeric antigen receptor T cells exhibited specific lysis of melanoma cell with gangliosides GD2 expression in vitro experiment.

Conclusion: These data demonstrate the expression of ganglioside GD2 are more frequent in acral and mucosal melanomas than in cutaneous melanomas in Chinese patients. The higher expression of gangliosides GD2 on metastatic melanomas compared to primary melanomas was in conformity with the viewpoint that GD2 expression was related to increased metastatic potential. Gangliosides GD2 chimeric antigen receptor T cells represents a clinically appealing treatment strategy for melanoma patients with ganglioside GD2 expression.

#2304

**Attenuated oncolytic virus HSV1716 increases** in vivo **expansion of GD2- targeting CAR T cells in murine solid tumor models.**

Samuel T. Haile,1 Joe Conner,2 Crystal Mackall1. 1 _NIH, Bethesda, MD;_ 2 _Virttu Biologics, United Kingdom_.

Neuroblastoma, osteosarcoma, and rhabdomyosarcoma are among the most prevalent childhood solid tumors. Each of these tumor types as well as melanomas exhibit increased levels of the tumor associated carbohydrate, GD2 on their cell surface making them ideal targets for chimeric antigen receptor (CAR) T cell-directed therapies. Despite the ability of GD2 CAR T cells to target tumor cells in vitro, there is great interest in improving tumor clearance in vivo, especially for solid tumors where current outcomes remain poor. We hypothesize that the immunosuppressive milieu present within the solid tumor microenvironment serves as a major factor limiting the effectiveness of GD2 CAR T cells. Additionally, we propose that administration of oncolytic viruses could induce inflammation within the tumor microenvironment that may enhance, rather than inhibit, the effectiveness of immune based therapies. GD2 CAR T cells composed of the 14G2a single chain variable fragment linked to the cytoplasmic signaling domains of CD28 and CD3 zeta (GD2-28z) were expressed in murine lymphocytes and evaluated for the ability to target and lyse GD2-expressing tumor cells. Additionally GD2-28z T cells were evaluated for their ability to secrete the proinflammatory cytokines IFNγ and IL-2. In order to determine the oncolytic ability of attenuated HSV1716, tumor cells were cultured in the presence or absence of HSV1716 and relative cell survival was measured. We observed specific lysis of GD2-expressing tumor cells when co-cultured with GD2-28z, but not mock T cells. Furthermore, GD2-28z T cells secrete IFNγ and IL-2 following co-culture with GD2-expressing tumor cells. Interestingly, melanoma cell lines were not susceptible to oncolytic lysis while rhabdomycosarcoma (RMS) cell lines were susceptible. The combination of GD2-28z and HSV1716 enhanced CAR expansion in both melanoma and RMS models. Given that the melanoma cells in our model are not susceptible to oncolytic lysis yet we observe an increased T cell expansion when used in combination with HSV1716, this supports our hypothesis that HSV1716 is inducing inflammation, which may then triggering T cell expansion.

#2305

Comparative evaluation of peripheral blood T cells and resultant engineered anti-CD19 CAR T cell products from relapsed/refractory non-Hodgkin's lymphoma (NHL) patients.

Timothy Langer,1 Emily Lowe,1 Arianne Perez,1 Steven Rosenberg,2 Steven A. Feldman,2 Lily Lu,2 John M. Rossi,1 Edmund Chang,1 Marika Sherman,1 Marc Better,1 James N. Kochenderfer,3 Adrian Bot1. 1 _Kite Pharma, Santa Monica, CA;_ 2 _NCI, NIH, Bethesda, MD;_ 3 _NCI - Experimental Transplantation and Immunology Branch, Santa Monica, CA_.

Introduction: Administration of autologous anti-CD19 CAR T cells prepared from peripheral blood mononuclear cells (PBMC) of patients with various B cell malignancies have mediated high rates of objective response (Kochenderfer et al. Blood 2012, J Clin Onc 2014, Mackall et al, Blood 2013). We analyzed characteristics of the starting material (PBMC) and resultant CAR T cells derived from 14 patients with relapsed/refractory NHL at the NCI Surgery Branch.

Methods: After apheresis collection, the T cell-containing PBMC fraction was enriched, activated with anti-CD3 antibody and cultured in serum-free medium for 2 days. Activated T cells were transduced with a retroviral vector encoding the anti-CD19 CAR gene and further expanded with IL-2 to achieve a target dose of CAR T cells. The CAR T cells and starting PBMC population were cryopreserved and analyzed after thawing by flow cytometry. Differences in cell composition between the starting PBMC population and CAR T cells were evaluated with paired t-tests, adjusted for multiplicity.

Results: Biologically active autologous CAR T cells were successfully prepared from all 14 NHL subjects. Each product was comprised of both CD4+ and CD8+ T cells, but showed considerable inter-subject variability. The CD4/CD8 ratio in the PBMC and product CAR T cell population were correlated (Pearson correlation coefficient=0.74, p=0.0027). While the starting T cell population generally showed a T cell profile comprised of similar proportions of effector T cells (Teff) (x̄ = 20%, std=16%) and naïve T cells (Tn) (x̄ = 20%, std=14%), there was a shift towards a less differentiated T cells population after CAR T cell production, with a decreased Teff (paired t test p = 0.0151) and elevated Tn (p = 0.0031). The percent of juvenile cells (Tn+Tcm) was higher in the final product (x̄ = 64%, std=21%) than in the starting T cell population (x̄ = 43%, std=20%, p* = 0.0031, *stepdown Bonferonni). These products were active in vitro and in subjects with NHL. Clinical responses occurred regardless of product characteristic differences such as CD4/CD8 T cell ratio and juvenile / differentiated T cells. Additional analyses are ongoing, including evaluation of PD-1 expression, % of myeloid cells and gene expression profile.

Conclusions: CAR T cells were successfully prepared from all subjects enrolled in the study notwithstanding the diverse nature of the subject starting lymphocyte population. The CAR T cells showed a less differentiated profile, compared to the starting lymphocytes. CAR T cell production by essentially the same process described here is currently being utilized in company-sponsored multicenter trials (NCT02348216, NCT02601313).

#2306

**Treatment with hTERT/survivin mRNA-loaded dendritic cells combined with autologous** ex vivo **expanded T cells improves progression free survival in stage IV melanoma patients when compared to dendritic cell vaccines alone.**

Iris Bigalke, Elin Aamdal, Siri Torhaug, Marianne Lundby, Else M. Inderberg, Gustav Gaudernack, Anne M. Rasmussen, Steinar Aamdal, Gunnar Kvalheim. _Oslo University Hospital, Oslo, Norway_.

In previous studies with tumor specific antigen loaded dendritic cells (DCs) we have shown that 50% of patients with metastatic malignant melanoma mount specific immune responses and clinical effects. However responses were only transient.

Here we investigated, in a phase I/II study, if the combination of autologous DCs loaded with mRNA hTERT and survivin followed by transfusion of autologous ex-vivo expanded polyclonal T cells can strengthen the immune response and improve survival.

Sixteen melanoma patients with non resectable metastases were included.

Monocyte derived DCs were generated according to standard protocol using Jonuleit maturation cocktail.

Patients received 4 vaccines in weekly intervals, a DTH-challenge in week 6, followed by monthly boosts with either 5 or 10 x10E+6 DCs.

Blood samples were taken before and during DC vaccination and used for testing of specific immune responses against hTERT and survivin.

Patients with immune responses were offered additional treatment with T cells.

Fourteen patients could be evaluated, seven showing specific immune responses against hTERT and/or survivin. Three of them received a dose of 3x1010 expanded T cells after pre-treatment with Fludarabin and Cyclophosaphamide. DC vaccination was continued the day after T cell transfer.

Patient (Pt) 1 and 2 showed an increase of hTERT specific T cells in peripheral blood after T cell transfer. Pt 1 lost the hTERT specific response 20 months after beginning of DC-vaccination correlating with progression of the disease.

Pt 2 lost the hTERT response 29 months after start of DC-vaccination and proceeded with a response against surviving. Thereafter progression of disease occurred.

Pt 3 had a response against survivin prior to T cell collection and at the same time progression occurred. Following administration of expanded T-cells a strong increase of survivin specific T cells in peripheral blood was detected. In spite of this the patient only had a progression free survival of 11 months.

Patients were given the checkpoint inhibitor Ipilimumab when progressing and Pt 1 and Pt 2 are still in stable disease.

Patients who did not mount any immune response during DC therapy had a mean PFS of 4,7 (2-7) months while patients with specific immune responses show a mean PFS of 9 (6-14) months.

Retrospectively we performed an extended analysis of the DCs and their impact on immune responses. Flow-analysis showed a patient individual expression of maturation markers and co-stimulatory molecules. Mimicking T cell encounter by CD40L stimulation showed individual differences of IL-10 release but no IL-12p70.

Differences in quality of the DCs did not have an impact on immune responses.

Our data show that patients receiving expanded T cells after mounting a specific immune response have a longer PFS when compared to patients with DC vaccination alone.

#2307

Improved CAR safety by a non-lethal switch to control CAR activity at the T-cell surface membrane.

Alexandre Juillerat,1 Alan Marechal,2 Jean-Marie Filhol,2 Philippe Duchateau,2 Laurent Poirot2. 1 _Cellectis Inc, New York, NY;_ 2 _Cellectis, Paris, France_.

Adoptive immunotherapy using engineered T-cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering. Novel specificities in T cells have been typically implemented through the genetic transfer of the so-called chimeric antigen receptors (CARs). CARs are synthetic receptors that associated an extracellular targeting moiety with one or more intracytoplasmic signaling domain derived from lymphocyte activation receptors. Present CAR architectures are designed to combine all relevant domains within a single polypeptide, thereby; they combines advantages of MHC unrestricted target recognition to the potent native effector mechanisms of the T cell. Although adoptive transfer of CAR T cells is proven to be an effective strategy to cancer therapy, potential adverse effects such as Cytokines Release Syndrome (CRS) and/or the risk of on-target off-tumor targeting are still a major concern. To date only suicide mechanisms that can eradicate the engineered T-Cell "at will" or mRNA CAR transfection approaches have been proposed to addresses this safety issue.

Here, we describe the development of a small molecule based switch-on technology to control the surface presentation of a chimeric antigen receptor in T-cells. By grafting protein domains that can interact upon addition of a small molecule drug in the hinge domain of the CAR architecture, we are able to turn a T-cell endowed with an engineered CAR from an off-state to an on-state, in term surface presentation as well as for its specific cytolytic properties. This system offers the advantage of a "transient CAR T-cell" for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. This non-lethal control system of CAR engineered T-cells represents an important advance for the safety of this technology.

#2308

Manufacturing and characterization of KTE-C19 in a multicenter trial of subjects with refractory aggressive non-Hodgkin's lymphoma (NHL) (ZUMA-1).

Marc Better, Vijay Chiruvolu, James Oliver, Maryam Sorkhabi, Jeff S. Aycock, Emily Lowe, Edmund Chang, Arianne Perez, Lynn Navale, John M. Rossi, Adrian Bot. _Kite Pharma, Santa Monica, CA_.

Introduction: KTE-C19 is an autologous anti-CD19 CAR T cell product that is being studied in a phase 2 multicenter trial (ZUMA-1, NCT02348216). We developed a robust and efficient manufacturing process to support this trial, and compared the characteristics of the starting lymphocytes to resultant CAR T cells.

Methods: After apheresis and PBMC enrichment, cells were activated with anti-CD3 antibody and cultured in serum free medium. T cells were transduced with a retroviral vector encoding the CAR gene, expanded to achieve a target dose and cryopreserved. The product CAR T cells and the starting PBMCs were evaluated by flow cytometry.

Results: 7 subjects were dosed in the phase 1 portion of the trial. KTE-C19 was successfully manufactured at a dose of 2 x 106/kg (minimum 1 x 106/kg) for all subjects. All lots contained mainly CD3+ T cells (median 97%; 94-99%). While there was inter-subject variability in PBMC and CAR T cell product characteristics, the CD8/CD4 T cell ratios in PBMC and corresponding CAR product were similar (Table 1). T cells in PBMC from patients with NHL contained a majority of effector memory (36%) and effector T cells (27%), however, T cells in KTE-C19 contained a majority of central memory (37%) and effector memory (42%) CAR+ T cells. These CAR T cells were active and objective responses occurred in 5/7 patients.

Conclusions: A KTE-C19 dose was successfully produced for all subjects. The optimized manufacturing process generated clinically active CAR T cells with a less differentiated phenotype than T cells in the starting PBMC population. Less differentiated cells likely confer preferred product characteristics based on preclinical studies. This manufacturing process is robust and well suited for multicenter clinical trials.

Table 1. Comparative analysis of T cells from KTE-C19 and PBMC from patients with refractory aggressive NHL.  | |  | |  | |

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

ID | Sample | Naïve (%) | Central Memory (%) | Effector Memory (%) | Effector (%) | CD8/CD4 ratio

Subject 1 | KTE-C19 | 11 | 58 | 30 | 1 | 4.6

|

PBMC | 16 | 12 | 46 | 27 | 2.3

Subject 2 | KTE-C19 | 14 | 47 | 35 | 4 | 0.37

|

PBMC | 26 | 30 | 34 | 9 | 0.27

Subject 3 | KTE-C19 | 6 | 34 | 56 | 4 | 2.3

|

PBMC | 4 | 5 | 36 | 55 | 2.9

Subject 4 | KTE-C19 | 11 | 15 | 52 | 22 | 2.0

|

PBMC | 1 | 2 | 24 | 72 | 1.8

Subject 5 | KTE-C19 | 15 | 40 | 38 | 7 | 1.0

|

PBMC | 9 | 15 | 50 | 26 | 1.1

Subject 6 | KTE-C19 | 19 | 34 | 42 | 5 | 2.3

|

PBMC | 10 | 13 | 59 | 19 | 2.0

Subject 7 | KTE-C19 | 15 | 26 | 45 | 14 | 0.5

|

PBMC | 12 | 13 | 36 | 39 | 0.5

Median

(Range) | KTE-C19 | 13 (6-15) | 37 (15-58) | 42 (30-56) | 5 (1-22) | 1.9 (0.4-4.6)

|

PBMC | 10 (1-26) | 13 (2-30) | 36 (24-59) | 27 (9-72) | 1.9 (0.3-2.9)

#2309

T-cell development from T cell-derived induced pluripotent stem cell.

Sjoukje J.C. van der Stegen, Maria Themeli, Justin Eyquem, Jorge Mansilla-Soto, Michel Sadelain. _Memorial Sloan Kettering Cancer Center, New York, NY_.

The ability to differentiate T cells of defined specificity and function from T-cell derived induced pluripotent stem cells (TiPSC) may be useful for the treatment of a range of pathologies, including cancer, infection and immune deficits. We recently reported that genetic engineering of TiPSC to express a Chimeric Antigen Receptor (CAR) is an effective strategy to combine the unlimited availability of TiPSC and facilitate reprogramming of the T cell specificity and functional potential. In vivo, CD19-retargeted human TiPS-derived CAR T cells (CARTiPSC-T) display therapeutic potency in a lymphoma model. Surprisingly, despite expression of the endogenous αβ TCR, the CARTiPSC-T cells possessed innate-like phenotype and function, most similar to γδ T cells. Although innate T cells have anti-tumor activity, they lack some vital features for therapeutic efficacy including long-term memory and in vivo persistence, which characterize mature CD8+ and CD4+ αβ TCR T cells.

Additional research into the mechanisms underlying in vitro T cell differentiation of TiPSC is required to improve the development of mature TCRαβ T cells and facilitate the employment of their therapeutic potential. T cell lineage determination depends in part on the balance between Notch and TCR signaling, we are investigating their respective roles, as well as that of the CAR, in determining lineage commitment. We hypothesize that the combined CAR and early CD3/TCRαβ expression disrupts the TCR/Notch signaling balance, prohibiting mature TCRαβ T cell development.

To study the effects of the different Notch ligands, TiPSC were differentiated on OP9 cells expressing one of four Notch ligands (DLL1, DLL4, Jagged-1 or Jagged-2). Preliminary data suggests that DLL4 is able to facilitate T cell development to CD4/CD8 double positive (DP) stage, however, this is hindered by CAR expression.

To determine the role of early TCRαβ expression, elimination of TCRαβ expression was facilitated by targeted disruption of the TCRα constant region using the CRISPR/Cas9 system. Elimination of early TCRαβ expression showed improved development towards DP stage of TiPSC on OP9-DLL1.

TiPSC are a valuable system for the study of human T cell differentiation. In addition, and most importantly they are further amenable to genetic engineering with TCRs or CARs, which may be useful for the generation of therapeutic "off-the-shelf", antigen-specific T cells.

#2310

With a little help from CD4 T cells in adoptive T-cell transfer.

Else M. Inderberg, Sébastien Wälchli, Marit R. Myhre, Kari Lislerud, Gunnar Kvalheim, Gustav Gaudernack. _Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway_.

Adoptive transfer of genetically engineered T cells offers great opportunities in cancer immunotherapy, however until recently, HLA-Class II-restricted TCRs have been largely ignored.

We have identified and cloned HLA class II-restricted CD4+ T cells isolated from patients vaccinated with long peptides derived from antigens such as hTERT, survivin and frameshift mutated TGFβRII. Several of these cancer patients demonstrated outstanding clinical responses to peptide- or dendritic cell based cancer vaccines with >10-year survival. In these patients strong T-cell responses against peptides other than those used for vaccination were detected, suggesting epitope spreading.

Responses against certain peptides associated with clinical benefit were identified and CD4+ T-cell clones recognizing such peptides were isolated. In depth studies of their specificities lead to the selection of HLA-DR and -DQ restricted T-cells having high functional avidity for the HLA-peptide complexes. We studied tumor cell and/or protein recognition and found one telomerase-specific CD4+ T-cell clone recognizing a melanoma cell line with the corresponding HLA allele.

The TCR sequences of the interesting clones were identified. The ribosome skipping 2A sequence technique was used to express these TCRs in an mRNA expression vector or retrovirus. When expanded T cells were electroporated with these TCRs, both redirected CD8+ and CD4+ T cells produced TNF-α, IFN-γ and the degranulation marker (CD107a) following co-incubation with specific peptide-loaded targets.

We have demonstrated that choosing highly functional CD4+ T-cell clones specific for tumor-associated or -specific antigens from patients with clinical responses after immunotherapy treatment is a successful method for identifying highly functional HLA class II restricted TCRs for adoptive T-cell transfer.

The use of HLA class II-restricted TCRs may be of therapeutic value both in haematopoietic malignancies and in melanoma where tumor cells often express HLA class II. In addition, combining the redirection of T cells with both HLA class I- and class II-restricted TCRs may provide a more powerful therapeutic effect in adoptive T cell therapy.

#2311

Adenovirus-transduced allogeneic dendritic cells for cancer immunotherapy.

Magnus Essand, Grammatiki Fotaki, Chuan Jin, Mohanraj Ramachandran, Jing Ma, Alex Karlsson-Parra, Di Yu. _Uppsala University, Uppsala, Sweden_.

Immunotherapy is currently being established as standard treatment for cancer. Immune checkpoint blockade antibodies can cure a large proportion of patients with refractory melanoma and lung cancer, and adoptive transfer of patient-derived CD19 chimeric antigen receptor (CAR) T cells results in complete remission in a majority of patient with refractory leukemia. Cancer vaccines based on patient-derived dendritic cells (DC) modified ex vivo to present tumor antigens are promising for cancer treatment especially when combined with immune checkpoint blockade antibodies. Ex vivo-modified DCs are however poor in migrating to secondary lymphoid organs after re-administration. In reality, it is more likely endogenous DC that endocytos the injected DC, mature and then migrate to lymph nodes where they present peptide fragments of tumor antigen to T cells together with required co-stimulation.

Herein, we present a new strategy utilizing adenovirus (Ad)-transduced allogeneic DCs (alloDCs) as immunostimulatory adjuvant to induce both innate and adaptive immune response. Ad/alloDC can be prepared, tested and made ready for release in advance and quickly be prepared for injection in cancer patients upon request, thereby minimizing GMP-processing and handling of cells. Besides encoding tumor antigens, adenovirus also provides stimulatory danger signals for DC activation. We show that Ad/alloDCs secret a panel of proinflammatory cytokines and chemokines including IL-12 and CXCL10 at high levels in a sustainable fashion. We also show that Ad/alloDC recruit and activate neutrophils and NKs to the site of injection. Ad/alloDC can also recruit and interact with allo-reactive T cells to create a proinflammatory milieu, which further recruit and activate endogenous DCs. We verified that endogenous DCs activated in situ by an Ad/alloDC injection migrate to draining lymph node, where they present the engulfed tumor antigen to T cells. We show that these migratory endogenous DCs facilitate T cell activation and proliferation. Moreover, when injected intratumorally, Ad/alloDCs modulate the immunosuppressive environment by reducing the ratio of monocytic to granulocytic myeloid-derived suppressor cells (MDSC). To evaluate the therapeutic efficacy, we use B16 melanoma tumor-bearing C57Bl/6 mice injected with adenovirus-transduced DCs from Balb/c origin to obtain an allogeneic reaction. We show that Ad/alloDC treatment could significantly delay tumor growth and prolong survival of tumor-bearing mice.

#2312

TEM8/ANTXR1 specific T cells co-target tumor stem cells and tumor vasculature in triple-negative breast cancer.

Tiara Byrd,1 Kristen Fousek,1 Antonella Pignata,1 Christopher Szot,2 Kevin Bielamowicz,1 Steven Seaman,2 Daniel Landi,1 Nino Rainusso,1 Poul Sorensen,3 Joachim Koch,1 Winfried Wels,1 Bradley Fletcher,1 Meenakshi Hegde,1 Brad St Croix,2 Nabil Ahmed1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _National Cancer Institute, Frederick, MD;_ 3 _Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada_.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no approved targeted therapies. Tumor endothelial marker 8 (TEM8), initially identified as a marker of tumor endothelial cells in colorectal cancer and other solid tumors has recently been shown to be upregulated in TNBC and breast cancer stem cells (BCSCs). We investigated whether TEM8 specific chimeric antigen receptor (CAR) T cells recognize and kill both tumor endothelial cells as well as TNBC tumor cells. TEM8 specific CAR molecules were generated using single chain variable fragment derived from the monoclonal antibody, L2. L2 CAR T cells selectively recognized TEM8, secreted immunostimulatory cytokines and effectively killed both TEM8 positive TNBC and tumor endothelial cell lines. Moreover, L2 CAR T cells targeted breast cancer stem cells significantly reducing the number of mammospheres relative to non-transduced T cells. In vivo, adoptive transfer of L2 CAR T cells induced regression of established vascularized TNBC xenografts. Hence, TEM8 may serve as an attractive target for immunotherapy of TNBC.

#2313

Improved killing of tumor cells by a novel flexible antibody-based modular T cell retargeting system.

Armin Ehninger,1 Marc Cartellieri,2 Anja Feldmann,3 Claudia Arndt,3 Stefanie Koristka,4 Simon Loff,1 Malte von Bonin,5 Gerhard Ehninger,5 Michael Bachmann3. 1 _GEMoaB GmbH, Dresden, Germany;_ 2 _Cellex Patient Treatment GmbH, Dresden, Germany;_ 3 _Institute of Radiopharmaceutical Cancer Research, HZDR Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany;_ 4 _Tumorimmunology, University Cancer Center (UCC), Medical Faculty Carl Gustav Carus, TU Dresden, Dresden, Germany;_ 5 _Medical Clinic and Policlinic I, University Hospital Carl Gustav Carus Dresden, Dresden, Germany_.

In recent years, bispecific antibodies (bsabs) and chimeric antigen receptors (CARs) emerged as promising candidates for an antigen-specific cancer immunotherapy. Bsabs and CARs redirect T cells in an antigen-specific manner towards tumor cells and unleash their enormous cytotoxic potential to attack the tumor. Nevertheless, several challenges have to be solved to pave the way for a more widespread application of both strategies. Both approaches are monotherapies bearing the inherent risk for development of antigen-loss tumor variants under treatment. In addition, due to their single antigen specificity each given bsab or CAR is only curative for a restricted number of tumor indications and long-lasting and demanding research is necessary to bring a new bsab- or CAR-based drug to patients in need. Moreover, current CAR T cell approaches do not allow for any control of the magnitude and duration of T cell reactivity, thus bearing the risk for overshooting anti-tumor reactions leading to severe side effects and ongoing destruction of healthy tissue carrying the target antigen after tumor clearance. To overcome these limitations, we recently introduced a novel flexible antibody-based modular platform (UniTARG) that can be rapidly and easily adapted for redirection of T cells to any TAA in both a bsAb- or CAR-setting. The UniTARG technology consists of a universal effector arm and individual targeting modules (TMs). The effector arm is either a universal CAR (UniCAR), which has specificity for a short peptide motif of 10 amino acids derived from a human nuclear protein, or a bsAb (UniMAB) with specificity for human CD3 on T cells and the respective peptide epitope. The antigen-specificity of the system is provided by TMs comprising a binding domain e.g., a tumor-antigen specific scFv, fused to the nuclear antigen motif recognized by the UniCAR/UniMAB binding domain. Here we provide first in vitro and in vivo prove of concept for these new approaches. Redirection of T cells armed with the universal effector modules was effective at picomolar concentrations of targeting modules directed against various antigens. The modular composition of both platforms prompted us to explore, if T cell retargeting against several antigens simultaneously might be feasible. Using either two single-specific TMs or single bi-specific TMs significantly enhanced specific lysis of tumor cells in vitro and durable responses in vivo were observed. Furthermore, our results proof that TMs against new targets can be developed in a couple of weeks and all TMs tested so far engaged effector module armed T cells with similar potencies. Taken together, the UniTARG platform technology represents a promising tool in the field of both bsAbs and CARs with the advantage of simultaneous or consecutive dual or even multispecific T cell retargeting. Furthermore the platform approach allows a rapid development of new therapeutic options against additional targets.

#2314

Different stimulation conditions affect the immune phenotype of GD2-specific chimeric antigen receptor-expressing T cells.

Laurin Ochs, Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Claudia Rossig, Silke Jamitzky. _University Children´s Hospital Münster, Muenster, Germany_.

The in vivo persistence of chimeric antigen receptor (CAR) modified T cells is a major prerequisite for their antitumor activity and was found to be associated with a less differentiated immune phenotype. Here, we compared two in vitro T cell stimulation conditions: coated anti-CD3/CD28 antibodies [3/28], and Dynabead stimulation of enriched CD3+ T cells [DB]. Peripheral blood T cells from three healthy donors were stimulated with either of the two methods, retrovirally transduced with the GD2-specific CAR GD2-BBz on day 2 or 3, and expanded in RPMI/AIMV medium with 50 IU/ml recombinant human interleukin-2 for 13 days. T cell expansion rates were comparable between the two stimulation conditions and independent of CAR gene expression. Transduction efficiencies, determined by staining with the GD2-CAR-specific antibody Ganglidiomab, were also comparable. The immune phenotype by expression of CD3, CD4, CD8, CD45RO and CD197 was determined by flow cytometry analysis on day 13 or 14 after initial stimulation. The proportions of central memory (TCM), effector memory (TEM) or naïve T cells (TN) within the two types of cultures were noticeably different (Table 1), with a higher proportion of non-transduced CD8+ T cells with a TCM phenotype after DB compared to CD3/CD28 stimulation (p=0.005 for DB d2, p=0.01 for DB d3). Compared to non-transduced T cells, CAR-expressing cells of all types of cultures had higher proportions of TCM cells (p=0.02 for CD4+ T cells, p<0.01 for CD8+ T cells). In conclusion, we found that the stimulation conditions have a strong impact on the T cell phenotype and that retroviral CAR gene transduction can also affect T cell differentiation. The optimal T cell culture conditions for a product with sustained persistence in vivo will ultimately emerge from clinical trials.

Table 1: Proportions of T cell subpopulations on day 13 or 14 (medians and ranges)

---

|

xy | TN: CD45RO-/CD197+ | TCM: CD45RO+/CD197+ | TEM: CD45RO+/CD197-

3/28 | NT | 37.9% CD4+ (31.8-41.3)

53.0% CD8+ (20.9-72.9) | 20.2% CD4+ (20.2-27.0)

6.1% CD8+ (3.7-6.9) | 35.2% CD4+ (32.8-45.4)

35.5% CD8+ (17.0-66.3)

|

CAR

d2 | 24.4% CD4+ (15.1-28.8)

32.9% CD8+ (23.9-48.9) | 33.6% CD4+ (29.9-49.3)

27.7% CD8+ (17.7-38.8) | 37.3% CD4+ (22.4-47.2)

27.1% CD8+ (25.4-32.7)

DB | NT | 33.1% CD4+ (15.0-45.6)

53.8% CD8+ (46.8-53.8) | 32.6% CD4+ (29.9-45.2)

19.6% CD8+ (17.8-26.3) | 23.3% CD4+ (16.6-51.7)

12.1% CD8+ (10.6-15.2)

|

CAR

d2 | 11.5% CD4+ (10.9-27.7)

37.1% CD8+ (24.7-41.8 | 60.7% CD4+ (53.0-69.7)

48.0% CD8+ (34.4-65.2) | 16.5% CD4+ (16.1-26.6)

8.7% CD8+ (5.7-10.3)

DB | NT | 33.2% CD4+ (26.5-55.2)

51.6% CD8+ (34.2-62.5) | 23.0% CD4+ (21.9-40.2)

14.2% CD8+ (11.2-16.9) | 19.1% CD4+ (17.6-48.6)

15.2% CD8+ (13.0-19.6)

|

CAR

d3 | 17.5% CD4+ (13.4-34.5)

36.6% CD8+ (28.5-46.5) | 45.7% CD4+ (36.6-57.5)

38.0% CD8+ (22.2-55.1) | 22.0% CD4+ (21.4-32.6)

10.8% CD8+ (8.5-19.6)

#2315

Dynamic SPECT imaging of PSMA-specific CAR T cells in mice bearing prostate cancer.

Nia Emami-Shahri,1 Julie Foster,2 Jane Sosabowski,2 John Maher,1 Sophie Papa1. 1 _King's College London, London, United Kingdom;_ 2 _Barts Cancer Institute, London, United Kingdom_.

Immunotherapy using CAR-T-cells is acquiring an expanding role in the treatment of malignant disease. However, efficacy of solid tumour CAR-T-immunotherapy has proven inconsistent. Limitations include poor T-cell trafficking to tumour deposits, insufficient T-cell longevity in-vivo and the occurrence of both predicted and unanticipated on-target and off-target toxicity. To refine this therapeutic approach, it is desirable to develop systems that allow the monitoring of T-cell location and persistence in-vivo.

A retroviral vector named PiN-4 was constructed, which co-expresses: (i) an interleukin (IL)-4-responsive chimeric cytokine receptor (4αβ); (ii) a prostate-specific membrane antigen (PSMA)-targeted CAR (P28z) and (iii) hNIS, which promotes the uptake of technetium-99m pertechnetate (99mTcO4-) in viable cells. IL-4 enriched human 4P28zN+ T-cells were administered intravenously to Nod Scid Gamma (NSG) mice bearing prostate tumour xenografts.

Measurement of the tumour xenografts by bioluminescent imaging (BLI) found that only PSMA-expressing tumours responded to 4P28zN+ T-cell treatment. Longitudinal SPECT-CT imaging further confirmed this with the preferential accumulation of 4P28zN+ T-cells in PSMA-expressing tumours compared to PSMA-negative tumours.

Use of NIS as a T-cell imaging reporter brings several potential advantages. It promotes receptor-mediated uptake of the inexpensive, low toxicity and clinically useful SPECT tracer, technetium-99m pertechnetate (99mTcO4-). In addition, the ectopic expression of NIS is well tolerated by host cells and hNIS functions only in viable cells. These data demonstrate proof of concept for the utility of hNIS as an imaging reporter in genetically engineered T-cells.

#2316

GPC3-specific chimeric antigen receptor T cell in combination with Sorafenib as a novel therapeutic treatment for hepatocellular carcinoma.

Thu Le Trinh,1 Qunfeng Wu,2 Lung-Ji Chang,2 Mitchell Ho,3 Chen Liu2. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of Florida, Gainesville, FL;_ 3 _National Cancer Institute, Bethesda, MD_.

FDA-approved targeted therapy for advanced hepatocellular carcinoma (HCC) is limited with the only option of Sorafenib treatment with severe side effects. Immunotherapy based on adoptive transfer of tumor-specific T cells have demonstrated a promising clinical outcome for solid tumors. In our study, we developed T cells expressing the fourth generation of GPC3-specific chimeric antigen receptor (α-GPC3 CAR) that efficiently eliminated GPC3-positive HCC cells (Huh7 and HepG2) without killing GPC3-negative primary hepatocytes. This CAR was constructed using a lentiviral vector containing CD28, CD27 and 41BB costimulatory domains, a CD3ζ signaling domain fused to a FKBP-iCasp9 apoptosis inducing gene, and a GPC3-specific sequence derived from HN3, a single-domain VH human monoclonal antibody (gift from Mitchell Ho, NCI). After 16h of incubation at the E (α-GPC3 CAR T cells ) : T (Huh7 or HepG2 cells) ratio of 1:1, α-GPC3 CAR T cells effectively lysed Huh7 (39%) and HepG2 (77%) but not primary hepatocytes ( 9%), while the control CAR T cells (α-CD30 CAR) could not initiate a specific lysis on those cells. At the E:T ration of 3:1, the specific killing increased to 78% (Huh7) and 99% (HepG2) while remaining low with primary hepatocytes (11%). Especially at the very low E:T ratio of 1:10, combination with Sorafenib priming (24h, IC10 concentration) prior to CAR T incubation boosted the specific lysis up to 25% (p=0.0416). These results indicate that combination of GPC3-specificCAR T cells transfer and Sorafenib regimen could be a promising therapeutic option for the treatment of HCC, with the doses of both CAR T and Sorafenib reduced up to 10 times, hence lower side effects while preserving the efficacy of tumor-specific cytotoxicity.

### Immune Checkpoints 1

#2317

PD-1 and PD-L1 expression patterns and DNA mismatch repair status for precision management of patients with gastric cancer.

Keisuke Kimura,1 Takeshi Nagasaka,1 Yoshiko Mori,1 Takashi Kawai,1 Tomokazu Fuji,1 Fumitaka Taniguchi,1 Kazuya Yasui,1 Toshiaki Toshima,1 Yuzo Umeda,1 Hiroshi Tazawa,1 Ajay Goel,2 Toshiyoshi Fujiwara1. 1 _Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Center for Gastrointestinal Cancer Research; Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX_.

BACKGROUND: Programmed cell death protein 1 (PD-1) and its ligand (PD-L1) downregulate T cell activation and are related to immune tolerance. Recently, microsatellite unstable (MSI) colorectal cancers have been shown to have good therapeutic response to PD-1 antibody immunotherapy, possibly due to release exhaustion of their ability to recognize high number of tumor neo-antigens. The aim of this study was to clarify the significance of PD-1 and PD-L1 expression, and to analyze the relationship between MSI status and MLH1 hypermethylation status in gastric cancer.

METHODS: A total of 105 patients who underwent curative gastrectomy for gastric cancer were included in this study. The PD-1, PD-L1, HER2, and mismatch repair proteins (MLH1/PMS2/MSH2/MSH6) expression were examined by immunohistochemistry. MSI status were examined by analyzing three mononucleotide repeat markers. MLH1 methylation was evaluated using a highly sensitive fluorescence-based PCR assay, as we reported previously (JNCI 2009). KRAS/BRAF/PIK3CA mutations were determined by conventional Sanger sequencing. Infection of Helicobacter Pylori (H. Pylori) and EB virus (EBV) were determined by amplifying H. Pylori and EBV specific sequence by PCR, followed by conventional Sanger sequencing.

RESULTS: PD-1 and PD-L1 were expressed in 40% (38/95) and 33% (31/95) gastric cancers, respectively. HER2 was expressed in 15% (14/96) gastric cancers. Infection of H.Pylori and EBV were observed in 71% (70/98) and 8% (8/98) gastric cancers, respectively. MSI was observed in 13% (13/98) gastric cancers. Among 13 MSI cancers, 11 cases (85%) showed extensive methylation in the MLH1 promoter region, suggesting their sporadic manifestation. KRAS (exon 2), BRAF (exon 15) and PIK3CA (exon 9 & 20) mutations were observed in 5% (5/98), 0% (0/98), and 4% (4/98), respectively. Among these, one case harbored simultaneous KRAS and PIK3CA mutation. Gastric cancers with KRAS/PIK3CA were more frequently MSI-positive (5/8, p=0.003). Among eight EBV infected cancers, only one case showed MSI and MLH1 methylation. PD-1 expression was significantly correlated with PD-L1 expression (p=0.04). Although both PD-1 and PD-L1 expression were associated with MSI (p=0.007 and p=0.008, respectively), only PD-L1 expression was significantly correlated with gastric cancers with MLH1 methylation (p=0.01). While expression of immune checkpoint molecules were specific for tumors with MMR deficiency, HER2 expression was exclusively observed in MMR-proficient tumors.

CONCLUSIONS: PD-1/PD-L1 expression is associated with MMR-deficient gastric cancers. In contrast, HER2 expression is observed exclusively in gastric cancers with MMR-proficiency. Our result highlight that expression analysis of genetic markers could lead the precision management of patients with gastric cancer.

#2318

Discovery and validation of a novel checkpoint target preferentially expressed in the tumor microenvironment.

Narendiran Rajasekaran,1 Amani Makkouk,1 Russel K. Pachynski,2 Aruna Azameera,1 Cariad Chester,1 Xing Zhao,1 Atsushi Yonezawa,1 Holbrook E. Kohrt1. 1 _Stanford University, Stanford, CA;_ 2 _Washington University School of Medicine, St Louis, MO_.

Immunotherapeutic approaches targeting a single immune checkpoint, programmed death ligand 1, have resulted in unprecedented response rates and survival benefit across multiple cancer histologies. The efficacy of PD-1 blockade is augmented when delivered in combination with other immune checkpoints, specifically anti-CTLA-4, despite a significant increase in toxicity. The current challenge is identification of novel immunomodulatory targets with equivalent efficacy to PD1 blockade as monotherapy, and which can be applied in combination with anti-PD1 with a superior therapeutic index.

As the majority of autoimmune toxicity occurs in healthy, non-malignant tissue, we developed a novel high throughput RNA screen to identify new immune checkpoint targets preferentially expressed in the tumor microenvironment. Across numerous tumor types including tumors targeted by monoclonal antibodies (Abs), ie rituximab, trastuzumab, among others, the screen reproducibly identified a list of novel targets for immunotherapy which through a secondary screen were refined for cell surface expression. By this approach we selected a novel type II transmembrane glycoprotein (TIITG) with dual function as an ectoenzyme and in signal transduction. Further experiments in-vitro and ex-vivo from tumor biopsies confirmed upregulation of the target on tumor-infiltrating, activated, immune cells including but not limited to innate immune effectors. In-vitro and in-vivo murine models using agonistic antibodies to the TIITG demonstrated (1) augmentation of natural killer cell spontaneous and antibody-dependent cytotoxicity, (2) inhibition of myeloid derived suppressor cell and regulatory T cell function, and (3) enhancement in depth and breadth of the adaptive response including post vaccination. Antagonistic Abs had minimal to no effect on the immune response, while agonistic Abs augmented type 1-cytokines (IFNg and TNFa) and cytotoxic markers, including CD107a, perforin, and granzyme. Surprisingly, in a murine autoimmune toxicity model established with prior immune checkpoint agonists, agonists of the TIITG elicited no apparent adverse effects, autoimmune or otherwise. Finally, human xenograft models support antitumor activity across over a dozen tumor histologies as monotherapy in addition to synergy with anti-PD1.

In summary, using a novel screening technique we uncovered promising immune targets for cancer therapy. The first of which, TIITG, has completed lead selection and is being prepared to enter a first-in-human clinical trial.

#2319

Selinexor, a selective inhibitor of nuclear export (SINE), enhances the in vivo efficacy of checkpoint blockade with antibodies targeting CTLA4 or PD-1/PD-L1 in melanoma.

Matthew R. Farren,1 Reena Shakya,1 Rebecca Hennessey,1 Thomas Mace,1 Jennifer Yang,1 Omar Elnaggar,1 Gregory Young,1 Yosef Landesman,2 Robert Carlson,2 Sivan Elloul,2 Marsha Crochiere,2 Christin Burd,1 Gregory B. Lesinski1. 1 _Ohio State University - James Comprehensive Cancer Center, Columbus, OH;_ 2 _Karyopharm Therapeutics Inc., Newton, MA_.

Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >1000 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. This small molecule targets exportin-1 (XPO1), a key nuclear export protein with >200 cargo proteins which include both tumor suppressors and cell cycle modulators. As a result, selinexor blocks nuclear export of proteins including IκB, NFAT1c, STAT1 and STAT3, which regulate expression of the inhibitory T cell receptors CTLA4, PD1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and thereby enhance the anti-tumor activity of antibodies targeting PD-1/PD-L1 or CTLA4. Human (A375, CHL-1) and murine (B16F10) melanoma cell lines expressed high levels of PD-L1 protein at baseline, and PD-L1 expression was induced following selinexor treatment in numerous other tumor cell lines (including HCT-116, MDA-MB-468, MV-4-11, OVCAR-8, and PC-3). Examination of lymphocytes revealed that selinexor also increased expression of PD-1 and CTLA4 by ~2-fold. Mice bearing syngeneic B16F10 melanoma tumors treated with selinexor (15 mg/kg 2 x weekly) and anti-CTLA4 (250 µg, 2 x weekly) demonstrated a significant reduction in tumor growth rate (p = 0.0065) while monotherapy had no significant effect on tumor growth. Similar results were obtained in mice bearing B16F10 melanoma treated with the combination of selinexor + anti-PD-1 (200 µg, 2 x weekly, p < 0.034) or selinexor + anti-PD-L1 (100-200 µg, 2 x weekly, p < 0.001). Importantly, no weight loss or signs of toxicity were evident in any in vivo study. Further immunophenotypic analyses have been completed in animals receiving selinexor alone or in combination with anti-PD-L1. In combination treated mice, we observed a significantly increased percentage of splenic NK cells (p ≤ 0.050), and a significantly increased percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p<0.001). These changes are indicative of increased anti-tumor immune activity; however, they were accompanied by significantly increased percentages of myeloid cell subsets in combination treated mice (p ≤ 0.050). The immunologic significance of this myeloid cell expansion is currently under investigation. These data indicate that the efficacy of selinexor may be enhanced by disrupting immune checkpoints in effector cells (T and NK cells). This provides data in support of novel, evidenced-based combinations involving immunotherapy with XPO1 inhibition that deserve further investigation for advanced cancer.

#2320

CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer.

Sandra Ortiz-Cuaran,1 Aurélie Swalduz,1 Maria A. Albaret,1 Christine Menetrier-Caux,1 Véronique Haddad,2 Arnaud Paré,1 Geneviève De Souza,1 Anne P. Morel,3 Maurice Pérol,4 Christophe Caux,1 Sylvie Lantuejoul,5 Alain Puisieux,3 Pierre Saintigny1. 1 _Centre Léon Bérard. INSERM U1052 - CNRS UMR 5286, Cancer Research Center of Lyon. University Lyon I., Lyon, France;_ 2 _Pathology Department, Centre Léon Bérard, Lyon, France;_ 3 _INSERM U1052 - CNRS UMR 5286, Cancer Research Center of Lyon. Centre Léon Bérard. University Lyon I., Lyon, France;_ 4 _Thoracic Oncology Unit, Cancer Centre Léon Bérard, Lyon, France;_ 5 _Centre Léon Bérard. CHU Grenoble, Department of Pathology. Université de Grenoble Joseph Fourier., Lyon, France_.

Non-small-cell lung cancers (NSCLC) account for 85% of lung cancers and are mostly diagnosed at advanced stage. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, have proven to unleash anti-tumor immunity and mediate durable cancer regressions in a portion of patients with NSCLC. Epithelial-to-mesenchymal transition (EMT) is a developmental process that enables reprogramming of polarized epithelial cells towards a mesenchymal phenotype with migratory and invasive properties. EMT has been involved in escape from oncogenic-induced senescence, resistance to chemotherapy and development of metastatic disease. Moreover, EMT promotes cancer cell plasticity and favors their adaptability to selective pressures. Here we made use of in silico approaches and further in vivo validation to evaluate the potential role of EMT as a major escape mechanism to tumor immunosurveillance in NSCLC. We performed an initial in silico analysis to assess the expression of immune checkpoint ligands (ICPLs) in 129 NSCLC cell lines (CCLE), in relation to their EMT status defined by a 72-gene EMT signature that was previously reported in NSCLC. Unsupervised hierarchical clustering analysis revealed that the expression of selected ICPLs was associated with the mesenchymal or epithelial phenotype. In particular, CD70, a member of the tumor necrosis factor superfamily, was significantly associated with the mesenchymal status (p < 0.001). We identified an E-box sequence (CANNTG) in CD70 gene promoter thus suggesting the possible regulation of CD70 by ZEB1 and/or SNAI. We extended the in silico analysis to a set of 488 adenocarcinomas (ADC) and 501 squamous cell carcinomas (SCC) from the TCGA and confirmed that CD70 expression was associated with a mesenchymal status (q-value <0.001). In a set of E-like (H441 and H1650) and M-like (H23 and HCC44) NSCLC cell lines we validated the association between an increased level of CD70 by flow cytometry and the mesenchymal status. Expression of CD70 by immunohistochemistry was observed in 5/51 lung ADC (10%), 6/45 (13%) lung SCC, 16/26 (63%) pulmonary sarcomatoid carcinomas and 2/10 (20%) small cell lung carcinomas. Interestingly, in pulmonary sarcomatoid carcinomas, CD70 expression was closely associated with the mesenchymal component. Our results suggest an association between the expression of CD70 and the mesenchymal phenotype, thus shedding light on the potential role of CD70 in immune escape of a subset of NSCLC.

#2321

Ibrutinib enhances antitumor activity in solid tumors when combined with checkpoint inhibitors.

Jeff Hsu, Yujun Huang, Chun-Te Chen, Jun Chen, Mint Sirisawad, Betty Y. Chang. _Pharmacyclics LLC, an AbbVie Company, Sunnyvale, CA_.

Introduction: Anti-CTLA-4 antibodies enhance antitumor immunity by activating antigen-specific cytotoxic T lymphocytes and depleting suppressive regulatory T cells (Treg) from the tumor microenvironment. Anti-PD-L1 or anti-PD-1 blocks negative regulators of T-cell activation and response, facilitating immune-mediated antitumor activity. These therapeutic antitumor approaches via immune checkpoint blockade may be enhanced by ibrutinib (ibr), a first-in-class oral inhibitor of Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK). A previous study suggested ibr may alter the Th1/Th2 T-cell balance by inactivating ITK and potentially enhancing antitumor immune responses in solid tumor models, including those that do not express BTK (Sagiv-Barfi. PNAS. 2015). Here we report the combined effects of ibr and CTLA-4/PD-1/PD-L1 blockade in a panel of established syngeneic solid tumor models.

Methods: Immunocompetent mice with matched genetic background were inoculated subcutaneously in the right front flank with syngeneic tumor cells. Treatment began when mean tumor size reached approximately 70-100 mm³. Tumor growth inhibition (TGI) and tumor growth delay (TGD) were the study endpoints. The mean and the standard error of the mean for each group were calculated at each time point. Statistical analysis of independent sample t-tests and one-way ANOVA were used for comparisons between and among groups. Potential synergistic effects between treatments were analyzed by two-way ANOVA. Tumor and blood samples were collected for immunologic analysis at the tumor site or in peripheral compartments by flow cytometry.

Results: Treatment with ibr alone resulted in ~15%-50% TGI across various syngeneic subcutaneous mice models, including MBT-2 (bladder cancer), MC38 (colorectal cancer), and Pan02 (pancreatic tumor), where BTK expression is not prevalent in the tumor lines and tumor cells are not sensitive to ibrutinib. Ibr in combination with checkpoint inhibitors demonstrated benefits in both TGI and TGD in the MC38 model. When ibr was combined with anti-CTLA-4 monoclonal antibody, complete regression of established MC38 tumors was observed.

Conclusion: The efficacy observed with ibr treatment alone in solid tumor models lacking BTK expression suggests ibr can lead to immunomodulation by altering the tumor microenvironment. Additionally, synergistic growth suppression was observed when ibr was combined with checkpoint inhibitors. To further understand the mechanism of action associated with combination ibr immunotherapy in syngeneic models, immunophenotypical analyses of tumor tissues are under investigation.

#2322

Intratumoral treatment with a highly interferogenic TLR9 agonist reverts tumor escape from PD-1 blockade.

Shu Wang, Jose Campos, Edwina Naik, Crain Chad, Gallotta Marilena, Coffman Robert L., Cristiana Guiducci. _Dynavax Technologies, Berkeley, CA_.

The induction of tumor specific T cell exhaustion by the PD-1/PDL-1 pathway is an important mechanism of immune escape in cancer. Therapies blocking or reversing T cell exhaustion can produce durable responses in metastatic cancers including melanoma, NSCLC, renal carcinoma and bladder cancer. However, only a small subset of patients experience response upon anti-PD-1/PDL-1 treatment, whereas many others are refractory, showing no response at all. Thus, a key challenge is transforming a non-permissive tumor microenvironment into an immunologically competent one, in order to increase the frequency of patients with durable responses to PD-1/PDL-1 blockade.

We have characterized a mouse tumor model with a reproducible dichotomous response to anti-PD-1 treatment analogous to that observed in human cancers. We show that the ability of tumors to be rejected in response to anti-PD-1 treatment is highly correlated with degree of T cell infiltration, expression of Type I IFNs and B cell activation signatures, in whole tumors and in tumor infiltrating leukocytes. We then evaluate whether immunomodulation by intratumoral vaccination with a TLR9 agonist, SD-101, would affect rate of response to anti-PD-1 blockade. SD-101, is a CpG-C class oligonucleotide, optimized to induce both very high levels of IFN-alpha as well as maturation of dendritic cells and B cells. Intratumoral injection of SD-101 in tumor bearing mice undergoing anti-PD-1 therapy leads tumor rejection in 100% of treated mice versus a 20% tumor rejection in mice treated with anti-PD-1 blockade alone. SD-101 alone, although efficacious in reducing tumor growth, did not lead to durable tumor rejection.

Immune cell subset depletion studies implicate CD8, but not CD4, T cells in the rejection of tumors treated with anti-PD1 plus SD-101. T cell response generated in one flank by IT injection of SD-101 synergizes with systemic PD-1 blockade to induce rejection of unvaccinated distal tumors. In addition, we show that mice receiving systemic anti-PD-1 plus SD-101 have a higher percentage of CD8+ T cells infiltrating the tumors when compared to anti-PD-1 responders, anti-PD-1 non-responders or SD-101 given alone. Infiltrating CD4 and CD8 T cells in tumors with combination treatment, show a higher proliferative capacity and a higher frequency of cells producing both TNF-a and IFN-g than tumors responding to anti-PD-1 monotherapy. This demonstrates the potential for SD-101 to significantly enhance the frequency of responders to anti-PD-1 therapy by increasing the number and functionality of anti-tumor T cells within the tumor.

#2323

**Immune checkpoint inhibition by anti-PD-1 or CD137 co-stimulation enhances cytotoxicity towards CD30** + **tumors mediated by the bispecific tetravalent CD30/CD16A TandAb AFM13.**

Xing Zhao,1 Narendiran Rajasekaran,1 Uwe Reusch,2 Jens-Peter Marschner,2 Martin Treder,2 Holbrook E. Kohrt1. 1 _Center for Clinical Sciences Research Stanford, Stanford, CA;_ 2 _Affimed GmbH, Heidelberg, Germany_.

AFM13 is an NK-cell engaging CD30/CD16A bi-specific tetravalent TandAb antibody currently in Phase 2 clinical development in Hodgkin Lymphoma (HL) and CD30+ malignancies. Immune checkpoints inhibitors have demonstrated clinical efficacy in a variety of cancers, including HL. NK-cells are also regulated by a number of check-points, prompting us to investigate the combination of AFM13 with several immuno-modulatory antibodies. In previous experiments we were able to demonstrate higher efficacy of AFM13 than several immuno-modulatory antibodies in monotherapy and strong synergy between AFM13 and an anti-PD-1 antibody in vitro, as well as in vivo PDX models with human CD30+ HL tumors. In order to investigate the underlying immunological mechanisms we employed the same PDX model by implanting tumor fragments derived from surgical specimens of HL patients in immuno-deficient mice. After establishing tumors, mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-PD-1 weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes, myeloid cells and intra-tumoral cytokines were evaluated on days 30, 44, and 58, i.e. 2, 16 and 30 days after treatment start. While monotherapy with AFM13 was reproducibly more potent than anti-PD-1, significant synergy was observed when both agents were combined. Analysis of the tumors on day 58 revealed a strong correlation between tumor growth inhibition and levels of tumor-infiltrating NK-cells, T-cells, myeloid cells and intra-tumoral cytokines such as IFNg. In contrast to anti-PD-1 monotherapy, which only induced T-cell infiltration, AFM13 monotherapy was able to induce infiltration of NK- and T-cells in the tumors, however the combination further enhanced infiltration of both, NK- and T-cells. AFM13 resulted in stronger infiltration of macrophages than anti-PD-1, which was also increased by the combination of both agents, therefore further supporting cross-talk between innate and adaptive immunity. Furthermore, tumor analyses at earlier time-points (days 30 and 44) showed that the initial immune response is characterized by NK-cell infiltration and activation, as well as infiltration of macrophages, whilst the adaptive immune response by T-cells and activated dendritic cells was more pronounced on day 58. Combining AFM13 and anti-PD-1 augments infiltration and activation of all immune subpopulations.

In conclusion, our data support strong synergistic anti-tumor efficacy when AFM13 is combined with anti-PD-1 checkpoint blockade in HL PDX models, mediated by tumor-infiltrating lymphocytes, macrophages and dendritic cells, and provide strong evidence for cross-talk between innate and adaptive immunity induced by AFM13-recruited human NK-cells.

#2324

FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells.

Mi-Heon Lee,1 Jane Yanagawa,1 Tonya C Walser,1 Jonathan W. Goldman,1 Edward B. Garon,1 Gang Zeng,1 Sherven Sharma,1 Boning Gao,2 John Minna,2 Steven M. Dubinett,1 Jay M Lee1. 1 _UCLA, LA, CA;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX_.

BACKGROUND: Activating KRAS mutations are common driver mutations in non-small cell lung carcinoma. Patients with mutated KRAS demonstrate less benefit from adjuvant chemotherapy and resistance to tyrosine kinase inhibitors. KRAS mutations are known to activate the RAF-MEK-ERK pathway. Fos-related antigen-1 (FRA1) is a MEK/ERK-dependent transcription factor and member of the AP-1 transcription factor superfamily. Immune checkpoint pathways including the PD-1/PD-L1 pathway are involved in immune-mediated tumor evasion. We hypothesize that KRAS mutation directly regulates the PD-1/PD-L1 pathway through ERK activation and FRA1 may be an important transcriptional mediator.

METHODS: In order to assess the role of KRAS mutation independent of other somatic mutations and to begin to understand potential immune suppressive pathways operative in pulmonary premalignancy, premalignant human bronchial epithelial cell lines (HBEC) were used instead of human lung cancer cell lines. Four immortalized HBEC (2, 3, 7, and 11 cell lines) with KRAS v12-mutation (HBEC-KRAS) compared to control (HBEC-vector) were used to assess mRNA and protein expression levels of PD-L1 by RT-qPCR, flow cytometry, and western blot. Cell-lines were treated with MEK (ERK kinase) inhibitor (PD0325901) for 24 hours (h) up to 1μM. FRA1 was silenced by transfection with siRNA at 100nM.

RESULTS: In comparing HBEC-KRAS to HBEC-vector (wild-type) cells, PD-L1 mRNA (1.3~3.4 fold) and surface protein expressions (1.2~2.3 fold by flow cytometry) were increased in all 4 cell lines and confirmed by western blot analyses. PD-L1 mRNA and protein levels were highest in HBEC3-KRAS. MEK inhibition resulted in decreased PD-L1 expression by 10 fold (HBEC3-vector) and 11 fold (HBEC3-KRAS) in mRNA levels and 3 fold (HBEC3-vector and HBEC3-KRAS) in surface protein levels. In comparing HBEC3-KRAS to HBEC3-vector, FRA1 increased mRNA (2.7 fold) and protein level expression (2.8 fold). In comparing HBEC3-KRAS to HBEC3-vector cells, FRA1 silencing (FRA1 siRNA) resulted in reduced PD-L1 (53% at 48 h; 73% at 72 h) and FRA1 (94% at 48 h; 99% at 72 h) by western blot. In comparing HBEC3-KRAS to HBEC3-vector cells, ERK phosphorylation increased 7.9 fold and FRA1 silencing led to inhibition of ERK phosphorylation by 2.8 fold at 72 h by western blot.

CONCLUSIONS: Here, we demonstrate that PD-L1 expression is elevated in premalignant KRAS mutated human bronchial epithelial cells, and ERK activation mediates KRAS mutation driven up regulation of PD-L1 and at least in part through FRA1 dependence. Our data suggest that KRAS mutation may directly regulate the PD-1/PD-L1 immune checkpoint pathway through FRA1 and MEK/ERK-dependent transcriptional regulation. Further understanding of KRAS driven molecular pathways that modulate immune checkpoints may elucidate therapeutic targets for potential new combination immune therapies.

#2325

Immune checkpoint(s) expression in AML patients enrolled in a phase 1-2 study with guadecitabine.

Carolina Fazio,1 Alessia Covre,1 Maria Lofiego,1 Pietro Taverna,2 Mohammad Azab,2 James N. Lowder,2 Sandra Coral,3 Michele Maio1. 1 _University Hospital of Siena, Siena, Italy;_ 2 _ASTEX Pharmaceuticals Inc., Pleasanton, CA;_ 3 _Epigen Therapeutics, Pordenone, Italy_.

Immune checkpoint(s) blockade is becoming the therapeutic mainstay in melanoma and lung cancer. Based on these important clinical achievements, immunotherapy with immunomodulating monoclonal antibodies (mAb) is being explored as a new therapeutic modality in most human malignancies, either alone or in combination with other immunomodulating agents. We have provided extensive evidence that the second generation DNA hypomethylating agent (DHA), guadecitabine (SGI-110), plays a promising role in potentiating the immunogenicity and the immune recognition of human malignancies through the up-regulation of the expression of different immune molecules on cancer cells. These findings led us to hypothesize that DHA could represent ideal partners for immune checkpoint(s) blocking agents to improve their therapeutic efficacy. Here, we studied the expression of programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), and 2 (PD-L2), and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) in peripheral blood mononuclear cells of acute myeloid leukemia (AML) patients (N=23), enrolled in a phase 1-2 study with guadecitabine. Patients included in this preliminary analysis were selected based on their responsiveness to therapy. Quantitative RT-PCR analyses showed a constitutive expression (gene/β-actin molecules >7.5E-05) of PD1, PD-L1, PD-L2 and CTLA-4 in 82.6%, 100%, 47.8% and 8.7% patients, respectively. Guadecitabine therapy up-regulated (≥2 fold) the expression of PD1, PD-L1, PD-L2 and CTLA-4 in 57.9%, 56.5%, 18.2% and 0% patients, respectively, at any of the time-points investigated during treatment. De novo expression of PD1, PD-L2 and CTLA-4 was observed in 100%, 41.6% and 38.1% of the immune checkpoint(s)-negative patients, respectively.

Though preliminary, these results further support the strong link between epigenetics/DNA methylation and immune response, in cancer patients, and strengthen the therapeutic potential of epigenetic immunomodulation, with "consolidated" and emerging immune checkpoint(s) blocking mAb.

#2326

LTX-315, an oncolytic peptide, increases anticancer immunity mediated by CTLA4 blockade in an interleukin-2 receptor beta-chain-dependent manner.

Takahiro Yamazaki,1 Marie Vetizou,1 Camila Flores,1 Aurelien Marabelle,1 Baldur Sveinbjørnsson,2 Øystein Rekdal,2 Guido Kroemer,1 Laurence Zitvogel1. 1 _Gustave Roussy Cancer Campus, Villejuif, France;_ 2 _Lytix Biopharma AS, Tromsø, Norway_.

Intratumoral immunotherapies aim at reducing local immunosuppression as well as reinstating and enhancing systemic anticancer T cell functions without inducing side effects. LTX-315 is a first-in-class oncolytic peptide -based local immunotherapy that meets these criteria by inducing a type of malignant cell death that elicits anticancer immune responses. Here, we show that LTX-315 rapidly reprograms the tumour microenvironment by decreasing the local abundance of immunosuppressive Tregs and myeloid derived-suppressor cells and by increasing the frequency of polyfunctional TH1/TC1 cells with a concomitant raise in their expression of CTLA4 and a drop in PD-1 expression. Logically, in tumours that were resistant to intratumoural or systemic CTLA4 blockade, subsequent local inoculation of LTX-315 cured the animals or caused tumour regressions with abscopal effects(meaning that even malignant lesions that were established in the opposite flank and were not treated by LTX-315 responded to the therapy). This synergistic interaction between CTLA4 blockade and LTX-315 was reduced upon blockade of the β-chain of the interleukin-2 receptor (CD122) which is required for signalling in response to IL-2 and IL-15. This preclinical study provides a strong rationale for administering the oncolytic peptide LTX-315 to patients who are receiving treatment with the CTLA4 blocking antibody ipilimumab.

#2327

IFNgamma regulates PD-L1 expression through activating IRF1 transcription in tumor cells.

Chunwan Lu, Amy V. Paschall, Priscilla S. Redd, Iryna Lebedyeva, Kebin Liu. _Georgia Regents University, Augusta, GA_.

The emerging clinical success of checkpoint blockade immunotherapy in human cancer patients has highlighted the critical importance of PD-L1, a ligand for the T cell inhibitory receptor PD-1, as a molecular target in cancer immunotherapy. PD-L1 is constitutively expressed in tumor cells and is also inducible by inflammatory cytokines such as IFNgamma. It has been proposed that tumor cells may sense the elevated IFNgamma from activated host T cells as a "threat" in the tumor microenvironment and adapt it by up-regulating PD-L1. The aim of this study is to elucidate the molecular mechanism underlying IFNgamma regulation of PD-L1 expression in tumor cells. We observed that PD-L1 is constitutively expressed, albeit at low level, in multiple types of cancer cells, including pancreatic, colon, breast, and sarcoma cells. IFNgamma treatment dramatically increased PD-L1 expression level and IFNgamma up-regulates PD-L1 expression through the Jak-STAT1 signaling pathway in vitro and in vivo. Chromatin immunoprecipitation assay did not identify IFNgamma-activated pSTAT1 binding to the pd-l1 promoter. Instead, we observed that IFNgamma activates IRF1 transcription and IRF1 is required for IFNgamma-induced PD-L1 expression. Chromatin immunoprecipitation analysis shows that pSTAT1 is associated with the irf1 but not the pd-l1 promoter. Analysis of the irf1 promoter DNA sequence revealed a pSTAT1-binding consensus sequence, and electrophoretic mobility shift assay indicates that pSTAT1 directly binds to this DNA element of the irf1 promoter. Furthermore, we demonstrated that IRF1 is associated with the pd-l1 promoter chromatin near the irf1 transcription initiation site. The pd-l1 promoter region contains a putative IRF1-binding consensus sequence and electrophoretic mobility shift assay shows that IRF1 binds to this DNA element of the pd-l1 promoter region. Taken together, our data indicate that IFNgamma activates pSTAT1 that binds to the irf1 promoter to activate irf1 transcription. IFNgamma-induced IRF1 then binds to the pd-l1 promoter to activate pd-l1 transcription in tumor cells.

#2328

ICOS signaling promotes CD4+ effector T-cell function during antitumor responses.

Todd C. Metzger, Hua Long, Shobha Potluri, John C. Lin, Reid M.R. Feldman. _Pfizer, Inc., South San Francisco, CA_.

Inducible costimulator (ICOS) is a CD28-related cell surface receptor which provides an important costimulatory signal to T cells during activation. Prior work has found that anti-ICOS antibodies can promote anti-tumor immunity in vivo, but it is unclear whether this effect results from ICOS signaling blockade and/or agonism, or whether direct antibody-mediated depletion of ICOS+ Tregs is responsible for enhancing anti-tumor immunity. Here, we generated anti-ICOS-L blocking antibodies in mice and engineered such antibodies to contain mouse IgG1 Fc regions with low Fc receptor engagement and good pharmacokinetics. These reagents were used in vivo to specifically block ICOS signaling in syngeneic mouse tumor models without potential depletion of immune cells through ADCC. The role of ICOS signaling in anti-tumor immunity was addressed both in isolation, and together with combination immunotherapy treatments of mouse syngeneic tumor models, followed by flow cytometry-based characterization of peripheral and tumor-infiltrating T cell subsets. Finally, CT26 tumor cells were engineered to express mouse ICOS-L in a doxycycline-inducible fashion to promote additional ICOS signaling within the tumor microenvironment.

Initial characterization of ICOS blockade in tumor models revealed that inhibition of ICOS signaling alone was sufficient to mediate a modest anti-tumor effect, and characterization of intratumoral T cell populations demonstrated a specific reduction of intratumoral Tregs with ICOS signal deprivation. A survey of previously identified immunomodulators found that, like anti-CTLA-4, anti-OX40 treatment of tumors was able to drive upregulation of ICOS on multiple T cell subsets, while anti-4-1BB therapy was not. Combining ICOS-L blockade with these additional immunotherapeutic approaches showed that inhibition of ICOS signaling had little impact on CD8+ T cell-mediated anti-tumor immunity induced by anti-4-1BB. However, the efficacy of anti-OX40 therapy, which depletes Tregs and relies to a large degree on CD4+ effector T cells responses, was significantly impaired with ICOS-L blockade. In contrast, the induction of additional intratumoral ICOS signaling in established tumors increased the frequency of tumor rejection and overall survival when combined with anti-OX40 therapy.

Taken together, these results show that ICOS signaling during the anti-tumor immune response acts on both effector and regulatory T cell subsets, and thus manipulation of ICOS signaling alone may be an inefficient strategy for immunotherapy. However, the use of ICOS agonists in combination with Treg-depleting therapies such as anti-OX40 should limit the potential to promote Treg function or expansion and instead result only in enhanced CD4+ effector T cell responses.

#2329

Glutaminase inhibition with CB-839 enhances anti-tumor activity of PD-1 and PD-L1 antibodies by overcoming a metabolic checkpoint blocking T cell activation.

Matt Gross,1 Jason Chen,1 Judd Englert,2 Julie Janes,1 Robert Leone,2 Andy MacKinnon,1 Francesco Parlati,1 Mirna Rodriquez,1 Peter Shwonek,1 Jonathan Powell2. 1 _Calithera Biosciences, South San Francisco, CA;_ 2 _Johns Hopkins School of Medicine, Baltimore, MD_.

Recent studies have highlighted the importance of the tumor metabolic environment for controlling immune activation. T-cells activated through the TCR/CD28 receptor switch to a highly glycolytic metabolism and increase their requirement for glucose and glutamine. Consequently, limited availability of glucose or glutamine can block T-cell activation and proliferation. Likewise, immune checkpoints proteins, PD-1 and CTLA-4, suppress T cell metabolism by inhibiting glycolysis, glutamine uptake and glutaminolysis (Patsoukis et al Nat Comm. 2015). Chang et al (Cell. 2015) recently demonstrated that glucose consumption by tumors restricts glucose availability and blocks activation of T cells, and that treatment with CTLA-4, PD-1, or PD-L1 antibodies can re-activate T cell glycolysis.

CB-839 is a glutaminase inhibitor currently in Phase 1 trials in patients with solid and hematological malignancies. CB-839 blocks glutamine consumption in tumors and causes a significant elevation of tumor glutamine levels. Therefore, we hypothesized that CB-839 might enhance the activity of immune checkpoint inhibitors via metabolic modulation of the tumor microenvironment. We first confirmed that T- cell proliferation is dependent on glutamine but is only minimally inhibited by CB-839. In the absence of glutamine, splenic mouse T cells stimulated with anti-CD3/CD28 had reduced glucose consumption and did not proliferate. In contrast, CB-839 treatment did not mimic the effects of glutamine withdrawal on T-cells. CB-839 had no effect on glucose consumption by activated T-cells and only a minimal effect on proliferation. We also confirmed in the OVA vaccinia model that CB-839 had minimal effects on CD4 and CD8 T-cell proliferation in vivo, while the non-specific glutamine inhibitor DON caused a dramatic reduction in the number of CD4 and CD8 T-cells.

To determine if CB-839 could enhance the anti-tumor efficacy of immune checkpoint inhibitors, we treated mice bearing syngeneic CT26 colon carcinoma tumors with anti PD-1 or anti PD-L1 alone or in combination with CB-839. The addition of CB-839 to either anti PD-1 or anti PD-L1 treatment enhanced anti-tumor activity, augmenting tumor regression and promoting survival. Depletion of CD8+ T-cells from CT26 tumors reversed the anti-tumor effects of PD-L1 and CB-839, demonstrating that the combination targets CD8+ T-cells in the immune microenvironment.

These data are the first demonstration that modulation of glutamine metabolism in tumors can enhance the activity of checkpoint inhibitors and provide a rationale for combining CB-839 with immune checkpoint inhibitors in the clinic. Overall, these data highlight a new therapeutic approach to treating cancer by targeting tumor metabolism as a means of enhancing the efficacy of immunotherapy.

#2331

HDAC6, new role as master regulator of PD-L1 and immune-related pathways.

Tessa Knox,1 Maritza Lienlaf,2 Patricio Perez,2 Mibel Pabon,2 Calvin Lee,2 Fengdong Cheng,1 Eva Sahakian,2 John Powers,2 Susan Deng,2 Smalley Keiran,2 Alan Kozikowski,3 Javier Pinilla,2 Amod Sarnaik,2 Ed Seto,1 Jeffrey Weber,4 Eduardo Sotomayor,1 Alejandro Villagra1. 1 _The George Washington University, Washington, DC;_ 2 _H. Lee Moffitt, Tampa, FL;_ 3 _University of Illinois at Chicago, Chicago, IL;_ 4 _New York University, New York, NY_.

Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well-documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immunobiology is still not completely understood, and the reported immunological outcomes when using HDACi are heterogeneous. Our group recently reported that the pharmacological or genetic abrogation of a single HDAC, HDAC6, modulates the expression of immuno-regulatory proteins, including PD-L1, PD-L2, MHC class I, B7-H4 and TRAIL-R1. We primarily focused in PD-L1, which is an important negative regulator of T-cell function and often over-expressed in cancer cells. In a mechanistic point of view, we have found that the pharmacological inhibition and genetic abrogation of HDAC6 inactivates the STAT3 pathway, impairs the nuclear translocation and the recruitment of STAT3 to the PD-L1 promoter and subsequently down-regulates the expression of PD-L1. Moreover, the in vivo abrogation of HDAC6 reduces tumor growth in melanoma models, effect that is enhanced in the presence of the immune check-point blocking antibodies anti-PD-1 and anti-CTLA4. These results provide a key pre-clinical rationale and justification to further study isotype selective HDAC6 inhibitors as potential immunomodulatory agents in cancer.

#2332

Patient-derived tumor xenografts in humanized NSG-SGM3 mice: a new immuno-oncology platform.

Li-Chin Yao,1 Mingshan Cheng,1 Minan Wang,1 Jacques Banchereau,2 Karolina Palucka,2 Leonard Shultz,3 Carol Bult,3 James G. Keck1. 1 _The Jackson Laboratory, Sacramento, CA;_ 2 _The Jackson Laboratory, Farmington, CT;_ 3 _The Jackson Laboratory, Bar Harbor, ME_.

Humanized mice engrafted with tumors enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently found that humanized NOD-scid IL2Rγnull (NSG) mice bearing patient-derived xenografts (PDX) allow efficacy studies of check-point inhibitors. Next generation NSG strains include triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSG-SGM3). Here we provide a direct comparison of check-point inhibitors evaluation in NSG and NSG-SGM3 mice engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from the same donor and implanted with PDX tumors. Corroborating earlier studies, reconstitution of human immune system in the blood was faster and more robust in NSG-SGM3 compared to NSG recipients throughout the course of the study (18 weeks). Human CD45+ cells reached 25% of total blood cells at week 4 in hu-NSG-SGM3 mice and at week 9 in hu-NSG mice. A majority of blood hCD45+ cells in hu-NSG-SGM3 at week 4 were CD33+ myeloid cells. Circulating hCD3+ T cells reached 10% at week 9 and included regulatory T cells (Tregs), consistent with earlier studies. Hu-NSG mice displayed comparable hCD3+ T cells in the blood only at 12-15 weeks and did not contain Tregs. PDX tumors were then engrafted into partially HLA-matched hu-NSG-SGM3 mice at 9 weeks post engraftment. Two PDX models previously shown to respond to anti-PD1 therapy in hu-NSG mice, BR1126 and LG1306, were used. Treatment with the anti-PD-1 receptor antibody pembrolizumab (Keytruda) significantly reduced tumor growth in both models. Thus, PDX-bearing hu-NSG-SGM3 mice might serve as a new and improved platform for preclinical immuno-oncology efficacy studies.

#2333

Possible prediction of tumor specific checkpoint inhibitor response based on TCGA somatic mutation load.

Leandro Machado Colli, Mitchell J. Machiela, Timothy Myers, Lea Jessop, Kai Yu, Stephen J. Chanock. _National Cancer Institute, Bethesda, MD_.

Immune checkpoint inhibitor therapy has been shown to be effective in a subset of patients with melanoma, non-small cell lung cancer (NSCLC) and kidney cancer. Recent studies have suggested that the number of non-synonymous mutations (NsM) can be used to select melanoma and NSCLC patients most likely to benefit from immune checkpoint inhibitor treatment. It is hypothesized that NsMs generate novel epitopes and gene products which can be detected by the immune system. The aim of this study is to apply prior information on NsM count and immune checkpoint inhibitor treatment to a range of tumor subtypes in the TCGA database to evaluate the proportion cases that could be possible responders. In our analysis of published studies of melanoma and NSCLC, the receiver operator characteristic (ROC) curve suggests a cutoff of 192 NsM would have a maximum combination of sensitivity (74%) and specificity (59.3%) for potential clinical benefit for patients. We conducted an analysis of 7,757 samples from 26 different TCGA tumor types and observed that approximately 16.2% of TCGA samples harbor more than 192 NsM, and thus could be in the category associated with a higher likelihood of response to immune checkpoint inhibitors. Based on NsM count, bladder, colon, gastric, and endometrial cancers each contained more than 30% of tumors with a high NsM and thus, could be possible candidates for checkpoint immune therapy. Although our model estimates clinical response to immune checkpoint treatment from NsM count is preliminary and needs future validation, information on NsM count can be useful for selecting tumor types most likely to benefit in clinical trials of immune checkpoint inhibitor treatment.

#2334

Anti-cancer efficacy of the combination with immunomodulatory antibodies and MEK inhibitor for Kras mutation and p53 knockout driven lung cancer.

Jong Woo Lee,1 Yu Zhang,1 Hee Sun Park,2 Sin Aye Park,3 JaSeok Peter Koo,1 Roy S. Herbst1. 1 _Yale School of Medicine, New Haven, CT;_ 2 _Chungnam National University, Daejeon, Republic of Korea;_ 3 _Seoul National Univeristy, Seoul, Korea, Republic of Korea_.

Lung cancer remains a major cause of cancer mortality. Malignant lesions are normally endogenously corrected by the immune surveillance system. However, tumors evade this immunity by inducing immunosuppressive microenvironments during cancer progression. It has been thought that there are two critical components of tumors making them capable of maintaining the immunosuppressive microenvironment. One is the inhibition of cytotoxic T lymphocytes (CTL) activity against the tumor through immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1), which are negative regulators of CTL, and the other is by the induction of tumor infiltrating immune suppressive cells, which can deactivate the CTL. Recent studies demonstrate that multiple cancer types, including melanoma, lung, kidney, bladder, and stomach, respond to immune checkpoint inhibitors, such as PD-L1 and PD-1 with 11-30% response rates and durable responses. However, a substantial number of patients still fail to respond to immunotherapy and the refractory mechanisms are largely unknown. In this study, we focus on KRas-driven lung cancers, as there are no clinically effective targeted drugs available for treating this type of lung cancer. We demonstrate that during the progression of tumors in KRas mutation and p53 knockout-driven lung cancer mouse models; KRasG12D/+;p53-/- (KP), there is a gradual increase in the number of myeloid derived suppressor cells (MDSC) and that the combination of either anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor, which targets downstream of KRas signaling, shows anticancer efficacy in these animal models. These combinations, in comparison to either single agent alone, effectively blocks the growth of subcutaneously (s.c.) injected syngeneic mouse lung cancer cells in immune competent transgenic KP mice, significantly increasing the survival rates: 37.5% (for anti-PD-1 antibody and MEK inhibitor), 62.5% (for anti-PD-L1 antibody and MEK inhibitor) vs. 0% single agents or control at the end of treatment. We find that the tumors in the control treated group harbor a substantial number of immune cells, including PD-L1 expressing MDSC. The combination treatment with either an anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor dramatically modulates the composition and the activity of tumor infiltrated immune cells. Tumors in the combined treated group show a significant decrease in PD-L1 expressing MDSC in comparison with control tumors. Additionally, a combined treatment would block the PD-L1 activity of the infiltrated PD-L1 expressing MDSC in malignant tumors and thus lead to improved survival. These results point to a potential therapeutic opportunity for currently untargetable KRas-driven lung cancers.

#2335

Imprime PGG triggers PD-L1 expression on tumor and myeloid cells and prevents tumor establishment in combination with αPD-L1 treatment in vivo.

Kathryn Fraser, Anissa Chan, Ross Fulton, Steven Leonardo, Adria Jonas, Xiaohong Qiu, Nadine Ottoson, Takashi Kangas, Keith Gordon, Jeremy Graff, Nandita Bose. _Biothera, Eagan, MN_.

Immune checkpoint inhibitors, including α-PD-1/PD-L1 antibodies, have revolutionized cancer therapy, yielding compelling long-term clinical responses in cancers resistant to traditional treatment. Translational studies have reported that expression of PD-L1 on the surface of tumor or infiltrating immune cells correlates to greater objective response. However, a large number of cancer patients are still refractory to treatment with checkpoint inhibitors, suggesting that the efficacy of anti-PD-1/PD-L1 immunotherapies may be improved by combination with agents that can both activate immune responses within the tumor microenvironment and induce PD-L1 expression. Imprime PGG (Imprime), a β glucan PAMP (Pathogen Associated Molecular Pattern) is currently in clinical development in combination with tumor-targeting antibodies, anti-angiogenic antibodies and immune checkpoint inhibitors. As a PAMP, Imprime is readily recognized by, and binds to, innate immune cells, triggering a coordinated immune response that includes neutrophil activation, repolarization of M2 macrophages and dendritic cell (DC) maturation. Co-culture of M2s or DCs with T cells elicits increased PD-L1 expression on the surface of these myeloid cells, drives T cell expansion and induces interferon gamma (IFNγ) production. When exposed to the IFNγ-rich media from these co-cultures, tumor cell lines from numerous cancers (lung, breast, and pancreas) routinely upregulate surface expression of PD-L1. In vivo treatment of Imprime in tumor free mice also repolarized splenic macrophages to M1 functionality, and furthermore, enhanced the ability of antigen presenting cells to prime antigen-specific CD8 T cells. These results suggest that Imprime has the potential to enhance the efficacy of checkpoint inhibitors. We therefore evaluated anti-tumor efficacy in two distinct syngeneic murine tumor models. In the CT-26 model, treatment began when tumors reached 40-100 mm3. Though neither Imprime nor αPD-1 (clone RMP1-14) was alone effective, the combination substantially repressed tumor growth. In the MC-38 model, tumors were injected subcutaneously. Three days later, mice were randomized to treatment groups. Tumors were evident at day 29 in 17/18 vehicle-treated mice, 16/18 Imprime-treated mice and 12/18 αPD-L1 (10F.9G2)- treated mice. Remarkably, though tumors grew initially, only 3/17 mice treated with αPD-L1 + Imprime showed palpable tumors at day 29. To assess the durability of this response, these mice were re-challenged by injection of MC-38 tumor cells on the opposite flank. These mice remained tumor-free even while MC-38 tumors readily grew in age-matched, tumor-naïve, control mice. Collectively, these data show that Imprime treatment can dramatically enhance the efficacy of immune checkpoint inhibitors in syngeneic tumor models.

#2336

**Use of T-cell receptor-engineered tumor infiltrating lymphocytes (TILs) in a novel** in vivo **model of adoptive T-cell therapy for lung cancer to study predictive biomarkers of checkpoint blockade response.**

Edmund K. Moon, Shaun O'Brien, Raghuveer Ranganathan, Evgeniy Eruslanov, Soyeon Kim, Kheng Newick, Albert Lo, Xiaojun Liu, Yangbing Zhao, Steven M. Albelda. _Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA_.

Introduction:

Lung cancer accounts for 30% of cancer-related deaths in the U.S. (www.cancer.org). Surgical resection (for the eligible 25% of patients) is the only true curative therapy, but is associated with relapse rates ≥ 40%. (Sugimura et al., 2007) In advanced stage lung cancer, systemic chemotherapy and/or irradiation can produce objective responses and palliation of symptoms, but with only modest survival benefits. (Shepherd et al., 2000) Recently, immunotherapy using PD1/PDL1 blockade has shown promise in patients with progressive lung cancer with some (10-20%) demonstrating remarkable and durable anti-tumor responses. (Brahmer et al., 2012; Topalian et al., 2012) Given this exciting success in a subgroup of patients, one key goal is to be able to identify which lung cancer patients will respond. Although there is some data to suggest that staining tumors for PDL1 might be predictive, these findings are preliminary and do not seem to be definitive. (Topalian et al., 2012) Based on analyses of human TILs isolated from a novel animal model of adoptive T cell transfer for lung cancer, we propose a more predictive set of biomarkers.

Materials & Methods:

Bead-activated human T cells were lentivirally transduced to express a high-affinity TCR clone (Ly95) conferring reactivity against the tumor associated antigen (TAA) NY-ESO-1 in the context of HLA-A2. They were injected intravenously in NOD SCID IL2rγ null (NSG) mice bearing A549-A2-ESO (A549 lung cancer cells expressing NY-ESO-1/HLA-A2) flank tumors. 30 days post transfer, TILs were isolated and analyses (FACs, effector function, mRNA seq) were performed comparing TCR+ vs. TCR- TILs. Details of T cell preparation, in vivo model, and TIL harvest were previously described. (Moon et al., 2015)

Results:

Ly95 TILs had significant hypofunction in their ability to lyse fresh tumor and secrete cytokine compared to non-injected Ly95 T cells. FACS staining of intracellular cytokine production upon restimulation with anti-CD3 antibody revealed intact function of TCR- "bystander" TILs but significant hypofunction of TCR+ TAA-reactive TILs. There was significantly more upregulation of PD1, Ox40L, 41BB, and TIGIT on the hypofunctional TCR+ TILs than the functional TCR- TILs.

Conclusions:

PD1/PDL1 blockade has a greater chance of inducing tumor response if TAA-reactive, hypofunctional TILs expressing PD1 are present. Even without knowing the TAA landscape or the TCR clonal repertoire of the TILs, identifying them is possible by analyzing the expression of a sensitive/specific biomarker set on those TILs. The TILs from our in vivo model may provide us with such a set of biomarkers. We will validate the this biomarker set on real patient lung cancer TILs. Some have already undergone checkpoint therapy, allowing us to study the ability of this biomarker set to predict response.

#2337

The adenosine A2A receptor antagonist CPI-444 blocks adenosine-mediated T-cell suppression and exhibits antitumor activity alone and in combination with anti-PD-1 and anti-PD-L1.

Stephen Willingham,1 Po Ho,1 Robert Leone,2 Emily Piccione,1 Carmen Choy,1 Andrew Hotson,1 Joseph Buggy,1 Jonathan Powell,2 Richard Miller1. 1 _Corvus Pharmaceuticals, Burlingame, CA;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Elevated levels of extracellular adenosine within the tumor microenvironment create an immunosuppressive niche that promotes tumor growth and metastasis. Adenosine signaling via the A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapies such as anti-PD-L1 or anti-PD-1 monoclonal antibodies (mAbs).

CPI-444 is an oral A2AR antagonist that has been evaluated previously in phase 1 clinical trials for non-oncology indications. CPI-444 demonstrates binding to A2AR with a Ki of 3.5 nM and > 50-fold selectivity over other adenosine receptor subtypes. We examined the immune activating and anti-tumor properties of CPI-444 alone and in combination with anti-PD-1/PDL-1 mAbs.

In vitro studies using activated primary human T cells demonstrated that CPI-444 fully inhibited the production of intracellular cAMP following incubation of the cells with 5'-N-ethylcarboxamidoadenosine (NECA), a stable analog of adenosine. A2AR agonists suppressed rapid TCR-mediated ERK phosphorylation and, subsequently, production of IL-2 and IFNγ by activated T cells; blockade of A2AR with CPI-444 restored T cell signaling and function.

The efficacy of CPI-444 was evaluated in MC38 and CT26 syngeneic mouse tumor models. In the MC38 model, daily treatment of mice with CPI-444 (1, 10, 100 mg/kg) led to a dose-dependent reduction in tumor growth, leading to full tumor elimination in 9/30 treated mice. New tumors failed to establish when the cured mice were re-challenged with MC38 cells, indicating that CPI-444 induced systemic anti-tumor immune memory. Combining CPI-444 with anti-PD-L1 mAb treatment in the MC38 model synergistically inhibited tumor growth and led to elimination of tumors in 9/10 treated mice. In the CT26 model, CPI-444 alone or anti-PD-1 alone led to non-significant reductions in tumor growth; however the combination of CPI-444 and anti-PD-1 led to a synergistic inhibition of tumor growth and prolonged survival compared to either agent alone. These results, and others, suggest that adenosine signaling may be an important resistance mechanism in tumors that incompletely respond to anti-PD-1/PD-L1 mAb therapy.

Based on these results and others, we plan to initiate a Phase 1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with anti-PD-L1 (atezolizumab) in patients with solid tumors.

#2338

CD39+ Treg cooperate with a CD73-expressing Th1/Th17 subset for Adenosine-mediated immunosuppression in human breast tumors.

Nicolas Gourdin,1 Céline Rodriguez,1 Selena Vigano,2 Isabelle Durand,1 Julien Faget,1 Camilla Jandus,2 Jean Yves Blay,1 Pedro Romero,2 Christine Menetrier-Caux,1 Christophe Caux1. 1 _CRCL Inserm U1052/CNRS5286, Lyon, France;_ 2 _Ludwig Cancer Research Center, Department of Oncology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland, Lausanne, Switzerland_.

Our group and others have previously reported that Treg infiltrating (Ti-Treg) breast tumors have a negative impact on patients' outcome. We further reported their selective recruitment in the tumor environment through CCL22 secretion and expansion upon ICOS engagement by plasmacytoïd dendritic cells.

Here, we investigated the mechanism of Ti-Treg mediated suppression and observed that Ti-Treg express high CD39 levels. CD39 is an ectonucleotidase that cooperates with CD73 to degrade the danger signal ATP into immunosuppressive Adenosine (Ado). The potency of Ado mediated suppression is illustrated in Adenosine-Deaminase (ADA)-deficient patients unable to degrade Ado and developing severe immunodeficiency.

We further show that, in contrast to murine Treg, human CD39+Treg do not express CD73 enabling them only to degrade ATP into AMP. Within T cells, CD73 expression was mainly associated with naïve CD8+ T cells and a subset of memory conventional CD4+ T cells (Tconv) that exhibit Th1/Th17 characteristics (CXCR3+CCR6+CCR4negIFNγhighIL17+). CD39+ Treg isolated from healthy donor blood, in presence of exogenous ATP, potently inhibit purified CD73+ but not CD73neg Tconv, proliferation and cytokine production (IFNγ, TNFα). By using enzymatic inhibitors, we demonstrated the involvement of CD39 and CD73 through Treg/Tconv cooperation for Ado mediated immunosuppression. Of importance, when integrated in [Treg-CD4+CD73+] high density coculture, CD4+CD73neg T cells expressing similar levels of Ado receptors (A2A, A2B) are also inhibited.

In conclusion, our findings support the existence of an Ado mediated immunosuppression loop in the tumor through cooperation between CD39highTreg and CD73-expressing Th1/Th17 subsets in the breast tumor environment.

The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200.

#2339

RNAi discovery platform to identify novel genes that prevent immune surveillance in pancreatic ductal adenocarcinoma (PDAC).

Antonio Sorrentino, Ayse Nur Menevse, Tillmann Michels, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Valentina Volpin, Sabrina Wagner, Rienk Offringa, Michael Boutros, Philipp Beckhove. _German Cancer Reseach Center (DKFZ), Heidelberg, Germany_.

BACKGROUND: Being one of the most treatment-resistant cancer types, pancreatic ductal adenocarcinoma (PDAC) is characterized by its ability to escape immune surveillance by developing many immunological obstacles. These include a plethora of mechanisms that either dampen immune cell functionality, or foster tumor cell resistance towards immune attack. Immunotherapeutic strategies, such as immune checkpoint blockade, have proven clinical success in many cancer entities, but showed little clinical benefit in PDAC patients, emphasizing the need to identify more key players that could radically improve immunotherapy.

AIM: We aim to systematically identify the whole arsenal of tumor-associated immune modulators by performing a high-throughput RNAi screen and subsequently validate novel therapeutic targets, whose blockade could potentially enhance anti-tumor immune response in PDAC patients.

METHODS: We generated a luciferase-expressing PANC-1 cell line and knocked down 2514 genes using a siRNA library. Our library includes G-protein coupled receptors, protein kinases and 1117 surface proteins. We co-cultured HLA-A201+ matched tumor infiltrating lymphocytes (TILs) derived from a PDAC patient with the transfected tumor cells. We then measured the remaining luciferase intensity of the tumor cells as an estimation of TIL-mediated cytotoxicity. In order to exclude genes whose knock-down affected cell viability per se, we cultivated tumor cells with the siRNA library in the absence of TILs.

RESULTS: We identified 155 candidate genes whose knock-down enhances TIL-mediated killing more efficiently than PD-L1 down-regulation. 35% of these genes are surface molecules and are most likely to directly mediate tumor immune evasion. Beside novel undescribed genes, our list contains well characterized immune modulators, supporting the reliability of our approach. Of note 13 of our hits were also found in a related melanoma screen and might play a role in the regulation of immune surveillance of different tumor entities. Among our candidates, 4 hits were chosen for further validation. We confirmed the expression of our selected candidates in several tumor cell lines and assessed the siRNA on-target effect using several non-overlapping siRNA sequences targeting the same hit. Transfection of PANC-1 with different siRNA sequences showed knock-down of the target gene as assessed via qPCR. Additionally we observed increased T-cell mediated killing as measured via luciferase-based killing assay and Chromium release assay.

CONCLUSION: We set up a robust and systematic method to unravel novel key players of pancreatic cancer immune surveillance. Further functional validation of our candidate genes will prove their potential to be used as relevant therapeutic targets in the clinic.

#2340

PD-1 blockade suppresses gastric cancer development by promoting antitumor immunity in mice.

Woosook Kim,1 Yoku Hayakawa,1 James G. Fox,2 Timothy C. Wang1. 1 _Columbia University, New York, NY;_ 2 _Massachusetts Institute of Technology, Cambridge, MA_.

Since advanced gastric cancer is difficult to cure with chemo and/or radiation therapy, new therapeutic approaches are clearly needed. Targeting immune checkpoints such as CTLA-4 and PD-1/PD-L1 has shown promising antitumor immunity in many malignancies by modulating T cell activity. PD-L1 and to a lesser extent of PD-L2 are expressed in human gastric cancer and expression of PD-L1 is associated with poor prognosis. Therefore, developing immunotherapies is an attractive strategy for the treatment of gastric cancer. We evaluated the efficacy of anti-PD-1 therapy in a mouse model of gastric cancer. Gastrin-deficient (GAS-KO) mice have been shown to develop large antral tumors with the combination of N-methyl-N-nitrosourea (MNU) and Helicobacter felis (H. felis) infection. Tumors developed in GAS-KO mice exhibited increased expression of PD-L1 and higher mutation rates than those in wild-type mice. To study the effect of anti-PD-1 antibody in tumor initiation or an early stage of progression, GAS-KO mice were induced with H.felis/MNU to develop tumors, one week later (10 weeks post-MNU) treated with anti-PD-1 antibody or isotype control (10 mg/kg IP qw) and sacrificed at 30 weeks post-MNU. We confirmed that GAS-KO mice treated with isotype control had moderate tumor burdens. Of interest, however, less and much smaller tumors were observed in mice treated with anti-PD-1. To compare immune cell populations in spleen tissue and peripheral blood from each group of mice, flow cytometric analysis was performed. Anti-PD-1-treated mice showed an increase in T cells including cytotoxic CD8+ T cells and a decrease in regulatory T cells and myeloid-derived suppressor cell (MDSC) populations compared with controls. In accordance with this, CD3-positive T lymphocytes were more frequently found in gastric tumors of anti-PD-1-treated mice than controls. Furthermore, qRT-PCR analysis revealed differential cytokine gene expression profiles including Il6, Il1b, and Il23 following anti-PD-1 treatment. Together, these data implicate that PD-1 blockade promotes immune infiltrates and therefore potentiates antitumor immune responses, suggesting targeting PD-1 as a potential new anticancer strategy in gastric cancer. Understanding anti-PD-1 therapy at the cellular and molecular level could provide new strategies for developing highly efficacious combination therapies.

#2341

Elevated immune activity following an anticancer combination therapy of a novel oncolytic immunotherapeutic agent, CAVATAK (Coxsackievirus A21), and immune checkpoint blockade.

Min Yuan Quah,1 Yvonne Wong,1 Robert Andtbacka,2 Gough Au,1 Darren R. Shafren1. 1 _Viralytics Limited, New Lambton, Australia;_ 2 _Huntsman Cancer Insitute, Salt Lake City, UT_.

Background: Coxsackievirus A21 (CAVATAKTM) is a bio-selected oncolytic immunotherapy virus. Following intravenous (i.v) infusion, CAVATAK preferential infects ICAM-1 expressing tumor cell, resulting in tumor cell lysis and a systemic immune-mediated anti-tumor response. A Phase I/II trial of i.v delivered CAVATAK (NCT01227551) in advanced cancer patients displayed signs of antitumor activity in some lesions. Blockade of programmed death protein-1 (PD-1) and or CTLA-4 in many advanced cancer patients has resulted in substantial tumor responses via a mechanism involving reversal of tumor induced T cell suppression and loosening the host "immunological handbrake". We investigated host immune activity in a B16-ICAM-1 melanoma immune competent mouse following a combination of intravenous delivered CAVATAK and PD-1 or CTLA-4 blockade.

Methods: Preclinical studies in C57BL mice were conducted to assess the antitumor activity of CAVATAK and anti-mouse PD-1 (mPD-1) mAb or anti-CTLA-4 (mCTLA-4) mAb in a B16-ICAM-1 melanoma immune competent mouse model. CAVATAK was administered i.v, while anti mPD-1 or mCTLA-4 mAbs were delivered via the intraperitoneal route. Following treatment of the primary cutaneous B16-ICAM-1 tumor with 4 cycles of CAVATAK injections and 4 cycles of anti-PD-1 or anti-CTLA-4 mAbs, mice were then re-challenged with an additional intradermal administration of B16 cells. Sequential serum samples were taken to monitor viral loads, inflammatory cytokines and anti-viral neutralising antibodies.

Results: Significant single agent antitumor activities against the primary B16-ICAM-1 tumors were observed in mice treated with either CAVATAK, anti-PD-1 or anti-CTLA-4 mAbs relative to saline controls. Interestingly, the depth of the anti-tumor activity appeared to be greater in treatments involving anti-CTLA-4 blockade. Surprisingly, levels of serum anti-CVA21 neutralising antibody were enhanced in both the anti-CTLA-4/ CVA21 and anti-CTLA-4/ anti-PD-1/CVA21 groups relative to mice treated with CVA21 alone of with anti-PD-1/CVA21. Despite the presence of higher levels of serum anti-CVA21 neutralising antibody, no reductions in anti-tumor activity from the combination therapy were observed in mice within the anti-CTLA-4 groups.

Conclusion: The significant anti-tumor activity mediated by the combination of CAVATAK and checkpoint inhibitor antibodies (anti-PD-1 and anti-CTLA-4) observed in the presented murine melanoma model supports clinical evaluation of such an immunotherapeutic combination treatment regime. Enhanced anti-CVA21 immune responses following immune-checkpoint blockade confirms the loosening of the host "immunological handbrake" and a general heightening of systemic immune surveillance.

#2342

NKG2A immune checkpoint blockade enhances the anti-tumor efficacy of PD1/PD-L1 inhibitors in a preclinical model.

Caroline Sola, Thomas Arnoux, Fabien Chanuc, Nicolas Fuseri, Benjamin Rossi, Laurent Gauthier, Corinne Leget, Cécile Bonnafous, Nicolai Wagtmann, Yannis Morel, Pascale André. _INNATE PHARMA, Marseilles, France_.

Monalizumab (IPH2201) is a novel, first-in-class humanized IgG4 targeting the immune checkpoint receptor NKG2A (Natural Killer Group 2A). NKG2A is expressed as a heterodimer with CD94 on the surface of subsets of cytotoxic lymphocytes: NK (Natural Killer) cells, γδ T cells and tumor infiltrating CD8 T lymphocytes. CD94/NKG2A is an inhibitory receptor that binds to HLA-E (Human Leukocyte Antigen-E) in humans and orthologous Qa-1b in mice. Upon ligand binding, CD94/NKG2A triggers inhibitory signaling that reduces NK and CD8 T cell responses. HLA-E is frequently up-regulated on cancer cells of many solid tumors or hematological malignancies, protecting from killing by NKG2A+ immune cells. By blocking the binding of CD94/NKG2A to HLA-E, monalizumab leads to enhancement of NK and cytotoxic T cell responses.

The immune checkpoint receptor programmed cell death 1 (PD-1) is another inhibitory receptor widely upregulated by tumor-infiltrating T lymphocytes (TILs). In many tumor types immune surveillance is hampered by the expression of PD-L1, one of the ligands of PD-1. Blocking the PD-1 pathway has proven efficient as anti-tumor therapy. Nevertheless many patients remain refractory to these therapeutics. Combination treatment with PD-1 blockers and mAb to a second checkpoint receptor, CTLA-4, have proven effective only for some patients, suggesting a need for combining with other checkpoint blockers.

The A20 cell line is a mouse B cell lymphoma line that expresses PD-L1, but not Qa-1b. Upon subcutaneous injection into Balb/c mice, A20 formed solid tumors in which PD-L1 expression was retained and where Qa-1b expression was induced. A20 tumor growth was controlled by NK and CD8 T cells. In spite of high expression of PD-1 on many immune infiltrating cells, and high expression of PD-L1 on tumor cells, monotherapy with anti-PD-1 or -PD-L1 mAb resulted in only moderate reduction in tumor growth.

Interestingly, more than 50% of A20 tumor infiltrating NK cells and about 10% of CD8 T cells expressed CD94/NKG2A. The NKG2A+ CD8 T cell population also co-expressed PD-1. Qa-1b expression was induced not only on the surface of tumor cells but also on infiltrating immune cells in vivo. Consistent with the hypothesis that Qa-1b protects tumor cells from killing by NKG2A+ effector NK and T cells, treatment with an antibody that blocks NKG2A significantly delayed A20 tumor growth. Mice were then treated with both anti-NKG2A and anti-PD-1 or -PD-L1 in combination. These combinations resulted in significantly higher anti-tumor responses compared to monotherapies, characterized by an increased frequency of complete tumor cell regression.

Together, these data indicate that blocking NKG2A in conjunction with PD-1/PD-L1 checkpoint inhibitors could provide synergistic anti-tumor efficacy and support the rationale for investigating this combination in clinical trials.

#2343

The immune modulatory roles of IAP inhibitor, LCL161, and its connection to immune-checkpoint molecules.

Maria Pinzon-Ortiz,1 William Hastings,1 Tyler Longmire,1 Pamela Shaw,1 Xianhui Rong,1 Masato Murakami,2 Benjamin H. Lee,1 Glenn Dranoff,1 Kenzie MacIsaac,1 Z. Alexander Cao1. 1 _Novartis, Cambridge, MA;_ 2 _Novartis, Basel, Switzerland_.

Tumor immunotherapy is a unique therapeutic modality in our fight against human cancers. The recent success of immune-checkpoint therapies highlights the value and potential of this approach. Previous studies have demonstrated positive immune-modulatory activities of the IAP inhibitor, NVP-LCL161. In this study, we sought to further explore the immune-modulatory activities of NVP-LCL161 in connection with immune-checkpoint molecules.

In PBMC stimulation assays, NVP-LCL161 potently induced IFN and enhanced proliferation, while down-modulating IL-10 production. Additionally, the role of NVP-LCL161 on immune-checkpoint modulation was revealed in CyTOF analysis of in vitro stimulated PBMCs. NVP-LCL161 treatment led to increases in TIM-3 expression on multiple T cell subsets, and on cells of the myeloid lineage. These observations illustrate potential modulation of specific immune-checkpoint molecule by NVP-LCL161, thus suggesting possible synergy between TIM-3 immunecheckpoint blockade and NVP-LCL161. NVP-LCL161 was further combined in vivo with anti-PD1 antibody in the murine syngeneic tumor model, MC38. At multiple dosing schedules, synergy was observed with this combination, which appeared to be independent of single agent efficacy. Expression profiling of MC38 tumor tissues by a customized Nanostring panel revealed elevation of gene signatures in T cells, dendritic cells, cytokines and chemokines, amongst others.

In summary, the preclinical data support the hypothesis that NVP-LCL161 is an active immune modulator. Its combination activity with immune_checkpoint inhibitors would warrant further exploration in both preclinical and clinical setting.

#2344

Discovery and characterization of new original blocking antibodies targeting the CD73 immune checkpoint for cancer immunotherapy.

Marc Giraudon Paoli,1 Severine Augier,1 Marilyne Royannez Blemont,1 Céline Rodriguez,2 Hélène Rispaud Blanc,1 Stéphanie Chanteux,1 Nicolas Gourdin,2 Laurent Gauthier,1 Christine Ménétrier Caux,2 Yannis Morel,1 Christophe Caux,1 Carine Paturel,1 Ivan Perrot1. 1 _Innate Pharma, Marseille, France;_ 2 _Centre Léon Bérard, Lyon, France_.

CD73 (NT5E) is a cell membrane ectoenzyme of the NTPDase family that plays a major role in the conversion of AMP into Adenosine (Ado). Within the tumor microenvironment, accumulation of Ado causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. CD73 expression in the tumor environment has been associated with poor disease outcome and/or with a pro-metastatic phenotype. Thus, targeting CD73 may promote anti-tumor immunity by reducing Ado accumulation and may block tumor cell metastasis by inhibiting CD73 on tumor cells. Here, we describe the generation and characterization of novel anti-human CD73 antibodies, intended for the treatment of a wide range of cancers. The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200. Antibodies were discovered that inhibited CD73 function by different mechanisms, including the direct blocking of CD73 enzymatic activity or the down-modulation of membrane CD73 expression. Epitope mapping revealed that antibodies acting by these different modes of action bound to distinct sites on CD73. All selected antibodies cross-react with cynomolgus CD73 protein and have strong avidity and affinity for membrane or recombinant CD73, by flow cytometry and Surface Plasmon Resonance, respectively. Antibodies that inhibit CD73 enzymatic activity strongly reduce AMP catabolism by both recombinant and cellular CD73 with IC50 in the nanomolar range. They also efficiently reverse ATP- and AMP-mediated T cell suppression in in vitro assays in presence of both CD39+ and CD73+ cells. The antibodies that induce down-modulation of cellular CD73 expression do not block recombinant CD73 enzyme activity and partially inhibit cellular CD73 activity; they reverse ATP- but not AMP-dependent T cell suppression. The antibodies displaying the most interesting features were humanized. Evaluation of their activity in animal models is ongoing.

#2345

SIRPαFc, a CD47-blocking cancer immunotherapeutic, triggers phagocytosis of lymphoma cells by both classically (M1) and alternatively (M2) activated macrophages.

Gloria H. Y. Lin, Vien Chai, Vivian Lee, Karen Dodge, Tran Truong, Mark Wong, Lisa D. S. Johnson, Xinli Pang, Penka S. Petrova, Robert A. Uger, Natasja N. Viller. _Trillium Therapeutics Inc., Toronto, Ontario, Canada_.

Macrophages are characterized by their heterogeneity and plasticity in response to the microenvironment. Although macrophages have the capacity to phagocytose cancer cells that express pro-phagocytic signals, tumor cells often evade macrophage-mediated destruction by increased cell surface expression of CD47, which delivers an anti-phagocytic ("do not eat") signal by binding the inhibitory signal-regulatory protein α (SIRPα) receptor on the surface of macrophages. We have previously shown that soluble SIRPα-Fc fusion protein (SIRPαFc) neutralizes the suppressive effects of CD47 and promotes macrophage-mediated phagocytosis of tumor cells both in vitro and in vivo. In an attempt to recapitulate the functional and phenotypic heterogeneity of tumor infiltrating macrophages, we have examined the ability of SIRPαFc to trigger phagocytosis of lymphoma cells by six distinctly polarized macrophage populations.

We generated human monocyte-derived macrophages (MDMs) and polarized them into M0, M1, M2a, M2b and M2c subsets. These MDMs varied greatly in their expression of myeloid surface markers including CD14, CD11b, CD80, CD86, HLA-DR, CD206, CD200R and CD163; as well as in expression of the Fc gamma receptors (FcγRs) CD16, CD32 and CD64. Next, the ability of SIRPαFc to trigger MDM phagocytosis of lymphoma cells was examined using a flow cytometry-based assay. Blockade of CD47 on the tumor cells using SIRPαFc dramatically increased phagocytosis of tumor cells by all subsets, with M1 and M2c MDMs being superior at phagocytosis. Moreover, we found that M0, M2a and M2b MDMs, which exhibited slightly lower phagocytic capabilities, were remarkably plastic in nature and could readily be re-polarized into highly phagocytic MDMs using a variety of agents. This suggests that SIRPαFc will be efficacious in triggering the destruction of cancer cells by the diverse population of MDMs found in vivo.

To further understand what drives the phagocytic capacity of polarized MDMs, we analyzed FcγR expression and observed a positive correlation between MDM expression of the high-affinity FcγRI (CD64) and phagocytic activity following SIRPαFc treatment. Moreover, re-polarization of M0, M2a and M2b MDMs resulted in upregulation of FcγRs and enhanced tumor cell phagocytosis. Finally, by individually blocking CD16, CD32 and CD64 on MDMs prior to the phagocytosis assay, we found that the low-affinity FcγRs CD16 and CD32 also contribute to SIRPαFc-mediated phagocytosis of lymphoma cells.

In conclusion, SIRPαFc triggered phagocytosis of lymphoma cells by a diverse panel of polarized MDMs, which required MDM expression of FcγRs. These data support the evaluation of SIRPαFc in cancer patients and a clinical study of SIRPαFc in patients with lymphoma and other hematological malignancies is currently in progress. 

### Therapeutic Antibodies and Vaccines

#2346

**Intratumoral administration of OncoVEX** mGM-CSF **results in local innate immune alterations and induces systemic anti-tumor effects in syngeneic mouse tumor models.**

Keegan Cooke,1 Karen Fitzgerald,2 Becky Yang,2 Juan Estrada,1 Brian Belmontes,1 Pedro Beltran,1 Achim K. Moesta2. 1 _Amgen, Inc., Thousand Oaks, CA;_ 2 _Amgen, Inc., South San Francisco, CA_.

Talimogene laherparepvec, is a modified oncolytic herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors, with lytic cell destruction promoting the local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally and is designed to recruit and stimulate antigen presenting cells to further enhance systemic antitumor immune response.

While available evidence suggests that talimogene laherparepvec can induce effects in distant tumors via an immune-mediated effect, additional mechanistic evidence is being sought to help better understand the putative systemic effect. For preclinical mechanism of action studies we employed OncoVEXmGM-CSF, an HSV-1 virus modified in the same manner as talimogene laherparepvec except that murine GM-CSF is expressed. We utilized this virus to further dissect the underlying innate and adaptive immunity following viral administration in syngeneic tumor models.

Intratumoral administration of OncoVEXmGM-CSF into established murine tumors induced regressions in the injected lesion but also resulted in significant anti-tumor effects in contralateral (uninjected) lesions. We have previously demonstrated by immunohistochemistry that the systemic effects observed in the contralateral lesions are not driven by systemic spread of the virus, but instead correlate with the infiltration of CD3+ T cell populations. Here we report the full phenotypic characterization of immune infiltrates by flow cytometry in both the injected and contralateral lesions. In a time-course fashion, this characterization revealed the initial infiltration of the injected tumor by innate effector cells and a concomitant induction of a potent type I interferon response. This in turn drives the local release of proinflammatory cytokines and chemokines, resulting in the recruitment of adaptive immune subsets to the injected tumor and enhancing systemic anti-tumor reactivity. Additionally, both the primary oncolytic replication of the virus and the activation of type I interferon pathway are directly correlated with STING activity.

In conclusion, in vitro and in vivo observations suggest that intratumoral injection of OncoVEXmGM-CSF activates multiple immune-mediated mechanisms of action leading to T-cell activation and induction of anti-tumor immunity in mice.

#2347

The amphiphatic β(2,2)-amino acid derivative, LTX-401 induces complete regression of experimental hepatocellular carcinomas.

Brynjar Mauseth,1 Ji-Hua Shi,2 Ketil Camilio,1 Øystein Rekdal,3 Baldur Sveinbjørnsson,1 Pål-Dag Line4. 1 _Department of Medical Biology, University of Tromsø, TROMSØ, Norway;_ 2 _Division of Cancer, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway;_ 3 _Lytix Biopharma, Oslo, Norway;_ 4 _Institute of Pathology, Oslo University Hospital, Oslo, Norway_.

Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and third cause of cancer-related deaths. Because HCC is a relatively chemotherapy-resistant cancer, and many patients are restricted to surgical resection or liver transplantation, there is an urgent need to develop new and improved therapeutic regiments. In the present study, we have investigated the therapeutic effect of a novel oncolytic compound, LTX-401, designed for the local treatment of solid malignancies. In vitro analysis revealed that LTX-401 displayed potent anticancer activity against a broad variety of cancer cell lines. In our model cell line (JM1 HCC), LTX-401 was found to induce necrotic (lytic) cell death followed by the release of potent immune stimulants in the form of Danger-Associated Molecular Pattern molecules (DAMPs) such as HMGB1 and ATP. Ultrastructural analysis by transmission electron microscopy also revealed that a substantial portion of LTX-401-treated JM1 cells contained significantly affected mitochondria. Additionally, cytochrome c was detected in cell supernatants after treatment. The anticancer effect of LTX-401 was investigated in a rat hepatocellular carcinoma model by inoculating JM1 cells into the liver (intrahepatic) of syngeneic Fischer 344 rats. LTX-401 induced a complete regression of HCC tumors by intratumoral injection. Tumor-specific protective immune responses were obtained in previously cured animals, as re-challenge with live and equivalent tumor cells did not lead to tumor growth. Furthermore, intratumoral treatment with LTX-401 was able to eradicate diffuse distant untreated metastasis in the liver. Immuno-histological analysis showed T-cell infiltration into treated tumors. Taken together, intrahepatic treatment with LTX-401 can induce complete regression and long lasting protective tumor-specific immune responses.

#2348

Nanovaccine : A novel immunotherapeutic strategy to treat bladder cancer.

Hyunjoon Kim, Lin Niu, Peter Larson, Katherine Murphy, Tamara Kucaba, David Ferguson, Thomas Griffith, Jayanth Panyam. _University of Minnesota, Minneapolis, MN_.

Introduction Bladder cancer is a significant healthcare problem. According to the National Cancer Institute, bladder cancer is the 6th most common type of cancer and represents 4.5% of total cancer patients in the U.S. Intravesical therapy with Bacillus Calmette-Guerin (BCG) vaccine is the gold standard for treating non-muscle invasive bladder cancer. However, BCG has several drawbacks. First, BCG is a tuberculosis vaccine, which often fails to trigger immune response against bladder cancer. This leads to low therapeutic efficacy and high recurrence rates. Secondly, intravesical injection is painful and causes urinary side effects, including burning sensation and increased frequency of urination. Therefore, a therapeutic regimen that can specifically treat bladder cancer and can be administered through other routes is highly needed. Immunotherapeutic strategy using nanoparticle can fulfill these needs. Polymeric nanoparticles encapsulating an immune adjuvant and co-injected with a tumor-associated antigen can trigger robust immune response against bladder cancer. Moreover, nanoparticles formulated using the FDA approved polymer poly (lactide-co-glycolide) (PLGA) is approved for human use and can be injected subcutaneously.

Methods Nanoparticles encapsulating a novel imidazoquinoline derivative that activates both Toll-Like Receptor (TLR) 7 and 8 was used as vaccine adjuvants. These novel nanoparticle-encapsulated TLR agonists were used in combination with a model antigen, ovalbumin, to create the vaccine. This is referred to as nanovaccine in this study. C57BL/6 mice were vaccinated subcutaneously with 1-5 doses of the nanovaccine or various control treatments. Two days after the final vaccination dose, mice were sacrificed and organs were harvested. Splenocytes and lymphocytes were analyzed using flow cytometry.

Results Nanovaccine improved dendritic cell activation in the lymph node. Frequency of co-stimulatory molecule CD80+ dendritic cells was two fold higher in the lymph node of vaccinated group. Greater activation of T cells and increased frequency of antigen-specific T cells were found in the spleen of vaccinated group. Antigen-specific CD44+ CD8 Tcell frequency was two fold higher in the vaccinated group. Nanovaccine elevated the frequency of both antigen-specific CD4 T cells and antigen-specific CD44+ CD4 T cells by ten fold.

Conclusion Nanovaccine generated antigen-specific immune response that can maximize the efficacy of immunotherapy. Moreover, nanoparticles were physically stable and remained active after subcutaneous injection, which can overcome the administration difficulties of current BCG therapy. Next step is to generate nanovaccine with bladder tumor-specific antigen or using whole cell lysate and test the therapeutic efficacy in tumor bearing mice. Generation of a bladder cancer-specific immune response will allow stronger and durable inhibition of tumor growth and diminished risk of progression.

#2349

Evaluation of the combination of the prodrug-mediated gene therapy vector AdV-tk and immune checkpoint inhibitor for glioblastoma treatment in a syngeneic mouse model.

Maria Carmela Speranza,1 Kazue Kasai,1 Johanna Kaufmann,1 Estuardo Aguilar-Cordova,2 Brian W. Guzik,2 E. Antonio Chiocca,1 Sean Lawler1. 1 _Harvard Medical School - Brigham and Women's Hospital, Boston, MA;_ 2 _Advantagene Inc., Auburndale, MA_.

There has been a recent surge of interest in cancer immunotherapies due to newly FDA approved immunologic treatments based on targeting immune checkpoint inhibition pathways. Despite the dramatic responses in animal models and early clinical human trials, particularly in melanoma, durable clinical responses are observed only in approximately 30% of patients, therefore combinatorial immune therapeutic approaches may be needed to improve outcomes further. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint (ICI) response, the combination of these two approaches offers a potentially advantageous interaction. AdV-tk is an immunostimulatory virus-based approach that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (AdV-tk) followed by administration of an anti-herpetic prodrug (ganciclovir - GCV). This approach has shown benefit in clinical trials of glioblastoma among other tumor types. The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the thymidine kinase protein (TK). This approach has consistently demonstrated anti-tumor immune stimulation and an increased intra-tumoral CD8+ T-cell infiltrate. Not surprisingly, however, this immune stimulation also leads to increased expression of immune checkpoint inhibitory ligands on tumor cells, including PD-L1. Our initial in vitro studies showed that the immune checkpoint ligand PD-L1 is expressed across a panel of human and mouse conventional and stem-like glioblastoma-derived cells (GSCs). After AdV-tk infection and GCV treatment PD-L1 is consistently up-regulated both at protein and mRNA levels, while no changes were detected after AdV-tk alone. In vivo experiments in immunocompetent mouse models with GL261 glioma cell lines demonstrate a significant improvement in survival when we combine AdV-tk+GCV and anti-PD-1 antibodies. This data suggests that combination of immune checkpoint blockade with AdV-tk treatment should be explored in glioblastoma clinical trials.

#2350

Vaccination enhances anti-tumor immunity in ovarian cancer following repolarization of the tumor microenvironment with CCR2 blockade.

Darren Cullinan,1 Pratibha Binder,1 Timothy Nywening,1 Ivy Wilkinson-Ryan,2 Brian Belt,3 Peter Goedegebuure,1 David Linehan,3 Matthew Powell,1 William Hawkins1. 1 _Washington University, Saint Louis, MO;_ 2 _Darmoth-Hitchcock, Lebanon, NH;_ 3 _University of Rochester, Rochester, NY_.

Introduction: Ovarian cancer (OC) expresses the tumor associated antigen mesothelin and contains a relative abundance of T-cells. However, ovarian cancer is also infiltrated by immunosuppressive tumor associated macrophages (TAM) that dominate the tumor microenvironment (TME). The CCL2/CCR2 chemokine axis is co-opted by various human malignancies to facilitate the recruitment of bone marrow (BM) derived inflammatory monocytes (IM) to the TME where they become immunosuppressive TAMs. Herein, we explore the rationale for combination of a CCR2 inhibitor (CCR2i) with a mesothelin peptide vaccine.

Methods: Monocyte counts were obtained from preoperative CBCs under IRB approval. Mice were vaccinated with a dual eight-mer peptide (50 nM/vaccination) on days 0 and 7 with an irradiated peptide pulsed dendritic cell boost on day 14. Mice were challenged with 4 million syngeneic OC cells (ID8) on day 15. CCR2 inhibitor (Tocris) and CCR2 KO mice were used.

Results: Preoperative monocyte counts of human ovarian cancer patients were stratified into low (>1 SD below mean), mid (within 1 SD of mean), and high (>1 SD above the mean) groups. Patients with a high monocyte count (n=15) had a significantly decreased median survival of 1.2 years compared to 4.8 years in the low monocyte group (n=15). The mid group (n=69) had a median survival of 3.5 years (p 0.001). The hazard ratio between the low and high groups was 0.24 (0.05-0.39). Flow cytometry of peripheral blood from these patients demonstrated that the majority of these monocytes were CCR2+ inflammatory monocytes. Human OC overexpresses CCL2 compared to normal ovarian tissue and analysis of the TME from resected human OC patients revealed an abundance of CCR2+ TAM, which greatly outnumbered tumor infiltrating lymphocytes (TIL). In a murine ID8 tumor model, which recapitulates features of human OC, CCR2i prevented IM egress from the bone marrow with a resultant decrease in TAM at the primary tumor site. Furthermore, there was an increase in TIL infiltrate following CCR2 blockade. Addition of vaccine to CCR2i caused an improvement of effector to suppressor ratio and prolonged survival compared to vaccine (p=0.02) or CCR2i alone (p=0.02) and control (p<0.0001).

Conclusion: Thus far vaccination has not provided durable patient responses in OC. Therapies targeting the immunosuppressive TME are an attractive treatment modality to enhance vaccination and facilitate anti-tumor immunity in OC.

#2351

Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models.

Keegan Cooke, Juan Estrada, Jinghui Zhan, Petia Mitchell, Yannick Bulliard, Pedro J. Beltran. _Amgen, Thousand Oaks, CA_.

Background:

Talimogene laherparepvec is an oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1), designed to kill neoplastic cells through two mechanisms: a) direct lysis and b) stimulation of a tumor antigen-specific adaptive immune response by way of GM-CSF expression. In order to support mechanism of action studies in a mouse melanoma model relevant to talimogene laherparepvec's approved U.S. indication, we expressed the HSV-1 entry receptor Nectin1 on B16F10 melanoma cells. B16F10 cells have previously been shown to be resistant to HSV-1 due to lack of entry receptor expression (Miller et al., Mol Ther, 2001). The goal of our work was to determine if mNectin1 expression in B16F10 melanoma cells renders them sensitive to talimogene laherparepvec treatment. For preclinical studies we used OncoVEXmGM-CSF, an HSV-1 modified similarly to talimogene laherparepvec except that murine GM-CSF is expressed instead of human GM-CSF. Herein we describe studies to assess the efficacy of OncoVEXmGM-CSF in a novel B16F10-mNectin1 syngeneic melanoma model.

Methods:

Parental B16F10 cells were transduced with lentivirus expressing either mNectin1, or eGFP as a control. In vitro sensitivity was determined by plating both cell types in 96-well plates, infecting with serially diluted OncoVEXmGM-CSF and then measuring ATP 72 hours later. For efficacy studies, B16F10 or B16F10-mNectin1 cells were injected subcutaneously on the right flank of female C57BL/6 mice. On day 10, mice were randomized into treatment groups based on tumor volume (n=10/group). Talimogene laherparepvec was delivered intratumorally (5x106 PFU/dose,) every 3 days during the first week. Tumor growth and body weight were measured twice per week throughout the experiment. Survival analysis was performed using a Kaplan-Meier estimator.

Results:

B16F10 cells were insensitive to OncoVEXmGM-CSF up to 100 MOI (multiplicity of infection). In contrast, two other syngeneic cell lines, A20 and CT-26, showed MOI IC50 of 1 and 20. B16F10 cells expressing mNectin1 showed high sensitivity to OncoVEXmGM-CSF with an MOI IC50 of 0.01. In contrast, B16F10 cells expressing eGFP were insensitive up to an MOI of 100. In vivo, B16F10 mNectin1 cells showed a similar growth pattern as the parental B16F10 when injected subcutaneously. However, OncoVEXmGM-CSF treatment of B16F10-mNectin1 tumors caused a highly significant (p<0.0001) inhibition of tumor growth and increase in median overall survival compared to the control group. B16F10 mNectin1 tumors that grew during or following OncoVEXmGM-CSF treatment were shown to maintain mNectin1 expression suggesting other mechanisms of OncoVEXmGM-CSF resistance were involved.

Conclusion:

Expression of mNectin 1 in the B16F10 melanoma cell line confers sensitivity to OncoVEXmGM-CSF in vitro and extends median overall survival of mice bearing B16F10-muNectin1 tumors.

#2352

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allow their intratumoral delivery and an improved tumor-growth inhibition.

Jean-Baptiste Marchand,1 Patricia Kleinpeter,1 Laetitia Fend,1 Christine Thioudellet,1 Michel Geist,1 Nathalie Sfrontato,1 Véronique Koerper,1 Renée Brandely,1 Dominique Villeval,1 Karola Rittner,1 Nathalie Silvestre,1 Philippe Erbs,1 Laurence Zitvogel,2 Eric Quemeneur,1 Xavier Preville1. 1 _Transgene, Illkirch-Graffenstaden, France;_ 2 _Institut Gustave Roussy, Villejuif, France_.

We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted in the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The 3 purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e. without tumor) confirming the virus tropism for tumoral cells and/or that tumoral microenvironment allows virus escape from immune surveillance. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mPD-1, than after IT administration of 10 µg of J43. Interestingly, in the MCA 205 tumor model, WR-mPD-1 (both mAb and scFv) induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that provided by an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. New generation of oncolytic vaccinia virus that will express several transgenes simultaneously may also be designed with the goal of providing to the patients enhanced therapeutic/toxicity ratio.

#2353

Combination immunotherapy: T-cell costimulation (OX40L, TL1A, 4-1BBL and ICOSL) secreted locally by Gp96-Ig vaccines, elicits robust antigen-specific, memory T cell responses and tumor elimination.

George J. Fromm, Suresh de Silva, Louise Giffin, Jason Rose, Aditi Goyal, Taylor H. Schreiber. _Heat Biologics, Inc., Durham, NC_.

Combination cancer immunotherapy incorporating T cell costimulation, vaccination, and checkpoint inhibition is anticipated to broaden clinical response compared to any single agent. Because T cell costimulation occurs at the site of immunization, we asked whether the delivery of a costimulator by a gp96-Ig secreting allogeneic vaccine would provide comparable costimulation to systemically administered agonist antibodies. As proof of concept, we engineered gp96-Ig vaccines that locally secrete Fc-OX40L and demonstrate that the priming of antigen-specific CD8+ T cells (peak of 13.3% of total CD8+) is significantly higher when compared to combinations with OX40 antibodies (8.4%) or vaccine alone (5.6%). Vaccine-expressed Fc-OX40L was associated with increased CD127+KLRG-1- memory precursor cells and antigen-specific CD4+ proliferation, with reduced off-target inflammation. Importantly, vaccine-expressed Fc-OX40L stimulated IFNγ+, TNFα+, granzyme-b+ and IL-2+ by antigen-specific CD8+ T cells, which enhanced rejection of established CT26 and B16.F10 tumors. We have subsequently expanded our repertoire of combination vaccines to secrete gp96-Ig along with either Fc-tagged TL1A, 4-1BBL or ICOSL. Each costimulator secreting vaccine cell line has a unique functionality, without the negative consequences of off-target inflammation associated with systemic administration of its agonist antibody counterpart. For example, costimulation with a TNFRSF25 (receptor for TL1A) agonist antibody synergized with gp96-Ig vaccination and generated a robust antigen-specific CD8+ T cell response. However, TNFRSF25 treatment also lead to a significant accumulation of FOXP3+ regulatory T cells (Treg). A gp96-Ig vaccine co-secreting Fc-TL1A resulted in similar antigen-specific CD8+ T cell production with no activation of the Treg compartment. Additionally, combining two separate gp96-Ig vaccines (one secreting Fc-OX40L and the other Fc-TL1A) leads to an additive increase in memory precursor production (CD127+KLRG1-), which may play a vital role in maintaining effective anti-tumor immunity. Together, we demonstrate that the magnitude and specificity of vaccination can be enhanced by locally secreted costimulatory molecules when delivered within a single product. This may simplify clinical translation and importantly, provide significant patient benefit by improving safety and lowering costs.

#2354

Cancer vaccine development for hepatocellular carcinoma - HEPAVAC.

Sarah Kutscher,1 Roberto Accolla,2 Yuk T. Ma,3 Regina Heidenreich,4 Francesco Izzo,5 Alfred Koenigsrainer,6 Markus Loeffler,6 Phillip Mueller,1 Andrea Mayer,1 Hans-Georg Rammensee,6 Bruno Sangro,7 Sven Francque,8 Danila Valmori,9 Toni Weinschenk,1 Harpreet Singh-Jasuja,1 Luigi Buonaguro5. 1 _Immatics Biotechnologies GmbH, Tubingen, Germany;_ 2 _University of Insubria, VARESE, Italy;_ 3 _University of Birmingham, Birmingham, United Kingdom;_ 4 _CureVac AG, Tubingen, Germany;_ 5 _National Cancer Institute, NAPLES, Italy;_ 6 _University of Tubingen, Tubingen, Germany;_ 7 _University of Navarra, PAMPLONA, Spain;_ 8 _University of Antwerp, ANTWERP, Belgium;_ 9 _INSERM, NANTES, France_.

The HEPAVAC Consortium aims to develop a highly innovative, novel cancer vaccine approach for hepatocellular carcinoma (HCC). The international project consortium consists of 9 European Partners from academia and the biotech industry with complementary and substantial expertise in developing immunotherapeutic strategies to treat cancer. The project has started in September 2013 and is supported by the European Commission's 7th Framework Program (www.hepavac.eu).

HCC/normal adjacent tissue matched samples have been collected for HLA immunopeptidome analysis. 17 HCC samples from HLA-A*02+ patients and 15 samples from HLA-A*24+ patients have been analysed by mass spectrometry (LC-MS/MS). RNA-expression profiles have been established for 12 HCC samples. HLA-presentation/expression of peptides and mRNA on primary HCC samples are compared to >140 normal tissue samples from relevant organs (including heart, brain, lung, kidney, liver, nerve, skin etc.) from Immatics' database.

A total of 9051 HLA-A*02-restricted different tumor-associated peptides (TUMAPs) have been identified from HLA-A*02+ samples, while a total of 3286 different HLA-A*24-restricted TUMAPs have been identified from HLA-A*24+ samples. Of these, 33 HLA-A*02+ TUMAPS and 33 HLA-A*24+ TUMAPs have been validated, including peptide synthesis, immunogenicity testing and pharmaceutical evaluation. Most promising TUMAP candidates show selective expression only in HCC samples and no expression in normal tissues. In parallel, more than 6600 HLA-DR TUMAPs have been identified in Hep3B cells transfected with CIITA as well as an average of 1500 HLA-DR TUMAPs have been identified in HCC samples.

A total of 16 newly identified HCC-specific epitopes have been selected for the HEPAVAC vaccine cocktail and are currently synthesized according to GMP standard. Of these, 7 are restricted to HLA- class I A*02; 5 HLA-A*24 and 4 HLA class II. In parallel, preclinical studies assessing the formulation and combination of the immunological RNA-based adjuvant (RNAdjuvant®) with peptide cocktails are underway. A multi Center phase I/II clinical trial in early HCC patients is predicted to start in July 2016.

#2355

Strong immune responses to pancreatic cancer cells induced by human tumor lysate vaccine remodeled to express α-gal epitopes and their implication for a universal vaccine.

Kenta Furukawa,1 Masahiro Tanemura,1 Eiji Miyoshi,2 Tomoki Hata,2 Kentaro Kishi,1 Hiroki Akamatsu,1 Hidetoshi Eguchi,2 Masaki Mori,2 Yuichiro Doki2. 1 _Osaka Police Hospital, Osaka, Japan;_ 2 _Osaka University Graduate School of Medicine, Japan_.

Pancreatic cancer (PC) is a lethal disease that remains one of the most resistant to traditional therapies. We are in urgent need of other therapies. Immunotherapy designed to target tumor-associated antigens (TAAs) is a promising treatment approach for PC. But vaccination against a single TAA seems to be insufficient. In our previous study, we showed effectiveness vaccination by whole PC cells, engineered to express α-gal epitopes (Cancer Res, 2010). Subsequently we investigated strong immune response of tumor lysate vaccine engineered to express α-gal epitopes (Int J Oncol, 2015). In this study, for developing clinical application of this immunotherapy, we investigated the effects of vaccination with human PC tumor lysate obtained from PC patients, expressing α-gal epitopes.

Tumor specimens were obtained from 11 PC patients at the time of surgical resection. To express α-gal epitopes, we cloned the α1,3galactosyltransferas (α1,3GT) from a New World Monkey and expressed it in a soluble form in the yeast expression system of Pichia pastoris. α1,3GT KO mice were immunized with pig tissue to generate anti-Gal Ab in their sera. These high anti-Gal KO mice were vaccinated intraperitoneally by either unsynthesized (control group; group C) or α-gal tumor lysate (α-gal group; group A).

Vaccination with α-gal PC tumor lysate elicited strong immune responses of anti-PC cell IgG and anti-PC TAAs, including MUC1 and Mesothelin. Productions of anti-PC cell IgG in group A were 8~32-fold higher than those of group C. Furthermore, productions of anti-MUC1 and of ant-Mesothelin Ab in group A were 4~8-fold higher than those of group C. Expansion of TAAs (MUC1 and Mesothelin)-specific B cells was significantly higher [MUC1: spots of group A vs. C= 151 vs. 28 (p<0.001), Mesothelin: 97 vs. 36 (p=0.03)]. The number of spots of IFN-γ secreting T cells in the presence of MUC1 and Mesothelin peptide stimulation was significantly higher in group A than in group C [MUC1: 828 vs. 146 (p<0.001), Mesothelin: 988 vs. 384 (p=0.02)].

To demonstrate in vivo tumor destruction, an animal experiment was performed. Splenocytes from vaccinated KO mice were prepared, and then transferred intraperitoneally into NOD/SCID mice. Followed by transferring, mice were challenged with 1×107 of live PANC-1 cells. In mice from group A, regrowth of tumors was significantly prevented and survival period was significantly prolonged [group A vs. C= 95.0 vs. 45.0 days (p<0.001)]. In the immunohistochemical analysis, the tumor created in NOD/SCID mice from group A was shown severe central necrosis and strong infiltration of CD4+ and CD8+ T cells and macrophages into the periphery. In contrast, the tumor obtained from NOD/SCID mice of group C indicated no lymphocytic infiltration.

We conclude that the use of human PC tumor lysate vaccine, remodeled to express α-gal epitopes can elicited strong immune responses to pancreatic cancer.

#2356

**Erythrocytes used as tumor antigen delivery system to target antigen-presenting cells embody an innovative approach for** in situ **cancer immunotherapy.**

Magali Cremel, Nathalie Guerin, Quitterie Barthe, Vanessa Bourgeaux, Willy Berlier, Françoise Horand, Yann Godfrin. _Erytech Pharma, Lyon, France_.

Introduction: In current vaccine therapy, efficient delivery of tumor antigens (TA) to antigen-presenting cells (APC) represents a major step toward the development of strong TA-specific immune response including cytotoxic T lymphocyte (CTL) induction. Our innovative approach is to use the property of erythrocytes to be naturally phagocytosed by APC when senescent. Indeed, TA can be encapsulated into erythrocytes that will be used as carrier to specifically deliver the TA to APC, which will ensure their degradation and presentation to T cells.

Materials and Methods: The proof of concept was established with Ovalbumin (OVA), tyrosinase-related protein 2 (TRP2 (melanoma TA) and prostate specific antigen (PSA, prostate TA). These TA were encapsulated into erythrocytes by a hypotonic lysis process. To stimulate the erythrophagocytosis by APC, the membranes of erythrocytes were coated with antibodies. Then, erythrocytes containing TA were intravenously injected to mice concomitantly with Poly(I:C) adjuvant. The CTL response was analyzed by IFNγ ELISPOT and in vivo lysis of TA-expressing target cells injected to immunized mice. Humoral response was assessed by the determination of TA-specific immunoglobulin titer in mouse serum.

Results: All TA were efficiently encapsulated in erythrocytes in a dose-dependent manner. A strong T-cell response was induced after 2 injections of TA encapsulated in erythrocytes: 2µg of OVA and 20µg of TRP2 induced the in vivo lysis of 97% and 96% of TA-target cells, respectively. By comparison, injections of free antigen induced less than 5% of lysis. Furthermore, a significant number of TA-specific IFNγ-secreting cells was generated (1585 and 286 cells / 106 cells for TRP2 and PSA, respectively) compared to the free TA (< 80 cells / 106 cells for both TA). In addition, we established a dose-dependent PSA-specific humoral response. Finally, a significant delay of tumor growth was obtained in mice more than twenty days after the subcutaneous implantation of EG7-OVA or B16F10 (melanoma) tumor cell lines, compared to injections with free antigen or control vehicle.

Conclusion: The use of erythrocytes as TA carrier to specifically deliver antigen to APC and induce efficient immune response against tumor can be a very promising strategy in cancer immunotherapy.

#2357

Therapeutic potential of the natural human IgG1k antibody pritumumab.

Ivan Babic,1 Rajesh Mukthavaram,1 Natsuko Nomura,1 Sandeep Pingle,1 Mark C. Glassy,2 Santosh Kesari3. 1 _UCSD Moores Cancer Center, San Diego, CA;_ 2 _Nascent Biotech, Inc., UCSD Moores Cancer Center, John Wayne Cancer Institute, San Diego, CA;_ 3 _John Wayne Cancer Institute, Santa Monica, CA_.

Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. A recombinant version of the mAb was made using the GPEx® system in CHO cells. In a series of comparable studies the CHO mAb was compared with the original hybridoma. Binding specificity with both flow cytometry and immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) activity confirms the comparability of the two versions. A concentration-dependent study of pritumumab-mediated ADCC suggests an EC50 of 39.6ng/ml. Further studies include establishing a xenograft model with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest pritumumab is suitable for development as an anti-tumor therapeutic.

#2358

KHK2805, a novel ADCC- and CDC-enhanced anti-FOLR1 antibody with AccretaMab® technology, shows a potent anti-tumor activity in combination with pemetrexed.

Munetoshi Ando, Keiko Nagata, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. _Kyowa Hakko Kirin Co., Ltd., Shizuoka, Japan_.

Introduction: Folate receptor 1 (FOLR1, FR alpha) is a folate transporter which is expressed in many cancers including ovarian cancer (OvC) and non-small cell lung cancer (NSCLC), and which is an attractive target for cancer therapy, is currently the subject of ongoing studies. We established KHK2805, a novel anti-FOLR1 monoclonal antibody with AccretaMab® technology to enhance both the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. It was demonstrated that KHK2805 exhibits markedly high ADCC and CDC activity levels in clinical samples from ovarian cancer patients, while a tolerable safety profile has been observed in preclinical models using cynomolgus monkeys (100 mg/kg weekly for 4 weeks, intravenously). A greater understanding of the biology of FOLR1 is important for providing a novel therapeutic option for KHK2805 for patients with cancer. Pemetrexed (PEM), a second-generation anti-folate which inhibits thymidylate synthase, glycinamide ribonucleotide transformylase and dihydrofolate reductase, is used as a standard therapy for patients with cancers such as NSCLC. It is not fully understood how PEM treatment affects the expression of FOLR1 on cancer cells. We therefore examined the level of FOLR1 expression in cancer cells after PEM treatment, and the anti-tumor activity of KHK2805 in combination with PEM.

Materials and Methods: The FOLR1 expression levels after PEM treatment were examined in OvC, NSCLC, and endometrial cancer cells by flow cytometry. The ADCC activity of KHK2805 against PEM-treated cells was evaluated. The anti-tumor activity of KHK2805 in combination with PEM was investigated in SCID mice.

Results: Flow cytometry showed that PEM treatment increased the FOLR1 expression of various cancers such as OvC (IGROV1, SKOV3, MCAS), NSCLC (NCI-H1437, NCI-H2228), and endometrial cancer (MESSA, HEC1A, HEC1B) cells. In addition, PEM treatment enhanced the ADCC activity of KHK2805 against MCAS, NCI-H1437, and NCI-H2228, in comparison to cells that did not receive PEM treatment. Furthermore, the anti-tumor activity of KHK2805 in SCID mice bearing subcutaneous MCAS tumors was enhanced by PEM treatment.

Conclusions: PEM induced further FOLR1 expression in OvC, NSCLC, and endometrial cancer, resulting in the enhancement of the ADCC activity of KHK2805. The use of KHK2805 with PEM might therefore be a potent therapeutic option.

#2359

A-337, a potent bispecific antibody targeting EpCAM×CD3, as a potential immunotherapeutic agent for human solid tumors.

Yumin Cui, Zhihua Huang, Hanyang Chen, Xinfeng Zhang, Bo Qi, Heng Liu, Xiaoqiang Yan. _Generon (Shanghai) Corporation, Shanghai, China_.

T cell bi-specific antibodies are potent molecules by binding and re-directing T cells to tumor cells through tumor associate antigens (TAAs), and triggering strong T cell activation and resulting in the killing of tumor cells. Bi-specific T cell engager antibody (BITE®) has demonstrated a remarkable clinical efficacy in liquid tumor. It remains to be a tremendous challenge for the development these types of bi-specific antibodies due to the induction of cytokine storm, thus limiting the dose-escalation in patients. A new immunotherapy antibody platform, (ITab TM), was developed to generate bi-specific antibody-like molecules. The molecules target TAA and activate human CD3 T cells simultaneously. A-337 is a bi-specific antibody targeting on EpCAM (Epithelial cell adhesion molecule) on tumor cells and CD3 in human T cells. In vitro, A-337 demonstrated high potency on activating human T cell measured by CD69 expression, and killing of EpCAM highly expressing tumor cell lines with EC50 values at < 100 pM in the presence of human peripheral blood mononuclear cells (PBMC). The human PBMC dependent cell killing activity of A-337 was correlated to the expression levels of EpCAM on the surface of tumor cell lines. In the mouse model, A-337 showed dose-dependent growth inhibition of SW480 xenograft. In cynomolgus monkey studies, A-337 by single IV infusion induced peripheral T cell re-distribution or activation and cytokine release and showed favorable pharmacokinetics features. The current studies reveal that A-337 has potent anti-tumor activities in vitro and in vivo. These results demonstrate that A-337 is a promising and potent immunotherapeutic agent to treat EpCAM-expressing solid tumors and warrants further development.

#2360

GP350 containing exosomes as DNA vaccine carriers.

Viswa Teja Colluru, Douglas G. McNeel. _University of Wisconsin - Madison, Madison, WI_.

Background: Plasmid DNA vaccination is a safe and economical vaccine modality that has been demonstrated to elicit cellular and humoral immunity in preclinical models and human clinical trials. We have previously shown that enriched mature naive B cells, and not myeloid/DC lineages, are capable of expanding Ag specific CD8 T cells in vivo in mice, and in vitro with human cells. In this study, we sought a means to directly deliver plasmid DNA to B cells as a means to specifically activate this antigen-presenting cell type.

Methods: Exosomes were isolated from EBV+ lymphoblastic cell lines (LCL) and from HEK293T cells that had been transfected to express the EBV protein GP350. Plasmid DNA was labeled with a fluorescent peptide nucleic acid (PNA) probe for detection by imaging and conventional cytometry. Exosomes were transfected with fluorescently labeled plasmid DNA and incubated with either human CD21 transgenic mouse splenocytes or human PBMC and assayed for uptake and activation by FACS. Plasmid DNA transfected exosomes were coincubated with human PBMC and evaluated for their ability to expand antigen-specific CD8+ T cells by different methods.

Results: EBV LCL and HEK293T derived exosomes were successfully transfected with plasmid DNA as verified by image assisted flow cytometry. Transient transfection of HEK293T cells with plasmids encoding a transgene led to release of exosomes containing the transgene product. EBV LCL and HEK 293T-GP350 derived exosomes increased delivery of plasmid DNA to both human B lymphocytes and transgenic hCD21 mouse B lymphocytes, when compared to naked DNA alone, in an hCD21 dependent fashion. At 24h, plasmid DNA containing B lymphocytes exhibited increased activation and upregulation of CD80/86 costimulatory molecules. Further, human B lymphocytes treated with exosome-plasmid DNA complexes led to antigen mRNA production and expansion of Ag specific CD8 T cells without the need for APC subset enrichment. Studies comparing the immunogenicity of GP350-exosome mediated delivery versus naked DNA delivery are underway in different mouse models.

Conclusion: GP350 containing exosomes can successfully increase delivery of plasmid DNA to B lymphocytes even in the presence of other phagocytic cells like macrophages or dendritic cells. This led to detectable antigen production and expansion of cognate Ag specific CD8 T cell expansion without the need for B cell enrichment. Studies examining in vivo immunogenicity of exosomal plasmid DNA delivery are underway, but results to date suggest this approach may be a simple method to boost vaccine efficacy through increased delivery of DNA directly to relevant antigen-presenting B cells.

#2361

Novel animal models for prediction of cancer vaccine response.

Heather Gibson, Richard F. Jones, Joyce D. Reyes, Wei-Zen Wei. _Wayne State University, Detroit, MI_.

Predicting cancer vaccine response is a challenge that must be addressed in immune competent animals that recapitulate human disease and human response to therapy. Furthermore, genetically heterogeneous humans respond differently to vaccination due, in part, to their varying MHC haplotypes and polymorphisms including those found in costimulatory or checkpoint genes. We previously showed that human HER2 transgenic (Tg) mice of different genetic backgrounds have distinct response to HER2 vaccines in the order of BALB > (BALBxB6)F1 > (B6xDR3)F1 > B6 (Radkevich-Brown 2010). We anticipate even greater variance among human populations, and seek genetically diverse animal models to project human cancer vaccine response.

Domestic felines develop mammary tumors with similar pathology and etiology as human cancer, and expression of EGFR, HER2 and HER3 genes that are highly homologous to their human counterparts. Intramuscular electrovaccination of healthy outbred domestic shorthair cats with heterologous (xenogeneic) or single point-mutated self HER2 DNA induces HER2-specific T cells in ~30% of vaccinated cats (Gibson 2015). Humoral immune responses were induced by both vaccine constructs, with point-mutated feline HER2 vaccine producing antibodies to unique epitopes on feline mammary carcinoma (FMC). The latter inhibits FMC growth in vitro and delays tumor growth in SCID mice. Vaccination with wild type feline HER2 DNA fails to induce specific immunity and even hinders feline HER2 immune activation by other vaccines. Studies in domestic cats provide novel insight on the range and nature of vaccine response in healthy cats which can also be tested in feline cancer patients. Strategic modification of minimal residues may be most effective in inducing reactivity to cancer associated self antigens.

To address genetic regulation of vaccine response, diversity outbred (DO) mice offer a pragmatic option. These mice were generated by cross-breeding 8 inbred mouse strains to ensure broad genetic diversity among the mice (Svenson 2012). The human HER2 gene is introduced by breeding DO mice with our human HER2 transgenic (Tg) mice. F1 mice represent a genetically heterogeneous population that expresses human HER2 as a self antigen. Specific genes impacting vaccine response will be identified by genome-wide quantitative trait locus (QTL) scan to associate haplotype or single nucleotide variants with vaccine response. Findings in these mice may lead to the identification of critical genes that regulate cancer vaccine response. Candidate vaccines identified in DO mice can be further validated in cats and expanded to human trials. With these novel animal models, cancer vaccines can be designed and tested with greater precision, and patients with reactive genotypes may be identified before vaccination. (Supported by CA76340)

#2362

Optimizing prostate cancer immunotherapy by reducing vaccine induced PD-1 expression.

Christopher D. Zahm, Douglas G. McNeel. _University of Wisconsin - Madison, Madison, WI_.

Through early phase clinical trials we have shown that a plasmid DNA vaccine can elicit antigen-specific CD8 T-cells that can persist for years. However, the persistence of antigen-expressing prostate tumors demonstrates that mechanisms of tumor escape are at play. To investigate these mechanisms we have focused on vaccine strategies using the synovial sarcoma, X breakpoint 2 (SSX2) protein as a model target antigen and utilized mice which express HLA-A2 but not murine MHC-I. We have shown that immunization with a DNA plasmid encoding SSX2 elicits robust CD8+ cells and that altering HLA-A2 affinity can augment this response. High affinity altered peptide ligand (APL)-encoding DNA vaccines elicited a greater frequency of specific T-cells; however, they fared worse in anti-tumor studies due to increased PD-1 expression on T-cells, an effect which could be abrogated with PD-1 blockade. Our current research has focused on why high-affinity epitopes elicit higher PD-1 expression, and whether this might be modulated at the time of antigen presentation. To that aim we have used the OT-1 mouse model, in which T-cells express a T-cell receptor that is specific for the dominant ovalbumin epitope SIINFEKL. We generated APLs of SIINFEKL and used them to stimulate OT-1 splenocytes ex vivo. The high affinity SIINFEKL epitope resulted in high, sustained expression of PD-1 following activation, while lower affinity APLs resulted in less, transient expression. However, lowered PD-1 expression can be indicative of T-cells that receive weaker signals via the T-cell receptor (TCR) and likewise display lowered effector function, therefore we also analyzed cytokine production and the in vivo effects of the APLs. In immunization experiments using B6 mice carrying adoptively transferred OT-1 T-cells the lower affinity peptide again resulted in transient PD-1 expression while the high affinity peptide led to sustained levels of PD-1 expression. One week after vaccination with the low affinity APL, PD-1 on tetramer+ CD8 T-cells had returned to baseline while cells from mice immunized with SIINFEKL maintained PD-1 expression. Furthermore, 38(±8)% of tetramer+ cells from the SIINFEKL immunized group were PD-1+ 4-1BB- indicating that PD-1 expression is maintained even after activation in vivo. We are currently assessing the ability of immunization with each peptide to prevent B16-OVA tumor growth in vivo. To assess the mechanism by which PD-1 is regulated after stimulation with each peptide we used a combination of live cell imaging and RNA sequencing to observe APC/T-cell interactions and T-cell gene expression respectively. This research indicates that while increasing antigen affinity may generate a more robust T-cell response it does not always result in better anti-tumor immunity and provides a model in which we can study the biology underlying PD-1 expression.

#2363

Combinational therapy with specific immunization plus checkpoint inhibition results in enhanced tumor regression and survival benefit.

Elizabeth Gabitzsch, Adrian Rice, Yvette Latchman, Joseph P. Balint, Frank Jones. _Etubics Corporation, Seattle, WA_.

Effective immunotherapeutic treatment of various cancers may require combinational approaches to induce specific immunity to the tumor as well as inhibition of immune check-points. We have developed a viral gene delivery platform to immunize against numerous tumor associated antigens (TAA) such as carcinoembryonic antigen (CEA), HER2/neu and HPV 16. We investigated the therapeutic benefit of the immunotherapeutics alone and in combination with check-point inhibitors such as programmed death-ligand 1 (PD-1) blockade and LAG3 blockade in murine tumor models. As single agents, immunization with the viral delivery platform encoding the TAA induced target-specific cell-mediated immunity and anti-tumor responses in the respective model. When the immunotherapies were combined with immune checkpoint blockade, an increased level of anti-tumor activity against was observed in addition to an improvement in survival. Tumor microenvironment analysis immunized mice revealed an increase in CD8+ tumor-infiltrating lymphocytes (TILs). In addition, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1+ TILs. When immunotherapy was combined with anti-PD-1 antibody, we observed CD8+ TILs at the same increased level but found that a smaller fraction of these were PD-1+. Furthermore, we observed a reduction in PD-L1 expression on tumor cells, providing a mechanism by which combination therapy favors tumor clearance and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials.

#2364

Spontaneous and vaccine-induced clearance of Mus Musculus Papillomavirus1 (MusPV1) infection.

Rosie T. Jiang, Joshua W. Wang, Shiwen Peng, Chien-Fu Hung, Richard B.S Roden. _Johns Hopkins University, Baltimore, MD_.

Human papilloma virus (HPV) high risk (hr) types cause several cancers, including cervical, vaginal, penile, and oropharyngeal, whereas low risk types cause benign diseases such as skin warts, anogenital warts and recurrent respiratory papillomatosis. Although hrHPV infection is one of the most common sexually transmitted diseases most infections remain asymptomatic and are cleared while a sub-population develops persistent HPV-related pathologies including cancer. Further, immune-compromised populations, notably organ transplant and HIV-positive patients, are particularly vulnerable to HPV-related pathologies. Here, we describe the first establishment of mouse papillomavirus-1 (MusPV1) disease in outbred and immune competent SKH1-Elite mice (Crl:SKH1-Hrhr). Challenge of SKH1 mice with MusPV1 resulted in three clinical outcomes: 1) persistent (>2 months) papillomas (~15%), 2) transient papillomas that spontaneously regress typically within 2 months (~10%), 3) no visible papillomas and viral clearance (~75%). SKH1 mice with persisitent papillomas were treated using a potentially preventive and therapeutic DNA vaccine composed of human calreticulin (hCRT) linked to MusPV1 early proteins, mE6 and mE7 and the late protein mL2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations (15 µg in PBS) via in vivo electroporation were administered weekly and immune responses were measured, including mE6/mE7/mL2 serum antibody response and T-cell epitope mapping studies. Previously persistent papillomas disappeared within 2 months after final vaccination. Clearance of MusPV1 was confirmed by loss of mE6/mE7 transcript staining using RNA-CISH on formalin-fixed, paraffin-embedded tail sections. Vaccination induced a strong mE6 and mE7 CD8+ T cell response in all mice. Interestingly, mL2 antibody responses were faster and stronger in asymptomatic challenged mice and in those that spontaneously cleared their papillomas after challenge as compared to mice with persistent papillomas. In sum, MusPV1 challenge of outbred, immune-competent SKH1 mice represents a promising model to further study potential immunotherapies for HPV-related disease and the relationship between spontaneous clearance of papillomavirus and host genetics.

#2365

Checkpoint inhibitors and a multivalent melanoma vaccine as a novel combinatorial therapy.

Rachana R. Maniyar, Neha Tuli, Robert Suriano, Jan Geliebter, Marc Wallack, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

Over 1 million cases of skin cancer are diagnosed in the US every year, of which 75% deaths result due to melanoma, one of the deadliest forms of skin cancer, arising from melanocytes. We have isolated five patient derived melanoma cell lines (MEL-V, 3MM, GLM2, Mel2, KFM) and characterized the expression of various melanoma associated antigens (MAAs). The presence of a multitude repertoire of common MAAs (Gp100, MART-1, MAGE-A1, NY-ESO-1, Tyrosinase, TRP-1 (Gp75), TRP-2, Melanotransferrin, CD71, CD146) enabled us to use composite cell membrane preparations of these cells as a multivalent vaccine. To this end, we designed a vaccinia virus vaccine that was composed of membrane lysate preparations of the five primary cells and recombinant IL-2. When tested in a pilot study, delayed type hypersensitivity and a multivalent immune response were noted. In an effort to correlate genetic lesions with MAA expression we observed that in BRAF mutant melanoma cells, treatment with a BRAFv600E inhibitor, PLX4032, enhanced the expression of MAAs making the melanoma cells more visible to the vaccination driven immune response. We also observed that melanoma cells constitutively expressed CTLA-4 as determined by Immunofluorescence and Western Blots. This expression was found on the membranes as well as in the cytoplasm in comparatively high levels on wild type as well as the BRAFV600E mutant melanoma cell lines. Further our results suggest that CTLA-4 expression on melanoma cells promotes the immunosuppressive tumor microenvironment enabling tumor evasion. Given that CTLA-4 monoclonal antibodies such as Ipilimumab are approved for clinical treatment, this study opens up a new avenue into a plausible combinatorial therapy for our vaccinia virus based vaccine with Ipilimumab for BRAF mutant as well as wild type melanoma cells.

#2366

Pilot study of DNA microseeding to activate immune rejection of canine melanoma.

Cindy L. Zuleger,1 Chulhi Kang,1 Erik A. Ranheim,1 Ilene Kurzman,1 Michael D. Macklin,1 Michael A. Newton,1 David M. Vail,1 Jedd D. Wolchok,2 Elof Eriksson,3 Mark R. Albertini1. 1 _University of Wisconsin, Madison, WI;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Harvard Medical School, Boston, MA_.

Background: Canine malignant melanoma provides a model to study DNA vaccine delivery systems. A xenogeneic human tyrosinase (huTYR) DNA vaccine delivered by Biojector2000 received United States Department of Agriculture licensure when it appeared to prolong survival of dogs with melanoma compared to historical, stage-matched controls and to stimulate immune responses in some dogs. The current study evaluates toxicity, transgene expression, and antibody responses to huTYR in companion dogs with spontaneously developing melanoma following delivery of huTYR DNA to the skin via a modified tattoo device, a method termed DNA microseeding.

Methods: Five companion dogs with melanoma were scheduled to receive huTYR DNA at two sites (Site A and Site B) on the inner thigh by DNA microseeding every 2 weeks for 4 administrations at a range of huTYR DNA doses; 2 dogs (50 μg [Site A] and 100 μg [Site B]); 2 dogs (200 μg [Site A] and 400 μg [Site B]); and 1 dog (83 μg [Site A] and 83 μg commercial huTYR plasmid [Site B]). Vaccine site biopsies were obtained to determine transgene expression 24 hours after the 1st and 3rd vaccination time-points, and 48 hours after the 2nd and 4th vaccination time-points. Blood samples were obtained at baseline and 2 weeks after the 2nd, 3rd, and 4th vaccinations to quantify TYR-specific antibodies via indirect ELISA.

Results: No toxicity, beyond local site irritation, related to the vaccine administration was observed. The 3 dogs with known disease at study entry received 3, 1 and 4 treatments before discontinuation of treatment due to progressive disease. The 2 dogs without evidence of disease at study entry received all 4 planned treatments and remained without evidence for recurrence after treatment for the duration of the study (6 weeks). Only rare huTYR+ cells with macrophage-like morphology were observed in some vaccine site biopsies. A significant increase in anti-huTYR IgG was detected at Day 57 compared to pre-treatment in 2 of the 4 evaluable dogs (p=0.03 Wilcoxon Mann Whitney), and these were the 2 dogs without evidence of disease at study entry. Baseline anti-huTYR IgG levels were also greater compared to IgG levels against an irrelevant control antigen.

Conclusions: While microseeding of huTYR plasmid DNA resulted in only rare transgene expression at DNA doses up to 400 μg, humoral responses against huTYR were substantially boosted in 2 of 4 evaluable dogs. Additional testing is needed to determine if DNA microseeding enhances huTYR DNA vaccine immunogenicity compared to Biojector delivery.

#2367

cGMP production of a chimeric virus-like particle vaccine for prevention of HPV-associated cancers.

George W. Buchman,1 Brian P. Howard,1 Nadia Abdallah,1 Bala Medicherla,1 Mary Anne Fisher,1 Jonathan M. White,2 Michelle L. Kennedy,2 Shizuko Sei,3 Richard B.S. Roden,4 Christina Schellenbacher,5 Reinhard Kirnbauer,5 Robert H. Shoemaker3. 1 _Paragon Bioservices, Baltimore, MD;_ 2 _MRIGlobal, Kansas City, MO;_ 3 _Division of Cancer Prevention, National Cancer Institute, Bethesda, MD;_ 4 _Johns Hopkins University, Baltimore, MD;_ 5 _Medical University of Vienna, Vienna, Austria_.

The NCI Division of Cancer Prevention's PREVENT Cancer Preclinical Drug Development Program supports preclinical development and clinical translation of novel cancer preventive interventions. One of the projects currently supported came from principal investigators, Drs. Reinhard Kirnbauer and Richard Roden, who requested cGMP production of a novel chimeric virus-like particle (VLP)-based human papillomavirus (HPV) vaccine, comprising HPV16 L1 VLP with HPV16 L2-RG1 epitope repetitively displayed on the capsid surface (RG1-VLP). The vaccine is designed to prevent infection by a broad range of HPV types, including mucosal and cutaneous types not targeted by current vaccines (J Invest Dermatol 2013, 133:2706), and thus has the potential to become a broadly effective next generation HPV vaccine. The cGMP process is currently being developed and validated by Paragon Bioservices in Baltimore, MD, under NCI contract with MRIGlobal. RG1-VLP is expressed in Sf9 insect cells infected with a triple-plaque-purified recombinant baculovirus. After cell culture clarification and capsid maturation steps, VLPs are purified by a series of chromatography steps to remove host cell derived impurities. The purified VLPs are analyzed by ELISA and Western blot immunoassays. Removal of host cell protein and DNA is confirmed by ELISA and qPCR, respectively. Assembly of a chimeric RG1-VLP protein into full size VLP structures (approximately 50-60 nm in diameter) is verified by transmission electron microscopy. cGMP-produced VLPs should be available during the first half of 2016. The PREVENT Program will perform the required toxicology studies and will assist with IND filing, for a first-in-human Phase I study of RG1-VLP vaccine. While the clinical protocol is still under development, it is anticipated that 15-20 healthy adult volunteers will be enrolled into each of three to four escalating dose groups. While the primary objective of the Phase I study will be to establish the safety of RG1-VLP, preliminary immunogenicity studies will be included. Immunogenicity of RG1-VLP will be evaluated by serum anti-L1 and anti-RG1 antibody levels as well as protective and cross-neutralizing antibodies against HPV16 and a broad panel of HPV types including at least one type not covered by marketed vaccines.

#2368

Inhibition of epidermal growth factor receptor pathway by epidermal growth factor antibodies in non-small cell lung cancer.

Jordi Codony-Servat,1 Miguel Angel Molina-Vila,1 Jordi Bertran-Alamillo,1 Rafael Rosell,2 Erik D'Hondt1. 1 _Pangaea Biotech, Barcelona, Spain;_ 2 _Institut Catala Oncologia, Barcelona, Spain_.

Introduction: The Epidermal Growth Factor Receptor (EGFR) signaling system is frequently unbalanced in non-small-cell lung cancer (NSCLC). Epidermal Growth Factor (EGF) could be an attractive target in these tumors. We used a "cancer vaccine" composed of human recombinant EGF conjugated to a carrier protein P64K. The vaccine is intended to induce antibodies against patients' own EGF that would block EGF-EGFR interaction.

Objectives: In NSCLC cell lines we assessed whether anti-EGF antibodies can inhibit the pathway activated by EGF-EGFR binding and whether sera from immunized patients can block downstream key signal transduction proteins. NSCLC cell lines were selected to determine whethergenetic alterations in EGFR, KRAS, ALK, BRAF or MET can have an impact on inhibition of EGFR pathway by anti-EGF antibodies.

Experimental Procedures: Western blotting was used to evaluate the capacity of antibodies and sera from patients to inhibit EGFR activation pathway. MTT assays were performed to evaluate the effects on tumor cell proliferation.

Results: The anti-EGF antibodies showed a dose-dependent inhibitory effect on EGF-induced activation of EGFR and downstream pathway molecules such as Akt and ERK 1/2. This inhibition was observed in all cell lines tested, including KRAS, EGFR and BRAF mutated cells as well as cells with ALK translocations or MET amplification. Sera from 10 patients immunized with the P64K-EGF vaccine were then tested. All sera reversed EGF-induced activation of EGFR and ERK 1/2, but some sera were significantly more potent than others. By contrast, two control sera from non-vaccinated patients failed to inhibit EGF-induced phosphorylation of EGFR and ERK 1/2. Finally, anti-EGF antibodies were also able to block the stimulatory effects of EGF on the growth of some NSCLC cell lines.

Conclusions: Anti EGF antibodies and sera from patients immunized with an "EGF vaccine", called otherwise EGF Pathway Targeted Immunization inhibits EGFR signaling in NSCLC cell line models. This effect is independent of cells' genetic background.

#2369

Molecular profile of a GM-CSF overexpressing breast cancer whole-cell vaccine with systemic anti-tumor activity.

Markus Daniel Lacher, Charles L. Wiseman, Joseph Wagner. _BriaCell Therapeutics Corp, Berkeley, CA_.

BACKGROUND AND PURPOSE OF STUDY: The allogeneic whole-cell cancer vaccine BriaVax(TM) (formerly SV-BR-1-GM) is an ER/PR negative, HER2/neu positive breast cancer cell line (SV-BR-1) we engineered to stably overexpress GM-CSF. BriaVax, rendered proliferation incompetent by irradiation, has thus far been applied to 4 advanced stage cancer patients (3 subjects with breast cancer, 1 subject with ovarian cancer). One breast cancer subject responded to BriaVax with complete remission of a measurable lung lesion and near complete remission of multiple breast lesions after only 3 inoculations. Nevertheless, she relapsed 3 months after completing the protocol, with brain metastases as well as multiple breast lesions. We obtained FDA permission to resume vaccinations, and, upon doing so, all metastatic sites responded with a prompt tumor regression after only 3 inoculations. To prospectively identify patients with a high likelihood of benefiting from BriaVax therapy we began a program to identify molecular factors of diagnostic potential. Here, we describe a gene expression signature that might both be informative about BriaVax' mechanism of action and helpful for developing diagnostic or monitoring biomarkers.

METHODS: To prospectively identify patients with tumors responsive to BriaVax we began a molecular analysis of both the BriaVax cell line and cells obtained from the special clinical responder's blood. BriaVax gene expression profiles were obtained through Illumina BeadArray and NanoString nCounter technologies and compared to gene expression data sets publically available through the Gene Expression Omnibus (GEO; National Center for Biotechnology Information) portal.

RESULTS: BriaVax expresses a gene signature consistent with a mechanism of action involving not only the activation of cytotoxic T cells but also the induction of a humoral response. In addition, BriaVax expresses known cancer antigens. Notably, blood-derived cells of the special clinical responder expressed genes complementing BriaVax' gene expression signature, thus possibly explaining the unusually prompt and robust clinical response to BriaVax.

CONCLUSIONS: Our findings suggest that BriaVax exerts its therapeutic effects via multiple modes. We identified both candidate immunogens overexpressed in BriaVax compared to normal breast cells and unraveled a potential mechanism of action explaining the encouraging clinical response observed.

#2370

Therapeutic nano-DC vaccine For HER2 positive breast cancer.

Haifa Shen, Xiaojun Xia. _Houston Methodist Research Institute, Houston, TX_.

Harnessing a patient's own immune capacity to eradicate malignant cells is a promising approach for cancer treatment, and therapeutic vaccine represents an important arm of cancer immunotherapy. Our research aims to develop a potent therapeutic cancer vaccine for breast cancer which is traditionally considered as poorly immunogenic. We have recently shown that porous silicon microparticles (PSMs) can stimulate type I interferon (IFN) expression in dendritic cells (DCs), and thus serve as a potent adjuvant for a therapeutic cancer vaccine (Xia et al: Cell Reports 2015, 11:957-966). This action is mediated by the TRIF/MAVS pathways, and is independent of the cell surface or endosomal Toll-like receptors. In addition, the nanometer-size pores in PSMs can serve as a reservoir for sustained antigen release. We have assembled a Nano-DC vaccine comprising of bone marrow-derived DCs internalized with HER2 antigen peptide-loaded PSMs. Treatment of murine models of HER2 positive breast cancer with this Nano-DC vaccine not only activates and expands antigen-specific CD8+ T cells, but also promotes a Th2-to-Th1 transition in the tumor microenvironment to favor anti-tumor activity. Thus, our result supports the development of a therapeutic Nano-DC vaccine for breast cancer.

## TUMOR BIOLOGY:

### Clonal Heterogeneity and Evolution

#2371

Multi-cell phylogenetic inference of copy number evolution in breast cancer.

Alexander J. Davis,1 Ruli Gao,1 Emi Sei,1 Pei-Ching Tsai,1 Anna Casasent,1 Amy Zhang,1 Xiuqing Shi,2 Yong Wang,1 Jill Waters,1 Funda Meric-Bernstam,1 Mary Edgerton,1 Nicholas Navin1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Chinese Medical College, Beijing, China_.

Aneuploidy is a hallmark of breast cancer, but little is known about how these complex genomic rearrangements evolve during tumor growth. To investigate this question, we developed a Highly-Multiplexed Single Nucleus Sequencing (HM-SMS) method to profile genome-wide copy number in individual tumor cells and compared multiple cells to infer evolutionary lineages. We applied this method to analyze thousands of single cells from 12 triple-negative breast cancer patients and 10 ductal-carcinoma-in-situ (DCIS) patients. Integer copy number profiles were calculated from a combination of sequencing data and ploidy estimation using flow cytometry. A multi-cell segmentation algorithm was used to identify common CNA events that are shared between tumor cells in each patient. Trinary event matrices were calculated for each patient to determine the presence or absence of individual copy number aberrations (CNAs) in individual cells. Using the maximum parsimony criterion, we inferred a phylogenetic tree for each tumor and estimated the temporal order of the CNAs.. Our data suggest that most tumors consisted of 1-4 major clonal subpopulations that shared a common evolutionary lineage, suggesting that they evolved from a single normal cell in the breast tissue. Furthermore our data support a punctuated model of copy number evolution, which which the majority of CNAs were acquired in a short evolutionary burst, followed by one or more stable clonal expansions to form the tumor mass.

#2372

The genetic evolution of melanoma.

Alan H. Shain,1 Richard Yu,1 Iwei Yeh,1 Jamal Benhamida,1 Ivanka Kovalyshyn,2 Aravindhan Sriharan,3 Eric Talevich,1 Reinhard Dummer,4 Jeffrey North,1 Laura Pincus,1 Beth Ruben,1 William Rickaby,5 Corrado D'Arrigo,6 Alistair Robson,5 Robert Judson,1 Nancy Joseph,1 Boris Bastian1. 1 _UCSF, San Francisco, CA;_ 2 _Cleveland Clinic, Cleveland, OH;_ 3 _Orlando Health, Orlando, FL;_ 4 _University Hosptial of Zurich, Zurich, Switzerland;_ 5 _St. John's Institute of Dermatology, London, United Kingdom;_ 6 _Dorset County Hospital, Dorchester, United Kingdom_.

The pathogenic mutations in melanoma have largely been catalogued, but the order of their occurrence is not known. We identified 82 cases of melanocytic neoplasia with two or more histopathologically distinct precursor and descendent portions. These portions spanned several histopathologic stages including benign nevi, intermediate neoplasms such as dysplastic nevi, melanoma in situ, invasive melanoma, and metastatic melanoma. In total, 277 histopathogically distinct areas were microdissected and sequenced using a panel of several hundred cancer-genes at an average of 275-fold coverage. In all cases precursor lesions harbored mutations known to activate the MAPK pathway, most commonly affecting BRAF or NRAS, and thus these were considered initiating mutations. TERT promoter mutations were the earliest secondary mutations to occur, emerging in intermediate neoplasms and in situ melanomas. Bi-allelic CDKN2A mutations and/or deletions arose predominantly at the transition to invasive melanoma. TP53 and PTEN mutations occurred comparatively later in thicker primary melanomas. In the 12 cases in which matched primaries and metastases were sequenced, no pathogenic mutations were specifically associated with the transition to metastatic melanoma, suggesting that metastatic capability was already present in the primary tumors. Unequivocally benign neoplasms, which mostly consisted of conventional nevi, disproportionately harbored BRAFV600E mutations as the only apparent pathogenic alteration. Lesions that were histopathologically intermediate, such as dysplastic nevi, had two or more pathogenic mutations and were enriched for NRAS mutations. These findings provide genetic support for the existence of an intermediate stage of melanocytic neoplasia, resolving a long-standing controversy. We also found that melanomas with different initiating mutations evolve through distinct evolutionary trajectories linked to specific histopathologic precursors. A mutational signature of UV-radiation-induced DNA damage predominated in lesions from all histopathologic stages, implicating UV-radiation in both the initiation and progression of melanoma. The point mutation burden increased steadily from one progression stage to the next, stabilizing once melanomas became invasive. By contrast, copy number alterations only became prevalent at the transition to invasive melanoma. Melanomas evolve from benign lesions linearly, whereas, they show patterns of branched evolution when they evolve from intermediate lesions; this indicates that evolution may accelerate once a melanocytic neoplasm reaches an intermediate stage. In summary, our study outlines a framework of the genetic evolution of melanocytic neoplasms from precursor lesions and unveils the rate-limiting homeostatic factors that become disrupted as neoplasms progress from one stage to the next, as well as the pathogenic factors driving this evolution.

#2373

Assessing intra-tumor heterogeneity and tracking longitudinal and spatial clonal evolution by next-generation sequencing.

Yuchao Jiang, Yu Qiu, Andy J. Minn, Nancy R. Zhang. _University of Pennsylvania, Philadelphia, PA_.

Cancer is a disease driven by genetic and epigenetic alterations that follows Darwinian evolution. Recently, there have been increasing efforts to sequence the tumor from the same patient at multiple time points and/or from multiple spatially separated resections. Different snapshots of the same tumor have proved invaluable for identifying subclonal populations and inferring the tumor's history.

We propose a method, Canopy, for estimating the clonal history and for longitudinal and spatial comparison of mutation profiles from one or more samples derived from a single patient. Canopy accounts for normal cell contamination and reconstructs subclonal phylogeny utilizing both somatic copy number alterations (CNAs) and somatic single nucleotide alterations (SNAs). Canopy provides a general mathematical framework that enumerates all possible CNA-SNA phases and temporal orderings. Taking as input the mutant and reference allele frequencies for SNAs, and depth of coverage for CNAs, Canopy gives confidence assessments of all possible configurations of the cancer's clonal evolution.

Canopy is applied to three cancer sequencing datasets of varying study design, as well as to an extensive simulation study. On a whole-exome study of a transplantable metastasis model derived from human breast cancer cell line MDA-MB-231, Canopy successfully deconvolutes the mixed cell sublines, using the single cell sublines as ground truth, and identifies DNA signatures that can be prognostic of distant metastasis. On a whole-genome sequencing dataset of the primary tumor and relapse genome of a leukemia patient, Canopy predicts phylogenetic histories in concordance with existing knowledge. On a whole-genome sequencing dataset of the breast cancer tumor and its subsequent metastatic xenograft, Canopy's inferred clonal phylogeny is concordant with genomic markers of major clonal genotype and is confirmed by single-cell sequencing. Finally, through simulations, we explore the effects of various parameters on deconvolution accuracy, and evaluate performance with comparison against existing methods. Collectively, Canopy provides a rigorous foundation for statistical inference on repeated sequencing experiments from evolving populations delineated temporally and spatially.

#2374

Reconstructing the evolutionary history of metastatic cancers.

Johannes G. Reiter,1 Alvin P. Makohon-Moore,2 Jeffrey M. Gerold,1 Ivana Bozic,1 Krishnendu Chatterjee,3 Christine A. Iacobuzio-Donahue,2 Bert Vogelstein,4 Martin A. Nowak1. 1 _Harvard University, Cambridge, MA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _IST (Institute of Science and Technology) Austria, Klosterneuburg, Austria;_ 4 _Johns Hopkins University School of Medicine, Baltimore, MD_.

The evolution of metastases is responsible for 90% of cancer-related deaths. Genome wide sequencing and phylogenomic methods enable the reconstruction of the evolutionary history of a patient's cancer at unprecedented depth. However, due to a lack of samples from multiple spatially-distinct metastases from untreated patients and a lack of phylogenomic tools applicable to noisy and impure sequencing samples, the evolutionary rules governing metastatic spread have remained poorly understood.

We performed whole-genome sequencing (coverage: median 51x) as well as deep targeted sequencing (coverage: median 347x) on 21 samples from multiple regions of the primary tumor and many distinct liver and lung metastases of two treatment-naïve pancreatic ductal adenocarcinoma patients. We developed a tool, called Treeomics, that leverages computational and statistical advances to reconstruct the phylogeny of a cancer with commonly available sequencing technologies. Treeomics employs a uniquely-designed Bayesian inference model to account for error-prone sequencing and varying low neoplastic cell content (estimated purities 16-44%) to calculate the probability that a specific variant is present or absent in each sequenced lesion. Based on Mixed Integer Linear Programming, a mathematically guaranteed optimal evolutionary tree is produced.

We obtained robust phylogenies consistent with the biological processes underlying cancer evolution. The reconstructed phylogenies show that advanced cancer cells of related subclones were equally capable of seeding lung and liver metastases. Treeomics identified sequencing and biological artifacts such as those resulting from insufficient coverage or loss of heterozygosity; almost 7% of the variants were misclassified by conventional methods. Among the identified false-negatives was the common clonal driver mutation in KRAS within a region that has low sequencing read alignability and a significantly reduced coverage. Such artifacts can skew phylogenies by creating illusory tumor heterogeneity among distinct samples. Additionally, we reanalyzed publicly available data from ovarian, prostate and skin cancers. We further illuminated evolutionary relationships among some samples in a conclusive fashion and show that classical distance-based phylogenetic methods can produce evolutionarily implausible results. Treeomics avoids these common pitfalls and infers robust phylogenies confirmed by high bootstrapping values.

The new approach described here efficiently reconstructs the evolutionary history of metastases, detects potential artifacts in noisy high-throughput sequencing data, and finds subclones of distinct origin. These phylogenies shed new light on seeding patterns and metastatic progression, which has significant implications for clinical decision making and may provide predictive value for a patient's prognosis.

#2375

Multiclonal invasion in breast cancer identified by single cell DNA sequencing.

Anna K. Casasent,1 Annalyssa N. Long,2 Aislyn Schalck,1 Emi Sei,2 Ruli Gao,2 Alexander Davis,1 Yong Wang,2 Mary E. Edgerton,3 Nicholas E. Navin2. 1 _Graduate School of Biomedical Sciences, University of Texas, Houston, TX;_ 2 _Department of Genetics, UT MD Anderson Cancer Center, Houston, TX;_ 3 _Department of Pathology, UT MD Anderson Cancer Center, Houston, TX_.

Ductal carcinoma in situ (DCIS) is the most common form of early stage breast cancer and is frequently detected by mammography. Only 10% of low-grade and 30% of high-grade DCIS patients will later present with an invasive carcinoma, making it difficult to determine which patients to treat aggressively. Although DCIS is often considered a precursor to invasive breast carcinoma, a major question in the field is whether invasive subpopulations evolve directly from the in situ populations or independently. We hypothesize that early tumor cells evolve over an extended period of time in the ducts, generating multiple subpopulations that migrate out of the ducts together. To test this hypothesis, we developed an approach that combines laser-capture microdissection (LCM) with single cell sequencing to perform DNA copy number profiling while preserving the topographic location of cells. We applied this method to 10 high-grade breast cancer patients with defined in situ, invasive, and normal regions. Using these data, we delineated the clonal substructure and location of the in situ and invasive subpopulations. We found that most high-grade tumors are composed of 1-4 major clonal subpopulations that are intermixed in both the in situ and invasive regions. These subpopulations share a common evolutionary lineage or cell of origin. We also performed deep-exome sequencing (100X) of matched microdissected regions of in situ, invasive and adjacent normal tissue from these breast cancer patients. In each tumor we identified a large number of shared somatic mutations between the in situ and invasive regions, further supporting our direct genomic lineage hypothesis. We also identified a few mutations confined to the invasive tumor regions that may play an important role in the invasive phenotype. These data support a multiclonal invasion model in which distinct clones evolve in the ducts and subsequently migrate into the adjacent tissues to establish invasive tumors. This model has important implications for the diagnosis and therapeutic treatment of DCIS breast cancer patients since multiple clones may need to be targeted to inhibit invasion.

#2376

Clonal evolution of skin carcinomas during tumor progression and metastasis.

Melissa Q. McCreery, Allan Balmain. _UCSF, San Francisco, CA_.

Tumor heterogeneity and the ongoing evolution of tumor subpopulations have presented considerable challenges to the success of cancer therapies, highlighting a critical need for greater understanding of these phenomena. We used a K5CreER-Confetti reporter mouse in combination with the DMBA (dimethylbenzanthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate) skin carcinogenesis model to study the genetics, clonality, and evolutionary behavior of tumor subpopulations from tumor initiation to metastasis. Treating the skin of K5CreER-Confetti mice with tamoxifen results in widespread labeling of the epidermis and hair follicles, which persists for at least 18 months. Papillomas that develop from Confetti-activated skin are of clonal origin, evidenced by each tumor being distinctly dominated by a single Confetti color (RFP, YFP, GFP, or CFP; or uncolored). Twelve weeks after initiation, the majority of tumors remain single-colored, with only 22% (36 of 133) showing any evidence of a secondary color population present. By 20 weeks after initiation, however, nearly all tumors (78%, 42 of 54) have incorporated one or more secondary populations, readily identifiable as streaks of a distinct color. FACS separation and sequencing of these different colored populations reveals these populations are genetically distinct: in preliminary analysis, the primary color population typically carries the expected driver Hras Q61L mutation while the secondary populations do not. The tumor thus appears to be incorporating pseudo-normal tissue from its microenvironment during early growth. Activation of the Confetti reporter in tumors post-initiation in papillomas results in labeling of single cells with all 4 Confetti colors, allowing subpopulations and their evolution to be studied at later stages. These highly multi-color papillomas subsequently give rise to single-color carcinomas and metastases, suggesting that the conversion from a benign to malignant tumor is clonally driven. The Confetti mouse is a valuable tool for studying the clonality and genetics of subpopulations present in tumors, which need to be better understood in order to improve the treatment of heterogeneous human tumors.

#2377

Clonal evolution and mutational signatures in urothelial carcinoma revealed by paired exome analysis.

Iver K. Nordentoft,1 Philippe Lamy,1 Karin Birkenkamp-Demtröder,1 Mathilde Borg Houlberg Thomsen,1 Søren Vang,1 Palle Villesen Fredsted,2 Jakob Hedegaard,1 Michael Borre,3 Jørgen Bjerggaard Jensen,3 Søren Høyer,4 Jakob Skou Pedersen,5 Torben Falck Ørntoft,5 Lars Dyrskjøt Andersen5. 1 _Aarhus university Hospital/Skejby, Skejby, Denmark;_ 2 _Aarhus university, Department of Bioinformatic Research, Aarhus, Denmark;_ 3 _Aarhus university Hospital, Department of Urology, Skejby, Denmark;_ 4 _Aarhus university Hospital, Department of Pathology, Aarhus, Denmark;_ 5 _Aarhus university Hospital, Department of Molecular Medicine, Skejby, Denmark_.

Background: Bladder cancer (BC) is the 5th most common cancer in the western world. 75% of patients are diagnosed with a single or multifocal non-muscle invasive bladder tumor. About 70% of patients have disease recurrence, and 10-30% will eventually have disease progression. Synchronous and metachronous tumors are mono- or oligo clonal of origin, pointing towards a common ancestry (field disease). The field disease is acquiring molecular alterations over time that may fuel disease progression and metastasis. Detailed knowledge of tumor heterogeneity, clonality and mutational dynamic of early clonal mutations with disease driving potential are needed in order to determine the possible effect of targeted treatment.

Methods: In this study we characterized genomic alterations in two to five metachronous tumors from 29 patients initially diagnosed with stage Ta disease. Fourteen patients (32 tumors) had non progressive disease (NPD) and 15 patients (34 tumors) had progressive disease (PD). Whole exome sequencing (WES, ~50x mean read depth), Ultra deep targeted sequencing (~6,809x mean read depth) and whole transcriptome RNA-seq was performed for all samples. In addition multiregional WES was performed on 8 adjacent regions from a single tumor.

Results: We observed more variation in the mutational spectrum of the tumors from PD patients compared to NPD patients (P=0.0013). The frequency of APOBEC-related mutations was also higher in tumors from PD patients, and intra-patient shift in APOBEC classification was observed in only one NPD patient (1/14), while it was seen in 53% (8/15) of PD patients (P=0.009). We also observed a higher proportion of clonal mutations in the ancestral branch compared to sample-specific branches (P=0.002), based on analysis of phylogenetic trees of disease evolution and calculation of cancer cell fractions. Analysis of paired tumors revealed a high degree of intra-patient mutational heterogeneity, whereas multiregional sequencing of 8 regions from a single tumor biopsy showed little intra-tumor heterogeneity. A low number of subpopulations were present in the individual tumors (PyClone), and overall we found the presence of a subclone in all cancerous cells from all patient samples and the presence of one or two subclones characterizing individual sample. Finally, allele specific expression (ASE) in Ta tumors was found to be higher in tumors from PD patients compared to NPD patients (P=1.18e-56), with tumor suppressor genes showing a larger ASE than oncogenes (P=0.0164 vs P=0.417).

Conclusions:The higher intra patient variation of the tumor mutation spectrum in patients with progressive disease suggests that a new mutational profile develops during progression. Furthermore, we found high temporal and low spatial mutational heterogeneity, suggesting that targeted treatment decisions ideally should be based on analysis of biopsies from several tumors.

#2378

Responses of AML patients to tailored drug regimens: monitoring cancer subclones by ultra-deep resequencing.

Poojitha N Ojamies,1 Mika Kontro,2 Henrik Edgren,3 Pekka Ellonen,1 Sonja Lagstrom,1 Henrikki Almusa,1 Timo Miettinen,1 Samuli Eldfors,1 David Tamborero,4 Krister Wennerberg,1 Caroline Heckman,1 Kimmo Porkka,2 Maija Wolf,1 Olli Kallioniemi1. 1 _Inst. for Molec. Medicine Finland (FIMM), Helsinki, Finland;_ 2 _Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland;_ 3 _Medisapiens Ltd, Helsinki, Finland;_ 4 _Biomedical Genomics Lab, IMIM Hospital del Mar Medical Research Institute, Barcelona, Spain_.

As part of our individualized systems medicine (ISM) program, personalized treatment options are provided to clinicians based on in-depth genomic and molecular profiling as well as ex vivo drug sensitivity and resistance testing (DSRT) of leukemia patients (Pemovska et al. Cancer Discovery, 2013). In chemorefractory AML patients (n=17), the ISM strategy has resulted in up to 35% response rate when individually selected targeted drugs have been applied in patient treatment. The responses achieved have, however, been transient and patients have typically relapsed quickly. Here, we aimed to understand the molecular basis of such treatment failures by quantitating the kinetics of individual cancer subclones before, during and after targeted treatments, as well as at the time of relapse and disease progression. Longitudinal serial samples from 13 AML patients were studied at multiple steps during leukemia progression and drug response. Clonal evolution of leukemic subclones was studied by both exome sequencing to get genome-wide overviews of disease progression, as well as by ultra-deep (>10,000x) amplicon resequencing with unique molecular identifiers to identify rare clones carrying specific cancer-relevant mutations.

Nine of the 13 patients (69%) had multiple clones by exome sequencing and displayed branching evolution. In five patients who received treatment with targeted inhibitors we observed a significant differential therapeutic response of the individual AML subclones during therapy. In some cases, this could be directly attributed to the molecular mechanisms of drug response and resistance, such as the loss of NF1 in a subclone leading to cytarabine resistance or the loss of the FLT3-positive subclone in a patient responding to sunitinib treatment. Despite a prominent drug response at the level of the subclone carrying the driver mutations, in all these patients a new subclone emerged that led to progression of the disease. In three of the patients, the dominant clone appearing at relapse was already detected as a minor subclone in the diagnostic sample by amplicon resequencing. Amplicon sequencing enabled us to detect these minor subclones (down to 0,5% frequency) that were missed by exome sequencing. The results suggest that relapses in AML may arise because the drug-resistant subclone exists already before the onset of therapy.

Overall, it is necessary to quantify tumor evolution and drug responses at the level of cancer subclones. Ultra-deep resequencing can be used to monitor drug responses at the subclone level, even at very low frequencies. This could facilitate early detection of small subclones with important prognostic implications, as well as the design of intelligent combinations of targeted drugs that could block such subclones.

#2379

Spatial and temporal clonal evolution during development of metastatic urothelial carcinoma.

Mathilde B.H. Thomsen,1 Iver Nordentoft,1 Philippe Lamy,1 Soren Hoyer,2 Soren Vang,1 Jakob Hedegaard,1 Michael Borre,3 Jorgen B. Jensen,3 Torben F. Orntoft,1 Lars Dyrskjot1. 1 _Department of Molecular Medicine, Aarhus University Hospital, Aarhus N, Denmark;_ 2 _Department of Pathology, Aarhus University Hospital, Aarhus C, Denmark;_ 3 _Department of Urology, Aarhus University Hospital, Aarhus N, Denmark_.

Background: To better treat metastatic bladder cancer, it is important to understand the clonal evolution in space and time and to identify aggressive subclones giving rise to metastatic spread. Patients with metastatic bladder cancer have a median survival of 3-6 months if untreated and 13-14 months median survival if they receive adjuvant chemotherapy. Targeted therapy using e.g. antibodies or small molecule cancer drugs has not yet shown convincing results - likely due to lack of genetic profiling prior inclusion to therapy. Here we performed a thorough characterization of the spatial and temporal tumor heterogeneity in three patients with metastatic bladder cancer by whole exome sequencing (WES) of multiple small cellular regions from primary and metastatic tumor biopsies.

Methods: Biopsies from primary tumors, lymph node metastases, and distant metastases from three patients were studied. DNA was extracted from small cellular regions procured by laser-microdissection or punctures along with matched germline DNA. WES was performed using either Nextera Rapid Exome Capture or TruSeq Nano DNA Library Prep combined with SeqCap EZ Exome Capture and sequenced on the Illumina HiSeq2000 or NextSeq500 platforms.

Results: Exome sequence information from 24 cellular regions and germline DNA was obtained. The average mean target coverage obtained for tumor samples was 68X(27-215X) and germline was 167X(95-301X). Mutation calling using MuTect identified 256, 265 and 378 somatic SNVs in tumors from the three patients. We observed low spatial intra-tumor heterogeneity but large inter-tumor (primary vs metastasis) heterogeneity. We further identified 6-12 SNVs in known disease driver genes per patient. Public (present in all sampled regions) disease driver mutations could be identified in all cases, however, over time regional driver mutations emerged, especially following metastatic spread. We observed low allele frequencies across all cellular regions, and based on this we hypothesize that the distribution of the individual clones is intermixed. Further, we observed a similar intermix of sub clones in the metastatic lesions, which may be explained by metastases seeding as cell clusters or in individual clones in parallel.

Conclusions: The three patients studied showed low spatial intra-tumor heterogeneity but large genetic diversity between primary tumor and metastatic lesions. The differences between primary tumors and metastatic lesions observed emphasize the challenges in designing rational targeted therapy solely based on the genetic profile of the primary tumor.

#2380

Head and neck patient derived xenografts acquire histopathological and growth rate changes over increasing passages.

Alexander Pearson,1 Kelsey A. Finkel,1 Kristy A. Warner,1 Felipe Nor,2 David A. Tice,3 Manoela D. Martins,2 Trachette L. Jackson,1 Jacques E. Nor1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;_ 3 _Medimmune, Gaithersburg, MD_.

The purpose of this work was to model the growth rate and histopathological features of patient derived xenograft (PDX) tumors across multiple in-vivo passages. PDX models are frequently deployed in translational cancer research, but the growth rate consistency over time is unclear. While growth rates are generally assumed to be stable, changes in PDX growth over time in the absence of treatment could significantly change the interpretation of translational studies. Here, we examined PDX tumors over time to determine if they have stable growth rates and histopathological features from initial implantation across multiple passages. We implanted and developed three distinct PDX models derived from primary human head and neck squamous cell carcinoma (HNSCC) and adenoid cystic carcinoma (ACC) through at least four passages. To compare growth rates over multiple mouse life spans, we developed a mathematical approach to merge growth data from different passages into a single measure of log relative tumor volume (log-RTV) normalized to study initiation size. We analyzed log-RTV over time with linear mixed effect models and found that tumor growth rate changed over time. In each of the three PDX models tested, the log-RTV was significantly better described by a positive quadratic function compared to a linear function (P < 0.0001 in all three models). This finding implies that the growth rate is not stable over long periods of time, and changes across multiple xenograft passages. To further investigate the etiologies of these changes, two oral pathologists were blinded and analyzed PDX tissues for SCC and ACC models to determine if histopathological features changed over in-vivo passages. We found a significant correlation between passage number and HNSCC nuclear pleomorphism (p = 0.01), ACC histopathological pattern (p < 0.0001), and other features indicative of higher tumor grade in both the squamous cell carcinoma and adenoid cystic models. Our log-RTV transformation of PDX data allows statistical analysis of tumor growth data over long periods of time, including over multiple xenograft passages. In conclusion, this new analysis method allows for the design of longer translational xenograft experiments that span across multiple in-vivo passages. Non-linear tumor growth in our regression models revealed the exponential growth rate of PDX models increased over time. The tumor growth rate changes corresponded with quantifiable histopathological features closely related to passage number in multiple types of head and neck cancer.

#2381

**A multiancestral model of colorectal cancer:** in vivo **evidence that early heterogeneity contributes to cancer progression.**

Alyssa A. Leystra, Brook Luers, Junbo Son, Chelsie K. Sievers, Amanda M. Wisinger, Alexander R. Schwartz, Christopher D. Zahm, Kristina A. Matkowskyj, Dawn M. Albrecht, Linda Clipson, Dustin A. Deming, Michael A. Newton, Richard B. Halberg. _University of Wisconsin, Madison, WI_.

Background: Intratumoral heterogeneity has been linked to tumor progression and chemotherapy resistance in the clinic. Recent models of colorectal tumor evolution indicate that heterogeneity likely arises early during the first few cell divisions and is maintained in a non-Darwinian fashion with discrete clones coevolving. However, how this heterogeneity arises is not fully understood. We propose that some cancers are derived from multiple unique ancestors, and that discrete clones enhance establishment, growth, progression, and resistance to therapy.

Methods: Mice carrying the Min allele of Apc and expressing a constitutively active form of PI3K in a subset of colonic epithelial cells develop multiple adenomas and adenocarcinomas in the colon. Cell lineage tracing and fluorescent endoscopy were used to follow the progeny of individual founding cells through establishment, growth, progression, and response to targeted therapy. Cell sorting and 3D tumor spheroid co-culture were used to further examine the growth behavior and treatment response in vitro.

Results: Nearly half (44%; 30/68) of the tumors were derived from at least two ancestral clones. The presence of multiple clones was associated with an increased likelihood of a tumor becoming invasive (p=0.006). Moreover, each clone was more invasive within multi-ancestral tumors than within their homotypic counterparts, indicating that the increased invasion is shared among clones rather than owing to a single dominant clone (p=0.05). Additionally, the presence of both clones appeared to protect susceptible clones from targeted therapy. In vitro experiments demonstrated that co-evolved clones adopted similar growth patterns, whereas independently evolved clones did not.

Conclusions: Taken together, these data strongly indicate that distinct tumor founding cells and their coevolving progeny can contribute to tumor establishment and moreover can enhance growth and survival during tumor progression and response to therapy.

#2382

Novel integration of genomic and morphological information reveals tumor tissue heterogeneity.

Chen-Chung Lee,1 Paul Predki,1 Christopher Raub,2 Darryl Shibata,3 Clive Taylor,1 Emil Kartalov,4 Kenna Anderes1. 1 _Curate Biosciences, San Diego, CA;_ 2 _Catholic University of America, Washington DC, DC;_ 3 _Keck School of Medicine of USC, Los Angeles, CA;_ 4 _University Southern California, Los Angeles, CA_.

Cancer treatment decisions today are predominantly based upon decades-old histological methods, which more recently have been combined with simple phenotypic or molecular tests. To advance this paradigm, we have developed a digital molecular morphology platform that integrates histological images with next-generation sequencing (NGS) or PCR data. This allows us to visualize DNA mutations and RNA expression levels across a histological image, and to correlate mutational or expression status to cell morphological features. Thus, providing a novel approach to mapping tumor heterogeneity. The basic approach is to overlay a microstructure on top of H&E-stained FFPE slides to create a large number of tissue-bottomed micro-wells, which can be as small as a single cell or contain clusters of cells. Reagents are added to the micro-wells and biochemical reactions are carried out and analyzed in place (PCR) or partially offline (NGS). In the case of NGS, barcodes are used to reference the resulting sequence back to the tissue image. Software is then used to analyze and visualize the resulting genetic information in the context of the original histological image. Our initial work has focused on FFPE cancer biopsies using targeted cancer genes and gene panels. Here we demonstrate the use of this approach to measure and visualize KRAS G12V mutational status in colorectal cancer tissues, using both PCR and NGS detection modalities. We show that with this method we are able to efficiently extract DNA from the tissue while maintaining a leak-proof physical integrity from one micro-well to another. Further, we are able to PCR-amplify DNA in the micro-wells for direct fluorescence detection, or to create libraries for sequencing. Detection of KRAS G12V correlates well with malignant morphological features in the H&E stained tissue, and appears to present a more sensitive way to detect mutations than PCR or NGS from bulk FFPE tissue alone. We are currently applying this technology to a variety of research and clinical applications, ranging from assessment of tumor heterogeneity and evolution, to prediction of patient outcome, to post-operative NGS-based characterization of surgical margins. This integration of cellular and molecular data promises to more fully enable precision medicine to guide the course of treatment and improve individual patient outcomes.

#2383

Multiregional RNA sequencing identifies intratumor transcriptomic heterogeneity in a subset of early-stage hepatocellular carcinoma.

Amanda Craig,1 Mehmet E. Ahsen,2 Ismail Labgaa,1 Ashley Stueck,3 Delia D'Avola,1 Stephen C. Ward,3 Maria Isabel Fiel,3 Ganesh Gunasekaran,4 Josep Llovet,1 Swan Thung,3 Myron Schwartz,4 Bojan Losic,5 Gustavo Stolovitzky,5 Augusto Villanueva1. 1 _Division of Liver Diseases, Liver Cancer Program, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _T.J. Watson Research Center, IBM, Yorktown Heights, NY;_ 3 _Department of Pathology, Liver Cancer Program, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY;_ 4 _Department of Surgery, Liver Cancer Program, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY;_ 5 _Division of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY_.

BACKGROUND: Hepatocellular carcinoma (HCC) is known to have histological intra-tumor heterogeneity. Little is known about the underlying molecular contributions to this observation, or its potential impact on resistance to therapies.

AIMS: 1) To evaluate intra-tumoral molecular heterogeneity of primary HCCs using multi-regional RNA-seq; 2) to correlate molecular alterations with histological features at distinct areas.

METHODS: We analyzed 55 fresh-frozen tissues from 10 patients with BCLC-A HCC treated with surgical resection. Multi-regional sampling included 38 HCCs and 16 adjacent non-tumoral tissues (average 5.4 samples per patient). We performed H&E staining and RNA-seq on all samples. Each sequenced section was evaluated for a panel of 11 histological features. Data analyses included clustering (consensus and multidimensional scaling (MDS)), sample ordering via linear graph modeling of expression, pathogen quantitation, gene set enrichment analysis (GSEA), and nearest template method (NTP) for gene signature prediction.

RESULTS: Patients enrolled were mostly males (70%), with an average age of 59, all with single-nodule HCC, and a median tumor size of 5.3 cm (range 3-16 cm). Background liver disease was HBV in 50% of cases without histological cirrhosis in 80%. MDS and consensus clustering identified heterogeneity in 40% (4/10) of patients as defined by at least one sample not clustering with their counterparts. Sample ordering uncovered dominant tumor heterogeneity evolution paths, including HBV specific ones. GSEA revealed differential enrichment (FDR<0.05) of Cancer Hallmark Gene Sets in the 4 heterogeneous tumors. Indeed, these 4 tumors showed concordant gene set enrichment (FDR<.05) in all regions only 19% of the time compared to 83% in homogenous tumors on average. NTP further confirmed heterogeneity in molecular classification predictions in 2/4 heterogeneous tumors (e.g., Proliferation class). There were no significant differences in size or regions sequenced between heterogeneous and homogenous tumors. Molecular outliers show distinct histological features such as high mitotic rate and poorer differentiation.

CONCLUSION: Transcriptomic intra-tumor heterogeneity is frequent in a subset of surgically resected HCC, and it correlates with histological features. Integrative analysis of CNVs, expressed mutations, allele specific expression, and gene fusions is ongoing.

#2384

High-complexity neutral genomic barcoding technology reveals extensive clonal dynamics in multiple human cancer model systems.

Allison ML Nixon,1 Kevin R. Brown,1 Jennifer Haynes,2 Laura K. Donovan,3 Michael D. Taylor,3 Catherine W. O'Brien,2 Jason Moffat1. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _University Health Network (UHN), Toronto, Ontario, Canada;_ 3 _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Increasing evidence of extensive intratumoral heterogeneity, along with advances in high-throughput in vivo functional genetic screening technologies, have together highlighted the need to observe growth in cancer models at the clonal level. To address this, we have designed, constructed and validated multiple high-diversity lentivirally delivered barcode libraries, which utilize next-gen sequencing technology to read out millions of clones in heterogeneous cancer populations. These libraries can be used to address a multitude of biological questions in many cancer model systems. To date, we have completed, sequenced and analyzed three different types of barcoding applications to explore clonal dynamics in a variety of human cancer models. First, we tested the serial limiting dilution analysis (LDA) assay for tumor initiating cells (TICs) in patient-derived colon tumor models. In the LDA assay, a range of cell dilutions, down to a single cell, are transplanted into immunocompromised mice. The TIC frequency is calculated using a single-hit model from the proportion of tumors established at each dose. However, quantifying minimum cell numbers required for tumor formation does not reveal the actual diversity of clonal contribution during tumour engraftment and progression. In fact, recent research suggests that the single-hit model, which assumes a static hierarchy of intrinsically determined initiating cells, may not be biologically relevant as some clones may have the potential to form or contribute to a tumor only in specific environment, or in cooperation with another clone. Our barcoded, serial LDAs show interesting and informative patterns of clonal dynamics. Second, we have performed clonal lineage tracing on established human cancer cell lines in vitro compared to in vivo. Our findings demonstrate the presence of TICs in standard cell lines and serve as precursors to future in vivo genetic screens by defining upper limits of library size. Finally, we used a patient-derived xenograft model of brain tumor metastasis to investigate the presence of pre-existing metastatic clones with site-specific homing abilities. In summary, given the extensive heterogeneity present in cancer models, we propose the use of high-complexity barcoding technology to validate novel cancer targets in xenograft models, including LDAs and metastasis models. Furthermore, we suggest the use of barcodes for careful optimization of the system before genetic screens in animal models.

#2385

Understanding the dynamic interplay between genetically different cancer cell clones in glioblastoma.

Min Guo,1 Susanne Heller,1 Jessie Thorslund,2 Lukas Orre,2 Janne Lehtiö,2 Monica Nistér,1 Daniel Hägerstrand1. 1 _Department of Oncology-Pathology, Karolinska Institutet, Solna, Sweden;_ 2 _Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institutet, Solna, Sweden_.

Glioblastoma is the most frequent and aggressive brain tumor in adults. Based on genomic data glioblastomas can be classified into at least four subclasses: proneural, neural, classical and mesenchymal. However, recent publications have shown that individual glioblastoma can be heterogeneous and contain a mixture of these four subclasses. Moreover, it has been shown in other studies that intratumoral communication between genetically distinct cancer cell subclones in glioblastomas can affect the overall tumor growth via secreted proteins. In this project we aim to investigate the difference between protein secretomes from different tumor cell clones from within genetically heterogeneous glioblastomas.

For this we have used the U343 cell culture system, consisting of U343MG, U343MGa, U343MGa-31L and U343MGa-Cl2:6. These cultures were derived from a single glioblastoma, and can be divided into at least two categories based on their mutually exclusive expression patterns of FN1 and GFAP. Here we show that these different cultures display different characteristics with regard to matrigel invasion capacity, neurosphere formation capacity, and gene and protein expression. Combinatorial co-culture and conditioned media based experiments showed that the U343MG culture elicit anti-proliferative effects on U343MGa-31L via secreted factors. To identify proteins that are secreted by U343MG we have used Secretome Protein Enrichment with Click Sugars (SPECS) followed by mass spectrometry analysis. We detected 150 proteins by more than 2 peptides in U343MG conditioned media. Several of these proteins were growth factors and matrix proteins, including FN1. We are now performing a combined secretome and gene expression analysis of all U343 cultures to identify candidate secreted proteins that most likely are involved in the observed signaling effects between U343MG and U343MGa-31L. Finally, a functional genomic approach will be taken to experimentally pinpoint mediators of these inter-clonal effects.

This study shows that subclones from a heterogeneous glioblastoma can display different phenotypic characters and affect each other via secreted factors. Further knowledge about cell-to-cell communication through secreted proteins in glioblastoma may provide novel therapeutic targets.

#2386

Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition.

Ioannis Ragoussis,1 Paul Savage,2 Yu-Chang Wang,1 Timothee Revil,1 Dunarel Badescu,1 Sadiq Saleh,2 Ernesto Iacucci,1 Nicolas Bertos,2 Anie Monast,2 Attila Omeroglou,3 Dongmei Zuo,2 Morag Park2. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _The Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada;_ 3 _Department of Pathology, McGill University, Montreal, Quebec, Canada_.

Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor, a finding replicated in several independent experiments. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. One population was characterized by high MYC and the other high EGFR/KRT14. These findings were validated using immunohistochemistry on PDX and primary tumor material. Further functional experiments showed that that EGFR subpopulation has stem cell character istics and forms metastasis in the animal model. Microfluidic whole genome amplification followed by whole exome capture of 81

single cells, along with exome sequencing of the germline, primary and post treatment tumor and whole PDX showed that BRCA1 and TP53 are mutated in all single cells, as well as a number of sub-clonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 known cancer driver genes. Careful comparison of the exome sequencing data allowed the association of driver gene mutation prevalence with tumor progression. These findings are important in our understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance.

#2387

Pan-cancer analysis of clonal evolution reveals the costs and adaptive benefits of genomic instability.

Noemi Andor,1 Trevor A. Graham,2 Marnix Jansen,2 Li C. Xia,1 Athena Aktipis,3 Claudia Petritsch,3 Hanlee P. Ji,1 Carlo C. Maley4. 1 _Stanford, Palo Alto, CA;_ 2 _Barts Cancer Institute, London, United Kingdom;_ 3 _UCSF, San Francisco, CA;_ 4 _Biodesign Institute, Arizona State University, Tempe, AZ_.

Cancers are a mosaic of clones of varying population sizes. Any single cancer sample encodes a tumor-metagenome, since it represents the aggregate genomes of diverse clones that coexist within the sample. We quantified genomic instability as the fraction of the tumor-metagenome affected by copy number variations (CNVs) and leveraged two tumor mixture separation algorithms, EXPANDS and PyClone, to quantify genetic intra-tumor heterogeneity (ITH) from single cancer samples. We tested the potential of measures of genomic instability and ITH as prognostic biomarkers across 1,165 exome sequenced primary tumors from 12 cancer types at TCGA. Our results suggest that a tradeoff between the costs and adaptive benefits of genomic instability governs differential radiotherapy sensitivity.

Between 1 and 18 clones were estimated to coexist per tumor sample at >10% cell frequency (median=4). Clone number varied considerably within and between cancer types, with melanomas representing the most heterogeneous group. 86% all analyzed tumor samples contained at least 2 clones. Across cancer types, the presence of >2 clones was associated with worse overall survival as compared to tumors in which <=2 clones were detected (Log-rank test: P=8.6E-4, HR=1.49). An exceptionally favorable outcome was observed when >75% genomic instability was shared among <=2 clones. The highest risk was observed among individuals with an intermediate number of 4 clones - additional diversification beyond 4 clones did not impart further risk. The highest risk was also observed among individuals with 50-75% genomic instability, in both the original exome sequencing dataset and an independent SNP array dataset. Genomic instability <25% or >75% predicted reduced risk (HR=0.15, 95% CI: 0.08-0.29).

We analyzed the relation between radiotherapy intensity and overall survival among 242 individuals (21%) treated with radiotherapy and found that not all individuals did benefit equally from therapy. In order to achieve the same benefit from therapy, individuals with 25-50% genomic instability required higher therapy intensity (regression slope=1.83; P=0.009) than individuals with 50-75% genomic instability (slope=2.09; P=0.005). In contrast, individuals with <25% genomic instability did not benefit from increasing therapy intensity (slope=0.71; P=0.311).

Radiotherapy may be particularly effective against tumors with intermediate CNV burden, by pushing them past the limit of 'tolerable' genomic instability. Our results from two independent pan-cancer cohorts suggest that this limit is exceeded when >75% of a tumor's metagenome is affected by CNVs. This upper limit of tolerable genomic instability may be responsible for the non-linear association we observed between genetic ITH and survival. Leveraging a clone's distance to the upper limit of tolerable genomic instability may represent a new strategy to optimize therapy intensity.

#2388

Molecular heterogeneity and trunk driver mutations in hepatocellular carcinoma.

Daniela Sia,1 Andrew Neelis Harrington,1 Sara Torrecilla,2 Zhongyang Zhang,3 Genis Camprecios,1 Agrin Moeini,2 Sara Toffanin,1 Maria Isabel Fiel,1 Ke Hao,3 Monica Higuera,1 Laia Cabellos,2 Helena Cornella,2 Milind Mahajan,1 Yujin Hoshida,1 Augusto Villanueva,1 Sander Florman,1 Myron Schwartz,1 Josep Maria Llovet1. 1 _Mount Sinai Liver Cancer Program (Divisions of Liver Diseases, Department of Medicine, Department of Pathology, Recanati Miller Transplantation Institute), Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Barcelona-Clínic Liver Cancer Group (HCC Translational Research Laboratory, Liver Unit, Hepato-biliary Surgery), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic, CIBERehd, Universitat de Barcelona, Barcelona, Spain;_ 3 _Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY_.

Background and aims: Molecular heterogeneity in hepatocellular carcinoma (HCC) is ill-defined since trunk drivers (early events; common to all cells), branch drivers (later events; present in a subset of cells) and passenger mutations (not relevant), have not been thoroughly described. Most FDA/EMA approved molecular drugs target trunk drivers. We explored heterogeneity by analyzing trunk vs branch mutations in different HCC regions within single and multinodular tumours.

Methods: Intra-tumoral heterogeneity was assessed in 21 patients with single HCCs (size > 4cm; 2 regions/tumour: 42 samples) and inter-tumoral heterogeneity was studied in 17 patients with multinodular HCCs (2-3 nodules/patient; total: 39 samples). Gene expression profiling, SNP array and deep-sequencing (coverage ~850x) assessing 6 oncodrivers (TERT promoter, TP53, CTNNB1, ARID1A, AXIN1-2 by TruSeqAmplicon, validated by sanger) were explored. Clonality differentiating metastatic (clonal) vs synchronic (non-clonal) tumours was defined by SNP profiles. Trunk mutations were defined as present in a) all regions of a given tumour, or b) in all nodules of metastatic-clonal tumours; all other were considered as branch.

Results: Intra-tumoral heterogeneity assessed by sequencing identified at least 1 oncodriver in 19/21 patients with single tumours. Among those, trunk mutations accounted for 17/19 (90%), and branch for 2/19 cases. Overall 63 mutations were identified, 56 (90%) were identical in different tumoral regions (i.e. truncal; TERT promoter most prevalent). Inter-tumoral heterogeneity explored by SNP profiles defined metastases in 35% (6/17 multinodular cases) and synchronous tumors in 65% (11/17 cases). Genetic proximity confirmed clonality in all metastatic nodules. Regarding molecular subclasses, half of clonal tumours retained identical molecular fingerprint, but the other half switched to more aggressive subclass. All non-clonal tumours belonged to distinct molecular subclasses. Driver oncogenes were explored in 9 patients (5 metastasis and 4 synchronic). Metastatic tumours showed 13 mutations, among which 11 (85%) were truncal. Mutations in non-clonal synchronic tumours were distinct.

Conclusions: Single large HCCs shared common trunk drivers at distinct regions (90%). Similarly, 40% of multinodular tumours were clonal (metastasis) and shared common trunk oncodrivers, while 60% were synchronic, with distinct genomic profile/oncodrivers. Further studies at single-cell sequencing level are recommended.

#2389

Microarray analysis of polysome-associated mRNAs from different regions of the same human glioblastoma reveals intratumoral heterogeneity.

Glaucia N. Hajj, Fernanda S. Lupinacci, Martín Roffé, Hellen Kuasne, Tiago G. Santos, Victor P. Andrade, Paulo Sanematsu, Vilma R. Martins, Silvia R. Rogatto. _AC Camargo Cancer Center, São Paulo, Brazil_.

Glioblastoma is among the most aggressive tumor type and less responsive to chemotherapeutic agents, thus a better understanding of the behavior of these tumors may help to develop new treatments for this disease. Currently, many genome-wide projects attempt to define general patterns of gene expression based on deep sequencing or microarray data from total mRNA populations. However, this approach provides little information about the molecular mediators of tumor biology, because the expression levels of mRNAs do not necessarily reflect the levels of proteins. The identification of mRNAs target of translational alterations in tumors can show gene expression profiles that better reflect the population of proteins. In this work we were able to identify mRNAs differentially translated in a human glioblastoma that presented histologically different parts. The sample was divided in 8 pieces that were classified as high or low grade based on histological characteristics. Actively translating ribosomes and their associated mRNAs were isolated biochemically through a polysomal profile. Total and polysomal mRNA was then extracted and submitted to a microarray analysis. By comparing high vs low grade tumor pieces, we were able to identify more differentially translated genes (138 genes) than differentially expressed genes (43 genes). Among the differentially translated mRNAs, there are many related to proliferation, development and cancer. By Integrated Pathway Analysis, we identified the TGF-β pathway among the the most relevant for grade progression. Thus, the technique of isolating mRNAs engaged in translation can be used to identify biomarkers of tumor progression, leading to new therapeutic approaches.

#2390

The polyclonal nature of multinodular goitre revealed by DICER1 sequencing.

Leanne de Kock,1 Ismaël Bah,2 Mona Wu,1 John R. Priest,3 Jiannis Ragoussis,4 William D. Foulkes5. 1 _Lady Davis Institute and Segal Cancer Centre, Jewish General Hospital; McGill University, Montreal, Quebec, Canada;_ 2 _Department of Pathology, McGill University Health Centre, Montreal, Quebec, Canada;_ 3 _Minneapolis, MN;_ 4 _McGill University and Genome Quebec Innovation Centre & Department of Human Genetics, McGill University, Montreal, Quebec, Canada; _5 _Program in Cancer Genetics, Department of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada_.

Nontoxic multinodular goitre (MNG) occurs frequently in the general population and is characterised by nodular enlargement of the thyroid gland. In 2011, Rio Frio et al discovered that familial MNG and MNG occurring with Sertoli-Leydig cell tumours were associated with germ-line mutations in the microRNA-processing gene, DICER1. We recently reported the first instance of biallelic DICER1 mutations in an MNG. This prompted us to further investigate the role of DICER1 mutations in MNG pathogenesis.

Given that MNG may either be a monoclonal or polyclonal disease, we set out to investigate whether the molecular landscape was correspondingly heterogeneous. We obtained core biopsies (n = 27) from 9 MNGs. 1mm cores were excised from morphologically distinct regions, including from areas of follicular hyperplasia (FH), hyperplasia within colloid pools (HC) and/or otherwise unremarkable areas. DNA extracted from each core was processed using a custom Fluidigm Access Array followed by deep sequencing. We focused our investigation on the RNase IIIb domain of DICER1 to identify known hotspot mutations. All mutations were validated by Sanger sequencing.

We identified 6 individually distinct DICER1 RNase IIIb hotspot mutations in 14 (of 27) cores consisting of FH or HC (Table 1). These mutations affected the metal-ion binding residues at positions 1705, 1709, 1810 & 1813. Two MNGs that arose in individuals without germ-line DICER1 mutations did not carry any RNase IIIb hits. Of the remaining 7 germ-line DICER1-mutated cases, 2 carried 1 RNase IIIb mutation; 4 carried 2 different RNase IIIb mutations and one case did not carry any successfully-validated RNase IIIb mutations.

We demonstrate that spatial heterogeneity of RNase IIIb DICER1 mutations exists in MNG. We postulate that cyclic hyperplasia followed by regression over time in response to external stimuli, particularly in germ-line DICER1 mutation carriers, contributes to the polyclonal accumulation of RNase IIIb mutations in MNG.

Table 1: DICER1 RNase IIIb Mutation Frequencies

---

Case | Germ-line DICER1 Status | Morphology | c.5113G>A

p.E1705K | c.5125G>A

p.D1709N | c.5126A>G

p.D1709G | c.5428G>T

p.D1810Y | c.5429A>T

p.D1810V | c.5437G>C

p.E1813Q

1 | Positive | HC | 41.7 | \-- | \-- | \-- | \-- | \--

FH | \-- | \-- | \-- | \-- | \-- | 22.1

2 | Positive | FH | \-- | \-- | \-- | \-- | \-- | \--

UR | \-- | \-- | \-- | \-- | \-- | \--

HC | \-- | \-- | \-- | \-- | \-- | \--

UR | \-- | \-- | \-- | \-- | \-- | \--

HC | \-- | \-- | Sanger | \-- | \-- | \--

HC | \-- | \-- | Sanger | \-- | \-- | \--

3 | Positive | HC | \-- | \-- | \-- | \-- | 29.2 | \--

FH | \-- | 43.7 | \-- | \-- | \-- | \--

FH | \-- | 28.8 | \-- | \-- | \-- | \--

4 | Positive | HC | \-- | \-- | 30.6 | \-- | \-- | \--

UR | \-- | \-- | \-- | \-- | \-- | \--

HC | \-- | \-- | 33.1 | \-- | \-- | \--

FH | \-- | \-- | \-- | 52.3 | \-- | \--

5 | Positive | HC | \-- | \-- | \-- | \-- | \-- | \--

HC with FH | \-- | \-- | \-- | \-- | \-- | \--

6 | Positive | FH | \-- | \-- | \-- | \-- | \-- | 39.3

FH | \-- | \-- | \-- | \-- | \-- | 13.1

FH | \-- | \-- | \-- | \-- | \-- | \--

7 | Positive | FH | \-- | \-- | \-- | 41.8 | \-- | \--

FH | 28.0 | \-- | \-- | \-- | \-- | \--

8 | Negative | FH | \-- | \-- | \-- | \-- | \-- | \--

HC | \-- | \-- | \-- | \-- | \-- | \--

9 | Negative | FH | \-- | \-- | \-- | \-- | \-- | \--

PTC | \-- | \-- | \-- | \-- | \-- | \--

FH | \-- | \-- | \-- | \-- | \-- | \--

Abbreviations: FH, follicular hyperplasia; HC, hyperplasia within colloid pools; UR, unremarkable; PTC, papillary thyroid ca. Notes: Each row corresponds to a separate core. Mutant allele frequencies are indicated in each cell (all >10%); ''--'' indicates mutation not observed in core DNA; Sanger, indicates mutations previously identified in an independent PCR from bulk MNG DNA, tested by Sanger sequencing. All Fluidigm positives were confirmed by Sanger sequencing from the same DNA that was used to do the Fluidigm analysis.

#2391

Flow cytometric sorting of subpopulations followed by RNASeq reveals distinct phenotypes in PDX model of basal breast cancer.

Warren Porter,1 Friedrich Hahn,1 Eileen Snowden,1 Mitchell Ferguson,1 Frances Tong,1 Shannon Dillmore,1 Joel S. Parker,2 Aaron Middlebrook,3 Smita Ghanekar,3 Rainer Blaesius1. 1 _BD Technologies, Research Triangle Park, NC;_ 2 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 3 _BD Biosciences, San Jose, CA_.

The recognition of tumor tissue as an interactive ecosystem of distinct cell types has recently emerged as a very promising basis for more successful treatment of cancer patients. While intratumor heterogeneity (ITH) as a phenomenon has been known for decades it is only recent that its functional significance can be investigated with effective tools. Starting with a correlation of cellular heterogeneity with aggressiveness, metastatic potential and drug susceptibility of a cancerous lesion the current focus on functional differences between various tumor cell compartments is revealing a number of distinct functions and interactions. The discovery of communication between various tumor cells through soluble factors as well as differences in implantation of homogeneous vs. heterogeneous cell populations into immune compromised mouse models suggest a "division of labor" among cell types within many tumor tissues.

We have investigated the surface marker distribution of more than 8 different PDX tumor models by flow cytometry and detected extensive immunophenotypic heterogeneity. Using a range of markers associated with cancer stem cells, EMT and invasiveness (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) we find heterogeneity with respect to many surface markers as well as individual immunophenotypic signatures for each model. Building on this characterization we chose several markers for sorting of subpopulations and performed gene expression analysis. Transcriptome analysis of a breast cancer model revealed two phenotypic signatures which suggest a strong proliferative population alongside a second population which is far less proliferative but much more active in angiogenesis, ECM organization and secretion of various soluble factors. This observation suggests a very clear example of distinct roles of multiple cell types to form a tumor tissue.

Our findings could have implications for therapeutic strategies directed at more than one cell type as well as development of better diagnostic tools which take into account the presence of various phenotypically distinct cell populations.

#2392

Genomic and immune heterogeneity in synchronous melanoma metastases is associated with differential tumor growth and response to therapy.

Alexandre Reuben, Christine N. Spencer, Peter A. Prieto, John P. Miller, Xizeng Mao, Wei-Shen Chen, Hannah Cheung, Hong Jiang, Cara Haymaker, Mariana Petaccia De Macedo, Haven R. Garber, Pei-Ling Chen, Vancheswaran Gopalakrishnan, Jacob Austin-Breneman, Courtney W. Hudgens, Jason Roszik, Patrick Hwu, Scott E. Woodman, Lynda Chin, Michael A. Davies, Rodabe N. Amaria, Sapna P. Patel, Alexander J. Lazar, Michael T. Tetzlaff, Karen C. Dwyer, Ignacio I. Wistuba, Padmanee Sharma, James P. Allison, Jianhua Zhang, Andrew Futreal, Zachary A. Cooper, Jennifer A. Wargo. _MD Anderson Cancer Center, Houston, TX_.

There have been significant advances in the treatment of metastatic melanoma through targeted and immunotherapy, however a significant proportion of patients still progress on these regimens with many experiencing mixed responses. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. The appreciation of genetic heterogeneity as a contributor to resistance to therapy has grown, though immune heterogeneity has been poorly characterized. The goal of the present study is to better understand the molecular and immune heterogeneity in synchronous melanoma metastases at the time of disease progression. In this study, we prospectively evaluated 32 tumors from 15 patients who were treatment-naïve (n=4), or had received prior targeted (n=4) or immunotherapy (n=7). Whole exome sequencing demonstrated between 4-41% of non-synonymous exonic mutations (NSEM) were restricted to individual tumors within a patient. Deep profiling of infiltrating immune cell subsets by flow cytometry and immunohistochemistry analyses confirmed the immune infiltrate between synchronous metastases to be highly heterogeneous, specifically in regards to CD4 and CD8 T lymphocytes. In aggregate, 92% of these T cell clones were unique to distinct tumors within the same patient, with limited overlap with clones detected in the blood. NetMHC3.4 neoantigen prediction demonstrated a large fraction of predicted neoantigens were restricted to individual tumors, with over 10% of these presenting extremely high predicted affinity. Importantly, analysis of RECIST measurements of individual lesions within the same patient suggested this molecular and immune heterogeneity could contribute to differential tumor growth and response to therapy within the same patient. This has important clinical implications, and suggests a single tumor biopsy may not be sufficiently representative of the molecular and immune landscape of multiple tumors within the same individual.

#2393

Kidneys of VHL patients reveal the origin of renal clear cell carcinoma.

Mayyan Mubarak, Samuel Sommaruga, David Voigt, Xiacao Xu, Steve Kim, Ailin Song, Alexander O. Vortmeyer. _Yale University, New Haven, CT_.

VHL disease is a classic tumor suppressor gene syndrome characterized by development of specific types of tumors in selective organs with nervous system and kidney being most consistently affected. Detailed studies on human spinal cord and cerebellum have previously revealed earliest stages of CNS tumorigenesis and the morphologic sequence resulting in development of frank tumors.

To elucidate earliest stages of renal clear cell carcinoma, we performed a similar approach in kidney tissues of four VHL patients and three sporadic control cases. From all cases, blocks of interest were procured, followed by serial sectioning and 3dimensional reconstruction of potential precursor lesions. The results reveal an abundance of foci with aberrant mesonephric clear cell proliferations that initially develop along the tubular lining, but have the potential to aggregate within individual tubules. This stage is followed by microscopic invasive clear cell aggregations which represent tumor precursor structures.

This study presents evidence for a consistent morphologic sequence for renal clear cell carcinogenesis. Molecular analysis of early steps within this sequence will allow for identification of earliest genetic and proteomic changes in the future. Therapeutic targeting of earliest changes may allow to develop preventive strategies for renal cancer development for VHL patients, potentially also for the non-VHL population.

#2394

Scalable, rapid and affordable low-pass whole genome sequencing method for single-cell copy-number profiling on LM-PCR based WGA products.

Genny Buson,1 Paola Tononi,1 Claudio Forcato,1 Francesca Fontana,1 Gianni Medoro,1 Rui Neves,2 Birte Möhlendick,2 Nikolas Stoecklein,2 Nicolò Manaresi1. 1 _Silicon Biosystems, Bologna, Italy;_ 2 _Department of General, Visceral and Pediatric Surgery, Medical Faculty, University Hospital of the Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany_.

Introduction: Genome-wide profiling of copy-number alterations (CNA) in single cells enables the characterization and investigation of genomic heterogeneity and evolution in tumor cell populations such as individual circulating tumor cells (CTCs), or disaggregated solid tumors. Ampli1™ WGA is a commercially available kit for whole-genome amplification from single cells suitable for array comparative genomic hybridization (aCGH), which is currently the gold standard for genetic diagnosis of CNA. However, labor and reagent costs limit aCGH applicability. Here we introduce for the first time a rapid, high-throughput low-pass Whole Genome Sequencing (WGS)-based method for CNA analysis (LPCNA) on single cells, enabling simultaneous profiling of multiple cells at lower cost and effort.

Methods: Six single cells from NCI-H23, NCI-H661 and NCI-H1650 cell lines and 19 white blood cells (WBCs) were amplified using Ampli1™ WGA Kit. Only 5µl (1/10th of total WGA product) were used as sample input for LPCNA. Purified WGA products were processed for each sample in a single-tube, one-step reaction resulting in IonTorrent-compatible indexed libraries. Samples were pooled and size-selected to recover fragments with a length ranging from 300 to 400 bp. Following purification and template preparation, pools were sequenced on Ion 318™v2 Chips. Array CGH was performed for comparison, using Agilent SurePrint 4x180k arrays as previously reported. CNA analysis was performed using Control-FREEC for low-pass WGS data, or Agilent Genome Workbench for aCGH.

Results: By exploiting the deterministic nature of Ampli1™ whole genome amplification, we devised a method to generate IonTorrent compatible libraries in a single reaction, drastically reducing time of bench work and cost.

Method accuracy has been assessed by comparing the copy number profiles generated by our approach with about 870,000 reads/sample (average genome coverage of 0.064x, range 0.051-0.073) with those obtained by a validated aCGH method. Very good agreement in gain and loss profiles was achieved for all single tumor cells analyzed. As expected WBC analysis resulted in normal copy-number profiles with autosomes copy number = 2, with minimal false-positive rate (<0.6% at a resolution of 760kb with 618k reads).

Highlights: This innovative low-pass sequencing approach for CNA detection on single cells enables high level multiplexing on different NGS platforms. The approach is highly scalable and flexible. From few (6-8) samples (IonTorrent PGM, 318™v2 Chip), up to 96 samples (Ion Proton, Ion PI™ Chip v3) can be processed in a single run. This approach meets the need for rapid results (<7h turnaround time for library preparation) and affordable cost required for massively parallel single-cell analysis such as multiple tumor cells or CTCs.

This work has been carried out within IMI CANCER-ID consortium (www.cancer-id.eu).

#2395

Direct assessment of sequence heterogeneity in human cancers by Duplex Sequencing.

Michael W. Schmitt,1 Jesse J. Salk,1 Edward J. Fox,1 Justin R. Pritchard,2 J Graeme Hodgson,2 Victor M. Rivera,2 Pamela S. Becker,1 Jerald P. Radich,3 Lawrence A. Loeb1. 1 _University of Washington, Seattle, WA;_ 2 _ARIAD Pharmaceuticals, Inc., Cambridge, MA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

The extent of heterogeneity in DNA sequence within human cancers has been difficult to fully assess, due to the absence of methods with sufficient sensitivity to detect minority variants. For example, next-generation sequencing could in principle detect sub-clonal mutations at any genomic position; however due to amplification errors and inaccuracies in the sequencing platform itself, such approaches are limited to detection of mutations present in >1% of cells. We have overcome this limitation by developing an approach, Duplex Sequencing, which improves the accuracy of next-generation sequencing by >100,000 fold. Duplex Sequencing is based upon separately tagging and sequencing the two strands of single molecules of duplex DNA. True mutations are present at the same position in both DNA strands and are complementary, whereas artifacts arising from amplification or sequencing errors are seen in only one strand. The calculated error rate of our approach is less than one artifactual mutation per billion nucleotides sequenced (ref. 1). We have also developed an enrichment approach based on sequential rounds of hybridization to biotinylated probes which enables efficient sequencing of targeted regions of the genome (ref. 2). Duplex Sequencing thereby enables exceptionally sensitive detection of sequence heterogeneity within any set of genes. We have now applied Duplex Sequencing to study of sequence heterogeneity in both normal and malignant samples. In acute myeloid leukemia (AML), we find multiple sub-clonal mutations which fall below the detection limit of conventional approaches, some of which encode mutations that can drive chemotherapy resistance and cancer progression. Our results indicate that prior studies have under-estimated the burden of sub-clonal mutations in AML by more than 1,000-fold. We have additionally studied chronic myeloid leukemia (CML); treatment of CML with inhibitors directed against the Abl kinase represents the prototypical targeted cancer therapy. With Duplex Sequencing, we find that Abl mutations are extremely uncommon at the time of CML diagnosis. In contrast, refractory patients who fail therapy frequently possess multiple sub-clonal mutations within the Abl kinase, most of which are below the resolution of conventional approaches but are readily detected with our method. In ongoing work, we have found multiple sub-clonal mutations within human prostate cancer and colon cancer, suggestive of widespread intratumor heterogeneity that may limit the efficacy of single-agent therapy in these diseases.

References:

1. Schmitt MW, Kennedy SR, Salk JJ, Fox EJ, Hiatt JB, Loeb LA (2012). Detection of ultra-rare mutations by next-generation sequencing. Proceedings of the National Academy of Sciences USA. 109(36):14508-13.

2. Schmitt MW, Fox EJ, Prindle MJ, Reid-Bayliss KS, True LD, Radich JP, Loeb LA (2015). Sequencing small genomic targets with high efficiency and extreme accuracy. Nature Methods. 12(5):423-5.

#2396

Heterogeneity of colorectal cancers and multiple liver metastases by mutation and copy number profiling.

Sharmini Alagaratnam,1 Tuva Høst Brunsell,1 Stine Aske Danielsen,1 Anita Sveen,1 Mette Eknæs,1 Merete Hektoen,1 Bård Røsok,2 Bjørn Atle Bjørnbeth,2 Arild Nesbakken,2 Ragnhild Lothe1. 1 _Dept for Molecular Oncology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway;_ 2 _Dept of Gastrointestinal Surgery, Oslo University Hospital, Oslo, Norway_.

To investigate genetic and genomic heterogeneity in liver metastases of colorectal cancer, multiple tumor foci in the liver per patient were identified on the basis of radiological images and samples from these systematically biobanked. For a total of 67 patients, 258 metastatic tumor samples were collected (minimum of 2 and maximum of 9 samples per patient, median of 4 samples per patient). Additionally, for n=14 patients, tumor samples from the corresponding primary colorectal cancer were also available. DNA isolated from these samples was subjected to mutation profiling by Sanger sequencing for the genes BRAF, KRAS, PIK3CA and TP53, as well as allele-specific copy number profiling by CytoscanHD SNP array. For the four genes investigated for mutations, 39/67 (58%) of the patients were homogeneous in their mutation profile, while 21/67 (32%) were heterogeneous (one or more samples had mutations not detected in all samples). For 7/67 patients (10%), no mutations were detected in any of these four genes. For 9/14 patients with matching primary tumors, there was complete concordance between the primary tumor and all liver metastases profiled for mutations, while the remaining 5/14 patients showed discordance in mutation profile between the primary tumor and at least one of the liver metastases profiled. High resolution copy number profiles of this cohort detected many of the aberrations previously described in primary colorectal cancer, specifically gain of chromosomes 7, 8q, 13q and 20q, and loss of chromosomes 5q, 8p, 15q, 17p and 18q. The extent of heterogeneity observed in patient-wise mutation profiles was largely mirrored in the copy number profiles of the same tumors. 16/27 (60%) of the patients for which both mutation and copy number profiles were available showed concordance in the degree of heterogeneity per patient. In summary, intra-individual heterogeneity varied from patient to patient, with results from the genetic and genomic data types largely concurring.

#2397

Mutation signatures and intratumor heterogeneity of esophageal squamous cell carcinoma in a Chinese cohort.

Yu Wang,1 Wenqing Yuan,2 Mengfei Liu,2 Jian Bai,3 Qingxuan Song,1 Zhen Liu,2 Jingjing Li,2 Amir Abliz,2 Changqing Zeng,3 Hong Cai,2 Yang Ke,2 Jun Li1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Peking University Cancer Hospital & Institute, Beijing, China; _3 _Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China_.

Esophageal squamous cell carcinoma (ESCC) is one of the most common and most aggressive cancers. The epidemiological features of ESCC are extremely complex, with strong geographic differentiation among world's populations. Rural Anyang in the Henan Province of China is a well-known high-incidence area, however the causal factors in this population remain elusive. We performed exome sequencing of 81 tumor-normal pairs, identified TP53, ZNF750 and NOTCH1 as significantly mutated genes, and observed highly recurrent aberrations in several other genes previously reported for ESCC (PIK3CA, KMT2D, FAT2 and FAT1). Our catalog of ~7,000 single-nucleotide mutations revealed two main signatures: C>T transitions at NpCpG due to spontaneous deamination of 5-methyl-cytosine, and C>T and C>G mutations at TpCpW attributed to the APOBEC family of cytidine deaminases. The latter signature points to HPV infection as one of the potential mutagenic sources, consistent with our previous studies that detected HPV DNA in tumor samples and anti-HPV-E7 antibody in patient's blood. To characterize intratumor heterogeneity we applied our newly developed method, CHAT, to estimate the clonal frequencies of copy number alternations and single nucleotide mutations in each tumor. Many tumors show a multi-modal distribution of clonal frequencies, suggesting extensive within-tumor diversity due to coexistence of multiple clones. Known ESCC-related genes tend to show mutations of higher clonality than those in other genes. To better understand the patterns of growth, migration and metastatic potential among different cells within a tumor we performing exome sequencing to compare multiple regions in 10 ESCC as well as 2 esophageal neuroendocrine carcinoma tumors. For each, we analyzed 4-6 sectors of the tumor, 2-4 of adjacent normal tissue samples, and 1-2 nearby lymph nodes. In many ESCC's, each local region still contains multiple clones, which are often shared with another region, suggesting extensive dispersal of the clonal populations and relatively slow sweep by the most dominant clone. We constructed "clone trees" to depict the most likely lineage relationship of the clones and the likely driver genes or pathways for each branch. Metastatic samples at lymph node often contain multiple clones, including those appearing in the early portions of the evolutionary tree, suggesting polyclonal seeding to the lymph nodes as well as invasion of early-stage tumor cells. The analysis of spatial heterogeneity of molecular lesions thus revealed likely temporal progression of tumorigenic events that may have driven the initiation, outgrowth as well as metastasis of ESCC.

(This work is supported by funding from the Joint Institute for Translational and Clinical Research of the University of Michigan Health System and Peking University Health Sciences Center.)

#2398

Colorectal primary tumors and metastases are highly homogeneous regarding driver mutations.

Miguel Silva, Carlos Resende, Rui Barranha, Sara Meireles, Fatima Carneiro, Jose L. Costa, Jose C. Machado. _Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal_.

Genetic mutations play a key role in the phenotypic and functional heterogeneity that arise among cancer cells within a tumor. The distinct tumor subclones can be intermingled or spatially separated and recent studies suggest this subclonal architecture varies dynamically throughout the disease course. As such, there is a need to accommodate this level of genetic heterogeneity in the classical notion that the carcinogenic process evolves in a stepwise manner through the accumulation of a specific set of mutations along time.

To genetically compare independent lesions from the same patient in the progression of colorectal carcinoma (CRC), we selected 16 patients with metastatic CRC with available primary tumor (PT), lymph node (LN) and liver metastases (LM), and corresponding normal mucosa (NM), resulting in 65 samples. Clinical data were available for all patients. DNA was extracted from FFPE tissues after morphologic evaluation by a pathologist. Sequencing was performed with the Ion PGM system using Ampliseq primer panels consisting of a multiplex PCR covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes frequently mutated in human cancers. Genes include APC and CTNNB1, considered the initial genetic event in CRC development. NM from all patients was sequenced to identify germline genetic variants. Data was analyzed on the Ion Torrent Server v4.2 using the Variant Caller and Coverage Analysis plugins. Ion Reporter Server and IGV were used for variant annotation and reads visualization, respectively.

A total of 1212 somatic variants were detected among the 65 samples. C/G>T transitions were the most prevalent alterations. In keeping with its initiating role, APC mutations showed the highest allelic fraction in the majority of samples. Seven samples exhibited a remarkably high number of variants (mean=145). Excluding these, an average of 3,5 variants were observed per sample. For each patient, putative driver mutations (in APC, CTNNB1, KRAS or TP53) detected in PT were also detected in LN and LM. In LM of 6/16 patients (38%) an average of 4 private mutations were observed. Mutations in other genes, empirically classified as passenger mutations, were also frequently found in this series. However, these mutations were heterogeneously distributed in the different lesions of the same patient. In most patients treated with chemotherapy before liver metastasectomy, additional private mutations were detected in the LM.

Contrary to what was previously described, CRC presents a high level of homogeneity concerning well-established driver mutations, since all were present in every tissue of each patient. Conversely, a higher level of heterogeneity was detected between different lesions of the same patient for passenger mutations. Relapse after chemotherapy seems to associate with the emergence of new mutations, reflecting its influence in the dynamics of tumor cell populations. 

### Intratumor Heterogeneity and Treatment Responses

#2399

System analysis of adapted and quiescent/dormancy(QD) models of drug resistance in melanoma cell lines.

Feng Liu,1 Parvita Paresh Panchal,1 Zi Wang,1 Ahmed Farhat,1 Angela Garcia,1 Tcharles Fagundes,2 Fabian Filipp,3 Frank L. Meyskens1. 1 _UC Irvine, Irvine, CA;_ 2 _Brown University, Providence, RI;_ 3 _UC Merced, Merced, CA_.

The biological basis for inherent and acquired drug resistance in melanoma is a prominent issue, highlighted by the recent discovery of various pathways leading to acquired resistance to the mutant BRAF inhibitor Vemurafenib (Plx4032). In order to dissect patterns of drug resistance, we treated 3 melanoma cell lines (A375, 1205Lu and SK-Mel28) under 2 diverse conditions: (1) Increasing drug concentrations over time( 4 months)- Melanoma cells treated with increasing concentrations of Plx4032 (initial and increasing concentrations of 0.1, 0.5, 1 and 5 μM PLx4032) exhibited an adaptation response. After an initial pause in proliferation, these cells proliferated in the continuing presence of the drug. Cells able to grow in the presence of 10 or 20 μM of Plx4032 were cultured as pools. RNA-Seq profiling and system biology analysis comparing the gene expression in the parental SK-Mel28 cells and adapted A2-1 revealed three major altered pathways: the PDGF-MAPK-DUSP-HIF2α, TGF-β-SMAD9-FGF1 and EDNRB-GNAI2-PLCB4-PKA-MITF pathway. Our qRT-PCR and western blot experiments have confirmed an up-regulation of PDGF which by-passed the BRAF gene and activated ERK1/2 kinases. These changes were accompanied by increase of NRAS accumulation in the adapted cells. (2) Using high initial drug concentrations of PLx4032 (>10 μM), > 99.9 of cells were killed, but some single cells (Q/D) survived for at least 4 weeks in the continuous presence of drug. After the drug was removed cellular proliferation reoccured 2-3 weeks later (designated post-dormant cells 1, PD1 cells). These cells did not show increased IC50s to Plx4032 after 72 hour of retreatment. However, when treated with high dose of drug again, the PD1 cells produced more dormant cells (PD2 cells), suggesting inheritance of the DQ trait. RNA-Seq profiling studies of the DQ cells are underway. The drug-induced dormancy/quiescence and adaptive response represent 2 different pathways of how cells survive treatment and develop resistance and provide a beginning to understanding the biological basis of the well established clinical phenomenon of long term clinical dormancy of melanoma and potentially other cancers.

#2400

Mechanisms of resistance to immuno and targeted therapies in acral melanoma.

Rebecca J. Lee,1 Maria Romina Girotti,1 Amaya Viros,1 Franziska Baenke,1 Amit Mandal,1 Garima Khandelwal,1 John Bridgeman,2 Elena Galvani,1 Gabriela Gremel,1 Milena Kalaitsidou,3 Garry Ashton,1 Isabel Peset,1 Matthew Smith,1 Jacqueline Swan,1 Kang Zeng,1 Haoran Tang,1 Caron Abbey,1 Robert Hawkins,4 Alberto Fusi,3 Crispin Miller,1 David Gilham,4 Nathalie Dhomen,1 Paul Lorigan,5 Richard Marais1. 1 _CRUK Manchester Institute, manchester, United Kingdom;_ 2 _Cellular therapeutics Ltd, manchester, United Kingdom;_ 3 _The Christie NHS Foundation Trust, manchester, United Kingdom;_ 4 _Institute of Cancer Sciences, The University of Manchester, Manchester, United Kingdom;_ 5 _The University of Manchester, Manchester, United Kingdom_.

Acral melanoma is a rare subtype of melanoma found on the non-hair bearing surfaces of the skin. It is associated with a worse prognosis than cutaneous melanoma, with higher proportions of patients developing metastatic disease. Combination treatments targeting the MAP kinase (MAPK) pathway and immune checkpoint inhibitors have improved overall survival in patients with stage IV disease. However, resistance to both modalities remains a challenge. This is exemplified by our comprehensive analysis of a patient presenting with acral melanoma that developed resistance to both immune and targeted therapy.

The patient developed metastatic disease in multiple sites and we obtained fresh tumour tissue and generated cell lines from a skin metastasis at baseline prior to nivolumab. Following an initial complete response, the patient developed new mediastinal lymph node and brain metastases, which were also resected. On further relapse, the patient commenced dabrafenib with a partial response. Despite ongoing extra-cranial control, the brain lesion progressed and was therefore resected.

To investigate resistance to immune therapy, we performed comprehensive genomic analysis and gene expression profiling on the lesions from the three sites, which identified common mutations in all three lesions and a low overall mutational burden consistent with acral melanoma. Heterogeneity of the MHC class I and II restricted antigen profile of the pre-treatment subcutaneous metastasis versus lymph node and brain progressions could not account for the development of resistance to immunotherapy. Gene expression profiling revealed up-regulation of known mechanisms of immune tolerance in both the lymph node and brain metastasis compared to the pre-treatment subcutaneous metastasis.

To investigate resistance to targeted therapy, we compared patient-derived cell lines from brain lesions taken pre (DabS) and on progression on dabrafenib (DabR), and showed ongoing sensitivity to dabrafenib despite the patient having progressed in the brain. When cells from the DabR cell line were cultured in cerebrospinal fluid (CSF) in the presence of dabrafenib, we observed a reduction in cell death. Thus, extrinsic factors present in CSF may have resulted in the progression of the brain lesion in this patient.

Using a combination of platforms to study patient derived tissues, we show that immune editing through selection of cells with an immune resistant phenotype may have resulted in the resistance of this patient's tumours to immune therapy whilst resistance to MAPK targeted therapy could have been mediated by extrinsic factors in the CSF.

#2401

RD3-loss orchestrates GC-signaling independent evolution of progressive neuroblastoma.

Singh Meghna, Wolansky Rachel, Terence S. Herman, Natarajan Aravindan. _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Recently we recognized the transcriptional/translational loss of Retinal degeneration Protein 3 (RD3) in high-risk progressive neuroblastoma (HR-NB) and defined its novel tumor evolution stabilization function. Studies have shown that RD3 competitively binds to guanylate cyclases (GCs) and maintains GC expression, stability, as well as restores GC localization. Accordingly, we investigated the role of GCs, if any, in RD3-loss associated evolution of HR-NB. Utilizing an in vivo mouse model of spontaneous HR-NB coupled with ex vivo approaches using metastatic site derived aggressive cells (MSDACs), we examined the transcriptional and translational expression of GCs and validated with high-content confocal immunofluorescence. Further, utilizing a cGMP competitive enzyme immunoassay, we investigated both intracellular and soluble GC activity. Whole genome microarray analysis revealed a differential activation of GC signaling transcriptional machinery in three different clones of MSDACs (compared to parental SH-SY5Y cells) ex vivo. Immunoblotting identified a consistent translation of GCs (GC-1 and GC-2) in a manifold of metastatic tumors (compared to non-metastatic primary xenografts) in vivo and in MSDACs ex vivo. Confocal immunofluorescence analysis validated the near-identical expression of GC1 and GC2 both in SH-SY5Y cells and MSDACs. cGMP immunoassay recognized indistinguishable intracellular and soluble GC activity in clones of differentiated/undifferentiated MSDACs as opposed to the clones of SH-SY5Y. For the first time, the results of this study define the transcription and translation blueprint of GCs in HR-NB and, further imply that the RD3-loss may dictate GCs-signaling independent alternate molecular flow-through in the evolution of HR-NB.

Acknowledgements: Stephenson Cancer Center, NIH COBRE 1P20GM103639-01, Broken Arrow Lady Elks Auxiliary, and Oklahoma TSET.

#2402

The impact of genomic heterogeneity in colorectal carcinoma on therapy response - a study on monoclonal primary colon cultures.

Rüdiger Meyer,1 Nicole E. McNeil,1 Ewa Krawczyk,2 Daniel W. Rosenberg,3 Zhongqiu Zhang,4 Richard Schlegel,2 Jens K. Habermann,5 Thomas Ried1. 1 _Section of Cancer Genomics, Genetics Branch, NCI, National Institutes of Health, Bethesda, MD;_ 2 _Center for Cell Reprogramming ,Georgetown University Medical Center, Washington, DC;_ 3 _Center for Molecular Medicine, University of Connecticut Health Center, Farmington, CT;_ 4 _Department of Surgery, Waterbury Hospital, University of Connecticut School of Medicine, Waterbury, CT;_ 5 _Section of Translational Surgical Oncology and Biobanking, Department of Surgery, University of Lübeck, Lübeck, Germany_.

Introduction: Individual response to chemotherapy in colorectal cancer patients is highly variable and the underlying mechanisms of treatment resistance of colorectal cancer cells are poorly understood. Recent studies revealed a considerable degree of genomic and genetic tumor heterogeneity. We hypothesize that the intra-tumoral heterogeneity has a direct impact on treatment response as sub-populations of cancer cells are resistant to currently used chemotherapeutics and facilitate tumor growth under treatment.

Experimental procedures: Patient-derived primary colorectal cell lines were established employing three different methodological approaches: (i) mouse xenografts and reintroduction of the graft to culture, (ii) conditional reprogramming by Rho kinase inhibition in combination with irradiated feeder cells and (iii) organoid growth in Matrigel with Wnt-, R-spondin and Noggin conditioned medium. Established primary cultures were characterized genomically by array CGH and spectral karyotyping (SKY).

Results: To date, we successfully established eight patient-derived primary colorectal cell lines. Culture conditions were optimized for long-term in vitro growth of colorectal primary tissue. Array CGH has identified patterns of chromosomal imbalances in these cell lines, i.e. gains of chromosomes 7, 13 and 20 as well as a loss of chromosome 18. Additionally, gene amplifications of MYC and EGFR were identified. As these aberrations are typically found in colorectal cancer, the established primary tissue derived cell lines closely reflect the nature of the tumor in vivo in terms of genomic instability. Array CGH findings were confirmed using SKY. Sub-cultivation of single cell derived monoclonal cell lines will facilitate addressing the tumor heterogeneity in terms of their genomic and genetic background using genome and transcriptome analyses. Single-cell derived lines will then be exposed to chemotherapeutic drugs currently used in clinical routine such as 5-FU and Oxaliplatin. The respective sensitivity will be correlated to the aberration profiles.

Conclusion: Exploration of tumor heterogeneity in terms of their genomic and genetic background in the context of treatment response might facilitate understanding of therapy resistance. Moreover, reliable prediction of the patient's response to the employed chemotherapeutic drug is a necessary step towards individualized treatment in colorectal cancer.

#2403

Molecular mechanisms orchestrating intratumoral heterogeneity in Kras-driven carcinomas.

Roxana Azimi. _Massachusetts Institute of Technology, Boston, MA_.

The considerable variability within tissue microenvironments as well as the multiclonality of cancers leads to intratumoral heterogeneity. This increases the probablility of cellular states that promote resistance to therapy and eventually lead to reconstitution of the tumor by treatment-resistant cancer cells, which in some cases have properties of normal tissue stem cells. Wnt signals are important in the maintenance of stem cells in various epithelial tissues, including in lung development and regeneration. We hypothesized that Wnt signals contribute to tumor heterogeneity in genetically engineered KrasG12D; Tp53Δ/Δ ("KP") mouse lung adenocarcinomas (LUAD). We observed that a subpopulation of LUAD cells exhibited high Wnt reporter activity and had increased tumor forming ability, which could be suppressed by silencing of Wnt signaling pathway components or by small molecule Wnt inhibitors in vitro and in vivo. KP LUAD cells show hierarchical features with two distinct populations, one with increased Wnt reporter activity and another forming a niche that provides the Wnt signal. Lineage-tracing experiments in the autochthonous KP tumors demonstrated that Wnt responder cells have increased tumor propagation ability in vivo. Strikingly, selective ablation of the Wnt responder cells resulted in tumor stasis. CRISPR-based targeting or small molecules targeting Wnt signaling reduced tumor growth and prolonged survival in the autochthonous KP mouse lung cancer model. Interestingly, we found that a similar Wnt-driven mechanism operates to establish an intratumoral hierarchy also in other Kras-driven carcinomas. These results indicate that maintenance of heterogeneity within tumors may be advantageous for the tumor cell population collectively, which may be utilized as a therapeutic target to homogenize cancer cell phenotypes within tumors.

#2404

Dysregulation of lung developmental pathways associated with LKB1 loss in lung cancer.

Jacob M. Kaufman,1 Cindy Lowe,2 Bradford Harris,2 Kelli Boyd,2 Joseph Amann,3 Rosana Eisenberg,2 David Carbone,3 Pierre Massion2. 1 _Duke University Hospital, Chapel Hill, NC;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _The Ohio State University College of Medicine, Columbus, OH_.

Loss of the LKB1 tumor suppressor influences differentiation phenotypes in mouse models of lung adenocarcinoma and in human tumors. We utilize molecular data from resected human lung adenocarcinomas characterized by the TCGA to demonstrate that LKB1 deficient tumors exhibit a higher prevalence of neuroendocrine and gut-like differentiation markers than do LKB1 wild-type tumors (41% vs 9%, odds ratio = 7.3, P = 5.9E-19 for neuroendocrine differentiation; 36% vs 12%, odds ratio = 4.3, P = 2.5E-11 for gut-like differentiation. Fisher's exact test). Using copy number analysis and transcription factor enrichment analysis, we link these phenotypes to activation of particular transcription factors, namely FOXA2, HNF1A, ASCL1, and TTF1. We observe similar associations among non-small cell lung cancer cell lines, which may represent appropriate ideal models for further experimentation. Furthermore, we examine these differentiation phenotypes using immunohistochemical analysis of a tissue microarray (TMA), combined with RNAseq expression analysis, and MS/MS proteomic characterization of the same cohort of 36 tumors. Our molecular characterization confirms the neuroendocrine and gut-like differentiation phenotypes observed in the TCGA dataset and their association with LKB1-loss. Moreover, our immunohistochemical analysis demonstrates that these distinct differentiation subtypes are heterogeneously expressed within single tumors. Co-expression of neuroendocrine, gut-like, and respiratory differentiation is significantly more likely to occur within LKB1 deficient tumors, and is also associated with increased expression of a c-Kit positive sub-population that appears to lack expression of more differentiated markers. Among the TCGA tumors, this tri-lineage expression pattern is seen in 12.5% of LKB1-deficient tumors, compared to 1% of LKB1-wild-type tumors (Odds ratio 11.5, P = 3.2E-8. Fisher's exact test). These findings give further support to the role of LKB1 in regulating differentiation phenotypes in lung cancer, and may suggest a hierarchy of progenitor cells and lineage-specific subpopulations within these tumors.

#2406

Identifying and targeting chemoresistant subclones in triple negative breast cancer.

Gloria V. Echeverria,1 Sahil Seth,1 Shirong Cai,1 Stacy Moulder,1 William F. Symmans,1 Timothy P. Heffernan,1 Jeffrey Chang,2 Helen Piwnica-Worms1. 1 _M.D. Anderson Cancer Center, Houston, TX;_ 2 _University of Texas Health Science Center, Houston, TX_.

Triple-negative breast cancer (TNBC) is an aggressively metastatic subtype that can only be treated by chemotherapy. Nearly 50% of patients have residual disease after neoadjuvant chemotherapy (NACT) and have extremely poor prognoses. Recent genome sequencing studies have revealed extensive intratumoral heterogeneity (ITH) in treatment-naïve TNBC. However, the functional contribution of ITH to chemoresistance in TNBC is unknown. To understand how this occurs, we are generating patient derived xenograft (PDX) models from treatment-naïve TNBC in order to identify and characterize tumor cell populations that are responsible for chemoresistance with the ultimate goal of selectively targeting resistant tumor clones.

Through an IRB-approved clinical trial using standard techniques, samples (primary tumor, skin metastasis, and blood for germline reference) were obtained from a patient with newly diagnosed, untreated metastatic TNBC. Subsequently, this patient was found to have disease resistant to chemotherapy. Tumor cells were implanted into the humanized mammary fat pad of NOD/SCID mice to establish PDX models of the primary (PIM1-P) and metastatic (PIM1-M) tumors. RNA sequencing and whole-exome sequencing (~300X) were performed on the patient's primary and metastatic tumors and the first and third passage PDX tumors. Mouse sequences were computationally subtracted from the PDX data and which was then processed according to Genome Analysis Toolkit best practices workflow. Variants were called using MuTect and copy number alterations were estimated by ExomeCN. This revealed high concordance between the genomic profiles of the patient and PDX models. While there were 81 somatic non-silent mutations shared in the patient and PDX, only a few fell in known cancer genes, including TP53 (V143fs), ELF4 (L593H), and ARID3A (R351P). Modeling of clonal clusters with ABSOLUTE revealed ITH in the patient tumor that is preserved in the PDX.

Both the patient and PDX model (PIM1-P) exhibited progressive disease when treated with paclitaxel. Only partial responses were observed in mice treated with doxorubicin plus cyclophosphamide (AC), consistent with residual disease after standard NACT for patients with TNBC. AC treatment resulted in an ~60% reduction in tumor volume which was not enhanced by repeated cycles. Tumors rapidly re-grew when treatment was halted or if treated tumor cells were engrafted into the mammary fat pads of new recipient mice. This suggests that a subpopulation of AC-resistant tumor cells is present in PIM1-P tumors. To dissect this subpopulation, we established conditions for simultaneous tracking of thousands of PIM1-P tumor clones in vivo using a high-complexity library of up to 30 million unique DNA barcodes. Mice engrafted with barcoded PIM-1P tumors are being treated with AC to identify and characterize AC-resistant tumor cells present in PIM1.

#2407

Neuroendocrine and luminal progenitors drive cancer progression in prostate cancer.

Maximilian Marhold,1 Erwin Tomasich,1 Simon Udovica,1 Gerwin Heller,1 Corinna Altenberger,1 Andreas Spittler,2 Reinhard Horvat,3 Peter Horak,4 Michael Krainer1. 1 _Medical University of Vienna, Department for Internal Medicine I - Oncology, Vienna, Austria;_ 2 _Medical University of Vienna, Department for Surgery, Vienna, Austria;_ 3 _Medical University of Vienna, Department for Pathology, Vienna, Austria;_ 4 _SMZ Ost, Department for Medicine II - Oncology, Vienna, Austria_.

Neuroendocrine differentiation of prostate cancer (PCa) occurs frequently during the development of castration resistance and rarely in primary tumors. Using the transgenic mouse model of prostate cancer (TRAMP) in a B6/C57 background, we were able to detect, isolate and further characterize basal, luminal and neuroendocrine subsets of cancer cells. We performed allograft experiments in NSG mice to ensure cellular stem and progenitor properties as well as metastatic potential, and thus created a murine model of neuroendocrine prostate cancer.

Whilst all three previously described cell populations were present in approximately three quarters of primary TRAMP tumors, some tumors lacked basal stem cells and showed a more aggressive phenotype. These tumors, mainly consisting of small cancer cells, expressed markers of neuroendocrine differentiation such as synaptophysin and chromogranin A as shown by immunohistochemistry and are further referred to as neuroendocrine carcinomas (NECs), as ruled by an experienced uropathologist of our institution. Adenocarcinoma-like tumors (ACs), in contrast, showed high expression of cytokeratins and retained glandular histology. Using fluorescence-activated cell sorting (FACS) against newly discovered NEC markers within the TRAMP model, we found a relative increase of neuroendocrine progenitors in prostate NECs compared to ACs (approximately 75 vs. 33%, respectively). In ACs, on the other hand, luminal progenitors were found to be the predominant drivers of cancer progression. To further evaluate this, we transplanted single-cell-suspensions into NSG mice without androgen supplementation and observed successful engraftment of both non-basal cell populations. Additionally, we were able to passage the resulted tumors for at least two generations and observed maintenance of histology and biological features for tumors of both luminal and neuroendocrine origin.

Based on a RNA sequencing, we were able to define gene signatures for neuroendocrine and luminal progenitors, uncovering a number of novel potential therapeutic targets. Ultimately, we were able to evaluate the prognostic value of the signatures obtained from mice in the human disease by in-silico analyses of publically available gene expression profile databases.

In conclusion, we created and characterized a murine model of neuroendocrine prostate cancer using flow cytometry and murine allografts. Further, we established gene expression signatures of luminal and neuroendocrine progenitors and translated them to the human disease. Our findings foster the understanding of neuroendocrine differentiation in prostate cancer and may help in developing new targeted approaches in this entity.

#2408

Melatonin action in xenograft model of breast cancer, comparing radiopharmaceuticals in the detection of intratumor heterogeneity by PET/CT confirmed by immunohistochemical markers.

André L. Mota,1 Bruna V. Jardim-Perassi,1 Thaiz F. Borin,1 Nathália M. Sonehara,1 Lorena Pozzo,2 Emerson S. Bernardes,2 Bruno Cogliati,3 Debora APC Zuccari1. 1 _Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, Brazil;_ 2 _Instituto de pesquisas energéticas e nucleares – IPEN, São Paulo, Brazil;_ 3 _Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, São Paulo, Brazil_.

Breast cancer is the most common cancer among women and has a high mortality rate. The rapid tumor growth makes difficult the perfusion of O2 mainly in the tumor center, and this hypoxia in tumor microenvironment can exerts selective pressure on the tumor cells, selecting subpopulations with advantage for survival in adverse conditions, characterizing the intratumor heterogeneity. In this context, melatonin, a hormone naturally produced by the pineal gland, has shown antitumor activity, as immunomodulatory, antioxidant, pro-apoptotic, anti-proliferative, antimetastatic, antiangiogenic and with potencial effects in intratumor heterogeneity. The aims of this study was to evaluate the effectiveness of melatonin in a xenograft model of breast cancer, focusing on intratumor heterogeneity verified by PET/CT and by immunohistochemical markers of hypoxia. The tumors were developed by the implantation of 5 million triple-negative breast cancer cells (MDA-MB-231) into the flank of female Balb/c nude mice (n=14). Treatment with melatonin (n=7) or vehicle (n=7) was initiated 10 days after tumor implantation and continued for 14 days. At the end, micro-PET/CT scanning was performed with 18F-FDG, which is an analogue of glucose, and with 18F-FAZA, a specific radiopharmaceutical for hypoxia detection. The radiopharmaceuticals were injected by retro-orbital via and the images were acquired using Albira PET/SPECT/CT Carestream Molecular Imaging. Then, markers of hypoxia were evaluated by immunohistochemistry in mammary tumors. The results showed that tumor growth in animals treated with melatonin was lower than those treated with vehicle (p<0.05). There was tumor regression in one animal treated with melatonin, showing 18.72 mm3 at start of treatment and no longer being detected after seven days of treatment. 18F-FDG can be used as hypoxia marker, however, studies show that can occurs overlapping areas of aerobic and anaerobic glycolysis in the tumor, difficulting the identification of hypoxic areas. 18F-FAZA has a hypoxia-specific uptake mechanism and a better spread by the tumor. Results of PET/CT showed that 18F-FDG had homogeneous uptake in breast tumors. In some tumors with high volume, there was no uptake in the tumor center, corresponding to areas of necrosis. In addition, there was an intratumor 18F-FAZA uptake heterogeneity, indicating possible hypoxic areas. Also, PET/CT results showed that there was less hypoxic areas in animals treated with melatonin, which was confirmed by immunohistochemical markers. Taken together, our results showed an important action of melatonin, in control of mammary tumor growth, decreasing hypoxia areas and controlling cancer progression. Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo

#2409

Inhibition of PLK1 induces growth arrest and apoptosis in tumorigenic OS cells.

Maria V. Guijarro, Elham Nasri, Padraic P. Levings, Steven C. Ghivizzani, C Parker Gibbs. _University of Florida, Gainesville, FL_.

Osteosarcoma (OS) remains a significant therapeutic challenge. At first diagnosis, patients without signs of cancer beyond the primary tumor show a 5-year survival rate of 60-80%; however, this falls to 20% in patients with evidence of metastatic disease. Toward development of treatments with greater therapeutic efficacy, we have been investigating the nature of intratumoral heterogeneity in OS. We have found that subpopulations of cells from primary OS biopsies are selectively capable of activating an exogenous reporter construct comprised of the human Oct4 promoter linked to the Green Fluorescent Protein (GFP). These Oct4/GFP+ cells are >100 times tumorigenic than GFP- cells from within the same tumor when injected into NSG mice.

Our objective in this study was to investigate the effects of Polo-like kinase 1 (PLK1) among heterogeneous cell populations within individual OS tumors. PLK1 is a serine/threonine-specific kinase that plays an important role in mitosis and the maintenance of genomic stability. While PLK inhibition has previously been shown to reduce tumor growth in OS cell lines, drug resistance and modest clinical efficacy have been reported in several studies.

Microarray analysis revealed an upregulation of PLK1 of more than 1.7-fold in the Oct4/GFP+ cells relative to the GFP- population in all three OS cell lines tested, OS156, OS521 and OS28 (p value=0.05). We next investigated the effects of the PLK inhibitors, BI2536 and BI6727, to determine the sensitivity of drug inhibition between 6 OS cell lines and among subpopulations within each tumor. IC50s showed that OS521, OS220 and OS28 OS cell lines were more resistant to the inhibitors than OS156, DCS0313 and D2712. Strikingly, all the Oct4/GFP+ populations of the tumors were more sensitive to drug treatment compared to GFP- cells. Cell cycle analysis showed that high doses (>0.5 µM) of either of the inhibitors induced apoptosis whereas low doses (<10 nM) induced G1 arrest. Within tumors, proliferation, clonability and migration were reduced in the GFP+ cell population relative to GFP- cells at doses of 10 nM. Consistent with these in vitro findings, tumor cell growth of Oct4/GFP+ pretreated cells injected in NSG mice was inhibited (by 50 %) at a low dose of 10 nM (p=0.005)

These findings offer evidence of the differential drug resistance as a consequence not only of the individual patient efficacy but also of the Oct4/GFP+ and GFP- populations from the same tumor, confirming that the intratumoral heterogeneity is a major obstacle for the treatment of OS, as the presence of minor populations that are insensitive to therapy can lead to disease relapse. Future studies will be conducted to understand what makes tumors resistant or vulnerable to PLK1 inhibition.

#2410

Recurrent pediatric ependymomas exhibit increased tumorigenicity in mouse brains of patient-derived xenograft models.

Sibo Zhao, Huiyuan Zhang, Laszlo Perlaky, Adekunle Adesina, Ching Lau, Donald W. Parsons, Murali Chintagumpala, Xiao-Nan Li. _Baylor College Of Medicine, Houston, TX_.

Ependymoma is the third most common malignant pediatric brain tumor, accounting for ~10% all intracranial tumors in children. Current standard treatment for ependymoma patients include maximally safe surgical resection followed by radiation therapy. The degree of gross total resection remains the best prognostic factor, although more molecular characterization has been completed in recent years demonstrating several subgroups of ependymoma that can more accurately predict clinical outcome. 5-year event free survival is 40-85% and varies depending on the extent of surgical resection, tumor grade, and other prognostic factors. Prognosis is even more dismal in those patients with recurrent ependymomas which occurs in nearly half of the patients, and there are no known curative options to offer these patients other than palliative re-irradiation and additional surgeries. Chemotherapies and molecular targeted therapies have not been proven to increase survival outcomes. Therefore, it is imperative to learn more about the biology of recurrent diseases and identify the cellular driver of ependymoma recurrence, as well as understanding the mechanisms of therapy resistance. We hypothesize that ependymoma recurrence is driven by a subpopulation of therapy-resistant tumors cells with enhanced tumorigenicity in SCID mice.

Between the years of 2005 to present, we have collected a total of 77 pediatric ependymoma patient tumor samples from across the country. Of those, we identified 9 patients with recurrent ependymomas in which we have collected tumor samples from each recurrence. The median age of these patients is 6 years old (ranges from 2 - 10 years) and has a 2:1 male to female ratio. The median time to first recurrence is 35 months (ranges from 9 - 61 months). 5 patients had one recurrence, 2 patients had two recurrences, 1 patient had 3 recurrences, and 1 patient had more than 5 recurrences and we have tumor tissues from this particular patient over the span of 6-year period. To study the tumorigenicity of these brain tumors at the different clinical stages, whether at diagnosis, first recurrence, second recurrence, or beyond, we directly implanted tumor cells (10,000 cells/mouse) from all 9 patients into the matched locations in the brains of SCID mice (e.g., human cerebral tumors to mouse cerebrum, human cerebellar tumors to mouse cerebellum). In 3 of the 9 models, tumor formation was confirmed in mouse brains from the latest recurrent patient tumor, while samples from the same patient from earlier stages either did not form tumors in mouse brains or are currently pending. Tumor formation in the remaining models are still pending.

In conclusion, our preliminary findings demonstrate progressive enhancement of tumorigenicity during ependymoma recurrence. This has become a very unique and extremely valuable platform to study brain tumor recurrence.

#2411

Evolution during propagation and treatment of patient-derived triple negative breast cancer xenografts.

Hyunsoo Kim,1 Pooja Kumar,1 Francesca Menghi,1 Joshy George,1 Guru Ananda,1 Susan Mockus,1 Chengsheng Zhang,1 Nicholas Larson,2 Henry C. Chen,3 Yan Yang,3 James Keck,3 R. Krishnamurthy Karuturi,4 Charles Lee,1 Carol Bult,4 Edison Liu,4 Jeffrey H. Chuang1. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _The Jackson Laboratory, Sacramento, CA;_ 4 _The Jackson Laboratory, Bar Harbor, ME_.

Individual tumors, including the aggressive and difficult to treat triple-negative (ER-/PR-/HER2-) breast cancers (TNBCs) are heterogeneous collections of cells with multiple subclonal populations each contributing to the tumor. While subclonal heterogeneity is likely responsible for the development of drug resistance, identification of how tumor cell populations change over time has been difficult, largely because of the challenges in resampling tumor tissue at close time points. Here we quantify tumor evolution in human patient-derived xenografts implanted into NSG mice, which we use to test subclonal heterogeneity as a function of location within a tumor, propagation time, and drug treatment. We used high-depth (~400x) sequencing of a targeted panel of 358 genes to quantify somatic mutation allele frequencies from 6 spatially-separated and 8 temporally-propagated xenograft samples derived from the same TNBC patient tumors. Samples ranged in age from 2-4 months post-engraftment. Although we observed a few low frequency mutations distinguishing samples, overall we found that allele frequencies of somatic mutations were well-preserved on this time scale. We then generated replicate xenografts from the same patient tumor and treated them respectively with cisplatin, doxorubicin, cyclophosphamide, docetaxel, or vehicle control for 25 days. Although again somatic mutations showed few differences in allele frequency across samples, substantial variations were seen when data were analyzed for copy number alterations. To confirm these effects we repeated the treatments for xenografts derived from two additional TNBC patients. Again we observed strong changes in tumor heterogeneity at the copy number level. This effect was particularly, but not exclusively, apparent in tumors with the greatest response to therapy. We further verified these measurements through Sanger and digital PCR sequencing on the treated mice and other mice in the same cohorts. Using a multi-sample xenograft propagation, dissection, sequencing, and computational analysis protocol, we have shown that tumor subpopulation changes in response to treatment can be quantified and distinguished from spatial or temporal effects, even for treatment time courses as short as 1 month. In triple negative breast cancer these variations are most apparent at the level of copy number variation. Our study demonstrates how patient-derived xenografts can provide detailed resolution of tumor population evolution during the manifestation of resistance.

#2412

Reconstructing intratumor heterogeneity: lessons from therapeutic intervention in patient-specific models.

Dirk Schumacher,1 Karsten Boehnke,2 Marlen Keil,3 Alessandra Silvestri,4 Maxine Silvestrov,4 Jens Hoffman,3 Iduna Fichtner,3 Reinhold Schaefer,1 Christian RA Regenbrecht5. 1 _German Cancer Consortium (DKTK), Heidelberg, Germany;_ 2 _Eli Lilly and Company, Madrid, Spain;_ 3 _EPO - Experimental Pharmacology & Oncology GmbH, Berlin, Germany; _4 _cpo - cellular phenomics & oncology GmbH, Berlin, Germany; _5 _Charité Berlin, Berlin, Germany_.

Precision medicine approaches seeking to predict individual susceptibility to single or combinatorial drugs for advanced and/or metastasized solid cancers heavily depend on biological model systems that maintain key morphological, phenotypic and genomic features of the original tumor.

Here, we present a case study of a female patient with metastasized colorectal cancer. Using tissue from five distinct areas of the primary tumor and its metastasis of the synchronously removed primary tumor and liver metastasis, we generated cell cultures. To address cell morphology and phenotypic features we performed immunohistochemistry (IHC) and immunofluorescence imaging showing that these cell cultures express colon-specific marker proteins/combinations CDX2 and CK20+/CK7-. Ultra-deep panel sequencing of original tumor tissue samples revealed damaging mutations in KRAS (G12D), PIK3CA (H1047R) and TP53 (C242F). Two cell cultures showed an additional SNP in the tumor suppressor SMAD4 (R361H), which was not detected in the original tissue, thus hinting at the existence of a small sub-population of cancer cells with a divergent mutation pattern.

In parallel we established patient-derived xenografts (PDX) models from each region and investigated the response to targeted therapeutics, aiming at key molecules of the MAPK, PI3K/AKT and mTOR pathways in these genetically heterogeneous populations. drug response in single and mixed cell populations to address differential growth characteristics and response to mono- and combinatorial drug treatment. To distinguish the cells-of-origin and to calculate distributions/contribution of each population to the in vivo tumor growth in this model, sub-populations were color-coded using lentiviral constructs, expressing either a green or red tag. FACS analyses after treatment were performed and ratios of SMAD4mut(green) vs. SMAD4wt(red) were determined for each PDX tumor. These analyses, together with confirmatory IHC staining for the respective tag, showed significant effects on both tumor growth and population distributions in a treatment-dependent manner, advocating for systematic evaluation of combinatorial treatment in patients.

#2413

Cell type-specific deconvolution of heterogeneous tumor samples with immune infiltration using expression data.

Zeya Wang,1 Jeffrey S. Morris,2 Chris C. Holmes,3 Jaeil Ahn,4 Bo Li,5 Wei Lu,6 Ximing Tang,6 Ignacio I. Wistuba,6 Wenyi Wang7. 1 _Department of Statistics, Rice University, Houston, TX;_ 2 _Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Department of Statistics, University of Oxford, Oxford, United Kingdom;_ 4 _Department of Biostatistics and Bioinformatics, Georgetown University, DC;_ 5 _Department of Statistics, Harvard University, Cambridge, MA;_ 6 _Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX;_ 7 _Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX_.

Tumor tissue samples comprise a mixture of cancerous and surrounding stromal cells. Understanding tumor heterogeneity is crucial to analyzing gene signatures associated with cancer prognosis and treatment decisions. Compared with the experimental approach of using laser micro-dissection to isolate different tissue components, in silico dissection of mixed cell samples is faster and cheaper. Numerous computational approaches previously developed have limited ability to deconvolute heterogeneous tumor samples. We have developed a deconvolution model, DeMix-T, that can explicitly account for a third component such as infiltrating immune cells, and address this challenging problem when the observed signals are assumed to come from a mixture of three cell compartments, infiltrating immune cells, the tumor microenvironment and cancerous tissues. The optimization-based algorithm for DeMix-T finds a local optimum for our estimation problem and is computationally feasible when it needs to compute high-dimensional integrals. DeMix-T therefore involves a novel two-stage filtering method that yields accurate estimates of cell purities and compartment-specific expression profiles. Simulations and real data analyses have demonstrated the good performance of our method. Compared with other deconvolution tools, DeMix-T can be applied more widely and provides deeper insight for cancer biomarker studies. It allows for a further understanding of immune cell infiltration in cancer and assists in the development of novel prognostic markers and therapeutic strategies.

#2414

ADAR1-dependent RNA editing is a mechanism of therapeutic resistance in human plasma cell malignancies.

Elisa Lazzari,1 Leslie A. Crews,1 Christina Wu,1 Heather Leu,1 Shawn Ali,1 Raffaella Chiaramonte,2 Mark Minden,3 Caitlin Costello,4 Catriona H.M. Jamieson1. 1 _Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center at University of California, San Diego, San Diego, CA;_ 2 _Department of Health Sciences, Univeristy of Milan, Milan, Italy;_ 3 _Princess Margaret Hospital, University Health Network, Toronto, Canada;_ 4 _Department of Medicine, Moores Cancer Center at University of California, San Diego, San Diego, CA_.

Introduction

Multiple myeloma (MM) is a plasma cell malignancy that accounts for more than 10% of all blood cancers and may progress to plasma cell leukemia (PCL). Despite treatment, virtually all patients become unresponsive to treatment. RNA editing is a post-transcriptional pre-mRNA processing activity that represents an unexplored potential source of clonal molecular heterogeneity contributing to therapeutic resistance. In particular, adenosine deaminase acting on RNA (ADAR) 1, which exists in two isoforms, one constitutive and one inflammation-responsive, has been associated with disease progression and cancer stem cell (CSC) maintenance. The aim of this study was to investigate whether enhanced ADAR1 expression and activity contributed to therapeutic resistance of MM and PCL.

Procedures

1) ADAR Quantification: Whole gene and isoform-specific qRT-PCR was used to detect ADAR1 expression in PCL and MM primary samples and in human MM cell lines (HMCL).

2) RNA Editing Detection: We developed a RNA editing site-specific qPCR (RESS-qPCR) assay to detect RNA editing in cancer stem-cell associated transcripts.

3) Therapeutic Resistance Assay. A MM cell line was exposed to lenalidomide continuously in vitro to establish a model of therapeutic resistance.

4) Development of a humanized PCL mouse model: We established novel in vivo PCL primagrafts by intrahepatic transplantation of primary total mononuclear cells into neonatal RAG2-/-gc-/- mice.

Results

Approximately, 30% of MM patients in the MM Genomic Initiative dataset harbor copy number amplifications of the ADAR locus on chromosome 1q21, which portends a poor prognosis. We observed significantly increased ADAR1 expression in primary PCL samples and aberrant RNA editing of the stem cell transcription factor GLI1 and the DNA cytidine deaminase APOBEC3D. Notably, high-ADAR1-expressing PCL cells successfully engrafted in RAG2-/-gc-/- mice. As the inflammation-responsive isoform of ADAR1 was upregulated in primary samples, we sought to explore the effects of the anti-MM agent and immunomodulatory drug lenalidomide on ADAR1 expression and activity. Continuous in vitro exposure to lenalidomide led to increased ADAR1 mRNA and protein level and a potent induction of RNA editing activity. Increased RNA editing was detected in several cancer and stem cell-associated transcripts, including GLI1, APOBEC3D, AZIN1 and MDM2. Notably, this aberrant RNA editing activity was associated with increased self-renewal capacity in vitro and a cancer stem cell phenotype.

Conclusions

ADAR1 overexpression and deregulated RNA editing represents a unique source of RNA and proteomic diversity, and may confer a survival and self-renewal advantage to MM cells. This research identifies ADAR1 as a new diagnostic and therapeutic target in MM, and establishes a robust humanized PCL primagraft model for future pre-clinical testing of ADAR1 modulatory agents.

#2415

GBM exhibits phenotypic microheterogeneity and harbors pre-existing multi- resistant clones with a mesenchymal transition signature.

Anna Segerman, Maria Niklasson, Caroline Haglund, Tobias Bergstrom, Malin Jarvius, Yuan Xie, Demet Caglayan Simov, Annika Hermansson, Marianne Kastemar, Zeinab Naimaie-Ali, Malin Berglund, Ann Westermark, Magnus Sundstrom, Goran Hesselager, Lene Uhrbom, Mats Gustafsson, Rolf Larsson, Marten Fryknas, Bo Segerman, Bengt Westermark. _Uppsala Universitet, Uppsala, Sweden_.

Glioblastoma multiforme (GBM) remains in the group of incurable malignancies, with a median survival of 15 months. Intratumoral heterogeneity is a key factor driving relapse by providing the basis for escape of therapy-resistant cells. While the heterogeneous genomic and transcriptomic landscape of cancers is currently thoroughly characterized, phenotypic heterogeneity within tumors is less well understood. The overall goal in this study was to identify pre-existing treatment resistant clones with disease relapse potential and biomarkers linked to resistance vs sensitivity. To enable functional studies of isolated tumor clones we adopted an in vitro subcloning strategy. An adherent glioma neural stem cell culturing protocol was used to enrich for cells with stem-like properties and to permit efficient phenotypic screening.

We established six libraries of clonal cell cultures from fresh GBM surgical specimens, corresponding to five different patient tumors. In all 708 clonal cultures were expanded. These were considered representatives of the self-renewing compartment within the tumor cell population. We found a remarkable variation in drug and radiation response within the clone libraries. A striking observation was that clones resistant to one drug, also tended to be resistant to most of the drugs we used, regardless of the drug's mechanism of action. This indicates that resistance in large parts is mediated by a general mechanism.

Most clones carried genetic aberrations that were unique or found only in a few clones but it was primarily the transcriptome data that demonstrated a clear link to the phenotypic data.

A continuous gradient between multi-resistance and sensitivity was found to be connected to a gradual transition between a mesenchymal (MES), in resistant clones, and a more proneural (PN) and proliferative character in sensitive clones. The continuous distributions and lack of discrete groups in clonal response phenotypes and in linked signatures points to a general cell-state related resistance mechanism.

We also found that resistant clones had a lower methylation level in promoters of known master regulators of MES subtype associated genes. This indicates that the phenotypic heterogeneity is driven by fluctuations in the epigenetic status.

Taken together, our findings imply that intratumoral heterogeneity in GBM includes general clonal resistance mechanisms among glioma- initiating cells driven by epigenetic mechanisms. This evokes hope that new therapeutic approaches involving epigenetic reprogramming can be applied to sensitize cells toward conventional treatment.

#2416

Important role of FTO in the survival of rare panresistant triple-negative inflammatory breast cancer cells under a severe metabolic challenge.

Balraj Singh,1 Hannah E. Kinne,1 Ryan D. Milligan,1 Laura J. Washburn,1 Mark Olsen,2 Anthony Lucci1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Midwestern University, Glendale, AZ_.

Cancer is an evolution-like process. Effectively dealing with the currently untreatable component of breast cancer (the cells that drive metastasis) is vital. In this regard, we have developed a model of panresistant cancer cells that allows us to evaluate therapeutic agents to eradicate them. We have previously shown that only 0.01% cells survive a metabolic challenge involving lack of glutamine in culture medium of SUM149 triple-negative Inflammatory Breast Cancer (TN-IBC) cell line. These cells, designated as SUM149-MA for metabolic adaptability, are resistant to chemotherapeutic drugs, and they efficiently metastasize to multiple organs in nude mice. The MA cells can survive a variety of challenges in the metastasis process because of their embryo-like nature. We hypothesized that obesity-related molecular networks, which normally help in cellular and organismal survival under metabolic challenges, may help in the survival of MA cells. We found that fat mass and obesity-associated protein FTO is overexpressed in MA cells. It was reported recently that obesity-associated cis-acting elements in non-coding region of FTO regulate the expression of IRX3 gene, thus activating obesity networks. Here we found that IRX3 protein is significantly overexpressed in MA cells (4 to 5-fold) as compared to the parental SUM149 cell line, supporting our hypothesis. We utilized MO-I-500, a pharmacological inhibitor of FTO to investigate its role in MA cells. When included in a glutamine-starvation medium in 1.5 to 2 µM range, MO-I-500 significantly (>90%) inhibited survival and/or colony formation of SUM149-MA cells as compared to untreated cells or those treated with a control compound MO-I-100. Interestingly, MO-I-500 treatment had no significant effect on cell growth of either the SUM149 or SUM149-MA cell line when added to a complete medium containing glutamine that does not pose a metabolic challenge. Furthermore, SUM149-MA cells that were initially selected in a glutamine-free medium were not affected by a subsequent treatment with MO-I-500, even in glutamine-free medium. These results are significant as they reveal linkages between tumor adaptability, the embryonic nature of cancer cells, and obesity-like networks at the roots of therapy-resistant TN-IBC. They also suggest a novel approach for evaluating potential anticancer agents that would halt cancer evolution and prevent development of resistance to currently offered therapies.

Supported by a State of Texas Grant for Rare and Aggressive Cancers.

#2417

Efficiency in recovery of pure tumor cell populations from limited tumor tissue specimens intended for clinical application.

Valeria Sero,1 Claudio Forcato,2 Chiara Bolognesi,2 Genny Buson,2 Giulio Signorini,2 Paola Tononi,2 Gianni Medoro,2 Nicolò Manaresi,2 Farideh Z Bischoff1. 1 _Silicon Biosystems Inc., San Diego, CA;_ 2 _Silicon Biosystems Spa, Bologna, Italy_.

Introduction: We have previously shown reliability in isolating pure populations of rare cells from complex tissues using the DEPArray™ system. Several molecular techniques can be applied to a variety of histological samples, for example Macrodissection is the gross manual dissection of FFPE samples used to isolate areas of interest within a specimen for optimal downstream analysis, while Fine Needle Aspiration (FNA) is a safe procedure routinely used to examine a lesion helping to make a diagnosis. These techniques are also used to assess the effect of treatment. Here we demonstrate preliminary results showing 100% efficiency in recovering pure tumor cell populations from different samples using the DEPArray™ platform to overcome the issue of tumor heterogeneity. Method: FFPE macrodissected sections (n=9;) and FNA (n=5;) originating from prostate, breast, pancreatic and lung tumors were evaluated. Each was processed using a tissue disassociation and staining procedure followed by DEPArray™ sorting based on cytokeratin (Ker), vimentin (Vim) and nuclear staining. The recovered cell populations were lysed in the collection tube prior to PCR-based target enrichment for next generation sequencing using IonTorrent™ AmpliSeq CHPv2. Results: DEPArray™ analysis allowed identification of well separated cell populations, including Tumor (Vim-/Ker+) and Stromal (Vim+/Ker-) cells in all the samples analyzed. In the Macrodissection samples we were able to estimate the % of tumor cells (mean 23% range 4-54%), demonstrating an unexpected low frequency of tumor cells remaining following macrodissection. In FNA specimens analyzed only 21% (4.3% to 42.7% range) of the total (mean of 6335) cells analyzed were of tumor (KER+) origin. For subsequent NGS analysis, groups of pure cells (mean 174 cells, range from 37 to 280) for each population were recovered. Among the tumor cells isolated from the macrodissected and FNA specimens, we observed non-synonymous somatic variants and LOH events for different genes. This situation can not be highlighted in unsorted population. Conclusions: DEPArray™ technology can be used to isolate pure tumor cells from heterogeneous FFPE samples used in diagnostic application, such as Macrodissection or FNA specimens. Thus, the DEPArray™ platform brings digital precision to detection, quantification and recovery of pure target cells for subsequent downstream molecular analysis that can improve cancer diagnosis and treatment decisions.

#2418

Microenvironment-on-chip: Development of a microfluidics-based tumor ecosystem.

Gonzalo Torga,1 Ke-Chih Lin,2 Robert H. Austin,2 Kenneth J. Pienta1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Princeton University, Princeton, NJ_.

Despite its limited clinical predictive capacity, conventional cell culture remains the most frequently used preclinical model in biomedical research. For many years, microfluidics-based organ-on-chips have been proposed to overcome conventional cell culture limitations but given their inability to provide reliable and reproducible data, their usage remains anecdotal.

Immune, stromal and cancer cells constitute a complex adaptive system in each tumor microenvironment. To study the causes and consequences of tumor heterogeneity in these complex adaptive systems, we have developed a platform to model and investigate tumor microenvironments at primary and metastatic sites in which multiple nuclear-labelled cell types can be grown in a multi-welled, communicating habitat with controlled gradients for temperature, pH, nutrients, and oxygen tension. In conjunction with labelled-nuclei, genetically-engineered cell lines with fluorescence-based biosensors can be generated to detect specific genetic expression changes in response to cell to cell interactions as well as varying environmental conditions over time.

One of the major caveats for microfluidics prototypes is reproducibility. The proposed design is easily adaptable to most of the commercially available incubation microscopy systems, allowing the experiments to be replicated by independent groups in an effort to provide reliable and reproducible data.

The ability to establish dynamic gradients within individual habitats longitudinally over time and space provides the capacity to study the effect of chemokines and drugs on the tumor ecosystem in order to discover key interactions between host cells and cancer cells and to develop improved therapeutic strategies.

In contrast to previous organ-on-chip models, our technology is not limited to a mere observational platform for automated cell counting and tracking but also provides flexibility for downstream capacities such as immunofluorescence, in situ hybridization or single-cell sequencing, as well as retrieval of single cells or specific subpopulations based on the fluorescent reporters and labelled nuclei.

The presented engineered microenvironment allows continued in vitro quantitative studies of the interactions of multiple cell types as well as varying environmental conditions. Here we propose a cancer-on-chip platform that is able to recapitulate key components and interactions to mimic different tumor microenvironments in a comprehensive manner, yet simple enough to provide reliable and reproducible data.

#2419

Anastomotic recurrences are clonally related to primary tumors in sporadic colorectal carcinoma.

Efsevia Vakiani, Ronak H. Shah, Christine Iacobuzio-Donahue, David B. Solit, Martin R. Weiser. _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Background:

Anastomotic recurrences occur in 2-10% of colorectal carcinoma (CRC) cases after surgical resection of the primary tumor. To date there are no molecular data investigating their genetic profile and multiple theories exist as to their pathogenesis including re-growth of tumor cells present within local lymphatics after initial surgery, seeding of anastomosis by metastatic tumor cells and a second primary tumor. The aim of our study was to compare the genomic profile of anastomotic recurrences to that of matched primary tumors and, where available, to that of distant metastases.

Design:

Thirty-six tumors from 14 patients were genotyped using a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes. All patients had resection of their primary tumor with clear margins and recurred either at the anastomotic line or in the peri-anastomotic area 1.1 to 7.0 years following resection of their primary tumor. In 3 patients 2 consecutive anastomotic recurrences were sequenced, while in 6 patients a distant metastasis that occurred 1.2 to 3 years prior to the anastomotic recurrence was also analyzed. All tumors were microsatellite stable except in one patient with genetically confirmed Lynch syndrome.

Results:

A total of 254 somatic mutations were detected including 140 mutations in the microsatellite stable cases. The most commonly mutated genes (mutated in > 3 patients) were APC, KRAS, TP53, PIK3CA, ATM and PIK3R1. In 13/14 patients the anastomotic recurrence(s) and primary tumor shared between 50-100% of mutations, including mutations in key driver genes such as APC, KRAS and TP53, consistent with these tumors being clonally related. In the patient with the Lynch syndrome the 2 tumors showed distinct somatic mutations suggestive of independent primaries. We identified eleven genetic events present in a distant metastasis and not in a primary tumor and none of these events was detected in the anastomotic recurrence arguing against re-seeding of the anastomotic site by a metastatic clone. All five patients with isolated anastomotic recurrence were free of disease 0.9 to 6.1 years following resection of their recurrent CRC.

Conclusions:

Anastomotic recurrences appear to be clonally related to the primary tumor in sporadic CRC cases. Our data also provide molecular data in support of the treatment of anastomotic recurrences as localized disease.

#2420

A population sampling approach for validating deconvolution of high throughput sequencing data to describe clonality in heterogeneous tumor samples.

Amy L. Paguirigan,1 Weston D. Christensen,1 Zaneta J. Holman,1 Jordan L. Smith,1 Emily A. Stevens,1 Brent Wood,2 Jerald P. Radich1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Unversity of Washington, Seattle, WA_.

This abstract describes an in silico model of a heterogeneous population sampling methodology that elucidates how to best determine which genetic variants in a population are likely to occur in the same or different clones along with an experimental system to validate the approach.

While the role of clonal evolution during leukemia development and therapy has been a focus for a number of avenues of research, our abilities to deduce the clonal composition of individual samples have been limited by the use of bulk samples. Leukemia populations of unknown heterogeneity are typically described by extracting nucleic acids from the entire sample, including cells of unrelated lineages, any residual normal cells, and those cells constituting the leukemic population. Bioinformatic approaches are employed to reconstruct the identities of the clones involved using clustering of feature frequencies in sequence data, but by definition this type of approach relies on a basic set of assumptions regarding the underlying biology which may or may not be accurate at all loci for all samples.

We have generated an in silico model of how to use random population sampling with either targeted high throughput sequencing (54 genes) or targeted pyrosequencing (4 genes) to better determine which assumptions are not accurate for a given patient. In addition, we have an in vitro leukemia system on which we can test the in silico model. Flow cytometric validation of three AML cell lines along with the genetic characterization of each cell type and verification of comparable growth rates in a normalized medium in vitro has been performed. Subsequent mixing of these cell lines, random sampling of cells (10-2,000) isolated via flow cytometry and DNA sequencing can be used to test our ability to deconvolve the mixture based on a bulk measurement with the knowledge of the true population composition obtained via flow cytometry.

Using bulk sequencing data, we can identify potential clonal structures likely to exist in a given sample via variant allele frequency densities and k-means clustering. Given the possible clonal models, we can select variants that appear to be in the same clone. The in silico sampling model then aids in determining the ideal replicate number and sample size to best discriminate if proposed variants are truly in the same clones or not. Using our experimental system, we then verified if the predicted variants can be deconvolved using specific sampling methods.

With this sampling approach in hand and a framework for testing the assumptions, we can perform a prospective analysis of genetic variants within a heterogeneous leukemia sample via high throughput sequencing, then selectively validate selected mutations for concurrence in individual cells via targeted pyrosequencing. These results suggest that we can better describe leukemia heterogeneity without the requirement for extensive single cell analyses.

#2421

Circulating tumor cells reveal the genetic evolution of metastatic breast cancer.

Simon A. Joosse,1 Anna Babayan,1 Katharina Prieske,1 Daniela Indenbirken,2 Malik Alawi,1 Eike C. Burandt,1 Adam Grundhoff,2 Volkmar Müller,1 Klaus Pantel1. 1 _University Medical Center Hamburg-Eppendorf, Hamburg, Germany;_ 2 _Heinrich-Pette-Institute for Experimental Virology, Hamburg, Germany_.

BACKGROUND. Anti-cancer treatment failure is caused by the genetic and phenotypic heterogeneous properties of the disease. Early dissemination of circulating tumor cells (CTCs) into the blood circulation of cancer patients allows for parallel genetic evolution of the primary tumor and metastases. This study investigated the genomic make-up of CTCs originating from metastasis as a so called "liquid biopsy" and compared these with the different clones of the archived autologous primary tumor on single cell level.

METHODS. From two breast cancer patients, individual CTCs were isolated from blood using Ficoll density gradient followed by micromanipulation of keratin positive cells. Single cells from archived primary breast tumors from the same patients were captured by laser microdissection. DNA was isolated and amplified by whole genome amplification and copy number alterations (CNA) were obtained by shallow, whole genome next generations sequencing (NGS).

RESULTS. From the first breast cancer patient, the genomes from 50 single cells from the primary tumor were sequenced. An unsupervised phylogenetic cluster analysis based on CNA revealed five distinct subclones that displayed increasing chromosomal instability from the first to the last cluster. Using support vector machine (SVM) learning, the CNA profiles of 42 CTCs were residing mostly to the first three clones with few chromosomal aberrations, whereas only one CTC resided to the fourth clone and none to the last with many chromosomal aberrations. The tumor from a second breast cancer patient displayed the presence of three genetically distinct clones with increasing chromosomal instability after sequencing 11 single cells. CTCs (n=12) from this patient at metastatic disease were classified to the last branch using SVM, however with low probability. After repeating unsupervised clustering on the genomes of the primary tumor tissue and CTCs, a fourth branch was formed with CTCs only.

CONCLUSION. Our results suggest that therapy resistant metastases in breast cancer patients can originate from tumor clones from early stages of tumor evolution and may genetically still be similar (patient 1). On the other hand, further genetic progression may also take place (patient 2) after which the genetic landscape of the metastasis does not resemble the primary tumor anymore. These results underline the importance of "liquid biopsy" in the diagnosis of metastatic cancer.

#2422

Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip.

David Chianese,1 Mark Connelly,1 Carrie Morano,1 Thai Bui,1 Steven Gross,1 Renouard Sanders,1 Tom Barber,2 Ravi Kapur,3 Shyamala Maheswaran,4 Mehmet Toner,2 Daniel Haber4. 1 _Janssen Diagnostics, LLC, Huntingdon Valley, PA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Harvard Medical School, Boston, MA;_ 4 _Massachusetts General Hospital and Harvard Medical School, Boston, MA_.

Today, circulating tumor cells (CTCs) can provide important prognostic information about a patient. However, to one day help guide decisions for precision medicine, more and increasingly sophisticated information will have to be extracted from each patient blood sample. We describe a new microfluidic CTC isolation technology, the Harpoon CTC Isolator and Chip (Hrp Isolator). From a single sample the Hrp Isolator can enable the analysis of plasma components such as ctDNA, as well as the ability to enrich all circulating tumor cell types, regardless of their antigenic expression. To enrich the CTCs from 7.5 mL of whole blood, the Hrp Isolator removes the red blood cells and platelets based on their small size and the white blood cells by negative depletion using magnetic particles. The time required to process 7.5 mLs of blood is approximately 90 minutes. In contrast, the CELLSEARCH® CTC Test (CS), the only US FDA-cleared test, uses antigens expressed by CTCs to target the CTCs and positively select them out of blood. To enumerate CTCs from patient samples, the output from the Hrp Isolator was then loaded into and run on the CELLSEARCH® System using the CS test (Hybrid method). CTCs were enumerated from 37 breast and prostate cancer patients by CS and by the Hybrid method. At least 1 CTC was found in 23 (62%) patient samples by the Hybrid method versus 17 (46%) by CS. The product of the Hrp Isolator enrichment contained on average the same numbers of CTCs as were found by CS (mean = 13.3 ± 18 by CS versus 12.0 ± 16 by Hybrid) in patient samples having <75 CTCs. For samples with high CTC counts (>75), the Hybrid method may have underestimated the total Hrp Isolator CTC count. An important feature of the Hrp Isolator is its ability to enrich CTCs that are negative or low expressers of epithelial cell adhesion molecule (EpCAM) or cytokeratin (CK). These cells could have been missed by positive-selection technologies such as CS and may not have been counted using the Hybrid method. CTC spiked samples were run on the Hrp Isolator and the CTC enriched product was spun onto microscope slides, stained, and examined using a Vectra Multispectral Imaging System (Hrp+Vec method). Using the Hrp+Vec method, 79% to 100% of spiked CTCs were recovered depending on the slide fixation method used. Also, using Hrp+Vec we confirmed the ability of the Harpoon Isolator to enrich EpCAM-/CK+ and EpCAM+/CK- CTCs from patient samples. This establishes the existence of "non-canonical" tumor cell types in circulation and adds to the biological diversity of the CTC population in circulation. These results emphasize the need to further characterize all tumor cell populations and to determine what, if any, clinical significance may be assigned to these diverse CTC types.

#2423

Deep phenotyping of dissociated solid tumor cells from breast cancer specimens.

Aaron J. Middlebrook,1 Peter Llontop,1 Mary Beth Hanley,1 Warren Porter,2 Friedrich Hahn,2 Eileen Snowden,2 Rainer Blaesius,2 Smita Ghanekar1. 1 _BD Biosciences, San Jose, CA;_ 2 _BD Technologies, Raleigh-Durham, NC_.

Exploring tumor heterogeneity with the goal of improving outcome has led to the need to glean as much information as possible at an individual cell level from these valuable specimens. Traditional approaches to solid tumor analyses fail to reveal the diverse range of cellular compartments beyond tumor cells that comprise the tumor microenvironment. A comprehensive approach to tumor interrogation requires efficient tissue dissociation to facilitate analysis at the single-cell level. Compared to current methods, single-cell analysis of tumor derived cell suspensions by flow cytometry has the potential to provide a more thorough understanding of the many subpopulations within the tumor microenvironment and the cell-to-cell interactions that govern this space.

Here, we demonstrate an efficient workflow that enables comprehensive cell analysis of solid tumors from breast cancers. After dissociating human breast cancer biopsies from primary tumors into cell suspension, we analyzed the immune compartment and the cancer/stromal cell compartment by multicolor flow cytometry, using 26 markers. A comprehensive analysis of this data set reveals the heterogeneity within the tumor microenvironment on a phenotypic level, which might have potentially significant correlations to the clinical status and molecular phenotype of the cancer. These results encourage the expansion of the use of flow cytometry as a means of solid tumor biopsy analysis, highlighting the potential clinical value of this approach in disease management.

### Pediatric Cancer Genomics, Genetics, and Epigenetics

#2424

Genomic landscape of aggressive tumor subtype in MYCN-non-amplified neuroblastoma.

Miki Ohira-Ichikawa. _Department of Cancer Diagnosis, Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan_.

Neuroblastoma is known to exhibit wide ranges of clinical phenotypes, from spontaneous regression to highly resistant to chemotherapy. One of the challenges in the clinic is to develop suitable therapeutic strategies for the intermediate risk-type of patients (stage 3 or 4 without MYCN amplification) whose prognosis sometimes tends to be poor in terms of long-term follow-up (10 years survival rate was 51% in our database, n=161). To identify key biological pathways involved in the unfavorable phenotypes in this patient group, we have analyzed 63 primary tumors (dead:32, alive:31) without MYCN amplification nor ALK alteration by integrated genomic analyses such as array CGH, whole exome sequencing (SureSelect XT Human All Exon V4, average read depth:157, x20 coverage:95%), RNA sequencing, as well as Methylome profiling (Infinium HumanMethylation450 BeadChip). By using the Karkinos and ANNOVAR software, we found so far 816 non-synonymous somatic single nucleotide variations (SNVs) in coding exons in total (approximately 20 per tumor in average) and number of alterations in each tumor strongly correlated with age at diagnosis (p<0.0001) and patient prognosis (p=0.0077). Recurrent somatic SNVs (n=2 to n=5) were found in 66 genes, 19 of those were involved only in the tumors from the 24 patients with poor outcome. Gene Ontology and pathway analysis (GeneMANIA) using the 66 genes showed that mutations occurred in the features including cytoskeleton, ion channel and extracellular matrix were significantly correlated with patient survival of the 63 MYCN-non-amplified tumors with logrank p-values of 0.0039, 0.0482 and 0.0011, respectively. ATRX mutations were observed in four tumors (6.3%), all of them were occurred in patients with unfavorable phenotypes (3 dead, 1 alive but relapsed twice). By methylome signature, the 63 tumors were subdivided into at least three clusters, which also showed strong correlation with patient prognosis (cluster-1 vs. cluster-2 and -3, p=0.0368). Unfavorable cluster-2 and -3 exhibited hyper-methylation phenotypes in CpG islands and the former was quite similar to that of MYCN-amplified tumors. Thus, these prognosis-related genomic features could be useful markers for risk classification and help understanding molecular mechanism for aggressive phenotypes of MYCN-non-amplified neuroblastomas.

#2425

Genome-wide mapping of MYCN, MYC, and MAX binding across neuroblastoma cell lines identifies novel transcriptional targets.

Jo Lynne Harenza, Robyn Sussman, Derek Oldridge, John Maris. _Children's Hospital of Philadelphia, Philadelphia, PA_.

Arising from the sympathetic nervous system, neuroblastoma (NBL) is the most common extracranial solid tumor in children. Although the 5-year survival rate for children in the low and intermediate risk groups ranges between 90 to over 95%, children with high-risk disease have only a 40-50% likelihood of survival and relapse of high-risk disease is currently largely incurable. The best-characterized genetic determinant of high-risk disease is amplification of MYCN. Members of the MYC protein family are highly homologous and share redundant biological functions, each able to form heterodimers with MAX at consensus E-box motifs (CANNTG) to regulate transcription. While MYCN is known to transcriptionally activate genes involved in malignancy and repress expression of genes that antagonize metastases, amplification of MYCN alone is likely not sufficient for transformation. To identify MYCN and MYC DNA binding partners and ultimately, therapeutic targets in the high-risk NBL subset, we performed chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq) for MYCN, MYC, and MAX across three MYCN amplified and three non-MYCN amplified cell lines (n=2 ChIPs per protein). Peak calling was performed using MACS2 and annotated using bedtools and/or ChIPseeker. MYCN displays genome-wide binding in all three amplified cell lines, suggesting global transcriptional regulation in NBL. ChIPenrich and TOPPgene were used to determine enrichment of peaks based on gene ontology. As expected, we show MYCN ChIP peaks were significantly enriched for MYC and MAX interactions, as well as DNA-binding, in the MYCN amplified subset of cell lines. Genes containing peaks within 1 kb of gene promoters were intersected with genes differentially expressed based on MYCN amplification status in high-risk NBL cases based on RNA-Seq analysis. This set of over 4000 genes was significantly enriched (p < 1E-10) for multiple categories of RNA binding and ribosomal proteins, as well as interactions with known oncogenes and/or cooperating partners of MYCN or MYC: CUL7, VCAM1, SUMO2, and CDK2 (p < 1E-20). Finally, we performed de novo motif discovery on MYCN ChIP peaks using Homer2 and found significant enrichment (p < 1E-50) of 15-20 motifs, including the canonical MYCN basic helix loop helix E-box motif (CCACGTGGCN, ~23% enriched), an ELK1 ETS motif (~44%), an AHR motif (~29%), and a SMAD4 (MAD) motif (~21%). To our knowledge, this combination of RNA- and ChIP-Seq represents the first global epigenomic analysis of MYCN, MYC, and MAX binding across multiple NBL cell lines, revealing MYCN-targeted transcriptional upregulation and/or putative repression. This and further epigenomic characterization of known and novel MYC and MYCN transcriptional targets will enable prioritization of potential therapeutic targets for treatment of the high-risk NBL subset.

#2426

Detection of somatic mutations associated with pulmonary metastasis of osteosarcoma by whole exome sequencing.

Shintaro Iwata,1 Yasutoshi Tatsumi,2 Tsukasa Yonemoto,1 Hiroto Kamoda,1 Takeshi Ishii,1 Hiroki Nagase,2 Miki Ohira3. 1 _Chiba Cancer Center, Chiba, Japan;_ 2 _Chiba Cancer Center Research Institute, Chiba, Japan;_ 3 _Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan_.

Objective: Pulmonary metastasis is a main cause of death for the patients with osteosarcoma (OS), although no specific genetic alteration associated with pulmonary metastasis has been reported. The objective of this study was to identify candidate somatic genetic alteration contributed to pulmonary metastasis in pediatric OS.

Methods: Five sample sets including the tissues taken from primary tumor (P), resected pulmonary metastatic tumor (M), and normal tissue (N) of the same pediatric OS patients were analyzed by whole exome sequencing with Ion Torrent Proton sequencer. 40ng of genomic DNAs extracted from each tissue were used for multiplex PCR amplification with Ion Ampliseq Exome Kit. Data analysis including alignment to the hg19 human reference genome and variant calling was done using the Torrent Suite Software. Obtained genomic data was validated by visualizing in Integrative Genomics Viewer or capillary sequencing. Candidate somatic genetic alterations were annotated by wANNOVAR database. Pathway analysis was performed using GeneMANIA.

Results: Mean read depths on the P, M, and N were 219, 212, and 109, respectively. We identified 1364 and 1311 candidate non-synonymous somatic single nucleotide variants (SNVs) among the P and M, respectively. After the validation and filtering, we detected 135 non-synonymous SNVs within the exon region in M. Among these, 32 SNVs were overlapped with those in P. Only 2 recurrent SNVs were identified within the whole SNVs. C:G>T:A transition mutations were frequently observed in both P and M, and the frequency were higher in M comparing to P. Pathway analysis showed a significant enrichment of the candidate SNVs in genes involved in extracellular matrix regulation (FDR;2.37E-03).

Conclusion: We have identified 137 SNVs associated with pulmonary metastasis from pediatric OS. Extracellular matrix-relating signature might contribute to the genesis of pulmonary metastasis.

#2427

Polycomb group protein BMI1 protects neuroblastoma cells from DNA damage-induced apoptotic death in cooperation with L3MBTL2.

Nobuhiro Akita,1 Hisanori Takenobu,2 Hiroshi Chikaraishi,2 Miki Ohira,2 Takehiko Kamijo2. 1 _RIKEN Research Center for Allergy and Immunology, Tsurumi-ku, Yokohama City, Japan;_ 2 _Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama, Japan_.

[Introduction] BMI1 is a polycomb protein and its overexpression has been correlated with cancer development and aggressiveness. We previously reported that v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN)-induced BMI1 positively regulated neuroblastoma (NB) cell proliferation via the transcriptional suppression of tumor suppressors in NB cells (ONCOGENE, 2010). The role of BMI1 in DNA damage repair pathways has recently been highlighted. The ubiquitination of H2AK119 is also up-regulated locally at DNA damage sites, namely those of double-strand breaks and UV-damaged lesions. BMI1 and other PcG proteins make foci at DNA damage sites and are involved in DNA damage repair pathways; however, the exact role of PcG proteins in DNA damage repair pathways remains to be elucidated.

[Results and Discussion] In order to evaluate the potential of BMI1 as a new target for NB therapy, we herein examined the effects of BMI1 reductions on NB cell differentiation and apoptotic NB cell death. BMI1 knockdown (KD) up to 7 days in NB cells significantly induced their differentiation. BMI1 depletion up to 14 days significantly induced apoptotic NB cell death along with the activation of p53, increases in p73, and induction of p53 family downstream molecules. BMI1 depletion in vivo markedly suppressed NB xenograft tumor growth. A pathological analysis using the TUNEL assay indicated the induction of apoptotic cell death. BMI1 reductions activated ATM and increased γ-H2AX in NB cells. The recently identified polycomb protein L3MBTL2 was clearly decreased in BMI1 KD NB cells. BMI1 KD-induced L3MBTL2 reductions occurred at the protein level. BMI1 interacted with Ring1b, but not with L3MBTL2. Furthermore, L3MBTL2 KD by siRNA effectively accelerated apoptosis induced by the BMI1 inhibitor, PTC-209, related to apoptotic cell death accompanied by Jun/EGR-1/p53 pathway activation in NB cells. In neuronal cells, NGF depletion induced JUN pathway activation and apoptotic cell death. Furthermore, JUN modulates myeloma cell apoptosis by interacting with EGR-1, which down-regulates Survivin and triggers caspase signaling; JUN up-regulates EGR-1 expression by binding its promoter, which suggests that EGR-1 is the direct downstream target gene of the JUN transcription factor. In solid tumors, EGR-1 regulates radiation-induced cell death via apoptotic pathways including p53-dependent apoptosis.

These results clarified, for the first time, the cooperation between PcG BMI1 and L3MBTL2 in DNA damage responses. Therefore, BMI1 and L3MBTL2 appear to be promising targets in new therapies for NB tumors.

#2428

Genetic aberrations in the DNA repair pathway among children with Philadelphia chromosome positive leukemias.

Nellina Andriano,1 Paola Bonaccorso,1 Valeria Iachelli,1 Manuela La Rosa,1 Emanuela Cannata,1 Luca Lo Nigro2. 1 _University of Catania, Catania, Italy;_ 2 _Azienda Policlinico - OVE, Catania, Italy_.

Background. Aberrations in the DNA repair pathway among children with leukemia are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. Germline aberrations of the DNA-repair machinery are crucial for generating chromosomal instability and occurrence of acute leukemia in children. In order to better understand this mechanism, we studied a specific subgroup of childhood leukemia, e.g. t(9;22) or chromosome Philadelphia positive (Ph+) acute lymphoblastic and chronic myeloid leukemias. We addressed our research to specific genes (Nijmegen Breakage Syndrome (NBS1) and Fanconi Anemia) involved in the DNA repair pathway, in the attempt to identify mutations and aberrant expressions, predisposing and/or cooperating with the leukemogenic process.

Materials and Methods. We analyzed diagnostic and remission samples of 12 children: 8 with a Ph+ acute lymphoblastic leukemia (ALL) and 4 with a chronic myeloid leukemia (CML) diagnosed and treated at our institution from 1999 to 2013. We performed RT-PCR analyses to detect mutations in exons 3-6 of the NBS1 gene, followed by a direct sequencing analysis (BMR - Padova - Italy). The status of FANCD2 and PALB2 genes was studied by a multiplex ligation-dependent probe amplification (MLPA) using the Salsa MLPA P057-025R FANCD2-PALB2 Probemix and SALSA MLPA EK1 kit (MRC-Holland, Amsterdam, the Netherlands). Samples from five healthy donors (HDs) were used as wild-type controls. For data elaboration, we used Coffalyser.Net software for MLPA. Moreover we performed a real-time PCR amplification of BRCA1 (exons 14-15) and BRCA2 (exons 15-16) genes, respectively, calculating the median fold-changes (MFC) in patients comparing with HDs.

Results. We detected the NBS1 E185Q polymorphism (c533G>C, rs1805794) in both diagnostic and remission samples in 3 out of 12 children with a Ph positive acute leukemia (2 ALL, 1 CML). We also found two pairs of deletions in FANCD2 (Δ32-35; Δ35-38) and PALB2 (Δ2; Δ2-6) in 4 out of 12 Ph positive cases (2 ALL and 2 CML). These aberrations were mutually exclusive. Thus a total of 7 cases (58%) showed an aberrant pathway. Moreover we determined that the MFC of BRCA1 expression among children with Ph positive ALL and CML was slightly higher than HDs (1,388 vs 0,433 and 1,625 vs 0,451, respectively). Surprisingly the MFC of BRCA2 expression was strongly higher than HDs in both children with Ph positive ALL (5,924 vs 0,002) and CML (54,504 vs 0,256).

Conclusion. Our preliminary findings strongly suggest that germline mutations or deletions in association with aberrant expression of specific genes that regulate the DNA repair machinery may contribute to the chromosomal instability in children with Ph positive leukemias. These data will be confirmed in a larger population of different subtypes of pediatric leukemia.

#2429

Exome and deep sequencing of clinically aggressive neuroblastoma reveal mutated genes at low frequency involved in cancer progression.

Vito Alessandro Lasorsa,1 Daniela Formicola,1 Piero Pignataro,1 Flora Cimmino,1 Francesco Maria Calabrese,2 Jaume Mora,3 Maria Rosaria Esposito,4 Marcella Pantile,4 Carlo Zanon,4 Marilena De Mariano,5 Luca Longo,5 Michael D. Hogarty,6 Carmen de Torres,3 Gian Paolo Tonini,4 Achille Iolascon,1 Mario Capasso1. 1 _University of Naples Federico II - CEINGE Biotecnologie Avanzate, Napoli, Italy;_ 2 _University of Bari, Bari, Italy;_ 3 _Hospital Sant Joan de Déu, Developmental Tumor Biology Laboratory and Department of Oncology, Esplugues de Llobregat, Barcelona, Spain;_ 4 _Pediatric Research Institute (IRP) – Fondazione Città della Speranza, Neuroblastoma Laboratory, Padova, Italy;_ 5 _U.O.C. Bioterapie, IRCCS AOU San Martino-IST, National Cancer Research Institute, Genova, Italy;_ 6 _Children's Hospital of Philadelphia, Division of Oncology, Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA_.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents as metastatic at the time of diagnosis that is under the age of 5 for 90% of children. Aggressive tumors show survival rates of less than 50%. The complete spectrum of somatic mutations of the most aggressive forms of neuroblastoma is still to be defined. Here we sought to identify additional potential cancer drivers in high-risk and ultra-high-risk (high-risk patients with any adverse event within 36 months from diagnosis) neuroblastoma.

Whole exome sequencing was performed for 17 ultra-high-risk germline and tumor pairs to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in an additional set of 48 germline and tumor pairs (62.5% were ultra-high-risk and high-risk), 17 ultra-high-risk tumors and 17 human-derived neuroblastoma cell lines.

Combining both cohorts we found 22 significantly mutated genes, many of which implicated in cancer progression processes. Of these, fifteen (68.2%) were highly expressed in neuroblastoma supporting the biological rationale for their involvement in this malignancy. CHD9, annotated as cancer driver in public databases, was the most significantly altered gene (4.0% of cases) after ALK. Other genes (PTK2, NAV3, NAV1, LRRC17, PXDN, FZD1, ARHGEF10L and ATRX) expressed in neuroblastoma and involved in cell invasiveness and migration were mutated at frequencies between 4% and 2%. Pathways implicated in cell survival, proliferation and motility (focal adhesion and regulation of actin cytoskeleton) were the most frequently disrupted affecting 14.1% of cases, suggesting potential novel therapeutic strategies to prevent disease progression. Rare potentially pathogenic germline variants were significantly enriched in BARD1, CHEK2 and AXIN2.

To conclude, the combination of whole exome and deep targeted sequencing in a discovery and validation cohort experimental design, identified novel cancer genes in clinically aggressive neuroblastomas. Our analyses demonstrate that infrequently mutated genes may have pathway-level implications in leading tumor progression and suggests possible novel strategies for therapeutic interventions in aggressive forms of neuroblastoma.

#2430

Neuroblastoma: Telomere elongation is responsible for aggressive behavior.

Niloufar Javanmardi,1 Susanne Fransson,1 Rose-Marie Sjöberg,1 Anna Djos,1 Per Kogner,2 Tommy Martinsson1. 1 _Univ. of Gothenburg, Gothenburg, Sweden;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

Neuroblastoma (NB) is a highly malignant pediatric tumor of the sympathetic nervous system that commonly displays low overall mutation frequency. We searched for new structural rearrangements in a series of 275 NBs of all stages using high density SNP microarrays. Exome sequencing was also performed on 15 tumor/constitutional DNA pairs and 25 additional tumors, mainly high-risk NBs, using paired-end sequencing on Illumina instrument. Normalized coverage data were used to generate array comparative genomic hybridization (CGH)-like profiles. Structural rearrangements leading to gain of TERT gene were observed in 15 of 275 neuroblastomas as judged by SNP microarray. 12 out of these 15 tumors with TERT rearrangement belong to 11q-deleted subgroup of NB patients that marks tumors with a poor prognosis. These rearrangements occurred only in high-risk NBs in an almost mutually exclusive fashion with MYCN amplifications, which is a known genetic event in this tumor type. Furthermore, in the small series of NBs analyzed by exome sequencing, we detect extensive structural rearrangements of chromosome 2, 5 and 7 leading to amplification of MYCN, TERT and CDK6 respectively in one tumor. The exome sequencing approach detected at average 14 (range 2-77) somatic protein-changing alterations per tumor. Combining structural and mutational data from the exome sequencing show recurrent alterations in ATRX gene. In our NB cohort we see alterations of ATRX in four non MYCN-amplified tumors: two focal deletions, one nonsense mutation and one deleterious missense mutation. ATRX inactivation results in alternative lengthening of telomeres (ALT). This means that high-risk neuroblastomas cancer cells can preserve their telomeres by ALT or by activation of telomerase reverse transcriptase (encoded by TERT). Alterations in TERT and ATRX were mutually exclusive, which is in agreement with the independent activation by these genes of telomere lengthening. Moreover, three cases with chromothripsis of chromosome 5 with rearrangements affecting the TERT were identified through SNP microarrays. These results identify TERT as at least one of the target genes activated by chromothripsis of chromosome 5. This remodeling of the genomic context likely abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumors. This study identifies recurrent TERT and ATRX rearrangements and telomere lengthening as an important mechanism characterizing high-risk tumors and it supports the pharmacological inhibition of these targets to improve the outcome for this patient group.

#2431

Enrichment of targetable mutations in the relapsed neuroblastoma genome.

Olivia M. Padovan-Merhar,1 Pichai Raman,1 Kaitlyn R. Rubnitz,1 Siraj M. Ali,2 Vincent A. Miller,2 Yael P. Mosse,1 Meaghan P. Granger,3 Brian D. Weiss,4 John M. Maris,1 Shakeel Modak5. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _Foundation Medicine, Inc., Cambridge, MA;_ 3 _Cook Children's Health Care System, Fort Worth, TX;_ 4 _Cincinnati Children's Hospital, Cincinnati, OH;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Neuroblastoma (NB) is a pediatric tumor responsible for 15% of pediatric cancer deaths. Patients with relapsed high-risk disease have less than a 5% chance of survival despite intensive cytotoxic chemotherapy regimens. Personalized therapies targeted against driver oncogenes may improve patient outcomes. However, genetic analyses of tumors biopsied at diagnosis generally harbor few, if any, targetable mutations [Pugh et al. Nat Gen 2013]. A recent study comparing the genetic profiles of tumors from 23 NB patients before and after disease relapse showed that relapsed tumors have a higher percentage of targetable mutations, particularly in the ALK/RAS/MAPK pathway [Eleveld et al. Nat Gen 2015]. We performed a retrospective study to further define the genetic landscape of diagnostic and relapsed NB. A total of 151 NB samples from 11 institutions were submitted for targeted sequencing of 236 genes commonly mutated in cancer using the FoundationOne assay; 40 at diagnosis, 67 at disease relapse, and 38 during primary therapy (i.e. second look surgery). Three patients were biopsied at both diagnosis and disease relapse. Patients were included in the study based solely on the availability of FoundationOne data. We identified 38 unique genes with known oncogenic mutations in this cohort. Of these, ALK was the most prevalent, with mutations occurring in 13.8% of patients, and there was a higher frequency of known oncogenic ALK mutations in relapsed disease (17% of patients) than at diagnosis (7.7% of patients). Further, there were more unique genes with known oncogenic mutations or gene amplifications in relapsed disease (25 mutated, 7 amplified) than at diagnosis (14 mutated, 2 amplified). Patients with relapsed disease were more likely to have at least one known mutation or gene amplification (60% vs. 41% of patients, P=0.07). ALK mutations in NB are targetable with available therapies, and of the 37 other mutated genes detected, 13 are "potentially actionable", falling within pathways that are targetable by drugs which are either currently available or in clinical trials. Patients with relapsed disease displayed a greater likelihood of having potentially actionable mutations (34% vs. 21% of patients). Of the 144 unique patients in this study, 21 were reported to have received targeted therapy based on the sequencing results and 14 outcomes were reported: while 10 patients showed progressive disease, one patient had a complete response, one had a partial response, and two had stable disease after targeted therapy. Our data confirm recent evidence suggesting that NBs undergo substantial mutational evolution during therapy, and as a result, relapsed disease is more likely to be driven by a targetable oncogenic pathway. These data support the conclusion that biopsy of relapsed NB has the potential to benefit the patient. Prospective clinical trials to match sequencing results to targeted therapies are required.

#2432

Epigenetic reprogramming of osteosarcoma tumor initiating cells alters histologic and metastatic phenotype.

Emma V. Hyddmark, Padraic Levings, Margaret White, Maria Guijarro, Elham Nasri, Ali Zarezadeh, Glyn Palmer, Steve Ghivizzani, Charles P. Gibbs. _University of Florida, Gainesville, FL_.

Osteosarcoma (OS) is a pediatric cancer with 40% mortality despite aggressive treatments. Major treatment challenges are that some tumors do not respond to therapies or develop resistance over time, processes partially attributed to cellular heterogeneity within a tumor. We have previously demonstrated such heterogeneity using a reporter consisting of an Oct4 promoter driving expression GFP. Cells capable of activating the reporter, the GFP+ cells, are 100-fold more tumorigenic than the GFP- cells. Pure populations of GFP+ cells injected into mice produces heterogeneous tumors, indicating that the GFP- cells arise from the GFP+ cells. The two populations display distinct gene expression profiles, which are reproducible between tumors and representative of global changes. These characteristics suggest that the emergence of a GFP- cell population is due to epigenetic changes. The objective of this study was to investigate to what extent tumorigenic potential in OS is governed at the epigenetic level and can be manipulated exogenously by agents that modify or re-pattern the epigenome.

To test this, sorted GFP+ OS cells were treated with the histone deacetylase inhibitor Trichostatin A (TSA) in vitro at a dose that increased histone acetylation without inducing cell death. Although TSA treatment showed promising antitumoral effects in vitro by inhibiting proliferation and inducing cell cycle arrest, however, global interrogation of the gene expression changes induced by TSA treatment revealed an activation of many cancer associated pathways. In fact, TSA treated Oct4-GFP+ cells injected into NSG mice generated tumors that displayed enhanced metastatic dissemination, a reduced GFP+ population, and histologic changes. Differential expression analyses between the GFP+ cells isolated from TSA tumors or tumors generated from DMSO treated cells showed that the new phenotype was associated with enrichment for cell cycle pathways and a decrease in extracellular matrix pathways. This was confirmed at the protein level and with proliferation assays. As rapidly proliferating cells are generally more sensitive to chemotherapy, we investigated the sensitivity of the GFP+ cells from TSA and Control tumors to Doxorubicin (Dox). Cells isolated from TSA tumors were significantly more receptive to Dox-induced cell death.

Our results suggest that OS malignancy is partially governed at the epigenetic level and that it is possible to reduce intratumoral heterogeneity. A reduction in intraturmoral heterogeneity can be exploited in cases of tumors that are refractory to treatment or have acquired epigenetic resistance. Intermittent treatment with epigenetic modifiers could potentially sensitize the tumors to the other treatments, as have been demonstrated for other cancer types.

#2433

Loss of chd5-mediated gene repression synergizes with MYCN to accelerate neuroblastoma tumorigenesis in zebrafish.

Mark W. Zimmerman,1 Shuning He,1 Jimann Shin,1 Shizhen Zhu,1 Feng Guo,1 Marc Mansour,1 Deepak Reyon,2 J. Keith Joung,2 Jinhua Quan,1 Timur Yusufzai,1 A Thomas Look1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

Neuroblastoma is a malignancy of the peripheral sympathetic nervous system (PSNS) and accounts for 10-15% of cancer deaths among children. For the 40% of patients presenting with high-risk disease, current therapeutic approaches are insufficient and long-term survival is less than 50%. Along with genomic amplification of the MYCN oncogene, hemizygous loss of the 1p36 chromosomal region is a major risk factor in neuroblastoma. The human CHD5 gene is a neuronal specific chromatin remodeling helicase that maps to 1p36, and is thus frequently lost in high-risk neuroblastoma. Our laboratory has previously generated a faithful model of pediatric neuroblastoma in the zebrafish driven by overexpression of the MYCN oncogene in the PSNS (dbh:MYCN). Additionally, zebrafish chd5 mutant alleles were created using the newly developed gene editing technologies TALEN and CRISPR-Cas9. The resulting chd5 mutant fish exhibit abnormal development of the PSNS in the form of expansion of the superior cervical ganglia and enlargement of the interrenal gland (adrenal medulla). Haploinsufficiency for Chd5 combined with dbh:MYCN expression accelerates the onset and increases the penetrance of neuroblastoma tumorigenesis in zebrafish, indicating a tumor suppressive function. Elevated p-ERK and PCNA+ cells in tumor tissue indicates that loss of Chd5, cooperates with MYCN overexpression to accelerate neuroblast proliferation in vivo. Chd5 (in addition to Chd3 and Chd4) is a core member of the epigenetic regulatory NuRD complex, which also contains HDAC1-2, MTA1-3, MBD2-3, GATAD2A/B and RBBP4/7. The conserved biological function of Chd5 is to silence gene expression through the maintenance of a repressed chromatin state. Tumors deficient for Chd5 expression exhibit reduced levels of the H3K27me3 histone modification, a marker of facultatively repressed genes. Future studies will further explore the mechanism and function of Chd5 so that the pathways mediating tumor suppression can be elucidated and that essential proteins in these pathways can be targeted in ways that exploit the synthetic lethal relationships that are established.

#2434

DNA methylation characterization of fusion-positive and fusion-negative rhabdomyosarcoma primary tumors and cell lines.

Wenyue Sun, Bishwanath Chatterjee, John F. Shern, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. _National Cancer Institute, Bethesda, MD_.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 or PAX7-FOXO1) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. The current study will explore these issues in a larger, separate cohort of RMS tumors and compare DNA methylation in RMS cell lines and primary tumors.

METHODS: DNA methylation was examined in 48 RMS tumors (21 FP and 27 FN) as well as 10 RMS cell lines (5 FP and 5 FN) on the Illumina HumanMethylation450 BeadChip platform.

RESULTS: Unsupervised clustering analysis using the most variable probes (top 1%) in the 48 RMS tumors revealed that patterns of DNA methylation segregated these tumors into two distinct subgroups; one subgroup contains all 21 FP cases along with 2 FN cases and a second subgroup contains 25 of the 27 FN cases. A principal component analysis confirmed this close association of methylation pattern and fusion status, and showed that the two "discordant" FN cases map in a region between the FP and FN clusters. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. Application of an 11-gene methylation signature developed in our earlier study classified these 48 cases into FP and FN categories with >95% accuracy. In contrast to our previous findings, there was a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Though unsupervised clustering analysis indicated that FP tumors and cell lines cluster as do the FN tumors and cell lines, a principal component analysis clarified these relationships by showing that the two groups of cell lines are located at a considerable distance from the two tumor subtypes. Analysis of the most varied probes in the tumors indicated that the vast majority of these probes are hypermethylated in all RMS cell lines. In a complementary analysis with the most variable probes (top 1%) in the cell lines, most of these probes are hypomethylated in all RMS tumors, though two smaller groups of probes show differential methylation between both groups of FP and FN samples.

CONCLUSIONS: This study provides an independent validation that FP and FN RMS tumors possess distinct and characteristic methylation profiles. The enrichment of PAX3-FOXO1 binding sites in genes that are differentially methylated between these FP and FN tumors suggests that the PAX3-FOXO1 fusion protein may contribute to this methylation pattern. These analyses also indicate that RMS cell lines do not faithfully recapitulate the DNA methylation patterns that characterize primary tumors.

#2435

Amplification of CDK4 and MDM2 is associated with atypical clinical features in high risk neuroblastoma patients.

Susanne M. Fransson,1 Hanna Kryh,1 Niloufar Javanmardi,1 Inge M. Ambros,2 Ana P. Berbegall,3 Ingrid Ora,4 Rosa Noguera,3 Jurate Asmundsson,5 Bengt Sandstedt,1 Peter F. Ambros,2 Per Kogner,6 Tommy Martinsson1. 1 _Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden;_ 2 _Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria;_ 3 _Department of Pathology, Medical School, University of Valencia, Valencia, Spain;_ 4 _Department of Pediatric Oncology and Hematology, Clinical Sciences, Lund University, Lund, Sweden;_ 5 _Karolinska University Laboratories, Unit for Pathology, Stockholm, Sweden;_ 6 _Department of Women's and Children's Health; Karolinska Institutet, Stockholm, Sweden_.

MYCN-amplification and 11q-deletion are important, although incomplete, markers of high-risk neuroblastoma. Thus, characterization of additional genomic alterations that can be used as prognostic and/or predictive markers is of clinical importance in order to provide best treatment possible.

By using SNP-microarrays we identified a small group of neuroblastomas with high grade amplification of one or multiple loci on 12q, commonly involving the potential oncogenic target genes CKD4 (12q13-14) and/or MDM2 (12q15). The CDK4 and MDM2 regions were co-amplified in 13/16 samples, two tumors had CDK4-amplification in absence of MDM2-amplification while one tumor had MDM2-amplification without CDK4-amplification. Exome sequencing was performed on seven tumors and whole genome sequencing (WGS) was performed on tumor and constitutional DNA from one patient with 12q-amplification. No novel protein altering SNVs were detected within the amplified regions except a rare INHBE mutation in one patient. The tumor examined with WGS show high degree of structural rearrangements including chromothriptic features on chromosome 12 leading to high grade amplification of CDK4 and MDM2. This sample displayed 21 somatic protein changing alterations although not in any gene with known function in chromatin stability or DNA repair.

Interestingly, the majority of the 12q-amplified neuroblastomas were of abdominal origin, some with renal location with initial suspicion of Wilms' tumor. Atypical metastatic pattern were also seen in this patient group showing low degree of bone marrow involvement favoring other metastatic sites such as lung.

The consistent co-amplification of two separate chromosome 12 regions in this subset of neuroblastoma suggests that there are one or more genes with importance in tumor development/progression. Our study indicates that CDK4 appears as main target in this 12q-amplified neuroblastoma subgroup although other genes such as MDM2 and FRS2 also could provide proliferative advantages. The 12q-amplified neuroblastomas exhibit distinct clinical features and may benefit from targeted therapy using a small molecule CDK4/CDK6 inhibitor such as LEE011 (Novartis).

#2436

Exploring genomic alterations in pediatric cancer using ProteinPaint.

Xin Zhou, Michael N. Edmonson, Mark R. Wilkinson, Aman Patel, Gang Wu, Yu Liu, Yongjin Li, Zhaojie Zhang, Michael Rusch, Matthew Parker, Jared Becksfort, James R. Downing, Jinghui Zhang. _St. Jude Children's Research Hospital, Memphis, TN_.

Current cancer genome data portals have focused primarily on presenting data generated from adult cancer studies. These portals typically lack features for exploring pathogenic germline mutations, somatic gene fusions, and gene expression profiling, all of which are important biomarkers for risk stratification of pediatric cancer. We have developed ProteinPaint (https://pecan.stjude.org/proteinpaint/), a web service hosting 30,000+ validated somatic SNV/indels and fusion transcripts detected in 1,654 pediatric tumor samples from 17 subtypes, 252 pathogenic or loss-of-function germline lesions detected in >1000 pediatric cancer patients of 21 subtypes, and gene expression profiles derived from RNA-Seq of 928 pediatric tumors. Cancer genomic alterations are shown on novel "disc-on-stem" skewer graphs which were designed to depict the diverse prevalence, complex allelic alteration, and temporal origin of sequence mutations and gene fusions. Adult somatic cancer mutation data from the COSMIC database can be displayed in parallel with pediatric cancer data sets for cross-study comparison. We will demonstrate examples of how ProteinPaint's integrative view of genomic alteration, gene expression and pediatric-adult data comparison has facilitated the evaluation of somatic and germline mutation pathogenicity in a clinical setting. Custom data including sequence mutations in the MAF format used by the Cancer Genome Atlas (TCGA) project, copy number alterations, and structural variations can all be imported and visualized alongside published pediatric and adult cancer data sets. Furthermore, ProteinPaint supports curation and annotation of fusion transcripts predicted from RNASeq data and analysis of tumor clonal evolution with a 2-D plot of mutation frequency of paired diagnosis and relapse samples. ProteinPaint delivers a premium user experience with animation and interactive features for visualizing large cancer mutation datasets, and can serve as a workbench to import, explore and interpret user data. Its framework continues to expand as its intuitive visualization has enabled non-bioinformatics scientists and clinicians to access and manipulate genomic data for discovery and clinical reporting.

### Pediatric Cancer Molecular Pathways

#2438

Genome-wide screen reveals a role for glucocorticoids in B cell development that can be exploited to improve treatment of B cell acute lymphoblastic leukemia.

Miles Pufall,1 Karina Kruth,1 Sarah K. Tasian2. 1 _University of Iowa, Iowa City, IA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Glucocorticoids (GCs) are a component of highly effective therapy for B cell acute lymphoblastic leukemia (B-ALL), the most common childhood cancer. Although successful in 90% of patients, the prognosis for the remaining 10% is dismal. Because sensitivity to GCs alone predicts outcome well, we hypothesized that enhancing or restoring GC sensitivity can improve outcome. To first determine how GCs kill B-ALL cells, we first measured the transcriptional response of sensitive B-ALL samples to dexamethasone (dex). In addition to apoptotic genes (BIM, BCL2), and metabolic genes (TXNIP) identified in other studies that contribute to cell death, we also found broad regulation of B cell development genes, including key checkpoints (ITGA4, IL7R, and BCL6). This suggested that high levels of GCs push arrested B-ALL cells through development, resulting in cell death. To validate these results, and to identify cellular pathways that modulate GC cytotoxicity, we used a next-generation shRNA screen to knock down every protein-coding gene in the genome. The screen identified 265 genes that significantly modulate GC sensitivity. In addition to validating the importance lymphoid development genes, the screen revealed which cofactors and chromatin modifiers collaborate with GR to orchestrate cell death gene programs. Further we were able to map critical signaling pathways with high resolution, including the pro-survival B cell receptor pathway. Inhibition of PI3Kδ at the apex of this pathway potentiated the response of GC-regulated genes to dex, and synnergized with dex to induce cell death in even the most refractory B-ALL tissue. These results indicate a previously unrecognized for GCs in B cell development that can be exploited to improve both the potency and treatment of lymhoid disease.

#2439

Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150 increases the chemosensitivity and decreases tumor-initiating potential of neuroblastoma cells.

Seiichi Odate, Shuang Yan, Veronica Veschi, Norris Lam, Zhihui Liu, Carol J. Thiele. _National Cancer Institute, Bethesda, MD_.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The long-term, event-free survival of high-risk NB remains ~ 50% despite intensive multi-modality treatments. Activated JAK/STAT3 pathway plays an important role in many human cancers. In NB patients, cytokines activating STAT3 have been associated with poor patient outcome and have been implicated in the survival of a rare population of NB tumor initiating cells. Thus targeting STAT3 may be a promising therapeutic strategy for high-risk NB. To evaluate the biologic consequences of genetic targeting of STAT3, we assessed the effects of inhibition of STAT3 in NB cell lines containing a tetracycline (Tet)-inducible STAT3 expression plasmid. Additionally we evaluated pharmacogenomic inhibition of STAT3 using AZD9150, a generation 2.5, 16-nucleotide, antisense oligonucleotide (ASO) that is now in Phase I/II clinical trials. Studies were conducted in 3 representative NB cell line models (AS (MYCN-WT) and NGP and IMR32 (MYCN-Amplified)). Both the Tet-inducible STAT3 shRNA and AZD9150 reduced endogenous STAT3 mRNA and protein levels causing decreased transcription and expression of STAT3 target genes, such as CyclinD1, D3, and MYC/MYCN. In functional in vitro studies Tet-inducible STAT3 shRNA and AZD9150 decreased NB cell migration and clonogenicity in soft agar. In vivo, STAT3 inhibition by Tet-inducible STAT3 shRNA or AZD9150 alone had little effect on the growth of established tumors nor did it alter the survival of tumor-bearing mice, despite decreases in STAT3, P-STAT3 and target gene expression in xenografts from AZD9150-treated mice compared to those from ASO-treated mice. To assess whether inhibition of STAT3 altered the tumor initiating potential of NB cells, NB tumor xenograft cells from ASO or AZD9150 treated mice were re-implanted and secondary tumor growth assessed. At 200,000 and 20,000 ASO-treated NB cells, 100% of mice had tumors while only 40 and 20%, respectively of AZD9150-treated mice had tumors. These results indicate that the inhibition of STAT3 decreased the frequency of tumor initiating cells in the NB xenograft from AZD9150 treated mice. Since tumor initiating or stem-like cells are frequently more resistant to cytotoxic agents, we next evaluated a combination therapy with cisplatin. We found that in vitro either genetic shSTAT3 or AZD9150 mediated pharmacogenomic inhibition of STAT3 significantly increased the sensitivity of NB cells to cisplatin. Furthermore, in established NB tumors xenografts, the combination of STAT3 inhibition with cisplatin caused a 30% decrease in tumor size (P=0.0092) and increased the survival of AZD9150 treated tumor-bearing mice compared to ASO-treated mice (P=0.026). Our study supports the development of strategies targeting STAT3 in combination with conventional chemotherapy for patients with high-risk NB.

#2440

FEM1A regulation of the NF-κB/YY1/miR-29 circuitry in rhabdomyosarcoma.

Samira C. Grifoni,1 Joseph F. Maher2. 1 _University of Mississippi Cancer Institute, Jackson, MS;_ 2 _University of Mississippi Cancer Institute, Madison, MS_.

Introduction: Rhabdomyosarcoma (RMS), a malignant tumor related to defective skeletal muscle differentiation, is the most common soft tissue neoplasm of children, and metastatic RMS continues to have a poor prognosis. The canonical form of transcription factor NF-κB, a p50/p65 heterodimer, regulates normal myogenic differentiation, and is dysregulated in RMS tumorigenesis. NF-κB is active in myoblasts and blocks myogenic differentiation; for myogenic differentiation to occur, NF-κB must be down-regulated, which leads to down-regulation of YY1 and de-repression of miR-29, a microRNA that promotes myogenic differentiation. Conversely, NF-κB is up-regulated in RMS, leading to activation of YY1, down-regulation of miR-29, and suppression of myogenic differentiation. The mechanisms of NF-κB regulation in these contexts are incompletely understood. FEM1A is a gene most highly expressed in cardiac and skeletal muscle. We have shown that FEM1A is up-regulated early during skeletal muscle differentiation, and is consistently down-regulated in RMS, both in human RMS and in mouse RMS genetic models. However, whether down-regulation of FEM1A plays a role in RMS tumorigenesis, or is just a marker of defective myogenic differentiation, is unknown. In macrophages, FEM1A has been shown to inhibit canonical NF-κB, both by directly binding p50, and by binding and preventing the degradation of p105, the precursor of p50 that also acts as a cytoplasmic inhibitor of NF-κB activation. Therefore, we tested the hypothesis that over-expression of FEM1A in RMS cells decreases NF-κB activation, which in turn results in decreased expression of YY1, leading to increased expression of miR-29, promoting RMS myogenic differentiation.

Methods: Cultured RMS13 rhabdomyosarcoma cells were used. To determine if FEM1A regulates NF-κB/YY1/miR-29 circuitry, resulting in RMS13 differentiation, we transfected RMS13 cells with HA-FEM1A using X-treme Gene HP (Roche).

Results: Using immunoblotting, we observed that FEM1A overexpression decreases p50 and p65 translocation to the nucleus, decreases YY1 protein levels, and increases myosin heavy chain expression, a marker of skeletal muscle differentiation, in RMS13 cells. Using qPCR, we observed that FEM1A overexpression increases miR-29 gene expression in RMS13 cells.

Conclusion: These data demonstrate that FEM1A negatively modulates NF-κB and YY1, and promotes miR-29 expression and myogenic differentiation, in RMS cells. This indicates that FEM1A down-regulation may play a role in RMS tumorigenesis, rather than just being a marker of defective differentiation. Results from our study have the potential to improve understanding of RMS tumor biology, and could allow the FEM1A/NF-κB/YY1/miR-29 circuitry to become a target for molecular-based therapy of RMS.

#2441

CDK12/13 inhibition cooperates with the Ewing sarcoma oncoprotein EWS/FLI to attenuate homologous recombination repair in Ewing sarcoma cells.

Amanda Balboni,1 Björn Stolte,1 Peter Kalev,1 Nicholas Kwiatkowski,2 Tinghu Zhang,1 Brian Abraham,2 Gabriela Alexe,1 Dipanjan Chowdhury,1 Richard A. Young,2 Nathanael S. Gray,1 Kimberly Stegmaier1. 1 _Dana-Farber Cancer Institute, Brookline, MA;_ 2 _The Whitehead Institute for Biomedical Research, Cambridge, MA_.

A therapeutic challenge in pediatric oncology is the paucity of readily "druggable" genetic events in many of the childhood malignancies. These tumors are frequently defined by sentinel abnormalities involving transcription factors in an otherwise quiet genomic landscape. One approach to treating these tumors would involve direct targeting of the aberrant transcription factor; however, this is a drug discovery challenge. A second approach would be to identify synthetic lethal relationships in the context of the aberrant transcription factor. THZ1, a covalent and potent inhibitor of CDK7, CDK12, and CDK13, kinases involved in transcriptional regulation, recently emerged as a targeted strategy to impair aberrant transcription. Extensive profiling of THZ1 against a diverse panel of >1,000 cancer cell lines revealed that the pediatric solid tumor, Ewing sarcoma, was exceptionally sensitive to this compound. We found that the anti-proliferative effects of THZ1 in Ewing sarcoma can be attributed primarily to CDK12/13 inhibition. Treatment of Ewing sarcoma cells with THZ531, a covalent and selective CDK12/13 inhibitor, decreased the phosphorylation of the C-terminal domain of RNA polymerase II, induced apoptosis, and markedly decreased colony formation capacity of Ewing sarcoma cell lines. In contrast, treatment with a selective CDK7 inhibitor had minimal effect. EWS/FLI is the transcription factor fusion protein that typically drives tumor establishment and maintenance in Ewing sarcoma tumors. Based on prior reports of this compound class inhibiting a small subset of highly expressed genes critical to tumor maintenance, we expected that these inhibitors would disrupt oncogenic EWS/FLI-driven transcription as the mechanism of inducing cell death. Surprisingly, however, global gene expression profiling revealed that THZ531 did not selectively repress EWS/FLI or EWS/FLI target genes. Rather, we observed that THZ531 preferentially repressed genes involved in DNA damage repair. Consistent with this finding, we found that THZ531 induced defects in DNA damage repair and highly synergized with DNA damaging agents that induce lesions repaired by homologous recombination (HR). Furthermore, we found that suppression of EWS/FLI attenuated sensitivity to THZ531 and the PARP inhibitor olaparib and abrogated synergy observed with this drug combination. Thus, we conclude that EWS/FLI establishes tumor cell synthetic lethality to CDK12/13 inhibitors by imparting sensitivity to DNA repair defects. This work establishes a novel mechanism of action of CDK12/13 inhibitors and gives further credence to the role of EWS/FLI in DNA damage response. Ongoing work is dedicated to the in vivo testing of THZ1 alone, and in combination with olaparib, as a novel targeted therapy for the treatment of Ewing sarcoma.

#2442

EWS/FLI1 activates GLI1 indirectly through GLI2 in Ewing Sarcoma.

Laura Christensen, William A. May. _USC/Children's Hospital Los Angeles, Los Angeles, CA_.

GLI1 expression in Ewing tumors has been shown to be enhanced by EWS/FLI1 expression in both model transformation systems and in Ewing cell lines. In Ewing cells, GLI1 has been shown to produce significant phenotypic effects consistent with a role in Ewing tumor formation and propagation. Also consistent with an important role is the demonstrated correspondence between the transcriptional target sets of EWS/FLI1 and of GLI1 in Ewing tumor cells. The principal physiologic developmental regulator of GLI1 is the diffusible Sonic Hedgehog ligand which binds the cell surface tumor suppressor Patched with consequent activation of the transmembrane protein Smoothened. In Ewing tumors, the enhancement of GLI1 expression has been shown to be Hedgehog independent and to occur downstream of Smoothened. Indeed, evidence has been published that EWS/FLI1 directly activates the GLI1 promoter. While there is experimental evidence that GLI1 inhibition may be clinically useful in Ewing tumors, preclinical evidence for one inhibitory approach has been disappointing. Interestingly, direct transcriptional activation of GLI1 has not been demonstrated to be of significance in other most other physiologic or pathologic contexts. We speculated that direct transcriptional activation of GLI1 by EWS/FLI1 might be only part of the story in Ewing tumors.

Since GLI2 is the most potent physiologic transcriptional activator of GLI1, we undertook to investigate the role of GLI2 in Hedgehog/GLI (HH-GLI) signaling in Ewing Tumors. We found that GLI2 is broadly expressed in Ewing tumors. Based on GLI2 overexpression and on shRNA knockdown of GLI2, we found that GLI2 expression increases GLI1 expression in Ewing cell lines. Via GLI2 knockdown, we have found that GLI2 has biologic effects similar to or greater than GLI1 in Ewing cells. Finally, we demonstrate that EWS/FLI1 enhances nuclear localization of GLI2, suggesting an additional indirect mechanism by which EWS/FLI1 activates HH-GLI signaling and GLI1 expression in Ewing tumors.

These data indicate that GLI2 is an active player in HH-GLI pathway activation in Ewing tumors. They also suggest that measures which target GLI2 may have equal or greater efficacy to those aimed at GLI1.

#2443

Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor.

Akanksha Mahajan, Sung-Hyeok Hong, Jason U. Tilan, Susana Galli, Congyi Lu, Jasmine Rodgers, Anju Duttargi, Rachel Acree, Luciane R. Cavalli, Joanna B. Kitlinska. _Georgetown University, Washington, DC_.

Ewing sarcoma (ES) is a tumor driven by EWS-ETS fusion proteins. Yet, the same fusions are present in localized and metastatic tumors that carry strikingly different prognoses. Despite low levels of genomic instability in primary ES tumors, the presence of complex karyotypes is one of a few adverse prognostic factors, implicating an acquired chromosomal instability (CIN) in ES progression. As transcriptional targets of EWS-ETS, neuropeptide Y (NPY) and its Y5 receptor (Y5R) are highly expressed in ES and further activated by hypoxia. We have found that overexpression of Y5R leads to defects in cytokinesis, followed by formation of polyploid cells, chromosome loss and CIN. Thus, the goal of our study was to determine whether CIN that is driven by hypoxia-induced activation of NPY/Y5R axis promotes ES metastases. ES cells were injected into gastrocnemius muscles of SCID/beige mice. Hypoxia in the resulting primary tumors was created by 72h ligation of the femoral artery. Then, the tumors were excised and mice were monitored for metastases. Tissues and cells derived from primary tumors and metastases were subjected to cytogenetic analyses. ES metastasis was associated with progressive genomic changes in tumor cells. Cells derived from primary tumors exhibited increases in nuclear sizes and ploidy, as compared to the original cells. Tumor hypoxia exacerbated this effect. This initial increase in ploidy was followed by a decrease in nuclear size, increase in mitotic errors and reduced chromosome numbers in cells from metastatic tissues, suggesting that ES progression associates with increased CIN and is triggered by cell polyploidization. This notion was confirmed by increased DNA copy number alterations in tissues from ES metastases observed in xenografts derived from 2 different cell lines and a clinical case of matched primary tumor and metastasis tissue (array-CGH). In SK-ES1 xenografts, these alterations involved gains in the locus of Y5R. Consequently, FISH identified an SK-ES1 clone with 3 copies of the Y5R gene. The percent of cells with Y5R gene amplification increased with the degree of SK-ES1 progression, with 16-24% cells in the original SK-ES1 cell line, 40-60% in primary tumors and 86-100% in metastases. This was associated with an increase in Y5R expression in metastatic tissues. Thus, the metastasis in SK-ES1 xenografts associated with a selection of the clone with amplified Y5R. SK-ES1 cells subjected to hypoxia in vitro presented with similar increases in nuclear sizes and enrichment in the clone with amplified Y5R (48%), as was observed in primary tumors. Y5R activation in normoxic SK-ES1 cells mimicked this effect. Our findings support the role for acquired CIN in ES progression and metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. Thus, Y5R antagonist may serve as an adjuvant treatment to prevent ES CIN and progression.

#2444

TSLP regulates expression of Bcl2 family proteins in Ph-like ALL with CRLF2 alterations.

Cornelia Stoian, Muhammad Omair Kamal, Olivia Francis, Rhaya Johnson, Simone Montgomery, Jacqueline Coats, Hannah Choi, Shania Aponte-Paris, Micheal Reed, Shanalee Martinez, Karina Mayagoitia, Evgeny Chirshev, Chunhua Song, Sinisa Dovat, Kimberly J. Payne. _Loma Linda University, Loma Linda, CA_.

B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood malignancy. A subset of children with B-ALL are at high risk for relapse and death. Gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome positive ALL. Approximately half of these Ph-like B-ALL are characterized by genetic alterations that result in overexpression of CRLF2. CRLF2, together with the IL-7 receptor α chain, forms a receptor complex for the cytokine, TSLP. When TSLP binds, the receptor initiates downstream JAK2/STAT5 and PI3/AKT/mTOR pathway activation. The activating JAK mutations found in some CRLF2 B-ALL led to speculation that TSLP stimulation is not a factor in this disease. However, we find that TSLP increases phosphorylation of STAT5, AKT and S6 (downstream of mTOR) in CRLF2 B-ALL cells, including those with JAK defects. Activation of these pathways has been associated with oncogenesis and chemoresistance and their downstream targets include members of the Bcl2 family. The Bcl2 family pro-survival molecule Bcl-XL is a down stream target of STAT5 in Ph+ B-ALL. Mcl-1, another BCL2 family pro-survival molecule is known to be upregulated by mTOR activation via post-translational mechanisms in B cell lymphoma. We hypothesized that TSLP-induced JAK2/STAT5 and PI3/AKT/mTOR pathway activation contribute to chemoresistance in high risk CRLF2 B-ALL by upregulating the expression of Bcl-XL and Mcl-1. To test this hypothesis we cultured human CRLF2 B-ALL cell lines (MUTZ5 and CALL4) with and without TSLP and evaluated expression of the Bcl2 family pro-survival proteins, Bcl-XL, Mcl-1, and Bcl2. We found that TSLP induced significant increases in Bcl-XL and Mcl-1 proteins, but not Bcl2 in CRLF2 B-ALL cells. These cell lines have activating Jak mutations and thus reflect the ability of TSLP to increase expression of the Bcl2 family proteins in cases where activating JAK mutations are present. Next we evaluated the effect of Mcl-1 inhibitor on MUTZ5 and CALL4 cells. Preliminary data from these experiments show that cell counts in cultures treated with Mcl-1 inhibitor are reduce by >90% and this reduction is maintained in the presence of TSLP. These data provide evidence that TSLP-induced CRLF2 signals increase expression of Bcl2 pro-survival proteins, even in CRLF2 B-ALL cells with activating JAK mutations. These data also suggest that Mcl-1 inhibitors could be an effective treatment for this disease. Ongoing studies will evaluate the effect of TSLP and Mcl-1 inhibitors in primary CRLF2 B-ALL samples.

#2445

Cytoplasmic mislocalization of p27 promotes metastasis and its autoantibody correlates with prognosis in osteosarcoma.

Manjula Nakka,1 Yiting Li,1 Aaron J. Kelly,1 Ching C. Lau,1 Mark Krailo,2 Donald A. Barkauskas,2 John Hicks,1 Tsz-Kwong Man1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Southern California, Los Angeles, CA_.

Development of metastasis is the major cause of death in osteosarcoma, which is the most common malignant bone tumor in children and young adults. However, detection of metastasis relies solely on imaging techniques and targeted treatments of metastatic patients are still not available to improve the dismal outcome. Using an immunoproteomic approach, we identified that p27 autoantibody was significantly elevated in the plasma of high-risk osteosarcoma patients. Results of immunohistochemistry showed that cytoplasmic mislocalization of p27 occurred in a majority of osteosarcoma cases and in highly metastatic osteosarcoma cell lines. We demonstrated that ectopic expression of cytoplasmic p27 and shRNA-mediated gene silencing promoted and inhibited, respectively, the migration and invasion of osteosarcoma cells. Additionally, a mutation in S10 or T198, but not T157, phosphosite abolished the pro-migration and invasion phenotypes of cytoplasmic p27, possibly through a RhoA-related mechanism. Furthermore, mice injected with cells carrying cytoplasmic p27 increased the development of pulmonary metastasis when compared to an empty vector control. Lastly, using a large cohort of serum samples (n=233), we showed that a higher level of the p27 autoantibody significantly correlated with poor overall and event-free survival of osteosarcoma (p < 0.05). Together, our results suggest that the circulating p27 autoantibody can be used as a prognostic biomarker at diagnosis and cytoplasmic mislocalization of p27 is a frequent event in osteosarcoma, which can promote tumor cell motility and invasiveness as well as metastasis formation. Targeting specific p27 phosphorylations may provide a novel therapeutic approach for osteosarcoma patients with metastasis.

#2446

Ezrin inhibition up-regulates stress response gene expression and blocks osteosarcoma metastasis.

Haydar Çelik,1 Gülay Bulut,2 Jenny Han,1 Garrett T. Graham,1 Tsion Z. Minas,1 Erin J. Conn,1 Sung-Hyeok Hong,1 Gary T. Pauly,3 Mutlu Hayran,4 Xin Li,5 Metin Özdemirli,6 Ayşe Ayhan,7 Michelle A. Rudek,8 Jeffrey A. Toretsky,1 Aykut Üren1. 1 _Department of Oncology, Georgetown University Medical Center, Washington, DC;_ 2 _Department of Molecular Biology and Genetics, Faculty of Engineering and Natural Sciences, Bahçeşehir University, Istanbul, Turkey;_ 3 _Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD;_ 4 _Department of Preventive Oncology, Cancer Institute, Hacettepe University, Ankara, Turkey;_ 5 _Department of Biostatistics, Bioinformatics, and Biomathematics, Georgetown University, Washington, DC;_ 6 _Department of Pathology, Georgetown University Medical Center, Washington, DC;_ 7 _Department of Pathology, Seirei Mikatahara Hospital and Hamamatsu University School of Medicine, Hamamatsu, Japan;_ 8 _The Johns Hopkins University School of Medicine, Department of Oncology and Department of Medicine, Division of Clinical Pharmacology and The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD_.

Ezrin is a member of the ezrin, radixin, moesin (ERM) protein family of membrane-cytoskeleton linkers. Ezrin has been implicated in many essential cellular functions including cell adhesion, motility, maintenance and determination of cell shape, cell proliferation and apoptosis, regulation of ion channels, morphogenesis and signal transduction. Ezrin promotes invasive and migratory capabilities of cancer cells. A high level of ezrin expression is associated with poor clinical outcome and metastatic behavior of pediatric solid tumors including osteosarcoma and rhabdomyosarcoma as well as multiple other tumor types. Ezrin, therefore, could be a promising molecular target for the prevention and treatment of cancer metastasis. We previously discovered two small molecule inhibitors, NSC305787 and NSC668394, which bind directly to ezrin and inhibit its activity in mediating the invasive phenotype of osteosarcoma cells in multiple in vitro and in vivo assays. In this study, we expand on our previous findings by demonstrating that NSC305787-treatment but not NSC668394 significantly reduces pulmonary metastasis in a genetically engineered mouse model of osteosarcoma. We assessed the pharmacokinetics of compounds in mice and demonstrated that NSC305787 has a more favorable pharmacokinetic profile compared with NSC668394. In order to uncover ezrin-mediated biological pathways that can be used for a specific pharmacodynamic marker(s) of response to ezrin inhibition, we profiled global gene expression in osteosarcoma cells after treatment with inhibitors. We identified several commonly up-regulated genes with functional relevance to integrated stress response, implicating that a common underlying mechanism may be shared by these compounds. We further validated the microarray data through extensive testing using real-time qPCR and verified the specificity of the transcriptional response using another novel ezrin inhibitor MMV667492 that we have identified recently from the MMV400 "Malaria Box" library. The effect of ezrin inhibitors on the expression of stress genes was recapitulated by siRNA-mediated depletion of ezrin. The up-regulation of stress genes was much weaker in cells with reduced ezrin levels compared to wild-type cells, indicating the specificity of the compounds on ezrin-mediated cellular responses. Analysis of the expression of stress genes in white blood cells and skin of NSC305787-treated mice demonstrated up-regulation of the DDIT4/REDD1, suggesting that DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of response to ezrin inhibition. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive, may have important functions regulating gene expression and inhibition of ezrin activity by NSC305787 in osteosarcoma could be an attractive therapy to prevent clinically significant metastasis.

#2447

PIN1 protects GLI1 from ubiquitination and promotes Hedgehog-driven medulloblastoma tumorigenesis.

Jean-Francois M. Rual,1 Tao Xu,1 Honglai Zhang,1 Sung-Soo Park,1 Sriram Venneti,1 Rork Kuick,1 Kimberly Ha,1 Lowell Michael,1 Mariarita Santi,2 Chiyoko Uchida,3 Takafumi Uchida,4 Ashok Srinivasan,1 Andrzej Dlugosz,1 Sandra Camelo-Piragua1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 3 _Fukushima University, Fukushima, Japan;_ 4 _Tohoku University, Sendai, Japan_.

Medulloblastoma is the most common malignant brain tumor of childhood. Therapeutic approaches to medulloblastoma have led to significant improvements, but are achieved at a high cost to quality of life. Aberrant upregulation of the hedgehog (Hh) pathway drives cerebellar tumorigenesis in ~30% of medulloblastoma patients. Hh pathway inhibitors such as the SMO antagonist Vismodegib are currently being tested in Hh-activated medulloblastoma and the preliminary results are encouraging. However, resistance to SMO inhibition can be acquired, leading to relapse. Alternative therapeutic approaches are needed. We aim to uncover novel Hh signal modulators that are essential in medulloblastoma to maintain tumorigenic potential. Using our proteomic platform for systematic protein interaction mapping, we discovered a novel interaction between GLI1, a key transcription factor for the mediation of Hh signals, and PIN1, a peptidylprolyl cis/trans isomerase that regulates the post-phosphorylation conformation of its substrates. Our results support a molecular model in which PIN1 protects GLI1 from proteasomal degradation, thus contributing to the positive regulation of Hh signals. Most importantly, our in vivo functional analyses of PIN1 in mouse models of Hh-driven medulloblastoma demonstrate that the loss-of-PIN1 impairs tumor development and increases survival by 3 fold, from 57 to 158 days (P < 0.0001), establishing PIN1 as a key factor in Hh-driven medulloblastoma tumorigenesis. Finally, in human medulloblastoma tumor samples, the GLI1 and PIN1 proteins are correlated in their expression, supporting the relevance of the GLI1/PIN1 interaction in this disease context. In summary, the discovery of the GLI1/PIN1 interaction uncovers PIN1 as a novel therapeutic target in medulloblastoma tumorigenesis. If our hypothesis is validated, i.e., PIN1 inhibitors can improve survival in mouse models of Hh-driven medulloblastoma, our project will strongly justify testing the clinical relevance of PIN1 blockade in medulloblastoma patients.

#2448

Suppression of MYCN-driven neuroblastoma malignant phenotype by telomerase-targeted tumor suppressor p53 transactivation.

Terutaka Tanimoto,1 Hiroshi Tazawa,1 Hiroshi Noso,1 Takanori Oyama,1 Yasuo Urata,2 Shunsuke Kagawa,1 Takuo Noda,1 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Oncolys Biopharma,Inc., Okayama, Japan_.

Background: Neuroblastoma (NB) is a primary malignant tumor in the peripheral sympathetic nervous system. High-risk group of NB has an unfavorable clinical course, even after treatment with current chemotherapy regimens and aggressive surgical resection. Therefore, a novel therapeutic strategy is needed for the treatment of high-risk group of NB. Recent accumulating evidences in whole-genome sequencing analysis have shown that human telomerase reverse transcriptase (hTERT) gene rearrangement and MYCN gene amplification are predictive biomarkers for the high-risk group of NB, in which massive transcriptional activation of hTERT mRNA was frequently observed. We recently developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific virus replication, and OBP-702 further expresses tumor suppressor p53 protein. In this study, we investigated whether OBP-301 and OBP-702 inhibit cell viability and MYCN protein expression in human NB cells with different MYCN status.

Methods: We used 3 human NB cell lines with or without MYCN amplification, including IMR-32 (MYCN-amplified), LA-N-5 (MYCN-amplified), SK-N-SH (MYCN-not amplified). The antitumor effects of OBP-301, OBP-702, and Ad-p53, a replication-defective adenovirus expressing p53 gene, were evaluated using XTT assay. Virus-induced apoptosis and MYCN suppression were analyzed by Western blot analysis.

Results: OBP-301, OBP-702, and Ad-p53 suppressed the viability of all 3 NB cell lines, whereas OBP-702 showed the most profound anti-tumor effect especially in MYCN-amplified NB cells. OBP-702 induced higher p53 expression than Ad-p53, resulting in the p53-mediated apoptotic cell death. Although MYCN expression in NB cells with MYCN amplification was downregulated after infection with all types of adenoviruses, the inhibitory effect of OBP-702 was the strongest among these adenoviruses.

Conclusions: These results suggest that a hTERT-driven oncolytic adenovirus OBP-702 is a promising antitumor agent to induce profound apoptotic cell death through p53 transactivation and MYCN suppression in human NB cells with or without MYCN amplification. In vivo experiments are under way to investigate the antitumor effect of OBP-702 against xenograft NB tumors.

#2449

Hedgehog pathway inhibition sensitizes embryonal rhabdomyosarcoma to standard chemotherapy.

Gabriele Manzella, Tina Kottarathil, Beat W. Schaefer. _University Children's Hospital, Zurich, Switzerland_.

Hedgehog (Hh) is one of the key pathways orchestrating embryonic development as well as regulating homeostasis of postnatal organs. Perturbation of Hh signaling activity is associated with the onset of different childhood cancer entities such as embryonal rhabdomyosarcoma (eRMS). According to this, previous findings from our laboratory revealed that the tumor initiating populations (TIPs) of eRMS retain high activity of the pathway, which boosts their tumorigenic potential and self-renewal ability. Moreover, TIPs are well known to be responsible for seeding recurrent tumors following standard chemotherapy. This phenomenon might explain the failure of cytotoxic drugs in high-risk eRMS patients for whom conventional therapeutic approaches remain inadequate.

Therefore, in this study we investigated the role of Hh pathway in the context of chemoresistance. We established several patient-derived xenograft (PDX) models and primary cells derived from diagnostic and relapsed tumor specimens. Histological and immunofluorescence analysis confirmed that both cellular and patient heterogeneity were preserved in our cohort. Interestingly, we found that Hh target genes were significantly upregulated in chemoresistant PDX tumors and demonstrated that the use of Hh antagonists could significantly sensitize eRMS cells toward debulking drugs such as doxorubicin, actinomycin and vincristine. Ongoing experiments aim at confirming these results in cells with selective knock- out of components of Hh pathway as well as at elucidating if chemotherapy might induce a selection of resistant clones or a transition of sensitive cancer cells toward a resistant phenotype with high Hh pathway activity.

In conclusion, our data indicate that inhibition of Hh signaling might represent a promising strategy to enhance the therapeutic window of standard cytotoxic agents.

#2450

MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis.

Chengyuan Xue,1 Denise M. Yu,1 Samuele Gherardi,2 Jessica Koach,1 Giorgio Milazzo,2 Laura Gamble,1 Bing Liu,1 Amanda Russell,1 Tao Liu,1 Belamy B. Cheung,1 Glenn M. Marshall,1 Giovanni Perini,2 Michelle Haber,1 Murray D. Norris1. 1 _Children's Cancer Inst. Australia, Sydney, Australia;_ 2 _University of Bologna, Bologna, Italy_.

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4), which plays important roles in cancer progression, has been reported to be a direct transcriptional target of MYC. In this study, we have shown that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome and furthermore that siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells impaired migration and colony formation, and led to an increased proportion of cells in G1/S phase of the cell cycle. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that TFAP4 expression is positively regulated by MYCN through direct promoter binding. In addition, when MYCN was overexpressed in neuroblastoma cells, TFAP4 was required for the observed increase in cell migration. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study unveils a complex regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying relevant therapeutic targets for aggressive forms of this disease.

#2451

Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2.

Abhinav Dey, Caroline Maier, Anshu Malhotra, Anna Kenney. _Emory University, Atlanta, GA_.

Sonic hedgehog (Shh)-mediated medulloblastoma growth requires IGF2 (Insulin-like Growth Factor 2) and we recently showed that Yes Associated Protein (YAP1) induces IGF2 expression by Y-box protein 1(YB1) in Shh-stimulated cerebellar granule neural precursors (CGNPs), proposed cells-of-origin for the Shh molecular subclass of medulloblastoma, and mouse Shh-medulloblastoma cells. We found elevated levels of YAP1, YB1 and IGF2 in tumor cells occupying the peri-vascular niche, a microenvironmental niche proposed to house so-called tumor re-populating cells that survive radiation and contribute to medulloblastoma recurrence, which is fatal. We have developed an ex vivo approach using organotypic brain tumor slice cultures to better understand how YAP1, YB1, and IGF2 regulate peri-vascular niche cell survival post-radiation. We observed that the perivascular niche cell population expressing stem cell markers increases markedly following exposure to radiation. Additionally, on targeting any component of the YAP1-YB1-IGF2 axis we observed increased level of cell death within the niche and compromised cancer stem cell niche expansion post-radiation. These findings strongly indicate that therapeutic approaches intended to impair the function of this pathway could be used to reduce the use of cranio-spinal radiation of medulloblastoma patients, which causes life-long side effects that drastically impair quality of life.

#2452

Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma.

Sandra E. Ghayad,1 Ghina Rammal,1 Farah Ghamloush,2 Hussein Basma,2 Raya Saab2. 1 _Lebanese University, Beirut, Lebanon;_ 2 _American University of Beirut, Beirut, Lebanon_.

Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells. Tumor exosomes contain intact and functional protein, mRNA and miRNA that may alter the cellular environment to favor tumor growth. Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, with two distinct subtypes, alveolar (ARMS) and embryonal (ERMS) histology. ARMS tumors are characterized in the majority of cases by a fusion oncoprotein PAX3-FOXO1, thought to be an initiating factor in tumorigenesis. To date, no studies have been performed to investigate the possible role or content of exosomes in paracrine signaling in RMS.

In the present study, we isolated and characterized exosomes secreted from RMS cells, using 3 ERMS (fusion oncoprotein negative) and 2 ARMS (fusion oncoprotein positive) cell lines. RNA analysis showed that small RNA were enriched in RMS-derived exosomes, therefore we focused on evaluating the miRNA content. Array expression analysis showed that exosome miRNA clustered together well, and to a higher extent than cellular miRNA, in ARMS cell lines. Similarly, in the 2 ERMS cell lines that had mutant p53, exosome miRNA clustered together and the pattern of expression was very different from the corresponding cellular miRNA. ARMS and ERMS exosome-enriched miRNA were different, and only 2 miRNA were found to be common among both; putative targets are implicated in cancer and inflammation. Functionally, using in vitro assays, we found that both ERMS- and ARMS-derived exosomes significantly increased the cellular migration and invasion of normal human fibroblasts, and had a positive effect on viability and proliferation of both fibroblasts and RMS cells. In vivo, Matrigel plug assay showed that RMS-derived exosomes had a positive effect on the migration and invasion of stromal cells, suggesting a positive role on angiogenesis.

We conclude that RMS-derived exosomes can exert specific paracrine effects on recipient cells, enhancing cell viability as well as invasive properties of fibroblasts and stromal cells, and therefore is likely to play a role in promoting RMS angiogenesis and metastasis. Ongoing studies are aimed at directly assessing the role of RMS-derived exosomes in clinical angiogenesis and metastasis, using in vivo murine models, in addition to dissecting the role of specific enriched miRNA on recipient cell biology.

#2453

Neuroblastoma is bi-phasic and includes classical neuro-epithelial cells and chemo-resistant mesenchymal cells.

Rogier Versteeg, Tim van Groningen, Linda J. Valentijn, Bart A. Westerman, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Johan van Nes. _Academic Medical Center, Amsterdam, Netherlands_.

Introduction

Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We investigated whether neuroblastoma tumors include divergent cell types that may underlie this plasticity.

Experimental procedures

Fresh tumor cells cultured in neural stem cell medium were analyzed by FACS, whole genome sequencing, Chip-seq, mRNA profiling, and motility and chemo-sensitivity assays. Inducible transgenes were used to test state-transitions. Tumors were analyzed by immunohistochemistry.

Results

New neuroblastoma cell lines always included two cell types, which share the same genetic defects but have highly divergent phenotypes. One type has a neuro-epithelial (NE) phenotype and expresses all classical neuroblastoma markers. The other type has a mesenchymal (MES) character, is motile and lacks all neuroblastoma markers. Immunohistochemistry (IHC) detected a small fraction of MES cells in most primary neuroblastoma.

In four isogenic cell line pairs, we found that MES cells were more chemo-resistant than their NE-type counterparts. Indeed, comparison of primary neuroblastoma lesions before and after chemotherapy showed an accumulation of viable MES-type cells in post treatment samples. Moreover, comparison of primary, pre-treatment tumors with relapses emerging 4-5 years later in the same patients showed a strong enrichment for MES cells in the latter.

As these data suggest a role for MES-type cells in relapse development, we analyzed their key regulatory pathways. The isogenic MES-NE cell line pairs showed consistent mRNA expression differences between both phenotypes, activating major signaling routes and transcription factors. Chip-seq identified divergent histone modifications. MES cells had high NOTCH pathway activity and PRRX1 expression. Induced expression of NOTCH or PRRX1 converted multiple NE-type cell lines into MES-type cells, including chemo-resistance. Further analysis of these routes reconstructed molecular wiring of MES-type cells. This identified key-players like MEK and PDGFRβ, which were successfully targeted by small molecules to specifically kill MES cells in vitro.

Conclusions

Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells differ in many characteristics, but can transdifferentiate into each other. MES and NE cells may correspond to developmental stages, i.e. mesenchymal migratory cells delaminated from the neural crest and more differentiated cells of the adrenergic lineage. MES cells accumulate after chemo-therapy and in relapses. They may survive classical therapy and over time seed relapses, that ultimately become heterogeneous again. Elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy. 

### Pediatric Cancer Targets, Models, Therapies, and Resistance

#2454

Omega-3 fatty acids DHA and EPA decrease oncogenic PGE2 in medulloblastoma in vitro and in vivo.

Linda Ljungblad, Filip Bergqvist, Teodora Andonova, Per-Johan Jakobsson, John Inge Johnsen, Per Kogner, Malin Wickström. _Karolinska Institutet, Stockholm, Sweden_.

Background: The embryonic brain tumor medulloblastoma is a highly malignant tumor of the cerebellum and the most common CNS malignancy of childhood. By combining neurosurgery, chemotherapy and radiation overall survival is about 70 % but the intensive treatment regimen often leads to severe neurological sequelae and the risk of resistant relapses is significant. Hence, there is a need to improve treatment to reduce and alleviate late side effects.

Inflammation plays an important role in the tumor microenvironment by suppressing immune response, promoting angiogenesis, facilitating tumor invasion, and metastasis. Prostaglandin E2 (PGE2) is a pro-inflammatory lipid mediator derived from the omega-6 fatty acid arachidonic acid (AA) through conversion by the key enzymes COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Previous work from our group showed that medulloblastomas express high levels of COX-2 and mPGES-1 as well as all four PGE2 receptors (EP1-4) and most importantly that PGE2 promotes tumor proliferation.

The ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) show anti-inflammatory characteristics in general and exert anti-tumoral properties in several cancer types. Here we investigate the potential role of DHA and EPA in treatment of medulloblastoma and their effect on prostaglandins in vitro and in vivo.

Methods: In six medulloblastoma cell lines the cytotoxic activity of DHA and EPA was analyzed using cell viability assays. PGE2 levels were assessed in three medulloblastoma cell supernatants after 24 hrs of DHA treatment by ELISA. The prostaglandins were evaluated in human medulloblastoma xenografts in nude mice treated with DHA or a combination of DHA and EPA for 30 days from time of tumor take. Control mice were fed with normal chow. The levels of prostaglandins in the tumor tissue were analyzed with LC-MS/MS. Furthermore, the incorporation of fatty acids in tumors, erythrocytes and brain tissue was analyzed with GC-MS/MS.

Results: Both DHA and EPA exerted cell toxicity in a dose dependent manner in vitro. DHA reduced the PGE2 production in all three cell lines investigated. In vivo, the treatment with DHA alone and the combination of DHA and EPA significantly increased the omega-3 index in tumors, erythrocytes and normal brain. The PGE2, and prostacyclin was significantly reduced in both treatment groups while thromboxane levels remained unchanged. The in vivo treatment was non-toxic.

Conclusions: Lowering the pro-inflammatory PGE2 levels could mitigate the ongoing inflammation in the tumor microenvironment. We show that the omega-3 fatty acids DHA and EPA exert toxic effects on medulloblastoma cells in vitro and reduce PGE2 both in vitro and in vivo. We propose that DHA and EPA are interesting candidates to improve current medulloblastoma therapy.

#2455

Development of patient-derived orthotopic xenograft mouse models of pediatric low grade gliomas.

Mari Kogiso,1 Lin Qi,1 Frank K. Braun,1 Xiumei Zhao,1 Zhigang Liu,1 Holly B. Lindsay,1 Yuchen Du,1 Huiyuan Zhang,1 Sibo Zhao,1 Ching-Hua Liu,2 Vidya P. Mehta,3 Xiaoyun Shen,1 Jeffrey Murray,4 Laszlo Perlaky,1 Jack Su,1 Patricia Baxter,1 Adekunle Adesina,3 Donald W. Parsons,1 Xinyan Lu,2 Murali Chintagumpala,1 Xiao-Nan Li1. 1 _Texas Children's Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX;_ 2 _Clinical Cytogenetics Laboratory, Department of Hematopathology, M.D. Anderson Cancer Center, Houston, TX;_ 3 _Department of Pathology, Texas Children's Hospital, Houston, TX;_ 4 _Cook Children's Hospital, Fort Worth, TX_.

Background: Low grade gliomas (LGGs) account for 1/3 of pediatric brain tumors. Despite favorable outcomes with current therapeutic strategies, some tumors still recur following total resection. New clinically-relevant pediatric LGG (PLGG) animal models that replicate this subset of aggressive tumors are therefore needed.

Methods: Fresh tumor tissues from 36 PLGGs were collected. Self-renewal capacity was examined in vitro in 11 tumors with a neurosphere assay. Putative glioma stem cells (CD133+ and/or CD15\+ cells) were analyzed in 23 PLGGs by flow cytometry (FCM). Formation of orthotopic xenograft tumors was examined by direct implantation of 25 PLGG tumor cells into the matched locations in the brains of SCID mice followed by serial tumor sub-transplantations. In addition to histo-pathological characterizations, BRAF V600E mutation was validated with pyrosequencing, and CDKN2A deletion with FISH.

Results: The yield of viable tumor cells was low. Neurosphere formation was absent in 10/11 PLGGs after 37.6 ± 15.2 days. FCM analysis of 20 pilocytic astrocytomas

and 2 gangliogliomas confirmed the presence of low levels (<1.5%) of CD133\+ (CD133+/CD15- and CD133+/CD15+) cells, and relatively high fractions of CD133-/CD15+ cells (12.9 ± 20.1% in pilocytic astrocytomas and 67.9 ± 26.1% in gangliogliomas). Unlike 21 pilocytic astrocytomas, 1 grade II astrocytoma and 2 gangliogliomas that did not formed xenograft tumors after 219.9 ± 70.4 days of observations, a pleomorphic xanthoastrocytoma (PXA) (WHO grade II) formed intra-cerebral (IC) xenografts that have been serially subtransplanted 3 times in vivo. Similar to the patient tumor, xenografts from this model (IC-3635PXA) exhibited low cell proliferation (Ki67\+ cells <10%), medium microvessel density, homozygous CDKN2A deletion and relatively low abundance of CD133+ (<2%). The GFAP+ and CD15\+ cells in xenografts, however, were slightly reduced while the vimentin positive cells dramatically increased from 50% to 100% compared to patient tumor. The allele frequency of BRAF V600E mutation increased from 28% in patient tumor to 33%, 70% and 67% in passage I, II and III xenografts, respectively, and to 69% and 67% in cells grown in FBS-based media and serum-free media, respectively. All these biological changes were accompanied by extensive single cell invasion along blood vessels into normal mouse brains.

Conclusion: This study demonstrated that the tumor take rate of PLGGs was low despite the presence of putative glioma stem cells. Nonetheless, we have successfully established a PXA xenograft mouse model and xenograft-derived 3635PXA cell lines which replicate key features of the original patient tumor and demonstrate both enrichment of the BRAF V600E mutant allele frequency and active invasive growth in mouse brains. This novel model system should facilitate biological studies and pre-clinical drug screenings targeting the BRAF V600E mutation in pediatric PXA.

#2456

Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain.

Shuning He,1 Marc R. Mansour,2 Mark W. Zimmerman,1 Dong Hyuk Ki,1 Hillary M. Layden,1 Koshi Akahane,1 Eric D. de Groh,3 Antonio R. Perez-Atayde,4 Shizhen Zhu,5 Jonathan A. Epstein,3 A. Thomas Look1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University College London, London, United Kingdom;_ 3 _Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA;_ 4 _Children's Hospital Boston, Boston, MA;_ 5 _Mayo Clinic Cancer Center and Mayo Clinic Center for Individualized Medicine, Rochester, MN_.

Earlier reports indicated that the role of Nf1 tumor suppressor gene in limiting sympathoadrenal cell growth during embryologic development is independent of its ability to down-modulate RAS-MAPK signaling. This finding raised the question of whether neuroblastoma pathogenesis was also accelerated by loss of a similar non-canonical function of NF1. To elucidate how loss of the NF1 tumor suppressor gene contributes to the development of high-risk neuroblastoma, we relied on a transgenic zebrafish model that overexpresses MYCN and harbors loss-of-function nf1 mutations. We show here that loss of nf1 leads to aberrant activation of RAS signaling in MYCN-induced neuroblastoma, promoting both increased tumor cell survival and rapid tumor cell proliferation. We demonstrate further that the GTPase-activating protein (GAP) activity of the (GAP)-related domain (GRD) is sufficient to suppress accelerated initiation of neuroblastoma in nf1-deficient zebrafish, even though this transgene is unable to restrict abnormal sympathoadrenal cell growth during embryologic development. Hence NF1 exhibits different activities in vivo in the normal development and tumorigenesis of the peripheral sympathetic nervous system. Our findings establish nf1-deficient zebrafish that overexpress MYCN as an ideal animal model system for investigating new strategies to improve treatment of very high risk neuroblastomas with aberrant RAS-MAPK activation. We are currently performing high-throughput in vivo drug analysis using these zebrafish with primary tumors.

#2457

Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma.

Lionel Blanc, Brian Dulmovits, Julien Papoin, Jessica Kang, Eva Atsidaftos, Jeffrey M. Lipton, Adrianna Vlachos. _The Feinstein Institute for Medical Research, Manhasset, NY_.

Background: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia and congenital anomalies, notably skeletal defects. DBA is known in the majority of cases to be caused by haploinsufficiency of one of the small or large ribosomal protein (rp) subunits. In addition, there is a cancer predisposition in DBA. Indeed, somatic and germline rp mutations have been associated with a number of malignancies. In DBA patients one of the common malignancies is osteogenic sarcoma (OS). It is proposed that selective pressure caused by faulty translation may lead to clonal expansion of cells with acquired interdicting mutations leading to cancer.

Purpose: To quantitate the incidence of cancer in patients with DBA and to examine osteoblast formation in vitro using a mouse embryonic stem (mES) cell model haploinsufficient for ribosomal protein Rps19 to identify putative bone defects that may promote tumorigenesis.

Methods: The DBA Registry of North America (DBAR), the largest established patient cohort with prospective follow-up since 1991, was interrogated to ascertain the incidence of cancer in DBA. For in vitro studies, mES cells were differentiated towards osteoblasts using 1,25-dihydroxy-vitamin D3, dexamethasone, ascorbic acid and β-glycerophosphate for 10 days. qRT-PCR and histologic stains (alizarin red, alcian blue and alkaline phosphatase) were used to assess osteoblast production in wild type and Rps19+/- cultures.

Results: The DBAR reported the first quantitative assessment of cancer incidence in DBA. The significantly elevated observed to expected ratios were 287, 36, 33 and 28 respectively, for myelodysplastic syndrome (MDS), colon carcinoma, OS and acute myeloid leukemia (AML). As of Nov 2015 (N=751), with a median patient age of 20 years, 11 patients with gastrointestinal carcinoma, 5 with OS, 2 with AML and 8 with MDS are reported to the DBAR. The incidence of gastrointestinal carcinoma and OS is highly significant in this young population. We elected to study OS due to the skeletal defects and poor growth encountered in DBA patients. Using mES cells induced to differentiation towards osteoblasts, we observed reduced mineralization and therefore, bone formation, by alizarin red staining in Rps19+/- cells. Furthermore, rp haplo-insufficient cultures displayed a significant increase in alcian blue staining, a marker for chondrogenesis; these results suggest that regulation of osteochondrogenic potential was altered. In support of this phenotype, qRT-PCR analyses revealed that Rps19 haploinsufficiency induced abnormal expression of the master transcription factors and p53 involved in osteoblast and chondrocyte development.

Conclusions: The most common cancers in DBA are AML, OS and adenocarcinoma of the colon. With regards to OS, we are now creating a conditional Rps19 CRISPR murine model, in order to clarify the contribution of both rp and p53 in the etiology of OS.

#2458

Targeting checkpoint kinase 1 (CHK1) with the small molecule inhibitor LY2606368 mesylate monohydrate in models of high-risk pediatric cancer yields significant antitumor effects.

Caitlin D. May,1 Richard Beckmann,1 Wayne Blosser,1 Michele Dowless,1 Alle VanWye,2 Teresa Burke,1 Gerard Oakley,1 Jennifer Stephens,1 Julie Stewart,1 Beverly Falcon,1 Louis Stancato1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Advanced Testing Laboratory, Indianapolis, IN_.

CHK1 is a serine/threonine protein kinase essential for S-phase and G2/M cell cycle checkpoint regulation following DNA damage. Targeted inhibition of CHK1 in several tumor types increases DNA damage and replication stress, culminating in cell death through mitotic catastrophe. Recent studies have identified CHK1 as a therapeutic target in several pediatric tumor types. We evaluated the antitumor efficacy of LY2606368 mesylate monohydrate ("LY"), a checkpoint kinase 1 (CHK1)/CHK2 inhibitor currently in early phase clinical trials for adult solid cancers, in a panel of pediatric tumor cell lines and mouse models of embryonal tumors and pediatric sarcoma. In vitro effects of LY were assessed via Cell Titer Glo, immunoblotting, and cell cycle analysis by flow cytometry. For in vivo studies, mice bearing cell-derived (CDX) or patient-derived xenografts (PDX) of several pediatric tumor types were treated with four weekly cycles of 10 mg/kg LY BID for 3 consecutive days, followed by a 4 day dosing holiday. Tumor volume and body weight were measured 2x weekly. Xenograft tumor health following LY, chemotherapy, or combination treatment was evaluated by fluorescent immunohistochemistry (IHC) for a panel of markers for cell proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (CD31, smooth muscle actin [SMA], MECA32). Single digit nanomolar sensitivity to LY was observed in the majority of pediatric cancer cell lines evaluated in vitro. A more detailed analysis of LY-treated neuroblastoma and pediatric sarcoma cell lines showed increased DNA damage, CHK1 phosphorylation, and MAPK pathway activation. Significant single agent LY activity was observed in mouse models of neuroblastoma and pediatric sarcoma, but not in models of hepatoblastoma or retinoblastoma. Acquired resistance to LY was observed in the ST162 and SJCRH30 models of alveolar rhabdomyosarcoma. Interestingly, more stroma was observed following LY single agent treatment as measured by CD31, SMA, and MECA32 IHC staining; co-treatment with chemotherapy reduced the amount of SMA expressing-cells. Overall, our data demonstrate that LY is highly effective as a single agent in murine in vivo models of human neuroblastoma and several pediatric sarcoma subtypes. Current studies include further evaluation of the LY mechanism of action; investigation into the mechanism of intrinsic and acquired resistance; and identification of possible biomarkers for LY sensitivity.

#2459

Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering.

Jorida Coku,1 Elizabeth O. Scadden,1 Kangning Liu,1 Annette Vu,1 David M. Booth,2 Michelle Chen,1 Sharon Kim,1 C. Patrick Reynolds,3 György Hajnóczky,2 Michael D. Hogarty1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Thomas Jefferson University, Philadelphia, PA;_ 3 _Texas Tech University Health Sciences Center, Lubbock, TX_.

Development of multidrug resistance poses a persistent problem in cancer treatment, yet its underlying causes remain obscure. A principal role for mitochondria has been sought as this organelle integrates diverse stress signals to impact cell fate. Endoplasmic reticulum (ER) and mitochondria (mito) interact at specialized coupling sites called mitochondria-associated ER membranes (MAMs). MAMs serve as micro-domains for the transfer of essential calcium and lipid signals to mitochondria and regulate apoptotic sensitivity. Tightening of ER-mito tethering constitutes an early response to cellular stress leading to apoptosis, and alterations in ER-mito tethering have been implicated in diabetes and neurodegeneration, suggesting that the deregulation of this process may have broad relevance in disease. Here, we define a novel mechanism for chemotherapy resistance due to selection for reduced ER-mito tethering. Most high-risk neuroblastoma patients initially respond to chemotherapy before relapsing with lethal therapy resistant disease acquired during the course of intensive multimodality treatment. We obtained isogenic neuroblastoma cell lines from the same 7 patients both at the time of diagnosis (chemosensitive) and at the time of relapse (chemoresistant). We evaluated mitochondrial biomass (citrate synthase activity), mtDNA content (qPCR), and mtDNA sequence (Affymetrix MitoChip v2.0) but identified no changes correlated with acquired resistance. Electron microscopy image analyses of ER-mito interfaces revealed that tumors at relapse contain up to 70% fewer ER-mito tether complexes than their matched at-diagnosis tumors, as confirmed by IB for organelle-specific proteins. Mitochondria isolated from all 7 post-relapse tumors show attenuated cytochrome c release in response to tBid and Bim BH3 peptide, terminal death effectors downstream of most therapeutic stress. This attenuated mitochondrial response can be phenocopied by limited protelolysis of mitochondria to reduce ER-mito tethers. Reduced mitochondrial apoptotic signaling in post-relapse tumors correlates directly with chemoresistance (up to 800-fold) across diverse agent classes. To functionally validate this relationship, Cyclosporine A (CsA), a cyclophilin D inhibitor, was used in tumors at diagnosis to reduce ER-mito tethering. This led to attenuated apoptotic responses in isolated mitochondria, and increased tumor cell IC50 to diverse chemotherapeutics, partially phenocopying the therapy resistant state. Our findings support a novel model of differential apoptotic signaling in therapy resistant cells that relies on the altered proximity and interactions of the mitochondria with ER that may be harnessed to design more effective anti-cancer drug therapies.

#2460

Characterizing PAX3-FOXO1 dependent cell death in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy.

Marco Wachtel, Johannes Ommer, Beat Schäfer. _Univ. Children's Hospital Zurich, Zurich, Switzerland_.

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often by t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1. Expression of this chimaeric transcription factor is critical for tumorigenesis and cell survival. However, the exact mechanism of cell death after loss of PAX3-FOXO1 activity has not been determined so far.

Our aim was thus to characterize the cell death pathways activated upon silencing of PAX3-FOXO1. In addition, we aimed at finding drugs further sensitizing aRMS cells to this mode of cell death.

We used combined shRNA and CRISPR approaches, as well as a small molecule screen to demonstrate that after shRNA-mediated silencing of PAX3-FOXO1 expression, aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner. In accordance, we can show that the BH3-mimetic ABT-263 sensitizes aRMS cells to PAX3-FOXO1 silencing via this cell death pathway. Interestingly, ABT-199 was less effective, suggesting that Bcl-xl plays a major anti-apoptotic role in aRMS. Furthermore, induction of cell death is dependent on PI3K activity and antagonized by GSK3, suggesting that inhibition of PI3K in this sarcoma might have an anti-apoptotic impact. In contrast, combination of ABT-263 or GSK-3 inhibitors with small molecule drugs affecting PAX3-FOXO1 activity such as PLK1 and aurora kinase inhibitors can cooperate to enhance cell death in aRMS cells. These studies demonstrate the importance of elucidating biological mechanisms to guide development of rational drug combinations.

#2461

Rhabdomyosarcoma tumors recur by a PAX3-FOXO1-independent mechanism in a human myoblast xenograft model.

Puspa R. Pandey, Bishwanath Chatterjee, Javed Khan, Stephen M. Hewitt, Markku Martti Miettinen, Frederic G. Barr. _NIH NCI, Bethesda, MD_.

The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation, and is a characteristic feature of a subset of rhabdomyosarcoma (RMS) with aggressive behavior and poor prognosis. This study utilizes an inducible expression system in human myoblasts to dissect the molecular mechanism of PAX3-FOXO1 action during RMS tumorigenesis and progression.Constitutive and doxycycline-inducible expression constructs were used to generate a human myoblast cell line that constitutively expresses MYCN and reversibly expresses PAX3-FOXO1, respectively. PAX3-FOXO1 expression was assessed by Western blotting, qRT-PCR and immunohistochemistry. Oncogenicity in these engineered myoblasts was studied in vitro by focus formation and in vivo by intramuscular injection of NOD-SCID mice. Cell proliferation, apoptosis and myogenic differentiation were assessed by western blot or immunohistochemical assays.In focus formation assays, doxycycline-treated myoblasts expressing constitutive MYCN and doxycycline inducible PAX3-FOXO1 showed a high level of oncogenic transformation. When doxycycline was removed during the course of this assay, smaller foci formed with prominent myogenic differentiation and cell death. Intramuscular injection of these engineered myoblasts resulted in RMS tumor formation when fusion protein expression was induced by feeding mice a doxycycline-supplemented diet. After small palpable tumors formed, doxycycline withdrawal resulted in decreased PAX3-FOXO1 expression and tumor regression. Microscopic examination of regressing tumors revealed widespread myogenic differentiation and cell death. In most cases, the tumors recurred several weeks later despite the absence of inducing agent. Analysis of recurrent tumor samples revealed that a subset emerged in the absence of PAX3-FOXO1 expression. A cell line generated from a PAX3-FOXO1-independent recurrence demonstrated transformation in vitro in the absence of doxycycline. Though cell lines derived from primary tumors were dependent on PAX3-FOXO1 and differentiated when doxycycline was removed, the recurrent tumor-derived cells did not differentiate under these conditions and instead proliferated continuously. Furthermore, reinjection of these recurrent tumor-derived cells resulted in tumor formation in mice in the absence of doxycycline, and even more rapid tumor formation in the presence of doxycycline.Our study provides evidence that the PAX3-FOXO1 fusion protein is necessary to develop primary tumors but recurrent tumors can develop by a PAX3-FOXO1-independent mechanism. The recurrent tumors are postulated to have acquired secondary oncogenic events that were activated or selected by initial PAX3-FOXO1 expression. These secondary events have an additive oncogenic effect with PAX3-FOXO1 expression, and then contribute to tumor recurrence in the absence of PAX3-FOXO1 expression.

#2462

**MondoA mediates** in vivo **aggressiveness of common ALL and may serve as a T-cell immunotherapy target.**

Alexandra Sipol,1 Thomas G. P. Grunewald,2 Juliane Schmaeh,3 David Schirmer,1 Monique L. den Boer,4 Rebeca Alba Rubío,2 Michaela Baldauf,2 Caroline Wernicke,1 Hans-Jochem Kolb,1 Martin Horstmann,5 Gunnar Cario,3 Günther Richter,1 Stefan Burdach1. 1 _Children's Cancer Research Center, Department of Pediatrics, Technische Universität München, CCCM Munich - Comprehensive Cancer Center and German Translational Cancer Research Consortium (DKTK), Munich, Germany;_ 2 _Laboratory for Pediatric Sarcoma Biology, Institute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany;_ 3 _Schleswig-Holstein University Medical Center, Kiel, Germany;_ 4 _Erasmus University Medical Center, Department of Pediatric Oncology, Rotterdam, Netherlands;_ 5 _Children's Cancer Research Institute and Department of Pediatric Hematology and Oncology, University of Hamburg Medical Center, Hamburg, Germany_.

Oncogene addiction provides ideal targets for immunotherapy. We previously described MondoA (also known as MLXIP, MAX like protein X interacting protein) as a metabolic stress sensor, required for leukemogenesis. Here we report on the expression of MondoA in common acute lymphoblastic leukemia (cALL) compared to other malignancies, its role in malignancy of cALL in vivo, downstream pathways and correlation with relapse risk. Given the non-accessibility of transcription factors by drugs or chimeric antigen receptor transgenic T cells (CARs), we tested the targetability of MondoA by allo-restricted, peptide specific T cells.

Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were lentivirally transduced with MondoA short hairpin RNA (shRNA). Upon successful MondoA knock down (KD), KD and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. MondoA specific T cells were generated by priming of donor HLAA0201 negative (A2-) T-cells with A2+ dendritic cells bearing MondoA peptides, multimer-based sorting and subcloning of A2-CD8+ T-cells. For priming of T cells, five MondoA peptides were chosen by SYMPEITHI, BIMAS and NetCTL1.2. analyses. Peptide 428 stabilized best A2 expression on TAP-deficient T2 cells. Specificity and functionality of T cell clones were tested by ELISpot interferon gamma (IFg) and granzyme B assays with six MondoA+ leukemia lines (A2+, A2-). Off target effects of MondoA specific T-cell clones were assessed by IFg reactivity against the MondoA expressing A2+ NALM6 cell line vs. A2+ and A2- EBV immortalized lymphoblastoid cell lines from six donors. Peptide homology was assessed with BLAST algorithms in SWISSPROT.

We found MondoA to be most strongly expressed in pediatric cALL and AML. Moreover MondoA expression was high in gastrointestinal stromal tumors and alveolar rabdomyosarcoma. MondoA KD in cALL cell lines and their subsequent analysis in xenograft mice resulted in a reduced number of leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized in treated mice after MondoA KD. Further microarray analysis revealed an induction of aerobic glycolysis switch genes and hypoxia-response by MondoA. Consequently, HIF1A stabilization required MondoA expression and tied to these results, MondoA overexpression correlated with relapse risk; its expression was 63% higher in the very high-risk group as compared to the non-high-risk group of cALL. Therapeutically, MondoA-derived peptide antigens and A2+ cALL lines were successfully recognized and killed by specific, allo-restricted CD8+ T cells.

In conclusion, our findings demonstrate that MondoA maintains leukemic burden and aggressiveness of cALL in vivo possibly by modulating metabolic and hypoxia stress response. Moreover, we identified MondoA as a promising target for immunotherapy of cALL.

#2463

PEGPH20 increases the anticancer activity of standard chemotherapy combinations, vincristine (VIN) and D actinomycin (DACT), in a Wilms' xenograft model.

Jessica Cowell,1 Susan J. Zimmerman,1 Mathieu Marrela,1 Ping Jiang,1 Peter J. Houghton,2 Michael J. LaBarre,1 Daniel C. Maneval,1 Curtis B. Thompson,1 Xiaoming Li1. 1 _Halozyme Therapeutics, Inc, San Diego, CA;_ 2 _University of Texas, Health Science Center, San Antonio, TX_.

Hyaluronan (HA), which accumulates in the tumor microenvironment of many solid tumors, is associated with tumor progression and negative clinical outcomes. Preclinical studies have demonstrated that PEGPH20-mediated HA removal from HA-rich xenograft tumors in mice decreases tumor interstitial fluid pressure and water content, resulting in decompression of tumor vasculature, increased tumor perfusion and enhanced chemotherapeutic activity. Accordingly, the HA degrading enzyme, pegylated recombinant human hyaluronidase PH20 (PEGPH20), is currently being evaluated in clinical trials with selected treatment regimens in several malignancies, including metastatic pancreatic adenocarcinoma, gastric cancer, breast cancer and non-small cell lung cancer. Advances in treatment for pediatric Wilms' tumor patients have significantly increased survival, but salvage chemotherapy for relapsed pediatric patients remains challenging. To evaluate the role of HA in this disease, 19 Wilms' tumor histological specimens were surveyed for HA status using a novel probe (Jadin 2014). 79% (15/19) of the samples stained strongly for HA when compared to non-tumor kidney tissue, prompting additional preclinical studies. In brief, a human Wilms' tumor cell line, WT-CLS1, was transduced with hyaluronan synthase-3 (HAS3). The subsequent WT-CLS1/HAS3 cells produced more HA and WT-CLS1/HAS3 xenograft tumors grew significantly faster than parental WT-CLS1 tumors. To evaluate the efficacy of PEGPH20 in combination with Wilms' tumor chemotherapy (CTX) combinations, WT-CLS1/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors reached ~250 mm3, mice were staged into treatment groups: vehicle control, PEGPH20, vincristine (VIN) plus Dactinomycin (DACT), and PEGPH20+VIN+DACT at different dose/frequency combinations. Average tumor growth inhibition (TGI) and overall survival for PEGPH20+VIN+DACT-treated mice were superior to TGI and survival of VIN+DACT treatment alone. In separate studies WT-CLS1/HAS3 tumor-bearing mice were staged into three groups: vehicle control, low dose PEGPH20 (0.0375 mg/kg, iv) and high dose PEGPH20 (1 mg/kg, iv). Animals were administered the hypoxyprobe pimonidazole (60 mg/kg, ip) three hours prior to sacrifice, and whole tumors were removed and processed via immunofluorescence. High dose PEGPH20 reduced this measure of tumor hypoxia by 5% (p=0.023). Taken together, these data demonstrate that HA accumulation in Wilms' tumor is common, and treatment with PEGPH20 hyaluronidase can increase CTX efficacy and reduce tumor hypoxia in a preclinical model of this disease. These results support further studies with PEGPH20 in combination with chemotherapy in preclinical models of Wilms' tumor and suggest investigation in this pediatric patient population may be warranted.

#2464

Disrupting the epigenetic modifier HMGA2 in DIPG and GBM inhibits tumor invasion, growth and tumorigenicity.

Harpreet Kaur, Marianne Hütt-Cabezas, Isabella Taylor, Melanie Weingart, Fausto Rodriguez, Charles G. Eberhart, Eric H. Raabe. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are lethal, incurable brain tumors with poor prognosis. Identifying and targeting novel molecular markers regulating tumor invasion and growth can lead to development of better therapies. HMGA2, a minor-groove DNA-binding protein, is a transcriptional modulator in normal and cancer stem cells. We hypothesized that HMGA2 contributes to pediatric high grade glioma invasion and tumorigenicity through its ability to alter the transcription of large numbers of genes. We found increased expression of HMGA2 in a subset of GBM and DIPG tumors by western blotting and immunohistochemistry. We confirmed this result in multiple patient-derived tumor cell lines, including 3 DIPG neurosphere cell lines. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 in GBM cells significantly reduced invasion in transwell assay and colony formation in soft agar assay (shHMGA2 vs. shControl, P<0.01). Similarly, shRNA mediated suppression of HMGA2 in DIPG cell lines reduced proliferation as measured by BrdU incorporation, reduced invasion by transwell assay and increased apoptosis by cleaved caspase-3 (CC-3) immunofluorescence (shHMGA2 vs. shControl, P<0.01). Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited growth (BrdU assay) and increased apoptosis (CC-3 immunofluorescence) of DIPG cell lines (P<0.01). Mice bearing orthotopic GBM xenograft cell lines transduced with HMGA2 shRNA lived longer (median survival = 108 days) compared to control shRNA (67.5 days, P<0.0001 by log-rank analysis). Our results suggest an oncogenic role of HMGA2 in promoting DIPG and GBM invasion and growth, identifying HMGA2 as a potential therapeutic target in these devastating tumors. Netropsin or other minor groove binding agents may be effective therapeutics for pediatric high-grade gliomas.

#2465

Spleen tyrosine kinase (SYK) in neuroblastoma tumorigenesis.

Conny Tuemmler,1 Gianina Dumitriu,1 Julia Cserna,1 Ugo L. Moens,1 Per Kogner,2 John Inge Johnsen,2 Baldur Sveinbjørnsson1. 1 _Molecular Inflammation Research Group, Department of Medical Biology, Faculty of Health Science, University of Tromsø, Tromsø, Norway;_ 2 _Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden_.

Tyrosine kinases play an important role in tumorigenesis by contributing to the regulation of cell proliferation and survival, and are therefore among the most attractive drug targets. SYK is a non-receptor tyrosine kinase highly expressed in cells of hematopoietic origin where it contributes to immune receptor signaling. The role of SYK in cancer is best understood in hematological malignancies where it promotes survival, and SYK inhibition has been shown to be a promising therapeutic approach. For solid tumors the role of SYK remains double-edged, as it has been found to promote but also suppress tumor development dependent on the cancer type. Besides its role in survival signaling SYK has also been found to contribute to drug resistance in several malignancies.

Neuroblastoma (NB) is a malignancy of early childhood. Although there have been great advances in the treatment of NB during the past 30 years, the prognosis for high- risk patients remains poor.

In this study we have evaluated SYK as a potential drug target in NB.

Examination of gene expression datasets indicated a correlation between high expression of SYK and poor prognosis in NB. Screening of NB cell lines showed that SYK mRNA was present in the majority of NB cells lines whereas the SYK protein was detected less frequently. However, SYK was detectable in NB tumor tissue using immunohistochemistry.

The efficacy of different, commercially available, SYK inhibitors was evaluated by comparing the neuroblastoma cell lines SH-SY5Y (high SYK expression) and SK-N-BE(2) (low SYK expression) using a cell viability assay. While one inhibitor displayed a markedly stronger effect on the viability of SH-SY5Y cells compared to SK-N-BE(2) the effect of the other inhibitors appears to be less specific. To confirm that SYK inhibition leads to decreased cell proliferation SYK specific siRNA was used. After 48h incubation the cell viability of SH-SY5Y cells was decreased by approximately 25%.

Our findings indicate that SYK may be a possible drug target in neuroblastoma. To further evaluate its potential, combination studies using SYK inhibitors and cytostatic drugs will be performed.

#2466

Development of a novel gene targeting and clone screening platform to engineer common pediatric glioma mutations into model cell lines.

Nathan R. Fons,1 Yulia Surovtseva,2 Gregory A. Breuer,1 Ranjini K. Sundaram,3 Ranjit S. Bindra3. 1 _Department of Experimental Pathology, Yale University, New Haven, CT;_ 2 _Yale Center for Molecular Discovery, Yale Unviersity, New Haven, CT;_ 3 _Department of Therapeutic Radiology, Yale University, New Haven, CT_.

The utility of CRISPR/Cas9 gene targeting tools to alter endogenous genomic loci has revolutionized biomedical research across numerous disease focus areas, with especially large gains made in the development of disease models. However, the introduction of specific mutations using CRISPR/Cas9 can be associated with low yields of single cell clones harboring the desired homozygous or heterozygous mutations. Conventional clone screening methods can be a labor intensive and expensive process and thus, better approaches are needed to identify cells with the desired genotype(s). In parallel, there is a dearth of human tumor cell model systems for pediatric cancers, especially for Diffuse Intrinsic Pontine Glioma (DIPG). To address these needs, we recently designed and validated a platform which utilizes a novel, inducible Cas9 expression system coupled with a high-resolution DNA melt (HRM) analysis protocol to rapidly identify mutant clones. We applied our platform to model several common DIPG mutations, including truncating mutations within exon 6 of the gene, protein phosphatase 1D (PPM1D). Using our HRM analysis protocol, clones containing activating mutations in PPM1D were detectable and grouped distinctly from wild-type and non-functional clones; as validated through conventional Sanger sequencing and Ion Torrent Next Generation Sequencing. Importantly, multiple PPM1D mutant clones were generated in an expedited manner (< 4 weeks), using our unique HRM process-flow. We subsequently validated these clones in a collection of secondary assays, and we confirmed that the mutant proteins were functionally active. Several unique findings were identified in these studies, including substantial cell cycle-associated defects and basal activation of key proteins in the DNA damage response network. To our knowledge, we have created the first set of astrocyte cell lines harboring engineered PPM1D mutations at the endogenous gene locus. We are now testing these cell lines in high-throughput synthetic lethal screens to identify novel inhibitors of PPM1D-mutant DIPG tumors. These findings highlight the utility and power of our unique approach to rapidly create pertinent mutant cell lines for use in mutation-targeted biological assays. Our platform likely will become an invaluable tool for the development of better pediatric glioma cell line models in the future.

#2467

Association of specificity protein 1 and survivin expression in medulloblastoma: Identifying effective therapeutic targets.

Umesh T. Sankpal,1 Don Eslin,2 W. Paul Bowman,1 Jeffrey C. Murray,3 Irene Sanchez,3 Michelle Jones,1 Sagar Shelake,1 Yazmin Hernandez,1 Anmol Wadwani,1 Hassaan Patel,1 Ashni Dudhia,1 Riyaz M. Basha1. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _Arnold Palmer Hospital for Children, Orlando, FL;_ 3 _Cook Children's Medical Center, Fort Worth, TX_.

Medulloblastoma (MB) is an aggressive malignant brain tumor diagnosed mostly in children. MB is typically treated using a multimodal approach consisting of surgery, craniospinal irradiation, and chemotherapy. These treatments cause delayed consequences in pediatric patients. In order to treat this malignancy effectively, it is important to identify critical markers associated with the disease and finding specific agents to target such markers. The transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin are over expressed in many cancers and associated with poor prognosis. Sp1 mediates the expression of several oncogenes including survivin. Even though some evidence exists for the expression of survivin, the information on Sp1 is still limited in MB. The primary objective of this study was to determine the association of Sp1 and survivin in MB and developing the strategies to target these candidates using less toxic compounds. A human MB tumor tissue array consisting of 20 tumor and 3 normal controls was stained for Sp1 and survivin using specific antibodies. Tumor specimens showed distinct expression for both Sp1 and survivin in majority of tumor tissues. Using small interference RNA (siRNA) technology, the expression of Sp1 and survivin was inhibited in MB cell line, DAOY, and the cell viability was determined at 24 and 48 h using CellTiter-Glo kit. We observed that the inhibition of Sp1 and survivin by siRNA correlated with a decrease in DAOY cell viability. Previously, our laboratory showed that a small molecule, Tolfenamic Acid (TA) inhibits cell proliferation and tumor growth in mice via targeting Sp1 and anti-apoptotic protein, survivin. Taken together these results highlight the significance of targeting Sp1 and survivin for inhibiting MB cell proliferation and tumor growth in mouse model. Survivin has also been demonstrated to play a role in chemoresistance. We therefore, tested the combination treatment(s) involving TA and standard chemotherapeutic agents, Vincristine (Vinc) and Cisplatin (Cis) using MB cell lines, DAOY and D283. Cells were treated with TA, Vinc, Cis and combination of TA+Vinc and TA+Cis and the effect on cell viability and apoptosis was determined. Each drug alone caused a time and dose dependent decrease in cell viability that was enhanced in the presence of TA. The combination treatment also resulted in a 2-3 fold increase in apoptosis as determined by annexin V and propidium iodide staining. It is plausible that targeting Sp1 and/or survivin induces the susceptibility of MB cells to chemotherapeutic drugs. These preliminary results demonstrate the efficacy of combining inhibitors of Sp1 and survivin with chemotherapeutic agents to enhance their therapeutic response while limiting the toxicity and side-effects.

#2468

Combined targeting of the EWS/ETS transcriptional program by BET bromodomain and PI3K pathway inhibition blocks tumorigenicity and increases apoptosis in Ewing sarcoma.

Tim Hensel,1 Chiara Giorgi,2 Fiona Becker-Dettling,1 Julia Calzada-Wack,3 Frauke Neff,3 Thorsten Buch,4 Oxana Schmidt,1 Beat W. Schäfer,2 Stefan Burdach,1 Günther HS Richter1. 1 _Children's Cancer Research Centre and Department of Pediatrics, Klinikum rechts der Isar, Technische Universität München, CCCM Munich - Comprehensive Cancer Center, and German Translational Cancer Research Consortium (DKTK), Munich, Germany;_ 2 _Department of Oncology and Children's Research Center, University Children's Hospital, Zurich, Switzerland;_ 3 _Institute of Pathology, Helmholtz Zentrum, Munich, Germany;_ 4 _Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany_.

Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations that give rise to chimeric proteins (EWS-ETS), which generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome.

By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor (BEZ235) of the PI3K pathway. Microarray analysis revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference of BRD3 or BRD4 expression but not by BRD2 blockade, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers.

JQ1 treatment not only suppressed an ES specific expression profile but also blocked contact dependent and independent proliferation of different ES lines. But subsequent analysis of proliferation after transient knockdown of either BRD3 or BRD4 did not recapitulate inhibition of proliferation as observed after JQ1 treatment, indicating the necessary simultaneous blockade of both proteins. However, inhibition of proliferation after JQ1 treatment was due to a partial G1 arrest and S phase elongation of the cell cycle. In addition, induction of apoptosis as demonstrated by PARP1-, CASP7-cleavage and increased CASP3 activity significantly contributed to the reduction of the proliferative ability of ES lines. Single or combination treatment with the PI3K/mTOR inhibitor BEZ235 increased apoptosis of ES cell lines although single treatment with BEZ235 was less effective than JQ1 application. Consequently, tumor development was dose dependently suppressed with increased formation of apoptotic bodies in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program.

#2469

The role of L1 in proliferation and chemoresistance of retinoblastoma.

Dong Hyun Jo,1 Kyungmin Lee,2 Jin Hyoung Kim,3 Hyoung Oh Jun,3 Young Hoon Kim,4 Young-Lai Cho,5 Young Suk Yu,6 Jeong-Ki Min,2 Jeong Hun Kim1. 1 _Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea;_ 2 _Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea;_ 3 _Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea;_ 4 _Department of Pathology, College of Medicine, Seoul National University, Seoul, Republic of Korea;_ 5 _Center for Nanosafety Metrology, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea;_ 6 _Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Republic of Korea_.

Retinoblastoma is the most common intraocular malignant tumor in children, affecting approximately 1 in 20,000 live births. In this study, we investigated the role of L1, a transmembrane protein, in proliferation and chemoresistance of retinoblastoma to figure out the potential for the novel therapeutic target. Retinoblastoma tissues demonstrated varying degrees of L1 positivity in 86.6% (26/30) of samples. In particular, the degree of L1 positivity was inversely related with that of Flexner-Wintersteiner rosette formation. Further in vitro studies using stable cell lines which showed up- or down-regulation of L1 demonstrated that L1 was associated with cell-cell adhesion and proliferation of retinoblastoma cells. Retinoblastoma cells with low expression of L1 showed decreased tumor formation in vivo. In addition, L1 expression was related with resistance to carboplatin, one of the most-widely utilized chemotherapeutic agents against retinoblastoma. In line with these results, retinoblastoma cells with higher expression of L1 demonstrated increased resistance to carboplatin in vivo. We also observed diffuse and dense expression of L1 in retinoblastoma tissues from 4 patients who underwent enucleation despite intensive chemotherapy based on carboplatin. Taken together, L1 was related with proliferation and chemoresistance of retinoblastoma and might be a potential therapeutic target of retinoblastoma.

#2470

Isolation and characterization of cancer stem cells from the human medulloblastoma cell line DAOY: adherent vs. suspension in vitro cell cultures.

Javier de la Rosa,1 Ander Sáenz Antoñanzas,1 Xing Fan,2 Bárbara Meléndez,3 Juan A. Rey,4 Javier S. Castresana1. 1 _University of Navarra, Pamplona, Spain;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _Virgen de la Salud Hospital, Toledo, Spain;_ 4 _La Paz University Hospital, Madrid, Spain_.

Brain tumor stem cells (BTSC) in vitro cultures have been made possible by the formation of neurospheres in suspension conditions with the use of serum free medium supplemented with specific growth factors, as B27, EGF, or b-FGF. In this work, we tested a more recent alternative method for the culture of BTSC, using laminin pre-coated flasks and the same medium used for the neurosphere growth, which leads to BTSC expansion attached to a layer.

BTSC cultures in adherence conditions by the use of laminin can afford a more homogeneous exposition to growth factors than sphere cultures do, which promotes a higher enrichment in stem-like cells. Also, it is known that the laminin interactions with integrines or other components of the cell matrix can lead to inhibition signals of cell differentiation. Moreover, the monolayer conditions can support a higher number of cells in the flask.

The medulloblastoma cell line DAOY was cultured in different conditions of normoxia and hypoxia, and several cell populations were obtained for in vitro and molecular experiments.

An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have BTSC with a higher probability than the CD133- fraction.

The colony formation assay presented similar results for the two cultured methods, but clonogenicity in soft agar was higher in the laminin culture cells. In the cell migration assay, medullospheres migrated similarly to the laminin cultured cells, since both cell populations lost their adherence capacity after being cultured with serum free medium. Gene expression of CD133, CD15 and EZH2 was compared as well, and significant differences were not detected between the two cell populations.

We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining BTSC in DAOY medulloblastoma cell line.

#2471

Evaluation of CD47 expression and effects of CD47-SIRPα fusion protein in patients with osteosarcoma.

Sajida Piperdi,1 Michael Roth,2 Nick Morriss,1 Christian Zinone,1 Wendong Zhang,1 Pratistha Koirala,1 David Geller,2 Bang Hoang,2 Rui Yang,2 Jonathan Gill,2 Richard Gorlick2. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Montefiore Medical Center and the Children's Hospital of Montefiore, Bronx, NY_.

CD47 is a commonly expressed trans-membrane protein which binds to signal regulatory protein α (SIRPα) expressed on the surface of the macrophages resulting in inhibition of phagocytosis and cell clearance. Several malignancies demonstrate high expression of CD47 and recent studies suggest interfering with the CD47-macrophage interaction may be therapeutic. A prior expression analysis performed on osteosarcoma (OS) patient samples reported marked expression of CD47. The current study evaluated the utility of targeting CD47 in Osteosarcoma by determining the prevalence of CD47 expression and the prognostic value of CD47 expression in patients with Osteosarcoma. An immunofluorescence phagocytosis assay is being performed to directly assess the therapeutic potential of the CD47-SIRPα fusion protein.

A previously created and described tissue microarray (TMA) was stained with a murine monoclonal anti-CD47 (B6H12) antibody. The intensity and location of tissue staining were assessed by a comparison between the positive and negative control slides. 81 specimens were evaluated on the TMA. Overall, CD47 was expressed in 87.7% of specimens, with 28.4%, 27.2%, and 32.1% demonstrating high, intermediate, and low expression, respectively. Almost all metastatic tumor specimens (85.7%) expressed CD47. To evaluate CD47 expression quantitatively, a real time PCR was performed on OS patient-derived primary samples, and cell lines. 71.4% of patient derived cell lines (n=15) showed higher level of CD47 expression compared to that of mesenchymal stem cells and an osteoblast cell line. Furthermore, flow cytometry was performed on human OS xenografts and cell lines to determine the surface expression level of CD47 prior to treatment with CD47-SIRPα fusion protein. 85% of the OS cell lines (n=13) showed levels of CD47 expression comparable to that of the positive control, leukemia cell line HL60. Survival analyses suggested that increased expression of CD47 may be associated with poorer 5 year event free survival (69.3% vs 42.4%, p=0.6 and 49.5% vs. 33.6%, p=0.16) when using thresholds of intermediate/high expression of CD47 and high expression of CD47, respectively. Results of an in-vitro phagocytosis assay assessing cytotoxicity of a CD47-SIRPα fusion protein on OS cell lines using laser scanning cytometry at present show some inhibition of phagocytosis upon adding 500 ug/ml of CD47-SIRPα fusion protein, but it does not reach the statistical significance. These findings demonstrate that CD47 is highly prevalent in Osteosarcoma and suggest CD47 has potential utility as a novel target for anti-cancer therapy in Osteosarcoma. Although the immunofluorescence assays have not demonstrated marked augmentation of activity with the CD47-SIRPα fusion protein, this may be attributable to limitations of the method with clinical trials necessary to define its true level of activity in treating patients with Osteosarcoma.

#2472

Mechanisms of resistance to IGFR-targeted therapy in pediatric sarcomas.

Terry J. Shackleford,1 Seethalakshmi Hariharan,1 Hemant K. Bid,2 Peter J. Houghton1. 1 _Greehey Children's Cancer Research Institute, San Antonio, TX;_ 2 _The Ohio State University, Columbus, OH_.

Background: Insulin-like growth factor-I receptor (IGFR) signaling promotes tumorigenic properties which makes targeting IGFR an attractive therapeutic target. IGF signaling has been linked to proliferation, survival, angiogenesis, metastasis and resistance to anticancer therapies, and contributes to pediatric malignancies including Ewing sarcoma and rhabdomyosarcoma. Recently, monoclonal antibodies targeting IGFR have shown promise in a limited number of sarcoma patients. A substantial proportion of patients demonstrate intrinsic resistance, and those that do respond will often lose clinical benefit (acquired resistance). Intrinsic and acquired resistance ultimately results in failure to respond to treatment and continued growth of the tumor. Understanding these resistance mechanisms will identify combinations of targeted therapy to optimize therapeutic efficiency of this targeted agent.

Methods: To test intrinsic and acquired mechanisms of resistance, we utilized Receptor Tyrosine Kinase (RTK) Arrays to analyze the phosphorylation status of 49 RTKs. For intrinsic resistance, we used a panel of Ewing Sarcoma (EWS) cell lines that are unresponsive to TZ1, a monoclonal antibody that targets IGFR. For acquired resistance, we developed a TZ1 resistant rhabdomyosarcoma (RMS) cell line from TZ1 sensitive RH41 cells. The cell lines were treated with TZ1, an anti-IGFR monoclonal antibody. Signaling changes were assessed by comparing phosphorylation status of the RTKs in the untreated versus treated cells in the inherent resistance model and in the parental versus resistant cells in the acquired resistance model. Results: TZ-1 treatment of the EWS cell lines with intrinsic resistance decreased pIGFR, but these cells were characterized by expression of multiple phosphorylated RTKs. In the Rh41 acquired resistance model, increased activation of signaling pathways was detected in the resistance cells including: AXL, PDGFR, FGFR4 and ALK. Treatment of parental Rh41 cells with TZ1 also rapidly induced several activated RTKs. In TZ1-resistant Rh41 cells combined treatment of TZ1 with the PDGFR inhibitor, crenolanib, and the pan-FGFR inhibitor LY2874455 resulted in significant decrease in cellular proliferation.

Conclusions: Intrinsic resistance to TZ1 in EWS cell lines appears to be due to redundancy through multiple alternative activated RTKs. Systematic knockdown to determine which activated RTKs confer resistance is being undertaken. In Rh41 RMS cells acquired resistance to TZ1 is characterized by induction of several alternative activated RTKs, some of which are induced rapidly after TZ1 exposure. Combined treatment with TZ1 and drugs targeting PDGFR and FGFR was effective at overcoming resistance. These results suggest that identification of signaling pathways that compensate for IGFR inhibition can lead to effective potent multi-target combination therapy. Supported by USPHS Grant CA165995.

#2473

Combined BET-bromodomain and CDK2 inhibition in MYC-driven medulloblastoma.

Sara Bolin,1 Anna Borgenvik,1 Camilla Persson,1 Gabriela Rosén,1 Anders Sundström,1 Jun Qi,2 James E. Bradner,2 William A. Weiss,3 Yoon-Jae Cho,4 Holger Weishaupt,1 Fredrik J. Swartling1. 1 _Uppsala University, Uppsala, Sweden;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA;_ 4 _Stanford University, Palo Alto, CA_.

Misexpression of MYC genes (MYC and MYCN) occurs commonly in medulloblastoma (MB), the most frequent malignant childhood brain tumor. We previously showed that tumors are addicted to MYCN and that MYCN stabilization is required for MB development in mice (Swartling et al, Genes & Dev, 2010; Cancer Cell, 2012). Targeted MYCN suppression completely depleted MYCN-driven MB cells in vivo. Immediate transcriptional changes from such MYCN blockade were found by RNA-Seq and showed similarities to changes that occurred after CDK2 suppression or when inhibiting BET bromodomains. CDK2 and BET inhibitors both inhibited MYC protein expression and effectively induced cell cycle arrest or apoptosis. Compared with either agent alone a sustained combination treatment over 7-10 days displayed synergy and effectively abolished tumor cell proliferation in vitro. The combined treatment further reduced tumor growth in orthotopical MB transplants and significantly prolonged survival as compared to single agent therapy. Our data suggest that dual inhibition of CDK2 and BET Bromodomains could be a novel treatment approach in suppressing medulloblastoma by targeting MYC proteins.

#2474

DFMO targets cancer stem cells in neuroblastoma.

Tracey Avequin,1 Ping Zhao,1 Abhinav Nagulapally,1 Jeffrey Bond,2 Ebrahim Azizi,3 Max Wicha,4 Giselle L. Saulnier Sholler5. 1 _Helen DeVos Children's Hospital, Grand Rapids, MI;_ 2 _University of Vermont, Grand Rapids, MI;_ 3 _University of Michigan, Grand Rapids, MI;_ 4 _University of Michigan, Ann Arbor, MI;_ 5 _Helen DeVos Children's Hospital and Michigan State University, Grand Rapids, MI_.

Background: Difluoromethylornithine (DFMO) has shown promise in recent clinical trials as a therapeutic agent in treating neuroblastoma (NB). High-risk NB patients have an approximately 50% relapse rate, after which survival rate drops to lower than 10%. By targeting the cancer stem cells (CSC) we could decrease relapse rate in high-risk patients and improve long-term survival. DFMO is known to inhibit ornithine decarboxylase (ODC) impeding key polyamine biosynthesis as well as the LIN28/Let7 pathway and disrupting the glycolytic metabolism pathways both of which have been shown to be important in CSC models. We propose that DFMO may be an effective treatment against neuroblastoma CSCs.

Methods: BE(2)C and SMS-KCNR NB cell lines were studied. Limiting dilution assays of xenograft studies were performed. Untreated, 10-day 5mM DFMO-pretreated, or 20-day 5mM DFMO-pretreated cells were injected into mice between 10-5000 cells/mouse and followed for tumor formation. Xenograft Be2C mice were treated with either vehicle or 2%DFMO in drinking water and harvested at 7-14 days for tumor analysis. Tumors and cell cultures of untreated and DFMO-treated cells were analyzed by RT-qPCR, Western Blot analysis, and GeneChip gene expression analysis measuring levels of ODC1, LIN28B, mirLet7, MYCN, SLC2A4 (a glucose transporter), and CSC markers such as CXCR4, POU5F1, NANOG, SOX2, and KLF4. Riboflavin induced autofluorescence (AF) was used to identify CSC. This method detected cells after a 24-hour bath in 30µM riboflavin and excitement at 488nm in a MoFlo cell sorter at the University of Michigan Flow Core, after which cells are sorted into AF- and AF+ sub-populations for imaging, neurosphere assays with or without DFMO, RT-qPCR and Western Blot analysis. Knock-down of ODC1 is achieved by siRNA transfection via Lipofectamine in cells and analyzed by RT-qPCR, Western Blot, and neurosphere assays with or without DFMO.

Results: Pretreatment with DFMO in BE(2)C and SMS-KCNR cells in vitro and in vivo inhibits tumor formation in neurosphere assays and in limiting dilution xenograft models respectively. Tumors harvested from xenografts show a decrease in expression of CSC biomarkers and neurosphere formation capability in those treated with DFMO. AF+ cells showed an increase in stem cell marker expression and increase neurosphere formation relative to AF- cells, identifying them as cancer stem cells. In addition, AF+ cells are more sensitive to DFMO treatment with a greater decrease in neurosphere formation. Cells transfected with ODC siRNA or subjected to DFMO show a reduction in CSC biomarker RNA and protein and a decrease in neurospheres formation.

Conclusion: These results suggest that DFMO targets the CSC subpopulation in neuroblastoma, disrupting cell growth pathways and inhibiting tumor formation. This concept is under further investigation in a Phase II clinical trial to prevent relapse in high risk neuroblastoma patients.

#2475

Bmi1 is a therapeutic target in recurrent medulloblastoma.

Sheila K. Singh,1 Neha Garg,1 Branavan Manornajan,1 David Bakhshinyan,1 Chitra Venugopal,1 Robin Hallett,2 Kevin Xin Wang,3 Vijay Ramaswamy,3 Yoon-Jae Cho,4 Siddhartha Mitra,5 David Kaplan,2 Thomas Davis,6 Michael Taylor3. 1 _McMaster Stem Cell and Cancer Research Institute, Hamilton, Ontario, Canada;_ 2 _Cell Biology Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada;_ 3 _The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada;_ 4 _Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA;_ 5 _Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA;_ 6 _11PTC Therapeutics, South Plainfield, NJ_.

We describe the epigenetic regulator Bmi1 as a novel therapeutic target for the treatment of recurrent human Group 3 medulloblastoma, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Through comparative profiling of primary and recurrent medulloblastoma, we show that Bmi1 defines a treatment-refractory cell population that is uniquely targetable by a novel class of small molecule inhibitors. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden and increased mouse survival, without neurotoxicity. As Group 3 medulloblastoma is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.

Current clinical trials for recurrent medulloblastoma patients who no longer respond to risk-adapted therapy are based on genomic profiles of primary, treatment-naïve tumors. These approaches will provide limited clinical benefit for patients since recurrent metastatic Group 3 medulloblastomas are highly genetically divergent from their primary tumor. Our experimental approach defines a tractable target, the epigenetic regulator Bmi1, which characterizes not only recurrent medulloblastoma, but many other metastatic and treatment-resistant cancers. As future clinical oncology trials will most likely begin with relapsed patients, therapeutic targets from comparative analyses in primary and matched-recurrent tumors offer the greatest clinical yield and may be readily translated to the patient bedside.

#2476

DiSCoVERing innovative therapies for rare tumors: Combining genetically accurate disease models with advanced in silico analysis to identify novel therapeutic targets.

Allison Rose Hanaford,1 Tenley C. Archer,2 Antoinette Price,1 Ulf Kahlert,3 Jarek Maciaczyk,3 Nikkhah Guido,4 Wlliam Kim,5 Tobias Ehrenberger,6 Paul Clemons,5 Vlado Dancik,5 Brinton Seashore-Ludlow,7 Vasanthi Viswanathan,,5 Michelle Stewart,5 Matthew Rees,5 Alykahn Shamji,5 William Hahn,5 Stuart Schreiber,5 Ernest Fraenkel,6 Scott Pomeroy,2 Jill Mesirov,8 Pablo Tamayo,8 Charles G. Eberhart,1 Eric H. Raabe1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _University Medical Center Düsseldorf, Dusseldorf, Germany;_ 4 _Universitätsklinikum Freiburg, Freiburg, Germany;_ 5 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 6 _MIT, Cambridge, MA;_ 7 _Karolinska Institute, Stockholm, Sweden;_ 8 _University of California San Diego, San Diego, CA_.

The lack of genetically accurate models that recapitulate the genetics of human tumors hampers the development of new treatments for rare tumor types. One strategy to create an accurate model would be to use stem cells from a cancer's tissue of origin and add key oncogenic driver elements in combinations that are seen in tumors. These elements should also drive oncogenic transformation relevant to that particular tumor type. To validate this strategy, we used human neural stem and progenitor cells derived from the cerebellar anlage to develop a model of Group 3 MYC-driven medulloblastoma, a rare and frequently fatal pediatric brain tumor. The cells were transduced with c-MYC, dominant-negative p53, constitutively active AKT and hTERT, key oncogenic driver elements associated with aggressive medulloblastoma and injected into moice as orthotopic xerographs. These cells formed tumors in the brains of mice and resulted in decreased survival to a median of 117 days. These tumors histologically, pathologically, and genetically resembled the MYC-driven Group 3 medulloblastoma tumors. In contrast, human neural stem cells immortalized with SV40 did not form tumors in mice. To facilitate rapid screening of a large number of available drugs, we developed an in silico analysis technique, DiSCoVER (Disease-model Signature vs. Compound-Variety Enriched Response) that uses the expression profile of the human neural stem cell models and existing drug sensitivity databases to identify novel therapeutic targets. The DiSCoVER analysis predicted aggressive medulloblastoma would be sensitive to CDK inhibitors. Among the top hits were the CDK4/6 inhibitor palbociclib. Indeed, treatment of Palbociclib in human neural stem cell medulloblastoma model and high-serum medulloblastoma cell lines led to decreased proliferation by an average of 62% as measured by MTS assay compared to vehicle control (p<0.01). Palbociclib treatment also increased the percentage of apoptotic cells by an average of 150% as measured by cleaved caspase-3 immunofluorescence and cleaved-PARP western blot compared to vehicle control (p< 0.04). Treatment with palbociclib significantly extended the survival of mice with orthotopic medulloblastoma xenografts (p=0.003). We validated our human neural stem cell model of Group 3 medulloblastoma and show that this model, as well as patient-derived high MYC medulloblastoma models, are sensitive to CDK4/6 inhibition in vitro and in vivo. Our results strongly suggest that Palbociclib can be an effective treatment for poor-prognosis MYC-driven medulloblastomas in carefully selected patients (Group 3/C1). In summary, we have presented here a new method of generating genetically accurate models for rare tumors using region-specific human stem and progenitor cells and a novel analysis technique to find therapeutics to target them.

#2477

An orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma.

David L. Tamang, Pengyu Huang, Olga Golonzhka, Jeffrey R. Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. _Acetylon Pharmaceuticals, Boston, MA_.

INTRODUCTION: Neuroblastoma (NB) is an extra-cranial solid cancer and is among the most common cancers in infants less than 1 year of age, with 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating an urgent unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) treatment. RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified, and chemical inhibition of HDAC1/2 was shown to induce a similar expression pattern.

METHODS: In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by RT-PCR, gene expression microarray and retinoic acid receptor (RAR) chromatin immunoprecipitation followed by high-througput sequening (ChIP-Seq).

RESULTS: HDAC1/2i and RA together caused increased localization of the RAR to its own RARα and RARβ promoter regions, and increased in RAR mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26, an enzyme responsible for clearing intercellular RA, was reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i enhanced apoptotic pathways and decreased E2F driven cell cycle signaling.

We confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage. Consistent with this finding, we observed reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies after combination treatment. Further, combination treatment reduced the E2F-activators CDK4 and CDK6 at the protein level while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress, as are HDAC1, HDAC2, acetylated histone H3K9 ChIP-seq experiments, and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB.

#2478

Tumor hypoxia promotes Ewing sarcoma metastases in a mouse xenograft model.

Jason U. Tilan, Sung-Hyeok Hong, Susana Galli, Rachel Acree, Katherine Connors, Meredith Horton, Akanksha Mahajan, Larissa Wietlisbach, Yi-Chien Lee, Olga Rodriguez, Christopher Albanese, Joanna Kitlinska. _Georgetown University Medical Center, Washington, DC_.

Ewing sarcoma (ES) is a pediatric tumor induced by EWS-ETS fusion proteins, most often EWS-FLI1. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear. Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of nonperfused areas within tumor tissue was associated with poor prognosis. In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes via changes in transcriptional activity of the EWS-FLI1 gene. However, despite this line of evidence, no direct proof for this hypoxia-induced ES progression and spread has been provided. Moreover, the mechanisms by which hypoxia could exert such effects are unknown. To fill this gap, we created an in vivo model of hypoxia in ES and tested its effect on tumor metastasis. SK-ES1 ES cells were injected into gastrocnemius muscles of SCID/beige mice. Once the tumors reached a volume of 250mm3, they were either excised (control) or subjected to femoral artery ligation (FAL) for 72h prior to excision, inducing ischemia of the lower hindlimb, thus creating tumor hypoxia. Then, the mice were monitored for metastases. The extent of the metastatic disease was assessed and compared between experimental groups based on periodic MRI, necropsy and histopathology findings. FAL resulted in profound tumor hypoxia, as evidenced by inhibition of primary tumor growth, severe tissue necrosis and positive staining for a hypoxyprobe, pimonidazole. However, despite the impaired growth of primary tumors, xenografts subjected to FAL were more metastatic. The involvement of hypoxic cells in metastases was evidenced by the accumulation of pimonidazole-positive cells (hypoxic at the time of FAL) in areas of tissue invasion and intravasation. Consequently, mice bearing FAL-treated tumors exhibited a decreased latency of metastases formation and an increase in their number from an average of 0.9 to 2.3 metastases per mouse in control and FAL groups, respectively. We also observed a change in the pattern of metastases, as FAL-treated tumors metastasized more often to distant organs (average of 0.3 organ metastases per mouse in control and 1.3 in FAL group). The hypoxia-induced metastases were most often observed in adrenal gland and spine (50% and 42% of mice in FAL group, respectively), while no such metastases were observed in the control group. Moreover, 100% of FAL-treated mice had signs of bone marrow invasion, while no tumor cells were detectable in bone marrow of control mice. This data provides the first-ever direct evidence for tumor hypoxia as a driver of ES metastases. Moreover, our model of tumor hypoxia in vivo provides an excellent opportunity to identify hypoxia-induced pathways involved in ES metastatic progression that subsequently may become novel therapeutic targets for this disease.

#2479

Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation.

Mehrdad Rahmaniyan,1 Li Li,1 Julio Garcia,1 A A. Qudeimat,2 Jacqueline M. Kraveka1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Saint Jude Children's Research Hospital, Memphis, TN_.

Background:

Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in "high risk" disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear.

Objectives:

In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells.

Methods:

SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined.

Results:

Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. Since perturbation in complex sphingolipid levels is associated with apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Curcumin has been demonstrated to induce ROS generation. Pre-treatment with the antioxidants N-acetylcysteine or glutathione abrogated curcumin mediated apoptosis and sphingolipid generation in SMS-KNCR cells. Furthermore, curcumin mediated SMS and GCS inhibition was blocked by these antioxidants.

Conclusions:

ROS plays a key role in sphingolopid and curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma.

#2480

Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells.

Manu Gnanamony, David Pinson, Reuben Antony, Karen S. Fernández, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. _University of Illinois College of Med. at Peoria, Peoria, IL_.

Neuroblastoma is the most common extra-cranial solid tumor in children. Despite aggressive therapy prognosis of high risk neuroblastoma remains guarded and also unpredictable because of heterogeneity of tumor response to treatment. Radio-resistance has been recognized as a significant contributor to aggressive clinical behavior of neuroblastoma. Characterization of genetic alterations have been used for predicting clinical outcome in neuroblastomas and one of the most studied genetic markers in aggressive neuroblastoma is nMyc/MYCN. The MYCN gene belongs to the MYC family of transcription factors and nMyc expression is to a large extent inversely correlated with cMyc expression and nMyc expression is also associated poor clinical outcomes. In this study, we have attempted to correlate the expression of nMyc in neuroblastoma cells NB1691 and SKNBE2 to radiation resistance. We exposed neuroblastoma cells NB1691 and SKNBE2 to chronic radiation doses with a cumulative high energy X-ray radiation dose of 25Gy achieved by dosing at 5Gy increments over a period of 15 days. Cells surviving after the 25Gy cumulative radiation dose were selected and designated as NB1691R and SKNBE2R. Expression levels of nMyc, cMyc and uPA were determined. We observed that the radiation tolerant cells showed at least 164 fold increase in the expression of uPA in NB1691R cells and at least 97 fold increase in SKNBE2R cells. We also observed that the expression levels of nMyc decreased in both NB1691R and SKNBE2R cells to almost undetectable levels. Further, the expression levels of cMyc revealed that NB1691R cells showed a 208 fold increase and SKNBE2R a 71 fold increase when compared to parental cells. We further determined whether the expression of uPA can modulate the expression of nMyc. We silenced uPA expression in NB1691 cells and observed that downregulation of uPA moderately rescued nMyc expression. Cell cycle analysis revealed that acute radiation of NB1691 cells induced accumulation of cells in the G2/M phase and un-radiated NB1691R cells continued to show cells accumulated in the G2/M phase similar to acute radiated NB1691 cells.

Our study shows that:

1. Chronic radiation exposure suppresses the expression of nMyc and induces the expression of cMyc.

2. cMyc expression was accompanied with the expression of uPA and suppression of uPA expression moderately rescued the expression of nMyc.

Our preliminary findings suggest that chronic exposure to radiation alters the nMyc/cMyc ratio in neuroblastoma cells and induces the expression of the pro-invasive molecule uPA.

#2481

Targeting XPO1 overexpression with selinexor disrupts the survivin pathway in neuroblastoma.

Raquel Castellanos,1 Basia Galinski,1 David Tauber,1 Yosef Landesman,2 Edward Attiyeh,3 Daniel Weiser1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Karyopharm Therapeutics, Newton, MA;_ 3 _The Children's Hospital of Philadelphia, Philadelphia, PA_.

Background: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and accounts for a disproportionately high fraction (12%) of childhood cancer deaths. More than half of patients with high-risk NB will not survive their disease despite intensive treatment. Therefore new therapies that rationally target unique vulnerabilities in NB are urgently needed. The nuclear export protein Exportin-1 (XPO1) is more abundant in primary refractory NB (PRN), the most aggressive form of disease, making it an attractive therapeutic target. Overexpression of XPO1 has been implicated in tumor development and progression of multiple cancers. Selinexor (KPT-330, Karyopharm Therapeutics) targets XPO1 and inhibits its normal function of translocating tumor suppressor and growth regulatory proteins out of the nucleus. Inhibition of XPO1 results in forced nuclear retention of such proteins, which decreases cellular proliferation. Selinexor has demonstrated efficacy across a range of adult malignancies and is being developed for childhood cancer clinical studies. In this study we look at the effects of selinexor treatment in NB, specifically looking at proteins involved in apoptosis.

Methods: Whole protein cell lysates were extracted from NB cell lines IMR5 and NB-EBC1 after treatment with selinexor. Cells were treated across a range of concentrations (100nM-1000nM) and evaluated at three time points (24, 48, and 72 hours). Cytotoxic effects were measured by MTT proliferation assays.. Expression of XPO1, nuclear exported proteins, and downstream targets of the Survivin pathway were evaluated by immunoblots.

Results: Proliferation defects were observed in all NB cell lines treated with selinexor as compared to vehicle (DMSO) control. Expression of XPO1 was decreased in treated cells after 24, 48, and 72 hours of drug exposure. Expression of the anti-apoptotic protein Survivin was also decreased, as were STAT3 and acetylated STAT3. Cleaved Caspase-3 levels were increased and associated with apoptosis and decreased cellular proliferation.

Conclusions: This study provides a mechanism for XPO1 overexpression contributing to tumor progression in highly aggressive NB and explores its potential as a prognostic biological marker and therapeutic target in PRN. Treatment of NB cell lines with selinexor leads to apoptosis and associated decreased cellular proliferation. Inhibition of XPO1 function may have implications aside from nuclear export. Survivin, a pro-apoptotic protein, is decreased in the presence of selinexor, and downstream targets in the Survivin pathway are also disrupted. These data provide additional rationale for clinical development of selinexor for pediatric solid tumors, and particularly for diseases such as PRN where XPO1 is known to be overabundant. This work also suggests rational strategies for the optimal combinatorial approaches using selinexor for refractory neuroblastoma.

#2481A

A novel chemically-modified curcumin (CMC 2.24) promotes chemosensitivity in neuroblastoma.

David Tauber,1 Basia Galinski,1 Raquel Castellanos,1 Joseph Scaduto,2 Francis Johnson,3 Lorne Golub,3 Daniel A. Weiser1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Traverse Biosciences Inc., Stony Brook, NY;_ 3 _Stony Brook University, Stony Brook, NY_.

Introduction

The chemically modified curcumin, CMC 2.24 (TRB-N0224, Traverse Biosciences Inc.), was designed to overcome the poor solubility, bioavailability, and biological potency of naturally occurring curcumin in an effort to achieve superior safety and efficacy in chronic inflammatory conditions and cancer. It has been demonstrated that CMC 2.24 can sensitize cancer cells that are otherwise refractory to chemotherapy. Patients with neuroblastoma, a highly lethal childhood cancer of the peripheral sympathetic nervous system, often have de novo chemotherapy resistance and therefore succumb rapidly to their disease. We hypothesize that chemoresistance in primary refractory neuroblastoma is mediated by anti-apoptotic mechanisms that can be overcome when conventional therapy is combined with CMC 2.24.

Experimental Procedures

A heterogeneous panel of neuroblastoma cell lines was cultured under routine conditions and subjected to varying concentrations of CMC 2.24 to establish IC50s. Subsequent studies were focused on NB-EBc1, a MYCN non-amplified cell line, and IMR-5, a MYCN amplified cell line. IC50s were also generated for cisplatin as a single agent, and then when administered in combination with CMC 2.24. Whole protein cell lysates were extracted at 24, 48, and 72 hours after treatment and cell viability was measured by MTT proliferation assays.

Results

Proliferation defects were observed in all neuroblastoma cell lines treated with CMC 2.24 as compared to vehicle (DMSO) control. Relatively cisplatin-resistant cell lines had diminished cell viability when CMC 2.24 was added in combination with cisplatin, suggesting a synergistic effect. Alterations in response to CMC 2.24 treatment occur in anti-apoptotic proteins survivin and BARD1, found to be upregulated in chemoresistant cell lines. Additional mechanistic evaluation is ongoing.

Conclusions

There is no known curative therapy for patients with primary refractory neuroblastoma. CMC 2.24 enhances chemosensitivity in a panel of neuroblastoma cell lines and may provide a new therapeutic approach that can overcome de novo chemotherapy resistance. Further mechanistic and in vivo studies are needed to facilitate clinical development of a promising novel anti-cancer agent.

### Stemness Properties and Therapeutic Targeting in Solid Tumors

#2482

The Calcineurin/NFAT pathway mediates tumor initiating cell and drug resistance in lung cancer through SOX2 enhancer binding and ALDH upregulation.

Zhi-Jie Xiao, Jing Liu, Vicky PC Tin, Maria P. Wong. _The University of HongKong, Hong Kong, Hong Kong_.

Tumor initiating cells (TIC) are a small population of cancer cells with enhanced tumorigenicity and drug resistance. Their stemness properties could be activated by stress-induced cell plasticity but the mechanisms remain to be studied. Calcium signaling is important for integrating cell responses to intra- and extracellular stresses. The role of its downstream pathway, calcineurin/NFAT, in lung TIC has not been explored. This study investigated the role and mechanisms of calcineurin/NFAT in regulating TIC and drug resistance by analyzing NFATc2 expression in multiple TIC populations and functional evaluation using in vitro and in vivo models. In tumor spheres and TIC isolated by marker-based flow cytometry, NFATc2 showed overexpression and higher luciferase reporter activities. Knockdown of NFATc2 reduced TIC marker proportions and suppressed TIC properties including pluripotency genes expression, sphere formation, cell mobility and drug resistance in vitro. Xenograft models and limiting dilution assay showed shNFATc2 effectively suppressed tumorigenesis and reduced TIC frequency. Stable NFATc2 knockdown sensitized tumors to cisplatin and gefitinib treatment. In contrast, TIC-supportive effects were observed in vitro and in vivo when NFATc2 was overexpressed. Further, NFATc2 was activated in stable gefitinib or cisplatin resistant cell lines, and knockdown of NFATc2 sensitized them to drug treatment. These data implicated NFATc2 is involved in lung TIC maintenance and drug resistance. To further investigate the molecular mechanisms of how NFATc2 mediate these roles, we analyzed pluripotency genes and observed a significant correlation between NFATc2 and SOX2 expression in lung cancer cell lines and human lung carcinomas. Computational analysis predicted NFATc2 binding sequences in SOX2 regulatory regions. Detailed studies including luciferase reporter assay, site-directed mutagenesis and CHIP-qRCR confirmed NFATc2 could regulate SOX2 expression through binding to its enhancer. Furthermore, immunohistochemistry on human lung cancers showed significant correlation between expressions of SOX2 and the anti-oxidative stem cell marker ALDH, suggesting SOX2 regulates ALDH expression. In vitro, NFATc2 knockdown suppressed ALDH1A1 expression but it was rescued by SOX2 overexpression. Vice versa, ALDH1A1 was upregulated in NFATc2 overexpressing cells but this was prevented by SOX2 knockdown. Functionally, reactive oxidative species (ROS) levels assessed by flow cytometry were suppressed by activated NFATc2/SOX2/ALDH1A1 axis, while treatment with the ROS inhibitor N-acetylcysteine reversed the sensitization of shNFATc2 on cisplatin treatment. Together, the data showed NFATc2 is involved in the regulation of TIC phenotypes in lung cancer. It increases drug resistance by ROS scavenging mediated by the SOX2/ALDH1A1 axis.

#2483

Generation of patient-specific and genome-edited MEN2A patient-derived induced pluripotent stem cells as novel tools for drug screening.

Julien Hadoux,1 Christophe Desterke,2 Olivier Féraud,3 Mathieu Guibert,3 Roberta Francesca De Rose,4 Paule Opolon,5 Dominique Divers,3 Emilie Gobbo,3 Frank Griscelli,6 Martin Schlumberger,5 Annelise Bennaceur-Griscelli,7 Ali Turhan8. 1 _Inserm UMRS 935, Villejuif, France;_ 2 _US-33, Villejuif, France;_ 3 _Inserm UMRS 935, ESTeam Paris Sud, Infrastructure INGESTEM, Villejuif, France;_ 4 _Department of Health Sciences, University of Catanzaro, Catanzaro, Italy;_ 5 _Gustave Roussy, Villejuif, France;_ 6 _Paris Descartes University & Gustave Roussy, Villejuif, France; _7 _APHP, Departement of hematolopgy, University Paris Sud, Villejuif, France;_ 8 _APHP, Departement of hematolopgy, University Paris Sud, Le Kremlin Bicêtre, France_.

Introduction

MEN2A is a cancer-predisposing syndrome that affects patients with germline RET mutations. Animal models do not faithfully recapitulate the full clinical spectrum of the disease. Moreover, it has been suggested that primitive malignant stem cells could be at the origin of resistances and relapses in patients treated with standard chemotherapy. In order to modelling MEN2A malignant primitive stem cells, we have induced to pluripotency MEN2A peripheral blood mononuclear cells from patients who developed medullary thyroid carcinoma harboring "high risk" or "moderate risk" RET mutation.

Methods

PBMC from patients with germline RETC620R and RetC634Y mutations were reprogrammed into pluripotent stem cells using Sendai-virus mediated Oct4, Sox2, Klf4 and c-Myc gene transfer. An isogenic IPSC line was generated by genome editing, using CRISPR-Cas9 to correct the "high risk" RETC634Y mutation. Hallmarks of pluripotency, genotype and phenotypes were characterized .

Results

Pluripotency behaviour of each IPSC line was confirmed in vitro (EBs, and pluripotent markers) and in vivo (teratoma assay in immunodeficient mice). Genome edition of the RETC634Y iPSC led to the generation of the RETY634C isogenic control iPSC. Ret expression was confirmed on both RETC634Y and RETY634C. In order to determine the accuracy and absence of off-target effects, we have performed a whole exome sequencing of both RET-mutated and RET-corrected IPSCs. These experiments revealed some off target effects of the CRISPR-mediated gene edition. Gene-array datas revealed an increased expression of neuronal development-related set of genes in RETC634Y iPSC, as compared to RETY634C control, that may account for the development of certain MEN2A features. Treatment of IPSC with vandetanib, a Ret inhibitor, led to a decrease of iPSC colonies formation in RETC634Y mutated iPSC compared to RETY634C, suggesting that RET-iPSC could serve as a plateform drug screening model. The comprehensive pathological assessment of IPSC RET-mutated and corrected derived-teratoma did not reveal, at this point, any neuroendocrine tumor-reminiscent structure.

Conclusion

We report here the first MEN2A-iPSC from "high risk" RETC634Y iPSC and its genome-edited isogenic normal IPSC, with their genomic profiling by whole exome sequencing. These well caracterized RET-IPSC will allow to develop cell-based assays in high throughput screening for drug discovery and to model MEN2A.

#2484

Non-canonical Hedgehog/Gli1 signaling drives lung adenocarcinoma stem cells survival and its targeting inhibits CSC-derived tumors.

Agnese Po,1 Marianna Silvano,1 Evelina Miele,2 Adriana Eramo,3 Matilde Todaro,4 Carlo Capalbo,1 Valentina Salvati,3 Giovanni Sette,3 Danilo Cucchi,1 Zein M. Besharat,1 Gianluca Canettieri,1 Lucia Di Marcotullio,1 Isabella Screpanti,1 Giorgio Stassi,4 Ruggero De Maria,5 Ann Zeuner,3 Enrico De Smaele,1 Elisabetta Ferretti1. 1 _Sapienza University of Rome, Roma, Italy;_ 2 _Italian Istitute of Technology, Roma, Italy;_ 3 _Istituto Superiore di Sanità, Roma, Italy;_ 4 _University of Palermo, Palermo, Italy;_ 5 _Regina Elena National Cancer Institute, Roma, Italy_.

Introduction: Lung Adenocarcinoma (AC) is the most frequent lung cancer histological subtype and is a leading cause of cancer-related death worldwide. Hedgehog/Gli (Hh/Gli) signaling pathway regulates lung development and its aberrant activation contributes to tumor pathogenesis and play a role in cancer stem cells (CSC) control.

We investigated oncogenic Hh/Gli signaling in AC-CSC.

Methods: human AC-CSC were derived from primary tumors. For in vitro studies AC-CSC were maintained in serum-free medium supplemented with EGF/bFGF. For in vivo experiments, immunocompromised mice were injected with AC-CSC. Gli1 inhibitor GANT61 was used both in vitro and in vivo (IP 40 mg/kg twice/we) treatments.

Results: Using mRNA datasets and tissue microarray of Non-small Cell Lung Cancer (NCSLC) samples we found that in AC Gli1 is expressed independently of its canonical activator Smoothened (Smo) expression level. Similarly, CSC were Gli1 positive regardless of Smo expression levels. Indeed, we found that SMO regulatory region is highly methylated in AC cells where Smo mRNA and protein are low.

Looking for Smo-independent regulation of GLI activity we found that NRP2/Erk and IGF1R/Erk axes regulate Gli1 in AC-CSC. Suppression of Gli1 with GANT61 in CSC resulted in impairment of cell growth and stemness features. We performed in-vivo experiments: CSC-derived xenograft from GANT61 treated mice showed impaired tumor growth, reduced proliferation and increased apoptosis. We next highlighted a tumor-stroma crosstalk. A positive autocrine oncogenic feedback loop sustains Gli1 in AC-CSC since we found that the CSC expressed NRP2 and its ligands VEGF-A and VEGF-C. A paracrine positive loop also sustain Hh/Gli signaling in AC where Cancer-Associated Fibroblasts (CAFs) harbor Hh/Gli1 canonical pathway that regulates the expression of VEGF-A, VEGF-C and IGF2 ligands of NRP2 and IGF1R respectively.

Conclusion: Our findings suggest a non-canonical activation of Hh/Gli pathway in AC. In this context, GLI1 transcription factor is effectors of NRP2/Erk and/or IGF1R/Erk signaling inputs.

Our results have implications for the understanding of AC development and sustain the inclusion of target therapy acting downstream of Smo level in the design of AC treatment.

#2485

Heterozygous deletion of RUNX3 promotes vasculogenesis by enhancing endothelial progenitor cells function.

Su Young Oh, Se-Hyung Lee, Sun Hee Lee, You Mie Lee. _Kyungpook National University, Daegu, Republic of Korea_.

Endothelial progenitor cells (EPCs) are bone marrow (BM) -derived stem cells to be committed and differentiated into mature endothelial cells. Runt-related transcription factor 3 (RUNX3) is a tumor suppressor involved in TGF-β signaling pathway and participate in cell cycle arrest and anti-angiogenesis. RUNX3 is frequently deleted or transcriptionally silenced in a variety of cancers by epigenetic mechanisms. The aim of study was to determine the role of RUNX3 in EPC function and vasculogenesis. Here we found that RUNX3 homozygous knockout (Rx3-/-) embryos had vascular defects in embryonic day (ED) 10 and developmental abnormalities in ED 16.5 organs. The number of EPC colonies and circulating EPCs (CD34+/VEGFR2+ cells), migration and tube formation capacity of EPCs derived from RUNX3 heterozygous (Rx3+/-) mice were increased in compared to those wild type (WT) mice, suggesting that deletion of RUNX3 can enhance vasculogenesis. Expression level of VEGF, VEGFR2, SDF-1, CXCR4 and endothelial nitric oxide synthase (eNOS) mRNA was significantly up-regulated in Rx3+/- EPCs. Protein expression of VEGF, p-VEGFR2, p-Akt, p-ERK, p-SAPK/JNK and p-eNOS was also augmented in Rx3+/- EPCs. Finally, we found that recovery of blood flow was highly stimulated in Rx3+/- mice using hindlimb ischemia mouse models. Taken together, our study revealed that RUNX3 might inhibit vasculogenic processes in physiological ischemia by inhibition of EPC functions.

#2486

Docetaxel resistant cells display cancer stem cell properties and regulate growth via PI3/Akt signaling.

Prithy C. Martis,1 Melissa A. Laramore,1 Hunter L. Gazda,1 Atira Dudley,1 Pradeep R. Dumpala,1 Allyson J. Ocean,2 Nathaniel Berman,3 Tapan Parikh,3 Zoe P. Andrada,3 Angelica Nazarian,3 Joanne Thomas,3 Eugene Akahoho,3 George Stoms,4 Alex Yaroshinsky,4 Thomas J. Fahey,5 David J. Wolf,3 Lawrence S. Gazda,1 Barry H. Smith3. 1 _The Rogosin Institute-Xenia Division, Xenia, OH;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _The Rogosin Institute, New York, NY;_ 4 _Vital Systems, Rolling Meadows, IL;_ 5 _NewYork-Presbyterian Hospital, New York, NY_.

Both normal organogenesis and tumor development follow a Gompertzian growth curve. An understanding of the mechanisms operating in these apparently discordant situations, in which growth rate slows as mass increases, may be useful for the treatment of cancer. We have previously shown that encapsulating murine renal adenocarcinoma (RENCA) cells in a double layer of agarose to form spherical macrobeads, undergo Gompertzian growth regulation as tumor colonies within the confines of the inner agarose matrix. As the colonies reach maximal size, the RENCA macrobeads (MBs) secrete factors that also inhibit the proliferation of freely growing target cells outside the MB.

In the current study, we report the ability of the tumor colonies within the MB to re-form following the debulking of tumor mass with docetaxel (0.5 μg/ml or 5.0 μg/ml). Docetaxel-resistant cells were assessed for the stem cell marker OCT4 using immunohistochemistry (Abcam ab19857), RT-PCR (Qiagen Cancer Stem Cell PCR Array) and cell migration/invasion (Corning Cell Migration Chemotaxis and Invasion Assay). Docetaxel-resistant cells are shown to be a discrete population of cells that exhibit stem cell-like properties, including prolonged quiescence (>16 wk), OCT4 staining, stem cell gene expression, increased migration and invasion, and in vivo tumor induction. These cancer stem-like properties are dose and time post-treatment dependent with the greatest expression observed in the few surviving cells at 6 wk post-treatment with 5 μg/ml. Tumor growth is observed in syngeneic BALB/c mice following transplantation of a single docetaxel-resistant cell at higher frequencies (≥ 6 of 9) as compared to the grafting of single monolayer cells (0 of 8) at 5 wk when using cells recovered at 6 wk from the 5 μg/ml dose treated MBs.

To understand the mechanism of colony growth regulation within the MB, we hypothesized that tumor cells external to the MB would use similar growth control signaling when exposed to RENCA MBs or MB conditioned media. We have previously shown an increase in the activity of the transcription factor MEF2 in cells exposed to RENCA MB conditioned media. To investigate PI3/Akt signaling, a known pathway for MEF2 regulation, in human DU145 prostate tumor cells exposed to MB conditioned media, we used In-Cell and Western blotting. Exposure to RENCA MBs resulted in Akt hyperphosphorylation (≥ 48 hours) and de-phosphorylation of MEF2D in DU145 target cells.

These findings support the hypothesis that RENCA MBs, as biological cell systems with the in vitro capability of inhibiting cancer cell proliferation, achieve this effect, at least in part, by signaling through Akt to regulate MEF2D. This effect is being investigated in colorectal patients with progressive disease, following informed consent, who underwent laparoscopic intraperitoneal implantations of RENCA MBs as part of ongoing clinical trials.

#2487

Nicotine-mediated regulation of Sox2 and stemness in non-small cell lung cancer.

Courtney Schaal, Srikumar Chellappan. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

Lung cancer is the leading cause of cancer deaths in both men and women, and is highly correlated with cigarette smoking. Nicotine, the addictive component of tobacco smoke, cannot initiate tumors, but can promote proliferation, migration, and invasion of cells in vitro and promote growth and metastasis of tumors in vivo. More recently, nicotine has been implicated in the promoting self-renewal of stem-like side-population cells from lung cancers. This subpopulation of cancer stem-like cells has been implicated in tumor initiation, generation of entire heterogeneous tumor population, metastasis, dormancy, and drug resistance. We have shown that nicotine promotes the expression of genes that promote stemness, such as Stem Cell Factor (SCF/c-kit ligand), in a specific receptor dependent fashion. Here we demonstrate that nicotine can induce the expression of embryonic stem cell factor Sox2, which is indispensable for self-renewal and maintenance of stem cell properties in non-small cell lung adenocarcinoma cells. High level of Sox2 expression also correlates with poor patient survival outcome in lung cancer, and metastatic lung adenocarcinoma shows higher levels of Sox2 expression than primary tumor or normal tissue, implicating Sox2 in this disease. Nicotine was found to induce Sox2 in A549 cells after 18 and 24 hours of stimulation, an effect which was diminished by 48 hours. This was seen at the mRNA and protein level as assessed by transient transfection and luciferase assay, real-time PCR, western blot, and immunofluorescence respectively. Experiments were conducted to elucidate the mechanism by which nicotine induces Sox2; it was found that siRNA mediated depletion of α7 nicotinic acetylcholine receptor (nAChR), the Hippo pathway effector Yap1, or the transcriptional repressor ID1 could abrogate nicotine-mediated induction of Sox2. Further, treatment with inhibitors of Src kinase or PI3K additionally abrogated the effect, suggesting these proteins play a role in nicotine-mediate induction of Sox2. Interestingly, nicotine could induce Sox2 expression in human mesenchymal stem cells (hMSCs) after 24 hours of stimulation as seen by immunofluorescence and real-time PCR, and this effect was abrogated by treatment with inhibitors to Src kinase, PI3K, Yap1, or α7 nAChR. Additionally, studies are under way to elucidate whether e-cigarettes or flavored tobacco, newer and less studied alternatives to traditional cigarettes, have similar effects on stemness. These studies can be expected to have a direct impact on our understanding of the molecular mechanisms involved in the initiation, growth, and metastasis of non-small cell lung cancer, especially in smokers and tobacco users.

#2488

Stat3 inhibitor (BBI-608) with radiation therapy is promising in malignant pleural mesothelioma.

Seiji Matsumoto,1 Hiroshi Doi,2 Tohoru Nakamichi,1 Ayumi Kuroda,1 Masaki Hashimoto,1 Teruhisa Takuwa,1 Nobuyuki Kondo,1 Seiki Hasegawa1. 1 _Department of Thoracic Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan;_ 2 _Department of Radiology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan_.

Background:

The prognosis of Malignant pleural mesothelioma (MPM) is very poor, the new drug for MPM is need. IL-6 in serum with MPM patients is high, IL-6/Stat3 pathway is activated. We investigated that Stat3 is the potential target for the treatment of MPM.

Methods:

Cell viability was assayed with Cell Counting Kit-8 (CCK-8: WST-8 Dojindo). MPM cells (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) were seeded into 96-wellplates. After the treatment with Stat3 inhibitor (BBI-608). Cell Counting Kit-8 solution was added to each well of the plate and measured the absorbance using a microplate reader. The levels of phosphorylated Stat3 (p-Stat3) were measured in cell lysates using an InstantOne ELISA assays (eBioscience). The translocated p-Stat3, c-Myc were analyzed by Confocal immunofluorescent. In vivo study, H226 cells were injected into the subcutaneous over the flank region of nude mice. Mice were randomly assigned into four groups (5 mice each group) 1) vehicle control 2) treated with BBI-608 3) Radiation 4) BBI608/RT. Tumor is measured twice per week.

Results:

BBI-608 inhibited MPM cell lines (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) viability in a dose-dependent manner. The level of p-Stat3 was decreased 90% by BBI-608 10μM treatment in H226. Untreated H226, p-stat3 was observed in cytoplasma and localized in the nucleus. As compared with untreated cells, p-stat3, c-Myc were decrease in cytoplasma and was not localized in the nucleus with BBI-608 treated cells. BBI-608 suppressed tumor growth (p<0.05) and completely suppressed tumor growth with radiation in mouse model.

Conclusion:

In this study, we have shown that BBI-608 inhibited the proliferation of all MPM cell (epithelial, biphasic, sarcomatoid) lines and inhibited Stat3 phosphorylation and blocked the translocation to the nucleus. We also demonstrated that BBBI-608 completely suppressed tumor growth with radiation in mouse model. Furthermore, Stat3 inhibitor with Radiation Therapy (START) is promising in MPM therapy.

#2489

Therapeutic implication of identifying pancreatic cancer stem cells possessing fructose metabolic signature.

Chia-Ning Shen,1 Chi-Che Hsieh,2 Wen-Shan Li,3 Michael Hsiao1. 1 _Academia Sinica - Genomics Research Ctr., Taipei, Taiwan;_ 2 _China Medical University - Ph.D.Program for Cancer Biology and Drug Discovery, Taichung, Taiwan;_ 3 _Academia Sinica - Institute of Chemistry, Taipei, Taiwan_.

The emergence of the cancer stem-cell theory in the last 20 years has shed light on many of the unresolved challenges. For example, the current cancer chemotherapy can mainly cause cancer remission but often fails to cure cancer due to the existence of cancer stem cells. Therefore, development of methods targeting cancer stem cells could facilitate the improvement of therapeutics for pancreatic ductal adenocarcinomas (PDACs). However, the key characteristics of cancer stem cells (CSCs) remain to be determined. Accumulating evidences revealed the similar features shared by pluripotent stem cells and cancer stem cells, we therefore hypothesize acquisition of stemness characteristics during the reprogramming process is superficially reminiscent of the dysplastic transformation proposed in cancers. In the current work, comparative genome-wide profiling analysis was initially conducted on embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and different subpopulation of pancreatic cancer cells. We initially revealed expression level of pluripotent transcriptional factors and surface markers such as Oct4, Sox2, Nanog, Lin28, CD133, and ABCG2 was significantly higher in ABCG2+CD44+ subpopulation of pancreatic cancer cells correlated with their drug resistance and high metastatic potentials suggesting ABCG2+CD44+ possess the features of CSCs. The further analysis was performed on metabolic pathway and revealed, very similar to ESCs/iPSCs, the metastatic ABCG2+CD44+ subpopulations had evaluated level of GLUT5 & KHK and retain the expression of critical enzymes involved in Krebs cycle suggesting ABCG2+ CD44+ CSC subpopulations can utilize fructose efficiently and possess the capability to be grown in different nutrient-supplying and high-oxygen environment. Utilizing tumor-engraft mice and spontaneous pancreatic cancer mice, we actually revealed fructose replacement enhanced cancer drug resistance and CSC-mediated cancer progression possibly via up-regulating ST6Gal1 as knockdown of ST6Gal1 or treatment of ST6Gal1 inhibitors can suppress self-renewal and metastasis capability of ABCG2+CD44+ CSC subpopulations. In summary, Otto Warburg proposed in 1920s that tumor cells product energy by glycolysis followed by lactic acid fermentation in cytosol rather than by oxidation pyruvate in mitochondria. However, accumulating evidence from recent work actually showed that many cancers exhibit the Warburg effect while retaining mitochondrial respiration. The current work actually elucidated that pancreatic CSCs possess metabolic features of pluripotent stem cells which can utilize fructose efficiently and possess the capability to be grown in different nutrient-supplying and high-oxygen environment. Importantly, these key metabolic features are critical in CSC-mediated cancer drug resistance and metastasis.

#2490

Smoking enriches cancer stem cell population and activates Sox9 through NF-kB signaling in pancreatic ductal adenocarcinoma.

Ramakrishna Nimmakayala, Parthasarathy Seshacharyulu, Seema Chugh, Imayavaramban Lakshmanan, Satyanarayana Rachagani, Surinder K Batra, Moorthy P Ponnusamy. _University of Nebraska Medical Center, Omaha, NE_.

Introduction: Recent studies have demonstrated a clear association between smoking and the incidence of pancreatic ductal adenocarcinoma (PDAC); however, the effect of cigarette smoke in the activation of stem cell (SC) or cancer stem cell (CSC) genes and their involvement in the initiation and progression of PDAC have not yet been studied. It is well known that CSCs are responsible for the drug resistance and aggressiveness of the disease including PDAC. In this study, we investigated the effects of smoking on enrichment of SC/CSCs in pancreatic normal and ductal adenocarcinoma cells, and we also examined whether smoking can activate NF-kB signaling, which is in part leads to enrichment of CSC and induction of CSC markers in PDAC.

Methods: Cigarette smoke extract (CSE) was prepared, and HPNE (Human pancreatic nestin positive cells) and Capan-1 pancreatic cancer (PC) cells were treated with CSE for up to ~15 weeks. Side population (SP) were analyzed by Hoechst staining using Flow-cytomer, and various CSC markers such as PD2 (a stem cell maintenance marker), CD44, ALDH-1, SOX-9 (a multipotent SC marker) and Oct-3/4 (a pluripotent marker), and NF-kB signaling molecules were analyzed by western blotting. ALDH1+ cells, CD44+CD24+ CSCs and G0/G1 phase low cycling quiescent cells were analyzed by flow cytometer. An in-vitro sphere culture was also performed to further confirm the smoke induced CSC properties. Smoke exposed pancreatic tissues excised from unfloxed littermate control (LSL-K-Ras G12D) pancreatic tissue sections were immunostained for SOX-9 using immunohistochemistry (IHC), and for SOX9 and CD44 using immunofluorescence.

Results: Our results showed increased SC/CSCs and more number of spheres by CSE treated cells as compared to their untreated controls and displayed elevated protein expressions of SC/CSC markers. We also observed an elevated CD44+CD24+ CSCs, increased ALDH1+ cells and increased G0/G1 low cycling quiescent cell population in CSE treated cells as compared to untreated controls. In addition, increased immunohistochemical staining for SOX9 and increased immunofluorescent signal for SOX-9 and CD44 were observed in smoke exposed animal tissues indicating that smoking may transform SOX-9+ multipotent SCs into SOX9+CD44+ CSCs. We also analyzed the expression levels of NF-kB signaling molecules in CSE treated HPNE and Capan-1 cells. As compared to their untreated controls, CSE treated cells showed elevated protein expression levels of phospho AKT, phospho RelA (SOX-9 promoter binding subunit of NF-kB complex) and phospho IKKα (a kinase that phosphorylates IKBα, an inhibitor of RelA) suggesting that smoking activates NF-kB signaling.

Conclusion: Our results illustrate that smoking enriches SC/CSCs populations in normal pancreatic cells as well as in pancreatic cancer cells, and activates SOX9 through NF-kB signaling in PDAC.

#2491

Effect of HSP90 inhibitor ganetespib on stemness in small cell lung cancer.

Roberto Gomez-Casal,1 Peng Zhang,1 Hong Wang,1 Paul Gardner,2 James Herman,1 Vera Levina1. 1 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 2 _University of Massachusetts Medical School , Worcester, MA_.

Small cell lung cancer (SCLC) is the most aggressive and metastatic form of lung cancer with a 5 year survival rate of less than 7%. Typically, patients are diagnosed with SCLC when the tumor has already widely metastasized. Combination chemotherapy, generally platinum-based plus etoposide is the mainstay first-line treatments for metastatic SCLC. Despite being highly sensitive to first-line therapy treatments, most patients with SCLC experience relapse with resistant disease within 2 years and die from systemic metastasis. Since there are no approved therapies for the treatment of SCLC, discoveries of therapies that can counteract the spread and relapse of SCLC are needed. Therapy resistance in SCLC can occur through a variety of mechanisms, one possible mechanism might be through the activities of cancer stem-like cells (CSCs) within the SCLC cell population. Different markers, such as ALDH+, CD133+, and uPAR+, are considered as markers of CSCs in SCLC. We used flow cytometry and the Aldefluor kit to separate cells with high and low ALDH activity; flow cytometry and fluorescently- labeled antibody against CD133 and uPAR to separate CD133+ and uPAR + cells. We confirmed that ALDH+/CD133+ and ALDH+/uPAR+ cells were capable of generating tumor spheres in the second and third generations. When we compared SCLC cell lines that were generated from untreated tumors (DMS-53 cell line) with those generated from tumors that were heavily treated (H69 and DMS-114 SCLC cell lines), we found that the percentage of CSCs in DMS-53 cell was significantly lower than that found in H69 and DMS-114 SCLC cell lines. CSCs in SCLC express a distinctive pattern of receptors, signaling molecules, and transcription factors such as ALDHA1, CD133, uPAR, OCT-4, SOX2, STAT3, PKM2, DNA-PK, HIF-1a, and HIF-2a; the upregulation of these factors was determined through analysis of genes and protein expression levels.

These proteins are associated with cancer stemness, resistance to conventional therapies, proliferation, and metastasis. Many of these proteins are known to interact with the molecular chaperone heat shock protein 90 (HSP90) and therefore can be targeted simultaneously with HSP90 inhibitors. We used a new HSP90 inhibitor ganetespib, which is showing considerable promise in late stage clinical trials for treatment of other cancer types. Ganetespib used as monotherapy induced cell cycle arrest and apoptosis and effectively inhibited growth of bulk SCLC cells and CSCs growing as tumor spheres. Ganetespib at low nanomolar concentrations significantly potentiates the effect of cisplatin and etoposide in vitro. Further in vivo studies are needed to unravel role played by HSP90 in therapy resistance and metastasis in SCLC.

#2492

Do disseminated tumor cells with stem cell character after therapy predict prognosis in primary ovarian cancer.

Issam Chebouti,1 Christina Blassl,2 Pauline Wimberger,3 Hans Neubauer,2 Tanja Fehm,2 Rainer Kimmig,1 Sabine Kasimir-Bauer1. 1 _Department of Gynecology & Obstetrics, University Hospital Essen, Essen, Germany; _2 _Department of Gynecology & Obstetrics, University Hospital Duesseldorf, Duesseldorf, Germany; _3 _Department of Gynecology & Obstetrics, University Hospital Dresden, Dresden, Germany_.

Background: We recently showed that the presence of disseminated tumor cells (DTCs) in the bone marrow (BM) of primary ovarian cancer patients (pts) significantly correlated with reduced progression free survival (PFS) and overall survival (OS). Here we analyzed whether the negative prognostic impact a) was related to the persistence of DTCs after platinum based chemotherapy and/or b) may arise from a cellular phenotype, being associated with stem cell character.

Patients and Methods: 79 pts were studied for DTCs before (BT) and after therapy (AT) using immunocytochemistry applying the pan cytokeratin (CK) antibody A45-B/B3. Eight pts harboring at least five DTCs AT were analyzed on two additional cytospin slides by four immunofluorescence staining for DAPI, CK (FITC), SOX-2 or LIN-28 (TRITC), CD45 and CD43 (Cy5), respectively. A DTC was classified as a stem-like tumor cell in case of Dapipos, CD45neg, CD34neg, SOX-2pos/LIN-28pos and Ckpos or Ckneg as evaluated by microscopy.

Results: DTCs were detected in 33/79 pts (42%) with a median number of four DTCs (range 1-37) BT and in 32/79 pts (41%) AT, median cell number eight DTCs (range 1-100), respectively. A persistence of DTCs was found in 13 pts, 20 pts were only positive before therapy, 19 pts after therapy and in 27 pts, no DTCs were detected at any time. Whereas the presence of DTCs BT significantly correlated with reduced OS (p= 0.024), pts initially DTCneg BT but DTCpos AT had a significant shorter PFS (p=0.025). The persistence of DTCs resulted in a shorter PFS and OS reaching borderline significance (p=0.06; p=0.07). AT, Ckpos/LIN-28pos cells were detected in 9/10 patients [median two cells (range 1-5)] and Ckneg/LIN-28pos cells in 7/10 patients [median three cells (range 1-11)], respectively. For SOX-2, the results were 6/8 patients [median two cells (range-0-4)] and 7/8 patients [median four cells (range 1-11)], respectively. Interestingly, two further pts characterized as DTCneg AT but positive BT harbored 1-2 LIN-28pos and SOX-2pos cells in their BM AT. In cases vice versa, a few LIN-28 as well as SOX-2-positive cells were also present even BT.

Conclusion: DTCs present AT express stem cell associated proteins and seem to be correlated with worse outcome. Furthermore, these cells were also detected in some cases BT which might explain worse outcome of pts who changed from DTCneg to DTCpos. Thus, additional therapeutic regimens may be necessary to eliminate these cells.

#2493

Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target in breast cancer.

PARTHASARATHY CHANDRAKESAN,1 Nathaniel Weygant,1 Dongfeng Qu,1 William Berry,1 Randal May,1 Naushad Ali,1 Sripathi Sureban,1 Eddie Bannerman-Menson,2 Michael Schlosser,2 Courtney Houchen1. 1 _UNIVERSITY OF OKLAHOMA HEALTH SCIENCES CENTER, Oklahoma City, OK;_ 2 _COARE Biotechnology Inc., Oklahoma City, OK_.

Introduction: Breast cancer is a heterogeneous disease, and is comprised of a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we targeted DCLK1 as a novel therapeutic strategy in breast cancer cells to reduce cancer cell self-renewal, tumorigenesis and metastasis.

Methods: Anti-DCLK1-siRNA was transfected into breast cancer cell lines (MCF7 [luminal] and MDAMB231 [basal-triple negative breast cancer cell line]). For analysis of circulating tumor cells, MDAMB231/MCF7 cells were stably transfected with RFP (red fluorescence protein). Tumor cell colony formation, mammosphere formation (self-renewal), cell migration and invasion were assessed to evaluate the effect of DCLK1 knockdown and or DCLK1 overexpression on breast cancer cells self-renewal and metastatic potential. Xenograft model was used to identify the role of DCLK1 on breast cancer cell growth and circulating tumor cells in vivo. The data were generated utilizing experimental protocols for clonogenic culture, circulating tumor cells, immunohistochemistry, RT-qPCR and Western blotting.

Results: Expression of DCLK1 was ~36% higher in human breast cancer tissues than in normal breast tissue. Silencing DCLK1 via RNA interference decreased the colony formation (~70%), and self-renewal ability (~85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to less (40-80%) breast cancer cells migration and invasion. Furthermore, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were markedly reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in xenograft tumors resulted in reduced tumor growth (~81%) and CTCs (~95%) in vivo. However, overexpression of DCLK1 in the non-metastatic MCF7 breast cancer cell line increased its in vitro migration and invasion potency and pluripotency along with in vivo tumor formation and progression.

Conclusions: DCLK1 expression is associated with breast cancer cell self-renewal, metastasis and tumorigenesis. Targeting DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis and tumor growth. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer.

#2494

Adhesive signature technology for tumor initiating cell purification in cancer research.

Efrain A. Cermeno, Austin P. Veith, Susan N. Thomas, Andres J. Garcia. _Georgia Institute of Technology, atlanta, GA_.

In spite of major therapeutic advances, cancer relapse and low rates of patient response persist. This failure is due in part to a small subpopulation of tumor initiating cells (TICs) with stem cell like properties that are responsible for the growth of the tumor and the progression of metastasis. Currently, no efficient and reliable methods to isolate TICs for study exist. Our lab has developed a methodology to isolate cells based on their unique adhesion binding strength to a matrix. The novel technology (micro-Stem cell High- Efficiency Adhesion based Recovery [μSHEAR]) consists of a microfluidic device that applies varying degrees of detachment shear forces on cells. Using this device, human pluripotent stem cells and their progeny have been isolated with high reproducibility, yield (>97%), purity (95-99%), and survival (>95%) rates (Singh et al, Nature Methods 2013). The process is fast (10 min), label free, and scalable.

The objective of this research is to isolate the rare TICs from the general cancer cell population by exploiting differences in adhesion strength. To study these differences, we measured the adhesion strength (adhesive signature) of a panel of breast cancer cell lines to fibronectin using a hydrodynamic assay. Based on this screen, we selected the MDA-MB- 231, MDA-MB-453, and MCF7 cell lines for purification of TICs using the uSHEAR technology. Briefly, microfluidic channels were sterilized and coated with fibronectin. MDA-MB- 231, MDA-MB-453 or MCF7 breast cancer cells were enzymatically disassociated, pipetted into the inlet reservoir, and cultured in the device for 24h before detachment experiments. Cells were exposed to different shear forces to selectively detach cell sub-populations. Recovered cell/colonies were counted, seeded into a mammosphere formation assay (MFA), and analyzed after 10 days

The ability to form mammospheres is characteristic of TICs. After uSHEAR mediated separation of breast cancer cells into three fractions, the strongest adhering fraction consistently produced larger and more mammospheres. Furthermore, as the selection adhesive force for the adhered fraction was increased, greater increases in mammosphere number and size were observed. After 10 days, the mammospheres were disassociated and the number of cells quantified. A 5-15 fold increase in the final number of cells was observed in the strongly adherent fractions of cells, compared to 0.8-1.7 fold increase in the unsorted controls. Our results show that cancer cells with a higher mammosphere formation potential, a hallmark of characteristic TICs, have a higher adhesion strength. Furthermore, these cells can be enriched via adhesion based separation giving rise to more and larger mammospheres than their less adherent counterparts. Our results suggest that TICs could be separated based on adhesive forces. Future studies are aimed at characterizing the isolated cells as TICs and testing the isolation strategy in primary tumors.

#2495

hPaf1/PD2 interacts with OCT3/4 in maintenance of the self-renewal process of ovarian cancer stem cells.

Saswati Karmakar, Parthasarathy Seshacharyulu, Arokia Priyanka Vaz, Imayavaramban Lakshmanan, Moorthy Palanimuthu Ponnusamy, Surinder Kumar Batra. _UNMC, Omaha, NE_.

Background: Ovarian cancer (OC) is the most lethal gynecological malignancy among women with more than 85% of the patients manifesting tumor recurrence. Emerging evidence suggests that a small population of cells within the tumor - the 'cancer stem cells (CSCs)' is capable to giving rise to the entire histopathology of the tumor and is responsible for mediating drug resistance, recurrence, and disease aggressiveness. Previously, hPaf1(human RNA polymerase II associated factor1)/PD2 (Pancreatic Differentiation2) - a core component of RNA polymerase II associated factor (PAF) complex, was shown to be overexpressed in pancreatic CSCs and involved in the maintenance of mouse ESCs. Hence, we hypothesized that hPaf1 is involved in the maintenance of self-renewal property of ovarian CSCs (OCSCs). In this study, we investigated the functional role of hPaf1 in OCSCs which has not been explored before.

Methods: Expression of hPaf1, cancer stem cell marker ESA, and self-renewal protein OCT3/4 was analyzed using confocal microscopy on OC tissue array. Side population (SP) was isolated from two OC cell lines OVCAR3 and A2780 by Hoechst staining using flow-cytometer, and characterized using tumor sphere assay. Immunoblotting was performed on characterized SP and NSP (non-SP) for OCSC and self-renewal markers. Transient knockdown of hPaf1 in SP was performed to understand how hPaf1 affects the CSC phenotype through immunoblotting, confocal microscopy, colony formation assay, and tumor sphere formation assay. To determine the interaction between hPaf1 and OCT 3/4, reciprocal co-immunoprecipitation was performed in SP/OCSCs.

Results: There was a significant overexpression and considerable co-localization of hPaf1/PD2 with ESA and OCT3/4 in OC tissues compared to normal ovary tissues. SP from OVCAR3 formed larger and greater number of tumor spheres compared to NSP cells. Moreover, SP isolated from OVCAR3 and A2780 showed higher expression of hPaf1 along with CSC markers (CD44, CD133, ESA, CD24), and self- renewal proteins (β-CATENIN, SOX2, OCT3/4, SHH, and HER2). Transient knockdown of hPaf1 resulted in a significant decrease in expression of CSC markers and self-renewal proteins. In addition, functional characteristics of CSCs such as in vitro tumor formation capacity in non-adherent media and colony formation ability were impaired upon knockdown of hPaf1 suggesting that hPaf1 is involved in maintenance of the CSC phenotype. We also observed that hPaf1 physically interacts with OCT3/4 in OVCAR3 SP cells which provides a mechanism for the maintenance of OCSCs by hPaf1.

Conclusion: Altogether, hPaf1/PD2 is overexpressed in OCSCs and its knockdown resulted in loss of OCSC phenotype. Moreover, hPaf1 is responsible for the maintenance of OCSCs through its interaction with OCT3/4. Hence, therapies that are able to abrogate hPaf1 mediated self-renewal of OCSCs represent potential therapeutic avenues to overcome tumor relapse in OC.

#2496

Novel role of YAP1 in tumor angiogenesis and vascular mimicry.

Namrata Bora Singhal, Srikumar Chellappan. _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that cancer stem-like cells (CSCs) contribute to the genesis and progression of NSCLC. These cells have been shown to form differentiated tumor cells upon receiving appropriate micro-environmental cues and can give rise to multiple components of the tumor, including the vasculature. We isolated the CSCs from NSCLC cell lines and PDX tumors based on the side population (SP) phenotype. SP cells displayed high self-renewal capacity, were highly drug resistant, and could form metastatic tumors in immunocompromised mice. The present study shows that the transcriptional co-activator YAP1, which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and the ability to form angiogenic tubule-like structures in matrigel, a unique feature of cancer stem-like cells that is also called as vascular mimicry. Inhibition of YAP1 by a small molecule inhibitor Visudyne or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of SP cells. The stem -like SP cells from NSCLC were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2) which are crucial genes during angiogenic tubule regression and growth in addition to YAP1. Further, depletion of YAP1 with siRNAs reduced the expression of VEGF, KDR and AngPT-2 mRNA levels. Overexpression of YAP1 resulted in increase in the promoter activity of both KDR and AngPT-2 in transient transfection experiments. This suggests that YAP1 might play a role in regulating VEGF, KDR and AngPT-2 mediated angiogenic functions in cancer cells.

In addition to vascular mimicry, YAP1 appears to play a unique role in regulating the expression of genes involved in angiogenesis. Hypoxia is known to contribute towards angiogenesis as well as cancer progression and metastasis. Preliminary experiments showed that cells grown in hypoxic conditions or treated with hypoxia mimetic compounds like DMOG resulted in increase in YAP1 mRNA and protein expression. However, such an increase was not observed in YAP1 orthologue, TAZ or other canonical Hippo pathway proteins. Hypoxia-inducible factor-1alpha (HIF-1α) is the key transcription factor that regulates the expression of various hypoxia response genes like VEGF. We find that YAP1 directly interacts with HIF-1α as detected by co-immunoprecipitation experiments and proximity ligation assays (PLA). YAP1 also associated with VEGF promoter as seen in ChIP RT-PCRs, and this interaction was elevated under hypoxic environment. Our data suggest a distinct role for YAP1 in regulating hypoxia response, promoting tumor angiogenesis and vascular mimicry.

#2497

ONC201 targets cancer stem cells in colorectal, prostate and glioblastoma multiforme tumors via modulation of stem cell-related gene expression.

Varun Vijay Prabhu,1 Joshua E. Allen,2 Dan Zhao,3 Amriti R. Lulla,1 Christina Leah B. Kline,1 A. Pieter J. van den Heuvel,4 Avital Lev,1 Tracy T. Batchelor,3 David T. Dicker,1 Andrew S. Chi,3 Wafik S. El-Deiry1. 1 _Fox Chase Cancer Centre, Philadelphia, PA;_ 2 _Oncoceutics, Inc, Philadelphia, PA;_ 3 _Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA;_ 4 _Penn State Hershey Cancer Institute, Department of Medicine (Hematology/Oncology), Penn State College of Medicine, Hershey, PA_.

ONC201 is a first-in-class anti-tumor agent that selectively targets cancer cells without observed toxicity. ONC201 is being tested in phase I/II clinical trials for patients with advanced solid tumors and hematological malignancies. Several tumors contain a rare population of cancer stem cells (CSCs) capable of self-renewal that contribute to tumor maintenance and resistance to therapy. We have previously demonstrated that ONC201 targets self-renewing, chemotherapy-resistant colorectal CSCs (Prabhu et al, Cancer Res, 2015). We hypothesized that anti-CSC effects of ONC201 involve changes in stem-cell related gene expression. A targeted network analysis of gene expression profiles of HCT116 p53-null and RKO human colon cancer cells treated with ONC201 revealed that several stem cell-related pathways and proteins are affected with potential implications for anti-CSC effects in diverse tumor types. Specifically, mRNA levels of ID1 (colon/glioblastoma CSC-regulation, 2.5 fold), ID2 (glioma stem cell regulation, 3.2 fold), ID3 (colon/glioma CSC-regulation, 2.9 fold), ALDH7A1 (prostate CSC marker, 2 fold) were significantly downregulated and KLF9 (glioblastoma stem cell inhibitor, 1.5 fold) was significantly upregulated in HCT116 p53-null cells upon ONC201 treatment. Also, mRNA levels of Wnt pathway related genes such as ligand WNT16 (haematopoietic stem cell/prostate cancer resistance-related, 13.5 fold), receptors FZD2 (2.98 fold), FZD4 (3.9 fold) and transcription factor TCF7L2 (3.55 fold) were significantly downregulated. Validation with qRT-PCR indicated that ID2, WNT16 mRNA levels were significantly downregulated while KLF9 mRNA was significantly upregulated in response to ONC201 treatment in HCT116 p53-null cells. Interestingly, ONC201 downregulated mRNA levels of ID1 (2.1 fold) and FZD4 (1.6 fold) in RKO cells but not in ONC201-resistant RKO cells, indicating that CSC-inhibition could serve as a biomarker of ONC201 response. To functionally validate anti-CSC effects of ONC201 in glioblastoma multiforme (GBM) and prostate cancer we determined effects on self-renewal using tumorsphere formation. ONC201 eradicated CSC-enriched 3-dimensional neurosphere culture models of primary GBM samples, including newly diagnosed and recurrent samples. ONC201 potently inhibited in vitro cell proliferation of all 5 lines, with IC50 values of 433 nM (MGG18), 1.46 µM (MGG4), 1.09 µM (MGG8), 3.97 µM (MGG67R) and 688 nM (MGG152). ONC201 significantly reduced tumorsphere formation of 22Rv1, DU145 and PC3 human prostate cancer cells. Additionally, western blotting revealed that Wnt16 was downregulated in LNCaP and 22Rv1 while CSC marker CD44 was downregulated in 22Rv1 cells. Ongoing studies are further validating these potential anti-CSC molecular targets of ONC201 and determining their contribution to the overall anti-CSC and anti-tumor effect of ONC201.

#2498

Tumorspheres cultured from circulating epithelial tumor cells (CETCs) overexpress stem cell markers in patients with solid cancers.

Katharina Pachmann, Monika Pizon, Dorothea Schott, Ulrich Pachmann. _TZB Bayreuth, Bayreuth, Germany_.

Background: Solid malignancies continuously shed tumor cells which may enter the circulation, spread to other tissues and initiate metastases, but it is assumed that only a small subpopulation among CETCs is capable of metastasis formation, the subpopulation of CETCs capable of forming tumorspheres in vitro and carrying stem cells properties. The aim of this study was to characterize the pattern of expression of the tumorspheres as compared to CETCs. Methods: Blood samples were obtained from patients diagnosed with solid tumors. CETCs were enumerated with the maintrac method and subsequently cultivated in epithelial stem cell-selective medium. Immunofluorescence and qRT-PCR were performed to examine the metastatic ability of tumorspheres in vitro. Results: Analysis of surface markers in non-adherent tumorspheres forming from CETCs under stem cell- selective conditions after a period of 14 days showed typical phenotype for cancer stem cells depending on type of cancer. Furthermore, spheres had high enzymatic activity for ALDH 1. Array qRT-PCR analysis revealed that putative stem cell markers, such as Oct4, Sox2, Nanog, EpCAM, ALDH1 and CD133 are overexpressed in relation to house-keeping genes RPL13a and GAPDH in tumor spheres in contrast to the significantly lower expression level of these stem cell markers in individually isolated CETCs. High expression level of pluripotency genes in tumorspheres was associated with aggressive tumor behaviour in terms of tumor progression and type of cancer. Conclusion: Here we show that stem cell markers are overexpressed in tumorspheres as compared to CETCs. Obviously only a small population of CETCs can be grown into spheres which possess stem cell properties and most probably are responsible for recurrence and treatment failure. Precise characterization of CETCs and tumorspheres on the single cell and sphere level will contribute to a better understanding of metastasis formation and lead to development of cancer stem cell based cancer therapy.

#2499

Sox2 enhances breast tumor angiogenesis by promoting the transition of cancer cell to CD31+ and LYVE-1+ cells.

Rongrong Wang,1 Xuefei Li,1 Yingxi Xu,2 Tongchao Sun,2 Weijun Su,2 Chenhua Yu,3 Xiaoli Dong,3 Rong Xiang,1 Na Li2. 1 _Key Lab of Tumor Microenvironment and Neurovascular Regulation, School of Medicine, Nankai University, Tianjin, China;_ 2 _The International Collaborative Laboratory for Biological Medicine of the Ministry of Education, School of Medicine, Nankai University, Tianjin, China;_ 3 _The 2011 Project Collaborative Innovation Center for Biological, School of Medicine, Nankai University, Tianjin, China_.

Angiogenesis plays an important role during the development of tumor. Sox2 is one of the transcription factors involved in tumorigenesis and metastasis. However, its function during the process of tumor angiogenesis and lymphogenesis is still poorly understood. Here, we demonstrated that down-regulation of Sox2 significantly reduced the tumor angiogenesis and lymphogenesis as reflected by attenuated immunofluorescence staining of CD31 and LYVE-1 and improved prognosis in xenograft models of breast tumor. Furthermore, Sox2 also promoted the recruitment of bone marrow derived endothelial progenitor cells (MDEPC) to tumor microenvironment. Most importantly, we demonstrated that some of CD31+ and LYVE-1+ cells were derived from Sox2+ cancer cells and silencing of Sox2 could inhibit this transition. We revealed that Sox2 promoted the expression of LYVE-1, VEGFs and enhanced the phosphorylation of VEGFR2 in breast cancer. ChIP-seq and dual-luciferase assay further revealed the binding of Sox2 protein on proximate promoter region of VEGFB and LYVE1 to regulate their transcriptional activities, disclosing the underlying molecular mechanism on the regulatory effects of Sox2 on angiogenesis and lymphogenesis. Our study indicated that Sox2 improved tumor angiogenesis and lymphogenesis through regulating the related genes and unveiled a novel function of Sox2 in tumor progression.

#2500

Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis.

Lavanya Reddivari, Venkata Charepalli, Ramakrishna Vadde, Sridhar Radhakrishnan, Joshua D. Lambert, Ryan J. Elias, Jairam KP Vanamala. _Pennsylvania State University, University Park, PA_.

We have previously shown that the grape bioactive compound resveratrol (RSV) potentiates grape seed extract (GSE)-induced colon cancer cell apoptosis at physiologically relevant concentrations. However, RSV-GSE combination efficacy against colon cancer stem cells (CSCs), which play a key role in chemotherapy and radiation resistance, is not known. Hence, we tested the anti-cancer efficacy of the RSV-GSE in an azoxymethane-induced mouse model of colon carcinogenesis. RSV-GSE suppressed tumor incidence similar to sulindac, without any gastrointestinal toxicity. Additionally, RSV-GSE treatment reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis. In in vitro mechanistic studies, RSV-GSE suppressed proliferation and sphere formation properties of colon CSCs (positive for CD44, CD133 and ALDH1b1), suppressed pGSK3β and nuclear translocation of β-catenin, a critical regulator of CSC proliferation, similar to sulindac. RSV-GSE, but not sulindac, suppressed nuclear levels of downstream proteins of Wnt/β-catenin pathway, c-Myc and cyclin D1. RSV-GSE also induced mitochondrial-mediated apoptosis in colon CSCs characterized by elevated p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-mediated knockdown of p53, a tumor suppressor gene that is mutated in advanced stages of colon cancer, in the colon CSCs did not alter efficacy of RSV-GSE. The suppression of Wnt/β-catenin signaling and elevated mitochondrial-mediated apoptosis in colon CSCs support potential clinical testing/application of grape bioactives for colon cancer prevention and/or therapy.

#2501

**Combination therapy with mebendazole, trametinib and metformin eliminates recalcitrant NRAS** Q61K **melanoma cells.**

Maryam AbdusSamad,1 Anirudh Gaur,2 Cynthia M. Simbulan-Rosenthal,2 Edward C. McCarron,1 Dean S. Rosenthal2. 1 _Division of Surgical Oncology, The Harry and Jeanette Weinberg Cancer Institute, MedStar Franklin Square Medical Center, Baltimore, MD;_ 2 _Department of Biochemistry and Molecular & Cellular Biology, Georgetown University School of Medicine, Washington, DC_.

Melanoma is an aggressive and lethal form of cancer with increasing incidence worldwide. In recent years, the FDA approved several promising treatments for malignant melanoma. However, treatment often fails due to the survival and regrowth of drug-resistant cells. Studies by our lab and others suggest that Melanoma Initiating Cells (MIC), a CD133+ population that forms xenograft tumors, may represent an intermediate stage in the acquisition of stable drug resistance. This study aims to determine the effect of CD133-expression and drug resistance in metastatic melanoma cells harboring difficult-to-treat mutation signatures by focusing on the two main downstream cascades of MAPK and PI3K/AKT/mTOR. Our results show that triple combination therapy using mebendazole (repurposed anthelmintic BRAF inhibitor), trametinib (MEK inhibitor) and metformin (repurposed anti-diabetic mTOR inhibitor) synergistically reduces cell viability of NRASQ61K melanoma cells when compared with mebendazole, trametinib or metformin alone. Further, the surviving fraction from each monotherapy is highly enriched for CD133+ cells. Thus, the multi-kinase inhibitor trametinib, along with repurposed anthelmintic and anti-diabetic drugs may inhibit MAPK and PI3K/AKT/mTOR pathways, which could potentially be an effective therapy for the resistant subpopulation of melanoma initiating cells.

#2502

Bioengineered-induced cancer-like stem cells: characterization and applications.

Sungpil Cho,1 Hongsuk Park,2 Elke A. Jarboe,1 C Matthew Peterson,1 You Han Bae,1 Margit Matthew Janat-Amsbury1. 1 _University of Utah, Salt Lake City, UT;_ 2 _Washington University in St. Louis, St. Louis, MO_.

Cancer stem cells (CSCs) are a small subset of tumor cells capable of self-renewal, tumor initiation, and growth. Isolation, expansion, and differentiation of CSCs in the proper stem niches remain a topic of great debate. To address some of the challenges, we developed bioengineered-induced cancer like stem cells (iCLSCs) through in vitro oncogenic reprograming of C57BL/6 mouse embryonic stem cells (mESCs). We constructed retroviral plasmids through integration of well-defined oncogenic elements such as SV40LTg and HrasV12 into a mouse stem virus long terminal repeat (MSCV-LTR) plasmid fused with either a GFP or RFP gene. We successfully acquired these bioengineered-iCLSCs through retroviral introduction alongside with FACS sorting of positively infected mESCs. We then cultured the bioengineered-iCLSCs on mouse embryonic fibroblast (mEF) feeder cells. Bioengineered-iCLSCs expressed the introduced oncogenic genes and showed enhanced proliferation similar to cancer cells. In addition, we ascertained the unhampered stem properties of the bioengineered-iCLSCs by examining the maintenance of alkaline phosphatase activity as well as the expression of mouse stem cell marker SSEA-1. Furthermore, the orthotopic inoculation of these bioengineered-iCLSCs into either the mammalian fat pad or the ovarian bursa of female C57BL/6 mice lead to the formation of teratomas in supportive niches. Interestingly, the teratomas formed in the ovarian bursa exhibited malignant and immature features, whereas the iCLSCs introduced to the mammary fat pad were not able to generate teratomas with similar features. These observations call for additional research to identify the dependency on a supportive niche and their role in metastasis based on a tumor-favorable microenvironment. Our study helped to establish the use of in vitro oncogenic reprograming to generate bioengineered-iCLSCs and suggests the potential application for the initiation of organ-specific, malignant tumor formation in an orthotopic small animal cancer model.

#2503

NQO1's role in maintaining the cancer stem cell phenotype in NSCLC.

Brian Madajewski, Erik A. Bey. _West Virginia University, Morgantown, WV_.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II detoxifying enzyme responsible for quinone reduction, where it scavenges quinone induced reactive oxygen species. In recent studies, NQO1 has been investigated as a possible drug target due to its overexpression in a number of solid tumors. To this end, treatment with the quinone-analog drug, β-lapachone (ARQ-761), has progressed to Phase II clinical trials. In an effort to better understand the necessity of NQO1 in the overall lifecycle of cancer, our earlier work demonstrated that NQO1 played a vital role in a number of tumorigenic processes including anoikis resistance, and most interestingly alteration of aldehyde dehydrogenase (ALDH) activity. Given that ALDH is a widely reported cancer stem cell marker, and loss of NQO1 leads to a decrease in ALDH activity, we have begun to investigate the effect of NQO1 expression on the non-small cell lung cancer (NSCLC) stem cell population. Here, we present data that demonstrates NQO1 is vital to tumorsphere formation as demonstrated by decreased spheroid formation following NQO1 knocked down with shRNA. We also show that NQO1 appears to be necessary for cancer stem cell renewal as illustrated by decreased serial tumorsphere formation. In addition to these aforementioned data, in extreme limited dilution assays we demonstrate a reduced cancer stem cell frequency in NQO1 knockdown cells as compared to controls. Interestingly, in NQO1 knockdown populations, those cells that do form spheres show a remarkable re-expression of NQO1 as well as a rescue of ALDH activity, further supporting NQO1's role in tumorsphere formation and stem cell maintenance. Future work on this project will involve use of the CRISPR-Cas9 system to generate NQO1 knockout cell lines, drug resistance studies, cancer stem cell marker validation, and in vivo limited dilution assays to definitively demonstrate NQO1's necessity in maintaining the NSCLC stem cell population.

#2504

Assessing the efficacy of the anti metabolic drug, CPI-613, for targeting of ovarian cancer stem cells.

Chiara Bellio, Rosemary Foster, Whitfield B. Growdon, Bo R. Rueda. _Massachusetts General Hospital, Boston, MA_.

Cancer stem cells (CSC) are believed to contribute to the recurrent and often platinum resistant ovarian cancer (OvCa). Moreover, it has been suggested that some anticancer treatments result in an increased frequency of CSC relative to non-CSC. More recently, we have determined that the PARP inhibitor olaparib increased the frequency of CD133, CD117 and ALDH positive cells in the established serous OvCa cell lines OVCAR4 and CaOV4 and in primary cell lines derived from serous carcinomas obtained with IRB approval at the time of initial surgery. We also showed that olaparib treatment increased sphere forming capacity, expression of stemness genes and CSC proliferation suggesting that olaparib has the unintended effect of stimulating CSC function. Previous work has shown that the metabolic inhibitor CPI-613 preferentially targets ovarian CSCs due to their altered metabolism. Our objective here was to determine whether CPI-613 could negate the effect of olaparib on CSC function.

OVCAR4 and CaOV4 cells were treated with 50 μM CPI-613 and the relative frequency of CD133+ and CD177+ cells was assessed at 72 hours post treatment by flow cytometry with cell viability analyzed by MTT. Additionally, the sphere forming capacity of CPI-613 treated cells relative to that of vehicle treated cells was determined in an extreme limiting dilution assay. Finally, the expression of stemness genes in the CPI-613 treated and vehicle treated cells was evaluated by RT-PCR.

Treatment of OVCAR4 and CaOV4 cells with CPI-613 decreased the frequency of CD133+ and CD117+ positive cells relative to untreated cells (p <0.001) without affecting cell viability. Additionally, sphere forming capacity was reduced in the CPI-613 treated cells. The decrease in both CD133+ and CD117+ cell frequency and sphere forming capacity suggests that CPI-613 is targeting the CSC population in these cells. Dual treatment with olaparib and CPI-613 blunted the increase in CD133+ and CD117+ cell frequency induced by single agent olaparib. Parallel treatment of CSC-rich spheres resulted in a considerable decrease in cell viability and frequency of CD133+ and CD117+ cells

Clinical trials with olaparib in OvCa have resulted in increased progression free survival but no significant difference in overall survival has been observed. This raises the question of whether treatment with olaparib selects for the recurrence of more resistant disease, driven in part by the proliferation of CSCs. Our results suggest that CPI-613-mediated disruption of the mitochondrial respiratory pathway preferentially targets CSC and suppresses olaparib's effects on CSC function. In conclusion, targeting CSCs with non-traditional strategies could augment clinical outcomes and CPI-613 represents a promising candidate for combination with FDA approved olaparib treatment in OvCa.

#2505

GNA13 induction of stemness and resistance to chemotherapy in breast and head and neck cancers.

Hui Sun Leong,1 Suhail Ahmed Kabber Rasheed,2 Manikandan Lakshmanan,3 Anandh Kumar Raju,3 Vinay Tergaonkar,3 Daniel shao-Weng Tan,1 Patrick Casey,2 N.Gopalakrishna Iyer1. 1 _National Cancer Centre Singapore, Singapore, Singapore;_ 2 _Duke-NUS Graduate Medical Scool, Singapore, Singapore;_ 3 _Institute of Molecular and Cell Biology Singapore, Singapore, Singapore_.

Despite advancement in cancer therapy, the ability of cancer cells to evade therapy often leads to tumor recurrence and distant metastasis, and ultimately to the death of the patient. Increasing evidence shows that these properties of recurrence can be attributed to a small population of cells within the tumor termed cancer stem cells (CSCs). Understanding the mechanisms that support the growth of CSCs could provide new tools to target these cells and improve therapeutic outcomes. G12 proteins, comprised of Gα12 (GNA12) and Gα13 (GNA13) are mediators of G protein coupled receptor (GPCR) signaling. These proteins are highly expressed in several cancers, and blocking their signaling can significantly inhibit cancer cell invasion and metastasis. Recently, we have shown that loss of GNA13 alone can suppress invasion of breast and prostate cancer cells. Moreover, we have recently obtained evidence that enhanced expression of GNA13 is capable of inducing a stem cell phenotype in cancer cells. Specifically, enforced expression of GNA13 in cancer cells containing low levels of GNA13 protein significantly induced CSC population as shown by increased aldehyde dehydrogenase (ALDH1) activity, increased CD44+/CD24\- cells, and enhancement of tumor sphere formation. In contrast, in cancer cells that express high levels of GNA13, knockdown of GNA13 suppressed ALDH1 activity, the CD44+/CD24\- cell population, and the formation of spheres. Using reporter assays we have identified that GNA13 induced CSC phenotype most probably via activating Jun Kinase - AP1 signaling pathway and blocking this pathway could significantly inhibit CSC phenotype induced by GNA13. Most importantly we have identified that GNA13 expressing cells are resistant to most commonly used chemotherapeutic agents and ionizing radiations. Blocking GNA13 using shRNAs sensitized these cells to both chemo- and radiotherapy significantly. We therefore hypothesize that GNA13 plays a pivotal role in the induction of CSCs and drug resistance, and the suppression of GNA13 activity may provide therapeutic advantage to cancer therapy.

#2506

Citral reduces breast tumor growth by inhibiting cancer stem cell marker ALDH1A3.

Margaret L. Thomas,1 Roberto De Antueno,1 Krysta Coyle,1 Brianne Cruickshank,2 Michael Giacomantonio,1 Roy Duncan,1 Carman Giacomantonio,1 Paola Marcato1. 1 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 2 _St. Mary's University, Halifax, Nova Scotia, Canada_.

Breast cancer stem cells (CSCs) can be identified by increased Aldefluor activity which is primarily due to aldehyde dehydrogenase 1A3 (ALDH1A3) expression. In addition to being a CSC marker, ALDH1A3 regulates genes expression via inducing retinoic acid (RA) signaling and plays an important role in mediating progression of cancers, including breast, melanoma, lung and glioblastoma. Therefore, ALDH1A3 represents a novel druggable anti-cancer target of interest. There are no currently characterized inhibitors of the ALDH1A3 isoform, so twelve general ALDH inhibitors were tested for their effectiveness in targeting ALDH1A3 activity. Ability to directly inhibit ALDH activity was quantified with the Aldefluor assay on a patient tumor xenograft and on breast cancer cell lines with known ALDH isoform activity. Inhibition of RA signaling was quantified by measuring expression of the RA-inducible genes RARRES1 and RARβ. To investigate the anti-cancer properties of these compounds, apoptosis was quantified with the annexin V assay. Citral significantly reduced ALDH1A3- and ALDH2-associated Aldefluor activity in breast cancer cell lines and also reduced Aldefluor activity of a patient-derived xenograft. Citral reduced expression of RA-inducible genes through reducing ALDH1A3 activity. Citral induced apoptosis in vitro in an ALDH-independent manner in breast cancer cell lines and reduced proliferation in the MDA-MB-231 breast cancer cell line. Nanoparticle (NP)-encapsulated citral was generated for in vivo use and administered intravenously to female NOD/SCID mice harbouring MDA-MB-231 ALDH1A3-overexpression tumors. 10mg/kg NP-citral significantly decreased the growth of ALDH1A3-driven MDA-MB-231 tumors. Nanoparticle-encapsulated citral shows promise as an adjuvant therapy for patients with tumors that have a large population of high-ALDH1A3 CSCs.

#2507

Mithramycin depletes stem-like cells in lung adenocarcinoma via repression of mastermind-like 2 and mastermind-like 3.

Haobin Chen, Mary Zhang, Sichuan Xi, Yin Xiong, Julie Hong, David Schrump. _National Cancer Institute, Bethesda, MD_.

Background: Targeting cancer stem-like cells is a potential strategy to prevent development of drug resistance and tumor recurrence. Our group has previously demonstrated that mithramycin attenuates induction of side population (a phenotype of cancer stem-like cells) by cigarette smoke condensate, and modulates expression of multiple genes regulating stem-cell related pathways in lung cancer cells. The present study was performed to further examine the effects of mithramycin on stem cell signaling pathways, and ascertain if mithramycin can eliminate stem-like cells in lung cancer following exposure to conventional chemotherapeutic or targeted agents.

Methods: Stem-like cell populations in lung adenocarcinoma cell lines (H2228, H358 and HCC827) were detected based on stem cell markers, CD133 or ALDH, using flow cytometry or ALDEFLUORTM assay. Sphere-formation assays were performed using low attachment plates and stem-cell media (serum-free DMEM/F12 media supplemented with epidermal growth factor and fibroblast growth factor). qRT-PCR and western blot techniques were used to evaluate gene and protein expression levels, respectively. SiRNA transfection was performed to knockdown target gene(s) of interest.

Results: A small CD133+ or ALDH+ cell fraction was detected in untreated H2228, H358 and HCC827 cells, respectively. The chemotherapeutic agent cisplatin enriched CD133+ fraction in H2228 cells and ALDH+ fraction in H358 cells, while the EGFR inhibitor erlotinib remarkably increased ALDH+ fraction in HCC827 cells. Consistent with the notion that stem-like cells are present in these cancer lines, H2228 and H358 cells formed pulmospheroids when cultured in stem cell media in low attachment plates, and these pulmospheroids showed increased expression of stem-cell marker genes (Aldh1a1 and Prominin) as well as stemness-related genes (Oct4, Sox2 and Nanog). Relative to untreated controls, mithramycin decreased the cancer stem-like cell fractions in all three cell lines, and abolished their enrichment after cisplatin or erlotinib treatment. Analysis of stem-cell signaling pathways revealed that maintenance of cancer stem-cell like fractions in H358 and H2228 cells requires Notch signaling. Mithramycin decreased gene and protein expression of Mastermind-like 2 and 3 (MAML2 and MAML3), both of which are important transcription co-activators of canonical Notch signaling. Knockdown of MAML2 and MAML3 remarkably decreased the stem-like cell fraction in H358 cells, which recapitulates the phenotype of mithramycin treatment.

Conclusion: Mithramycin depletes cancer stem-like cells via repression of MAML2 and MAML3 and inhibition of Notch signaling. These findings support evaluation of mithramycin as a strategy to eliminate stem-like cells emerging in lung cancers after cisplatin or EGFR targeted therapy.

#2508

A red-shifted fluorescent substrate for aldehyde dehydrogenase, AldeRed 588-A, for labeling viable ALDH-positive cells.

Nick Asbrock,1 Vi Chu,1 Martin Pomper2. 1 _EMD Millipore, Temecula, CA;_ 2 _Johns Hopkins, MD_.

Normal and cancer stem cells can be isolated based upon the enzymatic activity of aldehyde dehydrogenase I (ALDH1), a detoxifying enzyme responsible for oxidation of hazardous aldehyde byproducts. ALDH1 has been used to isolate cancer stem cells from various human malignancies including bladder, breast, cervical, colon, head and neck, liver, lung, pancreas, prostate and ovary. Currently, the ALDEFLUOR assay, is the only commercially available reagent for ALDH detection in live cells. The substrate used in this assay primarily emits in the green region of the electromagnetic spectrum (512nm). For researchers with valuable cell and transgenic animal models in which the target gene of interest has been tagged with eGFP, ALDEFLUOR therefore cannot be used. Selection of cells positive for aldehyde dehydrogenase (ALDH) activity from a green fluorescent background is thus difficult with existing reagents. We now describe a red-shifted fluorescent substrate for ALDH, AldeRed 588-A, that provides additional flexibility for utilizing ALDH as a marker for stem cell and cancer stem cell isolation. The activity of AldeRed 588-A was compared with the ALDEFLUOR reagent and demonstrated similar ability to fractionate ALDHpos cells in a number of cell lines tested.

#2509

Determining the functional and mechanistic role of homeobox transcription factor Meox1 in breast cancer and breast cancer stem cells.

Mari Gasparyan, Joseph P. Burnett, Lichao Sun, Duxin Sun. _University of Michigan, Ann Arbor, MI_.

Many mechanisms have been attributed to de novo and acquired trastuzumab drug resistance in patients with HER2-enriched breast cancer; common mediated resistance is inactivation of the tumor suppressor gene PTEN seen in approximately 40% of patients. Evidence show the HER2 gene plays a significant role in regulating breast cancer stem cells through PTEN deletion; increasing evidence show these cancer stem cells may be responsible for drug resistance, cancer recurrence and metastasis. Studies demonstrate how PTEN deletion in HER2-overexpressing breast cancer activates an IL6 inflammatory feedback loop expanding the cancer stem cell population displaying EMT properties, conferring trastuzumab drug resistance and metastasis.

While new therapeutic approaches for targeting HER2-enriched tumors with PTEN deletion are in progress, further understanding of molecular systems involving EMT cancer stem cell expansion as well as of de novo and acquired trastuzumab drug resistance is important for improving therapeutic options. Our lab conducted RNA sequencing to further explore functional mechanisms behind this aforementioned molecular system and found the upregulation of mesenchyme homeobox 1 (Meox1) as a potential transcriptional factor regulator.

Meox1 is a non-cluster homeobox transcription factor expressed during embryonic development, critical in somite morphogenesis. Recent evidence has linked Meox1 as a critical PBX1 cofactor in ovarian cancer. As of yet, the role of Meox1 has never been studied in the context of breast cancer in correlation with cancer stem cells. Therefore specifically, the purpose of this study is to elucidate the molecular role of Meox1 in HER2-amplified PTEN-deleted trastuzumab-resistant breast cancer in order to provide functional and mechanistic insight involving EMT cancer stem cell expansion as well as of de novo and acquired trastuzumab drug resistance.

RT-qPCR analyses of Meox1 expression using different breast cancer cell lines show correlation between increase in Meox1 expression with PTEN deletion. In vitro studies show siRNA knockdown of Meox1 decreases cancer stem cell properties of both colony and mammosphere formation in the BT474 HER2-enriched breast cell line that is PTEN-deleted and long term trastuzumab treated. Furthermore, lentiviral stable overexpression of Meox1 in normal immortalized MCF10A breast cancer cell line shows an increase in mammosphere formation compared to control infections.

These preliminary results suggest Meox1 may be a critical transcription factor responsible for cancer stem cell progression in PTEN-deleted trastuzumab-resistant breast cancer. As such, further functional and mechanistic investigation of this homeobox transcription factor may provide additional understanding for de novo and acquired drug resistance and disease recurrence, potentially offering advanced therapeutic options.

#2510

Iron control is a novel therapeutic target of cancer stem cells.

Toshiaki Ohara,1 Takayuki Ninomiya,1 Kazuhiro Noma,1 Hajime Kashima,1 Yuki Katsura,1 Takuya Kato,1 Yasuko Tomono,2 Hiroshi Tazawa,1 Shunsuke Kagawa,1 Yasuhiro Shirakawa,1 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Shigei Medical Research Institute, Okayama, Japan_.

Background: Iron overload is known to cause cancer. Iron depletion treatment has been reported to have an anti-cancer effect. However, it is unclear whether iron depletion treatment is also effective in cancer stem cells (CSCs) or not. Recently, new CSCs model, miPS-LLCcm, was epigenetically established from murine induced pluripotent stem cells (miPS cells) in Okayama university (Chen at el. PLOS ONE 2012). In this model, Green Fluorescent Protein (GFP) was designed under Nanog promoter lesion. Therefore, GFP positive cells indicated the undifferentiated cancer progenitor cells. The tumor tissue derived from miPS-LLCcm cells involved glandular structure, angiogenesis, and cytokeratin positive lesion in immunostaining. We hypothesized that CSCs depended on iron in proliferation and differentiation. Thus, we also hypothesized iron depletion treatment might have a novel therapeutic effect.

Methods: We used miPS-LLCcm cells as CSCs, colon26 and 4T1 cells as differentiated cancer cells. Iron depleted condition was simulated by iron free medium with 1% fetal bovine serum. Transferrin (Holo) and iron chelators (Deferoxamine and Deferasirox) additional examinations were done to reveal the dependency of iron. Cell viability was examined by XTT assay 48 hours after administration of transferrin and iron chelators. Western blot analysis was done to examine the effect of iron depletion on iron related markers and stemness markers including TfR-1, DMT-1, Nanog, SOX2, c-Myc, Oct3/4, Klf-4, E-cadherin. Subcutaneous tumor model of miPS-LLCcm cells was used in vivo. Deferoxamine (30mg/kg/day) and deferasirox (30mg/kg/day) were administrated directly into the tumor. Tumors were collected for immunostaining.

Results: Transferrin strongly promoted cell proliferation of miPS-LLCcm in iron depleted condition. However, Transferrin did not promote proliferation of colon26 and 4T1 in the condition. Iron depletion by iron chelators suppressed miPS-LLCcm proliferation and the expression of stemness markers including Nanog, SOX2, c-Myc, Oct3/4, Klf-4, and E-cadherin in vitro study. Deferasirox more strongly suppressed the expression of stemness markers than deferoxamine. Iron depletion by iron chelators suppressed subcutaneous tumor growth. The average tumor volume of the control group was 1270.8 ± 411.2 mm3 while that of deferoxamine treatment group was 502.5 ± 207.5 mm3 (p<0.05) and deferasirox treatment group was 360.2 ± 148.8 mm3 (p<0.01) on day 19. The expression of stemness markers was also more strongly suppressed in the deferasirox treatment group than deferoxamine treatment group.

Conclusions: Iron is a key element of the proliferation and stemness in CSCs model. Iron control therapy including iron chelators is a novel therapeutic target of CSCs.

#2511

Association of ALDH1A1 with cisplatin resistance via modulation of NEK-2 in ovarian cancer stem-like cells.

Md. Hafiz Uddin, Yong Sang Song. _Seoul National University, Seoul, Republic of Korea_.

Cisplatin (CDDP) resistance is the foremost obstacle in the treatment of ovarian cancer patients. According to the cancer stem cell hypothesis, the recurrence and chemoresistance are presumed to be linked to cancer stem/progenitor cells. Here, we investigated the cancer stem cell like phenotypes and mechanism of chemoresistance in CDDP resistant ovarian cancer cells.

A well-established CDDP sensitive ovarian cancer cell line A2780 and its resistant clone A2780 Cp were used and we also developed a supra resistant clone (SKOV3 Cp) from a naturally CDDP resistant cell line SKOV3 by six shocks treatment. Sensitiveness to CDDP was evaluated by MTT assay and self renewal efficiency by sphere formation, noble agar and clonogenic assays. A few cancer hallmarks and stem-cell markers were characterized using flow cytometry. Molecular analysis was performed using quantitative PCR and western blotting. For the silencing of selected genes small interfering RNA (siRNA) based approaches was utilized.

Both CDDP resistant A2780 Cp and supra resistant SKOV3 Cp cell lines showed significantly (P < 0.05) higher self-renewal ability in sphere formation, noble agar and clonogenic assays than their parental counterparts. They also showed significant resistance to apoptosis and sub-G1 arrest upon CDDP treatment. Stem cell marker ALDH1 positivity rates were higher both in A2780 Cp and SKOV3 Cp cell lines, quantified by ALDEFLUORTM assay kit using flow cytometry. Hoechst 33342 dye effluxing side populations were increased about five folds in A2780 Cp cells and two folds in SKOV3 Cp cells compared to A2780 and SKOV3 cells respectively. Among major stemness related genes (OCT4, SOX2, NANOG, NESTIN, BMI1, KLF4 and ALDH1A1) quantitative PCR analysis showed significantly higher expression of ALDH1A1 and KLF4 genes in both resistant and supra resistant cells than their parental clones. Of two genes, silencing ALDH1A1 in A2780 and A2780 Cp cells using siRNA greatly reduced the stem cell population as detected by ALDEFLUORTM assay and sensitized cells to CDDP. Moreover, silencing of ALDH1A1 reduce the level of its downstream target NEK-2. We also observed the downregulation of ABC transporters (ABCB1, ABCG2 and ABCC1) by silencing either ALDH1A1 or NEK-2 gene.

Taken together, present study showed for the first time that stemness gene ALDH1A1 can be involved in CDDP resistance via the upregulation of NEK-2 in ovarian or solid cancer. 

### Stemness Properties of Neuronal and Pediatric Tumors and New Approaches

#2512

Bmi1 identifies treatment-refractory stem cells in human glioblastoma.

Parvez Vora, Maleeha Qazi, Chitra Venugopal, Minomi Subapanditha, Sujeivan Mahendram, Chirayu Chokshi, Mohini Singh, David Bakhshinyan, Nicole McFarlane, Sheila Singh. _McMaster University, Hamilton, Ontario, Canada_.

Glioblastoma (GBM) is an aggressive and fatal primary adult brain tumor. Even with surgery, chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth and patient relapse are inevitable. Brain tumor initiating cells (BTICs), a rare subset of GBM cells with stem cell properties, were shown to be both chemo- and radio-resistant. We hypothesize that these treatment-resistant BTICs cause tumor relapse and a subset of neural stem cell genes regulate BTIC self-renewal, driving GBM recurrence.

Using patient-derived primary GBM samples, we designed an in vitro model of tumor recurrence by treating cells with TMZ and radiation. We also adapted the existing treatment protocol for adults with primary GBM for in vivo treatment of immunocompromised mice engrafted with GFP+ GBM cells. Post-chemoradiotherapy, GFP+ cells were recovered from mouse brains and profiled for self-renewal, proliferation and mRNA expression of important stem cell genes. Using in vitro and in vivo gain-of-function/loss-of-function experiments, we investigated the regulatory functions of Bmi1 in primary neural stem & progenitor cells (NSPCs) and GBM tumor formation. To understand the consequences of Bmi1 dysregulation on target gene expression, we performed global RNA-seq profiling on NSPCs and GBMs.

GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy, suggesting the presence of a treatment-refractory BTICs. GFP+ cells extracted from chemoradiotherapy treated human tumor xenografts showed increased self-renewal and elevated BTIC marker expression. Although treated mice responded to therapy with decreased tumor size, we observed tumor relapse post-chemoradiotherapy with increased Bmi1 protein expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis in xenografted mice, highlighting a critical role for Bmi1 in tumor initiation and maintenance. Conversely, over-expressing Bmi1 in NSPCs induced stem cell properties in vitro, but failed to initiate tumor formation in vivo. Using high-throughput sequencing data, we generated a map of signaling pathways dysregulated in GBM that may lead to tumor recurrence.

Our data confirms the existence of a rare treatment-refractory BTICs population that escapes therapy, and drives tumor relapse and recurrence with enhanced self-renewal capacity. Our human BTIC in vitro assays and human-mouse BTIC xenograft model provide fundamental tools to characterize the functional relevance and of key stem cell self-renewal genes in GBM recurrence.

#2513

Using ACT1, a connexin43 blocker, against glioblastoma stem cells.

Pratik Kanabur, Zhi Sheng. _Virginia Tech Carilion School of Medicine, Roanoke, VA_.

Background: The prognosis for patients with glioblastoma (GBM) is dismal, with a median survival time of 14.6 months following aggressive treatment. Recent evidence suggests that the cells driving GBM malignancy and recurrence are small populations of glioma stem cells (GSCs) which have been shown to be resistant to therapeutics that target the entire tumor.

Objective: To isolate GSCs from surgically resected tumor, determine capability of self-renewal and expression of stem cell markers, and gauge the therapeutic response to temozolamide and ACT1, an inhibitor of connexin43.

Methods: Surgically resected tumor was digested and individual tumor cells were isolated and incubated in serum-free stem cell media. Spheres were subject to the following assays for assessing their stem cell identity: (1) Serial dilution assay to assess self-renewal; (2) Western blot of stem cell markers (NESTIN, NOTCH1, and CD133) and the astrocyte marker GFAP. Verified glioblastoma stem cells were tested for their responsiveness to ACT1 and/or temozolomide using the sphere formation assay.

Results: Some glioblastoma stem cells had strong capability to form spheres (as low as 4 cells). Glioblastoma stem cells expressed a high level of NESTIN and NOTCH1, while levels of GFAP were low. Two stem cell lines VTC 036 and VTC 064 were selected for drug treatment. The sphere formation capability of these stem cells was significantly inhibited by ACT1 and temozolomide, but not each treatment alone.

Conclusion: Sphere formation is an effective way to isolate and enrich glioblastoma stem cells. The combinational treatment of ACT1 and temozolomide inhibits glioblastoma stem cell sphere formation.

#2514

Second generation HDAC inhibitor, Quisinostat reinstates RD3 in neuroblastoma CSCs and promotes stem cell differentiation.

DINESH BABU SOMASUNDARAM, Natarajan Aravindan. _University of Oklahoma Health Sciences, Oklahoma City, OK_.

Recently, we characterized the adaptive stemness and extreme plasticity of neuroblastoma (NB) cancer stem cells (CSCs). Further, new to science, we defined the loss of retinal degeneration protein 3 (RD3) in high-risk NB and identified its novel tumor evolution stabilization function. Moreover our studies identified definitive contribution of HDACs in the evolution of progressive NB. Herein, we investigated the potential of pyrimidyl-hydroxamic acid HDAC inhibitor, Quisinostat in restoring RD3 and NB-CSCs differentiation. Well characterized CD133+-CD34+ human NB CSCs exposed to increasing concentrations (2.5, 5, 10, 100, 200nM, 1, 2, 4, 8μM) of quisinostat were examined for inhibition of HDACs (HDACs 1-11, QPCR analysis), transcriptional (QPCR) and translational (immunoblotting) restoration of RD3, CSCs cell viability (automated trypan exclusion assay) differentiation (real-time live cell imaging) and formation of organized tumorospheres (tumorosphere formation assay). Quisinostat inflicted complete inhibition of NB-CSCs cell viability at 100nM concentration and beyond. QPCR analysis revealed a dose-dependent inhibition of HDACs in NB CSCs with quisinostat. We observed a significant transcriptional/translational restoration of RD3 selectively at 100nM and above of quisinostat treatment. Live-cell imaging demonstrated a dose-dependent -loss of stemness behavior and -increased differentiation of NB CSCs with quisinostat treatment. Tumorosphere formation assay demonstrated complete inhibition of organized tumorospheres at/after 100nM treatment. These results imply that Quisinostat restores RD3 and promotes NB-CSCs differentiation. More importantly, selective dose-dependent specificity of HDAC inhibition by Quisinostatnt and, restoration of RD3 and regulation of stemness physiognomies at/above 100nM concentrations identifies that HDAC 7 could serve as a critical player in this setting. Taken together, these results for the first time identify the potential of quisinostat in the regulation of NB evolution and with further studies may serve as a potential drug deliverable in the treatment and cure of high-risk NB.

Acknowledgements: Stephenson Cancer Center - Experimental Therapeutics Program, NIH COBRE 1P20GM103639-01.

#2515

Pharmacological WNT-inhibition acts synergistically with chemo- and radiotherapy by overcoming treatment-resistance in glioma stem cells.

Abigail K. Suwala, Ulf D. Kahlert, Jaroslaw Maciaczyk. _Department of Neurosurgery, Düsseldorf, Germany_.

Objective: Glioma Stem Cells (GSCs) are considered to be responsible for Glioblastoma's (GBM's) dismal prognosis. WNT signaling is one of the major players in GSCs that promotes their radio- and chemoresistance. In this study we tested whether LGK974, an inhibitor of both canonical and non-canonical WNT signaling, might sensitize GBM cells to chemotherapeutics and irradiation and determined possible mechanisms causing treatment-resistance in GBM.

Methods: At first we analyzed MGMT promoter methylation status of several GBM cell lines in order to define cells resistant to the standard chemotherapeutic drug Temozolomide (TMZ). For further investigations we chose two cell lines resistant or sensitive to TMZ, respectively. IC50 concentrations of TMZ and LGK974 as well as IC50 dose of γ-irradiation were tested via Titer Blue cell viability assay. Effectiveness of different concentration of each single therapy and its combination with LGK974 was determined to create a drug-response-curve due to the computerized simulation of synergism and antagonism in drug combination studies (Chou, 2006). A reporter assay for canonical WNT/β-catenin signaling was used to quantify the effect of TMZ and irradiation alone and in combination with LGK974 on the canonical and non-canonical WNT pathway. Western Blot and qPCR were applied to analyze the effects of combinatory therapy on protein and gene expression concerning WNT pathway activation.

Results: We observed a significant synergy of combined irradiation/TMZ with LGK974 in all cell lines. There was no difference in effectiveness between cell lines resistant or sensitive to TMZ. Interestingly, following TMZ treatment or irradiation cells showed slightly decreased canonical WNT pathway activity. Furthermore, non-canonical target genes and receptors were upregulated under chemo- and radiotherapy, suggesting a reciprocal activation of the non-canonical branch of WNT signaling pathway. This phenomenon might possibly be responsible for the development of resistance against standard chemo- and radiotherapy.

Conclusions: We propose that GSCs undergo a shift from canonical to non-canonical WNT signaling to become treatment-resistant under standard chemo- and radiotherapy. Treatment with LGK974 results in therapeutic synergy at least in part via suppressing the therapy-induced activation of non-canonical WNT signaling. Our results confirm the potential benefit of combining TMZ or irradiation with the WNT inhibitor LGK974 to overcome therapy-resistance in GBM.

#2516

TRIM8 modulates stem-like cells in glioblastoma.

Changming Zhang, Mukherjee Subhas, Tucker-Burden Carol, Monica Chau, Jun Kong, Daniel Brat. _Winship Cancer Institution, Atlanta, GA_.

Glioblastoma stem-like cells (GSC) contribute to rapid growth and clinical recurrence of these fatal tumors and are highly resistant to therapy. STAT3 is known to contribute to maintenance and proliferation of GSC. Here we report a novel protein known as Tri-partite Motif containing protein 8 (TRIM8) that modifies tumor growth through STAT3. TRIM8, previously referred to as "glioblastoma expressed ring finger protein" is expressed at equal levels in glioblastoma and normal brain samples, despite showing hemizygous deletion in 87% of cases in TCGA data. To uncover mechanisms, we knocked down TRIM8 in glioblastoma-derived neurospheres using sh-RNA and found that this reduced the levels of phosphorylated STAT3 (p-STAT3), C-MYC and SOX2. Overexpression of TRIM8 resulted in higher expression of p-STAT3, C-MYC, SOX2 and CD133. We also found that specifically blocking STAT3 in neurospheres by pharmacological inhibition reduced the expression of TRIM8. Conversely, upregulation of STAT3 induced by IL-6 results in increased expression of TRIM8. Together, our results indicate that TRIM8 could potentially regulate GSC by forming a positive feedback loop with STAT3.

#2517

Ewing sarcoma associated long non-coding RNA determines neural cell fate of the tumors.

Sheetal A. Mitra,1 Anirban P. Mitra,2 Jonathan D. Buckley,2 Timothy J. Triche1. 1 _Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _University of Southern California, Los Angeles, CA_.

BACKGROUND: Ewing sarcoma is a highly invasive pediatric bone and soft tissue malignancy with a 20% 5-year survival rate for patients with metastatic disease. 85% of Ewing sarcomas express a chimeric oncogene, mainly EWS-FLI1, which drives the gene expression signature in these tumors. The exact cell of origin for these primitive small round cell tumors is unknown. These undifferentiated tumors express some neuroectodermal, mesenchymal, and epithelial markers. Recently published work suggests that these tumors may have risen either from neural crest or mesenchymal stem cells. These genomic and phenotypic features may be responsible for the aggressive nature of this disease and understanding the tumor biology may help better treatment options for this metastatic cancer.

RESULTS: Our study on expression analysis of primary Ewing tumors using Affymetrix's Human Exon arrays identified a highly expressed novel long intergenic non-coding RNA (lincRNA), FEZF1-AS1 in these tumors. Its DNA sequence has no homology in most vertebrates. RNA-mediated knockdown of the lincRNA in Ewing cell lines decreased expression of some of the neural lineage genes as well as genes associated with cell adhesion and migration. In vitro matrigel assays detected a decrease in cell invasion capability of the lincRNA-knockdown cells. In the in vivo studies, the lincRNA-overexpressed cells had better engraftment and tumor growth in the liver than the normal controls. On attempt to differentiate the lincRNA-knockdown Ewing cell lines, we noted that a decrease in the lincRNA expression led to a decrease in the expression of neuroectodermal markers after a week of differentiation when compared to the control cells. We further detected temporal expression of this lincRNA during neuronal differentiation from embryonic stem cells.

CONCLUSIONS: Our study identified a unique lincRNA in Ewing sarcoma that may be partly responsible for inducing expression of the neural lineage genes in these tumors as its reduced expression impaired differentiation in the Ewing cell lines. Its prometastatic behavior helps the aggressive nature of these tumors. Its expression during neural differentiation along with its involvement in cell migration suggests that FEZF1-AS1 may be responsible for imparting neural stem cell like-characteristics to Ewing sarcoma.

#2518

Crosstalk between stem and non-stem cells in glioblastoma promotes radioresistance in a CD109-dependent manner.

Mutsuko Minata, Ichiro Nakano, Sunghak Kim, Marat PavliuKov, Jia Wang, Svetlana Komarova, Jun Wang. _University of Alabama at Birmingham, Birmingham, AL_.

Glioblastoma (GBM) is a lethal brain tumor that contains cancer stem cells that are bestowed with tumor initiating and treatment resistant properties. Patient-derived glioma stem-like cells (GSCs) that resemble the clinically relevant MES subtype harbor more aggressive and therapy resistant properties. Here we report that exposure of GSCs to ionizing radiation (IR) provokes inter-cellular signals from therapy-induced senescent non-stem cancer cells to stimulate compensatory growth in cancer stem cells resulting in persistent transcriptomic and phenotypic shift toward a more MES subtype. This IR-induced gain of MES features in GSCs is accompanied by conversion of CD133+ stem cell populations to CD133- /CD109+ cells. In both de novo and IR-induced MES GSCs, CD109+, but not CD109- populations are highly tumorigenic and multipotent in vivo. Inhibition of CD109 attenuates clonogenicity and radioresistance in MES GSCs GBM. Clinically, CD109 expression is significantly associated with the MES GBMs and poorer prognosis of GBM patients. Taken together, our data imply that radiation of GSCs induces direct transformation of cancer stem cells to the MES subtype accompanied by a loss of the widely adopted stem cell marker CD133 and a gain of a novel, tumor propagating, and clinically relevant MES stem cell marker, CD109.

#2519

A novel role for brachyury as a key regulator of sonic hedgehog signaling (Shh) and maintenance of stemness in gliomas.

Divya Kesanakurti, Alessandro Canella, Jihong Xu, Vinay K. Puduvalli. _Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Glioblastomas are the most common and aggressive form of adult primary brain tumors and are associated with a dismal prognosis. Accumulating evidences suggest that a subset of therapy-resistant cancer stem cells are responsible for recurrence in tumors targeting which may help in gliomas. We identified a significant overexpression of Brachyury (T-box transcription factor) in correlation with glioma histological grade when compared to non-tumor brain controls, with the highest expression being in grade IV gliomas. We also observed increased expression of Brachyury in several glioma cell lines and stem cells (GSCs) when compared to normal human astrocytes. Serum-induced differentiation of GSCs resulted in loss of Brachyury expression along with decreased stem-cell marker levels in these cells, which prompted us to study the potential role of Brachyury in glioma stemness and to delineate the underlying signaling mechanisms. Specific shRNA-mediated knockdown of Brachyury significantly decreased proliferation, migration and invasion in glioma cell lines. Brachyury suppression led to inhibition of sphere formation of GSCs and decreased stem cells marker expression including CD133, nestin and Nanog. Brachyury knockdown also inhibited the activation of sonic hedgehog (Shh) signaling and decreased Gli1 levels in these cells. On the other hand, stable overexpression of Brachyury resulted in the activation of Shh pathway and elevated stem cell marker expression in GSCs. Based on these in vitro findings, our future studies will be focused on determining the role of Brachyury in orthotopic tumor growth in vivo. In summary, our data reveals a novel role for Brachyury in the regulation of tumor invasiveness and stemness, and indicates a potential for therapeutic targeting of Brachyury in patients with gliomas.

#2520

IRS-1/LC3 nuclear structures and glioblastoma drug resistance.

Adam Lassak,1 Dorota Wyczechowska,1 Anna Wilk,2 Adriana Zapata,1 Mathew Dean,1 Luis DelValle,1 Jann N. Sarkaria,3 Augusto Ochoas,1 Francesca Peruzzi,1 Krzysztof Reiss1. 1 _Louisiana State University, New Orleans, LA;_ 2 _University of South Alabama, Cancer Institute, Mobile, AL;_ 3 _Mayo Clinic, Department of Radiation Oncology, Rochester, MN_.

Drug resistance and frequent tumor relapses are the major obstacles in glioblastoma therapy, and recurrent tumors are practically incurable. We previously reported that insulin receptor substrate 1 (IRS-1), which is a typical signaling molecule for insulin and insulin-like growth factor 1 receptors, can translocate to nucleus, and that nuclear IRS-1 (nIRS-1) was found in different tumor cells, including glioblastomas. To unravel its function, we employed glioblastoma cell culture, animal models, and clinical samples. Using confocal imaging, molecular cloning, subcellular fractionation, mass spectrometry, gene expression analysis, and different approaches to verify protein-protein interactions, we demonstrate for the first time that nIRS-1 can form complex nuclear structures in a restricted number of cancer cells in glioblastoma biopsies and in intracranial glioblastoma xenografts. We also demonstrated the formation of highly organized ring-like structures in several cell lines, following ectopic expression of IRS-1 cloned in frame with nuclear localization signal (NLS-IRS-1). In these nuclear structures IRS-1 localizes at the periphery, and the core of the structure harbors a key autophagy protein, LC3; however, other autophagy proteins or biological membranes were not detected. In living cells expressing NLS-IRS-1-GFP fusion protein, IRS-1/LC3 structures are highly dynamic. They rapidly exchange IRS-1 molecules with nucleoplasm and interact with other nuclear complexes including BMI1-positive Polycomb bodies, PML bodies and Cajal bodies. Importantly, clones and mixed populations of cells expressing the NLS-IRS-1 and capable of forming the IRS-1/LC3 ring-like structures undergo extensive remodeling of gene expression, which suggests a transition to stem-like phenotype and associated resistance to several different anticancer drugs, including temozolomide. This is the first demonstration of IRS-1/LC3 nuclear complexes, which are highly dynamic and may play a role in epigenetic remodeling of glioblastoma cells towards stemness. Further studies are required to determine detailed molecular composition and to explain how these new nuclear structures function.

#2521

STAT3 is a central regulator of proliferation, invasion and metabolism in GBM tumor initiating cells.

Ian Restall, H. Artee Luchman, Samuel Weiss. _University of Calgary, Calgary, Alberta, Canada_.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Currently, treatment for GBM involves surgical resection, chemotherapy, and radiation, resulting in a median survival of 15 months. This poor outcome highlights the need for improved therapeutic approaches to treat GBM patients. The signal transducer and activator of transcription 3 (STAT3) pathway is abnormally active in GBM, primarily in the mesenchymal subtype of GBM. STAT3 regulates various cellular processes including: proliferation, invasion and resistance to therapy. The Weiss lab has established a large collection of brain tumor initiating cell (BTIC) lines derived from GBM patients. These GBM BTIC lines are used to model the subpopulation of cells that are predicted to be the source of resistance and recurrence following treatment. Here, we are focused on the role of STAT3 as a central regulator of proliferation, invasion and metabolism in GBM BTIC lines. We show that activation of the STAT3 pathway using the ligand oncostatin M (OSM) or by stably expressing a constitutively active STAT3 mutant (STAT3C) in GBM BTIC lines leads to an increase in proliferation. STAT3 is known to regulate the expression of multiple matrix metalloproteinases (MMPs) that promote invasion. Using time-lapse imaging of 3D sphere invasion into matrigel, we observed that JAK2 (an activator of STAT3) and direct STAT3 inhibitors decrease invasion of GBM BTICs. Our analysis of gene expression data available from The Cancer Genome Atlas (TCGA) demonstrates that a glutamine metabolism gene expression signature correlates inversely with a STAT3 gene expression signature in GBM patient samples. Conversely, similar analysis reveals that a high glycolysis gene expression signature correlates with a high STAT3 gene expression signature. We hypothesize that targeting the STAT3 pathway will decrease proliferation and invasion while simultaneously sensitizing GBM BTIC lines to inhibition of glutamine metabolism. Glutaminase (GLS) is the first enzyme to act on glutamine, converting glutamine to glutamate, which is then further processed to α-ketoglutarate (αKG). αKG is important for the production of biosynthetic macromolecules that are essential for cell growth. Chemical inhibition of GLS decreases cell growth in culture with varying levels of sensitivity across multiple GBM BTIC lines. Interestingly, activation of the STAT3 pathway using OSM de-sensitizes GBM BTIC lines to GLS inhibition. Furthermore, chemical inhibition of JAK2 sensitizes a subset of GBM BTIC lines to GLS inhibition resulting in a further decrease in cell growth. Overall, we show that STAT3 is a central hub in GBM BTIC lines that modulates proliferation, invasion, and sensitivity to GLS inhibition. Currently, we are continuing these studies in vivo using GBM BTIC orthotopic xenografts in mice.

#2522

S100A4 is a critical regulator of stemness and the mesenchymal phenotype in GBMs.

KIn-Hoe Chow,1 Hee Jung Park,1 Joshy George,2 Andrew D. Gallup,1 Eric Neilson,3 Kyuson Yun1. 1 _The Jackson Laboratory Cancer Center, Bar Harbor, ME;_ 2 _The Jackson Laboratory Genomic Medicine, Farmington, CT;_ 3 _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Glioblastoma (GBM) is one of the most incurable human cancers, and the mesenchymal subtype of GBMs has the worst prognosis. The mesenchymal phenotype and glioma stem cells (GSCs) are associated with tumor recurrence and therapy resistance. While growing evidence suggests that tumor microenvironment modulates stemness and the mesenchymal phenotype, little is known about how these phenotypes are coordinately regulated. Here we report that S100A4 is a critical regulator of stemness and the mesenchymal phenotype in GBMs. First, we show that S100a4 is a novel marker and a regulator of GBM TICs. Using genetically engineered mice and patient derived GBM tumorspheres, we show that S100a4 expression is enriched in TICs and that S100a4 function is critical for self-renewal of these cells. Furthermore, we demonstrate that selective ablation of S100a4+ cells in vivo is sufficient to block tumor growth. Second, we show that S100A4 regulates expression of EMT regulators, SNAIL2 and ZEB1, in addition to master transcriptional regulators of the mesenchymal signature genes (CEBPB/D, RUNX1, FOSL2, and BHLHE40), suggesting that S100A4 is an upstream (master) regulator of mesenchymal transition in GBMs. Third, we show that acidosis, a common environmental stress in malignant tumors, induces S100A4 and epithelial-mesenchymal transition (EMT) regulator expression. Importantly, S100A4 is required for acidosis-induced EMT and mesenchymal transition. Consistently, S100A4 expression is strongly associated with the mesenchymal subtype of GBMs, and S100A4 is an independent prognostic indicator for the mesenchymal subtype GBM patient survival. In summary, we show that S100A4 is a critical node in the molecular network that controls glioma stem cells and the mesenchymal phenotype, and propose that targeting S100A4 may simultaneously suppress mesenchymal transition and stemness of glioma cells, particularly in stressful tumor environments.

#2523

Transcriptional profiling of glioblastoma stem-like cells reveals enrichment of ion channels with functional implications for malignancy.

Julia Pollak,1 Karan G. Rai,1 Patrick J. Paddison,2 Robert C. Rostomily,3 Jan-Marino Ramirez1. 1 _Seattle Children's Research Institute, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA_.

Glioblastoma Multiforme (GBM) is the most prevalent and aggressive form of cancer in the adult central nervous system. Ion channels are increasingly being linked to cancer progression through the regulation of cell proliferation and migration. However, the role of ion channels in GBM tumor formation and progression is not well understood. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence, suggesting that ion channel expression may be perturbed in this population. Here, we used RNA-sequencing to assess the expression patterns of ion channels and transporters and identify uniquely enriched ion channels in GSCs. Twenty-two patient-derived GSC samples were expression profiled along with human neural stem cell (NSC) and astrocyte control cell populations. Differential expression analysis revealed a set of ion channels highly enriched in GSCs compared with controls. Real-time PCR analysis confirmed the increased expression of ion channels of interest across selected GSC lines compared to NSCs. The expression pattern of ion channel candidates was also more likely to be associated with distinct GBM molecular subtypes. Next, an integrative approach was taken to further identify ion channels unique to GSCs; the abovementioned findings were compared to results from transcriptome analyses of GBM bulk tumor cells (The Cancer Genome Atlas) and region-specific GBM cells (Ivy Glioblastoma Atlas Project). Ion channels that were identified in these analyses were associated with altered clinical outcomes. The functional implications of these expression changes were further assessed with targeted drug screening of GSCs and NSCs. Pharmacological antagonists of GSC-enriched ion channels suppressed stem cell viability. These antagonists similarly hindered BrdU incorporation of dividing GSCs, suggesting that these channels play a role in stem cell proliferative capacity. Finally, calcium imaging was used to test the real-time functional responses of GSCs to channel blockers, and these outcomes will be discussed. Collectively, these findings suggest the presence of ion channels that uniquely identify GSCs from other neural cell types and influence GSC proliferation, a hallmark of GBM tumor recurrence.

#2524

STAT3 is a key regulator of an "EMT-like" process mediated by Slug in GBM.

Charles Chesnelong, H. Artee Luchman, J. Gregory Cairncross, Samuel Weiss. _University of Calgary, Calgary, Alberta, Canada_.

Glioblastoma Multiforme (GBM) is the most aggressive subtype of adult brain tumor with a median survival of 15 months. Despite a combination of maximal safe resection, radiation and chemotherapy, GBM invariably recurs, highlighting the need to better delineate the basis of recurrent disease and develop novel, more targeted and effective therapies. The Signal Transducer and Activator of Transcription 3 (STAT3) has been implicated in a proneural to mesenchymal shift associated with the emergence of a more aggressive, more resistant GBM phenotype at recurrence. Brain Tumor Initiating Cells (BTICs), defined by the key features of self-renewal, multipotency and tumorigenic potential, are integral players of recurrence post-treatment and represent a "reservoir of disease" that needs to be specifically targeted if GBM outcome is to be improved. Analysis of GBM patient transcriptomic data from The Cancer Genome Atlas (TCGA) shows that an Epithelial to Mesenchymal Transition (EMT) gene signature is particularly enriched in the mesenchymal subtype, tightly correlates with STAT3 activity and is associated with shorter survival. The key EMT transcription factor Slug is also highly expressed in mesenchymal samples and associated with poorer prognosis. Interestingly, we found stronger expression of EMT transcription factors Snail, Slug and Twist in faster proliferating, more aggressive BTIC lines. Noteworthy, Slug was more highly expressed in both BTICs and parental tumors compared to Snail and Twist and positively correlated with pSTAT3. We also found that Slug expression correlates with faster growth in vitro and shorter survival in orthotopic xenografts. Conversely, higher E-cadherin expression correlates with slower growth and longer survival of xenografted mice. While Slug is not a known STAT3 target gene (unlike Twist and Snail), we show that Slug expression is decreased after pharmacological inhibition of STAT3 signaling in BTICs. In contrast, activation of the STAT3 pathway via growth factor/cytokine treatment (EGF, OSM), as well as expression of a constitutively active form of STAT3 promotes Slug expression. We have identified a potential STAT3 consensus binding site in the Slug promoter and preliminary Chromatin Immuno Precipitation (ChIP) experiments suggest that Slug is a novel direct transcriptional target of STAT3. Over-expression of Slug in BTIC lines triggered down-regulation of E-cadherin and resulted in increased Cyclin D1 and pRB protein levels. However, we found that Cyclin D1 RNA levels remain unchanged suggesting that overexpression of Slug leads to the post-transcriptional stabilization of Cyclin D1 potentially via repression of UbcH5C. To conclude, STAT3 is a key regulator of an EMT-like process in GBM BTICs, mediated at least in part by Slug. Our results suggest that STAT3 and the key regulator Slug may be involved in the promotion of a more aggressive GBM phenotype and represent interesting therapeutic targets in GBM.

#2525

Sema3C promotes glioma stem cell survival and radioresistance.

Jianghong Man, Jocelyn Shoemake, Qiulian Wu, Jennifer Yu. _Cleveland Clinic, Cleveland, OH_.

Introduction: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined.

Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed.

Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NF-κB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of Plexin receptors were more resistant to radiation, and Sema3C and Plexin receptors were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance.

Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index.

#2526

Regulation of angiogenic factors in glioblastoma progression.

Carolina Larrain, Umadevi V. Wesley, Paul Clark, John S. Kuo, Robert Dempsey. _University of Wisconsin - Madison, Madison, WI_.

Background & Purpose: Glioblastoma is the most malignant primary brain tumor in humans and has a very low survival rate. The aggressiveness of this cancer can be attributed to the presence of glioblastoma stem cells (GSC), which are highly invasive and therapy resistant. Increased aggressiveness of GSCs is attributed to the release of angiogenic factors, which promotes the formation of new blood vessels that enhances tumor cell survival and proliferation. The neovascularization also provides a scaffold for tumor cells to migrate, enabling metastasis. Therefore, identifying the altered expression of angiogenic factors is of clinical importance. In this study, we examined the changes in the expression of pro-angiogenic factors in GSCs as compared to the original tumor from which they were derived. We also determined if differentiation of GSC towards a less invasive phenotype decreased the levels of these pro-angiogenic factors. Methods: GBM tumor cells were grown in complete media containing 10% Fetal Calf Serum (FCS) and GSCs were grown in media containing B27, heparin, EGF, and FGF. GSCs were differentiated by growing them in media containing 10% FCS and B27 with vitamin A, for about one week. We examined the expression of a panel of pro-angiogenic factors in GSCs (33-GSC) and in the original tumor cells (33-T) from which GSCs were derived. We then examined if differentiation of 33-GSC decreased the levels of these pro-angiogenic factors. Changes in protein levels were evaluated by immunofluorescence staining. The mRNA levels were quantitated using real time Q-PCR. Results: Immunofluorescence staining showed that the GSCs express higher levels of pro-angiogenic factors examined (11 out of 16). GSCs were successfully differentiated into astrocytes as indicated by morphology and GFAP expression. The differentiation of GSCs into astrocytes was sufficient to cause decreased expression of VEGF and c-MYC. Our data showed 27 fold increase in VEGF mRNA levels, 63 fold increase in MMP2 mRNA levels, 11 fold increase in MMP9 mRNA levels, and 2 fold increase in c-MYC mRNA levels in GSCs compared to the tumor line, indicating the regulation of these pro-angiogenic factors at transcriptional level. Conclusions: Increased expression of pro-angiogenic factors in GSCs may render these cells to be more aggressive and invasive, and suggests that differential expression of angiogenic factors in GSC cells may play an important role in the GBM progression, and therapy resistance.

#2527

MDA-9/Syntenin (SDCBP) promotes self renewal and malignancy in cancer stem cells.

Sarmistha Talukdar, Swadesh Das, Anjan K. Pradhan, Luni Emdad, Xue-Ning Shen, Jolene J. Windle, Devanand Sarkar, Paul B. Fisher. _Virginia Commonwealth University, Richmond, VA_.

Cancer metastasis is a complex process that culminates in the majority of patient deaths. A key component of the cancer paradigm involves cancer stem cells (CSCs), the primary mediators of cancer progression, resistance to therapy and recurrence of cancer after remission/latency. Although the clinical significance of CSCs is well accepted, the key upstream regulatory mechanisms that define these unique subsets of cancer cells requires further clarification. Previous studies demonstrate that MDA-9/Syntenin or syndecan binding protein (SDCBP) levels are elevated in large spectrum of tumors, including melanoma, breast carcinoma, gastric carcinoma and glioblastoma multiforme (GBM), versus normal cellular counterparts. We now uncover a unique function of MDA-9 as a facilitator of cancer stemness and survival.

Expression profiles of mda-9, Nanog, Oct4, Sox2 and c-myc were studied in different clinical samples and cell lines for correlations. Cells were sorted by FACS into stem and non-stem populations and the same genes were assayed for correlative expression. Stemness gene expression array profile analysis was performed on control and mda-9 silenced CSCs. Genetic (gain-of-function, loss-of-function) approaches were used to modify mda-9 expression in normal human astrocytes and prostate cells, non-stem cancer cells (NSCCs) and CSCs from breast prostate and GBM. Recovery-of-function approaches were also applied to elucidate the pathways responsible for MDA-9-mediated regulation of CSCs. The impact of MDA-9 on CSC-mediated tumorigenicity and progression was evaluated in vivo by intracranial glioma and subcutaneous xenograft mice models. Apoptosis was assayed by caspase activation and Annexin V staining.

A positive and statistically significant correlation was observed between mda-9 and stemness regulating genes (Nanog, Oct4, Sox2 and c-myc), in 50 clinical samples and 3 cell culture systems, including breast, prostate and GBM. CSCs expressed elevated levels of mda-9 vs. non-stem cancer cells or normal stem cells from a total of 11 cell lines including breast, prostate and GBM and two clinical GBM samples. Over-expression of mda-9 in NSCCs increased populations of CSCs as well as expression of stemness regulating genes. Loss of MDA-9 affected 84 genes linked to CSC regulation. Control intracranial glioma xenograft mouse models had large tumors with spongioblastic morphology typical of human gliomas, whereas the loss of mda-9 caused a large decrease in tumor size. Mechanistically, MDA-9 regulated multiple stemness genes and knockdown of MDA-9 caused a loss of stemness and tumorigenicity with initiation of apoptosis.

Our data uncover a previously unidentified relationship between MDA-9 and CSCs. CSCs appear more dependent on MDA-9 expression than normal stem cells. MDA-9 regulates CSCs on multiple levels of stemness and survival, reinforcing the relevance of this gene as a potential therapeutic target.

#2528

**Mechanism and role of** SOX2 **repression in human germline specification.**

Kushwaha Ritu, Nirmala Jagadish, George Bosl, Raju Chaganti. _MSKCC, New York, NY_.

The core pluripotency regulatory master transcription factor SOX2 is repressed in human primordial germ cells (PGCs) and in the seminoma (SEM) subset of human germ cell tumors (GCTs), which arises by transformation of PGCs. Neither the mechanism of this repression not its significance to GC and GCT development have so far been clarified. Here we show that SOX2 repression in TCam-2 SEM cells is mediated by the presence of the Polycomb repressive complex (PcG) and by the H3K27me3 chromatin mark at the SOX2 transcription start site. Moreover, repression can be abrogated by recruitment of the H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling. SOX2-activated TCam-2 cells express neuronal and other lineage genes, consistent with the gene's function as a neurectodermal effector. Based on the recent discovery that SOX17 initiates human PGC specification, with its downstream target PRDM1 acting to suppress mesendodermal genes, we propose that SOX2 repression is required for PGC specification to suppress neuroectodermal genes. Overt pluripotency is initiated in latently pluripotent transformed PGCs presenting as the SOX2 upregulated embryonal carcinoma (EC) subset of GCTs; by gene expression profiling analysis we characterize the functional pathways that distinguish PGC-like unipotent SEM from blastocyst-like pluripotent EC.

#2529

Expression of ABCF1 with stem cell markers CD133/GEP/beta-catenin in fetal liver and liver cancer cells.

Sze Wai Fung,1 Phyllis Fung Yi Cheung,2 Chi Wai Yip,1 George Sai Wah Tsao,1 Siu Tim Cheung2. 1 _The University of Hong Kong, Pokfulam, Hong Kong;_ 2 _The Chinese University of Hong Kong, Shatin, Hong Kong_.

Understanding stem cell biology in relation to cancer is of growing importance with the cancer stem cells (CSCs) notion and recurring deregulated stem cell pathways reported in cancers. ATP-binding cassette (ABC) transporter ABCB1 (also named P-glycoprotein and MDR1) has shown to be CSC marker, mediate multidrug-resistance in model systems though with controversial clinical relevance. We have systemically examined the expression profiles of ABC genes in hepatocellular carcinoma (HCC) clinical samples. ABCF1 was significantly over-expressed in HCCs compared to the adjacent non-tumor liver tissues (P<0.001) and associated with recurrence-free survival after curative partial hepatectomy (log rank, P=0.010). Recurrences have conventionally been attributed to aggressive cancer features and, recently, CSC features, as CSCs in small load could grow to tumor bulk. We therefore further explored the significance of ABCF1 in both fetal liver and liver cancer cells in respect to stem cell signaling. Expression of ABCF1 in fetal, neonatal and adult mouse livers were examined by immunohistochemistry, western blot and flow cytometry. Consistently, elevated ABCF1 expression was demonstrated in early embryonic stages [embryonic day 13 (E13): 42.4%; E15: 24.6%] but rarely in neonates and adult livers [1.5% and 1.2%, respectively]. Also, ABCF1 co-stained with stem cell-related marker β-catenin and hepatic CSC markers CD133 and GEP in fetal hepatocytes and in HCC cells (Table 1). ABCF1 suppression by siRNA enhanced chemo-sensitivity of the HCC cells Hep3B compared to control cells on cisplatin (cell apoptosis 7.5% vs 1.7%, P<0.001) and 5-Fluorouracil (11.7% vs 4.7%, P<0.001). Subcellular fractionation analysis on HCC cells showed that ABCF1 protein was present in both the cytoplasmic and membrane fractions. The current study highlighted the functional significance of ABCF1 in liver development and liver cancer as stem cell-related molecule regulating chemo-resistance.

Table 1.Co-expression of ABCF1 and hepatic stem cell markers in mouse fetal livers and HCC cells

---

|

Fetal liver cells

(E15) | Liver cancer cells

(Hep3B)

ABCF1+ | 24.6% | 34.5%

CD133+

ABCF1+ CD133+ | 16.3%

20.9% | 88.8%

34.3%

GEP+

ABCF1+ GEP+ | 45.7%

24.0% | 65.7%

29.7%

β-catenin+

ABCF1+ β-catenin+ | 40.6%

13.5% | 20.9%

17.5%

#2530

Live cell detection of intracellular biomarkers maintains cancer stem cells for further experimentation.

Don Weldon,1 Yuko Williams,1 Amish Patel,1 Steven McClellan2. 1 _EMD Millipore, Temecula, CA;_ 2 _Mitchell Cancer Institute, Mobile, AL_.

Cancer stem cells are widely studied as a potential therapeutic target in cancer research. Their expression of cell surface biomarkers such as EpCAM, CD44, and CD133 have been well characterized as a method for identification and enrichment from heterogeneous populations. Additional biomarkers with more functional relevance to self-renewal such as the transcription factors Sox-2 and Nanog, are intracellular and traditionally require fixation and permeabilization of the sample. Here we describe a method for identification and enrichment of live cancer stem cells based on intracellular biomarker expression.

Live human head and neck squamous cell carcinoma (HNSCC) cell lines, UM-SCC-47 and UM-SCC-104 were used as a model for Cancer stem cells in a mixed population. They were assayed for their expression of EpCAM, CD44, and CD133 by flow cytometry. Additionally, Aldehyde dehydrogenase (ALDH) activity was also detected in a population of the HNSCC cell lines further corroborating the presence of a cancer stem cell population. Live cell probes specific to Nanog and Sox-2 were used to enrich for cells expressing the targets of interest through Fluorescent Activated Cell Sorting (FACS).

This live cell biomarker approach was also recently demonstrated on solid tumors which were digested and cultured as single cells where they were then incubated with the live cell probes. FACS sorting was utilized to enrich for a cancer stem cell population. More importantly after detection these cells remain live and intact allowing them to be returned to culture and further studied.

#2531

Marker free isolation and expansion of cancer stem cells from small cell lung cancer.

Tessa M. DesRochers, Melissa Millard, Alina Lotstein, Lillia Holmes, Hal E. Crosswell. _KIYATEC, Inc., Greenville, SC_.

Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancer cases and, contrary to the advances in diagnostics and therapeutic options for non small cell lung cancer, SCLC long term survival has failed to materially improve over the last 3 decades. Early dissemination with hematogenous metastasis, advanced stages at diagnosis, dramatic response to chemotherapy with early and aggressive relapses are the clinical hallmarks. Limited surgical samples, early palliation and limited second line treatment options negating the benefit of repeat biopsies have limited tissue available for research to understand the biology of this relatively rare tumor. Recent data has suggested the presence of rare populations of cells within the primary SCLC tumors which have stem cell-like properties. We hypothesize that novel in vitro culture platforms and methods can be used to isolate and expand cancer stem cells (CSCs) within SCLC tumors and that the expanded CSCs will yield a renewable cell source for research applications and for predictive drug response profiling. Many groups have relied upon cell surface markers such as CD133 and CD24 to identify the CSC population. Our approach is unique in that we have employed a marker-free approach utilizing a combination of chemical and functional isolation. We then use a combination of growth factors, extracellular matrix proteins, and oxygen tension in a 3D perfusion culture system to further isolate, purify and expand isolated cells with the goal of having quantities of functional SCLC stem cells for molecular, proteomic and functional in vitro and in vivo assays. Initial cell sources will include immortalized cell lines and patient derived xenograft lines, with eventual adaptation to primary patient samples. Using numerous techniques, we have qualified these cells based upon marker expression (flow cytometry, RT-PCR), dye exclusion, drug response, and limited dilution assays. Future experiments will confirm stemness with limited dilution tumorigenicity assays in vivo. Initial data suggest poor correlation of canonical CSC marker expression with clonogenic growth in cell lines, supporting the need for unbiased and functional isolation methods. These expanded populations of CSCs may be used to identify novel CSC markers and/or targets, mechanisms of resistance and screen and develop novel therapeutics Our ultimate goals are to be able to use our novel culture platforms and methods to expand CSCs for real time precision medicine applications to identify novel therapies in real time that can eradicate the CSC population and improve outcomes of patients with SCLC.

#2532

Alginate-based 3D system for the enrichment and culture of cancer stem cells.

Karen A. Simon, Sylaja Murikipudi, Harry A. Rogoff, Chiang J. Li. _Boston Biomedical, Inc., Cambridge, MA_.

Cancer stem cells (CSCs)—cancer cells which have the ability to undergo "self-renewal" are considered to be responsible for the recurrence and metastasis of tumors. The development of a culture system which can mimic aspects of the tumor microenvironment within the cancer stem cell niche is important to elucidate the mechanisms that underlie the growth and survival of CSCs and to identify compounds that can target and eliminate this highly malignant cell population.

Here we demonstrate the use of an alginate-based 3D cell culture as an in vitro system for enriching and maintaining the stemness properties of cancer cell lines. In this work, we used alginate gels as a three-dimensional (3D) matrix to culture cancer cell lines. Alginate is an ideal system for CSC because of its inertness and ability to provide a hypoxic environment essential for maintaining the stem-like properties of these cells. We found that cells cultured in the alginate 3D matrix have increased CSC-related gene expression and alterations in metabolic-related gene expression that are consistent with changes reported to occur in CSC that help them to maintain their drug resistant and invasive properties.

In order to be useful as a drug screening platform to identify CSC targeting agents, we verified that the high stemness characteristics of the cells cultured in the alginate 3D matrix can be maintained over multiple passages and many months of continuous culture. The CSC cultures in the alginate 3D matrix were found to be resistant to both conventional chemotherapy and targeted therapeutics, while remaining sensitive to CSC targeting agents BB608 and BB503. These results support the potential of the alginate 3D culture system in the enrichment and expansion of CSCs, and provide a reliable in vitro system for the development and evaluation of CSC-targeting agents.

#2533

Interlukin-1 (IL-1) may induce prostate cancer (PCa) stem-like cells.

Shayna Thomas, Sana Merchant, Rachel Meade, Afshan Fathima Nawas, Nikki Delk. _The University of Texas at Dallas, Richardson, TX_.

Inflammation is implicated in prostate cancer initiation and progression. Our lab found that the inflammatory cytokine, interleukin-1 beta (IL-1β) concomitantly upregulates the pro-survival scaffold protein, Sequestome-1 (SQSTM1/p62) and represses androgen receptor accumulation in a relatively small cell subpopulation within various prostate cancer cell lines. Given that cancer stem cell subpopulations have been isolated from isogenic cell lines and given that prostate cancer stem cells are androgen receptor negative, we hypothesized that the IL-1β-responsive subpopulation might be stem-like cells. Cancer stem cells are significant in prostate cancer because they are resistant to chemo-radiation and androgen-targeted therapies and can contribute to the tumor cell pool through asymmetric division. Thus, we analyzed the mRNA and protein accumulation of several stem cell markers in various prostate cancer cells lines exposed to IL-1β. We also have experiments underway to analyze stem cell biology in order to determine the functional significance of the IL-1β-responsive subpopulation. Thus far, we have discovered that IL-1β can induce stem cell marker mRNA and protein accumulation, suggesting that IL-1β alters prostate cell fate and supports the notion that inflammation contributes to prostate cancer initiation and progression.

#2534

Low glucose and hypoxia environment promote the enrichment of liver cancer stem cells.

Chih Chung Lai, Tsu-Hsiang Shia, Yu-Peng Liu, Chia-Hung Yen. _Kaohsiung Medical University, Kaohsiung, Taiwan_.

Cancer stem cells (CSCs), a subpopulation possess tumor initiation and self-renewal capacity, are responsible for recurrence, metastases and drug resistance. Thus, it is important to understand their biology for identification of new therapeutic approaches. Liver surgery and/or transarterial (chemo) embolization (TAE/TACE) therapy usually generate a microenvironment that fosters aggressive tumor recurrence. These areas are characterized by chronic hypoxia and possibly low glucose concentration. Here, we tested the hypothesis that whether hypoxia along with low glucose environment promote the expansion of CSC population in HCC cells. We cultured human liver cancer cell line-Huh7 cells in normoxia or hypoxia condition with high or low glucose medium. The EMT markers (N-cadherin and Vimentin) but not stemness genes (CD44 and CD133) were upregulated in hypoxia condition. However, all genes were significantly induced under low glucose/hypoxia condition. In addition, results from sphere assay showed that low glucose/hypoxia condition dramatically improved the sphere formation ability of Huh7 cells, while low glucose or hypoxia along cannot promote sphere formation. Moreover, Huh7 cells cultured in low glucose/hypoxia condition displayed a lower sensitivity to cisplatin and doxorubicin treatment compared to cells maintained in high glucose/normoxia condition. Therefore, our findings suggested that low glucose concentration and hypoxia environment remarkably promote the enrichment of liver CSCs which could be useful for developing novel therapeutic modalities.

#2535

EpCAM-positive cancer stem cells acquired chemoresistance in hepatocellular carcinoma.

Harshul Pandit, Yan Li, Guozhen Cui, Xuanyi Li, Suping Li, Yingbin Yang, Jack Rostas, Robert C.G. Martin. _University of Louisville School of Medicine, Louisville, KY_.

Introduction: Recurrence of Hepatocellular carcinoma (HCC) after definitive tumor treatment occurs in over 70% of patients within 14 months of initial treatment. The recurrent HCC cells demonstrate a more aggressive tumor growth pattern when compared with the initial HCC cells. Recurrent HCC attains resistance to chemotherapy which accounts for most of the therapeutic failures and is one of the major factors for poor overall survival (OS) in this disease. Underlying molecular mechanism(s) responsible for acquired chemotherapy resistance and tumor recurrence has not been well defined. Cancer stem cells (CSCs) are a sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation and recurrence in tumor microenvironment. We hypothesize that in HCC, Wnt/β-catenin mediated CSC activation is responsible for acquired chemoresistance. Here, we aimed to study that enriched culture of EpCAM positive HCC cells with cancer stem-cell like properties could become resistant to anti-cancer drugs. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. To confirm CSCs, we analyzed CSC biomarkers (EpCAM, CD90, CD44, CD133) using flow-cytometry and Immunocytochemistry (ICC), and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance property of CSCs was studied using Doxorubicin (anthracycline antibiotic) and Sorafenib (multikinase inhibitor) by MTT assay. Tumorigenic potential of enriched CSCs was studied in orthotopic HCC mouse models. Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets i.e. C-Myc, Cyclin-D1, TCF1. Lentivirus mediated overexpression and knockdown of Wnt/β-catenin signaling components confirmed effect of Wnt/β-catenin signaling on activation of CSCs and drug resistance. Total 24 paired human HCC specimens were obtained and analyzed to confirm in vitro and in vivo findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression (EpCAM+) and significant higher chemoresistance compared with control HCC cells. ABCG2 expression (and its efflux activity) was found to be increased in CSCs, which further supports acquired chemoresistance. EpCAM+ CSCs have shown significant higher tumor proliferation rate in the mouse models compared with control. Wnt/β-catenin signaling activity was found to be increased in EpCAM+ CSCs compared with control, and associated with chemoresistance. Human HCC specimens confirmed concomitant increase/decrease in EpCAM and β-catenin expression. Conclusions: Subpopulation of EpCAM+ CSCs, (1) contribute to acquired chemoresistance in HCC, and (2) showing increased Wnt/β-catenin signaling. We believe, this EpCAM+ CSCs population is primarily responsible for HCC recurrence and therapeutic failure.

#2536

AP-1/Fra-1 as a target for therapy promotes chemoradiosensitivity of cancer and cancer stem cells.

Bhudev C. Das,1 Abhishek Tyagi,2 Bal Gangadhar Roy,3 Alok C. Bharti4. 1 _Amity Institute of Molecular Medicine & Stem Cell Research (AIMMSCR), Amity University Uttar Pradesh, Noida, India; Department of Molecular Oncology, B.R. Ambedkar Centre for Biomedical Research (ACBR), University of Delhi, New Delhi, India; _2 _Department of Molecular Oncology, B.R. Ambedkar Centre for Biomedical Research (ACBR), University of Delhi, New Delhi, India; Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, India; _3 _Institute of Nuclear Medicine and Allied Sciences, Defence Research Development Organization, Delhi, India;_ 4 _Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, India_.

Transcription factor activator protein-1 (AP-1) super-family is known to modulate expression of array of genes during development of many cancers. It is also an indispensable factor for efficient expression of high risk HPV oncogenes E6/E7. Recent studies demonstrated the role of AP-1 in promoting stemness/quiescence of cancer stem cells. We have recently reported that HPVE6 oncogene controls stemness and self-renewal of cervical cancer stem cells through Hes1/NOTCH-1expression but little is known about factor(s) responsible for chemo- radioresistance of cancer stem cells. To understand this we have dissected the role of AP-1and its family members by using a herbal compound, curcumin and UV irradiation in cervical cancer. We isolated cervical cancer stem cells and enriched them by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133). The isolated cervical cancer stem cells (SP+ve→CD49+veCD71-ve→CD133+ve) designated as CaCxSLCs displayed spheroid forming and self-renewal property with increased expression of HPVE6, AP-1 transcripts and a higher AP-1 DNA-binding activity compared to non-stem cancer cells (NSP-ve→CD49-veCD71+ve→ CD133-ve) or unsorted parental cells. Treatment with UV-radiation alone resulted in clonogenic survival of CaCxSLCs but not non-stem cancer cells with enhanced AP-1 expression and activation. In contrast, CaCxSLCs treated with UV in combination with curcumin, resulted in complete loss of AP-1 DNA-binding activity followed by decrease in sphere formation and percentage of SP cells but an increased expression of the fos-related antigen-1 (Fra-1) which has been earlier shown to us have tumor suppressor activity in cervical cancer.

Our study demonstrates a critical role of AP-1/Fra-1 signaling in imparting radiosensitization of cervical cancer stem cells. Curcumin, which down regulates almost all signalling pathways, including all members of AP-1 except Fra-1 indicating its role in chemo-radiosensitization of cancer stem cells. Thus combining curcumin with conventional cancer drugs may make cancer treatment most effective.

#2537

Role of N-cadherin in connexin 43 mediated gap junctional formation between dormant breast cancer cells and bone marrow mesenchymal and stromal cells.

Garima Sinha. _Rutgers Biomedical Health Sciences, Newark, NJ_.

Breast cancer (BC) remains a clinical problem. This is partly due to the existence of dormant BC cells (BCCs) in the bone marrow (BM) that could resurge decades after cancer remission. Cancer stem cells (CSCs) establish themselves in cycling quiescence within the BM microenvironment by forming gap junctional intercellular communication (GJIC) with mesenchymal stem cells (MSCs) and the hematopoietic supporting stroma. MiRNAs can be exchanged between the CSCs and other BM cells through the gap junction to impart cycling quiescence of the CSCs. Although the CSCs express several members of the connexin (Cx) family of proteins, GJIC between CSCs and BM cells requires Cx43. Preventing the formation of GJIC between CSCs and endogenous BM cells enhanced cycle of the BCCs with enhanced chemosensitivity. Since Cx43 is also involved in hematopoietic regulation, in order to identify targets to reverse dormancy, this study investigated the molecular mechanisms by which Cx43 is regulated, including the role of N-cadherin as a facilitator of GJIC between CSCs and BM cells. CSCs are epithelial-mesenchymal cells (EMT) and therefore express N-cadherin. In other studies Cx43 has been shown to interact with N-cadherin to facilitate the movement of Cx43 to the cell membrane. To this end, we hypothesize that the intracellular complex formed by N-cadherin and Cx43 in CSCs is regulated by the chemokine CXCL-12. MDA-MB-231, MDA-MB-468 and T47D were stimulated with different levels of CXCL12. At different times, the expression of N-cadherin was studied by real time PCR, confocal microscopy and flow cytometry. The results showed CXCL12 regulated both N-cadherin and Cx43. However, the outcome on their expression depended on the invasiveness of the tumor. Direct interaction between N-cadherin and Cx43 was demonstrated by time-line tracking for co-localization, molecular modeling and immunoprecipitation. In summary, this study showed that co-localization of N-cadherin and Cx43 is controlled by low level of CXC12. Ongoing studies are investigating how these findings affect GJIC between CSCs and BM stroma/MSCs.

#2538

The emerging role of SOX2 in cancer cell plasticity and EGFR-TKI resistance.

Yu-Fan Chiu, Ming-Han Kuo, An-Chun Lee, Yu-Ting Chou. _National Tsing Hua University, Hsinchu, Taiwan_.

Activating mutations in the epidermal growth factor receptor (EGFR) lead to the aberrant growth of lung adenocarcinoma while endowing cancer cells with sensitivity to EGFR-tyrosine kinase inhibitors (TKIs). Nonetheless, EGFR-TKI resistance often occurs in less than one year. The intriguing phenomenon that cancer cells have biological features similar to stem cells suggests that cancer and stem cells may share the same regulatory signaling pathway, but the involvement of stem cell signaling in EGFR-TKI resistance is not clear. Herein, we observed that SOX2, a master transcriptional factor controlling self-renewal of lung stem cells, was highly expressed in lung adenocarcinoma and inversely correlated with epithelial-to-mesenchymal transition (EMT). Clonogenic analysis showed that SOX2-silenecing attenuated cell growth in EGFR-mutated lung cancer cells. The selection of EGFR-TKI resistant HCC827 (delE746_A750) cells induced EMT while attenuating the expression of SOX2 and spheroid forming ability. Preselection of HCC827 (delE746_A750) cells with EMT features endowed cells with TKI resistance but suppressed SOX2 expression. Pharmacological inhibition of HDACs in EGFR-mutated lung cancer cells attenuated SOX2 expression but induced EMT, thus enhancing the development of EGFR-TKI resistance. Our findings support the notion that the switching expression between SOX2 and EMT plays a critical role in EGFR-TKI resistance, with the potential as biomarkers for lung cancer prediction.

#2539

**Cell cycle regulation influences the phenotype of breast and prostate cancer stem cells** in vitro **.**

Joseph Feduska, Carnellia Lee, Selvarangan Ponnazhagan. _University of Alabama at Birmingham, Birmingham, AL_.

The dissemination of metastases from the primary tumor accounts for over 90% of cancer-associated mortality. Despite advances on treatment modalities involving chemotherapeutic drugs, radiotherapy, and surgery, metastasis almost invariably arises by persistence of unresolved cancer cells following initial treatment. One intriguing explanation for this resiliency is the cancer stem-cell (CSC) hypothesis. According to the CSC model, phenotypic heterogeneity exists within the tumor, and a minority fraction of cells are able to reconstitute the tumor following ablation by current treatments. In like manner to their "normal" stem-cell counterparts, CSCs are capable of unlimited self-renewal, and the ability to "differentiate" to reconstitute the phenotypic heterogeneity of the initial tumor. Recent studies have sought to identify cancer stem cells in numerous types of solid tumors, including breast and prostate. Among the cell surface antigens utilized to identify the CSC subpopulations are CD24, CD44, CD133, and other factors associated with epithelial-to-mesenchymal transition (EMT).

In this study, we sought to investigate the phenotypic changes associated with cell-cycle in breast (BCa-SC) and prostate (PCa-SC) cancer. We characterized in vitro the malignant and osteolytic human breast cancer cell line MDA-MB-231 and human prostate cancer cell line PC3, with the goal of identifying an optimal checkpoint where the CSC subpopulation would be most vulnerable to subsequent targeted chemotherapy. We found that common CSC markers are expressed in a time-dependent manner due to changes in the cell cycle. Our hypothesis is that temporal inhibition of the cell cycle when the CSC phenotype is least prominent will circumvent the chemo-resistance inherent to CSC.

#2540

Disruption of a TGF-β-CTCF regulated pathway leads to cancer stem cells.

Jian Chen,1 Zhixing Yao,2 Jiun-Sheng Chen,3 Young Jin Gi,1 Yun Seong Jeong,1 Wilma Jogunoori,4 Mitchell Belkin,4 Bibhuti Mishra,4 Jon White,4 Abhisek Mitra,3 Shulin Li,3 Milton Finegold,5 Marta Davila,3 Jerry Shay,6 Keigo Machida,7 Hidekazu Tsukamoto,8 Lopa Mishra2. 1 _UT M.D. Anderson Cancer Ctr., Houston, TX;_ 2 _George Washington University, Washington, DC;_ 3 _UT MD Anderson Cancer Center, Houston, TX;_ 4 _Institute of Clinical Research, VA Medical Center, Washington, DC;_ 5 _Baylor College of Medicine, Houston, TX;_ 6 _UT Southwestern Medical Center, Dallas, TX;_ 7 _University of Southern Calinfornia, Los Angeles,, CA;_ 8 _University of Southern California, Los Angeles, CA_.

Background: Sporadic cancer formation from stem cells, and molecular switches leading to this remain poorly understood. Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder with an 800-fold increased risk of developing tumors. Imprinting of the IGF2/H19 and the CDKN1C/KCNQ1 loci on chromosome 11p15.5 is mediated by the chromatin insulator CCCTC-binding factor (CTCF). This regulation is lost in BWS, leading to aberrant overexpression of the growth promoting genes. Epigenetic silencing of β2-spectrin (β2SP, gene SPTBN1), a SMAD adaptor for Transforming Growth Factor-beta (TGF-β) signaling, is causally associated with BWS. With this new model for BWS, the mechanisms though which disruption of TGF-β signaling leads to tumorigenesis could be elucidated further. We further sought to determine the roles of the TGF-β-mediated β2SP/SMAD3/CTCF complex in stem cell biological function.

Experimental procedures: 1. Tumorigenesis analysis in Sptbn1+/− /Smad3+/− mice. 2. Whole-transcriptome sequencing were performed in the BWS cells and Sptbn1+/−; Sptbn1−/−; Smad3+/−; and Sptbn1+/−/Smad3+/− MEFs. 3. Tumor-initiating-cell (TIC) tumorigenesis, sphere formation assays were performed for stem cell like characteristics.

Results: 1. We observed that double heterozygous Sptbn1+/−/Smad3+/− mice with defective TGF-β signaling develop multiple tumors phenotypically similar to those of BWS. 2. CTCF is TGF-β-inducible and facilitates TGF-β-mediated repression of hTERT transcription via β2SP/SMAD3/CTCF interactions. This regulation is abrogated in the TGF-β defective mice and BWS, causing hTERT overexpression. 3. Whole-transcriptome RNA sequencing of MEFs isolated from these three TGF-β-deficient strains displayed increased levels of stem cell-associated genes, including PDPN, EPAS1, CXCL1 and ALDH2. 4. CTCF, β2SP, and SMAD3, and modulate stem cell like characteristics such as CD133+/CD49+ TIC tumorigenesis, sphere formation and nanog expression. 5. Further, knock-down of β2SP, Smad3, or CTCF in HepG2 cells resulted in an increase of sphere formation, further supporting a role of these elements in regulating stemness.

Conclusions: Loss of CTCF-dependent imprinting of tumor promoter genes such as IGF2 and TERT is caused by a defective TGF-β pathway, giving new insight into BWS-associated tumorigenesis and sporadic human cancers that are associated with mutations in SPTBN1 and SMAD3. Dysfunction of the β2SP/SMAD3/CTCF complex increases stem-like properties and enhances TICs. Our analysis provides important insight into the switches involved in sporadic cancer formation, particularly those associated with this human stem cell disorder.

#2541

Zebrafish adult cell dedifferentiation as a noncancer model of cancer.

Ke'ale Louie, Alfonso Saera-Vila, Phillip E. Kish, Alon Kahana. _University of Michigan, Ann Arbor, MI_.

Cancer stem cells (CSCs) are those cells within a malignant tumor that can self-renew and initiate new tumors, with the ability to produce all the cell lineages that comprise the tumor. Recent studies have revealed that cancer treatments that target the tumor without addressing CSCs might reduce the tumor load but be unable to achieve a cure. Generation and maintenance of CSCs through genetic and epigenetic reprogramming is essential for tumor maintenance and resistance to therapy. Therefore, developing therapies targeted at CSCs is critical to achieving cures for cancer1. Yet the biology of CSCs, and particularly the process of cellular reprogramming, is still not well understood, in part because of the many mutations carried by these cells, only a few of which are deterministic.

In order to develop a better understanding of the mechanistic underpinnings of adult cell dedifferentiation, we developed a zebrafish model that utilizes adult myocyte reprogramming in the context of muscle regeneration. Our model utilizes the lateral rectus muscle of the eye, which can recover from >50% myectomy by reprogramming the residual (i.e. "post-mitotic") myocytes to reenter the cell cycle, remodel the extracellular matrix, proliferate, migrate and then redifferentiate into myocytes that reform a functional muscle (as determined by an optokinetic response). The proliferative burst of reprogrammed myocytes is particularly robust, with >70% of cells undergoing mitosis during the regeneration process. Such a robust in vivo model of cell dedifferentiation represents a unique opportunity to study the cellular, genetic and epigenetic processes involved.

We find that the dedifferentiation process begins with activation of epigenetic regulators to remodel chromatin, followed by activation of autophagy to remodel the cytoplasm, followed by cell cycle reentry. Epigenetic changes include changes in DNA and histone methylation, along with activation of genes encoding transcription factors that are frequently found to be involved with oncogenesis, such as myc, jun, fos, and twist. A transcriptome analysis using deep sequencing reveals significant similarities between the genetic signatures of dedifferentiated zebrafish myocytes and human cancer cells. This analysis also identifies long non-coding RNAs that are induced early during the dedifferentiation process, and work is ongoing to annotate these lncRNAs with reference to human cancer.

In summary, an in vivo zebrafish non-cancer model of cancer can provide unique mechanistic insights into the biology of CSCs. In addition, as an aquatic species this model is particularly well suited for drug screening, providing an innovative new approach to developing drugs that target reprogramming/dedifferentiating cells.

## EPIDEMIOLOGY:

### Genetic Contributions to Cancer Epidemiology: Familial Cancers and GWAS

#2542

Chromatin remodeling gene ARID1B is linked to familial lung cancer.

Anthony M. Musolf,1 Claire L. Simpson,1 Mariza de Andrade,2 Diptasri Mandal,3 Colette Gaba,4 Ping Yang,2 Ming You,5 Elena Y. Kupert,5 Marshall W. Anderson,5 Ann G. Schwartz,6 Susan M. Pinney,7 Christopher I. Amos,8 Joan E. Bailey-Wilson1. 1 _National Human Genome Research Institute, National Institutes of Health, Baltimore, MD;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 4 _University of Toledo Dana Cancer Center, Toledo, OH;_ 5 _Medical College of Wisconsin, Milwaukee, WI;_ 6 _Karmanos Cancer Institute, Wayne State University, Detroit, MI;_ 7 _University of Cincinnati College of Medicine, Cincinnati, OH;_ 8 _Geisel School of Medicine, Dartmouth College, Lebanon, NH_.

Several papers have implicated mutations in the AT-rich interactive domain containing protein 1B (ARID1B) in carcinoma formation in multiple tissue types, including the lung. The ARID1B protein functions as part of SWI/SNF, a multi-subunit chromatin remodeling complex that repositions nucleosomes and may act as a tumor suppressor in cancer. ARID1B is located in a region in 6q that was previously shown to have strong evidence of linkage with lung cancer risk. As it is one of the more promising candidates within the target region, this study presents the viability of ARID1B as a candidate gene for familial lung cancer. Here, we chose individuals from families in our previous linkage study that showed the strongest linkage signal on 6q. 75 individuals from 9 families were sequenced for 37Mb of chromosome 6 from 130Mb to 167Mb using Illumina technology and a custom Agilent kit. Genotypes were imported into GoldenHelix SVS 7 for quality control and dropped if read depth < 10, quality score < 10 or a quality score to read depth ratio < 0.5. Variants with more than 1 Mendelian error were dropped. Variants with a single Mendelian inconsistency were dropped only for that family. Two-point parametric linkage analysis at theta=0 was performed using R and an implementation of the Elston-Stewart algorithm in the paramlink package using a penetrance model of 0.01/0.1/0.1 (dd/Dd/DD) and a disease allele (D) frequency of 0.01.

Family 102 showed strong evidence for linkage near ARID1B. The highest LOD score (1.34) out of all sequenced variants was located near this gene in a known conserved intergenic region between ARID1B and NOX3. Four other LOD scores > 0.7 were found in the intergenic region between these two genes. Two are transcription factor binding sites; two are conserved genomic elements. We also observed two linked intronic variants of ARID1B in this family; an enhancer (LOD = 0.89) and a transcribed region (LOD = 0.73). Two other families (47 and 59) displayed evidence of linkage in ARID1B. Here, the interest is not the value of the LOD scores (top scores were 0.64 and 0.54), but the centering of the linked haplotype around the gene. Family 47 has 60 variants with LODs between 0.64 and 0.61; 45 located within ARID1B or known regulatory regions. Family 59 has 61 variants with LODs between 0.54 and 0.51; 29 located within ARID1B or known regulatory regions The variants appear to be spread evenly throughout ARID1B, suggesting the presence of linked haplotypes. Analyses are currently underway to reconstruct possible haplotypes in these families. The results of this study show that variants in the ARID1B gene at least partially account for the linkage signal on 6q in three of the nine most strongly linked families in our cohort and thus we believe that ARID1B is a plausible candidate gene for further studies involving familial lung cancer. Replication studies using 27 new extended families are currently underway to confirm this initial result.

#2543

A family affair: Prostate cancer risk and family history of breast or prostate cancer.

Lauren E. Barber, Travis A. Gerke, Sarah C. Markt, Giovanni Parmigiani, Lorelei A. Mucci. _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background - There is suggestive evidence of familial clustering of breast and prostate cancer in first-degree relatives; women with a family history of prostate cancer are at increased risk of breast cancer. Few studies have investigated joint family history of breast and prostate cancer and prostate cancer risk, and no study to date has examined lethal prostate cancer.

Methods - We studied 42,672 from the Health Professionals Follow-up Study between 1996 to 2012. During follow-up, 4,258 prostate cancer cases were diagnosed, of whom 380 were lethal disease. Men self-reported family history of breast and prostate cancer, including in siblings or parents. Using cause-specific hazards regression, we estimated hazard ratios (HR) and 95% confidence intervals (CI) of the association between family history and prostate cancer risk and progression.

Results - About 8% of men had a family history of prostate cancer, 8.7% had a family history of breast cancer, and 1.4% of men had a family history of both breast and prostate cancer. A family history of prostate cancer was significantly associated with an increased risk of prostate cancer (HR: 1.69; 95% CI: 1.54-1.85). Increased risk was higher for men with a father diagnosed with prostate cancer (HR: 1.64 95% CI: 1.49-1.80) than for men with a diagnosis in a brother(s) (HR: 1.51 95% CI: 1.27-1.80). A positive family history of breast cancer was associated with a small, but significant 22% increased risk (HR: 1.22; 95% CI: 1.11-1.35). Familial breast cancer in a sister (HR: 1.22 95% CI: 1.06-1.41) increased risk more than familial breast cancer in a mother (HR: 1.12; 95% CI: 0.99-1.25). Men with a family history of both prostate and breast cancer had a 52% (HR: 1.52; 95%CI: 1.23-1.88) increased risk of prostate cancer compared to men with no family history of either cancer. Risk of lethal prostate cancer was also significantly increased for men with a positive family history of prostate cancer (HR: 1.64; 95% CI: 1.20-2.24), as well as for men with a family history of breast cancer (HR: 1.47; 95% CI: 1.07-2.01).

Conclusions - These results support the findings of familial aggregation of breast and prostate cancer, and for the first time suggest an association between familial breast cancer and lethal prostate cancer. Data from this prospective study have translational relevance for family counseling of cancer patients.

#2544

Association between flame-broiled fish consumption and breast cancer: A case-control study in women with high familial risk.

Alpana Kaushiva,1 Betty May,1 Deborah Armstrong,2 Jennifer Axilbund,2 Kala Visvanathan3. 1 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 2 _Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD;_ 3 _Johns Hopkins Bloomberg School of Public Health; Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD_.

Background: Heterocyclic amines (HCAs) are mutagenic pro-carcinogens formed when meat is cooked using high temperature methods that involve direct heat over an open flame (i.e. pan-fried, grilled or broiled). A number of studies have reported a positive association between flame-broiled (FB) red meat intake and breast cancer (BC) incidence, however data regarding FB chicken and fish are less consistent. Therefore, we examined the associations of FB chicken, meat, and fish with BC incidence in a case-control study among women with a family history of breast or ovarian cancer and/or a BRCA1/2 mutation.

Methods: Cases and controls were identified from the BOSS cohort (N =1400) at Johns Hopkins, a clinic-based cohort of individuals at high familial risk. Women diagnosed with incident stage 0-III BC within 2 years prior to enrollment (N=212) were matched 1:1 to cancer-free controls on age and year of enrollment (+/-1 year). Current and past intake of FB chicken, meat, and fish were ascertained at baseline. Data was also collected on BC risk factors (age at menarche, BRCA status, family history, physical activity, hormone therapy, oral contraceptive use) and fruit/vegetable intake. Multivariable conditional logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (CI) for the association between overall FB meat intake per day and BC risk, and separately for FB chicken, meat, and fish.

Results: The mean age of cases and controls was 49 years; 55% of controls and 42% of cases were premenopausal and 16% of cases and 14% of controls were positive for a BRCA1/2 mutation. Fifty two percent of cases and 56% of controls ate FB chicken, meat, or fish ≥once/wk. In multivariable analyses, BC risk was significantly elevated among women who consumed FB fish >once/wk (O.R. 2.68, 95% CI 1.24, 5.82) compared to those who ate it <once/wk (p-trend =0.004), particularly among women with ER+ BC (O.R. 2.07, 95% CI 1.27, 3.43). The association between FB intake and BC was significantly modified by BMI (p-interaction = 0.07). The odds of developing BC among obese women who ate FB fish ≥once/wk was 3.68 (95% CI 1.61, 8.39), and there was no association in women with a BMI<25. Additionally, there was no significant association between FB chicken and meat intake and BC in this study. Analyses are ongoing to assess concentrations of PhIP, MeIQx, and DiMeIQx. Of note, cases and controls reported a similar increase in the intake of FB fish and chicken and a decrease in FB meat consumption over the prior 2 years.

Conclusion: This is the first study to examine FB chicken, meat, and fish consumption, modifiable risk factors, in women with a familial risk of BC. Our findings that FB fish is associated with an increase in BC are consistent with laboratory findings and are particularly important given the high prevalence of frequent FB fish intake (>50%) in our high risk population. Further prospective studies are needed to validate our findings.

#2545

Identification of germline pathogenic alleles in 143 cancer predisposing genes in Mexican patients with hereditary breast cancer by massive parallel sequencing.

Felipe Vaca-Paniagua,1 Rosalía Quezada-Urban,1 Clara Estela Díaz-Velásquez,1 Rina Gitler,2 María Patricia Rojo-Castillo,2 Max Sirota-Toporek,2 Andrea Figueroa-Morales,2 Oscar Moreno-García,2 Fernando Mainero-Ratchelous,3 Lizzette Sabrina De Hoyos-Arévalo,3 Ivan Delgado-Enciso,4 Victor Hugo Garzón-Barrientos,5 Luis Ignacio Terrazas1. 1 _Facultad de Estudios Superiores Iztacala, Mexico, Mexico;_ 2 _Fundacion Alma, Mexico, Mexico;_ 3 _Instituto Mexicano del Seguro Social, Mexico, Mexico;_ 4 _Instituto Estatal de Cancerología de Colima, Colima, Mexico;_ 5 _Hospital General de Chilpancingo, Guerrero, Mexico_.

Breast cancer is the neoplastic disease with the highest incidence and mortality worldwide and nearly 10% of the cases are due to inherited pathogenic alleles in cancer predisposing genes such as BRCA1 and BRCA2. However, the spectrum of inherited breast cancer susceptibility not caused by BRCA1/2 pathogenic alleles has not been explored in the Mexican population. In this work we evaluated the presence of germline pathogenic alleles in the coding sequence and splice sites of 143 cancer susceptibility genes in 69 Mexican female patients with cancer that were selected for family cancer history. Inclusion criteria followed the guidelines of the National Comprehensive Cancer Network for Genetic/Familial High-Risk Assessment of Breast and Ovarian Cancer. Data from international databases of normal populations and cancer patients, as well as annotation information and technical parameters were used to define pathogenic variants. The genes with the highest frequency of pathogenic alleles (stopgain/loss, frameshift indels) were BRCA1 (8.7%, 6/69), BRCA2 (2.9%, 2/69), FANCC (2.9%), MSR1 (2.9%), FANCL (1.4%, 1/69), SDHB (1.4%) and TSC2 (1.4%). New or rare missense variants with unknown clinical significance (VUS), but defined as pathogenic in ClinVar or by algorithms assessing evolutionary conservation and deleterious structural changes at protein level, were found in single patients in the genes AIP, ANTXR1, APC, ATR, CD96, CYP21A2, ERCC3, ERCC6, FANCA, FANCB, FANCE, LIG4, LYST, MSH6, MSR1, MTAP, PDE11A, PDGFRA, PMS2, POLE, PTCH1, RAD50, RHBDF2, RUNX1 and WRN. These patients did not have pathogenic alleles, suggesting a potential contribution of these VUS to disease susceptibility. This work contributes to identify new susceptibility alleles that can predispose to hereditary breast cancer in the Mexican population. Further studies need to be conducted to define the clinical impact of the VUS identified here, which together with international efforts will better define genetic susceptibility to breast cancer. This work was supported by the National Autonomous University of Mexico, PAPIIT (IA204215).

#2546

With increasing age at tumor diagnosis in families with cancer, cancer is limited to fewer organs.

Hakan L. Olsson. _Department of Cancer Epidemiology, Clinical Sciences, Univ. of Lund, Lund, Sweden_.

Hereditary cancer that has monogenic inheritance affects every tenth patient, on average, who is diagnosed with cancer, and it has been suggested based on twin studies, that approximately 30% of all cancer patients have a genetic predisposition to developing cancer. The author posited that familial syndromes become more organ specific with increasing age at tumour presentation to the point that very late in life, only a few organs are affected by tumours disease. The reason for this could be that the tumour originates from a more differentiated, organ-specific progenitor/stem cell later in life, while the progenitor/stem cell might be involved in organogenesis in different organs earlier in life. Examples are given for skin cancer and breast cancer.

Summary: Patients with familial cancer who present with cancer at an older age at tumour presentation have a more organ restricted disease. This could be because the tumor has a more differentiated progenitor/stem cell origin. Examples are given for families with breast cancer, melanoma, and non- melanoma skin cancer.

References: Olsson H. A hypothesis about tumour development and the clinical features of hereditary cancer. European Journal of Cancer 2001 2023-2029.

Olsson H. With increasing age at tumor diagnosis in families with cancer, cancer is limited to fewer organs. Journal of Cancer and Therapy (in press).

Examples of the median age at diagnosis for melanoma (*) and breast cancer (**) in familial settings

---

Families with melanoma. CDKN2A positive melanoma cases. (melanoma, pancreas, smoking associated cancer) | 39*

Families with melanoma. CDKN2A negative melanoma cases. (melanoma and skin cancer) | 48*

Four or more tumors in the cancer registry, at least one of which is a melanoma. Multiple melanoma cases with other tumors and CDKN2A mutations. | 60*

Four or more tumors in the cancer registry, at least one of which isa melanoma. Melanoma, other tumours and adenocarcinomas. | 67*

Four or more tumors in the cancer registry, at least one of which is a melanoma. Melanoma and skin cancer. | 78*

Li Fraumeni's syndrome | <40**

BRCA1 | 44**

BRCA2 | 45**

BRCAX (majority breast only families) | 48**

Vertical inheritance | 52**

Horizontal inheritance (possible recessive inheritance, breast cancer only families). | 63**

#2547

Molecular insights into OGG1 gene, a modifier of cancer risk in BRCA1 and BRCA2 mutations carriers.

Carlos Benitez-Buelga,1 Tereza Vaclova,1 María Sofia Ferreira,1 Nora Soberon,2 María Antonia Blasco,2 Ana Osorio,3 Javier Benitez3. 1 _Human Genetics Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain;_ 2 _Telomere and Telomerase Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain;_ 3 _Human Genetics Group, Spanish National Cancer Research Center (CNIO), and Spanish Network on Rare Diseases (CIBERER), Madrid, Spain_.

Oxidative stress is a source of DNA damage which can lead to genomic instability and telomere shortening. The OGG1 glycosidase plays an important role in the Base Excision Repair (BER) pathway that counteracts the oxidative DNA damage caused by endogenous cell stress. Recently, we have demonstrated that a single nucleotide polymorphism (SNP) in the OGG1 gene can modify cancer risk in carriers of germline mutations in the BRCA1 and BRCA2 genes1. Hence, we aimed to explore the possible role of this variant in DNA damage and telomere shortening to explain the association of this cancer risk variant.

We used 2 independent set of samples with a heterogeneous BRCA mutational status.

First of all, a familiar breast and ovarian cancer (FBOC) set of 154 BRCA patients (BRCA1, BRCA2 and BRCAX) and 68 controls, were used to measure the mRNA OGG1 expression levels in blood and the leukocyte telomere length (TL).

Second, we used a panel of 23 Lymphoblastoid Cell Lines (LCL) from patients harboring germline mutations in BRCA1 and non-carrier controls to validate previous results regarding OGG1 mRNA expression levels and telomere shortening. Additionally we measured gammaH2AX intensity levels in LCL to evaluate the role of the SNP on DNA damage.

In the FBOC series, we found that the carriers of the variant had significant decreased expression levels of the OGG1 transcript compared with the non-carriers (p=0.013). Regarding TL studies, we found that the variant may exert a synergistic effect together with BRCA1/2 mutations on telomere shortening (p<0.0045). TL was not associated to higher cancer risk in our FBOC series, pointing to this phenotype not as the cause of the cancer risk association, but maybe as a hallmark of a higher DNA damage environment when both genetic events are present in the cell.

Using the LCL panel we were able to detect OGG1 mRNA down regulation due to the presence of the variant and a significant telomere shortening exclusively in the BRCA1 defective cells harboring the OGG1 SNP after 55 passages. The DNA damage studies, confirmed significant higher intensity levels of gammaH2AX in the damaged LCL harboring the SNP compared to those not harboring it (p<0.0001).

These results, may explain the molecular insights behind this modifier variant. Under the prism of a deleterious interaction, in cells harboring mutations in BRCA genes, the presence of the SNP itself, seems to affect OGG1 mRNA down regulation. This could lead to a defective performance of the BER pathway that may lead to genome instability, characterized by higher gammaH2AX intensity in damaged cells and telomere shortening when both genetic events are present, and as consequence, higher cancer risk.

1. Osorio A, et al (2014) PLoS Genet

Funding:

J.B's laboratory, INNPRONTA 2012, Spanish Ministry of Health (PI12/00070) and (CIBERER). C.B is granted by PI12/00070

M.A.B.'s laboratory, Spanish Ministry of Science and Innovation (SAF2008-05384;2007-A-200950) (TELOMARKER), ERCA (GA#232854)

#2548

Cancer in first-degree relatives of women with early-onset breast cancer: a comparison of self-reported and cancer registry data.

Annelie Augustinsson, Ulf Kristoffersson, Håkan Olsson. _Lund University, Lund, Sweden_.

Background

Breast cancer (BC) is the most common cancer in women, both world-wide and in Sweden. However, only about 2% occur in women ≤35 years of age. The aim of this study was to compare self-reported and registry data of family history of cancer, tumor characteristics and BRCA germline mutation status in women with early-onset BC.

Materials and methods

Between January 1st 1990 and December 31st 2013, a total of 779 women aged ≤35 years were diagnosed with BC in the South Swedish Health Care Region. Of these women, 231 women sought, or were referred for, genetic counseling at the Oncogenetic Clinic, Lund, Sweden, and were included in the study. Subsequently, 224 women were screened for germline mutations. Self-reported information about family history of cancer in first-degree relatives (parents, siblings and children) was obtained from the participants and was compared with data from the Swedish Cancer Registry. When available, hormone receptor (ER and PR) and HER2 status of the tumors was collected from pathology reports. Fisher's Exact Test was performed to estimate odds ratios (ORs) with 95% confidence intervals (CIs).

Results

Mutation analyses revealed 143 (63.8%) cases without mutations (BRCAX), 46 (20.5%) with BRCA1, 19 (8.5%) with BRCA2 and 16 (7.1%) with other mutations (TP53, CHEK2, PTEN, PALB2, CDH1 and MSH2/MSH6). Family history of BC and ovarian cancer (OvC) in first-degree relatives was self-reported in equal numbers as registry data. Family history of BC was found in ~30% of the cases, and of OvC in ~3%. However, family history of other types of cancer was self-reported in 13 (5.6%) and registry-reported in 58 (25.1%) cases (OR, 18.8; 95% CI, 3.91-180; p<0.001). Of the registry-reported cancers, cervical and skin cancers were the most common when diagnosed before the genetic counseling sessions, and when diagnosed after, prostate and skin cancers were the most common. When comparing tumor characteristics of BRCA1 with BRCAX cases, mutation carriers more often showed receptor negative tumors: ER- (OR, 13.4; 95% CI, 3.77-73.5; p<0.001), PR- (OR, 47.9; 95% CI, 7.34-2011; p<0.001) and HER2- (OR, 17.1; 95% CI, 1.81-inf; p<0.004).

Conclusion

The main findings in this study of early-onset BC were that cases with germline mutations in the BRCA1 and BRCA2 genes were exceptional high, that cervical cancer was the most common not self-reported cancer in first-degree relatives diagnosed before the genetic counseling, and that BRCA1 carriers had a higher rate of ER, PR and HER2 negative tumors, which tends to indicate a less favorable prognosis. When assessing the need for mutation screening, as shown previously by others, oncologists and genetic counselors need to consider both early-onset BCs and receptor negative tumors as predictors for BRCA1 mutations, and when obtaining information about family history of cancers, patients might lack information about cervical cancers in relatives.

#2549

Familial risk of upper gastrointestinal cancers by tumor location.

Elham Kharazmi,1 Masoud Babaei,1 Mahdi Fallah,2 Thianhui Chen,1 Kristina Sundquist,3 Kari Hemminki1. 1 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _National Center for Tumor Diseases (NCT), Heidelberg, Germany;_ 3 _Center for Primary Health Care Research, Lund University, Malmö, Sweden_.

Background: Upper gastrointestinal (UGI) cancers are within the most common malignancies worldwide. Familial clustering of UGI cancers and the significance of family history have been addressed in the past. We aimed to further elucidate the familial risk based on specified tumor locations.

Methods: Using the Swedish Family-Cancer Database, we determined the familial risk of UGI cancer patients diagnosed between 1958 and 2012. Standardized incidence ratio (SIR) was used for calculating concordant and discordant familial cancers risks, stratified by tumor locations. All results were adjusted for sex, age group, period, and socioeconomic status.

Results: Risk of esophageal cancer in first-degree relatives (FDRs) of esophageal cancer patients was 2•4-fold higher than that in those without such a family history. Familial risk for concordant cancer in upper and middle third of esophagus was 3•2- and 5•6-fold, respectively. Family history of esophageal cancer in lower part was associated with increased risk of gastric cardia cancer in FDRs and vice versa. Family history of both adenocarcinoma and SCC of esophageal cancer increased the familial risk of concordant (similar) and discordant subtypes. Occurrence of gastric cancer in the body and the antrum of stomach in a FDR were associated with an increased risk of gastric cancer 6- to 7-fold in the same location in family members, respectively.

Conclusion: Family history of UGI cancers in some specific locations like upper and middle third of esophagus, body and antrum of stomach can be considered as an important predictive evidence for the same cancers in relatives.

#2550

Identification of novel BRCA founder mutations in middle eastern breast cancer patients using capture and sanger sequencing analysis.

Rong Bu, Abdul K. Siraj, Khadija A. S. Al-Obaisi, Shaham Beg, Mohsen Al Hazmi, Yan Kong, Dahish Ajarim, Fouad Al-Dayel, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital & Res. Ctr., Riyadh, Saudi Arabia_.

Ethnic differences of breast cancer genomics have prompted us to investigate the spectra of BRCA1 and BRCA2 mutations in different populations. The prevalence and effect of BRCA 1 and BRCA 2 mutations in Saudi population is not fully explored. We conducted this study to characterize the prevalence of BRCA mutations in Middle Eastern breast cancer patients from Saudi Arabia. A total of 486 clinically high risk breast cancer patients were recruited from the King Faisal Specialist Hospital & Research Centre from 1990 and 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using capture sequencing and/or Sanger Sequencing of all coding exons of BRCA1 and BRCA2 in 190 cases. Thereafter, the validation was done using a panel of detected mutations in an additional 296 high risk breast cancer cases. Among the total 486 cases analyzed, 9 deleterious BRCA mutations were identified in 28 (5.8%) cases, comprising 25 cases in BRCA 1 and three cases in BRCA 2. The six BRCA 1 recurrent mutations (c. 1140dupG, c.4065_4068delTCAA, c.4136_4137delCT, c.4524G>A, c.5251C>T and c.5530delC) accounted for 96% (24/25) of all BRCA 1 mutant cases in this cohort. The one recurrent BRCA 2

mutation (c.7007G>A) accounted for 66.6% (2/3) of all BRCA2 mutant cases. Haplotype analysis was performed to confirm two BRCA1 recurrent mutations are novel putative founder mutation. In this study our findings suggest that BRCA mutations account for substantial proportion of high risk breast cancer cases in Middle Eastern population. Knowing the spectrum and frequency of founder mutation in this population will help in developing cost-effective rapid screening assay which can facilitate genetic testing and counseling for breast cancer risk assessment.

#2551

Identifying hereditary gastric cancer predisposition in the clinical cancer genomics community research network.

Thomas P. Slavin, Kai Yang, Sharon Sand, Tanya Chavez, Danielle Castillo, Joseph Herzog, Ilana Solomon, Christina Rybak, Mariana Niell-Swiller, Bita Nehoray, Aaron Adamson, Kathleen Blazer, Susan Neuhausen, Jeffrey Weitzel, Clinical Cancer Genomics Community Research Network. _City of Hope, Duarte, CA_.

Genetic Cancer Risk Assessment (GCRA) clinical referral and testing guidelines are limited for individuals and families with gastric cancer. In part, this is due to a lack of knowledge regarding hereditary gastric cancer susceptibility. The Clinical Cancer Genomics Community Research Network registry includes over 15,000 families seen for GCRA at City of Hope and 45 other collaborating sites. The purpose of this research was to identify variants conferring inherited gastric cancer susceptibility for individuals and families in our registry without previously known genetic predisposition. Adult research participants from our IRB-approved registry with a DNA sample and a personal history of gastric cancer were selected. Those with a previously identified genetic predisposition were excluded (n=8). In families with more than one eligible individual, the individual with earliest onset of disease was selected. All histologies were included (i.e., intestinal and diffuse adenocarcinomas, as well as, gastrointestinal stromal tumors). Of 47 eligible participants, 22 had previously uninformative clinical testing. Germline sequencing was completed using a novel 706 candidate cancer gene panel, which included genes involved in DNA damage response, cell cycle regulation, apoptosis, and the Fanconi anemia, mTOR, JAK-STAT, and RAS-MAPK pathways; known cancer susceptibility genes; and genes frequently mutated in gastric tumors (data from the Catalog of Somatic Variants in Cancer database). Based on 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology Standards and Guidelines, 18 out of 47 research participants had pathogenic or likely pathogenic germline variants. Of the 18, 11 had a first- or second-degree relative with gastric cancer. Variants were identified in established cancer susceptibility genes, such as BRCA2, BRIP1, RAD51C, ATM, FLCN, as well as in genes from rare autosomal recessive conditions, such as FANCC. In conclusion, using a 706 gene panel to test a GCRA cohort, we were able to identify potentially pathogenic variants in 38% of participants with gastric cancer, with nearly half of these variants in clinically actionable genes. The variants identified in this study will need to be further evaluated using segregation studies, tumor studies, and in larger cohorts to establish causality.

#2552

Influence of age, sex, colorectal cancer status, and mutation carrier status on physical activity in families with Lynch syndrome.

Mala Pande,1 Stephen Glombicki,2 Karen Basen-Engquist,1 Mark A. Jenkins,3 Aung Ko Win,3 Y Nancy You,1 Susan K. Perterson,1 Patrick M. Lynch1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Texas A &M University, College Station, TX; _3 _Melbourne School of Population and Global Health, The University of Melbourne, Melbourne, Australia_.

Lynch syndrome (LS) is due to inherited mutations that impair DNA mismatch repair (MMR) and predispose mutation carriers to an increased risk of colorectal (CRC) and other cancers. Physical activity (PA) reduces risk for sporadic CRC but little is known about the effect of PA on CRC risk in LS. We examined the influence of demographic and clinical characteristics on PA in LS mutation carriers and their unaffected relatives.

Self-reported PA data by decades of life: during the 20's (early), 30-40's (mid), and 50's and older (late) decades, from a retrospective cohort of LS subjects and their relatives enrolled in the Colon Cancer Family Registry were analyzed. Metabolic equivalent (MET) PA values were derived for each activity and standardized to MET hours/week. Mean PA by decade was compared across 3 groups: MMR mutation carriers (MMR+) with and without cancer, and non-carrier relatives (MMR-).

We analyzed 3,261 subjects (1,413 MMR-, 1052 MMR+ with no CRC and 796 MMR+ with CRC). PA was inversely associated with age. Men had higher levels of PA in the early and mid-decades than women. In their 20s, MMR- women had higher levels of PA on average than MMR+ women but there were no significant differences by MMR or CRC status in the mid and later decades. For men, there was no difference in mean PA by MMR+ status in the early or mid-decades, but in the later decades, MMR- men had higher PA than MMR+. Furthermore, mean PA in the mid-decades was significantly higher in MMR+ men with CRC than MMR+ with no CRC.

In this descriptive analysis, men and women had higher levels of PA at younger ages, although men exercised more than women in the early and mid-decades. Levels of mean PA were comparable for MMR+ men and women with and without CRC except in the mid-decades where MMR+ men with CRC had higher levels of PA than MMR+ men without CRC. Recall bias for higher PA in CRC cases cannot be ruled out.

Comparison of mean physical activity in early, mid and late decades of life by sex, MMR mutation carrier status, and colorectal cancer status  | |  | |  | |

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

|

Mean MET hours/week of Physical activity | |

|

Early decade (20s) | Mid-decades (30s-40s) | Later decades (50s and above)

|

Male | Female | Male | Female | Male | Female

Sex | 33.5* | 25.6* | 20* | 16.7* | 11.1 | 10.8

|  | |  | |

|

Non-carrier | 33.9 | 27.1* | 20.9 | 17.3 | 12.8* | 11.1

MMR carrier | 33.2 | 24.4* | 19.2 | 16.2 | 9.8* | 9.3

|  | |  | |

|

MMR carrier no cancer | 33.8 | 24.5 | 16.3* | 15.8 | 8.8 | 8.9

MMR carrier with cancer | 32.6 | 24.5 | 22.2* | 16.9 | 10.8 | 9.8

*starred pairs indicate a significant (p<0.05) difference in mean MET hours/week of PA between the groups

#2553

Prevalence of BRCA1 germline mutations in Algerian population: implications for genetic testing and counseling.

Farid Cherbal,1 Hadjer Gaceb,1 Chiraz Mehemmai,1 Rabah Bakour,1 Kamelia Yatta,1 Ikram Boukoufa,1 Hassen Mahfouf,2 Kada Boualga3. 1 _Unit of Genetics, LMCB, Faculty of Biological Sciences, USTHB, Algiers, Algeria;_ 2 _Public Hospital Academic Medical Oncology Services, School of Medicine, University of Algiers, Rouiba, Algeria;_ 3 _Radiation Therapy Services, Anticancer Center of Blida, Blida, Algeria_.

Background: Breast cancer is currently the leading cause of cancer morbidity and mortality among Algerian women.In this study, we estimated the prevalence of BRCA1 germline mutations in 192 patients with hereditary breast and/or ovarian cancer and sporadic triple-negative breast cancer, respectively.

Materials and Methods: 192 Patients and their families were referred through several public hospitals and private medical clinics which provide oncology services throughout Algeria. Patients were selected for family history of breast and/or ovarian cancer. In addition, in the present study, all women (age under 50 years) with triple-negative breast cancer (TNBC) were considered eligible. BRCA1 was screened by PCR-direct sequencing in all patients including all exons where a mutation was previously found in Algerian population (exons 2, 3, 5 and 11).

Results: The analysis of DNA samples of 192 individuals revealed that 11 patients carried pathologic germline mutations in BRCA1 gene (5.7%).Five distinct pathogenic mutations: c.19_47del, c.83_84delTG, c.181T>G, c.798_799delTT and c.2125_2126insA were indentified. Interestingly, the BRCA1 mutation c.83_84delTG detected in previous studies in two unrelated Algerian breast cancer families has been identified in this study in 4 patients with a frequency of 2 % (4/192): 3 TNBC patients and one patient with a bilateral ovarian cancer, respectively. The four patients had a strong hereditary breast and/ or ovarian cancer history. The c.181T>G/p.Cys61Gly mutation has been detected here in two young breast cancer patients, a bilateral breast cancer patient with a family history of breast cancer and a young TNBC patient (diagnosed at age 36 years), respectively. To date, the Cys61Gly pathogenic variant is one of the most frequent founder mutation identified in Central European populations, has been previously reported for the first time in two Algerian and Moroccan families. The BRCA1 mutation c.798_799delTT was identified here in a young TNBC patient with a family history of breast cancer. Interestingly, this mutation has been previously detected in several unrelated families from Algeria, Tunisia, Morocco and Italy, respectively. As Mediterranean countries share a common history and migration flow history, BRCA1 mutation c.798_799delTT could be a Mediterranean founder mutation. The BRCA1 mutation c.2125_2126insA has been detected here for the first time in three young TNBC patients with a family history of breast cancer and prostate cancer.

Conclusions: The accumulating knowledge about the prevalence and nature of BRCA1 mutations in Algerian population and the identification of specific mutations will contribute in the future to the implementation of genetic testing and counseling for families at risk. Based on our currents results, we recommend using the TNBC immunophenotype as criterion to screen for BRCA1 germline mutations in patients with early onset breast cancer.

#2555

Promoter DNA methylation patterns in breast tumor and matched adjacent non-tumor tissues in the breast cancer family registry.

Hui-Chen Wu, Hanina Hibshoosh, Regina Santella, Mary Beth Terry. _Columbia University School of Public Health, New York, NY_.

Alterations in DNA methylation occur frequently in breast carcinogenesis, but little is known about the DNA methylation changes in familial breast tumors. To examine whether the DNA methylation profiles in breast cancer tissues from high risk women from breast cancer families are similar to breast tumor tissues from The Cancer Genome Atlas project (TCGA), we measured DNA methylation by next generation sequencing in 48 genes identified in TCGA in 40 formalin-fixed, paraffin-embedded tumor tissues, and the corresponding adjacent non-tumor tissues from women with invasive breast cancer at the New York site of the Breast Cancer Family Registry (BCFR). We observed differences in DNA methylation patterns between tumor and normal adjacent tissue for five of the selected genes (RYR2, C1orf14, SLC7A14, SEPW1, and RPTOR). The methylation levels in tumor and adjacent tissues, respectively were 24% and 14% for RYR2, (p=0.01), 55% and 29% for C1orf14 (p=0.01), 32% and 21% for SLC7A14 (p=0.002), 53% and 28% for RPTOR (p=0.01) and 74% and 38% SEPW1 (p<0.0001). We only confirmed 5 of the 48 loci identified in TCGA in our sample of high risk women, suggesting genes identified from TCGA may be different than those in high-risk women. As tissue-specific methylation may prove to have strong prognostic significance, this suggests a critical need to engage in large-scale studies examining genome-wide DNA methylation profiles in high risk women in order to understand the etiology of familial breast cancer.

#2556

Family history of colorectal cancer in half-siblings as important as in siblings.

Mahdi Fallah,1 Elham Kharazmi,1 Kristina Sundquist,2 Hermann Brenner,1 Kari Hemminki1. 1 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 2 _Center for Primary Health Care Research, Lund University, Malmö, Sweden_.

Background: None of population-based epidemiological studies to date have investigated the familial risk of colorectal cancer (CRC) in half-siblings. We aimed to compare lifetime (0-79 years) cumulative risk (LCR) and relative risk of CRC between siblings and half-siblings of patients with CRC.

Methods: A nationwide cohort of first-degree relatives and half-siblings of CRC patients diagnosed in 1958-2012 were extracted from the Swedish Family-Cancer Database, the world's largest of its kind, with >15.7 million individual records. LCR as a tangible measure of absolute risk was also calculated. Standardized incidence ratios (SIRs) were adjusted for age, sex, period at diagnosis, and socioeconomic status. All family histories reported below are exclusive, meaning that risk reported for one affected half-sibling does not include those with both an affected half-sibling and any other first/second-degree relatives.

Results: The overall LCR of CRC in siblings of a patient with CRC was 7.2% (8.3% in men; 6.0% in women), which represents 1.8-fold (95%CI=1.7-1.1.9, n=1333) increase over the risk in those without any family history of CRC (men 4.5%; women 3.4%). Similarly, significantly increased LCR (5.9%) was found among half-siblings of CRC patients (SIR=1.7, 95%CI=1.3-2.0, n=88; maternal half-sibling: 1.6, 95%CI=1.1-2.1; paternal half-sibling: 1.7, 95%CI=1.3-2.3). If a parent and a half-sibling both had CRC, the risk in other half-siblings (SIR=4.4, 95%CI=2.8-6.5, n=24) was closer to that of those with both an affected parent and an affected sibling (SIR=3.2, 95%CI=2.9-3.6, n=281) rather than an affected parent alone (SIR=1.6, 95%CI=1.5-1.7, n=4250). Highly increased risk of CRC was also found in those with two (SIR=2.4, 95%CI=1.8-3.1, n=59) or three (SIR=9.8, 95%CI=5.1-17, n=12) affected siblings, in twin brothers (SIR=4.0, 95%CI=2.2-6.6, n=14), and presumably in men with two affected half-siblings (SIR=5.3, 95%CI=1.1-16, n=3). Other second-degree relatives such as grandparents (SIR=1.2, 95%CI=1.1-1.3, n=318), aunts/uncles (SIR=1.3, 95%CI=1.0-1.5, n=107) without an affected first-degree relative showed minor contributions to the familial risk of CRC, but we found higher risks for those with both an affected first-degree relative and a grandparent (SIR=3.2, 95%CI=2.3-4.2, n=46 ) or an aunt/uncle (SIR=2.3, 95%CI=1.2-4.0, n=13).

Conclusions: This study provides novel information, which is useful for the genetic counseling. In the cancer risk estimation for relatives of CRC patients, a family history of CRC in a half-sibling (even in the absence of an affected first-degree relative) is as important as a family history of CRC in a sibling.

#2557

Germline mutations in renal cancer predisposing genes: Analysis of the Geisinger MyCode population.

Heinric Williams,1 Raghu Metpally,1 Mahmoud Mohamed,1 Sarath Krishnamurthy,1 Sertac Kip,1 David J. Carey,1 Aris Baras,2 John Overton2. 1 _Geisinger Health System, Danville, PA;_ 2 _Regeneron Pharmaceutics, Inc, Rensselaer, NY_.

Renal cell cancer (RCC) is not a single disease but is made up of a number of cancers, each with a unique histology, biology, clinical course and response to therapy. Alterations in at least 16 hereditary genes have been attributed to the risk of developing RCC. In this study, we describe the prevalence and spectrum of germline variants among these genes and highlight correlations between germline genotype with tumor phenotype. Using the Geisinger MyCode cohort, we sequenced the whole exomes of 42,933 subjects. Subjects were divided into those with a renal cancer diagnosis, other cancer diagnosis and no cancer diagnosis. We analyzed the DNA sequences of 16 hereditary renal cancer genes from each of these groups. The damaging mutations was determined by following ACMG guidelines. Among the 42,933 subjects in the Geisinger MyCode cohort, 168 were diagnosed with RCC. None of these subjects had a family history of RCC. Clear cell RCC (ccRCC) was the most predominant histology (77%), followed by Type 1 papillary RCC (7%), chromophobe RCC (6%), and Type 2 papillary RCC (5%). Mutations in all the predisposing genes were identified in all the renal cancer subtypes but only a subset were deemed damaging. The top 2 ccRCC predisposing genes with damaging or likely damaging mutations were TSC2 (8%) and SDHD (4%). While TSC2 damaging variants were also found in the other histologies, novel TSC2 variants were differentially associated with high grade and metastatic disease in ccRCC as compared to those with low grade or non-metastatic disease. The genes harboring damaging or likely damaging variants in Type 1 and Type 2 papillary RCC were MET and FH respectively. Predisposing RCC germline mutations were found in a significant number of subjects with sporadic RCC. Family history was unhelpful in predicting the affected subjects. Knowledge of these mutations would be beneficial in counseling patients and their families as well as improving our understanding of the disease to direct patient care.

#2558

A retrospective analysis of the spectrum of genetic variations associated with hereditary cancers in Indians.

Lakshmi Mahadevan, Aditya Nair, Vedam L. Ramprasad. _MedGenome, San Francisco, CA_.

The hereditary cancer burden in India is on the rise due to an increase in awareness of the disease, decrease in the cost of genetic testing and opportunities for genetic counseling. More and more individuals from high risk families are undergoing genetic testing to assess their likelihood of developing the disease. On the other hand, the clinical community is facing a significant challenge on how to interpret and classify a variant as disease-causing or benign. This is because a large fraction of the variants identified in the affected individuals from India have not been reported in hereditary disease databases such as the Breast cancer Information Core (BIC), Clinvar and Swissvar. These variants of unknown significance (VUS) present in individuals from high risk families create a great deal of uncertainty on clinical follow up and disease management. We performed a retrospective analysis on more than 300 affected cases of hereditary ovarian and breast cancer tested at our genomics center in the last three years. We compiled all the known pathogenic variants and compared their distribution in the Indian cohort with what has been reported worldwide. The most prevalent mutation in BRCA1 p.Glu23Valfs*17 (185delAG) was detected at a frequency of 20.34% in line with what has been found worldwide. In addition, frameshift mutations in BRCA1 and BRCA2 were most frequently detected in breast and ovarian cancer in line with previous findings. Additionally, our analysis identified 13 novel previously unreported and potentially pathogenic variants in BRCA1 and BRCA2 genes. Variants were also detected in several moderate penetrance genes such as CHEK2, BARD1 and ATM involved in DNA damage response and in MSH2 and MSH6 genes regulating epigenetic control through chromatin modification. In this study, we report novel pathogenic variants in genes associated with hereditary breast and ovarian cancer in the Indian population.

#2559

Genome-wide haplotype association tests identified three candidate loci associated with sporadic colorectal cancer risk in Singapore Chinese.

Peh Yean Cheah,1 Lai Fun Thean,1 Yik Ying Teo,2 Woon-Puay Koh,2 Jian-Min Yuan,3 Min Hoe Chew,1 Choong Leong Tang1. 1 _Singapore General Hospital, Singapore, Singapore;_ 2 _National University of Singapore, Singapore, Singapore;_ 3 _University of Pittsburgh, Pittsburgh, PA_.

Colorectal cancer (CRC) is one of the most frequent cancers and leading cause of cancer mortality in the developed world. Twin study has attributed about 35% of the etiology of sporadic CRC to genes. Hitherto, more than 20 single nucleotide polymorphism (SNP) loci from genome-wide association studies (GWAS) were associated with CRC risk. Most of these index SNPs however have small effect sizes and often located in gene deserts. Thus they are unlikely to be causal. Haplotype-based methods may offer a more powerful approach to disease gene mapping. We previously performed a GWAS on 2,000 ethnicity-, age-, and gender-matched Singapore Chinese CRC patients and healthy controls using the Affymetrix SNP 6 platform. Sporadic patients were defined as aged 50 or more at the time of surgery and with no dominant family history of CRC. Haplotype block detection was performed in Golden Helix SVS after this SNP6 dataset was filtered for genotype call rate (>95%), Hardy-Weinberg equilibrium in the controls and principal component analysis. Block detection was performed requiring strong linkage disequilibrium between block pairs, minor allelic frequencies (MAF) of SNPs more than 0.01, the maximum number of markers and length of the block at 30 and 160kb respectively. Genome-wide, 73,333 blocks were computed using 336, 466 markers on 22 autosomes. Haplotype frequencies were estimated using the EM algorithm. Chi-Squared and logistic regression with odds ratio tests of haplotype association were performed using these pre-computed blocks on a per-haplotype basis. Three candidate loci at chromosomes 3, 15 and 20, each with 6 to 9 SNPs and sizes ranging from 4 to 15 kb achieved genome-wide significance and encompass potential novel disease genes. These loci are currently being validated in an independent replication panel using the Fluidgm SNPtype assays and pooled analyses will be performed.

#2560

Significant association between a fatty acid-related genetic variant and the risk of lung cancer.

Cheng Wang, Na Qin, Meng Zhu, Zhibin Hu, Hongbing Shen. _Nanjing Medical Univ., Nanjing, China_.

Systematically study the metabolism can help us understand the etiology of diseases. Recent metabolism GWAS provided us a chance to explore the association between serum metabolites and risk of diseases through genetic instruments. Based on the idea of mendelian randomization, we studied the associations between metabolites-related SNPs and lung cancer risk in 13,945 cases and 12,939 controls. We identified a fatty acid (FA) related SNP that was significantly associated with lung cancer risk (odd ratio (OR)=0.87, P=1.76×10-15) and this effect was stronger in females. We further validated a significant association between this SNP and serum levels of this FA (Beta=-0.57, P=1.68×10-3) in 253 Chinese healthy individuals by LC-MS system. The association between the SNP and the expression of FA-related gene was also observed in the liver tissues (Beta=-0.84, P=6.49×10-3). Together, we organized a mendelian randomization study which provided a FA related SNP as a female concentrated and smoke independent genetic determinant of lung cancer from the aspect of metabolites and proposed that higher level of this FA was a causal risk factor of lung cancer.

#2561

Integrated analysis of ESCC tumor/normal tissues and patient blood samples using expression, genotypes and DNA somatic change data.

Howard H. Yang,1 Hua Su,1 Huaitian Liu,1 Nan Hu,1 Chaoyu Wang,1 Lemin Wang,1 Ti Ding,2 Carol Giffen,3 Alisa M. Goldstein,1 Philip R. Taylor,1 Maxwell P. Lee1. 1 _NIH, Bethesda, MD;_ 2 _Shanxi Cancer Hospital, Taiyuan, China;_ 3 _Information Management Services, Inc., Silver Spring, MD_.

Esophageal cancer is the eighth most common and sixth most frequent fatal human cancer in the world, and the fourth most common incident cancer in China. Shanxi Province, a region in north central China, has among the highest esophageal cancer rates in China, and nearly all of these cases are esophageal squamous cell carcinoma (ESCC). Understanding the molecular mechanisms underlying esophageal carcinogenesis and its molecular pathology will facilitate the development of biomarkers for early detection to reduce ESCC mortality. Genome-wide association studies (GWAS) have found many loci associated with the development of ESCC, however, most of the significant SNPs identified by GWAS are noncoding variants with unknown functions located in intergenic regions far from genes and are difficult to study functionally. As an initial effort to identify causal variants for ESCC, we focused here on loci near genes with known biological functions. Our analysis consists of three steps. We identified some functional variants for ESCC in the second and third steps of the analysis. First, from the GWAS analysis we selected those loci in or around the genes in three important pathways - inflammation, immunity, and DNA repair - using information from KEGG, Biocarta, and GO databases. We selected loci which had their 95% confidence intervals of the odds ratios not including OR=1 based on GWAS analysis. Second, we integrated the expression and genotype data from ESCC somatic and germline samples and computed eQTLs using the normal tissues for n=100 cases which had both gene expression data and GWAS genotype data. We found some interesting results from the second step analysis. The top 20 most significant genes included XPC, TP73, CASP8, DAPK1, IGF1R and LEPR, which were implicated in different cancers (Cancer Genetics Web: http://www.cancer-genetics.org). CASP8 was known to be involved in apoptosis processes. It was significantly associated with early onset of esophageal adenocarcinoma (Wu et al, Neoplasia. 2011 Apr;13(4):386-92). It had been shown that IGF1R had a key role in malignant progression of esophageal cancer (Kalinina et al, Int J Cancer. 2010 Oct 15;127(8):1931-40; Doyle et al, Am J Gastroenterol. 2012 Feb;107(2):196-204). Finally, we further intersected these significant loci in the eQTL analysis with the loci that had significant association between genotype and the somatic DNA copy number alterations. One of the significant genes was ST6GAL1. It had been reported this gene may promote malignant progression by promoting the TGF-beta induced EMT through down-regulation of E-cadherin-mediated cell adhesion and up-regulation of integrin-mediated cell migration (J. Lu et al, J Biol Chem. 2014 Dec 12;289(50):34627-41). We also found some novel genes in the analyses and their relation to ESCC will be investigated in future.

#2562

Bladder cancer GWAS signal at 4p16.3 affects response of TMEM129 to chemically-induced endoplasmic reticulum stress.

A. Rouf Banday, Eniko Kiss, Wusheng Yan, Candace Middlebrooks, Ludmila Prokunina-Olsson. _National Cancer Institute, NIH, Gaithersburg, MD_.

Background: SNP rs798766 within the 4p16.3 region was associated with bladder cancer risk in several GWAS. This SNP is located within a linkage disequilibrium block that includes TMEM129, TACC3, FGFR3 and SLBP. We aimed to explore molecular mechanisms of this GWAS signal.

Methods: Expression analysis of all isoforms of TMEM129, TACC3, FGFR3 and SLBP was performed in the Cancer Genome Atlas (TCGA) bladder cancer dataset (N=412). Expression was analyzed by linear regression in relation to rs798766 genotypes adjusting for age, sex, race, DNA CpG methylation status and copy number variation (CNV). Expression in additional bladder tumors (N=42) was tested with TaqMan expression assays and with splicing form-specific in situ hybridization (ISH) assays in a tissue microarray that included 7 normal-tumor bladder tissue pairs. Allele-specific TMEM129 exontrap assays in 5 bladder cancer cell lines were used to evaluate alternative splicing. TMEM129 isoforms were cloned and overexpressed in cell lines for functional characterization by confocal microscopy, reporter assays, RNA-seq and qRT-PCR arrays to explore their effects on cell signaling and cell viability. Endoplasmic reticulum (ER) stress was induced by treating bladder cancer cell lines with chemicals dithiothreitol (DTT), tunicamycin and MG132.

Results: Of all the isoforms of four genes tested the strongest association with rs798766 genotypes was observed for TMEM129-b transcript (padj =1.65E-06); expression was decreased with bladder cancer risk allele. Exontrap analysis revealed that allele G of a SNP rs2236786 located within exon 3 of TMEM129 (r2=1.0 with GWAS SNP rs798766) is responsible for alternative splicing of TMEM129. Allele-specific alternative splicing also results in lower expression of TMEM129-b and creation of a new TMEM129-a2 transcript. TMEM129-a2 is predicted to undergo nonsense mediated decay (NMD) except when under ER stress. TMEM129 is an E3 ligase and its protein isoforms are predicted to be functionally different. Confocal imaging showed that all isoforms are located in ER and thus can compete with each other. Overexpression of TMEM129 isoforms resulted in differential activation of genes from the main ER stress response pathways - IRE1, PERK and ATF6. Chemically-induced ER stress increased expression of all TMEM129 isoforms, although the induction was lower in cell lines with risk G allele of rs2236786.

Conclusions: The risk of bladder cancer is known to be increased by environmental exposures. Our results suggest that bladder cancer risk could be modulated by a genetic variant that affects the function of TMEM129, an E3 ligase. This mechanism could be important for response to environmentally-induced ER stress and subsequent development of bladder cancer.

#2563

Analysis of a novel gene in relation to bladder cancer GWAS signals within the 20p12.2 region.

Rouf Banday, Wusheng Yan, Candace D. Middlebrooks, Eniko Kiss, Ludmila Prokunina-Olsson. _NCI-DCEG, Bethesda, MD_.

Background: Genome-wide association studies (GWAS) of bladder cancer risk recently identified several signals on chromosome 20p12.2 marked by single nucleotide polymorphisms (SNPs) rs6104690, rs62185668 and rs6108803 (Figueroa et al, HMG, 2015). Interestingly, associations of SNPs rs62185668 and rs6108803 were significantly stronger for the risk of muscle-invasive (stages T2-T4) compared to non-muscle-invasive (stages Ta and T1) bladder cancer, suggesting that detailed analysis of these signals could further our understanding of aggressive bladder cancer.

Methods: We used data from The Cancer Genome Atlas (TCGA) bladder cancer set (n=412) and an independent RNA-seq dataset for 12 bladder tissues. qRT-PCR analysis was performed with custom TaqMan assays in an additional set of 40 tumor and 40 adjacent normal bladder tissue samples. RNAscope in situ hybridization analysis with custom probes was used to evaluate mRNA expression in bladder tissue microarrays that included 7 pairs of normal-tumor bladder tissues.

Results: At a distance of more than 300 and 800 Kb, JAG1 and BTD3 are the closest genes to the GWAS signals in the 20p12.2 region. Analysis of TCGA data did not show significant association of JAG1 and BTD3 expression with genotypes of 20p12.2 GWAS markers analyzed through TCGA-genotyped proxies SNPs rs6040291 and rs6074214. Using RNA-seq data in an independent set of 12 bladder tissue samples, we discovered a novel putative gene in the 20p12.2 region. Cloning of this gene from bladder tumor tissue and annotation by several methods showed two alternative splicing forms, designated as major and minor transcripts. Expression of the major transcript in bladder tumors was significantly increased with the rs6104690-A allele (P = 0.014), which is associated with increased risk of bladder cancer. SNPs with stronger association with muscle-invasive bladder cancer (rs62185668 and rs6108803) showed weaker or non-significant associations with expression of this novel transcript (P = 0.037 and P = 0.42, respectively). This transcript might be protein-coding as it is predicted to have several open reading frames for short peptides, or it may be a long non-coding RNA (lncRNA). RNAscope analysis in bladder tumors showed nuclear expression of the major transcript. Functional analysis of this novel gene in relation to bladder cancer is underway.

Conclusions: A novel gene might be related to the GWAS signals within the 20p12.2 region and the risk of bladder cancer.

#2564

Cis and trans quantitative trait loci for MGMT promoter hypermethylation detected in lung exfoliated cells collected in sputum from smokers.

Shuguang Leng,1 Hector Guillen,2 Carmen Tellez,1 Maria Picchi,1 James Swenberg,3 Frank Gilliland,4 Steven Belinsky1. 1 _Lovelace Respiratory Research Institute, Albuquerque, NM;_ 2 _Texas Biomedical Research Institute, San Antonio, TX;_ 3 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 4 _University of Southern California, Los Angeles, CA_.

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. Concomitant methylation of a panel of cancer-relevant genes including MGMT detected in lung epithelial cells present in sputum predicts risk for subsequent lung cancer incidence in moderate and heavy smokers. In addition, MGMT methylation is also a prognostic biomarker for response of glioblastoma patients to the alkylating agent temozolomide. A single nucleotide polymorphism (SNP, rs16906252, G/A) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. The predisposition of the "A" allele for methylation in heterozygotes was due to the reduced gene transcription in luciferase reporter assay. In silico bioinformatics analysis suggested that rs16906252 was the causal cis-acting methylation quantitative trait locus for MGMT. We are current applying a cutting edge technology "Hybridization Capture of Chromatin-Associated Proteins for Proteomics" to identify the transcriptional factors whose binding was disrupted by rs16906252. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells, while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells. In addition, genetic polymorphisms that affect DNA methylation of the DNA repair gene MGMT have strong clinical relevance in smokers, not only for cancer risk assessment but also for stratification of lung cancer patients for alkylating agent chemotherapy. (Supported by NIH grant R01 CA097356 and NIH/NCI grant P30 CA118100).

#2565

Prostate cancer GWAS from more than 89,000 men identifies more than 30 novel prostate cancer susceptibility loci.

Fredrick R. Schumacher,1 Ali A. Al Olama,2 Sonja I. Berndt,3 Fredrik Wiklund,4 David V. Conti,1 Mahbubl Ahmed,5 Sarah Benlloch,2 Douglas F. Easton,2 Peter Kraft,6 Stephen J. Chanock,7 Brian E. Henderson,1 Zsofia Kote-Jarai,5 Christopher A. Haiman,1 Rosalind A. Eeles5. 1 _USC/Norris Comprehensive Cancer Center, Los Angeles, CA;_ 2 _Univeristy of Cambridge, Cambridge, United Kingdom;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Karolinska Institute, Stockholm, Sweden;_ 5 _Institute of Cancer Research, London, United Kingdom;_ 6 _Harvard School of Public Health, Boston, MA;_ 7 _National Cancer Institute, Bethesda, DC_.

Genome-wide association studies (GWAS) and fine-mapping efforts have identified over 100 loci associated with prostate cancer susceptibility. Combined multiplicatively, these loci capture 33% of the familial risk for prostate cancer among European-ancestral populations. In order to identify novel prostate cancer susceptibility loci we conducted a GWAS involving over 53,000 prostate cancer cases and 36,000 controls using the OncoArray that includes a 260K GWAS backbone and a custom portion of 310K SNPs developed from previous GWAS and fine-mapping studies of multiple cancer types (http://epi.grants.cancer.gov/oncoarray/). Following standard quality control procedures, the prostate cancer OncoArray genotyped data were imputed using the October 2014 release of the 1000 genomes project data as a reference and analyzed by participating study for overall and advanced prostate cancer using logistic regression. The study-specific results were combined using inverse variance fixed effect meta-analysis. Regions surrounding previously associated variants (+/- 500kb of the reported index variant) were excluded to identify novel associations. In the OncoArray meta-analysis, we identified more than 30 novel loci significantly associated (P<5.0x10-8) with overall, advanced (Gleason ≥8, death from PrCa, PSA>100, or disease stage "Distant"), or early-onset (≤55 years of age) prostate cancer. Previous GWAS of prostate cancer are being imputed to the latest release of the 1000 genomes project to enhance our statistical power for novel discovery. In total our overall meta-analysis will incorporate over 176,000 individuals of European ancestry - more than 95,000 prostate cancer cases and 79,000 controls. The current results, as well as the larger ongoing meta-analysis, provide further insight into the underlying mechanisms of prostate cancer carcinogenesis and will improve the utility of genetic risk scores for targeted screening and prostate cancer prevention.

#2566

Comprehensive analysis to identify functional basis of prostate cancer risk SNPs.

Mridu Middha,1 Xing Xu,2 Riina-Minna Vaananen,3 James Hayes,1 Pekka Taimen,4 Xiaoni Gao,1 Hans G. Lilja,5 Kim Pettersson,3 Robert J. Klein1. 1 _Icahn Institute for Genomics and Multiscale Biology, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Program in Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Division of Biotechnology, University of Turku, Turku, Finland;_ 4 _Department of Pathology, University of Turku and Turku University Hospital, Turku, Finland;_ 5 _Department of Laboratory Medicine, Surgery and Medicine, Memorial Sloan Kettering Cancer Center, New York, NY_.

Introduction:

Genome-wide association studies (GWAS) have identified numerous common single nucleotide polymorphisms (SNPs) associated with the risk of developing prostate cancer. Many of these prostate cancer GWAS hits occur in intergenic regions, distant from any annotated gene. Little is yet known about how these SNPs function to alter an individual's risk of prostate cancer. Here we test the hypothesis that many of these SNPs, or SNPs with which they are correlated, alter regulatory regions and thus gene expression in the prostate.

Materials and Methods:

To test if prostate cancer risk SNPs are correlated with gene expression changes, we conducted expression quantitative trait locus (eQTL) association analysis. Using genome-wide high-density genotypes and gene expression data from 56 tumor and 58 adjacent normal prostate tissues we asked if any of the prostate cancer risk SNPs correlate with expression changes in nearby genes using linear regression, adjusting gene expression levels for principal components of ancestry and different batches. We also asked if individuals who have a higher genetic risk of prostate cancer based on the genotype of all of their risk SNPs show different gene expression patterns in the prostate than those at lower risk of prostate cancer.

Results:

We have identified several novel cis-eQTLs in prostate tissue for prostate cancer risk SNPs including rs1933488 with RGS17. We have also replicated several previously known cis-eQTLs, including those for IRX4, PPP1R14A, and FOXP4. Individuals at higher genetic risk of developing prostate cancer have altered expression of 37 genes (p<0.001) in their tumor tissue compared to individuals at lower genetic risk.

Conclusions:

These data demonstrate that several prostate cancer risk SNPs are associated with expression changes in nearby genes in both pathologically malignant and pathologically benign prostate tissue from patients who underwent radical prostatectomy. Additional genes, including HMOX1, CSGALNACT1, and WDR36 show altered expression in patients with different levels of genetic risk of prostate cancer.

#2567

Characterizing histologic subtypes in lung cancer using principal component analysis.

Jinyoung Byun, Younghun Han, Christopher I. Amos. _Dartmouth College, Hanover, NH_.

Lung cancer is the leading cause of cancer death in the United States and worldwide. The two major forms of lung cancer are small-cell lung carcinoma (SCLC) and non-SCLC (NSCLC). NSCLC is divided into three major histologic subtypes: Squamous-cell carcinoma, adenocarcinoma, and large-cell. Histological subtypes of cancers vary with respect to the genes that shows altered regulation, compared to normal bronchioepithelial cells. Lung cancer histologic diagnosis is clinically relevant because there are histology-specific treatment indications and contradictions.

Principal component analysis (PCA) is one of the useful statistical tools for analyzing multivariate data and recently high-dimensional data in genetics and genomics. We demonstrated the classification between two major subtypes of lung cancer: 488 squamous cell carcinoma and 788 adenocarcinoma cases. We selected the top 20k significant SNPs using logistic regression and then conducted PCA as the unsupervised approach.

We observed a distinct pattern between two major subtypes of NSCLC in genome-wide association study (GWAS). There were several publications to classify the subtypes of histology in lung cancer using gene expression data. This enables to understand the patterns for other subtypes of lung cancer in GWAS.

#2568

Meta-analysis of genome-wide association studies identifies multiple lung cancer susceptibility loci in never-smoking Asian women.

Qing Lan,1 Zhaoming Wang,2 Wei Jie Seow,1 Kouya Shiraishi,3 Hongbing Shen,4 Chao A. Hsiung,5 Keitaro Matsuo,6 Jie Liu,7 Kexin Chen,8 Taiki Yamji,9 Yang Yang,10 I-Shou Chang,5 Chen Wu,11 Meredith Yeager,1 Takashi Kohno,3 Wei Zheng,12 Xiao-Ou Shu,12 Pan-Chyr Yang,13 Tangchun Wu,14 Dongxin Lin,11 Baosen Zhou,15 Jinming Yu,7 Michiaki Kubo,16 Stephen J. Chanock,1 Nathaniel Rothman1. 1 _NCI-DCEG, Bethesda, MD;_ 2 _Cancer Genomics Research Laboratory, Rockville, MD;_ 3 _National Cancer Center Hospital, Tokyo, Japan;_ 4 _Nanjing Medical University, Nanjing, China;_ 5 _National Health Research Institutes, Taipei, Taiwan;_ 6 _Aichi Cancer Center Research Institute, Aichi, Japan;_ 7 _Shandong Cancer Hospital and Institute, Jinan, China;_ 8 _Tianjin Medical University Cancer Institute and Hospital, Tianjin, China;_ 9 _National Cancer Center, Tokyo, Japan;_ 10 _Shanghai Pulmonary Hospital, Shanghai, China;_ 11 _Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China;_ 12 _Vanderbilt Epidemiology Center, Nashville, TN;_ 13 _National Taiwan University Hospital, Taipei, Taiwan;_ 14 _Tongji Medical College, Shanghai, China;_ 15 _China Medical University, Shenyang, China;_ 16 _RIKEN Center for Integrative Medical Sciences, Yokohama, Japan_.

Background: The incidence rates of lung cancer among never-smoking females in some parts of East Asia are among the highest in the world. Genome-wide association studies (GWAS) of lung cancer in Asian never-smoking women have previously identified six susceptibility loci associated with lung cancer risk including 3q28, 5p15.33, 6p21.32, 6q22.2, 10q25.2 and 17q24.3. This study aims to discover additional lung cancer susceptibility loci among never-smoking Asian females.

Methods: We imputed data from four GWAS of Asian non-smoking female lung cancer (6,877 cases and 6,277 controls) using the 1,000 Genomes Project (Phase 1 Release 3) data as the reference and genotyped additional samples (5,878 cases and 7,046 controls) for potential replication.

Results: In our meta-analysis, three new loci achieved genome-wide significance, marked by the single nucleotide polymorphisms rs7741164 at 6p21.1 (per-allele odds ratio (OR) = 1.17; P = 5.8 × 10-13), rs72658409 at 9p21.3 (per-allele OR = 0.77; P = 1.41 ×10-10), and rs11610143 at 12q13.13 (per-allele OR = 0.89; P = 4.96 ×10-9).

Conclusions: We identified new genetic susceptibility alleles for lung cancer in never-smoking women in Asia. These findings merit follow-up to understand their biological underpinnings and potential interactions with environmental exposures.

#2569

A genome wide association study of lung cancer identifies 11 novel susceptibility loci.

James Dowling Mckay,1 Yafang Li,2 Younghun Han,2 Xiangjun Xioa,2 John Field,3 Xuchen Zong,4 Heike Bickeböller,5 David C. Christiani,6 Paul Brennan,1 Maria-Teresa Landi,7 Rayjean Hung,4 Christopher I. Amos,2 on behalf of the OncoArray Lung Cancer Group. 1 _International Agency for Res. on Cancer, Lyons, France;_ 2 _Dartmouth College, NH;_ 3 _University of Liverpool, United Kingdom;_ 4 _Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, Ontario, Canada;_ 5 _University Medical Centre Göttingen, Göttingen, Germany;_ 6 _Harvard School of Public Health, Boston, MA;_ 7 _Division of Cancer Epidemiology & Genetics, National Cancer Institute, Bethesda, MD_.

Background

Genome wide association studies (GWAS) of lung cancer have identified susceptibility loci at 15q25, 5p15, 6p21, 13q13 and 22q12 that contain relevant candidate genes such as CHRNA3/5, TERT, HLA, BRCA2 and CHEK2, respectively. Many of these alleles appear more relevant to a particularly histological type of lung cancer.

With the aim of identifying novel lung cancer susceptibility loci, The Transdisciplinary Research in Cancer of the Lung (TRICL), the International Lung Cancer Consortium (ILCCO) and Lung Cancer Cohort Consortium (LC3) have undertaken a very large collaborative GWAS across 29 lung cancer studies, including analysis of the predominant lung cancer histological subtypes.

Methods

In collaboration with the Center for Disease Research (CIDR), TRICL, ILCCO and LC3 have genotyped 18,000 case-control pairs on the GAME-On Oncoarray. SNP imputation was undertaken using the 1,000 Genomes v3 reference panel, followed by logistic regression of each genetic variant with lung cancer and considering ancestry inferred by genetic profile to correct for cryptic population structure. The OncoArray and our previous GWAS results were combined using meta-analysis. This allowed for a GWAS of 10,155,682 SNPs for 25,655 lung cancer cases and 52,451 controls, as well as histology specific analysis of 6,629 squamous cell and 9,817 adenocarcinomas. Alternate genotyping techniques (Affymetrix, Taqman) were used to confirm the fidelity of the genotyping for variants of interest.

Results

We have identified common genetic variants exceeding genome wide significance (p<5x10-8) at eleven novel susceptibility loci. This included two associated with overall lung cancer (1p31, 19q13), eight with lung adenocarcinomas (3q28, 6p25, 8p12, 9p21, 10q25, 11q23, 15q21, 20q13) and one with squamous-cell lung carcinomas (10q24). These genetic variants are located near several intriguing candidate genes, such as telomere function genes (OBFC1, RTEL1), nicotine metabolism genes (CYP2A6), genes somatically mutated in lung cancer (NRG1) and genetic susceptibility loci linked to other cancers (IRF4, CDNK2A). In additional, we noted several borderline (p<10-6) associations with common variants located near nicotine addiction genes (CHRNB2, CHRNA2, CHRNA4, DBH) and other genes somatically translocated in lung cancer (ROS1). Integration of eQTL databases suggests that many of the associated genetic variants influence gene expression levels of these candidate genes.

Conclusion

We have identified eleven novel lung cancer susceptibility loci, doubling the number implicated by GWAS. These genetic variants were common (MAF 0.05-0.49) with modest to small genetic effects (OR's 1.10-1.17). Further expansion of GWAS efforts, particularly within histological subtypes of lung cancer, is likely to identify additional susceptibility loci and further increase our understanding of lung cancer aetiology.

#2570

Identification of genetic polymorphisms associated with myelodysplastic syndromes by genome-wide association study.

Kathy McGraw,1 Chia-Ho Cheng,1 Ann Chen,1 Giulio Genovese,2 Thomas Cluzeau,3 Hui-Yi Lin,4 Bartlomiej Przychodzen,5 Mar Mallo,6 Leonor Arenillas,7 Azim Mohamedali,8 Lionel Ades,9 Ashley Basiorka,10 Brittany Irvine,1 David Sallman,1 Eric Padron,1 Lubomir Sokol,1 Andrea Pellagatti,11 Chimene Brest,12 Sophie Raynaud,3 Bjorn Nilsson,13 Jacqueline Boultwood,11 Benjamin Ebert,14 Francesc Sole,6 Pierre Fenaux,9 Ghulam Mufti,8 Jaroslaw Maciejewski,5 Peter Kanetsky,1 Alan List1. 1 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL;_ 2 _Broad Institute of the Massachusetts Institute of Technology and Harvard Medical School, Boston, MA;_ 3 _French Group of Myelodysplasia and CHU of Nice, Nice, France;_ 4 _Louisiana State University, Baton Rouge, LA;_ 5 _Cleveland Clinic, Taussig Cancer Institute, Cleveland, OH;_ 6 _Institut de Recerca Contra la Leucemia Josep Carreras, Barcelona, Spain;_ 7 _Laboratori de Citologia Hematologica, Hospital del Mar, Barcelona, Spain;_ 8 _Kings College London, London, United Kingdom;_ 9 _French Group of Myelodysplasia and Hospital Saint Louis, Paris, France;_ 10 _H. Lee Moffitt Cancer Center and Research Institute and University of South Florida, Tampa, FL;_ 11 _Leukaemia & Lymphoma Research Molecular Haematology Unit, University of Oxford, Oxford, United Kingdom; _12 _CHU of Nice, Nice, France;_ 13 _Lund University, Lund, Sweden;_ 14 _Harvard Medical School and Brigham and Women's Hospital, Boston, MA_.

Background: The myelodysplastic syndromes (MDS) are senescence-dependent stem cell malignancies. Although germ line mutations in RUNX1, CEBPA, ETV6, or GATA2 may underlie familial cases, inherited genetic polymorphisms influencing MDS susceptibility has not been evaluated. We performed the first genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) linked to MDS predisposition.

Methods: DNA from 1361 MDS patients from 9 international centers was genotyped on the Affymetrix Genome-Wide Human SNP Array 6.0; all patients signed informed consent. Data on 4597 healthy controls genotyped on the same platform were obtained from the Database of Genotypes and Phenotypes (dbGaP). Applying standard quality control metrics and limiting to individuals of European ancestry, we analysed 999 MDS patients and 4,309 controls at 545,924 markers. We used logistic regression to determine associations with MDS after appropriate adjustment assuming an additive genetic model.

Results: We identified 15 SNPs at 9 loci associated with MDS, including rs6780298 (3q13; p=5.0x10-6) and rs1206819 (20q13; p=1.54x10-6) that mapped to the intragenic regions of MORC1 and EYA2, respectively; and rs2473784 (1p31; p=3.32x10-7) that mapped downstream of DEPDC1. Other hits (p≤5x10-6) at 4q28, 5p14, 9p22, 6p22, 10q21 are in or near SLC7A11, GUSBP1, SH3GL2, MOG and TRIM27, respectively. Using publicly available gene expression profiling data, we confirmed all of these genes are expressed in the hematopoietic compartment and MORC1, EYA2, DEPDC1, and SH3GL2 are increased in leukemic samples suggesting that variation in these genes may be functionally relevant in myeloid malignancies. Notably, EYA2 (20q13) flanks the commonly deleted region in del(20q) MDS and has oncogenic properties in solid tumors. SH3GL2 is very highly upregulated in leukemic samples and interacts with Dynamin-1, a GTP-binding protein involved in cellular trafficking and is similarly associated with solid tumorigenesis. To validate our observed associations, we analysed the 15 SNP markers in an independent set of 12,385 individuals of whom 117 developed hematologic malignancy (including MDS) during follow up. The markers at 1p31 including rs2473784 and 4 others in strong linkage disequilibrium were associated with individuals who developed hematologic malignancy (p<0.05). One other SNP (rs404660) showing an association in both the MDS GWAS analysis and the hematologic malignancy dataset is also currently under further investigation.

Conclusions: These data provide the first genome-wide identification of germline variants associated with MDS. Current work is underway to replicate these findings in an independent sample set of MDS patients and healthy controls. If confirmed, these SNPs may serve as biomarkers to identify individuals at risk for MDS and potentially support new prevention strategies.

#2571

In silico **and c** is **-expression QTL analysis of Barrett's esophagus and esophageal adenocarcinoma GWAS variants link cholesterol trafficking to Barrett's esophagus susceptibility.**

Paula L. Hyland,1 Hyuna Sung,1 Howard H. Yang,2 Lei Song,1 Nan Hu,1 Bin Zhu,1 Alisa M. Goldstein,1 Philip R. Taylor1. 1 _NCI-DCEG, Bethesda, MD;_ 2 _NCI-CCR, Bethesda, MD_.

Eight high-evidence susceptibility loci for Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) have been identified from genome-wide association studies (GWAS), but the functional relevance of these variants in the esophagus has not been studied. We used cis-expression quantitative trait loci (cis-eQTL) and in silico functional annotation analyses in esophageal tissues to annotate each variant. We observed that five variants influenced the expression of other neighboring coding and noncoding genes in esophageal mucosa, muscularis, and gastroesophageal junction tissues using NIH Genotype-Tissue Expression (GTEx) data. We identified and independently validated rs3072 (G) (BE odds ratio [OR]: 1.14, 95% CI: 1.09-1.18, P=1.8E-11 [Palles et al. Gastroenterology 2015;148:367-78]) as a negative cis-eQTL for the cholesterol hydrolase gene C2orf43 (alias lipid droplet associated hydrolase [LDAH]) (beta= -0.55, P=4.3E-17) in esophageal mucosa. In silico analysis suggests rs13394027 (or rs9306894) may be the more functionally relevant BE risk variant influencing a CCCTC-binding factor (CTCF) barrier to C2orf43 repression in esophageal tissues. Our results suggest a potential link between cholesterol homeostasis and/or trafficking in the esophagus and BE susceptibility which warrants investigation in future studies.

### Risk Prediction, Screening, and Comparative Effectiveness Research

#2573

Cancer diagnoses and patient use of electronic health records: results from the health information national trends survey.

Alexandra J. Greenberg,1 Angela Falisi,1 Lila J. Finney Rutten,2 Wen-Ying Sylvia Chou,1 Richard P. Moser,1 Bradford W. Hesse1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Mayo Clinic, Rochester, MN_.

The increasing adoption of electronic health records (EHRs) in the United States, spurred largely by the Centers for Medicare and Medicaid's EHR Meaningful Use Incentive Program, has potential to increase patient engagement and improve quality of healthcare delivered, especially for those diagnosed with cancer or other chronic conditions. Meaningful Use Stage 2 criteria require that providers give patients the ability to view, download and transmit their medical records; this is of particular importance for cancer patients, whose care is often delivered by teams of different specialists and for whom accurate communication of medical and treatment history between patients and providers is critical. To investigate whether there is a difference in patient access to and utilization of EHRs based on history of cancer diagnosis, we analyzed data from the National Cancer Institute's Health Information National Trends Survey (HINTS 4, Cycle 4). The survey was fielded between July and November 2014 via postal mail; 3,370 complete surveys were returned (response rate=34.4%). Of the respondents, 6.4% reported having a prior diagnosis of a cancer other than non-melanoma skin cancer. Among those with a self-reported history of cancer, 48.2% reported being offered access to their EHRs and 30.8% accessed their EHRs at least once, compared with those who did not have cancer (41.8% and 27.2%, respectively; p > 0.05). After adjusting for sociodemographic and healthcare-related characteristics, no significant difference was found in the odds of being offered EHR access based on cancer diagnosis status. Those with a history of cancer had a statistically significantly higher odds of accessing their EHRs at least once compared to those without a diagnosis of cancer after adjustment, (OR = 1.75, 95% CI = 1.08-2.81, p = 0.02). Other significant predictive factors included in the model were age, education, health insurance, having a regular provider, and internet access (p < 0.05). A borderline association was also seen between a previous cancer diagnosis and increased frequency of EHR access (p = 0.08). These findings suggest that, if offered access to their records, those with a diagnosis of cancer have higher odds of using their EHRs; further investigation is needed to determine how patients are using EHRs to help manage their care, the impact of this use on cancer-specific outcomes, and associations with quality of care.

#2574

Outcomes of contralateral prophylactic mastectomy in relation to familial history: a decision analysis.

Kalatu R. Davies, Abenaa Brewster, Isabelle Bedrosian, Patricia Parker, Melissa A. Crosby, Susan K. Peterson, Yu Shen, Robert Volk, Scott B. Cantor. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Purpose

Family history of breast cancer is associated with an increased risk of contralateral breast cancer (CBC) even in the absence of mutations in the breast cancer susceptibility genes BRCA1/2. We compared quality-adjusted survival outcomes after contralateral prophylactic mastectomy (CPM) with surveillance only (no CPM) among women with breast cancer in relation to degree of family history.

Methods

We created a microsimulation model for women with a first-degree, second-degree and no family history treated for a stage I, II, or III estrogen receptor (ER)-positive or -negative breast cancer at the ages of 40, 50, 60, and 70 years. The model incorporated a 10-year post-treatment period for risk of developing CBC and/or dying of the primary cancer or CBC. For each patient profile, we used 100,000 microsimulation trials to estimate the quality-adjusted life expectancy for two clinical strategies: CPM and no CPM.

Results

CPM had a minimal improvement on quality-adjusted life expectancy among women age 50 to 60 with no or a unilateral first or second-degree family history (range -.06 to 0.31) and was unfavorable for the majority of women age 70 with stage III breast cancer regardless of degree of family history (range -.08 to -.02). Sensitivity analysis showed the highest predicted benefit of CPM with 95% risk reduction was 0.57 QALY for a 40-year-old woman with stage I breast cancer who had a first-degree relative with bilateral breast cancer.

Conclusion

Women age 40 years with stage I breast cancer and a first-degree relative with bilateral breast cancer have a QALY benefit from CPM similar to that reported for BRCA1/2 mutation carriers. For the majority of women, having a family history does not improve the minimal effect of CPM on quality-adjusted life expectancy.

#2575

Adherence to oral oncolytics in patients with lung cancer.

Lisa M. Hess,1 Anthony Louder,2 Katherine Winfree,1 Yajun E. Zhu,1 Ana B. Oton,1 Radhika Nair2. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Comprehensive Health Insights, Louisville, KY_.

Introduction

With the growth in availability of oral agents for cancer treatment, maintaining adherence is key to achieve positive patient outcomes (such as improved survival rates) and also may reduce healthcare costs. The objectives of this study were to examine oral oncolytic usage patterns among lung cancer patients and to understand the relationship with healthcare resource utilization (HCRU), costs, and survival.

Procedures

Medical and pharmacy data from Humana were used to identify members with lung cancer (ICD-9-CM 162.2-162.9) who initiated oral oncolytic therapy between 1/1/2008-6/30/2013 without prior oral chemotherapy use. The study was focused on erlotinib due to infrequent use of other oral agents. Medication adherence (medication possession ratio - MPR) and persistence (time to discontinuation based on a 60 day gap in therapy) were evaluated across lines of therapy. Demographic and clinical characteristics and erlotinib usage of the study population were summarized descriptively. Generalized linear models were used to examine the association between erlotinib use and healthcare cost and utilization. Cox regression models were used to evaluate the association between erlotinib use and survival. In all the models adherence and persistence were grouped into quintiles.

Results

The 1452 members meeting eligibility criteria had a mean age of 71.9 (±9.1) years, were 45.9% male, and 88.9% were insured by Medicare; 62% received intravenous chemotherapy before initiating erlotinib. Nonadherence (MPR < 80%) was identified in 12.5% of members with ≥2 claims (n=1088). The highest adherence quintile was associated with both higher total healthcare costs ($5218 vs. $4028 per month; p<0.0001) and total pharmacy costs ($2618 vs. $1915 per month; p<0.0001) compared to lowest adherence quintile. Overall, the average duration of treatment was 204 (± 256) days. The highest persistence quintile was associated with fewer outpatient visits (2.9 vs. 3.6 per month; p=0.0126), inpatient stays (0.31 vs. 0.07 per month; p<0.0001) and emergency room visits (0.14 vs. 0.02 per month; p=0.0086), than the lowest persistence quintile. Persistence, but not adherence, was associated with improved survival outcomes [persistence quintiles 4 and 5 vs quintile 1, adjusted hazard ratio (adjHR) 0.21 and 0.51, respectively, p<0.0001; MPR quintiles 4 and 5 vs quintile 1, adjHR 1.73 and 1.94, respectively, p<0.0001].

Conclusions

Adherence to erlotinib was high in lung cancer, limiting detailed investigation into factors associated with reduced adherence. This study is limited in that all conclusions are based on the observed population without making any bias adjustment. We hypothesized that improved adherence would be associated with improved survival and reduced costs; this was not found to be the case. Those who remained on oral oncolytics for a longer period of time had a greater survival regardless of line of therapy.

#2576

**Radiation therapy delay and risk of ipsilateral breast tumors in women with ductal carcinoma** in situ **.**

Ying Liu,1 Shumei Yun,2 Min Lian,1 Graham Colditz1. 1 _Washington University School of Medicine in St. Louis, St. Louis, MO;_ 2 _Office of Epidemiology, Missouri Department of Health and Senior Services, Jefferson City, MO_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2016 Official Press Program. It will be posted online following its presentation.

#2577

A comparison of parent and provider verified HPV vaccination initiation and completion in US adolescents: findings from the National Immunization Survey - Teen, 2014.

Eric Adjei Boakye,1 Nosayaba Osazuwa-Peters,1 Kahee A. Mohammed,1 Christian J. Geneus,2 Betelihem B. Tobo1. 1 _Saint Louis University, Saint Louis, MO;_ 2 _Tulane University, New Orleans, LA_.

Introduction:

Several studies have examined predictors of HPV vaccine initiation and completion. Two common methods to measure HPV vaccine initiation and completion are parental recall and provider verified vaccine history. Some studies use parental recall because it is cost-effective and time-consuming. Others argue that the provider report should be used because it is the gold standard, although it is more costly and time-consuming. Yet, few studies compare the accuracy of parental report versus provider report for HPV vaccine initiation and completion.

Objective:

To compare the accuracy of parental reported and provider reported data on HPV vaccine initiation and completion among a national sample of adolescents in the United States.

Methods:

Data from the 2014 National Immunization Survey-Teen (NIS-Teen) were analyzed for 38,703 adolescents. HPV vaccine initiation and completion were assessed using parental recall (shot cards) and provider verified (medical records) vaccine history. Adjusted, weighted multivariable logistic regression compared the accuracy of parental and provider reported data on HPV vaccine initiation and completion.

Results:

HPV vaccine initiation (a) and completion (b), by subgroup analysis, is similar for parental recall and provider report. However, subgroup analyses for parental report were slightly lower than provider report, although not statistically significant (Table 1).

Conclusion:

This study showed that parental report is comparable to provider report when measuring HPV vaccine initiation and completion among adolescents in the US. For more cost-effective and timely assessment of vaccine uptake, an accurate and practical method of measuring HPV vaccine initiation and completion is paramount. Utilizing parental report may be a more cost-effective and timely alternative to provider report for clinical evaluation of vaccine uptake.

Table 1: Subgroup analysis of HPV vaccine initiation and completion, Odds Ratios (95% CI)

---

|

HPV Vaccine Initiation (a)

|

|

Parental Recall | Provider verified

Gender (Ref: Boys)  | 1.98 (1.81, 2.15) | 2.02 (1.81, 2.26)

Race (Ref: Caucasian) | |

African American | 1.19 (1.03, 1.37) | 1.31 (1.10, 1.56)

Hispanic | 1.38 (1.20, 1.58) | 1.51 (1.27, 1.79)

Region (Ref: South) | |

Northeast | 1.31 (1.18, 1.46) | 1.46 (1.27, 1.67)

Midwest | 1.11 (1.01, 1.22) | 1.15 (1.02, 1.30)

West | 1.25 (1.09, 1.43) | 1.42 (1.20, 1.69)

|

HPV Vaccine Completion (b)

|

|

Parental Recall | Provider verified

Gender (Ref: Boys)  | 2.24 (2.02, 2.48) | 2.37 (2.09, 2.69)

Race (Ref: Caucasian) | |

African American | 0.91 (0.77, 1.07) | 1.05 (0.87, 1.28)

Hispanic | 1.06 (0.91, 1.23) | 1.40 (1.17, 1.68)

Region (Ref: South) | |

Northeast | 1.39 (1.23, 1.57) | 1.55 (1.34, 1.80)

Midwest | 1.14 (1.02, 1.27) | 1.24 (1.08, 1.41)

West | 1.20 (1.03, 1.40) | 1.36 (1.12, 1.66)

#2578

Trust in health information source and colorectal cancer screening uptake among U.S. adults.

Adeyinka O. Laiyemo, Hamidat Segunmaru, Jessica Rogers, Mohammad Daremipouran, Clinton Burnside, Florencia Gonzalez, Cherie Spencer, Carla D. Williams. _Howard University College of Medicine, Washington, DC_.

Background: In order to promote positive health behavior of the population, public health outreach is often carried out in various forms to reach the target stakeholders. However, the effectiveness of these outreach programs is unclear.

Aim: To determine up-to-date adherence to colorectal cancer (CRC) screening guidelines among US adults according to the trust they have in various sources of health information.

Methods: We used the 2007 Health Information National Trends Survey (HINTS) and identified 4342 respondents (weighted population size = 82,811,829) who were at least 50 years old. Respondents expressed the degree of trust they have in various sources of health related information. We considered a source as trusted when it is trusted "a lot" or had "some trust" and regarded as not trusted if it was trusted "a little" or "not at all". We defined being current with CRC screening as the use of fecal occult blood testing (FOBT) within 1 year, sigmoidoscopy within 5 years, or colonoscopy within 10 years. We used logistic regression models to calculate odds ratios (OR) and 95% confidence intervals (CI). Survey weights were used in all analyses. Results: Approximately 94.3% of respondents trusted their doctors, 59.3% trusted family or friends, 50.2% trusted newsmagazines, 32.7% trusted radio, 66.2% trusted internet, 40.6% trusted television, 68.5% trusted government sources, 44.3% trusted charity organization and 34.5% trusted religious organizations. When compared with those who did not trust the source of health information, trust in radio (64.7% vs 60.7%; OR=1.24; 95%CI: 1.01-1.52), internet (64.0% vs 58.3%; OR=1.26; 95%CI: 1.04-1.53), television (64.4% vs 60.9%; OR=1.23; 95%CI: 1.02-1.48), and government sources (64.0% vs 58.2%; OR=1.32; 95%CI: 1.09-1.60) were associated with being up-to-date with colorectal cancer screening whereas trust in doctors (63.2% vs 49.2%; OR=1.53; 95%CI: 0.92-2.55), family or friends (61.5% vs 63.1%; OR=0.98; 95%CI: 0.84-1.15), newsmagazines (64.0% vs 60.8%; OR=1.14; 95%CI: 0.98-1.32), charity organizations (62.0% vs 62.1%; OR=1.04; 95%CI: 0.86-1.24) and religious organizations (59.7% vs 63.3%; OR=0.90; 95%CI: 0.74-1.09) were not.

Conclusion: Even with modest trust in the source of health information, outreach modalities which engage people repeatedly and those that they can readily access over and over again may exert substantial influence on the uptake of colorectal cancer screening. Multiple outreach methods should be used to promote colorectal cancer screening.

#2579

Longitudinal predictors of adherence to cervical cancer cotesting guidelines.

Katharine A. Rendle,1 Mark Schiffman,1 Li C. Cheung,2 Walter K. Kinney,3 Barbara Fetterman,4 Nancy E. Poitras,4 Philip E. Castle5. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Information Management Services, Inc., Calverton, MD;_ 3 _Kaiser Permanente Medical Care Program, Oakland, CA;_ 4 _Kaiser Permanente Northern California, Berkeley, CA;_ 5 _Albert Einstein College of Medicine, Bronx, NY_.

Introduction: Although clinical guidelines recommend cervical cancer screening using human papillomavirus (HPV) combined with Papanicolaou (Pap) testing for average-risk women, little is known about the longitudinal adoption of cotesting in clinical care, or if interval extension resistance is evident in practice. Using data from Kaiser Permanente Northern California (KPNC), which switched from annual Pap to 3-year interval cotesting in 2003, we examined predictors of cotesting guideline adherence.

Methods: We included all female patients aged 30-64 years who underwent baseline cotesting between 2003-2007 and completed at least one additional screening within 5.5 years. We excluded patients with a documented history of >CIN2, or positive HPV test at baseline or subsequent screenings (leaving 335,822 patients for analysis). We categorized interval length between cotesting into 3 categories: early (<2.5 years), adherent (2.5-3.5 years), and late (3.5-5.5 years). We used multinominal logistic regression models to examine the association (adjusted odds ratio [aOR]) between interval length from baseline cotesting to first follow-up cotesting (Interval 1) and the following predictors assessed at baseline: year of first cotesting, age, previous hysterectomy, and previous low-grade abnormal pap (<CIN2). We also examined predictors of persistent early screening in women classified as early screeners in both Interval 1 and Interval 2 (defined as time between first and second follow-up cotesting).

Results: Compared with the earliest cohort of women (baseline cotesting in 2003), the 2007 cohort was 66% and 52% less likely of being screened early (aOR=0.34, 95% CI: 0.33, 0.35) or late (aOR=0.48, 95% CI: 0.46, 0.50). However, among women classified as persistent early screeners, we found no clear trend in the relationship between year of initial cotesting and interval length. The strongest predictor of persistent early screening was older age, whereby women aged 60-64 years had the greatest likelihood of being a persistent early screener (aOR=2.1, 95% CI: 1.9, 2.3) versus women aged 30-34 years. Despite subsequent negative screens, women reporting a previous low-grade abnormal Pap at baseline had a greater likelihood of being a persistent early screener compared with women who did not (aOR=1.8, 95% CI: 1.6, 2.0).

Conclusions: Overall, women who underwent baseline cotesting in more recent years were significantly more likely to adhere to the recommended interval length than women in earlier years, indicating an increase in adoption of cotesting guidelines over time. However, there remain subgroups, including older women, which appear less likely to follow extended cotesting intervals. Further research is needed to understand these HPV screening practices in other healthcare settings and identify additional (potentially modifiable) patient and provider factors that are associated with screening practices.

#2580

Optimizing cervical cancer screening for HIV-infected women: A risk-based approach.

Hilary A. Robbins,1 L. Stewart Massad,2 Christopher B. Pierce,1 Lisa Flowers,3 Teresa M. Darragh,4 Howard Minkoff,5 Lisa Rahangdale,6 Marla J. Keller,7 Joel Milam,8 Margaret Fischl,9 Sadeep Shrestha,10 Christine Colie,11 Howard Strickler,7 Gypsyamber D'Souza1. 1 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 2 _Washington University School of Medicine, St. Louis, MO;_ 3 _Grady Memorial Hospital and Emory University School of Medicine, Atlanta, GA;_ 4 _University of California San Francisco, San Francisco, CA;_ 5 _Maimonides Medical Center, Brooklyn, NY;_ 6 _University of North Carolina School of Medicine, Chapel Hill, NC;_ 7 _Albert Einstein College of Medicine, Bronx, NY;_ 8 _University of Southern California, Los Angeles, CA;_ 9 _University of Miami Miller School of Medicine, Miami, FL;_ 10 _University of Alabama at Birmingham School of Public Health, Birmingham, AL;_ 11 _Georgetown University Medical Center, Washington, DC_.

Background: Women living with HIV (WLHIV) have increased cervical cancer risk. Guidelines suggest screening WLHIV with negative cervical cytology annually, and managing WLHIV with ASC-US cytology by general population (GP) guidelines (1 year return if HPV testing unavailable).

Methods: We used risk benchmarking to compare cervical precancer risk in WLHIV to the GP. For WLHIV ages 21-65 years in the Women's Interagency HIV Study (WIHS), we evaluated the first cytology result from 2000 or later. We used parametric survival models to calculate HSIL risks after negative or ASC-US cytology, overall and by CD4 cell count. Separately, we synthesized HSIL risk estimates among GP women in 13 published studies using mixed-effects models. We then benchmarked risks in WLHIV to the 3-year GP risks, which for negative cytology represent the threshold for re-screening.

Results: Among 2,653 WLHIV, 1,982 (75%) had negative cytology and 377 (14%) had ASC-US cytology. Most WLHIV with negative (72%) and ASC-US (52%) cytology had CD4≥350 cells/μL. We observed 95 cases of HSIL (CIN2+; 44 were CIN3+ including 1 cancer) within 5 years of cytology. After negative cytology, 3-year GP risk "benchmarks" were 0.83% (CIN2+) and 0.60% (CIN3+). WLHIV with CD4≥350 met both benchmarks by 2 years (1.1% and 0.68% respectively), while WLHIV with CD4<350 exceeded the CIN2+ benchmark at only 1 year (1.1%). After ASC-US cytology, 3-year GP benchmarks were 8.7% (CIN2+) and 4.4% (CIN3+). For WLHIV with CD4≥350, 3-year risks were similar to the benchmarks (9.4% and 4.0%), but WLHIV with CD4<350 approximated these risks at only 1 year (8.8% and 3.9%).

Conclusions: For WLHIV with CD4≥350, these data suggest that the interval for re-screening after negative cervical cytology can be lengthened from 1 to 2 years, and that inclusion in GP guidelines for managing ASC-US cytology is appropriate. In contrast, WLHIV with CD4<350 remain at increased risk and should be screened annually after negative cervical cytology and referred to colposcopy after ASC-US.

Cervical precancer risk after normal or ASC-US cytology in WLHIV compared to the general population

---

|

HSIL (CIN2+) risk, % | HSIL (CIN2+) risk, % | HSIL (CIN2+) risk, % | HSIL (CIN3+) risk, % | HSIL (CIN3+) risk, % | HSIL (CIN3+) risk, %

|

1 year | 2 years | 3 years | 1 year | 2 years | 3 years

NEGATIVE CYTOLOGY: | |  | |  | |

General population benchmark | |  | 0.83 | |  | 0.60

HIV+, overall (N=1,982) | 0.62 | 1.3 | 2.0 | 0.33 | 0.68 | 1.0

HIV+, CD4≥350 (N=1,393) | 0.47 | 1.1 | 1.7 | 0.38 | 0.68 | 0.96

HIV+, CD4<350 (N=542) | 1.1 | 2.0 | 2.8 | 0.24 | 0.65 | 1.1

ASC-US CYTOLOGY: | |  | |  | |

General population benchmark | |  | 8.7 | |  | 4.4

HIV+, overall (N=377) | 7.3 | 10.1 | 12.2 | 3.2 | 4.3 | 5.2

HIV+, CD4≥350 (N=191) | 5.5 | 7.8 | 9.4 | 2.5 | 3.3 | 4.0

HIV+, CD4<350 (N=175) | 8.8 | 12.8 | 15.9 | 3.9 | 5.5 | 6.8

#2581

Determinants for compliance to colorectal cancer screening in Korea: A multilevel analysis.

Aesun Shin, Hyeongtaek Woo. _Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea_.

BACKGROUND: This study aimed at investigating the individual and area level factors associated with colorectal cancer screening attendance.

METHODS: Colorectal cancer screening participation as well as other individual level data were gathered from the fifth Korea National Health and Nutrition Examination Survey, 2010-2012. The area level data were extracted from 2009 Health promotion strategies and programs development for health inequalities alleviation (Shin et al.,2009). Multilevel model was used for data analysis.

RESULTS: Information on a total of 8,783 individuals (3,777 men and 5,006 women) aged 50 years and older and deprivation index of each areas were used in the final analysis. Among individual level variables, men were more likely to participated in colorectal cancer screening within 5 years than women (58.0% for men vs 53.9% for women)(Odds Ratio (OR)=0.85; 95% CI 0.73 to 0.99), and highest among age 60-69 (63.3%). Area level variables's explained class-level variance was 6.4%.

CONCLUSION: In this study, by applying the multilevel analysis we can find that colorectal cancer screening attendance rate is associated with both individual and community factors.

#2582

Cervical cancer screening in rural East Africa.

Autumn Cummings,1 Brianna Rooney,1 Robert Chamberlain,1 Amr Soliman,1 Crispin Kahesa2. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Ocean Road Cancer Institute, Dar es Salaam, United Republic of Tanzania_.

Purpose: Cervical cancer screening (CCS) programs in rural east African communities often go unused by a majority the women residing in these areas. Most HIV-positive and HIV-negative women don't go for cervical cancer screening despite referrals at Bagamoyo District Hospital (BDH), Tanzania. The focus of this study was to understand what the patient barriers are to attending cervical cancer screening clinics.

Methods: Questionnaires were distributed to 114 female patients over the age of 18 within the BDH's Care and Treatment Center (CTC). A small sample of 15 patients was also approached from within the reproductive health clinic (RHC). Participants were asked about their age, religion, parity, marital status, village of birth, village of residence, education, and number of previous screens, why they decided to go (or not go) to screening, and how they first heard of cervical cancer as well as cervical cancer screening. We also gathered data on the number of times each patient had been previously screened, the date of the most recent screen, as well as the referral date for the next screen. Using this information, we then attempted to match the individual's records with that of the CCS clinic's records.

Results: The average age of women varied significantly between HIV-Positive and HIV-Negative patients (43 and 31, respectively). HIV-Positive women significantly varied among their education status. Parity and age varied significantly when comparing the screened women to those never before screened. When comparing by screen history, 22% of HIV-positive women had never been screened and all 15 (100%) of HIV-negative women had never had prior screening. Of the reasons given as to why women had not attended prior screening HIV-positive women reported that 36 (31.58%) think they were at risk; 25 (21.93%) had no reason; 20 (17.54%) didn't know when to go; and 19 (16.67%) didn't know where to go. As for the HIV-negative women, 11 (73.33%) said they didn't know where to go; 4 (26.67%) reported they didn't know when to go; 6 (40%) didn't think they were at risk. When analyzing reasons for not attending screening by whether or not prior screening had occurred, we found that most of the women who had never been screened reported to not know when or where to go for screening (36%).

Conclusion: There are many potential explanations that play a role in the low cervical cancer screening attendance rates. There are issues surrounding education, accessibility, and funding. Due to the high prevalence of HIV in these areas, it is important to address these issues in order to minimize the impact of cervical cancer. We found lack of education and knowledge about the screening program at BDH was the main explanation for why women did not utilize the screening services.

#2583

Mammography decision-making: Trends, predictors and effects of provider communication in the Health Information National Trends Survey 2011-2015.

Laura Spring,1 Megan R. Marshall,1 Erica T. Warner2. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Harvard School of Public Health, Boston, MA_.

Introduction: In 2009 the US Preventive Services Task Force (USPSTF) recommended against routine annual mammograms among women aged 40-49. However, mammography utilization appears unchanged since that time. We examined whether women, particularly those ages 40-49, are having discussions about mammography with their healthcare providers and how this affects screening behavior.

Methods: Data were drawn from the 2011-2014 Health Information National Trends Survey (HINTS). We included female respondents age 40 or older with no personal history of breast cancer who reported a medical visit in the previous two years, and responded to the following question: 'Has a doctor or other health professional ever told you that you could choose whether or not to have a mammogram?' (N=5085). We examined trends in the proportion of women reporting provider communication on mammogram choice according to 10-year age group. We used logistic regression to generate odds ratios (OR) and 95% confidence intervals (CI) for predictors of provider communication and also assessed whether provider communication was associated with mammography screening in the previous two years.

Results: Less than half of the women reported provider communication on mammogram choice in each year (2011:44.3%; 2012: 37.5%; 2013: 37.0%; 2014: 43.6%). In every year except 2014, women age 40-49 were most likely to report provider communication, though this difference was only significant in 2013 (45% vs. 30.2-36.6%; p=0.02). In multivariable-adjusted models, women age 40-49 were 27% more likely to report provider communication on mammogram choice (OR: 1.27, 95% CI: 1.02-1.59) compared to women age 50-59. Non-Hispanic black women were 33% less likely to report such communication (OR: 0.67, 95% CI: 0.52-0.88) compared to non-Hispanic Whites. The youngest (40-49 years) and oldest (≥70 years) women were most likely to report no mammogram in the previous two years. Women who reported provider communication on mammogram choice were 17% more likely to report no mammogram in the past two years (OR: 1.17, 95% CI: 0.93-1.46), though this was not statistically significant. When stratified by 10-year age group, provider communication was only associated with higher risk of no mammogram among women age 70 and older (OR: 1.93, 95% CI: 1.29-2.89).

Conclusions: Between 2011-2014 women age 40-49 were the most likely to receive healthcare provider communication on mammogram choice. Still, the majority of women in that age group did not receive such communication despite the 2009 USPSTF recommendation that the decision to start screening mammography prior to age 50 should be an individualized one that takes into account patient values regarding specific benefits and harms. Provider communication on mammogram choice can influence screening behavior, particularly for older women.

#2584

HPV prevalence and acceptability of HPV self-sampling for cervical cancer screening in the community of Santiago Atitlan, Guatemala.

Anna Gottschlich,1 Rafael Meza,1 Alvaro Rivera-Andrade,2 Edwin Grajeda,3 Christian Alvarez,1 Carlos Mendoza Montano4. 1 _University of Michigan School of Public Health, Ann Arbor, MI;_ 2 _Braun School of Public Health and Community Medicine, The Hebrew University, Jerusalem, Israel;_ 3 _School of Medicine and Surgery, Universidad Rafael Landivar, Guatemala City, Guatemala;_ 4 _Institute of Nutrition of Central America and Panama-INCAP and Faculty of Medical Sciences and Health, Universidad Mariano Galvez de Guatemala, Guatemala City, Guatemala_.

BACKGROUND: Incidence and mortality rates of cervical cancer in Guatemala are considerably higher than rates in developed countries, potentially due to the extremely low rates of cervical cancer screening in many Latin American countries, specifically in indigenous populations. Current estimates suggest that less than 40% of women in Guatemala have ever been screened for cervical cancer, with much lower rates in indigenous communities. We therefore proposed that reliance on Pap and VIA screening might not be the most effective methods for controlling and preventing cervical cancer in indigenous communities in Guatemala, and thus investigated self-collection HPV tests as an alternative method of screening.

METHODS: A randomly selected group of women (N=202), ages 18-60, residing in a predominantly indigenous community approximately 130 kilometers west of Guatemala City were surveyed during July 2015 to assess risk factors and knowledge of HPV and cervical cancer. Eligible women were then asked to self-collect a vaginal sample to be tested for HPV to assess the prevalence in the community, as well as the acceptability of HPV self-sampling as an option for cervical cancer screening. We further investigated the relationship between sexual history and past use of health services with two outcomes, positive HPV test and prior Pap or VIA screening, using logistic regression.

RESULTS: Of the 202 women who completed the survey, 188 (93%) were interested in providing a self-collected vaginal sample, and 178 (88%) were eligible for self-collection. After collection, 100% of these women stated that they would be willing to perform this test periodically as a method of cervical cancer screening, and 80% reported that they would prefer self-collection in the home to physician collected samples in a doctors office. Of the 178 self-collected samples, 37 (21%) women tested positive for any type of HPV, including 31 (17%) for high risk HPV.

We found a positive, statistically significant association between use of health services and prior Pap or VIA screening (OR=4.845; 95% CI: 1.553, 15.120; p=0.007), after adjustment for age, education, and HPV test result. We also found a positive, however non-significant, association between lifetime sexual partners and HPV infection (OR=1.770; 95% CI: 0.606, 5.167; p=0.267), after adjustment for age, education, age at first sexual experience, and age at first childbirth.

CONCLUSIONS: Self-collection HPV testing as a form of cervical cancer screening was very well accepted in this indigenous community. Further testing needs to be done to assess the rate at which women receive a follow-up Pap or VIA screening after receiving a positive result on their HPV test. Furthermore, past use of health services is significantly associated with prior cervical cancer screening, suggesting that comprehensive strategies to enhance healthcare access are critical to improve screening outcomes.

#2585

Mammography screening adherence after a false positive test result.

Mary W. Marsh, Thad Benefield, Mikael Anne Greenwood-Hickman, Laura Jones, Anne Marie Meyer, Louise M. Henderson. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Mammography screening has been shown to be effective in reducing breast cancer mortality, but it also generates a significant number of false positive test results. We sought to compare mammography guideline adherence after a false positive versus a true negative screening mammogram, looking for differences by age, race, family history of breast cancer, time since prior mammogram, and mammographic breast density.

Linked data from the Carolina Mammography Registry and privately insured beneficiaries in North Carolina from 2004-2009 were used to evaluate screening adherence among 14,071 women aged 40 to 64 years who had a screening mammogram with a false positive or true negative result. Women were classified as being adherent to mammography screening guidelines if they obtained a screening mammogram in the 9 to 24 months after their index screening mammogram. We compared characteristics of women with a false positive versus true negative screening mammogram result using the chi-square test. To determine if the index mammogram result (false positive or true negative) was predictive of subsequent adherence to screening mammography, we employed a random effects logistic regression, adjusting for age, race, menopausal status, first-degree family history of breast cancer, personal history of breast biopsy, mammographic breast density, time since prior mammogram, and comorbidities. We tested for interactions of these characteristics with the index mammogram result using backwards selection and considered interaction terms with p-values <0.05 significant.

Of the 14,071 screening mammograms 92.1% were true negative and 7.9% were false positive. Women with a false positive were more likely to be younger, pre-menopausal, have dense breasts, and have had their last mammogram 3 or more years ago. Women with a false positive result were also less adherent to screening mammography in the subsequent 24 months compared to women with a true negative result (81.8% vs. 84.2%, p-value=0.031). Women with a prior mammogram within 1 to 3 years and a false positive result were less likely to be adherent (adjusted odds ratio (OR)=0.77, 95% CI: 0.64-0.94) than women with a true negative result. In contrast, for women who had a prior mammogram 3 or more years ago, those with a false positive result were more likely to be adherent (adjusted OR=1.80, 95% CI: 1.13-2.87) than those with a true negative result.

Our findings suggest that a false positive screening mammogram may reduce the likelihood of a woman returning for her next planned screening mammogram if her prior mammogram was within 1 to 3 years, but it may increase her likelihood to return if her prior mammogram was more than 3 years prior. Future studies should expand beyond observational data to incorporate qualitative interview data with women to better understand the psychological distress experienced after a false positive screening mammogram result.

#2586

Evaluation of risk-based colposcopy in the ALTS trial.

Angela H. Liu,1 Mark Schiffman,1 Julia C. Gage,1 Philip E. Castle,2 Nicolas Wentzensen1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Albert Einstein College of Medicine, Bronx, NY_.

Objective: Women referred to colposcopy have a wide spectrum of underlying risk of precancer that could influence colposcopic practice and management. We sought to evaluate important risk-strata based on HPV status, cytology, and colposcopic impression in women undergoing colposcopy.

Methods: Among participants in the ASCUS-LSIL Triage Study, we stratified women from the immediate colposcopy and HPV triage study arms based on enrollment HPV typing, study cytology, and colposcopic impression. In each group, absolute risk of CIN3+ at enrollment colposcopy and cumulative risk over 2-year follow-up were estimated. Immediate CIN3+ risk of LSIL and HSIL cytology were used as thresholds for colposcopy referral and immediate treatment, respectively.

Results: We observed substantial differences in risk of precancer across strata. Women with HSIL cytology, who were HPV16-positive, and high-grade colpscopic impression had a 71% baseline and 78% 2-year risk of CIN3+, respectively. HPV16-positive women with HSIL cytology and low-grade impression, and women with high-grade impression and either HSIL cytology or HPV16 infection, also showed significant CIN3+ risk exceeding the risk of HSIL. In contrast, women with normal colposcopy, <HSIL cytology, and absence of HPV16 infection had the lowest baseline CIN3+ risk (3.2%), below the colposcopy referral threshold in ALTS.

Conclusion: Risk assessment at colposcopy can inform colposcopy-biopsy practice and guide management. In the low-risk group, biopsy yields little additional disease and a normal colposcopy may confer higher reassurance that CIN3+ is not present; while in the highest risk groups, immediate treatment without biopsy confirmation could be considered according to current guidelines. Analyses evaluating the benefit of multiple biopsies in these risk strata are underway and will be presented at the meeting.

#2587

Comprehensive colorectal cancer risk prediction to inform personalized screening and intervention.

Jihyoun Jeon,1 Sonja I. Berndt,2 Hermann Brenner,3 Peter T. Campbell,4 Andrew T. Chan,5 Jenny Chang-Claude,3 Mengmeng Du,6 Graham Giles,7 Jian Gong,8 Stephen B. Gruber,9 Tabitha A. Harrison,8 Michael Hoffmeister,3 Loic LeMarchand,10 Li Li,11 John D. Potter,8 Gad Rennert,12 Robert E. Schoen,13 Martha L. Slattery,14 Emily White,8 Michael O. Woods,15 Ulrike Peters,8 Li Hsu8. 1 _University of Michigan, Ann Arbor, MI;_ 2 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 3 _German Cancer Research Center (DKFZ), Germany;_ 4 _American Cancer Society, Atlanta, GA;_ 5 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 6 _Memorial Sloan Kettering, New York, NY;_ 7 _Cancer Council Victoria, Australia;_ 8 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 9 _Keck School of Medicine, University of Southern California, Los Angeles, CA;_ 10 _University of Hawaii Cancer Center, Honolulu, HI;_ 11 _Case Western Reserve University, Cleveland, OH;_ 12 _Carmel Medical Center, Israel;_ 13 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 14 _University of Utah Health Sciences Center, Salt Lake City, UT;_ 15 _Memorial University of Newfoundland, Newfoundland and Labrador, Canada_.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States, despite the fact that it is one of the most preventable and treatable cancers when detected early via screening. The current screening guidelines for CRC are based on age, family history of CRC, and previous screening results. However, multiple environmental and lifestyle risk factors have been established or suspected for CRC, as have many common genetic susceptibility loci. It is critical to utilize this information to better stratify individuals into low- and high-risk groups for optimized and personalized screening and intervention recommendations.

Using data from two large consortia (8421 CRC cases and 9767 controls): the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and the Colorectal Transdisciplinary study (CORECT), we developed risk prediction models for men and women based on family history, environmental and lifestyle risk factors, and known CRC susceptibility loci identified through genome-wide association studies. We constructed an environmental risk score (E-score) as a weighted sum of 19 established or potential environmental and lifestyle risk factors for CRC with weights obtained from a multivariate logistic regression analysis. Compared to the model that includes only family history, the E-score significantly improves the discriminatory accuracy for both men (AUC = 0.62 vs. 0.53, p-value < 1e-5 ) and women (AUC = 0.60 vs. 0.52, p-value < 1e-5 ). Similarly, we also constructed a genetic risk score (G-score) using 50 common variants associated with CRC risk, and the G-score also significantly improves the discriminatory accuracy for both men (AUC = 0.60 vs. 0.53, p-value < 1e-5 ) and women (AUC = 0.59 vs. 0.52, p-value < 1e-5 ) over the family history-only model. Compared to the model with family history and E-score, the inclusion of the G-score in the model further improves the discriminatory accuracy for both men (AUC = 0.65 vs. 0.62, p-value = 0.0152) and women (AUC = 0.63 vs. 0.60, p-value = 0.0005).

Our risk prediction models are the first to incorporate both comprehensive environmental and lifestyle risk factors, and known CRC common genetic variants. The E- and G-scores are independent risk predictors for CRC, and models that incorporate both scores improve the discriminatory accuracy significantly compared to family history-only models. Using risk-factor distributions available from nationally representative data (e.g., NHANES), we will provide absolute-risk estimates of CRC using both the E- and G-scores. We expect our comprehensive models incorporating both environmental and genetic risk factors to provide more accurate estimation of CRC, which will be useful for recommending individually tailored screening and intervention strategies to prevent this common cancer.

#2588

Prediction of the 5-year risk of hepatocellular carcinoma during long-term antiviral therapy in the general Korean population.

Jae-Jun Shim,1 In Hwan Oh,2 Jung Wook Kim,1 Chang Kyun Lee,1 Jae Young Jang,1 Ju-Seog Lee,3 Byung-Ho Kim1. 1 _Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Republic of Korea;_ 2 _Department of Preventive Medicine, Kyung Hee University School of Medicine, Seoul, Republic of Korea;_ 3 _Departments of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX_.

Oral antiviral therapy using nucleos(t)ide analogues can decrease risk for hepatocellular carcinoma (HCC) in patients with chronic hepatitis B. However, wide use of antiviral therapy is limited by stringent treatment indications in many countries. We compared preventive effect of antiviral therapy on HCC development between current and new extended indication for the treatment. We established Korean cohort with chronic hepatitis B virus (HBV) infection using the fifth National Health and Nutrition Examination Survey (KNHANES). The cohort represented 993,872 subjects with chronic HBV infection more than 40 years in Korea. We estimated five-year risk of HCC before and after antiviral therapy using a HCC prediction score (REACH-B). The National Health Insurance (NHI) in Korea, the European Association for the Study of the Liver (EASL) guideline, and a new extended indication were compared. During a 5-year period, predicted HCC cases were 2,725 per 100,000 individuals (0.55% / year). When the cohort was treated under the Korean NHI indication, EASL, and new extended indication, HCC risk decreased to 2,531 (-7.1%), 2,089 (-23.3%), and 1,122 (-58.8%) cases per 100,000 individuals, respectively (P < 0.0001). As participation rate in antiviral therapy among eligible patients increased every 10%, HCC risk decreased to -0.74%, -2.39%, and -5.87%, respectively. New extended indication could decrease HCC risk eight times more than the current Korean NHI indication. Prediction of HCC development among chronic HBV infection is feasible in the virtual population level. Considering the large preventive effect of antiviral therapy, the treatment indications should be revised to increase eligible patients.

Indications for antiviral treatment used in this study and predicted reduction of HCC risk

---

|

Korean NHI | EASL | New extended indication

Liver enzyme | AST or ALT ≥ 80 IU/L | ALT ≥ ULN x 1 | Any

HBV DNA level | ≥ 20,000 IU/mL | ≥ 2,000 IU/mL | ≥ 2,000 IU/mL

HCC risk reduction, mean (SD) (%) | -7.14 (0.14) | -23.33 (0.26) | -58.82 (0.07)

#2589

Dissecting extrinsic and intrinsic cancer risk.

Song Wu, Scott Powers, Wei Zhu, Yusuf Hannun. _Stony Brook University, Stony Brook, NY_.

Cancers were once thought to originate from mature tissue cells that underwent dedifferentiation in response to cancer progression. Today, cancers are proposed to originate from the malignant transformation of normal tissue progenitor and stem cells, although this is not uniformly accepted. Nevertheless, recent research has highlighted a strong correlation between tissue-specific cancer risk and the lifetime population size and cumulative number of cell divisions of tissue-specific stem cells. However, there has been extensive controversy regarding the conclusion that this correlation implies that there is a very high unavoidable risk for many specific cancers that are due solely to the intrinsic baseline population size of tissue-specific stem cells. This issue has become a key public health debate with dissemination of the 'bad luck' hypothesis. Here we provide strong evidence that unavoidable intrinsic risk factors contribute only modestly (<10-30%) to the development of many common cancer types. In an initial approach, we demonstrate that the correlation between stem-cell division and cancer risk does not distinguish between the effects of intrinsic and extrinsic factors. We then develop a novel data-driven approach to estimate intrinsic risk by considering the lower bound risk value for most cancers as a limiting condition for contribution of intrinsic processes. Furthermore, we present an independent mathematical model that capitalizes on the accumulation of endogenous mutations in stem cells. Using mutation rates as a starting point, we found that rates of intrinsic mutations are not sufficient to account for the observed lifetime cancer risk, even in the conservative estimate that all these mutations are due to intrinsic processes alone. Our results are consistent with vast epidemiological data at population level and also mutational signature data at molecular level. The consensus suggests that cancer risk is likely to be heavily influenced by extrinsic factors, and therefore is possibly modifiable and preventable. These results carry immense consequences for strategizing cancer prevention, research, and public health. [Note: this work is in press in Nature.]

#2590

Development and validation of a breast cancer risk prediction model for black women: findings from the Nigerian breast cancer study.

Shengfeng Wang,1 Temidayo Ogundiran,2 Adeyinka Ademola,2 Oluwasola A. Olayiwola,3 Adewunmi Adeoye,3 Adenike Adeniji-Sofoluwe,4 Imran Morhason-Bello,5 Stella Odedina,6 Imaria Agwai,6 Clement Adebamowo,7 Millicent Obajimi,4 Oladosu Ojengbede,6 Olufunmilayo I. Olopade,1 Dezheng Huo8. 1 _Center for Clinical Cancer Genetics & Global Health, Department of Medicine, University of Chicago, CHICAGO, IL; _2 _Department of Surgery, College of Medicine, University of Ibadan, Nigeria;_ 3 _Department of Pathology, College of Medicine, University of Ibadan, Nigeria;_ 4 _Department of Radiology, University College Hospital, Nigeria;_ 5 _Center for Population and Reproductive Health, College of Medicine, University of Ibadan, Nigeria;_ 6 _Center for Population and Reproductive Health, University of Chicago, CHICAGO, IL;_ 7 _Department of Epidemiology and Preventive Medicine, Institute of Human Virology and Greenebaum Cancer Center, University of Maryland, Baltimore, MD;_ 8 _Department of Public Health Sciences, University of Chicago, CHICAGO, IL_.

Background: Breast cancer (BC) risk prediction models, such as the Gail model, have been developed and widely used to identify women at higher risk of having breast cancer in developed countries. However, no model exists for Black women of sub-Saharan Africa (SSA). Because African women have different risk profiles, it is of public health importance to develop a Black women-specific model.

Methods: A total of 1,880 hospital-based cases and 2,166 population-based controls from the Nigerian Breast Cancer Study (NBCS, 1998~2015) were included in the analysis. Subjects were randomly divided into the training (2/3 of the data) and validation sets (1/3 of the data), and multivariate logistic regressions were used to derive the model. Risk factors were selected based on previous NBCS findings and literature review. Calibration and discrimination performances were assessed using the observed/expected ratio (O/E) and concordance statistic (C-index), respectively.

Results: The final model included age, age at menarche, parity, duration of breast feeding, family history of BC in first degree relatives, height, body mass index, benign breast diseases, oral contraceptive use, and alcohol drinking. The model performed well in the validation set with an O/E of 1.01 (95% CI: 0.93~1.09) and C-index of 0.694 (95% CI: 0.666~0.722). The odds ratios for developing BC by percentiles of the predicted chance of cases, relative to women in the middle quintile, showed a monotonic increasing trend.

Conclusion: In Nigeria, the most populous country in SSA with 175 million people, this study developed and validated a risk prediction model for BC that is specific to Black women. It can be used to identify individuals at high risk of BC for cancer prevention. Using the population incidence rates in Nigeria, an absolute risk prediction model will be further developed.

Table 1 Validation analyses in both training set and validation set

---

Percentile of predicted chance being cases | Training set (n=2699)

OR (95%CI) | Validation set (n=1347)

OR (95%CI)

<5% | 0.18(0.10-0.30) | 0.24 (0.11-0.55)

5%~20% | 0.31(0.23-0.41) | 0.53(0.36-0.78)

20%~40% | 0.62(0.48-0.79) | 0.83(0.60-1.16)

40%~60% | 1.00(referent) | 1.00(referent)

60%~80% | 1.50(1.18-1.90) | 1.74(1.25-2.44)

80%~95% | 2.76(2.10-3.62) | 3.68(2.48-5.44)

95%~ | 8.53(4.93-14.77) | 7.43(3.76-14.69)

C-index | 0.724(0.705-0.742) | 0.694(0.666-0.722)

#2591

Risk prediction for stomach cancer using helicobacter pylori infection and atrophic gastritis, fruits and vegetables, smoking and GWAS-identified genetic polymorphism (PSCA-rs2294008) in a Japanese population.

Hidemi Ito, Isao Oze, Satoyo Hosono, Miki Watanabe, Hideo Tanaka, Keitaro Matsuo. _Aichi Cancer Ctr. Research Inst., Nagoya, Japan_.

[Background] Helicobacter pylori (H. pylori) infection and atrophic gastritis (AG) following H. pylori infection are well-established risk factors worldwide. Smoking is a convincing risk factor and fruits and vegetables intake is a possible protective factor in Japanese populations. PSCA-rs2294008 is one of single nucleotide polymorphisms (SNPs) with the susceptibility of a stomach cancer risk identified by Genome-wide association studies in Japanese populations. The advances in molecular evidence may have a potential to impact cancer prevention. However, it has never been clear how much it may contribute to cancer risk of the stomach on a population level in combination with environmental factors. In this study, we established a risk prediction model of stomach cancer using these environmental, clinical and genetic risk factors as a potential practical application in cancer preventive intervention.

[Methods] We conducted two age- and sex- matched case-control studies, one for model derivation (697 cases and 1,372 controls) and the second (678 cases and 678 controls) for external validation. Based on data from the derivation study, a prediction model was developed by fitting a conditional logistic regression model using the following predictors: age, ABCD category defined by H.pylori infection and AG, smoking, fruits and vegetable intakes and PSCA genotype. Performance of the model was assessed in terms of discrimination (C statistic), calibration (calibration plots and Hosmer-Lemeshow test) and Integrated discrimination improvement (IDI) index. Cumulative risks were obtained by combining odds ratios estimated from the risk model with the age-specific incidence rate and population size.

[Results] The risk model, including a combination of PSCA genotype, smoking, fruit & vegetable intake and alcohol consumption, provided high discriminatory accuracy and good calibration in both the derivation and validation studies: C statistics were 0.78 (95% confidence intervals 0.76-0.80) and 0.80 (0.77-0.82), respectively, and the calibration plots of both studies stayed close to the ideal calibration line. IDI indices are 0.11 and 0.12 (p<0.00001), respectively.

[Conclusion] The risk prediction model that includes these environmental, clinical and genetic factors could be useful to classify Japanese into relevant risk groups of stomach cancer for personalized prevention programs.

#2592

The role of surgery in the treatment of epithelioid trophoblastic tumor: A single institution case series.

Abigail Winder, Janelle Sobecki-Rausch, Kruti Maniar, Karen Novak, Julie Kim, John Lurain. _Northwestern University, Chicago, IL_.

Objective: Epithelioid trophoblastic tumor (ETT) is a rare variant of gestational trophoblastic neoplasia (GTN) that develops from chorionic-type intermediate trophoblasts, simulates carcinoma, often presents many years following a pregnancy event, is associated with low or normal human chorionic gonadotropin (hCG) levels, and is relatively resistant to chemotherapy. Our aim was to identify the role of surgery in the management of both localized and metastatic ETT.

Methods: A retrospective review of women with ETT treated at a gestational trophoblastic disease center from 2010-2015 identified five women. Medical records were abstracted for demographic data, clinical presentation, hCG levels, prior pregnancy information, disease sites, and treatment with chemotherapy and surgery.

Results: The five women with ETT ranged in age from 28 to 48 years. Clinical presentation was abnormal uterine bleeding prompting dilation and curettage, which established the diagnosis of ETT in three women. One woman presented with repeated spontaneous pneumothorax requiring thoracoscopic resection, which identified a 6 cm ETT in the lung. Another woman was initially treated with methotrexate for a presumed ectopic pregnancy without a response. Two women had no identifiable antecedent pregnancy, two women had spontaneous abortions 19 and 72 months prior to the diagnosis of ETT, and one woman had a normal term delivery 48 months earlier. Four of five women had metastatic disease to the lungs. Three of these women underwent pulmonary wedge resection as well as hysterectomy followed by paclitaxel-cisplatin/paclitaxel-etoposide (TP/TE) chemotherapy, while the other woman was treated with etoposide, methotrexate, actinomycin D/etoposide, cisplatin (EMA/EP) chemotherapy and hysterectomy. The one woman with nonmetastatic disease was treated with hysterectomy only. All five women are currently without evidence of disease.

Conclusions: Surgery, including hysterectomy and resection of metastatic disease, is an important component in the treatment of women with ETT. Adjuvant chemotherapy with a platinum-containing regimen, such as TP/TE or EMA/EP, should be used in women with metastatic disease. All five women with ETT in this series were cured using this approach, including the four with metastatic disease.

#2593

Association of mammographic density measures and breast cancer 'intrinsic' molecular subtypes.

Aaron D. Norman,1 Rulla M. Tamimi,2 Christopher G. Scott,1 Kimberly A. Bertrand,2 Matthew R. Jensen,1 Daniel W. Visscher,1 Fergus J. Couch,1 John Shepherd,3 Bo Fan,3 Yunn-Yi Chen,3 Lin Ma,3 Andrew Beck,4 Vernon S. Pankratz,5 Karla Kerlikowske,3 Celine M. Vachon1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Harvard Medical School and Harvard School of Public Health, Boston, MA;_ 3 _University of California, San Francisco, CA;_ 4 _Harvard Medical School, Boston, MA;_ 5 _University of New Mexico, Albuquerque, NM_.

Percent mammographic density (PMD) is a risk factor for estrogen receptor (ER)-positive and ER-negative invasive breast cancer (BC). Gene expression profiling has identified molecular signatures that classify invasive BC into distinct subtypes that vary in their clinical behavior, response to treatment and likely, etiology. Immunohistochemical (IHC) staining of tumor sections using antibody panels can be used to classify these 'intrinsic' molecular subtypes. We evaluated whether density measures [PMD, absolute dense area (DA) and non-dense area (NDA)], are associated equally with all 'intrinsic' molecular subtypes.

Pooled analysis of six cohort or case-control studies included 3492 women with invasive BC and 10,148 without, who underwent screening mammography a median 4 years prior to diagnosis (for cases). PMD, DA, and NDA were assessed from digitized film-screen mammograms using a computer-assisted thresholding technique, and categorized as 0-10%, 11-25%, 26-50% and 51%+ (PMD) or into quartiles (DA and NDA). Receptor status was abstracted from pathology records and supplemented by IHC staining. We classified tumors as Luminal A (ER+ and/or PR+ and HER2- and grade 1 or 2), Luminal B (ER+ and/or PR+ and HER2+ or Luminal A and grade 3), HER2 expressing (HER2+/ER-/PR-) and triple negative (TN) (ER-/PR-/HER2-). For TN, we also differentiated basal-like tumors (positive for EGFR and/or CK 5/6) from unclassified (negative on both markers). We used polytomous logistic regression to calculate the odds ratio (OR) of each 'intrinsic' subtype of BC by categories of PMD, DA or NDA, adjusting for age, body mass index and study. We tested for statistical heterogeneity of associations by subtype.

Of 3492 invasive BC cases, 2217 (63%) were classified as Luminal A, 747 (21%) as Luminal B, 159 (5%) as HER2 expressing, and 369 (11%) as TN. Of TN, 203 were evaluated for CK 5/6 and EGFR, with 167 (82%) classified as basal-like and 36 (18%) unclassified. PMD was associated with BC risk across all subtypes. For Luminal A, compared to women with 11-25% PMD (reference), women with 0-10% had a reduced risk of BC (OR=0.63 [95% confidence interval: 0.55, 0.74]) while women with 26-50% had an OR=1.5 [1.3, 1.7] and women with 51%+ had the highest risk, OR=2.3 [2.0, 2.7]. Similar BC associations were seen across PMD categories when comparing the five subtypes (P-heterogeneity=0.63). Similar trends were seen for DA and BC across the five subtypes (P-het=0.25). NDA was inversely associated with BC across subtypes, and there was suggestion of a stronger inverse trend among HER2-expressing BC compared to other subtypes (P-het=0.09).

Our results suggest mammographic density measures are associated with all 'intrinsic' molecular subtypes. However, NDA may be more strongly inversely associated with HER2-expressing than other subtypes. Understanding the importance of density measures for BC subtypes has significance for subtype-specific risk models.

#2594

Clinical pathways as a platform to support clinical research.

Peter G. Ellis, Aurora Health Care, Baystate Health, Broward Health, Carroll Hospital, IU Health, TCCBD, UPMC. _Via Oncology, Pittsburgh, PA_.

Introduction

Clinical trials are essential to advancing cancer care; however, actual patient accrual rates generally fall well below the Institute of Medicine's stated goal of 10% of all newly diagnosed patients. To support the goal of increasing clinical trial accruals, Via Oncology (VO) uses its clinical pathways decision support tool (Via Portal or VP) to promote awareness of locally available clinical trials and provide associated analytics for its cancer center (CC) customers.

Methods

VO works continuously with each cancer center to identify and embed their CC specific clinical trials in the applicable disease within the VP. In their daily practice, providers navigate the VP by entering information about each patient's specific state and stage of disease. If a clinical trial is locally available anywhere within the CC's network, that trial is presented as the first treatment recommendation before any standard of care pathway recommendations. Links to trial specific documents are also presented. If the provider feels the patient might be appropriate for that trial, a secure email notification is generated for the trial coordinator. If the patient is not accruing to the trial, the provider must select the reason for non-accrual from a structured list of reasons. Additionally, the VP can be interfaced with trial management systems for timely opening and closing of trials within the VP.

Data captured in the VP for the twelve months ended October 31, 2015 was retrospectively analyzed to calculate accrual rates for patients potentially eligible for a clinical trial. The distribution of reasons for not accruing patients to clinical trials was also calculated and analyzed.

Results

During this time period, 10,000 patients had a high level

clinical presentation that matched an open clinical trial embedded in the VP

(the patients were not specifically matched for trial inclusion/exclusion

criteria). Of the total, 1,391 (13.9%) of patients were documented as accrued to a clinical trial. A total of 9,661 reasons for not accruing the patient to a trial were captured and distributed as follows: patient not eligible (34.4%), patient not interested (14.7%), patient accrued to other trial (0.8%), financial burden or insurance does not cover (0.5%), trial not available at site (25.6%), trial already selected (0.8%), other reason(23.2%).

Conclusions

VP also acts as a tool to promote clinical trial accruals and measure reasons for non-accruals. The reasons for not accruing provide insight to the barriers to accrual that patients and practices face. Additional reasons could be defined in the future to reduce the incidence of other non-defined reasons.

#2595

Texture variation on a mammogram and risk of breast cancer.

Megan Rice,1 Kimberly Bertrand,2 John Heine,3 Bernard Rosner,1 Rulla Tamimi1. 1 _Brigham and Women's Hospital, Boston, MA;_ 2 _Boston University, Boston, MA;_ 3 _Moffitt Cancer Center, Tampa, FL_.

Mammographic density is one of the strongest risk factors for breast cancer. The most widely used measure of mammographic density is based on the percentage of dense tissue on a mammogram (percent mammographic density). However, there is additional information in mammographic images not captured by current mammographic density measurements including heterogeneity in patterns of breast density, often referred to as 'texture'. More recent research suggests that textural features on a mammogram are associated with breast cancer risk independent of percent mammographic density. Therefore, we evaluated an automated measure called V, which measures the grayscale variation within a mammogram, in relation to subsequent breast cancer risk in 834 breast cancer cases (426 premenopausal and 408 postmenopausal) and 1805 controls (978 premenopausal and 827 postmenopausal) from a nested case-control study of breast cancer in the Nurses' Health Studies (NHS). Among all women, V was modestly correlated with percent mammographic density (r=0.48, p<0.01); this correlation did not vary substantially by menopausal status. Among premenopausal women, those in the highest quartile of V had a significantly higher risk of breast cancer (OR= 1.84, 95%CI: 1.30-2.61) compared to those in the lowest quartile. This association was similar, though more modest, among postmenopausal women (OR=1.54, 95%CI: 1.08-2.19). These associations somewhat attenuated after adjustment for percent mammographic density in both premenopausal women (OR comparing extreme quartiles=1.41, 95%CI: 0.97-2.03) and in postmenopausal women (OR comparing extreme quartiles=1.21, 95%CI: 0.81-1.81). Future work will examine the associations between V and risk of breast cancer subtypes (e.g., by ER/PR tumor expression) as well as examine the associations between other measures of texture including markovian, run-length, laws, wavelet, fourier, and power features with breast cancer risk.

#2596

Time to first morning cigarette and lung cancer in National Lung Screening Trial.

Fangyi Gu, Hormuzd Katki, Neal Freedman, Pam Marcus, Neil Caporaso. _NCI-DCEG, Rockville, MD_.

In the National Lung Screening Trial (NLST), the effectiveness of lung cancer screening by low-dose computed tomography (CT) depended on individual lung cancer risk. Time to fist cigarette (TTFC) is an easily assessed component of nicotine dependency that may be an independent predictor of lung cancer risk. We validated the independent association of TTFC with lung cancer and assessed whether including TTFC into risk prediction models might improve CT screening effectiveness.

We examined associations between TTFC and lung cancer incidence and mortality in the 18,729 participants of the ACRIN component of the NLST, which included 773 lung cancer diagnoses and 299 lung cancer deaths. Multivariable cox-proportional hazard regression models, adjusted for other metrics of smoking use and lung cancer risk factors, were used. In terms of screening effectiveness, we predicted each participant's 5-year cumulative lung cancer death risk using multivariable models that excluded or included TTFC. We then investigated 3 possible screening strategies based on risk scores from each model (for a total of six strategies): in each model scenario, those with a 5-year risk score above one of three thresholds (0.5%, 0.75%, and 1%) would be screened, whereas others would not. For each risk threshold, we compared screening sensitivities (the percentage who would have been screened among smokers who died of lung cancer) and specificities (the percentages who would not have been screened among smokers without lung cancer) between models without and with TTFC. We also estimated the absolute reduction in lung cancer death risk, due to screening, within each TTFC group (<15, 15-59, ≥60 minutes (min)).

Compared to smokers with TTFC≥60 min, smokers with shorter TTFC had higher lung cancer incidence [hazard ratios (HR) and 95% confidence intervals (CI) for TTFC 30-59 min=1.69(1.19-2.41), HR15-29min=1.72(1.22-2.43), HR6-14min=2.05(1.48-2.84), HR<5min=2.19(1.59-3.02), p-trend<0.001] and mortality [HR30-59min=2.10(1.11-3.96), HR15-29min=1.91(1.02-3.59), HR6-14min=2.59(1.43-4.69), HR<5min=2.77(1.54-4.97), p-trend<0.001]. With regards to lung cancer screening, fewer lung cancer deaths would be avoided by screening smokers with TTFC≥60 min compared to screening smokers with TTFC 15-59 min or TTFC<15 min (2.03 vs. 4.99 vs. 7.38 lung cancer deaths avoided per 10000 person-years, respectively). Adding TTFC to the multivariable risk prediction algorithm increased the specificity of screening for all three risk thresholds by 1.6-2% (p<0.01) while maintaining sensitivity.

We confirmed the independent inverse association between TTFC and lung cancer in NLST. Smokers with a TTFC greater than 60 min had less benefit from CT screening than smokers with a shorter TTFC. By increasing specificity and maintaining sensitivity, including TTFC in risk prediction models may more precisely identify those who benefit less from CT lung screening.

#2597

Breast and ovarian cancer risks associated with cancer predisposition gene mutations identified by multigene panel testing.

Fergus J. Couch,1 David E. Goldgar,2 Steven N. Hart,1 Emily Hallberg,1 Raymond Moore,1 Huong Meeks,2 Robert Huether,3 Holly LaDuca,3 Elizabeth Chao,3 Jill Dolinsky3. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Utah, Salt Lake, UT;_ 3 _Ambry Genetics, CA_.

Multigene panel testing (MGPT) for hereditary cancer is increasing in popularity in the USA. Many panels include genes identified as hereditary breast and/or ovarian cancer (HBOC) genes despite limited data regarding the precise cancer risks associated with mutations in these genes. Here we report on results from BreastNext and OvaNext panel testing of 20 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MRE11A, MSH2, MSH6, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, TP53) in a cohort of 15,083 individuals. The majority of individuals were from high-risk breast and/or ovarian (Br/Ov) cancer families, with 92.4% of all probands meeting National Comprehensive Cancer Network HBOC testing criteria. Pathogenic mutations were identified in 9.4% of the overall cohort.

To estimate gene-specific breast and ovarian cancer risks, case-control analyses were performed comparing the frequencies of pathogenic mutations from Caucasian breast or ovarian cancer cases from BreastNext and OvaNext with frequencies from Caucasian, non-Finnish, non-TCGA controls from the Exome Aggregation Consortium (ExAC) database. Mutations in the well studied ATM and CHEK2 genes were associated with moderate risks (OR>2) of breast cancer and mutations in PALB2 were associated with high-risks (OR>5) of breast cancer, consistent with previous reports. In addition, the study suggested that pathogenic mutations in MSH6, RAD51D, CDH1, and NF1 are associated with moderate to high risks of breast cancer. In contrast, RAD51C, RAD51D, and BRIP1 mutations were associated with high risks of ovarian cancer, PALB2 mutations were associated with moderate risks, but ATM and CHEK2 mutations were not associated with increased ovarian cancer risk. In addition, modeling of missense mutations in the predisposition genes using in silico prediction algorithms suggested that missense mutations in CDH1, CHEK2, MSH2, and MSH6 are associated with moderate risks of breast cancer and missense mutations in RAD51C increase risks of ovarian cancer. This large breast and ovarian cancer case-control analysis provides useful data for many predisposition genes previously lacking risk estimates, and should prove useful for clinical risk management of patients after clinical panel testing.

#2598

Predicting breast and ovarian cancer risks for BRCA1 and BRCA2 mutation carriers using polygenic risk scores.

Karoline Kuchenbaecker,1 Jacques Simard,2 Kenneth Offit,3 Fergus J. Couch,4 Douglas F. Easton,1 Georgia Chenevix-Trench,5 Antonis C. Antoniou,1 Consortium of Investigators of Modifiers of BRCA1/2. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _CHU de Quebec Research Center, Quebec City, Quebec, Canada;_ 3 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 4 _Mayo Clinic, Rochester, MN;_ 5 _QIMR Berghofer Medical Research Institute, Brisbane, Australia_.

Women who carry a pathogenic mutation in the BRCA1 or BRCA2 gene are at high risk of breast (BC) and ovarian cancer (OC). Their clinical management usually includes invasive risk-reducing interventions with substantial side effects. Improved personalized cancer risk estimates may help to identify women at particularly high risk or with high risk of disease at early ages who may benefit from early intervention as well as women at lower risk who may opt to delay surgery or chemoprevention. Genome-wide association studies have identified >100 common genetic variants that are associated with BC or OC risks. Several of these variants are also individually associated with risk of BC or OC for BRCA1 and BRCA2 mutation carriers. However, no study has evaluated the combined effects of all the known common genetic variants on BC or OC risk for BRCA1/2 mutation carriers.

We constructed polygenic risk scores (PRS) based on results of genetic association studies conducted in the general population. Each PRS was formed by the sum of the number of risk alleles across the variants weighted by their log-Odds Ratio estimate from population-based studies of BC or OC. We investigated 3 PRS for BC (overall, estrogen receptor (ER) positive, and ER-negative) and one PRS for OC. We used data for 15,252 BRCA1 and 8,211 BRCA2 female carriers. The association of each PRS with BC or OC risk was evaluated using a weighted cohort analysis with time to diagnosis as the outcome and estimated the Hazard Ratios (HR) per standard deviation increase in the PRS.

All PRS were significantly associated with cancer risks for BRCA1/2 carriers. The PRS for ER-negative BC displayed the strongest association with BC risk in BRCA1 carriers (HR=1.29 [1.25-1.33], p=8x10-64). In BRCA2 carriers, the strongest association was seen for the overall BC PRS (HR=1.26 [1.21-1.31], p=3x10-27). The OC PRS was strongly associated with OC risk for both BRCA1 and BRCA2 carriers. These relative risks translate to large differences in absolute risks for carriers: e.g., the OC risk was 6% by age 80 for BRCA2 carriers at the 10th percentile of the OC PRS compared with 19% risk for those at the 90th percentile of PRS.

Our findings demonstrate that BC and OC PRS derived from studies in the general population are predictive of cancer risks in BRCA1 and BRCA2 carriers. Incorporation of the PRS into risk prediction models would improve risk prediction and hence inform decisions on cancer risk management.

#2599

Association of periodontal disease and breast health in women undergoing screening mammography.

Hannah Lui Park, Teofilia Acheampong, Kathleen Nguyen, Cindy Nguyen, Argyrios Ziogas, Richard Kelly, Andrea Alvarez, Kathryn M. Larsen, Deborah Goodman, Hoda Anton-Culver. _University of California, Irvine, Irvine, CA_.

It is well established that chronic and persistent inflammation contributes to cancer development. Chronic inflammation is often associated with periodontal disease, or gum disease. Periodontal disease, which can be prevented or ameliorated by following proper oral hygiene, is known to be associated with various systemic disorders including coronary heart disease and some cancers, including head and neck cancer and pancreatic cancer. However, little is known about its potential association with breast cancer, with only one report in which periodontal disease was a positive predictor for breast cancer in a Swedish cohort. To examine if a potential link exists between periodontal disease and breast cancer in a separate cohort, mammography patients from the UC Irvine Athena Breast Health Network cohort were recruited to participate in a survey that included questions about their periodontal health. Diagnosis of invasive breast cancer, DCIS, and benign breast diseases was determined through data extraction from electronic medical records. There was no association between periodontal disease and DCIS or invasive breast cancer. However, there was a significant difference in the frequency of breast cysts among women with periodontal disease compared to women without periodontal disease (p<0.01). Women with periodontal disease were at least 4-fold more likely to have breast cysts, even after adjusting for potential confounders including age, race/ethnicity, and smoking history (p<0.05). To our knowledge, this is the first report on an association between periodontal disease and breast cysts. Our data suggest that further studies on potential links between periodontal disease and breast health are warranted.

#2600

Improving breast cancer risk prediction models: the addition of a genetic risk score, mammographic density, and endogenous hormones.

Xuehong Zhang,1 Megan Rice,1 Shelley S. Tworoger,1 Bernard A. Rosner,1 A. Heather Eliassen,1 Rulla M. Tamimi,1 Jing Qian,2 Graham A. Colditz,3 Walter C. Willett,4 Susan E. Hankinson2. 1 _Harvard Medical School, Boston, MA;_ 2 _School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA;_ 3 _Washington University School of Medicine, St. Louis, MO;_ 4 _Harvard T. H. Chan School of Public Health, Boston, MA_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2016 Official Press Program. It will be posted online following its presentation.

## PREVENTION RESEARCH:

### Targets, Markers, and Agents in Cancer Prevention

#2601

Gingerol aspirinate derivatives as novel chemopreventive agents for colon cancer.

Yingdong Zhu, Fang Wang, Yantao Zhao, Pei Wang, Shengmin Sang. _North Carolina A &T State University, Kannapolis, NC_.

Regular aspirin use has been convincingly shown to reduce the risk of colorectal cancer. However, long-term use of aspirin leads to gastrotoxicity. Herein, we designed and synthesized a novel class of gingerol- and shogaol-based aspirin prodrugs to simultaneously release aspirin and gingerol or shogaol to attenuate the side effects caused by aspirin. Prodrug GAS exerted enhanced anticancer activities, which are better than a physical mixture of aspirin and 6-gingerol as well as each individual. In addition, GAS eliminated the gastric toxicity induced by aspirin in mice. Metabolism of GAS in mice showed that the majority of GAS is decomposed to release 6-gingerol and aspirin or salicylic acid either in the intestine or after absorption. Mechanistic studies demonstrate GAS could dose-dependently inhibit COX pathway. These findings highlighted the improved anticancer properties of gingerol-based aspirin prodrugs. GAS may represent novel and safe alternatives of aspirin for the purpose of daily use in the future.

#2602

The nonclinical toxicology profile of pacritinib, a JAK2/FLT3 inhibitor with no dose-limiting clinical myelosuppression.

Rebecca Watson, Suliman Al-Fayoumi. _CTI BioPharma, Seattle, WA_.

Pacritinib is an orally active kinase inhibitor being developed for the treatment of myelofibrosis (MF). Pacritinib is a particularly potent inhibitor of wild-type and mutant isoforms of JAK2 and FLT3 (IC50 values < 15 nM), and it does not suppress JAK1 at clinically relevant concentrations. Additional kinases targeted by pacritinib include IRAK1 (IC50 = 13.6 nM) and c-fms (or CSF1R; IC50 = 39.5 nM). Pacritinib is pharmacologically active in nonclinical tumor models driven by JAK2 or FLT3 overexpression, and clinical trials have demonstrated that pacritinib can ameliorate symptoms in MF patients. A key differentiating feature of pacritinib over currently available JAK2 inhibitors is its lack of dose-limiting clinical myelosuppression, allowing for treatment of patients with cytopenias. The comparatively reduced myelosuppressive activity of pacritinib is thought to be due to its lack of pharmacological activity on JAK1, and/or its inhibition of IRAK1 and c-fms, which may contribute to anti-inflammatory activity. Nonclinical pacritinib data from rodent and canine studies were evaluated in comparison to publicly available information for other JAK2 inhibitors to determine if differences in nonclinical endpoints might correlate with the observed clinical differences in myelosuppressive effects. The qualitative nonclinical findings seen in rodents were similar for all JAK2 inhibitors, and included decreases in leukocytes, red cell mass, and reticulocytes; lymphoid depletion in the spleen, thymus, and/or lymph nodes; and hypocellularity of the bone marrow. Interestingly, platelet decreases were not observed in rodent or canine nonclinical models, even with JAK2 inhibitors associated with clinical thrombocytopenia. However, in the dog, pacritinib is unique for its minimal myelosuppressive effects in the pivotal 30-day and 39-week studies, despite being performed at higher exposure levels relative to other similar JAK2 inhibitor studies. With pacritinib, non-adverse lymphoid reduction was seen in the spleen, lymph nodes, Peyer's patches, and/or thymus; but there were no clinical pathology findings indicative of marked myelosuppression, nor were there any treatment-related findings in detailed bone marrow evaluations. Overall, these comparisons suggest that nonclinical bone marrow data and clinical pathology in the dog may be informative endpoints for estimating the myelosuppressive potential of JAK2 inhibitors in the clinic.

#2603

Discovery of chemopreventive agents from licorice (Glycyrrhiza Uralensis).

Shunan Tang,1 Shuai Ji,2 Yongrui Wang,2 Ye Min,3 Siwang Yu,4 Tony A-N. Kong5. 1 _State Key Laboratory of Natural and Biomimetic Drugs, Beijing, China;_ 2 _State Key Laboratory of Natural and Biomimetic Drugs, Peking University School of Pharmaceutical Sciences, Beijing, China;_ 3 _Department of Natural Medicines, Peking University School of Pharmaceutical Sciences, Beijing, China;_ 4 _Department of Chemical Biology, Peking University School of Pharmaceutical Sciences, Beijing, China;_ 5 _Rutgers University School of Pharmacy, Piscataway, NJ_.

Traditional Chinese Medicine (TCM) provides a rich resource of potential chemoprevention agents; however, the complexity arise from its multi-components and multi-targets nature hindered in-depth investigations and translations. Licorice is one of the most commonly utilized TCM herbal medicine which possesses many pharmacological activities including cancer prevention. We isolated over 120 licorice-derived compounds and systemically screened these compounds using cellular and molecular models including cytotoxicity, nitric oxide production, NF-κB and ARE luciferase reporters, and other models.

Isoangustone A (IAA) is an isoprenyled isoflavonoid which has been reported to induce apoptosis and cell cycle arrest in prostate cancer cells, and it was identified as a NF-κB inhibitor in our assays. IAA significantly inhibited NF-κB signaling in SW480 colorectal adenocarcinoma cells, and decreased the expression of Cox-2 and iNOS. Overexpression of constitutively activated IKKβ potently stimulated NF-κB signaling and reversed IAA-mediated inhibition. Oral administration of IAA significantly inhibited dextran sodium sulfate-induced NF-κB signaling and the expression of Cox-2 and iNOS in mouse colon tissue, and alleviated symptoms of colitis without observable acute toxicity.

IAA and similar compounds also induced autophagy in various cancer cells, and inhibition of autophagy significant reversed IAA-induced cell death, suggesting the existence of autophagic cell death. IAA caused cleavage and aggregation of LC-3 accompanied with p62 degradation, and typical autophagic morphology was observed by transmission electronic microscopy. Overexpression of dominant negative AMPK or treatment with AMPK inhibitor demonstrated that activation of AMPK was important for IAA-induced autophagy, while inhibition of Akt was not. Further studies suggest that metabolic stresses is essential for the pharmacological activities of IAA, and primary molecular target were identified. Notably, IAA significantly inhibited tumor growth in nude mice xenograft model.

These results suggest that IAA is potentially an anti-inflammatory bowel disease and anti- colorectal cancer agent. Based on the structural similarity, several other compounds were found to possess even stronger anti-inflammation, pro-apoptotic and autophagy-inducing activities. The present work discovered several promising chemopreventive compounds from licorice that deserve future study, and provides an example of study on TCM in chemoprevention.

Several other compounds were identified as Nrf2/ARE signaling modulators or apoptosis/autophagy inducers and further investigated.

The present works were supported by the National Natural Science Foundation of China (No. 81272468, 81472657).

#2604

Chemoprevention of colorectal cancer by LFA-9, a novel dual mPGES-1/5-LOX inhibitor: safer approaches to chemoprevention.

Naveena B. Janakiram,1 Altaf Mohammed,1 Gopal Pathuri,1 Venkateshwar Madka,1 Rebekah Ritchie,1 Taylor Bryant,1 Yuting Zhang,1 Qian Li,1 Stan Lightfoot,1 Hariprasad Gali,1 Steele E. Vernon,2 Chen S. Suen,2 Chinthalapally V. Rao1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Oklahoma City, OK_.

Colorectal cancer (CRC) protective effects of NSAIDs and COX-2 inhibitors are well established; however, they are associated with gastrointestinal (GI) bleeding and cardiovascular risk. Mechanistic studies suggest that sparing COX-1/2, prostaglandin (PG)I2 synthase and selectively targeting microsomal PG synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) would inhibit PGE2 and Leukotrienes (LTs), respectively. To design and develop selective inhibitors of mPGES-1/5-LOX, we used insilico small molecular docking simulation approaches, and identified LFA-9 as a novel selective dual mPGES-1/5-LOX inhibitor among >20 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized to assess inhibitory effects of LFA-9 on mPGES-1 and 5-LOX ex-vivo. At 7.5 μM, LFA-9 inhibited the mPGES-1 and 5-LOX activities by ~57% and ~68%, respectively. Maximum tolerable dose (100 -1,600ppm) of LFA-9 in AIN-76A diet on male C57Bl/6 mice were tested and observed that <1,600 ppm dietary LFA-9 to be safe and free from liver, GI and hematological toxicities. In AOM/DSS-induced colonic inflammation in rat, LFA-9 at ≥200ppm abolished the inflammation and suppressed mPGES-1 and 5-LOX activities in a dose-response manner.

Potential CRC preventive efficacy of LFA-9 was assessed in AOM-rat carcinogenesis with colonic Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. In rats, colonic ACF were induced by AOM/DSS treatment and two week after the AOM/DSS treatment, LFA-9 (200, 400, and 600ppm) was fed by diet and ACF were evaluated after six-weeks. LFA-9 showed dose-response (P<0.0001) inhibitory effect on ACFs formation. At 600ppm, LFA-9 significantly inhibited colonic total ACF and multi-crypts by >60% (P<0.0001). In APCMin/+ mice study, six-week-old male and female mice (10/group) were fed diet containing 0, 350, and 700 ppm LFA-9 for 14 weeks. Male and female APCMin/+ mice fed control diet developed 58±3.8 (Mean±SEM) and 59±5.9 polyps, respectively. LFA-9 administration at 350 and 700ppm in APCMin/+ mice significantly (p<0.0001) reduced total intestinal tumor multiplicity and size dose-dependently (31.6± 5.9 and 24.3±3.7, male mice; and 26.2±5.6 and 15.5±1.8, female mice, respectively). At the high dose both male and female mice showed a > 80% suppression of polyps with >2mm size. Mice fed 350ppm LFA9 showed colon tumor inhibition of 60% (male) and 90% (female). It is noteworthy that both male and female mice fed 700ppm LFA-9 showed 91% inhibition of colon tumors. LFA-9 showed significant suppression of markers of proliferation and inflammation. Overall, above results suggest LFA-9 as a novel dual mPGES-1/5-LOX inhibitor, a safer agent than other NSAIDs and has a potential for prevention of CRC in high-risk patients.

(Grant Support: NCI N01-CN 25001-26).

#2605

Metformin reduces growth of tumors generated from neuroblastoma stem cells in a xenograft mouse model; role of Cdc42 in mediating the effects.

Ambrish Kumar, Donald J. DiPette, Ugra S. Singh. _University of South Carolina School of Medicine, Columbia, SC_.

Neuroblastoma, the most common malignant childhood cancer of the postganglionic sympathetic nervous system, is derived from the neural crest cells. Despite the standard therapy, the mortality rate remains high in children with neuroblastoma. Growing evidence confirm that cancer stem cells are responsible for drug resistance, and disease relapse. Hence targeting of cancer stem cells is an effective strategy to cure cancer. In the present study we tested if anti-diabetic drug metformin (N', N'-dimethylbiguanide) has anti-survival effects against neuroblastoma stem cells.

Using a panel of human neuroblastoma cell lines of different genetics- SH-SY5Y, SK-N-BE(2), IMR-32, NGP, and SK-N-F1 cells, we demonstrated that metformin dose-dependently reduced the protein expression of stem cell-specific transcription factors- sox2, oct4 and nanog. Neuroblastoma cells, in stem cell specific medium, formed compact and distinct spheroids which were enriched in stem cells. Addition of metformin (0.5 mM and onwards) significantly inhibited initiation of spheroids, and no spheroid formation was observed at 20 mM metformin. We further examined if metformin interfered with self-renewal and differentiation capacity of neuroblastoma stem cells. Our results demonstrated that metformin-treated primary spheroids lost their ability to form secondary spheroids in a metformin depleted medium (drug withdrawal experiment). However, addition of pharmacological inhibitor of Cdc42 (ML141) along with metformin increased the numbers of spheroids suggesting involvement of Cdc42 in spheroid formation. To further test the inhibitory effect of metformin on the tumorigenicity of neuroblastoma stem cells, we generated subcutaneous tumors by inoculating spheroids in athymic mice. Metformin (10, 30, and 100 mg/kg per mouse) was given daily by oral gavage, and tumor volume was measured. The size of tumors collected from mice fed with 30 and 100 mg/kg metformin was significantly reduced compared to tumors from metformin-untreated mice. In these tumors, metformin induced DNA fragmentation and apoptosis by activating caspase-3. The presence of cleaved caspase-3 signal in both sox2-expressing and sox2-nonexpressing cells indicated that metformin induced cell death in both normal tumor cells and tumor stem cells. These data validate the anti-survival activity of metformin against neuroblastoma stem cells.

The fact that metformin is non-toxic and already approved by FDA to treat type 2 diabetes in children suggest metformin is a novel therapeutic drug to treat neuroblastoma.

#2606

Chemo-preventive efficacy of a natural product anti-cancer agent which surpasses Taxol.

Carolyn B. Howard. _Jackson State Univ., Jackson, MS_.

Breast cancer (BC), a major public health problem, is one of the leading causes of cancer-related deaths among women. In particular, triple-negative breast cancer (TNBC), the most aggressive form of BC poses significant therapeutic challenges. TNBCs are most common in women with BRCA1 gene mutations, younger women, and in pre-menopausal African American (AA) women compared to White women, significantly contributing to health disparities. Consisting of many heterogeneous, undifferentiated cell types including very high mammary cancer stem cell (MCSCs) content, TNBCs are diseases with high recurrence rates. Combining therapeutics to target a variety of growth pathways has inhibited growth and shrunk aggressive TN tumors. Yet, a major drawback of such therapy is the severe side effects commonly used chemotherapeutic agents have on normal cells. Therefore there is a critical need to develop new combinations of therapeutics that target MCSCs and improve the anticancer effects, while reducing harmful side effects. We present compelling preliminary findings comparing the anticancer effects of aqueous extracts of a plant, Vernonia amygdalina (VA), alone or in combination with Paclitaxel (TAX) in vitro and against TNBC cells injected into athymic mice intra-peritoneally. The effects of TAX alone or VA extracts alone or with TAX, were evaluated in Hsd:Athymic Nude-Foxn1nu, age 5-6 weeks, female mice inoculated subcutaneously with Hras MCSCs. Animals were grouped as follows: 1-Control, inoculated with Hras cells only; 2-VA treatment (10 mg/kg) simultaneously with injection of Hras cells, followed by 16-day VA treatment; 3-replicate of 2; 4-pretreatment with VA 10 days before injection of Hras cells, followed by 16-day VA treatment; 5-IP inoculation of TAX (10 mg/kg ) simultaneously with injection of Hras cells; and 6-subcutaneous inoculation of VA (5 mg/kg ) plus IP injection of TAX (5 mg/kg) simultaneously with injection of Hras cells. Studies revealed that though there was a lag in tumor growth for all treatment groups, the most significant reduction in tumor size was observed in group 4 animals, those which had been pretreated with VA each day for 10 days prior to inoculation with Hras cells. In the second round of experiments, we evaluated the efficacy of VA extracts (10-day pretreatment, high dose, 20 mg/kg) administered simultaneously with injections of both Hras cells and AA woman-derived MDA-MB-468 cells, at opposing sites, versus combined treatment with very low doses of VA and TAX (5 mg/kg of each agent, subcutaneously) toward inhibition of growth of tumors in our nude mice model. Based upon our findings, VA extract exhibits chemopreventative efficacy, addressing a major health problem which is the dominant biological cause of population-based disparities in BC mortality in the U.S. Our expectation is that this work could direct further in vivo studies and eventually translate to human clinical trials.

#2607

Honokiol, a phytochemical from magnolia plant, prevents UV radiation-induced immune suppression in mice through inhibition of PGE2 and DNA methylation.

Santosh K. Katiyar, Tripti Singh, Ram Prasad. _University of Alabama at Birmingham, Birmingham, AL_.

The exposure of ultraviolet radiation (UVR) to murine skin suppresses the development of allergic contact hypersensitivity (CHS) response which is considered to be a prototypic T-cell mediated immune response. UVR-induced suppression of immune system has been implicated in skin cancer risk. Honokiol has been shown to have anti-skin carcinogenic activity, and here we have assessed the effect of honokiol on UVR-induced immunosuppression and its possible mechanism of action. Our CHS experiments revealed that exposure of C3H/HeN mouse skin with UVB radiation (150 mJ/cm2) for 4 consecutive days resulted in significant suppression of CHS response (75%, P<0.001) to the skin contact sensitizer, 2,4-dinitrofluorobenzene (DNFB), which was associated with the enhancement in the levels of cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. Topical treatment of mice with honokiol (0.5 and 1.0 mg/cm2 skin area) in hydrophilic-cream based topical formulation or indomethacin (50 µg/mouse), a COX-2 inhibitor, significantly inhibits UV radiation-induced suppression of CHS response (P<0.01-0.005). The exposure of mice with UVB resulted in DNA hypermethylation, increase in DNA methyltransferase (Dnmt) activity, and elevated levels of Dnmt proteins in mouse skin, while treatment of mice with honokiol before each UVB exposure reduced the levels of DNA methylation as well as Dnmt activity compared with non-honokiol-treated control mice. The COX-2 deficient mice are resistant to UV-induced suppression of CHS response. Treatment of UVB exposed COX-2-deficient mice with PGE2 (50 µg/mouse/day) results in UV-induced suppression of CHS, increase in DNA methylation and Dnmt activity in the skin. Further, treatment of honokiol reversed the effect of PGE2 on UV-induced suppression of CHS response in COX-2-deficient mice. Treatment of C3H/HeN mice with 5-aza-2'-deoxycytidine (5-Aza-dc, 1.0 mg/kg body weight, i.p.), a DNA demethylating agent, after each UVB exposure restored the CHS response which was associated with a reduction in Dnmt activity and global DNA methylation levels compared to the UV-exposed mice which were not treated with 5-Aza-dc. We also compared the effect of honokiol with commercially available anti-cancer drugs, such as imiquimod and 5-fluorouracil, on CHS response in UV-exposed mice. Topical treatment of mice with equi-molar concentrations of honokiol and cancer drugs did not show significant difference in terms of inhibition of UVB induced immune suppression and thus suggests honokiol as a substitute for these drugs. These findings provide evidence that honokiol acts through inhibition of inflammatory mediators and subsequently DNA demethylation in UVR-exposed mouse skin.

#2608

Dietary polyunsaturated fatty acids modulate adipose inflammation and the adipokine secretome to influence mammary stem cell self-renewal.

Raymond M. Esper,1 Evan Hill,1 Yan Jiang,2 Nadeem Aslam,1 Becky Simon,3 Max Wicha,1 William Smith,1 Dean E. Brenner1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _MD Anderson, Houston, TX;_ 3 _BioCentury Publications, Redwood City, CA_.

Obesity strongly increases risk for multiple malignancies. One potential contributor may be the chronic low-grade inflammation that is likely driven by dysfunctional adipose characterized by aberrant adipokine production and macrophage activation/polarization. In adipose tissue, free fatty acids and their lipid mediators serve as important regulators of adipokine gene transcription. The adipocyte fatty acid composition is influenced by dietary availability. Here we present data demonstrating that dietary fish oil supplementation in obese rats (achieving a 2.5:1 ω6:ω3 PUFA ratio, comparable to a Mediterranean diet) attenuates adipose inflammation characterized reduction in the M1/M2 macrophage polarization ratio compared to a typical Western diet with a 20:1 ω6:ω3 PUFA ratio. We developed a novel adipose explant culture system to directly assay the secretome, and demonstrate that quenching adipose inflammation in obese animals is associated with a favorably rebalanced adipokine milieu, most notably increased adiponectin concentration and decreased leptin secretion. We previously showed that these two adipokines differentially modulate mammary stem cell self-renewal; here, we studied the effect of adipose conditioned media from obese rats on mammary stem cells using the mammosphere formation assay. Adipokines from obese rats fed the high-ω6 diet increased mammary stem cell symmetric self-renewal by 86.7 ± 7.5% ( ± SEM), whereas adipose-derived factors from the obese animals fed a low-ω6 diet supplemented with fish oil reduced the number of secondary mammospheres by 30.3 ± 10.1% by promoting either apoptosis, quiescence, or symmetric division-differentiation of the primary stem cells. Using a neutralizing antibody and a soluble receptor, we demonstrate that leptin contributes only 19% of the adipose-derived effect on mammary stem cell self-renewal. Taken together, our data suggests that a simple dietary intervention to modify the dietary PUFA ratio is an effective method to quench adipose inflammation and rebalance the adipokine milieu, which may modulate the mammary stem cell pool. If normal stem cells can give rise to breast cancer as has been theorized, then dietary interventions to quench adipose inflammation and reduce the rate of symmetric stem cell self-renewal might protect against the increased incidence of breast cancer seen in obese post-menopausal women.

#2609

Epigenetic reactivation of TIMP-3 in human prostate cancer cells by green tea polyphenols.

Gauri Deb, Vijay S. Thakur, Eswar Shankar, Sanjay Gupta. _Case Western Reserve University, Cleveland, OH_.

Metastasis is responsible for more than 90% of prostate cancer-associated mortality in the United States. Metastasis is facilitated by remodeling of the extracellular matrix by a family of proteolytic enzymes known as matrix metalloproteinases (MMPs). These zinc-dependent proteases elicit pivotal role in the proteolytic degradation of structural components of extracellular matrix contributing to the metastatic process. High levels of MMP-2/9 in the plasma and urine has been correlated with metastasis. MMPs are produced in a latent form (pro-MMP) that require activation and are inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). TIMPs inhibit MMPs by binding in a 1:1 stoichiometry through strong noncovalent interactions. In prostate cancer, low levels of TIMP3 has been associated with aggressive tumor phenotype and poor disease-free survival. Loss of TIMP3 in prostate cancer is attributed to epigenetic silencing tipping the balance towards active MMPs driving metastasis. Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) has been shown to suppress prostate cancer metastasis, however the mechanism has not been fully elucidated. We determined whether treatment with GTP/EGCG has ability to restore induction of TIMP3 and play a key role in suppressing invasiveness in prostate cancer. Treatment of prostate cancer LNCaP and DUPRO cells with 10µg/mL GTP and 20µM EGCG for 72 h significantly induced TIMP3 mRNA and protein levels. Silencing of enhancer of zeste homolog 2 (EZH2) and class I histone deacetylases (HDACs) significantly increased the expression of TIMP3 which was independent of DNA hypermethylation. Treatment of prostate cancer cells with GTP/EGCG significantly reduced EZH2 and class I HDAC protein levels; H3K27 trimethylation activity; and transcriptional activation of TIMP-3 was found to be associated with decreased EZH2 localization and H3K27 trimethylation enrichment at the TIMP3 promoter with a concomitant increase in histone H3K9/18 acetylation. EGCG/GTP treatment also caused a global reduction in histone methylation thereby increasing histone acetylation to revive and activate TIMP3 levels. Furthermore, EGCG/GTP exposure significantly reduced MMP2/9 gelatinolytic activity and abrogated invasive and migration capabilities in prostate cancer cells. Taken together, our findings suggests that TIMP-3 induction could be a key epigenetic event modulated by GTPs in restoring MMP: TIMP balance to delay prostate cancer invasion and metastasis.

#2610

Targeting the mTOR pathway for the prevention of ER-negative breast cancer.

Abhijit Mazumdar, Yun Zhang, Jamal Hill, Powel Brown. _UT MD Anderson Cancer Center, Houston, TX_.

Background:

Prevention of estrogen receptor (ER)-positive breast cancer prevention is now possible antiestrogen drugs; however, this treatment is ineffective against ER-negative breast cancers. ER-negative breast cancer occurs more often in younger women and is associated with a poor prognosis. Dysregulation of PI3K-mTOR pathway, specifically upregulation of mTOR and loss of phosphatase and tensin homolog (PTEN) has been commonly associated with ER-negative breast cancers and been shown to be associated with poor prognosis. We hypothesized that by targeting mTOR, will suppress the growth of ER-negative and triple negative breast cancers. To test the hypothesis, we used MMTV-erbB2, C3(1)/SV40T, and p53 null mammary gland mouse models to determine whether mTOR inhibitor everolimus is effective in preventing growth of ER-negative mammary tumors.

Methods

p53-null Balb/C donor mice were transplanted into both cleared inguinal mammary fat pads of Balb/C p53 wild-type mice. All mice were separated into 2 groups 1) sesame oil control and 2) everolimus (5 mg/kg). All treatments were given by oral gavage, five days a week from 3 months of age for MMTV-erbB2 and 2 months of age for SV40T mice, and in case of p53 mice, two months after transplantation. Mice were observed daily for tumor formation, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the generalized Wilcoxon test.

Results:

In MMTV-erbB2 mice the mTOR inhibitor, everolimus reduced tumor incidence and was associated with an increase in median survival time from 240 days to 410 days in. At 365 days, when all mice in control group died, only half of the mice treated with everolimus had developed mammary tumors. Long term treatment of everolimus was associated with mild toxicity that includes weight loss (<10%) and skin changes (matted hair, skin erythema). Everolimus also reduced tumor incidence in C3 (1)/SV40 mice, with an increase in median survival time from 130 days to median survival which has not reached yet (>180 days). At 150 days, when all mice from the control group developed tumor only 35% mice from treatment group had developed tumors. Everolimus also significantly reduced tumor incidence in p53 null mammary gland mice. At 420 days, when approximately in 50% of the control mice developed mammary tumors compare to only 7 % of the everolimus treated mice developed tumors.

Conclusion:

The mTOR inhibitor everolimus prevented ER-negative mammary in two mouse models (MMTV-erbB2, C3 (1)/SV40, and p53 null mammary gland mice). Based on our results Everolimus is an effective cancer preventive drug. Our results suggest that, studies with reduced everolimus dose alone or in combination with other targeted therapies such as selective estrogen receptor modulators are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients.

#2611

Effects of chalcones, nicotinamide, and resveratrol on PPAR gamma activation in oral cancer cells.

Jennifer Diaz, Beverly Wuertz, Art Galbraith, Frank G. Ondrey. _University of Minnesota, Minneapolis, MN_.

Several classes of natural products have cancer prevention potential and PPAR gamma is an intriguing chemoprevention target as a nuclear receptor that controls multiple growth and differentiation processes. We have previously tested many classes of natural products in A/J mice and have found that the following chemoprevention compounds had no significant dietary toxicities: inositol, eucalyptol, chalcones, nicotinamides, and resveratrol. We tested these compounds for their capacity to activate PPAR gamma reporter genes in CA-9-22 oral cancer cells. In several repeated experiments we found that Inositol hexaphosphate did not activate PPAR gamma reporter genes at high concentrations of 1-4 mM. We found that eucalyptol, trans-Chalcone, 4 methoxy chalcone, Nicotinic Acid, and Nicotinamide had the capacity for statistically significant activation of PPAR gamma reporter genes in oral cancer cells from 20-60% at concentrations of 1-100 uM. We found that resveratrol was the most potent agent and could activate PPAR gamma reporter genes 2 fold. As a reference, this was about half the activity of the prototypic PPAR gamma activator pioglitazone. We performed additional experiments with resveratrol and found that it could activate the PPAR gamma dependent squamous differentiation gene Involucrin in oral cancer cells and BEAS 2B cells. Additionally, resveratrol significantly decreased BEAS 2 B proliferation from 48-96 hours at concentrations of 10 uM. From this data we conclude that resveratrol has the capacity to activate PPAR gamma in vitro and resveratrol and its derivatives may also function as PPAR gamma activators as an additional mechanism of action. We also conclude that inositol, eucalyptol, chalcones, and nicotinamides do not activate PPAR gamma.

#2612

Zinc inhibits Orai1-mediated Ca2+ signals and proliferation in esophageal cancer cells.

Sangyong Choi,1 Chaochu Cui,2 Yanhong Luo,3 Jae-Kyun Ko,4 Jianjie Ma,1 Liwu Fu,2 Irina Korichneva,5 Zui Pan1. 1 _The Ohio State University, Columbus, OH;_ 2 _Sun Yat-Sen University Cancer Institute, Guangzhou, China;_ 3 _The Children's Hospital of Chongqing Medical University, Chongqing, China;_ 4 _Rutgers University-Robert Wood Johnson Medical School, Piscataway, NJ;_ 5 _University of Picardie Jules Verne, Amiens, France_.

Intracellular Ca2+ signals, including oscillations, regulate proliferation, migration, and other cellular events in cancer cells. Zinc (Zn), an essential micronutrient, has been studied for its chemopreventive effects in several cancers, including esophageal cancer. Although there is a growing research interest in the cross-talk between Ca2+ and Zn signaling, it remains elusive as to how Zn prevents tumor growth and whether Ca2+ signaling is involved. Our previous report demonstrated that Orai1, a store-operated Ca2+ entry (SOCE) channel, is highly expressed in esophageal squamous cell carcinoma (ESCC) compared to normal tissues, and that the elevated expression of Orai1 is strongly associated with poor prognosis in patients. In the current study, we show that physiological levels of Zn can significantly suppress cell proliferation in KYSE-150, a human ESCC cell line. We also show that Zn is able to inhibit Orai1-mediated SOCE and intracellular Ca2+ oscillations, both which are known as proliferation signals. Based on the topology information of Orai1, we hypothesized that the histidine residue in linker region between transmembrane 1 and 2 of Orai1 (H113) as well as three cysteine residues may play a critical role in Zn-inhibitory effects. Using a point mutation approach, we exhibit that the Zn-inhibitory effects on both SOCE and cell proliferation are vanished in KYSE-150 cells containing Orai1 H113A mutant. Furthermore, KYSE-150 cells expressing Orai1 with any mutation in cysteine residues (C126A, C143A and C195A) display significant loss of Zn-inhibitory functions. These results indicate that the four amino acid residues are likely to be involved in Zn-inhibitory effects on SOCE and cell proliferation.

Taken together, our data suggest that dietary Zn may inhibit Orai1-mediated SOCE and intracellular Ca2+ signaling, which in turn suppresses cell proliferation in ESCC. Further studies are required to search for novel and effective prevention strategies as well as therapeutic options targeting on Zn and Ca2+ signaling in ESCC.

#2613

MEK is a therapeutic and chemopreventative target in squamous cell carcinoma.

Charles H. Adelmann, Kimberly Truong, Roger Liang, Varun Bansal, Rachael Saporito, Woojin Lee, Lili Du, Courtney Nicholas, Marco Napoli, Elsa R. Flores, Kenneth Y. Tsai. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Purpose: While the prognosis of cutaneous squamous cell carcinoma (cuSCC) is excellent overall, advanced metastatic disease represents a substantial mortality burden for which no standard targeted therapy exists. Findings from genomic and proteomic studies and the observed induction of cuSCC by BRAF inhibitor driven MEK/ERK pathway engagement suggest that MEK/ERK activation is essential for cuSCC tumorigenesis and tumor proliferation. Most cuSCC arise from a clinically and histopathologically defined preneoplastic precursor, the actinic keratosis (AK). Molecular studies of early events in cuSCC pathogenesis strongly implicate MEK/ERK signaling at the proteomic and transcriptional level. This occurs at the earliest recognizable transitions from chronically UV-exposed skin to AK, with sharp elevation of ETS2/ELK1 transcriptional target expression. With these compelling rationales in mind, we tested whether MEK inhibitors (MEKi) are a clinically actionable treatment and chemoprevention approach for cuSCC. Given that several MEKi are approved, this is a readily translated strategy.

Experimental Design: Preclinical testing was performed in 10 cuSCC cell lines and two mouse models of cuSCC using two distinct MEK inhibitors, trametinib and cobimetinib. We show that two MEKi, trametinib and cobimetinib are highly effective against cuSCC cell lines in culture, effectively engage MEK/ERK signaling in cells and in vivo, and potently induce cell cycle arrest and senescence. Both established and new tumors are powerfully inhibited in both xenograft and UV-driven autochthonous mouse models. This model, which utilizes immunocompetent SKH-1E mice exposed to chronic, low-dose, solar simulated UV light (12.5 kJ/m2 UVB weekly administered across three doses) more faithfully recapitulates human cuSCC molecularly than chemical carcinogenesis models. Lesions in this model exhibit heterogeneity in latency and responses to therapy, as do human tumors.

Results: MEK inhibitor treatment of cuSCC lines strongly reduces proliferation and induces senescence markers in cells, including p21 and beta-galactosidase. This response was universal, but highly heterogeneous. Sensitivity to MEKi was determined, in part, by modulation of AKT activity, and combination MEKi and AKTi . In-vivo, an SRB1 xenograft model was exquisitely sensitive to oral trametinib treatment, and in our spontaneous UV-driven Hairless mouse model of cuSCC, treatment with the MEK inhibitors trametinib and cobimetinib strongly reduced tumor growth and almost completely abrogated tumor induction. We confirmed target engagement in-vivo showing that ERK signaling was significantly suppressed.

Conclusions: Overall, we conclude MEK signaling is critical for cuSCC tumor induction and maintenance, and that MEK inhibition is an attractive approach for both advanced cuSCC treatment as well as chemoprevention.

#2614

Surgical weight loss via sleeve gastrectomy, but not a low-fat diet, reverses the pro-tumorigenic effects of obesity.

Emily L. Rossi,1 Laura W. Bowers,1 Subreen A. Khatib,1 Laura A. Smith,1 Steven S. Doerstling,1 Alfor Lewis,2 Randy J. Seeley,2 Stephen D. Hursting1. 1 _The University of North Carolina, Chapel Hill, NC;_ 2 _The University of Michigan, Ann Arbor, MI_.

Background: Obesity is associated with increased incidence of basal-like breast cancer (BLBC), the most aggressive and lethal breast cancer subtype. Epidemiological data is conflicting regarding whether weight loss offers protection against BLBC in obese women; only interventions that typically result in significant sustained weight loss, such as bariatric surgery, produce a consistent anti-cancer benefit.

Purpose: We sought to determine the differential effects of surgical and non-surgical weight loss interventions on inflammation, metabolic hormones and tumor burden in a mouse model of pre-menopausal breast cancer.

Methods: Mice were fed a low fat control or high fat diet-induced obesity (DIO) regimen for 15 weeks to model chronic obesity. Diet-induced obese mice (n=75) were randomized to receive either a surgical weight loss intervention (sleeve gastrectomy) or dietary weight loss intervention (switch to low fat control diet), resulting in formerly obese (FOb)-Surg or FOb-Diet mice, respectively. Additionally, a subset of mice remained on the DIO diet (Obese, n=25), with another subset of normoweight control mice (Con, n=25) maintained on a low fat diet throughout the study. FOb-Surg and FOb-Diet mice lost a nearly identical amount of weight and body fat; both groups had significantly lower weight and percent body fat than Con. Four weeks after weight stabilized, all mice on study were orthotopically injected with E0771 mammary tumor cells, which model BLBC.

Results: At study endpoint, the average tumor weight in FOb-Surg mice was statistically equivalent to normoweight control mice. However, the average tumor weight in FOb-Diet mice was significantly greater than controls and statistically equivalent to the Obese mice. Furthermore, FOb-Surg mice had statistically lower levels of serum interleukin-6 and insulin compared to FOb-Diet, suggesting that in obese mice sleeve gastrectomy, relative to diet-induced weight loss, more effectively reduced obesity-associated inflammation, hyperinsulinemia and mammary tumor growth.

Conclusion: Our results suggest that the strong anti-cancer benefits seen with bariatric surgery may be related to a significant reduction in systemic inflammation and growth factor signaling, which did not occur with non-surgical weight loss despite an equivalent amount of weight and body fat loss in FOb-Diet mice. Identifying the mechanisms underlying the protective effects of bariatric surgery against breast cancer could help identify new targets and strategies for breaking the obesity-cancer link.

#2615

Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

Rahul K. Lall, Deeba N. Syed, Mohammad Imran Khan, Vaqar M. Adhami, Yuansheng Gong, John A. Lucey, Hasan Mukhtar. _University of Wisconsin, Madison, WI_.

With an estimated 220,800 new cases and 27,450 deaths in 2015, prostate cancer (PCa) continues to be the second leading cause of cancer related deaths in men in the US alone. Accumulation and turnover of extracellular matrix (ECM) is a hallmark of tissue injury, repair and remodeling in human diseases including PCa. Hyaluronan (HA), an extracellular glycosaminoglycan, is a major component of the ECM and plays an important role in regulating reepithelization, tissue healing and wound repair. Elevated HA levels have been shown to be associated with aggressiveness and disease progression in certain cancer types including PCa. Biological responses triggered by HA mainly depend on the HA polymer length. High molecular mass (HMM)-HA represses mitogenic signaling and has anti-inflammatory properties while low molecular mass (LMM)-HA is pro-angiogenic and promotes proliferation and inflammation. Diet-derived agents, such as flavonoids, are of particular interest for cancer chemoprevention as they offer a relatively favorable safety profile. Using HPLC-MS analysis, we identified HA as a novel target of the dietary flavonoid fisetin in tumor xenografts of human PCa NB11 and NB26 cells. Fisetin treatment decreased HA levels in PCa cells both in-vitro (40µM:40h) and in-vivo (1mg/animal). We then evaluated the effect of intraperitoneal administration of fisetin on HA size accumulation and prostate carcinogenesis using the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Using the combination of Size Exclusion Chromatography (SEC) with Multi-Angle Laser Light Scattering (MALS) analysis that overcomes limitations of column calibration, we evaluated intracellular and extracellular molar mass distribution of HA in fisetin treated cell culture and animal models. We observed lower levels of HMM-HA and higher levels of pro-angiogenic LMW-HA fragments in human PCa PC3 cells. In contrast, we found that fisetin treatment increased HMM-HA and reduced LMM-HA levels in PCa cells. Remarkably, the HA size profile of fisetin treated cells resembled those of normal RWPE1 cells, which predominantly exhibited higher abundance of anti-angiogenic HMM-HA and no detectable LMW-HA. Likewise, secreted HA in fisetin treated PCa cells comprised predominantly of LMM-HA, suggesting greater accumulation of anti-angiogenic HMM-HA within the cells. Molar mass analysis of fisetin treated TRAMP tumor tissues showed an abundance of HMM-HA and reduced levels of LMM-HA when compared to untreated controls. Similarly, secreted HA in fisetin treated TRAMP animals exhibited increased pool of pro-angiogenic LMM-HA, suggesting greater accumulation of anti-angiogenic HMM-HA within the tissues. Our findings suggest that fisetin confers resistance to PCa oncogenesis by increasing the abundance of anti-angiogenic HMM-HA which is associated with delayed disease progression.

#2616

Inhibition of TGFß to prevent brain metastases of breast cancer.

Chris E. Adkins, Afroz S. Mohammad, Emma Dolan, Jessica Griffith, Tori Terrell-Hall, Paul R. Lockman. _West Virginia University, Morgantown, WV_.

Introduction: Because systemic chemotherapy fails to effectively treat brain metastases, the need for novel strategies designed to prevent brain metastasis is urgent. Recently, transforming growth factor beta (TGFß) has been shown to influence the development of brain metastases of breast cancer and may offer a therapeutic target to prevent metastatic disease in brain. This study seeks to evaluate the efficacy of TGFß inhibition, using losartan, at preventing metastases in an experimental model of brain metastases of breast cancer.

Methods: Human breast cancer cells (MDA-MB-231Br-Luc) were inoculated into female NuNu mice after TGFß knockdown or treatment with inhibitor; after 1-week mice were sacrificed, their brains removed and sliced, and metastatic cells counted and localized to determine seeding. Survival studies were then initiated with daily low (10mg/kg) or high (25mg/kg) doses of losartan, or SB431542 (5mg/kg) prior to the injection of one of two human breast cancer cell lines (MDA-MB-231Br-Luc or JIMT-1-Br3-Luc) transfected with luciferase. Treatments resumed daily until neurological symptoms developed. Animals were sacrificed, brains removed, and sliced to evaluate the number of metastases and their respective sizes.

Results: The number of metastatic cells seeding brain was reduced after knockdown of TGFß (from 3,331 ± 263 cells to 1,079 ± 495 cells; p<0.01) and after treatment with galunisertib (808 ± 82 cells; p<0.01). There was a 33% reduction (p<0.05) in extravasated metastatic cells after TGFß knockdown and a 23% reduction (p<0.05) after galunisertib treatment. Mice inoculated with MDA-MB-231Br-Luc had a median survival of 32 days while losartan treatments groups exhibited extended median survival (38 days low, 43 days high) and inhibitor survival extended to 46 days. The MDA-MB-231Br-Luc TGFß knockdown median survival increased to 65 days. Median survival of mice inoculated with JIMT-1-Br3-Luc was 28 days and no difference (p>0.05) in median survival was observed for the JIMT-1-Br3-Luc losartan (30 days, low; 26 days, high) or inhibitor (29 days) treatment groups.

Conclusions: Inhibition of TGFß reduced the total number of metastatic MDA-MB-231Br cells seeding brain and simultaneously reduced the percentage of cells that had extravasated the vessel. While losartan did not increase survival for animals inoculated with JIMT-1-Br3-Luc cells, the MDA-MB-231Br model exhibited enhanced survival with losartan (15% survival rate; high dose), inhibitor (20% survival rate) treatment, and after TGFß knockdown (38% survival rate). The data indicate that TGFß offers a potential therapeutic target to reduce the risk of brain metastases of breast cancer.

#2617

Protective effect of the β-blocker carvedilol on UVB-induced skin damage.

Kevin M. Huang, Kristan H. Cleveland, Steven Yeung, Bradley T. Andresen, Ying Huang. _Western University of Health Sciences, Pomona, CA_.

The response to ultraviolet (UV) radiation is the primary etiologic factor leading to carcinogenesis of the skin. The effects of UV radiation on the skin is generally transient, but the long term consequences can be carcinogenic. Despite growing public awareness and the use of sunscreen to prevent skin cancer, the incidence continues to rise, and therefore there is a need to develop innovative chemoprevention strategies. Previously, we showed that the receptor subtype nonselective β-adrenergic receptor antagonist (i.e., β-blocker) carvedilol prevented EGF and UVB-mediated neoplastic transformation of the mouse epidermal cell line JB6 P+, indicating its chemopreventive potential against skin cancer. To demonstrate the in vivo effects of carvedilol, in the present study, hairless SKH-1 mice were irradiated for two weeks with 200 mJ/cm2 of UVB and drugs were topically applied immediately after the radiation. Since carvedilol is able to absorb UV, its analogue 4-hydroxycarbazole, which has the same UVB absorption profiling as carvedilol but does not function as a β-blocker, was included as a sunscreen control. Significant skin thickening measured by bi-fold skin thickness on the dorsal skin was observable within a week and over the course of the two-week period. Treatment with carvedilol, but not the analogue, reduced UVB-induced skin hyperplasia, reddening and inflammation. Furthermore, carvedilol, but not the analogue, reduced H2O2 and UVB- induced cytotoxicity and reactive oxygen species production in JB6 P+ cells. To elucidate the molecular mechanism(s) for carvedilol's chemopreventive activity, the signaling profile for JB6 P+ cells treated with EGF and/or carvedilol for 15 min was examined using a Phospho Explorer Antibody Microarray containing 1318 site and phosphor-specific antibodies from over 30 signaling pathways. The antibody array data suggested that the phosphorylation of the intracellular signaling pathways involved in skin carcinogenesis, AP-1 and NF-kB, were increased by EGF but attenuated by carvedilol. These results were validated using luciferase reporter assays. The array data also suggested that carvedilol was able to induce the expression of tumor suppressors p53 and PTEN. In addition, the checkpoint kinases CHK1 and CHK2 and other cell-cycle regulators such as cyclins were also upregulated by carvedilol, suggesting a role in carvedilol's protective effect. Since highly abundant mutations are observable in sun-exposed skin, our current work is to examine the effects of carvedilol on the production of cyclo-pyrimidine dimers (CPDs) induced by UVB. In conclusion, our study has demonstrated novel mechanisms underlying carvedilol's chemopreventive activity against skin cancer. Since carvedilol and other β-blockers are FDA approved drugs and relatively safe, they may offer a new approach of prevention for UVB-induced skin cancer.

#2618

Oncogenic mutant KRAS modulates CREB activation through MEK-ERK and AKT signaling in pancreatic cancer.

Jason Castellanos,1 Supriya Srinivasan,2 Kumar Honnenahally,1 Chanjuan Shi,1 Michael VanSaun,2 David Robbins,2 Nipun Merchant,2 Nagaraj Nagathihalli2. 1 _Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN;_ 2 _Department of Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL_.

Introduction: Cyclic AMP (cAMP) response element binding (CREB) overexpression in pancreatic ductal adenocarcinoma (PDAC) is associated with poor outcome, however, the mechanism(s) driving CREB overexpression in PDAC are not fully understood. We investigated the association of CREB activation with oncogenic KRAS, MEK-ERK and AKT signaling pathways.

Experimental procedure: Mouse lines derived from the Ptf1aCre/+; LSL-KrasG12D/+ (K518), Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT), LSL-KrasG12D/+;Pdx1Cre/+ (PanIN) and LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ (PDA) mouse models of PDAC, human immortalized pancreatic ductal epithelial lines (HPDE6-E6E7 or H6c7, HPNE E6/E7 and HPNE E6/E7/KRAS) were immunoblotted for phospho-CREB (pCREB) and total CREB expression. KRAS mutant PDAC cell lines and CREB shRNA flank xenografts (PKT GEMM and nude mice) were treated with CREB (ICG-001), MEK (AZD6244) and AKT (MK-2206) inhibitors. The effects on downstream signaling targets were interrogated with cell cycle, apoptosis, survival and anchorage-independent growth analysis. Human CREB shRNA cells were treated with AZD6244 and MK-2206 to confirm the molecular mechanism of the CREB inhibition together with MEK and AKT.

Results: The expression of pCREB was higher in cells with KRAS mutation. MEK inhibition resulted in activation of AKT, while combined inhibition of CREB and MEK prevented AKT reactivation. Treatment with the combination of MEK and CREB inhibitors significantly decreased tumorigenic potential and increased cell apoptosis. Combined MEK, AKT and CREB inhibition synergistically enhanced these effects further, more so in KRAS mutant cell lines. To explore the relationship of CREB, MEK and AKT signaling in vivo, PKT mice were treated with their respective inhibitors individually and in combination. Either CREB/MEK or CREB/AKT two drug combinations significantly extended the median survival compared with individual agents. Lysates from MEK and AKT inhibited tumors showed decreased phosphorylation of CREB, confirming that CREB is activated through both, MEK and AKT signaling in vivo.

Conclusions: Our study demonstrates that oncogenic KRAS activation enhances the expression of CREB through MEK and AKT signaling. CREB inhibition results in increased sensitivity to MEK and AKT targeted therapy in KRAS mutant PDACs.

#2619

Novel biomarkers and molecular alterations for breast cancer initiation and susceptibility.

Natascia Marino, Mariah L. Johnson, Hiromi Tanaka, Xi Wu, Theresa Mathieson, Jill E. Henry, Mircea Ivan, Yunlong Liu, Connie Rufenbarger, Anna Maria V. Storniolo. _Indiana University, Indianapolis, IN_.

Background: Despite significant advances in diagnosis and treatment, breast cancer remains the leading cause of cancer-related death in women worldwide. Therefore, there is a critical need to identify molecular mechanisms responsible for cancer initiation and progression. To date, there have been attempts to identify biomarkers that can predict progression of pre-malignant lesions (i.e. DCIS) to invasive carcinoma. Our study aims to identify earliest markers of breast cancer initiation.

Methods: We evaluated local (breast tissue) and systemic (serum/plasma) molecular alterations associated with breast cancer susceptibility using the unique resources available at the Komen Normal Tissue Bank at IUSCC. Human specimens (serum/plasma and breast tissue) donated by women before their cancer was clinically detectable were used to identify circulating biomarkers that are associated with breast cancer risk. Specimens from age-matched controls were used for comparison. Sera/plasma were analyzed for circulating miRNAs and telomeric level in the cell-free circulating DNA (cfDNA). We employed next generation sequencing to obtain transcriptome in the "normal" breast of women who eventually developed breast cancer and age-matched control normal breast.

Results/Discussion: Serum/plasma of women who developed breast cancer showed variation in circulating miRNAs as well as telomeric cfDNA levels as compared to the healthy control group. Among 385 microRNAs detected in circulation, 10 miRNAs were present at different levels in the susceptible group. In addition, susceptible group displayed reduced levels of plasma telomeric cfDNA and telomere shortening in breast epithelium.

Transcriptome analysis of microdissected breast epithelium and stroma revealed molecular alterations in the "normal" breast tissue associated with cancer development. Pathway analyses of these differentially expressed genes are underway to determine the mechanisms associated with breast cancer initiation.

Conclusion/Impact: Functional analysis of biomarkers identified in this study may help in cancer risk assessment and improvement of preventive therapy.

#2620

The relationships between necroptosis, apoptosis, and inflammation in human colorectal tissue.

Timothy Su,1 Chang Yu,2 Martha J. Shrubsole,1 Xiangzhu Zhu,1 Xinqing Deng,1 Eugene Shubin,1 Wei Zheng,1 Harvey J. Murff,3 Douglas L. Seidner,4 Reid M. Ness,4 Qi Dai*1. 1 _Department of Medicine, Division of Epidemiology, Vanderbilt University Medical Center, Nashville, TN;_ 2 _Department of Biostatistics, Vanderbilt Institute for Clinical and Translational Research, Vanderbilt University Medical Center, Nashville, TN;_ 3 _Department of Medicine, Division of General Internal Medicine and Public Health, Vanderbilt University School of Medicine, Nashville, TN;_ 4 _Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, Vanderbilt University Medical Center, Nashville, TN_.

Background: Formerly, apoptosis was considered the only form of regulated cell death and evading apoptosis was considered a hallmark of cancer. However, in recent years, necroptosis has been discovered as a new pathway of regulated cell death. Animal studies revealed very critical roles of necroptosis in many human inflammatory diseases and tumors. Early studies examining apoptosis by using TdT-mediated dUTP nick end labelling (TUNEL) assays cannot distinguish between apoptotic and necroptotic cells. Bax, which is a pro-apoptoic member of Bcl-2 family, is essential for mitochondrion-mediated apoptosis. Very recently, mixed lineage kinase domain-like (MLKL) is identified to be necessary for the execution of necroptosis. Cyclooxygenase (Cox), including Cox-1 and Cox-2, are the key enzymes responsible for the conversion of arachidonic acid to prostaglandins, which are important mediators of human physiology and pathophysiology, particularly inflammation. High expression of Cox-2 plays a critical role in the development of colorectal neoplasms. No study has examined how necroptosis biomarker (i.e. MLKL) and pro-apoptosis biomarker (i.e. Bax) are related to inflammation biomarker (Cox-2), TUNEL and cell proliferation biomarker (Ki-67). Methods: In our ongoing randomized trial (R01CA149633) "Personalized Prevention of Colorectal Cancer Trial (PPCCT)" conducted among colorectal polyp patients or those at high risk of colorectal cancer, we newly investigated the relationships between markers in these pathways using rectal biopsies (n=110) collected prior to intervention in the trial. The formalin-fixed and paraffin-embedded tissue from colorectal biopsy was serially sectioned, and three levels of the serial sections spaced 50 µm apart. were mounted on to one slide for each tissue block. The protein expression levels of Cox-2, Bax, pMLKL, and Ki-67 were detected immunohistochemically following EnVision+ System-HRP (DAB) rabbit or mouse kit (DAKO). Apoptotic cells in situ were detected with DeadEnd™ Colorimetric TUNEL System (Promega). Results: Cox-2 expression in both epithelium and stroma had strong positive correlation with Bax expression in the same zone (p-trend = 0.00186 and 0.00004, respectively) but was not significantly correlated with pMLKL. The positive associations were stronger in the upper zone than the bottom of the crypts. The upper zone also showed more TUNEL positive cells than the bottom zone, and had significant positive correlation with pMLKL intensity (r = 0.458, p = 0.005). Bax expression was not significantly related to MLKL expression. Conclusion: Cox-2 expression in non-tumoral colonic tissue may activate apoptotic pathway through Bax upregulation, but is not correlated with necroptotic pathway marker MLKL. In the epithelium, the necroptotic pathway may be more common in the upper zone of colonic cryps than the bottom zone,and may more strongly contribute to TUNEL than apoptosis pathway.

#2621

Disruption of mucin synthesis by targeting GCNT3 inhibits pancreatic cancer progression.

Altaf Mohammed, Naveena B. Janakiram, Venkateshwar Madka, Gaurav Kumar, Scott Edgar, Gopal Pathuri, Taylor Bryant, Hannah Kutsche, Yuting Zhang, Laura Biddick, Hariprasad Gali, Yan Daniel Zhao, Stan Lightfoot, Chinthalapally V. Rao. _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Pancreatic cancer (PC) is a lethal disease, and its management is an ongoing challenge. PC is the fourth leading cause of deaths due to cancer in the United States. It is a highly aggressive cancer that is usually diagnosed at an advanced stage, and has the worst prognosis of any malignancy, with a five year survival of <7% due to high chemoresistance. This chemoresistance is due in part to altered expressions of mucins, which form a mesh that makes target sites inaccessible to drugs. Clinical and preclinical studies have shown aberrant expression of mucins during PC development. The mucins may prevent drugs from accessing their sites of action. Although several mucins that lead to chemoresistance have been targeted, to date, no mucin synthesizing genes have been identified as targets. Using human pancreatic cancer patient survival data (90 cases of tumor and matched normal adjacent tissue), next-generation sequencing (NGS) of genetically engineered Kras mouse pancreatic tumors (N=6/group), human PC cells, we identified novel core mucin synthesizing gene target GCNT3 (core 2 beta 1,6 N-acetylglucosaminyltransferase). NGS revealed that GCNT3 upregulation (103-fold; p<0.0001) was correlated with increased mucins Muc4 (50-fold; p<0.04), Muc5ac (87-fold; p<0.01), Muc6 (67-fold; p<0.008), Muc1 (5-fold; p<0.009), Muc16 (5-fold; p<0.0003) and Muc20 (17-fold; p<0.007)]. Aberrant GCNT3 expression was associated with increased mucin production and aggressive tumorigenesis and reduced patient survival. Patients with low expression of GCNT3 had a longer survival time than patients with high expression of GCNT3 (median survival: 17.5 vs. 10.5 months, p=0.036). Further, using in-silico approaches of small molecular docking simulations, we identified talniflumate as a novel inhibitor that specifically binds to GCNT3. Our blind docking simulations reveal that talniflumate binds to GNTC3 with a docking affinity of -8.3 kcal/mol and deeper in the pocket of GCNT3. The docking predictions suggest three notable hydrogen bonds between talniflumate and GCNT3: Arg192 (3.0 Angstroms), Try288 (3.5 Angstroms), and Ala287 (2.9 Angstroms). Pancreata from 6-week-old Kras mice treated with talniflumate for 1 week showed a significant decrease in GCNT3 and mucin expression in PanIN lesions. mRNA expression of GCNT3 was also observed to be lower in pancreatic tissues from talniflumate-treated mice. CRISPR knock-out of GCNT3 in PC cells reduced proliferation and spheroid formation. Further, talniflumate alone and in combination with low-dose gefitinib reduced GCNT3 leading to disruption of mucins in vivo and in vitro. Hence, mucin disruption might enhance targeted therapy. These findings suggest a prominent role for Kras activation and aberrant mucin synthesis leading to PC pathogenesis, and warrant consideration of GCNT3 and EGFR inhibitors as a combination treatment for PC. (Grant Support: COMAA, Kerley-Cade Endowed Fund).

#2622

A LC-MS/MS method to quantify specific dietary isothiocyanates in human urine for epidemiological studies.

Marcin Dyba,1 Jian-Min Yuan,2 Jennifer Adams-Haduch,2 Fung-Lung Chung1. 1 _Georgetown University Medical Center, Washington, DC;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Epidemiological studies have demonstrated an inverse relationship between the intake of cruciferous vegetables and risk of cancers. The protective effects may be attributed to glucosinolates and glucobrassicins in these vegetables. While glucosinolates and glucobrassicins are not chemopreventive, their metabolites, isothiocyanates (ITCs), indole-3-carbinol (I3C), and 3,3′-diindolylmethane (DIM), have been shown to inhibit tumorigenesis in rodent models. In the past studies the total ITCs intake was estimated through food frequency questionnaires and/or urinary total ITC by a HPLC-based method. Self-reported intake of cruciferous vegetables has inherent limitations in estimating the intake of ITCs because of differences in the contents of their precursors present in different kinds of cruciferous vegetables, different locations and methods of cultivation, or different methods of food preparation. Animal and cell culture studies have shown that different types of ITCs have different anticancer properties and potency, suggesting that they are not equal in protecting against the development of cancers. The lack of a reliable method to quantify specific ITCs derived from dietary intake of cruciferous vegetables hinders the investigation of the role of specific ITCs against the development of cancer in human populations. To address this deficiency, we developed a flexible, highly sensitive, fast and reliable quantitative liquid chromatography mass spectrometry method (LC-MS/MS) for detecting and quantifying common dietary ITCs plus DIM in human urine. Urinary ITCs are measured as mercapturic acid conjugates (ITCs-NAC), predominant metabolite of ITCs, using multiple reaction monitoring (MRM) technique and deuterated [2H3]-ITCs-NAC as internal standards. DIM is quantified in separate experiment using [2H2]-DIM internal standard. The method was validated by a small set of previously analyzed samples from Singapore Chinese Health Study. We found an excellent correlation between previously reported total ITCs levels and sum of ITCs detected by newly developed method. The combination of minimal sample preparation, ultra-performance liquid chromatography, and highly sensitive mass spectrometer resulted in a method that is fast (below 10 min for ITCs and 12 min for DIM) and sensitive (low femtomolar LOQ) amenable to population studies.

#2623

Common genetic variants associated with breast cancer risk used in the Athena study to enhance models identifying women for breast cancer chemoprevention.

Sarah Theiner,1 Sarah D. Sawyer,1 Paige Kendall,1 Alexandra S. Perry,1 Denise Wolf,1 Scott Huntsman,1 Bo Pan,2 Jeffery A. Tice,1 David A. Pearce,3 Thomas Cink,3 Laura Esserman,1 Elad Ziv,1 Laura van 't Veer1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Peking Union Medical College Hospital, Beijing, China;_ 3 _Sanford Health, Sioux Falls, SD_.

Background: The U.S. Preventive Services Task Force recommends that women with a >3% five-year risk of developing breast cancer consider taking selective estrogen receptor modifiers (SERMs) or aromatase inhibitors (AIs) to reduce their risk. Polygenic risk score (PRS), calculated by adding the individual breast cancer risk association for each common genetic variant (SNP), has been found to predict women at low- to high-risk of breast cancer. We analyze associations between SNP risk alleles and known breast cancer risk factors (ethnicity, family history of breast cancer and number of biopsies); furthermore, we quantify the likely impact on chemoprevention recommendations by adding the PRS to known risk models in a subset of women participating in the University of California 100,000 women Athena Breast Health Network.

Methods: Our research cohort included 838 women with no previous diagnosis of breast cancer from the University of California, San Francisco, and was enriched for women determined to be at elevated risk for developing breast cancer by the Gail model. A panel of 75 breast cancer risk SNPs were evaluated on saliva and blood samples (Akesogen Inc; COGS oncochip array). The PRS for each patient was calculated by converting the odds ratio for each SNP into a likelihood ratio (LR) and combining LR's across SNPs. Breast Cancer Surveillance Consortium (BCSC), Gail, BCSC-PRS and Gail-PRS scores (risk models incorporating PRS within a Bayesian framework), were evaluated for each patient. Associations between variables were assessed using t-test or ANOVA. A threshold of p<0.05 was used to assess significance.

Results: Women in this study carry an average of 65 risk allele SNPs (of 150, 2 per locus). By ANOVA, there is a statistically significant association between the SNPs risk allele count and ethnicity (p=0.014), with a trend towards association with a family history of a first-degree relative with breast cancer (p=0.053). PRS is significantly associated with a family history breast cancer (p=0.031); neither SNP allele count nor PRS associates with previous biopsy status.

We found by adding PRS that 12% (86/707) and 13% (104/776) of patients with a prior BCSC or Gail score <3% five-year risk, respectively, changed classifications and would be eligible for chemoprevention. Conversely, 37% (36/98) and 36% (22/62) of patients with a BCSC or Gail score of >3% five-year risk, respectively, changed classifications by adding PRS and would no longer be eligible for chemoprevention.

Conclusion: The addition of SNP based PRS to BCSC and Gail models significantly changes how women are classified and as a result changes whether risk reducing agents are recommended.

PRS will be combined with BCSC and genetic test results for 9 breast cancer genes to calculate a women's breast cancer risk in the PCORI-funded Athena WISDOM study of 100,000 women, comparing risk-based vs. annual mammography screening.

#2624

Possibility of prostate cancer prevention based on cancer stem cell theory.

Nobuya Shiozawa,1 Ryosuke Sugahara,1 Ayami Sato,2 Masako Ota,1 Tomohiro Yano1. 1 _Toyo Univ., Gunma, Japan;_ 2 _Chiba Univ., Chiba, Japan_.

Cancer stem cells are considered to be one of the causes of cancer. In recent years, stemness (stem like) of gene expression patterns in the development process of cancer has been observed. Stemness and pathology of cancer are closely related. Elucidation of the stemness regulation of cancer is an important key to the development of new cancer prevention methods. However, as far as we know, there exist no cancer prevention studies focusing on cancer stem cells. In this study, we have investigated the possibility of prostate cancer prevention with a focus on cancer stem cells. We utilized prostate cancer cell lines PC3, DU145 (androgen-independent) and LNCaP (androgen-dependent) cells. Then, using spheroid formed cells, mRNA expression levels of cancer stem cells markers were estimated by RT-real time-PCR. Cancer cells have several malignant phenotypes under hypoxia, and we investigated for the cancer stem cells induced malignant alteration under hypoxia. In addition, under hypoxia, we investigated cell viability effect under tocotrienol-rich fraction (TRF) from annatto. TRF almost consists of delta- tocotrienol (δ-T3) that has the strongest anti-cancer activity. We observed spheroid formation capacity and compared the spheroid sizes and mRNA levels of DU145, PC3 and LNCaP cells. Size of DU145 spheroid is smaller in comparison to spheroids of PC3 and LNCaP. mRNA expression levels of cancer stem cells marker were elevated in PC3 and LNCaP cells. This means that PC3 and LNCaP cells have higher concentration of cancer stem cells. Furthermore, stemness decreases with increase of passage number, the number of cultures. We confirmed the presence of stemness in the formation capacity of spheroid until passage 4. Under hypoxia, cell viability of cancer stem cells was elevated compared to normal cultured prostate cancer cells. In addition, under hypoxia and TRF treatment, cell viability of cancer stem cells decreased with increase in TRF concentration compared to control (0 µg/ml). Our results suggest that TRF suppress cell viability of prostate cancer stem cells under hypoxic states and is effective in prostate cancer prevention.

#2625

Simultaneous targeting of ODC and 5-LOX/COX block the tobacco carcinogen-induced lung adenoma progression to adenocarcinoma in A/J mice.

Gaurav Kumar,1 Jagan Mohan R. Patolla,1 Venkateshwar Madka,1 Altaf Mohammed,1 Li Qian,1 Yuting Zhang,1 Laura Biddick,1 Anil Singh,1 Allison Gillaspy,1 Stanley Lightfoot,1 Levy Kopelovich,2 Vernon E. Steele,2 Chinthalapally V. Rao1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _National Cancer Institute, Bethesda, MD_.

Increased polyamine synthesis and inflammation have long been associated with intraepithelial neoplasia and their progression to malignant tumor growth, including lung cancer. Targeting multiple pathways simultaneously with low-dose combinations may be an effective approach to modulate different pathways and their downstream signaling, which may result in an increased efficacy and reduced side effects than a single-agent high dose strategy. The aim of the present study was to investigate the effects of DFMO (ODC inhibitor) and licofelone, a dual 5-LOX-COX inhibitor, individually and in combination, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone NNK-induced lung adenoma and progression to adenocarcinoma in female A/J mice. At 6 weeks of age, mice (25 /group) were fed AIN-76A-modified diet, and one week later, lung tumors were induced with a single intraperitoneal (i.p.) injection of 10 µmol NNK/mouse. Three weeks after the NNK treatment, groups of mice were fed with either control or experimental diets containing DFMO (1500 or 3000 ppm) or Licofelone (200 or 400 ppm) or combination of low doses of DFMO and Licofelone. Mice were killed after 17 or 34 weeks of drug exposure and tumors were evaluated via histopathology and lung tumors were assayed for modification of various biomarkers of proliferation and apoptosis. Results suggest that both DFMO and licofelone showed dose-dependent inhibition of NNK-induced lung adenoma progression to adenocarcinoma. At high dose DFMO and Licofelone showed 46% and 55% of adenocarcinoma inhibition. Importantly, low dose combination of DFMO and licofelone showed more pronounced effects at both 17 or 34 weeks in inhibiting the total adenocarcinomas (adenoma and adenocarcinoma progression) by >65% and somewhat in a synergistic manner as compared to individual low doses of DFMO and licofelone. Combination-treated lung tumors exhibited

modulation of ODC pathway key components (Arg1, Oat, Oaz, SRM, SMS, and SAT) along with decreased proliferation (PCNA, cyclin D1 and Cyclin A) and increased expression of p53, p21 and p27 compared to tumors from mice fed with control diet. These data suggest that targeting ODC plus 5-LOX/COX decreases the progression of adenoma to adenocarcinoma. Furthermore, adenoma progression delay by combination of DFMO and Licofelone is associated with decreased tumor invasive markers such as MMTPs and EMT markers. In conclusion, targeting two or more pathways is an effective chemopreventive approach for high-risk lung cancer individual's particularly former tobacco smokers with lung hyperplasia and adenomas.

(Supported by Kerley-Cade Chair Endowment and NCI-N01-CN-53300)

#2625A

Damnacanthal and its nanoformulation suppress cyclin D1 expression.

Pakin Sukamporn,1 Pleumchitt Rojanapanthu,2 Seung Joon Baek3. 1 _Faculty of Pharmacy, Bangkok, Thailand;_ 2 _Thammasat University, Pathumthani, Thailand;_ 3 _University of Tennessee, Knoxville, TN_.

Damnacanthal is an anthraquinone isolated from the root of Morinda citrifolia L. (Noni) and exhibits many pharmacological properties including anti-cancer activity. It has been reported that Damnacanthal targets several signal transduction proteins related to cell growth inhibition or apoptosis. However, the molecular mechanisms by which Damnacanthal affects anti-cancer activity has not been elucidated in details. Cyclin D1 is the important regulatory protein in cell cycle progression and overexpressed in many cancer cells. In this study, we investigated the molecular mechanism of damnacanthal on cyclin D1 expression. We found that damnacanthal inhibited growth of several cancer cells (HCT-116, HT-29, MCF-7 and PC-3) in a dose- and time- dependent manner with decreasing in cyclinD1 protein expression. Damnacanthal did not change mRNA of cyclin D1, rather it suppressed cyclin D1 expression at the post-translational level. Subsequent experiments with dominant negative mutant cyclin D1 suggest that cyclin D1 at the lysine sites plays a pivotal role in damnacanthal-mediated cyclin D1 suppression. Furthermore, damnacanthal was encapsulated in self-assembled chitosan nanoparticle to improve both physicochemical and biological activities. Our results suggest that encapsulated damnacanthal exhibits better activity in cell growth inhibition, compared to non-encapsulated damnacanthal. Damnacanthal nanoformulation has potential to be a candidate for development of chemoprevention or therapeutic agent for cancers.

Grant support: This work was supported by grant from the University of Tennessee Center of Excellence in Livestock Diseases and Human Health and The Thailand Research Fund (TRF) and Faculty of Pharmacy, Mahidol University (IRG5780007).

#2626

Metabolic profiles and potential pharmacodynamic biomarkers in female Sprague-Dawley rats on a standard (4% fat) or Western (20% fat) diet and treated with known active or inactive chemopreventive agents.

Matthew D. Thompson,1 Clinton J. Grubbs,2 Vernon E. Steele,1 Mark S. Miller,1 Fariba Moeinpour,2 Edward D. Karoly,3 Ronald A. Lubet1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Alabama at Birmingham, Birmingham, AL;_ 3 _Metabolon, Triangle Park, NC_.

The methylnitrosourea (MNU) - induced model of ER+ mammary cancers in female Sprague-Dawley rats has been routinely used in our laboratories for screening chemopreventive agents. In this study, we evaluated multiple known effective [tamoxifen, Targretin (an RXR agonist), Iressa (EGFR1 inhibitor)] or ineffective agents (Lipitor, metformin) in rats placed on either standard diet (20% fat) or a Western diet (20% fat, low calcium) at 43 days of age (DOA). Rats were given MNU at 50 DOA and administered the various agents or vehicle beginning at 57 DOA until the end of the study. Palpation of the rats showed that agents yielded the same results in rats on either diets. Somewhat surprisingly, metformin was ineffective in rats on either diet; confirming our lack of efficacy in a standard diet (Thompson, et al, Cancer Prev Res., 2015). To look for potential pharmacodynamics biomarkers, serum was obtained from the various groups at 78 days of age and at sacrifice; when a large tumor burden was observed in rats given vehicle, Lipitor, or metformin. Levels of approximately 500 metabolites were compared in the serum (Metabolon Research, Research Triangle Park, NC). We initially looked for metabolite changes related to different diets and found differential expression of alpha 10-undecanoate, 13-methylmyristic acid, 4-hydrox-benzoate, 2-amino-heptanpate, tocopherol and nicotinamide when comparing serum from rats on Western vs standard diets. Interestingly, each of the highly effective agents (tamoxifen, Targretin, and Iressa) yielded metabolic profiles that were strikingly different from rats given vehicle; based on unsupervised analysis. These metabolites become potential pharmacodynamic biomarkers for the highly effective doses of these agents. In contrast, the two negative agents did not yield a similar dichotomy between treated and vehicle serum. One could determine, however, metabolites which differed between metformin or Lipitor treated rats vs vehicle treated rats when performing a supervised analysis. We also compared serum for the early and late time points with either diet since the latter serum was from animals with a significant number of mammary cancers. We observed a number of metabolite changes including 4-OH butyrate, acetyl carnitine, oxalate, and threonate. These cancer related profiles will be discussed at greater length; in addition to the altered profiles caused by the administration of the various chemopreventive agents. Supported by NCI contract HHSN261201200021I.

#2626A

A lignan compound arctigenin inhibits prostate tumor growth in vitro and in vivo.

Piwen Wang,1 Susanne M. Henning,2 Jaydutt V. Vadgama1. 1 _Charles Drew University of Medicine and Science, Los Angeles, CA;_ 2 _University of California Los Angeles, Los Angeles, CA_.

Arctigenin is a novel anti-inflammatory lignan derived mainly from the seeds of Arctium lappa, an herb widely used in traditional Chinese medicine to treat inflammation related diseases such as cough, cold and swelling of throat. The present study was designed to investigate whether arctigenin is a potent inhibitor of prostate cancer using both in vitro and xenograft mouse models. The treatments with arctigenin at lower doses (< 2µM) for 48h dose-dependently inhibited the proliferation of two androgen-sensitive prostate cancer cell lines, LNCaP and LAPC-4, by 40-50% compared to control. There was no cytotoxicity observed in normal prostate epithelial PrEC cells with arctigenin doses up to 10µM. An intervention study was then conducted in severe combined immunodeficiency (SCID) mice to confirm the tumor-inhibitory effect of arctigenin in vivo. Male SCID mice (n=10 per group) were inoculated with 5x10^5 LAPC-4 cells subcutaneously. One week later when tumors were palpable the intervention started. Mice were administered with arctigenin at 50mg/kg (LD) or 100mg/kg (HD) or vehicle control daily for 6 weeks by oral gavage. After 4 weeks of intervention the tumor growth was significantly inhibited by both of the arctigenin doses. After 6 weeks of intervention, the tumor volumes were reduced by 48% (LD arctigenin) and 67% (HD arctigenin) compared to control. There was no significant difference in food or water consumption or body weight among groups. An ELISA assay of growth factors demonstrates that arctigenin treatments significantly decreased the concentrations of VEGF, EGF, NGF-β, TNF-α, and FGF-β in tumor tissues compared to control. Western blot analysis revealed a significantly reduced expression of galectin-4 in tumor tissues by arctigenin treatment. Previous studies have shown that strong expression of galectin-4 can be induced in different types of cancer, associated with tumor growth and progression. A microRNA PCR array assay was performed to measure the tumor concentrations of 84 microRNAs involved in prostate cancer development and progression. A panel of microRNAs including miR-126 and miR-135 were identified to be responsive to arctigenin treatment. Further mechanistic investigation using tissue microarray assay is ongoing to measure the protein markers of proliferation and apoptosis in tumor tissues. This study provides a promising non-toxic agent to enhance chemoprevention of prostate cancer. These results warrant future clinical trial studies to confirm the anti-carcinogenic effect of arctigenin in humans.

## CLINICAL RESEARCH:

### Genomic Landscapes

#2627

Molecular analyses of histopathologic morphologic features in breast cancer.

Yu Jing Jan Heng,1 TCGA Breast Cancer Expert Pathology Committee, Jong Cheol Jeong,1 Deena M.A Gendoo,2 Benjamin Haibe-Kains,2 Giovanni Ciriello,3 Katherine A. Hoadley,4 Charles M. Perou,4 Andrew H. Beck1. 1 _Beth Israel Deaconess Medical Center Harvard Medical School, Boston, MA;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _University of North Carolina, Chapel Hill, NC_.

Traditional histopathologic analysis of breast cancer phenotypes has played a central role in the diagnosis, prognosis and clinical management of breast cancer. This study integrated molecular data with breast cancer histopathologic annotations to elucidate the molecular basis of these common morphologic features.

We constructed a large, comprehensive histopathologic database of 850 invasive breast cancer cases from The Cancer Genome Atlas (TCGA). We integrated the consensus assessments of 11 morphologic features (nuclear pleomorphism, mitotic count, epithelial tubule formation, inflammation, DCIS, LCIS, lymphovascular invasion, necrosis, fibrotic focus, apocrine features and proportion of epithelium in invasive portion by area) with TCGA's genomic, transcriptomic and proteomic data. Using this highly annotated dataset, we identified molecular profiles associated with morphologic features, constructed Omics-based multivariate models to predict morphologic features and provided insights into their molecular etiology. The association of morphologic features' signatures with survival in ER-positive and ER-negative breast cancer was assessed using six independent datasets. All data are publicly accessible at http://pathology.ai/tcga_breast.

Morphologic features were associated with PAM50 subtypes, PAM50 proliferation scores, genomic alterations and gene expression (p<0.05). Clustering of morphologic features and genomic alterations produced two clusters of morphologic features and their separate were driven by TP53, CDH1 and PIK3CA mutations and chr12p13.3, ch8q24.21 and chr3q26.3 amplifications. The clustering of morphologic features and gene sets/pathways also produced two clusters of morphologic features characterized by "proliferation" or "inflammation". The transcriptomic signatures of nuclear pleomorphism and epithelial tubule formation were independently prognostic in ER-positive breast cancer. No signatures were prognostic in ER-negative.

Our detailed morphologic data enrich and complement TCGA's existing molecular data, increase our understanding of the molecular basis of breast cancer pathologic phenotypes, can facilitate the refinement of breast cancer classification, and enhance our understanding of breast cancer biology.

#2628

Molecular diagnosis for pediatric cancer through integrative analysis of whole-genome, whole-exome and transcriptome sequencing.

Jinghui Zhang, Michael Rusch, Joy Nakitandwe, Zhaojie Zhang, Michael N. Edmonson, Matthew Parker, Xiaotu Ma, Jared Becksfort, Andrew Thrasher, Jiali Gu, Yongjin Li, Erin Hedlund, Aman Patel, John Easton, Donald Yergeau, Bhavin Vadodaria, Xiang Chen, Tanja A. Gruber, Rose McGee, David Ellison, Sheila Shurtleff, James R. Downing. _St. Jude Children's Research Hospital, Memphis, TN_.

Next-generation sequencing (NGS) of the whole genome, whole exome, and transcriptome has enabled characterization of genetic landscapes of multiple cancers. By analyzing over 2,000 pediatric cancer patients, we have developed a comprehensive database for recurrent somatic alterations and pathogenic germline mutations as part of the St. Jude/Washington University Pediatric Cancer Genome Project. However, there is no systematic evaluation on whether NGS is able to identify germline and somatic lesions reported by existing molecular diagnostic assays and what combination of NGS platforms is best suited for clinical sequencing. Here we report the first comprehensive study that employs whole-genome sequencing at 30-45X coverage, whole-exome sequencing at 100X coverage and transcriptome sequencing using matched tumor/normal samples from cancer patients. A pilot study was carried out to perform NGS analysis on 78 children of leukemia, solid tumor or brain tumor with a total of 112 diagnostic or prognostic biomarkers previously characterized by multiple molecular diagnostic assays. We implemented an analysis pipeline that integrates the genetic lesions detected by all three NGS platforms to characterize somatic and germline single nucleotide variations (SNVs), short insertions and deletions (indels), structural variations including fusions, karyotypes, copy number alterations, loss of heterozygosity, tumor purity and tumor-in-normal contamination. The turn-around time for data analysis is 2 weeks with an overall sensitivity of 99% on detecting known biomarkers. Extensive validation of >3,000 somatic sequence mutations or structural variations from 38 cases shows that the specificity for somatic SNV, indel and structural variation is at 98%, 95% and 84% across the genome. We demonstrate that in addition to providing cross-validation, multi-platform NGS is required for detecting all genetic lesions of pathological significance including complex re-arrangements such as chromothripsis. In addition to known pathogenic or likely pathogenic mutations, our analysis has also unveiled novel pathogenic mutations (e.g. a germline deletion in TP53 in one patient with medulloblastoma) and identified multiple variants of unknown significance that may be worth further exploration (e.g. an in-frame deletion of exons 3-9 of DNMT3A in one neuroblastoma). Our study demonstrates that NGS is able to detect a wide range of genetic lesions currently characterized by multiple molecular diagnostic assays, providing critical insight into the design of clinical sequencing for ongoing studies.

#2629

The landscape of tumor mutation load across the entire spectrum of human cancer derived from 60,000 patients.

Garrett M. Frampton, Riley C. Ennis, Zachary R. Chalmers, Roman Yelensky, Doron Lipson, Philip J. Stephens. _Foundation Medicine, Cambridge, MA_.

INTRODUCTION

Tumor mutation load is a biomarker of emerging significance in cancer immunotherapy. Both mutation load and neoantigen load, as measured by whole exome sequencing, have been shown, in several tumor types, to correlate with patient response to both CTLA4 and PD1 inhibition. Consequently, understanding the factors associated with increased tumor mutational burden is critically important to cancer patient treatment decisions. We sought to better understand the landscape of tumor mutation load and potential response to immunotherapy based on data from comprehensive genomic profiling (CGP) of ~60,000 tumors from patients across ~400 cancer types.

METHODS

CGP profiling by hybridization capture of exonic regions from 236 or 315 cancer-related genes and select introns from 19 genes commonly rearranged in cancer was applied to ≥ 50ng of DNA extracted from >60,000 clinical FFPE cancer specimens. These libraries were sequenced to high, uniform median coverage (>500x) and assessed for base substitutions, short insertions and deletions, copy number alterations and gene fusions/rearrangements. Mutation load was accessed as the number of somatic, coding, base substitution and indel mutations, per megabase of genome examined.

RESULTS

We first validate that mutation load calculated based on CGP of the entire coding region of 315 genes (~1.3 MB) provides a representative measurement of genome-wide mutational load. We quantify and provide detailed data describing mutation load across common tumor types and identify recurrent somatic mutations that are associated with significant increase in tumor mutation load. Our analysis expands significantly upon existing data that quantifies mutation load in hundreds of additional cancer types. Lower grade and pediatric malignancies were observed to have the lowest somatic mutation load, while diseases with significant known mutatgenic exposure such as lung and skin cancers were most highly mutated. Finally, the genomic alterations most associated with increased mutation load were loss of function mutations in mismatch-repair genes (MSH2, MSH6, MLH1 and PMS2), DNA replication genes (POLD1, POLE) and in TP53.

CONCLUSION

These data demonstrate that tumor mutational load can be accurately quantified using targeted CGP with a CLIA-certified assay that is already integrated into routine patient care. As the role of mutation burden as a biomarker for patient response to immune therapy becomes established, this approach can be used to identify both targeted and immune therapeutic options that are either approved or in clinical trial. Additionally, by characterizing the landscape of mutation load across the full spectrum of human cancer, we provide new data for the rational expansion of the patient population that can potentially benefit from immunotherapy.

#2630

Integration of tumor microenvironment and molecular subclassification of colorectal cancer identifies patient subsets with poor prognosis.

Jesper B. Bramsen,1 Mads H. Rasmussen,1 Halit Ongen,2 Søren Vang,1 Philippe Lamy,1 Manel B. Esteller,3 Emmanouil T. Dermitzakis,2 Torben F. Orntoft,1 Claus L. Andersen1. 1 _Aarhus University Hospital, Department for Molecular Medicine (MOMA), Denmark;_ 2 _[1] Department of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, Switzerland [2] Institute for Genetics and Genomics in Geneva (iGE3), University of Geneva, 1211 Geneva, Switzerland [3] Swiss Institute of Bioinformatic, Switzerland;_ 3 _Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain_.

The patho-molecular diversity of colorectal cancer (CRC) complicates the prediction of patient postoperative prognosis and response to treatment. This calls for methods for CRC stratification beyond clinical features and the few molecular biomarkers currently implemented. Recent studies have highlighted the importance of the tumor microenvironment in CRC. Here we show by integrative transcriptome/methylome analysis of >300 patient samples that CRC generates five arche-typic microenviroments encompassing three molecularly distinct types of cancer cells. The existence of the microenviroments, which we denote "Secretory", "Stroma", "Immune", "ARE", and "CIN", could be independently identified in large external CRC datasets and are in concordance with the CRC subtypes recently defined by the CRC Subtyping Consortium (1). Detailed analysis of the five microenvironments reveals striking differences in cell composition and pathway activity. We observe that the microenvironment, particularly immune and stromal cell activity, is closely associated with patient prognosis. In particular, tumors associated with low anti-tumoral immune activity due to poor recruitment of immune cells or immune inhibition by activated stromal cells have poor prognosis. Importantly, we find that microenviroment subtyping is in fact pivotal for molecular prediction of patient prognosis as no single molecular marker carries robust prognostic information in all CRC subtypes and environments. Instead, potent prognostic biomarkers are environment-specific: As an example interferon signaling is deregulated in several microenviroment types, yet the specific interferon response genes deregulated (i.e. biomarker candidates) differ between microenvironments in accordance with their cellular composition. Our approach has allowed us to identify superior, single prognostic biomarkers for each CRC type and, importantly, their prognostic value in external datasets is only observed after environment subtyping. Collectively, these observations may well explain the poor validation rate of prognostic biomarker candidates in the past. In conclusion: we provide a CRC microenvironment framework that has the potential to guide future clinical decision-making primarily in relation to prognosis, but likely also in relation to therapy choice. This work is performed within framework the SYSCOL consortium funded by EU FP7

(1) Guinney, J. et al. "The consensus molecular subtypes of colorectal cancer", Nature Medicine (2015) doi:10.1038/nm.3967

#2631

Restrictions on access to systemic therapy limit the application of whole genome sequencing in clinical care.

Janessa J. Laskin,1 Yaoqing Shen,2 Daniel Renouf,1 Martin Jones,2 Howard Lim,1 Alexandra Fok,2 Cheryl Ho,1 Balvir Deol,1 Karen A. Gelmon,1 Stephen Chia,1 Richard Moore,2 Andrew Mungall,2 Stephen Yip,3 Steven Jones,2 Marco Marra2. 1 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 2 _Canada's Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada;_ 3 _Vancouver General Hospital, Vancouver, British Columbia, Canada_.

Background: Whole genome analyses have the potential to identify the full landscape of activating and inactivating genomic abnormalities at work within cancers, and can thus be used to provide rationales for selection of treatment agents or clinical trials in a broad range of patients.

Patients & Methods: Eligible patients (pts) with metastatic cancers were recruited within a general oncology practice across the province of B.C., Canada. Each pt underwent a fresh tumor biopsy and a blood sample and had comprehensive DNA (80X) and RNA sequencing. In-depth bioinformatic analyses were preformed to identify genomic changes that may be cancer "drivers" or therapeutically actionable targets. Aberrant pathways were matched to drug databases and manual literature reviews undertaken to identify drugs or clinical trials of potential utility for the individual pt.

Results: Between July 2012 - Oct 2015: 380 pts (358 adult + 22 peds) consented; 227 have completed whole DNA and RNA sequencing and analysis to date (remainder ongoing). For this analysis, data is available on 160 pts. Genome bioinformaticians assessed the genomic data to be potentially druggable in all cases. Medical Oncologists assessed this data to be directly clinically actionable in 135 (84%); the difference being that clinicians did not agree that some putative "druggable" drivers (such as p53) or pathways with no current drugs available (MMP, AURA, WNT) were "actionable".

Of the 135 cases, 58 (43%) pts received therapy based directly on this genomic information; 6 on a phase 1 clinical trial. The most common reasons for 77 pts defined as actionable but who have not received genomically-informed therapy were: drug only available on a clinical trial but trial not available to pt - 22 (26%); drug approved but not available off label - 18 (23%); pt presently on first-line therapy that is working - 14 (18%); or death/too unwell 13 (17%). The limited availability of clinical trials was primarily because of highly restrictive trials entry criteria, primarily limiting patients to one primary tumour type or narrowly defined biomarker entry criteria.

The most commonly mutated cancer genes identified by the genome analysts were: p53 as the predominant driver in 42%; APC in 16%; KRAS and PI3KCA mutations in 14%. Going forward it is essential to distinguish what driver mutations might be clinically actionable from those that are still only theoretically druggable; and similarly learn to distinguish which targets are actionable but not truly drivers.

Conclusions: Genomic DNA and RNA sequencing data were found to be clinically actionable in 84% of pts with advanced cancers in a population cancer care setting. However, the ability to act on this information is limited by the restrictive nature of clinical trials and the lack of accessibility of off-label drugs despite an identified biomarker. As genome sequencing becomes integrated into cancer management these drug access issues need to be addressed.

#2632

Clinical validation of circulating DNA analysis for the detection of point mutations and of the longitudinal metastatic colorectal patient follow up for detecting emergence of resistance to targeted therapy.

Alain R. Thierry,1 Safia El Messaoudi,2 Caroline Mollevi,3 Brice Pastor,1 Cynthia Sanchez,1 Jean-Luc Raoul,4 Rosine Guimbaud,5 Denis Pezet,6 Pascal Artru,7 Muriel Mathonnet,8 Christelle De La Fouchardiere,9 Christophe Borg,10 Eric Assenat,11 Olivier Bouche,12 Céline Gavoille,13 Scott Kopetz,14 Marc Ychou3. 1 _IRCM/INSERM U1194, Montpellier, France;_ 2 _IRCM/INSERM U1194 and DiaDx SAS, Montpellier, France;_ 3 _ICM/INSERM U1194, Montpellier, France;_ 4 _Institut Paoli Calmettes, Marseille, France;_ 5 _CHU Toulouse - Hôpital Rangueil-Purpan, Toulouse, France;_ 6 _CHU Estaing, Clermont-Fd, France;_ 7 _Clinique Jean-Mermoz, Lyon, France;_ 8 _CHU Limoges - Hôpital Dupuytren, Limoges, France;_ 9 _Centre Léon Bérard, Lyon, France;_ 10 _CHU Jean Minjoz, Besançon, France;_ 11 _CHU Montpellier - Hôpital St. Eloi, Montpellier, France;_ 12 _CHU Robert Debre, Reims, France;_ 13 _Instit.de Cancéro. de Lorraine Alexis Vautrin, Vandœuvre-lès-Nancy, France;_ 14 _University of Texas M.D. Anderson Cancer Center, Houston, TX_.

Clinical validation of circulating DNA analysis as liquid biopsy for the detection of point mutations and of the longitudinal metastatic colorectal patient follow up for detecting emergence of resistance to targeted therapy

We carried out two clinical studies in metastatic colorectal cancer patients (i) the clinical real-time evaluation of circulating tumor DNA (ctDNA) analysis for detecting point mutations in comparison with tumor tissue analysis before initiation of anti-EGFR therapy and (ii), the study of the emergence of associated resistance mutation in refractory patients under treatment.

(i) We realized a real-time blinded prospective multicentric clinical study on 140 patients comparing KRAS (exon 2,3 and 4) and BRAF V600E mutations determination by plasma and tissue analysis carried out in the conditions of standard management care. On 121 patients where both analysis were made, 43% were found mutant by tumor tissue analysis while 57% were found KRAS mutant by ctDNA analysis. 7.2% of patients were found BRAF mutant by tumor tissue while 14.4% were determined mutant by ctDNA analysis. 13% of plasma samples carried multiple KRAS point mutations and 4% of plasma samples exhibited at least one KRAS point mutation combined to the presence of BRAFV600E point mutation. Median data turnaround time was 16 [3-273] days for tumor tissue while it was 2 [0.5-10] days for ctDNA analysis. CtDNA analysis appears much more sensitive than tissue analysis and may be more accurate in respect to the intra-tumor or inter-tumor heterogeneity and the clonal evolution dynamics. We present the first RAS and BRAF mutation distribution which takes into account the presence of multiple mutations in the same mCRC patient.

(ii) In the other blinded clinical study, we retrospectively analyzed RAS (KRAS and NRAS) and BRAF mutations in serial plasma samples from 42 refractory mCRC patients to Folfox + cetuximab or dasatinib. 98% of the plasma were found mutant before or during treatment. 50% of KRAS mutant samples were missed by tumor tissue analysis before treatment. 4.8% of patients were found BRAF mutant by tumor tissue analysis while 7% were found mutant by ctDNA analysis. Longitudinal plasma analysis shows that 80% of initially WT patients acquire at least one RAS or BRAF mutation during treatment and that 38% of initially mutant patients acquire at least one newly KRAS or BRAF point mutation during treatment. CtDNA analysis allows tracking acquired resistance by studying the real-time clonal evolution of the tumor

Patients may harbor mutations at very low frequency on ctDNA down to 0.01% before initiation or during treatment revealing the need of a high sensitive technique to detect mutant subclones. Our results indicate that plasma analysis could advantageously replace tumor tissue analysis and enables longitudinal examination of tumor molecular profiling.

#2633

Radiogenomics of breast cancer using DCE-MRI and gene expression profiling.

Albert C. Yeh, Stephanie McGregor, Hui Li, Yuan Ji, Yitan Zhu, Tatyana Grushko, Alexandra Edwards, Fan Lui, Jing Zhang, Qiu Niu, Yonglan Zheng, Toshio Yoshimatsu, Galina Khramtsova, Karen Drukker, Gregory Karczmar, Hiroyuki Abe, Jeffrey Mueller, Maryellen Giger, Olufunmilayo Olopade. _University of Chicago, Chicago, IL_.

The utilization of radiomics (high-throughput extraction and analysis of imaging features) to abstract underlying genomic features of an evolving malignancy is an emerging field that has the potential to detail biological information of a tumor in a non-invasive and more accessible manner. Furthermore, applying radiomics confers a comprehensive spatial view of the tumor, a potential advantage over the limitations of sampling a small region of tissue that may not accurately represent the underlying complexity of the entire tumor. Most studies to date that incorporate radiogenomics in the analysis of breast cancers have focused on few basic clinical data or individual genetic mutations such as BRCA or HER2 status.

Here, we use an automated quantitative radiomics analysis platform developed at the University of Chicago that enables computerized feature extraction of tumors to analyze magnetic resonant imaging scans of 50 breast cancer patients (mean age of diagnosis [range]: 54 [24-89]; receptor status: HER2+: 14, Triple negative: 7; stage [1 through 4]: 10%, 40%, 42%, 8%) who have had comprehensive gene expression profiling performed using Agilent Human Gene Expression arrays. Our imaging platform extracts 38 features across six major phenotypes (size, shape, morphology, enhancement texture, kinetic curve assessment, and enhancement variance kinetics) (see Table for listing of 24 selected features). Existing radiomic analysis derived from a TCGA/TCIA dataset suggests that there are many correlations between imaging phenotypes and various genetic pathways, such as VEGF signaling and volume of enhancing voxels, base excision repair and enhancement texture entropy, and TGF-beta signaling and enhancement texture variance. We confirm these relationships as well as establish novel associations using a robust imaging dataset. By associating specific radiomic features with gene expression profile of tumors, we have the opportunity to extract detailed biological information non-invasively through clinical imaging.

Selected imaging phenotypes extracted from MRI scans

---

Phenotype category | Image phenotype | Description

Size | Volume | Volume of lesion

Size | Effective diameter | Diameter of a sphere with the same volume as the lesion

Size | Surface area | Lesion surface area

Shape | Sphericity | Similarity of the lesion shape to a sphere

Shape | Irregularity | Deviation of the lesion surface from the surface of a sphere

Shape | Surface area / volume | Ratio of surface area to volume

Morphology | Margin sharpness | Mean of the image gradient at the lesion margin

Morphology | Variance of margin sharpness | Variance of the image gradient at the lesion margin

Morphology | Variance of radial gradient histogram | Degree to which the enhancement structure extends in a radial pattern originating from the center of the lesion

Enhancement Texture | Contrast | Local image variations

Enhancement Texture | Entropy | Randomness of the gray-levels

Enhancement Texture | Difference variance | Variations of difference of gray-levels between voxel-pairs

Enhancement Texture | Angular second moment | Image homogeneity

Enhancement Texture | Maximum correlation coefficient | Nonlinear gray-level dependence

Enhancement Texture | Sum average | Overall brightness

Enhancement Texture | Sum of squares | Spread in the gray-level distribution

Kinetic Curve Assessment | Maximum enhancement | Maximum contrast enhancement

Kinetic Curve Assessment | Time to peak | Time at which the maximum enhancement occurs

Kinetic Curve Assessment | Uptake rate | Uptake speed of the contrast enhancement

Kinetic Curve Assessment | Curve shape index | Difference between late and early enhancement

Kinetic Curve Assessment | Total rate variation | How rapidly the contrast will enter and exit from the lesion

Enhancement-Variance Kinetics | Maximum variance of enhancement | Maximum spatial variance of contrast enhancement over time

Enhancement-Variance Kinetics | Time to peak maximum variance | Time at which the maximum variance occurs

Enhancement-Variance Kinetics | Enhancement variance increasing rate | Rate of increase of the enhancement-variance during uptake

## EPIDEMIOLOGY:

### Biomarkers and Other Epidemiologic Factors in Cancer Prognosis

#2634

Prognostic value of obesity-related genes methylation in localized renal cell carcinoma patients.

Julia Mendoza-Perez,1 Jian Gu,1 Nizar M. Tannir,2 Surena Matin,3 Jose A. Karam,3 Maosheng Huang,1 Shu Xiang,1 David W. Chang,1 Christopher G. Wood,3 Luis A. Herrera,4 Xifeng Wu1. 1 _Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Department of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Instituto Nacional de Cancerología (INCan)-Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico_.

Obesity is an established risk factor for renal cell carcinoma (RCC): more than 40% of RCC cases in US are attributed to excessive body weight. Growing evidence suggests that obesity may also be associated with the prognosis of RCC. The molecular mechanism linking obesity and RCC is not well understood. In the present study, we evaluated the association between promoter CpG island methylation of 20 obesity-related genes and RCC. A total of 275 newly diagnosed and previously untreated Caucasian RCC patients were included. For the discovery population, 75 tissue pairs of RCC tumors and normal adjacent tissues from the surrounding kidney were used and for the validation population an additional 200 pairs were included. Pyrosequencing was used to determine CpG site methylation on bisulfite-treated genomic DNA. We used Cox proportional hazards model to estimate hazard ratios (HRs) and 95% confident intervals (CIs) for the association of CpG methylation with recurrence and Kaplan-Meier curve to plot disease free survival (DFS) time by different CpG site methylation. The average age of the patients was 59 years and they were largely males; ~50% were never smokers. Most of the cases were clear cell RCC (ccRCC) and clinical stage I. Among the 20 markers, LEP, LEPR and NPY were significantly hypermethylated in tumor tissues compared to normal tissues (p<0.0001) in the discovery set. Similar results were obtained in the validation set. Moreover, LEPR methylation in tumors was associated with recurrence. When patients were dichotomized into high and low methylation groups according to the median value of LEPR methylation, high LEPR methylation was associated with a significantly higher risk of recurrence (HR=2.4, 95% CI, 1.03-5.8), adjusted by age, gender, clinical stage, grade, smoking status, BMI, hypertension and histology. The Kaplan-Meier analysis showed a shorter 5-year DFS probability for the patients with high LEPR methylation level (34.7%) compared with low methylation (41.7%) (LogRank, p=0.032). The MST was 39 months in high methylation group and 47 months in low methylation. Our results suggest that LEPR hypermethylation in tumors is associated with recurrence in RCC patients and thus LEPR may provide a functional link between obesity and RCC. Future studies are needed to elucidate the biology underlying the association of LEPR methylation and RCC recurrence. This work was supported in part by the National Institutes of Health (grant R01 CA170298) and the Center for Translational and Public Health Genomics, Duncan Family Institute for Cancer Prevention, The University of Texas MD Anderson Cancer Center.

#2635

Methylation in benign prostate and risk of disease progression in men subsequently diagnosed with prostate cancer.

Benjamin A. Rybicki,1 Andrew Rundle,2 Oleksandr Kryvenko,3 Dhananjay Chitale,1 Steven Belinsky,4 Deliang Tang2. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Columbia University, New York, NY;_ 3 _University of Miami, Miami, FL;_ 4 _Lovelace Respiratory Research Institute, Albuquerque, NM_.

In DNA from prostate cancer tumors, methylation patterns in gene promoter regions can be a biomarker for disease progression. It remains unclear whether methylation patterns in benign prostate tissue—prior to malignant transformation—may provide similar prognostic information. To determine whether early methylation events predict prostate cancer outcomes, we evaluated histologically benign prostate specimens from 353 men who eventually developed prostate cancer and received "definitive" treatment (radical prostatectomy [58%] or radiation therapy [42%]). Cases were drawn from a large hospital-based cohort of men with benign prostate biopsy specimens collected between 1990 and 2002. Risk of disease progression associated with methylation was estimated using time-to-event analyses. Average follow-up was over 5 years; biochemical recurrence (BCR) occurred in 91 cases (26%). In White men, methylation of the APC gene was associated with increased risk of BCR, even after adjusting for standard clinical risk factors for prostate cancer progression (adjusted hazard ratio (aHR)=2.26; 95%CI 1.23-4.16). APC methylation was most strongly associated with a significant increased risk of BCR in White men with low prostate specific antigen at cohort entry (HR=3.66; 95%CI 1.51-8.85). In additional stratified analyses, we found that methylation of the RARB gene significantly increased risk of BCR in African American cases who demonstrated methylation of at least one of the other four genes under study (HR=3.80; 95%CI 1.07-13.53). These findings may have implications in the early identification of aggressive prostate cancer as well as reducing unnecessary medical procedures and emotional distress for men who present with markers of indolent disease.

#2636

Tumor-infiltrating cytotoxic T-cells are associated with improved survival of colorectal cancer patients.

Anna E. Prizment,1 Robert A. Vierkant,2 Thomas C. Smyrk,2 Lori S. Tillmans,2 Heather H. Nelson,1 Charles F. Lynch,3 Stephen N. Thibodeau,2 Timothy R. Church,1 James R. Cerhan,2 Kristin E. Anderson,1 Paul J. Limburg2. 1 _University of Minnesota School of Public Health, Minneapolis, MN;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _University of Iowa, University of Iowa, IA_.

Background: The immune response has been shown to impact the course of colorectal cancer (CRC). Our goal was to examine the survival of CRC patients in relation to tumor-infiltrating cytotoxic (CD8+) T lymphocytes (CTL). We quantified CTLs in tumor epithelial and stromal tissues and correlated them with clinicopathological characteristics and survival of CRC patients in the Iowa Women's Health Study.

Methods: Paraffin-embedded tissue samples were available from 465 post-menopausal women diagnosed with incident CRC in 1986-2002. Tumor microarrays were constructed and immunostained with a CD8 antibody (Clone 144B; Dako). CTLs in tumor epithelial and stromal tissues were categorized by an experienced pathologist into non-detected; mild (1-10 cells per core); moderate (11-29 cells); and strong infiltration (≥30 cells per core), and averaged over cores per each person. We used Cox regression to estimate the hazard ratio (HR) and 95% CI for total and CRC death in relation to each CTL score after adjusting for age of diagnosis, SEER stage, grade, body mass, smoking history and integrated molecular pathway for CRC. The tumors were categorized by integrated pathway (traditional, alternate, serrated, unassigned) based on the combination of phenotypes: microsatellite instability (MSI) status, CpG island methylator (CIMP) phenotype; and mutations in BRAF and/or KRAS genes (described in Samadder, 2013).

Results: During follow-up until 2011, 31% of participants died from CRC, 36% from all other causes, and 33% were alive (median follow-up: 8.4 y). The Spearman correlation coefficient between epithelial and stromal CTL scores was 0.52. Both scores were inversely correlated with stage at diagnosis and were higher in tumors characterized by MSI-high, CIMP-high and BRAF mutation-positive status. The highest categories in the epithelial and stromal scores were associated with statistically significant decreases in total death (by 44% and 40%, respectively) and CRC death (by 68% and 56%, respectively) compared to lowest categories. After summing epithelial and stromal CTL scores, the combined HRs (95% CI) in the highest versus lowest category were 0.55 (0.38-0.78) for total death (p-trend=0.0002) and 0.36 (0.20-0.63) for CRC death (p-trend<0.0001). Additional adjustment for surgery, chemotherapy, radiation, and comorbidities did not markedly change the associations. Finally, there was a statistically significant quantitative interaction between stromal score and MSI status in relation to total and CRC death (P for interaction were 0.02 and 0.0001, respectively); HRs were lower for MSI-high than for MS-stable tumors. There was also a significant interaction between smoking status (current versus never) and epithelial CTL score: the HRs for total and CRC death were lowest among never smokers with the highest score.

Conclusions. Our study further supports infiltration of tumors with CTLs as an important prognostic factor in CRC.

#2637

Impact of pre-diagnosis weight loss on outcomes in a prospective cohort of esophageal cancer patients.

Sherry Shen,1 James L. Araujo,2 Nasser K. Altorki,3 Joshua R. Sonett,4 Adriana Rodriguez,2 Kivilcim Sungur-Stasik,4 Alfred I. Neugut,2 Julian A. Abrams2. 1 _Columbia University College of Physicians & Surgeons, New York, NY; _2 _Columbia University Medical Center, Department of Medicine, New York, NY;_ 3 _Weill Cornell Medical Center, Department of Thoracic Surgery, New York, NY;_ 4 _Columbia University Medical Center, Department of Thoracic Surgery, New York, NY_.

Background: Esophageal cancer survival rates remain extremely low and factors influencing outcomes for this malignancy are not well understood. Tumor cachexia is a poor prognostic factor for certain tumor types, but is not well-studied in esophageal cancer. Weight loss in esophageal cancer is likely multifactorial; it can be due to tumor cachexia as well as dysphagia from obstructing tumors. In this present study, we aimed to investigate the relationship between weight loss and overall survival in a cohort of esophageal cancer patients and to determine whether these associations differed with tumor size.

Methods: We prospectively enrolled subjects with recently diagnosed esophageal cancer at two tertiary care centers. Using a baseline questionnaire, we assessed demographics, medical history, medication use, and lifestyle factors. We recorded self-reported height and weight one year prior to and at diagnosis, which we used to calculate body mass index (BMI) and percent weight change, categorized by tertile. We ascertained from the medical records tumor characteristics including T stage (T1/2 or T3/4), location, pathology, and metastasis status, and collected follow-up data on treatment, imaging, and death. We used Cox regression to assess the association between percent weight loss and all-cause mortality.

Results: We included 134 subjects in the analyses, the majority of whom were male (81.3%) and had adenocarcinoma (82.1%). The median BMI one year prior to diagnosis was 28.3 (IQR 24.4 - 31.0) and median percent weight loss was 4.7% (IQR 0 - 10.9%). There was no association between BMI one year prior and all-cause mortality (HR 0.98, 95% CI 0.93 - 1.03). Increasing percent weight loss was associated with increased risk of all-cause mortality (unadjusted HR 2.74 for highest vs. lowest tertile, 95% CI 1.34 - 5.58, Ptrend = 0.005) and this remained significant when adjusted for BMI one year prior (HR 2.87 for highest vs. lowest tertile, 95% CI 1.40 - 5.89, Ptrend = 0.003). We found significant interaction between weight loss and T stage. Percent weight loss was significantly associated with all-cause mortality, adjusted for BMI one year prior, among patients with T stages 1 or 2 (HR 6.49 for highest vs. lowest tertile, 95% CI 1.30 - 32.4, Ptrend = 0.012), but not T stages 3 or 4 (HR 1.42 for highest vs. lowest tertile, 95% CI 0.54 - 3.71). In the final multivariable model, there remained a significant association between percent weight loss and all-cause mortality among patients with T stages 1 or 2 (HR 6.34 for highest vs. lowest tertile, 95% CI 1.22 - 33.1, Ptrend = 0.022).

Conclusions: In this cohort of esophageal cancer patients, we found that pre-diagnosis weight loss was associated with increased risk of all-cause mortality in patients with earlier stage tumors, independent of baseline BMI. We suspect that weight loss in early stage esophageal cancer may be due to tumor cachexia, a potential marker of more aggressive disease and worse prognosis.

#2638

Cardiovascular comorbidity and ovarian cancer patient survival: history of hypertension, heart disease, and diabetes, evidence from the Ovarian Cancer Association Consortium.

Albina Minlikeeva,1 Jo L. Freudenheim,1 Kirsten B. Moysich,2 Ovarian Cancer Association Consortium. 1 _University at Buffalo, Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: The role of previously diagnosed comorbidities in relation to prognosis for ovarian cancer patients has not been studied extensively, particularly examining histological subtype. We explored the association between pre-existing hypertension, heart disease, and diabetes and overall survival (OS) and progression-free survival (PFS) among women diagnosed with ovarian cancer in a large international study.

Methods: Using pooled data from 14 case-control studies participating in the Ovarian Cancer Association Consortium (n=7076), we examined associations between self-reported pre-existing hypertension, heart disease, and diabetes, and OS and PFS among patients diagnosed with invasive epithelial ovarian carcinoma. We used Cox proportional hazards regression models adjusted for age and stage to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). We also examined associations separately for the main histological subtypes, high-grade serous, low-grade serous, mucinous, endometrioid, and clear cell tumors. Analyses were also conducted in strata of menopausal status, body mass index (BMI) categories, stage of disease, and ever use of medications specific for each condition.

Results: No significant associations were observed for overall cancer for risk of death with history of hypertension (n=5847; HR=0.93; 95% CI 0.85-1.02), history of heart disease (n=4178; HR=1.11; 95%CI 0.91-1.36) or history of diabetes (n=7043; HR=1.07; 95%CI 0.95-1.20). Similarly, there was no association of these comorbidities with PFS. However, among cases with endometrioid tumors, history of hypertension was associated with lower risk of both OS and PFS (n=869; HR=0.70; 95%CI 0.51-0.96, and n=246, HR=0.54; 95%CI 0.33-0.90 correspondingly). There were no significant associations observed between history of the comorbidities and PFS among the other histological subtypes. None of the examined associations differed in strata defined by menopausal status, BMI categories, stage of disease, or categories of medications intake.

Conclusion: Pre-existing hypertension may be inversely associated with survival among women diagnosed with invasive ovarian cancer, particularly for those with endometrioid tumors. Understanding of the mechanism for this observation could provide for treatment strategies. Future studies are needed to explore the role of chronic diseases and their management in relation to prognosis of ovarian cancer patients and investigate the difference of tumor microenvironment among various histologic subtypes.

#2639

Survival prognosis of MGUS patients by clinical and risk subgroup: a result from a nationally representative prospective cohort.

Hyun-Seok Kim,1 Jieqi Liu,1 Brittany L. Gladney,1 Neil Kothari,1 Noa Biran,2 Victor Chang,3 David S. Siegel2. 1 _New Jersey Medical School, Newark, NJ;_ 2 _Hackensack University Medical Center, Hackensack, NJ;_ 3 _East Orange VA Medical Center, East Orange, NJ_.

Introduction:

Monoclonal gammopathy of unknown significance (MGUS) is a premalignant disorder preceding multiple myeloma that is present in 2.4% of the general population ages 50 years or older. MGUS can be categorized into conventional MGUS that includes IgM MGUS and non-IgM MGUS, and light chain MGUS (defined as those with abnormal free light chain ratio with complete lack of immunoglobulin heavy chain expression on immunofixation). Our objective was to investigate the prognosis of MGUS patients by clinical subgroup and risk stratification suggested by Mayo Clinic.

Methods:

Data is obtained from National Health and Nutrition Examination Survey (NHANES) III and NHANES 1999-2004, a nationally representative health survey with follow-up mortality data updated in December 2011. Subjects who were diagnosed with MGUS were included in the study. They were divided into three groups; IgM MGUS, non-IgM MGUS, and light chain MGUS (LC-MGUS). Median overall survival of three groups was obtained using log-rank test and age-adjusted hazard ratio for death between the groups was obtained using cox-proportional regression model. Finally, we also analyzed median survival and age adjusted hazard ratio on conventional MGUS subjects by Mayo clinic MGUS risk stratification based on risk factors of M-protein>1.5g/dL, non-IgG MGUS, and abnormal free light chain (FLC) ratio. Analyses were performed in R software version 3.2.2, and a P-value <0.05 was considered statistically significant.

Results:

22,523 subjects in the cohort were screened for MGUS with serum protein electrophoresis, serum protein immunofixation, serum FLC assay, and M-protein typing. There were 483 subjects with MGUS (47 IgM-MGUS, 385 non-IgM MGUS, and 51 LC-MGUS). The median ages of total MGUS subjects, and subgroups were 70, 72.3, 69 and 73 years old, respectively. Median follow-up was 116 months. Median overall survival was 117 months for IgM MGUS, 173 months for non-IgM MGUS, and 109 months for LC-MGUS (p= 0.03). However, the age adjusted hazard ratio (aHR) did not show significant difference between the sub-groups. Likewise, median overall survival of Mayo Clinic MGUS risk group 0, 1, 2 and 3 was 185 months, 163 months, 137 months, and 76.5 months, respectively (p <0.001). Only high-risk group (risk of 3) showed statistically significant aHR 2.9 (95%CI 1.5~5.6) compared with low risk group (risk of 0).

Conclusion:

There was no statistically significant mortality difference between MGUS subgroup. Only high-risk group defined by the Mayo Clinic MGUS risk stratification showed inferior survival and higher risk of death compared to rest of MGUS subjects. Further prospective studies are needed to validate our findings and to investigate whether early interventions for MGUS patients by clinical subgroup and risk stratification are needed rather than the current standard management of watchful-waiting.

#2640

Clonal hematopoiesis identified by matched-normal blood sequencing of solid tumor patients without hematologic malignancy is common and is associated with decreased overall survival.

Catherine Coombs, Ahmet Zehir, Sean Devlin, Sumit Middha, Donavan Cheng, Ashwin Kishtagari, David Hyman, David Solit, Mark Robson, Jose Baselga, Maria Arcila, Martin Tallman, Ross Levine, Michael Berger. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Recent genomic studies have identified somatic mutations in leukemia genes in asymptomatic individuals without hematologic malignancy. A subset of patients (pts) with clonal hematopoiesis (CH) subsequently develops hematologic malignancies. However, the incidence of CH in pts with solid tumors has not been extensively studied. We sought to assess for CH and its associated clinical impact in pts with solid tumors who were profiled using paired tumor/blood sequencing.

Methods: This study included pts who were consented on protocol NCT01775072. All had tumor and blood genomic profiling using the MSK-IMPACT hybridization capture-based next-generation sequencing assay, encompassing all protein-coding exons of 410 cancer-associated genes. In matched blood, we investigated for hotspot mutations from COSMIC database (v71) and non-hotspot mutations in leukemia-associated genes and genes reported in prior CH studies. Mutations were scored as present if variant allele frequency (VAF) in blood was greater than 2% and at least twice the VAF seen in tumor. For cases where at least one mutation exceeded these thresholds, we reduced the VAF threshold in blood to 1% to detect subsequent events. Mutations with VAF >35% in both blood and tumor were excluded as likely germline events.

Results: We analyzed 2,146 pts, including 239 pts (11%) with CH. Most commonly identified mutations were in DNMT3A, TET2, and TP53, seen in 167, 52, and 18 pts, respectively. The mean age at time of testing was 62.6 years in pts with CH and 55.4 years in pts without CH (p< 0.001). When comparing baseline blood parameters, there were no statistically significant differences except for higher MCV in CH vs. non-CH pts (91 vs. 90, p= 0.047). 51% of pts with CH had previous radiation therapy compared to 45% of pts without clonal mutations in their blood (p= 0.057). There was no statistically significant difference in the proportion of pts who had received previous chemotherapy (71% of pts in each group). On prospective follow up of pts with CH, no pts have progressed to develop overt hematologic malignancy, with limited follow up (median 13.1 months). Overall survival (OS) was estimated for pts without CH (n= 1907; 1-yr OS: 0.70), CH with 1 mutation (n= 184; 1-yr OS: 0.64), and CH with >1 mutation (n= 55; 1-yr OS: 0.59). The univariate hazard ratios were 1.16 (p= 0.269) and 1.63 (p= 0.015) for 1 mutation and >1 mutation, respectively, when compared to pts without CH. After adjusting for age and sex, a marginal OS association persisted for >1 mutation compared to no CH (HR: 1.46, p= 0.060)

Conclusions: CH is common among solid tumor pts without known hematologic malignancy, occurring in 11% of pts, and appears to be associated with decreased OS. Analyses will be replicated in a larger cohort with longer follow up to determine the clinical impact of these mutations in cancer pts.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Hitting the Target Harder: Preclinical Development of Potent and Selective Inhibitors

#2641

The development of potent, selective RET inhibitors that target both wild-type RET and prospectively identified resistance mutations to multi-kinase inhibitors.

Rami Rahal, Erica K. Evans, Wei Hu, Michelle Maynard, Paul Fleming, Lucian DiPietro, Joseph L. Kim, Michael P. Sheets, Doug P. Wilson, Kevin J. Wilson, Nicolas Stransky, Jason D. Brubaker, Timothy Guzi, Nancy E. Kohl, Christoph Lengauer. _Blueprint Medicines, Cambridge, MA_.

Introduction: RET/PTC1 was one of the first gene fusions identified from a solid tumor. In the past 3 years, oncogenic RET fusions have been identified in additional cancer types, most notably NSCLC and colon carcinomas. Activating germline RET mutations are also well established drivers of multiple endocrine neoplasia while somatic RET mutations are the most prevalent alterations in sporadic medullary thyroid cancers. In light of these findings, a number of approved multi-kinase inhibitors (mKIs) with in vitro activity against wild-type (WT) RET, such as cabozantinib, vandetanib, and lenvatinib, have been repurposed for treating RET-driven diseases. We have designed a next-generation inhibitor specifically tailored to target RET, while sparing other closely-related kinases, such as KDR/VEGFR2. Given that secondary mutations are a common resistance mechanism to kinase inhibitors, we prospectively identified resistance mutations that abrogate mKI activity and crafted our RET-selective inhibitors to also target these mutations.

The structures of several mKIs bound to RET were analyzed and amino acid substitutions that would disrupt the protein-inhibitor interactions were predicted. In vitro resistance screens with cabozantinib, ponatinib, and vandetanib in a Ba/F3 KIF5B-RET cell line were conducted and confirmed these predictions. The proprietary Blueprint Medicines' kinase inhibitor library was used to identify inhibitors of WT and resistance mutant RET as starting points for lead optimization.

Results: Both structural analysis and in vitro screening revealed that only a handful of positions within the RET kinase domain enable resistance mutations to mKIs, suggesting a narrow mutational spectrum. Using our library of kinase inhibitors, we identified potent, orally bioavailable inhibitors of RET that target both the WT and resistance mutants in KIF5B-RET-driven cell lines while sparing the majority of the kinome. These inhibitors also suppressed the proliferation of thyroid cancer cell lines harboring RET fusions or activating RET mutations and demonstrated in vivo activity in xenograft models. Our analysis of the PK-PD-efficacy relationship revealed that over 70% target suppression is required for maximal efficacy. Finally, our RET-selective inhibitors induced dose-dependent tumor growth inhibition in a KIF5B-RET fusion positive lung adenocarcinoma PDX model at well-tolerated doses, further validating RET fusions as oncogenic drivers in NSCLC.

Conclusion: This work describes the identification of potent inhibitors that specifically target WT RET and resistance mutations predicted to arise upon mKI treatment. By sparing kinases with known toxicity profiles, these molecules are predicted to robustly inhibit RET at tolerated doses and may provide patients with RET-driven diseases an opportunity for more durable and effective therapies.

#2642

Evaluation of the mechanism of MET-dependent cellular transformation and potent cytoreductive activity of MGCD265 in novel MET exon 14 mutation positive cancer models.

Lars D. Engstrom, Ruth W. Tang, David M. Briere, Harrah Chiang, Peter Olson, James G. Christensen. _Mirati Therapeutics, San Diego, CA_.

MET splice site mutations that result in the deletion of exon 14 (METex14del) are implicated as oncogenic drivers in a subset of non-small cell lung cancer (NSCLC). MET exon 14 contains the Y1003 CBL ubiquitin ligase regulatory binding site that normally mediates CBL-dependent MET degradation and signal attenuation. Deletion of this exon results in sustained activation of MET and its downstream signaling pathways. The diverse splice site mutations leading to exon 14 skipping comprise a unique and unprecedented class of RTK activating mutations and the molecular mechanism by which these genetic alterations transform cancer cells is not fully understood. One major challenge in understanding the utility of MET inhibition of the METex14del class has been the lack of available pre-clinical models.

In the present study, we generated and characterized multiple METex14del-driven cancer models to study the mechanism of MET-dependent cellular transformation as well as the response to MGCD265, a small molecule inhibitor of MET and AXL. METex14del models were identified via mining patient-derived xenograft (PDX) databases or were engineered using genome editing techniques to generate isogenic pairs of METex14del and WT cell lines.

The METex14del cell lines formed increased size and number of colonies in anchorage independent growth assays compared to their WT counterparts. The transformation of METex14del cells was associated with an increase in durable HGF-dependent activation of MET and downstream signaling pathways potentially due to dysregulated MET processing and signaling attenuation. MGCD265 was shown to effectively inhibit this growth and MET-dependent signal transduction in a concentration-dependent manner. When evaluated in the amplified METex14del-driven gastric xenograft model Hs746T, significant tumor regression was observed following MGCD265 treatment. In addition, MGCD265 demonstrated substantial regression of large established tumors, in two novel NSCLC METex14del-positive PDX models.

Together, these data confirm METex14del mutations are bona fide oncogenic drivers and sensitive to targeted therapeutics. Moreover, the models described in this study represent a relevant pre-clinical platform to further study receptor hyper-activation and drug action that is clinically actionable. Identification, development, and understanding of METex14del models will likely help further guide precision medicine strategies to treat NSCLC patients harboring these mutations.

#2643

Discovery and characterization of a selective TrkA inhibitor for the treatment of TrkA-driven cancers.

Vijaya G. Tirunagaru, Kumaragurubaran Nagaswamy, Sayan Mitra, Srinivas Maddi, Ram Sudheer Adluri, Hemant Joshi, Jang B. Gupta. _GVK Biosciencies, Hyderabad, India_.

Kinase gene fusions have been implicated as oncogenic drivers promoting cell survival and proliferation. TrkA gene fusions have been reported in a number of cancer types including lung, colorectal, papillary thyroid, sarcoma, cholangiocarcinoma and other tumors. We have identified novel, potent, selective and orally bioavailable TrkA inhibitors for the treatment of TrkA-driven cancers. One of the inhibitors, GVK-TrkI showed potent activity with an IC50 of 0.8 nM and 1.5 nM in the cell based assays for WT TrkA (AD293 cells expressing TrkA) and TPM3-NTRK1 fusion (KM12 cells) respectively. Potent activity of 12.5 nM IC50 was observed in TrkA Z-lyte kinase assay. Significant change in TrkA Z-lyte kinase IC50 was not observed at multiple ATP concentrations ranging from 100 uM to 1 mM suggesting non-ATP competitive mode of inhibition. Excellent selectivity (>1000x) was observed towards TrkB and TrkC in the cell based assays. GVK-TrkI inhibited TrkA autophosphorylation in a concentration dependent manner up to 24 h in KM12 cells in vitro. GVK-TrkI inhibited cell proliferation in KM12 cells with a GI50 of 34 nM while exhibiting no activity in cell lines without TrkA fusion. Comparable activity was observed in Ba/F3 cell line expressing MPRIP-NTRK1 fusion, demonstrating inhibition of TrkA driven cell proliferation irrespective of the N-terminal fusion with a different gene, MPRIP. In the KM12 xenograft model, treatment with GVK-TrkI at various doses and dosing regimens of 20 mpk bid, 50 mpk bid and 100 mpk qd resulted in dose dependent inhibition with almost 100% tumor growth inhibition (TGI) at 100 mpk qd with 3 out of 8 mice showing complete tumor regression. Besides, at 50 mpk in various dosing regimens of qd, bid and bid alternate day showed good correlation of TGI with compound exposure. GVK-TrkI was well-tolerated, causing no weight loss or death compared with vehicle control. Based on the evidence of ATP binding site mutations contributing to clinical resistance with kinase inhibitor drugs, GVK-TrkI is predicted to be effective as a second generation TrkA inhibitor with a non-ATP binding site mechanism compared to pan-Trk inhibitors currently being pursued in clinic. These results suggest that GVK-TrkI is an effective therapeutic option in TrkA driven cancers with TrkA gene fusions, activating mutations and cancers with NGF/TrkA overexpression resulting in constitutive activation of TrkA pathway.

#2644

AP32788, a potent, selective inhibitor of EGFR and HER2 oncogenic mutants, including exon 20 insertions, in preclinical models.

Francois Gonzalvez, Xiaotian Zhu, Wei-Sheng Huang, Theresa E. Baker, Yaoyu Ning, Scott D. Wardwell, Sara Nadworny, Sen Zhang, Biplab Das, Yongjin Gong, Matthew T. Greenfield, Hyun G. Jang, Anna Kohlmann, Feng Li, Paul M. Taslimi, Meera Tugnait, Yongjin Xu, Emily Y. Ye, Willmen W. Youngsaye, Stephan G. Zech, Yun Zhang, Tianjun Zhou, Narayana I. Narasimhan, David C. Dalgarno, William C. Shakespeare, Victor M. Rivera. _ARIAD Pharmaceuticals, Inc., Cambridge, MA_.

In non-small cell lung cancer (NSCLC), multiple classes of activating mutations have been identified in EGFR and HER2 that vary widely in their sensitivity to available tyrosine kinase inhibitors (TKIs). Erlotinib, gefitinib, and afatinib are approved for use in patients with the most common forms of EGFR activating mutations (ie, exon 19 deletions or L858R substitutions). However, no TKIs are approved for patients with EGFR activated by any other mutation, including exon 20 insertions or other uncommon substitutions, or for patients with any class of HER2 activating mutation (including exon 20 insertions). As inhibition of wild-type (WT) EGFR is associated with dose-limiting toxicities, a TKI that inhibits oncogenic EGFR and HER2 variants more potently than WT EGFR is more likely to be able to be dosed to efficacious levels. AP32788 is a potent inhibitor of all oncogenic forms of EGFR and HER2, including exon 20 insertions, with selectivity over WT EGFR.

Activity of AP32788 and other TKIs was assessed by measuring viability of Ba/F3 cell lines engineered to express 20 mutant variants of EGFR (n=14) or HER2 (n=6): 4 EGFR variants containing a common activating mutation with or without a T790M resistance mutation, 8 EGFR/HER2 variants containing an exon 20 activating insertion (eg, EGFR ASV, HER2 YVMA), and 8 EGFR/HER2 variants containing other uncommon activating mutations (eg, EGFR G719A, HER2 G776V). Inhibition of WT EGFR was assessed by measuring effects on EGFR phosphorylation in cells (A431) that over-express WT EGFR. Consistent with their clinical activity, erlotinib and gefitinib generally only inhibited the 2 EGFR variants with common activating mutations more potently than WT EGFR (IC50s 71 and 56 nM, respectively), and afatinib generally only inhibited EGFR with common activating mutations or uncommon substitutions more potently than WT EGFR (IC50 4 nM). In contrast, AP32788 inhibited all 14 mutant variants of EGFR (IC50s 2.4-22 nM), and all 6 mutant variants of HER2 (IC50s 2.4-26 nM), more potently than it inhibited WT EGFR (IC50 35 nM), including all 8 variants with exon 20 activating insertions. In mice implanted with a patient-derived tumor containing an EGFR exon 20 activating insertion, or with engineered Ba/F3 cells containing a HER2 exon 20 activating insertion, once daily oral dosing of AP32788 induced regression of tumors at doses that were well tolerated (30-100 mg/kg). In vivo efficacy was associated with inhibition of EGFR signaling in the tumor.

AP32788 potently inhibited all activated forms of EGFR and HER2 tested, including exon 20 insertions, more potently than WT EGFR, suggesting it may have the selectivity necessary to achieve efficacious levels of exposure in patients. A phase 1/2 clinical trial of AP32788 in NSCLC patients is planned.

#2645

BAY 1436032: A highly selective, potent and orally available inhibitor of mutant forms of IDH1.

Olaf Panknin,1 Stefan Pusch,2 Lena Herbst,2 Stefan Kaulfuss,1 Katja Zimmermann,3 Hartmut Rehwinkel,1 Roland Neuhaus,1 Sven Ring,1 Michael Brüning,1 Claudia Stark,1 Katja Prelle,3 Martin Michels,1 Michael Jeffers,4 Holger Hess-Stumpp,1 Karl Ziegelbauer,1 Michael Brands,1 Alwin Krämer,2 Andreas von Deimling2. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _Bayer Pharma AG, Wuppertal, Germany;_ 4 _Bayer Healthcare Pharmaceuticals, LLC, Whippany, NJ_.

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme that is frequently mutated in certain cancers, with incidence rates ranging from 7-90% for glioma, chondrosarcoma, intrahepatic cholangiocarcinoma and AML. Wildtype IDH1 (wtIDH1) catalyzes the conversion of isocitrate to α-ketoglutarate (αKG), while tumor-associated mutant IDH1 (mIDH1) catalyzes a rogue reaction: the production of 2-hydroxyglutarate (2-HG) from αKG. 2-HG therefore represents an "oncometabolite" that is believed to play a role in cancer by interfering with αKG-dependent enzymes, which in turn causes hypermethylation of histones/DNA and a block of normal cellular differentiation. Mutant IDH1 is a "driver" oncogene and the inhibition of this altered enzyme will decrease the growth of mIDH1 dependent tumors.

We report for the first time the preclinical profile and structure of BAY 1436032, a novel selective mIDH1 inhibitor. An optimization program based on a high throughput screening resulted in the identification of the clinical candidate BAY 1436032 for the treatment of mIDH1 dependent cancer. BAY 1436032 is a double-digit nanomolar and selective pan-inhibitor of the enzymatic activity of various IDH1-R132X mutants in vitro and displayed potent inhibition of 2-HG release (nanomolar range) in patient derived and engineered cell lines expressing different IDH1 mutants. In line with the proposed mode of action, a concentration-dependent lowering of 2-HG was observed in vitro accompanied by differentiation and maturation of mIDH1 tumor cells. Furthermore, BAY 1436032 showed a favourable selectivity profile against wtIDH1/2 and a large panel of off-targets in vitro.

To the best of our knowledge we were able to show for the first time single agent in vivo efficacy in mIDH1 patient derived glioma and intrahepatic cholangiocarcinoma solid tumor models with this clinical candidate along with monitoring of intratumoral 2-HG levels as a predictive biomarker. The BBB penetration profile of BAY 1436032 is further supported by preclinical data on in vivo brain-plasma ratios.

In conclusion, our data provide in vitro and in vivo proof of concept for BAY 1436032 as a potent and highly selective inhibitor of mutant forms of IDH1. The start of a Phase I study with BAY 1436032 is currently in preparation to determine the safety, tolerability, pharmacokinetics and preliminary anti-tumor and pharmacodynamic biomarker responses in patients with solid tumors.

#2646

The unique binding mode of NTRC 0066-0, a novel inhibitor of the spindle assembly checkpoint kinase TTK (Mps1), leads to long target residence time and potent antitumor activity.

Jos de Man, Joost C.M. Uitdehaag, Nicole Willemsen-Seegers, Jan Gerard Sterrenburg, Joeri J.P. de Wit, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Rogier C. Buijsman, Guido J.R. Zaman. _Netherlands Translational Research Center BV, Oss, Netherlands_.

[purpose] An abnormal number of chromosomes, or 'aneuploidy', is a common feature of solid human tumors and a predictor of poor prognosis in breast, lung, brain and colorectal cancer. Aneuploidy is caused by malfunctioning of the Spindle Assembly Checkpoint (SAC), a surveillance mechanism that ensures the fidelity of chromosome segregation. The protein kinase TTK (commonly referred to as Mps1) is a component of the SAC. Inhibition of TTK gene expression by RNA interference and inhibition of TTK kinase activity by small molecule kinase inhibitors causes chromosome missegregation and cancer cell death.

[experimental procedures] A novel class of compounds was identified that potently inhibits TTK enzyme activity and cancer cell line proliferation [1]. Its binding mode and that of reference inhibitors was characterized by protein crystallography. Binding kinetics and target residence time were determined by surface plasmon resonance using Biacore T200. Anti-proliferative activity was measured on a broad panel of cancer cell lines [2].

[results] The clinical candidate, NTRC 0066-0, inhibits TTK with subnanomolar potency (IC50) in a kinase enzyme assay and is more than 200 times selective over 276 kinases examined, including mitotic and cell cycle dependent kinases (CDKs). X-ray structures of the TTK kinase domain in complex with NTRC 0066-0 and analogs indicate that this class of compounds induces a large conformational shift in the glycine-rich loop, invoking an inactive kinase conformation. In surface plasmon resonance experiments, NTRC 0066-0 exhibited slow dissociation kinetics, resulting in a long target residence time. Parallel surface plasmon resonance experiments with mitotic kinases confirmed the exquisite selectivity of NTRC 0066-0 for TTK over Aurora and Polo-like kinases. NTRC 0066-0 potently inhibited the proliferation of a wide variety of human cancer cell lines with potencies in the same range as marketed cytotoxic agents. The crystal structure, binding kinetics and cellular potency of NTRC 0066-0 were compared to that of other TTK inhibitors such as Mps1-IN-2, AZD-3146, Mps-BAY2b and Bay 1161909 as well as analogs from the NTRC 0066-0 series. This suggest that the unique binding mode of NTRC 0066-0 results in long target residence time which contributes to its strong anti-tumor activity. In subsequent mouse xenograft models of human cancer cell lines, NTRC 0066-0 inhibited tumor growth as a single agent after oral administration at 20 mg per kg.

[conclusions] NTRC 0066-0 is a novel TTK inhibitor with outstanding in vitro properties and potent anti-tumor activity in mouse xenograft models. Our data suggest that long target residence time corresponds with potent cellular activity for TTK inhibitors.

References

[1] Maia et al. (2015) Annals of Oncology 26, 2180-2192; [2] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146.

#2647

Merestinib (LY2801653), targeting several oncokinases including NTRK1/2/3, shows potent anti-tumor effect in colorectal cell line- and patient-derived xenograft (PDX) model bearing TPM3-NTRK1 fusion.

Bruce W. Konicek, Steve M. Bray, Andrew R. Capen, John N. Calley, Kelly M. Credille, Philip J. Ebert, Gary Heady, Bharvin K. Patel, Victoria L. Peek, Jennifer R. Stephens, Suzane L. Um, Melinda D. Willard, Isabella H. Wulur, Yi Zeng, Richard A. Walgren, Sau-Chi Betty Yan. _Eli Lilly and Company, Indianapolis, IN_.

In cancer, the formation of chimeric gene fusions by genomic rearrangement causes aberrant receptor tyrosine kinase activation resulting in sustained oncogenic signaling driving tumorigenesis. Neurotrophic tyrosine receptor kinase 1 (NTRK1), the cognate receptor for nerve growth factor (NGF), has been reported in 7 tumor types as a NTRK1 kinase domain fused with several reported partners including the 5' coiled-coil domain of the tropomysin TPM3 gene. The resultant NTRK1 fusion protein is present in about 1.5% of colorectal cancer (CRC), 3% of lung and 12% of papillary thyroid cancers. In addition, gene fusions involving NTRK2 and NTRK3 are present in about 19 different tumor types. Thus pharmacologically targeting NTRK kinase in cancers bearing NTRK fusions may provide treatment options to patients who otherwise might be resistant to standard oncolytic regimens. Merestinib (LY2801653) is an orally bioavailable small molecule inhibitor of several oncokinases, including MET, AXL, ROS1 and MKNK1/2. Merestinib and its two primary metabolites, M1 (LSN2800870) and M2 (LSN2887652) were shown in scanMaxSM kinase binding assays to inhibit all three NTRKs with an IC50 ranging from 15-320 nM, and in the cell-based PathHunter® NTRK1 assay with an IC50 ranging from 12-92 nM. Merestinib, M1 and M2 were evaluated in vitro in TPM3-NTRK1 fusion bearing CRC cells (KM-12). Merestinib, M1 and M2 reduced p-NTRK1 levels, cell proliferation (IC50 of 11 nM, 18 nM and 100 nM respectively) and anchorage independent growth (IC50 of 45 nM, 79 nM and 206 nM respectively). Crizotinib previously reported (Nat Med. 2013;19:1469-72) to have moderate activity against NTRK1, was used to treat a patient with NTRK1 fusion resulted with transient response. Crizotinib was shown here to also reduce p-NTRK1 levels, cell proliferation (IC50=88nM) and anchorage independent growth (IC50 = 276nM) in vitro in KM-12 cells. Merestinib treatment at 24 mg/kg once daily arrested tumor growth (T/C=4%) in KM-12 xenograft tumor bearing mice. Crizotinib administered at 25 mg/kg twice daily in this same model did not result in tumor growth arrest (T/C=39.5%). Merestinib treatment at 24 mg/kg once daily led to tumor regression in a CRC PDX xenograft model (EL1989) bearing the TPM3-NTRK1 fusion. Crizotinib treatment at 25 mg/kg twice daily in this model did not show tumor regression. Further pre-clinical studies of Merestinib inhibition of NTRK2 and NTRK3 gene fusion are ongoing. These data support the clinical evaluation of Merestinib in patients with tumors harboring NTRK fusion. Merestinib is currently being studied clinically in advanced cancers (NCT01285037).

## IMMUNOLOGY:

### Immunotherapy Trial Results and Correlates

#2648

Chimeric antigen receptor T-cell therapy against anaplastic lymphoma kinase (ALK) is limited by target antigen density and CAR surface expression.

Robbie G. Majzner, Alec J. Walker, Meera Murgai, Ling Zhang, Adrienne H. Long, Kelsey M. Wanhainen, Rimas J. Orentas, Crystal L. Mackall. _National Cancer Institute, Bethesda, MD_.

ALK is overexpressed on the surface of neuroblastoma (NB) and is associated with high risk disease. We developed a second generation CAR based on a monoclonal antibody against ALK. ALK CAR T-cells significantly delay the growth of human NB cell lines in murine xenograft models, but animals eventually succumb to tumors.

In order to understand how target antigen density limits the effectiveness of this CAR, we created a library of NALM-6 B-cell leukemias with variable amounts of ALK expressed on the surface of each clone. In vitro, ALK CAR T-cells lyse high ALK expressing leukemias but have reduced activity against leukemias with low expression of ALK. In co-culture assays, we found that there is a minimum target antigen density required for CAR T-cells to produce appreciable amounts of Th1 cytokines. This threshold of ALK expression is above the expression on human NB lines. There is also a threshold target antigen density at which maximum cytokine production occurs. Above this density, no additional cytokines are produced. In xenograft models, ALK CAR T-Cells are significantly more effective against high ALK expressing leukemia than against low ALK expressing leukemia. To our knowledge, this is the first report of greater in vivo efficacy of CAR T-cells against tumors with higher antigen expression.

The level of surface expression of the CAR on T-cells also alters the function of CAR T-cells. We have identified a phenomenon in which both ALK and CD19 CAR T-cells lose surface expression of CAR over time in culture. Additionally, both the ALK CAR and the CD19 CAR downregulate quickly after T-cells are exposed to antigen. Using a fluorescently tagged CD19 CAR, we demonstrate that CARs are rapidly internalized upon antigen encounter.

We created an assay to understand the interplay of target density and CAR surface expression. We transduced T-cells with different amounts of supernatant to achieve different ALK CAR surface densities and then exposed these T-cells to varying amounts of immobilized protein-L in order to crosslink the CAR. T-cell activation (measured by an NFAT reporter) is greatest when both CAR surface expression and target antigen density are highest. As both variables are decreased, there is a significant loss of T-cell activity. Thus, diminished CAR surface density due to antigen-dependent and independent CAR downmodulation may limit CAR T-cell efficacy, especially in the context of low tumor antigen expression.

In conclusion, we have created a novel CAR targeting ALK that demonstrates in vivo efficacy against NB. Efficacy is limited by low target antigen density on NB and low CAR surface expression. We have identified phenomena in which CAR T-cells lose surface expression of CAR over time and also quickly internalize their receptors in response to antigen. These factors contribute to the efficacy of other CAR T-cells and this data provides important insights into future CAR target selection.

#2649

PD-L1 expression as a predictive factor for recurrence pattern and prognosis in curatively resected gastric cancer.

TOSHIAKI MORIHIRO, Shinji Kuroda, Tetsushi Kubota, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. _Okayama university, Okayama, Japan_.

Interaction of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) induces functional impairment of antigen-specific T cells, which leads to immune evasion of tumors. Immune checkpoint therapy including PD-1/PD-L1 blockade is an emerging treatment strategy which brings great improvement in patient's prognosis. Many reports have mentioned that PD-L1 expression on tumors correlates with poor prognosis in various types of cancers. While there is a few report about correlation between PD-L1 expression and prognosis in gastric cancer, one of the most common cancers in the world especially in Asia, it still remains controversial. In addition, almost no report has referred to the correlation of PD-L1 expression with metastatic or recurrence pattern of cancers. Thus, we investigated the correlation of PD-L1 expression with prognosis and recurrence pattern in gastric cancer patients. In this study, 255 gastric cancer cases which had curative surgical resection at Okayama University Hospital between 2002 and 2009 were analyzed retrospectively in terms of correlation of PD-L1 expression on tumors with patient's prognosis and recurrence pattern in addition to clinicophathological features. When PD-L1 expression was classified into 4 groups according to PD-L1 positive rate on tumors on immunohistochemical staining (0: < 1%, 1+: 1-5%, 2+: 5-10%, 3+: ≥10%) and 2+ and 3+ were defined as PD-L1 positive, PD-L1 positive rate was 15% (37/255). PD-L1 positive cases had significant correlation with advanced stage (p=0.0424), lymphatic invasion (p=0.0028) and venous invasion (p=0.0254), and tendency to be more observed in intestinal type than diffuse type (p=0.0599) in terms of histological types of cancer. Overall survival (p=0.0109) and disease-free survival (p=0.0052) were significantly poor in PD-L1 positive group. Moreover, PD-L1 expression had significant negative correlation with peritoneal dissemination (p=0.0081) and had tendency to cause hematogenous recurrence (p=0.1002) in terms of recurrence pattern after curative surgical resection, which might be reflected by the correlation of PD-L1 expression and histological types of cancer. Multivariate analysis revealed that PD-L1 expression was an independent risk factor for recurrence (p=0.0160, odds ratio: 3.19), along with diffuse type and advanced stage. In conclusion, PD-L1 expression on tumors strongly correlated with poor prognosis and negatively correlated with peritoneal dissemination in recurrence pattern in gastric cancer patients who had curative surgical resection.

#2650

In depth immune profiling of the response of melanoma to MAPK inhibition.

Sarah J. Welsh,1 Suzanah Boyd,1 Helen McGuire,2 Maria Gonzales,3 Alexander M. Menzies,3 Hojabr Kakavand,3 Elisa James,3 Elizabeth Smith,3 Barbara Fazekas,2 Richard F. Kefford,1 Alex Guminski,3 Richard A. Scolyer,3 Georgina V. Long,3 Helen Rizos1. 1 _Macquarie University, Sydney, Australia;_ 2 _Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia;_ 3 _Melanoma Institute Australia, Sydney, Australia_.

Melanoma patients with clinically palpable regional lymph node metastases (AJCC Stage III) carry a risk of relapse and death that approaches 70% at 5 years. The NeoCombi study is a pilot phase II study of neoadjuvant dabrafenib plus trametinib in patients with resectable Stage IIIB-C BRAFV600 mutation positive melanoma. Patients receive 12 weeks of combined drug treatment, followed by surgical resection of all disease. Patients then continue combination drug treatment for a further 40 weeks. We aimed to identify host and tumour biomarkers that predict response to MAPK inhibition.

Blood and tumour samples were obtained at baseline, early during treatment (3-7 days on therapy), at completion resection after 12 weeks on therapy, and, if relevant, on progression. Additional blood samples were also collected every 2 weeks up to 12 weeks of therapy. Samples were processed to generate plasma, peripheral blood mononuclear cells (PBMCs), fresh frozen tumour and where possible, tumour-dissociates and cell lines (tumour and immune cells). We have profiled tumour-specific and peripheral immune responses using a combination of flow cytometry, mass cytometry and multiplex cytokine/chemokine arrays. We also profiled circulating tumour-associated DNA (ctDNA) in plasma.

To date, we have accrued 17 patients onto the NeoCombi study. Primary analysis of the first 12 patients has been completed. Decreases in ctDNA and tumour viability, in addition to immunophenotype changes, were seen within 1 week of starting treatment. Changes in immunophenotype continued throughout the first 12 weeks of treatment with conversion of CD8+ and CD4+ naive (CD45RA+) cells to effector cells (CD45RA-) in most patients. We also noted that activation of regulatory T cells (CD4+CD25+CD127-HLA-DR+) increased over the first 12 weeks of treatment in patients with partial pathological responses compared to those with complete pathological responses. Interestingly, the cytokine profile remained relatively stable across treatment within each patient, suggesting that factors already present at baseline may determine response to treatment. A pro-inflammatory cytokine signature has been identified as a potential biomarker of patients likely to relapse and in vitro validation studies are currently underway. Additionally, although ctDNA was only detectable in 42% of patients (compared to over 70% in patients with stage IV disease), high levels of ctDNA identified patients at highest risk of relapse and, if detectable, proved a useful marker to monitor treatment response.

In conclusion, we have identified both host and tumour-specific biomarkers which may be useful to predict (and to monitor) response to MAPK inhibition in melanoma patients.

#2651

Clinical activity and immune correlates from a phase Ib study evaluating atezolizumab (anti-PDL1) in combination with FOLFOX and bevacizumab (anti-VEGF) in metastatic colorectal carcinoma.

Jeffrey Wallin,1 Michael J. Pishvaian,2 Genevive Hernandez,1 Mahesh Yadav,1 Suchit Jhunjhunwala,1 Lelia Delamarre,1 Xian He,1 John Powderly,3 Christopher Lieu,4 S. Gail Eckhardt,4 Herbert Hurwitz,5 Howard S. Hochster,6 Janet Murphy,7 Vincent Leveque,1 Edward Cha,1 Roel Funke,1 Daniel Waterkamp,1 Priti Hegde,1 Johanna Bendell8. 1 _Genentech, Inc., South San Francisco, CA;_ 2 _Lombardi Comprehensive Cancer Center., Washington, DC;_ 3 _Carolina BioOncology Institute PLLC, Huntersville, NC;_ 4 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 5 _Duke University Medical Center, Durham, NC;_ 6 _Yale Cancer Center, New Haven, CT;_ 7 _Massachusetts General Hospital, Boston, MA;_ 8 _Sarah Cannon Research Institute, Nashville, TN_.

Genetic and epigenetic alterations can distinguish cancer cells from their normal counterparts, allowing tumors to be recognized as foreign by the immune system. The immune system's ability to detect and destroy these abnormal cells is the foundation of cancer immunotherapy. In order for this to occur immune cells must traffic and persist in the tumors. Programmed death-ligand 1 (PD-L1; also called B7-H1 or CD274), which is expressed on many cancer and tumor-infiltrating immune cells, plays an important part in blocking immune-mediated tumor cell destruction by binding B7.1 (CD80) and programmed death-1 (PD-1), which is a negative regulator of T-lymphocyte activation. PD-L1 blockade has previously been shown to increase CD8+ T cell infiltration and PD-L1 protein levels, as well as the expression levels of immune genes in tumors. Vascular endothelial growth factor A (VEGF-A) is a secreted factor that specifically acts on endothelial cells to stimulate angiogenesis and has also been shown to exert immunosuppressive effects. Although some cytotoxic agents have been proposed to induce immunogenic cell death, the effects of chemotherapy and anti-angiogenic therapy on the tumor immune milieu have not been extensively studied. This study was designed to evaluate the safety, activity and biomarkers of combined FOLFOX treatment combined with PD-L1 and VEGF-A inhibition in 1L colorectal (CRC) patients using the humanized antibodies atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF-A), respectively. RECIST (v1.1) responses by the cutoff date of 09/01/2015 were observed in 52% (95% CI 30.6-73.2) of the patients (n=23) with a median progression free survival of 14.1 months (95% CI 8.7-17.1) and a median duration of response of 11.4 months (95% CI 7.6-15.9). No unexpected toxicities were observed. We found that CD8+ T cells and PD-L1 expression by IHC were increased in tumors following administration of FOLFOX alone as well as after combined administration of FOLFOX, atezolizumab and bevacizumab. Elevations in cytotoxic T-cell signatures (e.g. CD8A, IFNγ, GZMB, EOMES) with treatment were also noted in several of the patient tumors. Patients with elevations in tumor-infiltrating CD8+ T cells consistent with increased expression of cytotoxic T-cell signatures and PD-L1 exhibited sustained responses or prolonged disease control. These data suggest that the combination of FOLFOX, atezolizumab, and bevacizumab promote immune-related activity in CRC potentially resulting in enhanced efficacy.

#2652

Pan-cancer analysis of programmed death-ligand 1 (PD-L1) amplifications.

Jan Budczies,1 Michael Bockmayr,1 Frederick Klauschen,1 Wilko Weichert,2 Carsten Denkert,1 Albrecht Stenzinger3. 1 _Charité - Universitätsmedizin Berlin, Berlin, Germany;_ 2 _Technical University Munich, Munich, Germany;_ 3 _Massachusetts General Hospital, Harvard Medical School, Boston, MA_.

PD-L1, located in the chromosomal region 9p28, is a ligand of programmed cell death protein 1 (PD-1) and has been described as immune checkpoint regulator that allows cancers to evade the host immune system. To stratify patients for treatment with PD-1 inhibitors, PD-L1 expression is currently intensively studied on RNA and protein levels. However, no comprehensive study of PD-L1 amplifications including many cancer types has been performed so far.

We investigated PD-L1 amplifications in eighteen cancer types that were copy number profiled, gene expression profiled and clinically characterized by the TCGA consortium: Acute Myeloid Leukemia (LAML), Bladder Urothelial Carcinoma (BLCA), Brain Lower Grade Glioma (LGG), Breast invasive carcinoma (BRCA), Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), Colorectal adenocarcinoma (COADREAD), Glioblastoma multiforme (GBM), Head and Neck squamous cell carcinoma (HNSC), Kidney renal clear cell carcinoma (KIRC), Liver hepatocellular carcinoma (LIHC), Lung adenocarcinoma (LUAD), Lung squamous cell carcinoma (LUSC), Ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach adenocarcinoma (STAD) and Uterine Corpus Endometrial Carcinoma (UCEC).

We found a diverse landscape of focal PD-L1 amplification, 9p chromosome arm amplifications and polysomy 9. Focal amplifications had the highest incidence in OV (10.7%), LUSC (9.8%), HNSC (8.6%), BLCA (8.3%), SARC (8.2%), CESC (7.1%), STAD (6.8%) and BRCA (5.3%). 9p amplifications were most frequently found in OV (12.6%), BLCA (7.4%), BRCA (6.8%) and LUAD (5.2%). Polysomy 9 had the highest incidence in COADREAD (14.0%), HNSC (13.6%), CESC (11.2%), SARC (9.4%) and STAD (7.2%), BLCA (5.9%) and GBM (5.5%). In a pooled analysis of focal and non-focal alterations, PD-L1 amplifications positively correlated with gene expression in nine cancer types and significantly (p < 0.05) shortened overall survival in five cancer types.

In summary, PD-L1 amplifications represent a frequent genetic alteration that occurs in many cancer types and could potentially be exploited for patient stratification and immune therapy.

#2653

Tremelimimab plus tumor ablation for patients with hepatocellular carcinoma: Clinical results, immunomonitoring analysis of peripheral T cells and tumor biopsies.

Ashish Uppala, Mei ElGindi, Firouzeh Korangy, Drew Pratt, David Venzon, Austin Duffy, Oxana Rusher, David Kleiner, Elliot Levy, Bradford Wood, Tim Greten. _NCI, Bethesda, MD_.

Introduction: Ipilimumab, an anti-CTLA4 mAb, is the first immune-checkpoint inhibitor, to be approved by the FDA for the treatment of patients with melanoma. We performed a phase I/II clinical trial in patients with advanced hepatocellular carcinoma, who progressed on standard of care. Patients were treated with tremelimumab, another anti-CTLA4 mAb, and tumor ablation (radiofrequency ablation, cryoablation, and transcatheter arterial chemoembolization) to augment anti-tumor immunity. Peripheral blood mononuclear cells, viral load (HBV/HCV) and tumor biopsies were studied and clinical efficacy was correlated with immune monitoring results.

Methods: Patients with HCC were enrolled in a study of tremelimumab combined with tumor ablation performed on week 6. Tumor biopsies were performed at baseline and at time of RFA/TACE and analyzed by immunohistochemistry. Gene expression analysis was performed using Nanostring technology and QIAGEN's Ingenuity Pathway Analysis (IPA) software. Eleven-color flow cytometry was performed to study peripheral blood mononuclear cells obtained at baseline, after 4 and 8 weeks using the following antibodies: CD4, CD3, CD4, CD8, CD11c, CD14, CD19, CD20, CD25, CD38, CD45RA, CD56, CD123, CD127, CCR7, CCR4, CXCR3, PD-1, 4-1BB, TIM3, CTLA4, PD-L1, ICOS, HLA-DR, AFP- and survivin-HLA-A2 tetramer. HCV and HBV viral load was determined in serum samples.

Results: 27 patients were enrolled. 12 patients received TACE and 13 underwent tumor ablation during week 6 of tremelimumab therapy. Of N=10 patients evaluable for response outside of TACE/RFA-treated lesion, 4 (40%) achieved confirmed partial responses. 6-week tumor biopsies showed clear increase in the number of CD3+ and CD8+ T cells in patients showing a clinical response only. Multi-color flow cytometry of PBMC revealed statistically significant changes after the 1st cycle in activated CD4+ and CD8+ T cells. The CD4/Treg and CD8/Treg ratio increased only in patients showing a clinical response. Both AFP and survivin specific CD8+ T cells were detected in HLA-A2+ patients, but there was no change in the frequency of these cells upon treatment. However PD1 expression increased over time both on AFP and survivin specific T cells. Finally, eight of 9 patients with quantifiable HCV experienced a marked reduction in viral load. 100% (N=4) of HBV patients experienced a reduction in quantitative HBsAg. Pathway analysis conducted through IPA revealed that the top canonical pathways upregulated by treatment were T-cell receptor, Ephrin-A, IL-1, G-coupled protein, and IL-6 signaling.

Conclusions: Tremelimumab in combination with subtotal TACE or RFA leads to an accumulation of intratumoral CD8+ T cells, activation of CD4+ and CD8+ T cells in peripheral blood and reduction in HCV viral load and HBsAg in patients showing clinical response to therapy.

#2654

GAPVAC-101 phase I trial: First data of an innovative actively personalized peptide vaccination trial in patients with newly diagnosed glioblastoma.

Norbert Hilf,1 Katrin Frenzel,2 Sabrina Kuttruff-Coqui,1 Sandra Heesch,2 Sebastian Kreiter,3 Arie Admon,4 Valesca Bukur,2 Sjoerd van der Burg,5 Cecile Gouttefangeas,6 Judith R. Kroep,5 Marij Schoenmaekers-Welters,5 Jordi Piro,7 Berta Ponsati,7 Hans Skovgaard Poulsen,8 Ulrik Lassen,8 Francisco Martinez-Ricarte,9 Jordi Rodon,9 Juan Sahuquillo,9 Monika Stieglbauer,6 Stefan Stevanovic,6 Per thor Straten,8 Marco Skardelly,6 Ghazaleh Tabatabai,6 Michael Platten,10 David Capper,10 Andreas von Deimling,10 Valérie Dutoit,11 Hideho Okada,12 Christian Ottensmeier,13 Randi Kristina Feist,1 Jens Fritsche,1 Karoline Laske,1 Peter Lewandrowski,1 Martin Löwer,14 Regina Mendryzk,1 Miriam Meyer,1 Carsten Reinhardt,1 Bernhard Rössler,1 Anna Paruzynski,2 Nina Pawlowski,1 Colette Song,1 Stevermann Lea,1 Toni Weinschenk,1 Christoph Huber,3 Hans-Georg Rammensee,6 Pierre-Yves Dietrich,11 Wick Wolfgang,10 Ugur Sahin,2 Harpreet Singh-Jasuja1. 1 _Immatics Biotechnologies GmbH, Tuebingen, Germany;_ 2 _BioNTech AG, Mainz, Germany;_ 3 _CIMT - Association for Cancer Immunotherapy, Mainz, Germany;_ 4 _TECHNION - Israel Institute of Technology, Haifa, Israel;_ 5 _Leiden University Medical Center, Leiden, Netherlands;_ 6 _Eberhard Karls Universität Tübingen, Tuebingen, Germany;_ 7 _BCN Peptides S.A., Barcelona, Spain;_ 8 _Center for Cancer Immune Therapy, Copenhagen, Denmark;_ 9 _Vall d'Hebron University Hospital, Barcelona, Spain;_ 10 _University Hospital Heidelberg, Heidelberg, Germany;_ 11 _Université de Genéve, Geneva, Switzerland;_ 12 _University of California San Francisco, San Francisco, CA;_ 13 _University of Southampton, Southampton, United Kingdom;_ 14 _TRON -Translational Oncology at the University Medical Center Mainz, Mainz, Germany_.

The Glioma Actively Personalized Vaccine Consortium (GAPVAC; funded by the European Union Framework 7 Program) aims at treating newly diagnosed glioblastoma (GB) patients with two distinct actively personalized vaccines (APVACs).

Resected tumor material is analyzed for multiple biomarkers to characterize the tumor in depth and to enable the design of APVACs tailored to each individual patient: Tumor-specific mutations, the HLA peptidome and gene expression profile are assessed by next-generation sequencing, mass spectrometry and RNA microarray analysis, respectively. Further, the patient-individual immune status is investigated by assessment of leukapheresis samples utilizing an in vitro immunogenicity platform. Data are integrated to define two distinct APVACs for each patient: APVAC1 is composed of up to ten peptides selected from a pre-manufactured "warehouse". The warehouse contains 59 HLA class I-binding and three class II-binding tumor-associated peptides frequently over-presented in GB. APVAC2 is composed of one or two peptides that are de novo synthesized for a given patient and preferentially represent mutation-bearing neo-epitopes.

After a preparation phase in which the warehouse was generated and setup of APVAC selection and manufacturing processes took place, the GAPVAC-101 phase I clinical trial was initiated. Primary endpoints of the study are assessment of safety, feasibility of APVAC manufacturing and biological activity.

The trial is conducted at six European centers and recruits HLA-A*02:01 or A*24:02-positive patients with newly diagnosed GB after gross total resection. Patients receive APVAC1 and APVAC2 vaccinations plus immunomodulators (poly-ICLC and GM-CSF) three and six months post study enrolment, respectively, and concurrent to maintenance temozolomide (TMZ).

As of November 2015, 11 patients have been enrolled, of whom six already received APVAC vaccines. Composition and manufacturing are ongoing for four patients. All APVACs were generated in time without ultimate failures. APVAC1 vaccines differ substantially with 31 out of 59 warehouse peptides have been selected at least once, indicating the need for personalization due to tumor heterogeneity even for non-mutated epitopes. In patients' tumor samples an average of 40 non-synonymous mutations (including known driver mutations) were identified. Injection site reactions were the most frequent toxicities so far. One brain edema (Grade 3) and one allergic reaction (Grade 4)were observed, both potentially related to the vaccinations. First data on biological activity of APVACs and updated clinical data will be presented at the Annual Meeting.

In conclusion, the GAPVAC concept has been successfully translated into the clinics and so far demonstrated to be safe and feasible with its level of personalization matching the observed tumor heterogeneity.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Epigenetic Alterations in Cancer

#2655

Oncogenic chromatin factors drive cell type-specific transcription within megadomains in NUT midline carcinoma.

Mitzi I. Kuroda,1 Artyom A. Alekseyenko,1 Erica M. Walsh,1 Xin Wang,2 Adlai Grayson,1 Peter T. Hsi,1 Peter V. Kharchenko,2 Christopher A. French1. 1 _Brigham & Women's Hospital, Boston, MA; _2 _Harvard Medical School, Boston, MA_.

NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we find correlate with ~100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These 'megadomains' appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones. Megadomains drive transcription of underlying DNA in NMC patient cells and in naïve cells induced to express BRD4-NUT. Here we characterize the constituents of BRD4-NUT chromatin complexes using a crosslinking approach, BioTAP-XL. We find many transcriptional activating proteins known to associate with BRD4, along with novel interactors including p300/CBP and a previously uncharacterized BRD4-NUT Megadomain Associated Protein (BMAP1). BMAP1 is expressed in primary NMC tissue and a subset of more common head and neck squamous cell carcinomas (HNSQC). Concurrently, we discovered a patient-derived NMC harboring a novel BMAP1-NUT fusion. BMAP-NUT blocks differentiation, and like BRD4-NUT recruits p300 to form hyperacetylated megadomains, including at the MYC locus. Thus, our proteomic and genetic approaches have converged on a novel mechanism that involves reprogramming very large regulatory regions to drive oncogenic transcription.

#2656

Loss of SIRT6 reactivates the oncofetal RNA-binding protein Lin28b to drive pancreatic cancer.

Sita Kugel,1 Carlos Sebastián,1 Julien Fitamant,1 Kenneth N. Ross,1 Supriya K. Saha,1 Alon Goren,2 Sridhar Ramaswamy,1 Vikram Deshpande,1 Nabeel Bardeesy,1 Raul Mostoslavsky1. 1 _Massachusetts General Hospital Cancer Center, Boston, MA;_ 2 _Broad Technology Labs (BTL), The Broad Institute of Harvard and MIT, Cambridge, MA_.

Chromatin remodeling proteins are frequently dysregulated in human cancer, however little is known about how they control tumorigenesis. The NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (PDAC) relative to normal tissue. Thus, we aimed to determine the role of SIRT6 in PDAC and to determine how the loss of this chromatin modifier could influence PDAC formation, progression and metastasis.

Here, we uncover an epigenetic program mediated by SIRT6 that is critical for suppression of PDAC. We demonstrate that SIRT6 inactivation cooperates with oncogenic KRAS to greatly accelerate the development of lethal pancreatic tumors regardless of p53 status. In addition to developing PDAC and high-grade pancreatic intraepithelial neoplasia (PanIN) at an earlier age, SIRT6-deficient tumors had a greater propensity to metastasize to the lung, compared to their SIRT6 wild-type (WT) counterparts. Through genome-wide profiling of SIRT6 WT and knock-out (KO) PDAC cell lines, we identified the oncofetal RNA-binding protein Lin28b as a target of Sirt6-mediated transcriptional repression. Loss of SIRT6 in both murine and human PDAC cell lines resulted in hyperacetylation of histone 3 lysine 9 and lysine 56 at the Lin28b promoter, MYC recruitment, and pronounced induction of Lin28b expression. Knocking down Lin28b resulted in potent suppression of cell proliferation and tumor sphere formation in Sirt6null/low murine and human cell lines, while murine and human Sirt6WT/high PDAC lines were completely insensitive to the same treatment. Functionally, Lin28b binds to and induces the degradation of the let-7 family of microRNA, thus resulting in increased expression of let-7 target genes such as the Igf2bps, which have been correlated with increased agressiveness and metastasis in pancreatic tumors. Furthermore, Gene Set Enrichment Analysis (GSEA) comparing PDAC tumors and cell lines with high versus low expression of LIN28B revealed that LIN28Bhigh tumors were strongly enriched for the expression of let-7 targets.

Thus, our critical findings define SIRT6 as a tumor suppressor in PDAC, working as a transcriptional repressor of the oncofetal RNA-binding protein Lin28b. We demonstrate that loss of Sirt6 creates a permissive hyperacetylated chromatin state, allowing for MYC-dependent transactivation of Lin28b. This work not only provides new insights into the epigenetic mechanisms governing the reactivation of oncofetal proteins in cancer but also demonstrates that this pathway is well-conserved in human PDAC, where reduced SIRT6 expression defines a specific subset of tumors which may benefit from novel therapeutic approaches aimed at targeting components of the LIN28B/let-7 pathway.

#2657

Target specificity of epigenetic therapy in cancer.

Takahiro Sato,1 Matteo Cesaroni,1 Shoghag Panjarian,1 Anthony Tran,1 Jozef Madzo,1 Yasuyuki Okamoto,1 Hanghang Zhang,1 Xiaowei Chen,2 Jaroslav Jelinek,1 Jean-Pierre J. Issa1. 1 _Temple University, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

A major question facing the use of epigenetic therapies in cancer is specificity in modulating gene expression. In addition, combined targeting of DNA and histone methylation remains largely unexplored despite the promising synergistic effects observed from combining DNA methyltransferase inhibitors with HDAC inhibitors. To address these questions, we performed RNA-seq, DNA methylation analysis and ChIP-seq (H3K4me2, H3K9me2, and H3K27me3) to study the effects of inhibitors of DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343) in three different cancer models (colon cancer, breast cancer, and leukemia). In colon cancer cells (YB5), DAC affected 3% of the transcriptome and 93% of the effect was gene upregulation. DAC had a greater effect on genes expressed in normal tissues and silenced in cancer (443 genes) compared to genes that do not change in cancer (194 genes). 90% of DAC targets genes showed no promoter DNA methylation in normal colon but gained methylation in cancer. Depsi changed the expression of 35% of the transcriptome and showed little specificity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 had limited effects on their own (<1.5% of the transcriptome), but UNC0638 and GSK343 preferentially targeted genes with H3K9me2 or H3K27me3, respectively. DAC combined with histone methylation inhibitors led to synergistic gene upregulation while still maintaining specificity for DNA methylated and silenced genes. These synergistic genes had limited overlap, indicating the possibility to target distinct sets of genes based on different epigenetic therapy combinations. IPA analysis demonstrated that these genes are enriched in cancer pathways, and consistent with this analysis, the combination therapies were able to decrease cancer cell proliferation more effectively than monotherapy. Broadly similar results were seen with genome wide studies in both breast cancer (MCF7) and leukemia (HL-60) cells. These results demonstrate that DNA methyltransferase inhibitors preferentially target cancer relevant genes, and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining specificity.

#2658

Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas.

Robert Nakayama, Robert T. Williams, Seth H. Cassel, Mingxiang Teng, Rafael A. Irizarry, George D. Demetri, Cigall Kadoch. _Dana Farber Cancer Institute, Boston, MA_.

SMARCB1/BAF47/INI1 is a core subunit of the mammalian SWI/SNF (BAF) family of ATP-dependent chromatin remodeling complexes, which remodel nucleosome architecture to achieve coordinated regulation of gene expression. Genetic loss of SMARCB1 has been identified in several cancer types, including malignant rhabdoid tumor (MRT, 98%) and epithelioid sarcoma (EpS, 90%), strongly implicating this event as the oncogenic driver in these malignancies. However, the precise mechanism underpinning the tumor suppressive function of BAF47 to date remains unclear. In order to elucidate the underlying mechanism and to identify direct genetic targets of aberrant BAF complexes in this context, we comprehensively evaluated the effects of BAF47 reintroduction in BAF47-deficient sarcomas with respect to complex subunit and associated protein factor composition and stability, global chromatin structure, and gene regulation.

Reintroduced BAF47 stably integrated into BAF complexes, and remarkably, stabilized a highly specific set of BAF subunits, resulting in an increased complex molecular weight and stoichiometric nuclear abundance. These biochemical changes inducing the formation of wild-type complexes in MRT and EpS cell settings were directly linked to reproducible changes in BAF complex localization genome-wide, particularly, in the targeting to H3K4me3-marked promoter regions of direct target genes to establish DNA accessibility. Importantly, changes in BAF47-dependent BAF complex targeting between oncogenic and induced wild-type conditions were reproducibly associated with differential chromatin architecture and gene expression signatures hallmark to both MRT and EpS, and uniformly resulted in proliferative senescence of MRT and EpS cell lines in culture.

These studies highlight, for the first time, the full spectrum of structural and functional contributions of the BAF47 subunit, implicating its role as a keystone in heteromorphic BAF complex assembly; BAF47 is required for the stable integration of several BAF subunits and novel interacting factors, which we determine govern specific genome-wide targeting mechanisms and chromatin-templated activities. These results reveal the mechanisms underlying the oncogenesis of BAF47-deficient sarcomas and point toward novel therapeutic strategies for this group of human sarcomas.

#2659

A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer.

Charly R. Good, Jozef Madzo, Shinji Maegawa, Nora Engel, Jaroslav Jelinek, Jean-Pierre Issa. _Fels Institute at Temple University School of Medicine, Philadelphia, PA_.

TET1 is a DNA demethylase that regulates DNA methylation, hydroxymethylation and gene expression patterns. It has a catalytic domain that oxidizes methylated cytosine into 5-hydroxymethylcytosine and can then be further oxidized or converted to un-methylated cytosine. TET1 has a CXXC domain that allows it to recognize and bind to long stretches of CpG dinucleotides, known as CpG islands (CGIs). Our previous work found TET1 to protect CGIs from aberrant methylation and that the CXXC domain limits its ability to regulate genes outside of CGIs. However, many of the methylation changes in cancer are outside CGIs. To determine if TET1 could possibly play a role in this hypomethylation, we searched for alternatively spliced forms of the protein. We identified a novel isoform of TET1 (TET1ALT) that lacks the CXXC domain but retains the demethylase domain. TET1ALT has a unique transcription start site from a strong alternate promoter upstream of exon 3, yielding a protein with a unique translation start site. We confirmed promoter activity using Luciferase constructs, and confirmed TET1ALT translation and function through overexpression experiments. Gene expression analyses by qPCR found that TET1ALT is silenced in mESCs but becomes activated in select embryonic and adult tissues while full length TET1 is favored in mESCs, but is repressed in adult tissues. Thus, there is an isoform switch during differentiation and TET1ALT is the major isoform expressed in adult cells. TET1ALT is over expressed in several cancer cell lines, including breast cancer. In conclusion, we have identified a novel isoform of TET1 that has the potential to regulate DNA methylation outside of CGIs and is the predominant isoform expressed in adult cells. We are investigating the hypothesis that TET1ALT may contribute to the methylation changes observed in cancer outside of CGIs, such as in gene bodies and in enhancers.

#2660

**A renal CpG island methylator phenotype (R-CIMP) in kidney tumors associated with germline mutations of** FH **and** SDHB **.**

Christopher Ricketts, J. Keith Killian, Cathy D. Vocke, Carole Sourbier, Youfeng Yang, Maria J. Merino, Paul S. Meltzer, W. Marston Linehan. _National Cancer Institute, Bethesda, MD_.

Germline mutations of the genes encoding the Krebs cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDHB, SDHC, SDHD) have both been associated with an increased risk of renal cell carcinoma (RCC). These renal tumors demonstrate complete loss of enzyme activity and significantly increased levels of intracellular fumarate or succinate that are predicted to infer with the actions of multiple cellular processes primarily through the inhibition of 2-oxoglutarate-dependent dioxygenases, such as the prolyl hydroxylases that regulate the HIF pathway and the TET enzymes that regulate DNA methylation.

This study evaluated and compared the genome-wide methylation profiles of 33 tumor samples (including 9 FH -/- and 7 SDHB -/- tumors), 10 associated normal samples and 13 cell-lines (including cell-line models derived from FH -/- and SDHB -/- tumors) using the Illumina HumanMethylation450 BeadChip arrays. Tumors derived from patients

with germline FH or SDHB mutations demonstrated a distinct pattern of hypermethylation across the genome with enrichment for hypermethylation within the CpG island regions. SDHB mutation associated tumors demonstrated that was more variable and less extensive pattern of hypermethylation than the tumors associated with germline FH mutations. Cell-line models derived from HLRCC or SDHB tumors demonstrated patterns of hypermethylation similar to the patterns observed in the tumors, yet re-expression of either wild-type FH or SDHB within these cell-line models did not alter the methylation profiles. All the HLRCC and SDHB-RCC cell lines, including the cell-line re-expressing of either wild-type FH or SDHB, demonstrated growth inhibition and reduced invasion rates in response to treatment with 5-aza-2′-deoxycytidine.

Thus, renal cell carcinomas associated with FH or SDHB germline mutation demonstrate a specific R-CIMP hypermethylation profile that differentiates these tumors from tumors derived from other RCC susceptibility syndromes. These tumors represent the most aggressive

types of familial-associated RCC and this could provide a useful biomarker for identifying these tumors and de-methylating agents, such as 5-aza-2′-deoxycytidine, may be effective as part of a therapeutic regime for the treatment of these tumors.

#2661

The genomic landscape and clinical relevance of A-to-I RNA editing in human cancers.

Han Liang. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional mechanism, but its genomic landscape and clinical relevance in cancer have not been investigated systematically. We characterized the global A-to-I RNA editing profiles of >6000 patient samples of 20 cancer types from The Cancer Genome Atlas and revealed a striking diversity of altered RNA-editing patterns in tumors relative to normal tissues. We identified an appreciable number of clinically relevant editing events, many of which are in noncoding regions. We experimentally demonstrated the functional effects of several RNA editing events and provide the evidence that RNA editing could selectively affect drug sensitivity. These results highlight RNA editing as an exciting theme for investigating cancer mechanisms, biomarkers and treatments.

### LncRNAs, MicroRNAs, and Non-coding RNAs in Cancer

#2662

Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance.

Yashar Niknafs, Sumin Han, Teng Ma, James Rae, Arul Chinnaiyan, Felix Feng. _University of Michigan, Ann Arbor, MI_.

Background: We previously performed a bioinformatics-based analysis on 7,256 RNA sequencing libraries from tumors, normal tissues, and cell lines to delineate the landscape of long noncoding RNAs (lncRNAs) in the human transcriptome. This analysis identified 58,648 lncRNAs, including over 45,000 novel transcripts (Iyer and Niknafs et al, Nature Genetics, 2015). We now interrogate this lncRNA compendium to identify top candidate estrogen receptor (ER)-associated lncRNAs in breast cancer and characterize their association with disease progression.

Methods: To identify and prioritize lncRNAs that were differentially expressed in cancer vs normal tissue, and in ER-positive vs ER-negative disease, we performed differential expression testing on over 1000 RNA sequencing libraries including breast cancer and normal tissue samples from the The Cancer Genome Atlas (TCGA) project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cell lines, and via mouse xenograft studies. The mechanism by which this lncRNA promotes tumor progression was investigated by identifying its top protein interactors and its subdomains responsible for function, and then studying the effects of disrupting function of this lncRNA on cancer phenotypes.

Results: Differential expression analyses nominated BRCAT431 as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that shRNA-mediated knockdown of BRCAT431 significantly decreased proliferation, colony formation, and invasion (by >50% in most assays). Tamoxifen resistance was associated with significantly increased BRCAT431 levels in both MCF7 and T47D cells, and knockdown of BRCAT431 reversed tamoxifen resistance. Xenograft studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by >50%. RNA pulldown followed by mass spectrometry identified the RNA binding protein hnRNPL as a key protein interacting with BRCAT431. Deletion studies identified a 27 base-pair region of BRCAT431 necessary for its interaction with hnRNPL, and loss of this region abrogated BRCAT431-induced invasion. Finally, guilt-by-association studies demonstrate a strong association between BRCAT431 overexpression and tumor grade, recurrence, and metastases.

Conclusion: In this study, we develop the largest reported compendium of breast cancer lncRNAs. We prioritize BRCAT431 as the top lncRNA upregulated in ER-positive breast cancers, and demonstrate that it confers aggressive oncogenic phenotypes in vitro and in vivo. We identify a novel mechanism by which this lncRNA functions through interaction with hnRNPL. Our results suggest that by promoting tamoxifen resistance, this lncRNA increases the clinical risk of recurrence and metastases in breast cancer.

#2663

Insights into the oncogenic basis of long non-coding RNA PVT1 in human cancer.

Kojiro Tashiro,1 Robert Klink,1 Jon Zetterval,1 Veronica Diedrich,1 Yuen-Yi Tseng,2 Anindya Bagchi1. 1 _Univ. of Minnesota Masonic Cancer Ctr., Minneapolis, MN;_ 2 _Broad Institute of MIT and Harvard, Cambridge, MA_.

Though gain of 8q24.21 is a common mutation in human cancers, its functional annotation is limited to studying myelocytomatosis (MYC), the prominent oncogene in the amplicon. However, MYC is co-gained with an adjacent long non-coding RNA gene plasmacytoma variant translocation 1 (PVT1), the CCDC26 gene candidate and gasdermin C GSDMC. Whether copy number gain of one or more of these genes drives neoplasia is not known. We developed chromosome engineered mouse strains with an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. When rat Neu was introduced to test the change in the latency in mammary tumor development, only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc (but not with an extra copy of Myc or Pvt1, Ccdc26, Gsdmc) developed adenocarcinomas with reduced latency, suggesting that while an extra copy of Myc gene failed to measurably advance cancer, it may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the tumors, and in SK-BR-3 and MDA-MB-231, two human breast cancer cell lines with 8q24 amplification. Ablation of PVT1 markedly decreased MYC protein levels, suggesting a PVT1-dependence of MYC protein in MYC amplified cancer cells. Analysis of the cancer genome atlas suggested 99% of tumors with increased MYC copy-number also harbor co-gain of PVT1. Tissue microarray analysis revealed that PVT1 RNA and MYC protein expression are correlated in primary human tumors. These data suggests that PVT1 can potentiate MYC in human cancers. CRISPR mediated deletion of PVT1 in colorectal cell line HCT116 impaired tumor formation in xenografts, and significantly reduced MYC levels. We further confirmed the oncogenic potential of PVT1 in cancers even without supernumerary 8q24. Additionally, we have identified the putative functional domain of PVT1 which confers its oncogenic potential. We propose that the dependence of high levels of MYC on lncRNA PVT1 provides a much-needed therapeutic window against MYC protein, known to be refractory to small molecule inhibition.

#2664

Eradication of neuroblastoma by suppressing the expression of a single long noncoding RNA.

Andrew E. Tee,1 Pei Y. Liu,1 Giorgio Milazzo,2 Jo Vandesompele,3 Glenn M. Marshall,1 Giovanni Perini,2 Pieter Mestdagh,3 Marcel Dinger,4 Tao Liu1. 1 _Children's Cancer Institute Australia, Sydney, Australia;_ 2 _Department of Pharmacy and Biotechnology, University of Bologna, Italy;_ 3 _Center for Medical Genetics Ghent, Ghent University, Belgium;_ 4 _Garvan Institute of Medical Research, Sydney, Australia_.

Neuroblastoma is the most common solid tumour in early childhood, and accounts for approximately 15% of all childhood cancer death. N-Myc gene amplification occurs in one quarter to one third of human neuroblastoma tissues, and the majority of patient with neuroblastoma due to N-Myc gene amplification die of the disease. We performed RNA sequencing experiments, and identified 5 transcripts, including RP1X, which were considerably differentially expressed between N-Myc gene amplified and non-amplified human neuroblastoma cell lines. Affymetrix microarray studies revealed that DEPD was one of the few genes considerably down-regulated in neuroblastoma cells after RP1X depletion. Chromatin immunoprecipitation assays showed that knocking-down RP1X expression reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the DEPD gene promoter. Depletion of RP1X or DEPD significantly reduced ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, N-Myc protein stabilization, neuroblastoma cell proliferation and survival. Clonogenic assays showed that knocking-down RP1X with doxycycline completely abolished colony formation capacity of neuroblastoma cells stably transfected with doxycycline-inducible RP1X shRNA. Importantly, treatment with doxycycline in mice xenografted with neuroblastoma cells stably transfected with doxycycline-inducible RP1X shRNA, led to tumour eradication. In human neuroblastoma tissues from 600 neuroblastoma patients, high levels of RP1X gene expression correlated with DEPD gene expression and poor patient prognosis. In conclusion, this study identifies the novel long noncoding RNA RP1X as an important regulator of N-Myc protein stability and neuroblastoma tumourigenesis.

#2665

SHOT-RNAs: A novel class of tRNA-derived functional RNAs expressed in hormone-dependent cancers.

Shozo Honda,1 Megumi Shigematsu,1 Phillipe Loher,1 Juan P. Palazzo,1 Issei Imoto,2 Isidore Rigoutsos,1 Yohei Kirino1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _The University of Tokushima, Tokushima, Japan_.

Sex hormones and their receptors play critical roles in the genesis and progression of breast and prostate cancers. We here present that sex hormone signaling pathways promote the expression of specific tRNA halves termed Sex HOrmone-dependent TRNA-derived RNAs (SHOT-RNAs).

Screenings using 94 cancer cell lines indicated that SHOT-RNAs are specifically and abundantly expressed in estrogen receptor-positive (ER+) breast cancer and androgen receptor-positive (AR+) prostate cancer. None of the ER− breast cancer, AR− prostate cancer, or other examined cancer cell lines from other tissues exhibited abundant expression of SHOT-RNAs. ER-dependent accumulation of SHOT-RNAs is not limited to a cell culture system, but it also occurs in luminal-type breast cancer patient tissues.

The expression specificity allowed us to hypothesize that sex hormones and their receptors are the crucial molecular factors for SHOT-RNA generation. Our hypothesis has been confirmed by the following three observations: (1) siRNA-mediated reduction of ER or AR decreased SHOT-RNA levels, (2) hormone-depletion from the culture medium decreased SHOT-RNA levels, and (3) addition of the sex hormone, estrogen or androgen, to the medium increased SHOT-RNA levels. These results indicate the clear dependency of SHOT-RNA expressions on sex hormones and their receptors.

SHOT-RNAs are produced from aminoacylated mature tRNAs by angiogenin (ANG)-mediated anticodon cleavage. Resultant 5′- and 3′-SHOT-RNAs, corresponding to 5′- and 3′-tRNA halves, bear 3′-terminal modifications, cyclic phosphate (cP) and amino acid, respectively. Thus, SHOT-RNAs would not be captured by standard RNA-seq methods because the modification inhibits adapter ligation steps. This could be the reason why SHOT-RNAs had not been discovered in spite of their abundance and expression specificity.

To identify a comprehensive repertoire of SHOT-RNAs, we developed a method named "cP-RNA-seq" in which 3′-termini of all RNAs, except for those containing a 3′-terminal cP, were cleaved by a periodate treatment, thereby selectively amplifying and sequencing only cP-containing RNAs. The application of cP-RNA-seq to breast cancer cells identified eight cytoplasmic tRNA species as major sources of 5′-SHOT-RNAs.

We further investigated the functional significance of SHOT-RNA expression. Intriguingly, siRNA-directed reduction of each one of the three different species of 5′-SHOT-RNA severely impaired cell proliferation, whereas 3′-SHOT-RNA reduction showed no impact. These results strongly suggest that at least 5′-SHOT-RNAs are expressed as functional RNA molecules promoting cell growth.

Our results have unveiled a novel tRNA-engaged pathway in tumorigenesis of hormone-dependent cancers and implicate SHOT-RNAs as potential candidates for biomarkers and therapeutic targets. We will discuss our working model and future challenges.

#2666

The miR-424/503 cluster is a breast cancer tumor suppressor with a role in chemoresistance.

David Llobet Navas,1 Francois Bertucci,2 Ruth Rodriguez-Barrueco,1 Jose Silva1. 1 _Mount Sinai School of Medicine, NYC, NY;_ 2 _Centre de Recherche en Cancérologie de Marseille, Marseille, France_.

Recently, we have identified the miR-424(322)/503 cluster as an important regulator of mammary epithelial homeostasis. The miR-424(322)/503 cluster was identified as one of the few miRNAs significantly upregulated during involution after pregnancy. By generating a knock-out mouse model, we found that regression of the mammary epithelium after pregnancy was compromised in the absence of miR-424(322)/503. Mechanistically, our studies unveiled that miR-424(322)/503 is induced by the canonical TGF-β-SMAD pathway, and that it orchestrates changes in the mammary epithelium by downregulating the expression of key components of signal transduction (IGF1R) and apoptosis (BCL2) Llobet et al. Genes&Development 2014).

Remarkably, our new studies have revealed that miR-424(322)/503-/- female mice develop hyperplasia and mammary tumors that are promoted by pregnancy. Thus, we investigated the status of this cluster in human breast cancers. For this we analyzed the METABRIC dataset. These studies revealed that the miR-424(322)503 cluster was heterozygously deleted in ∼16% of breast cancers and that its deletion correlates with lower expression levels of the mature miRNA forms. Importantly, miR-424(322)/503 is located on the X-chromosome and we have confirmed that is monoallelically expressed due to X-chromosome inactivation. Thus, the mutation of the active allele strongly impacts the expression of the miR-cluster. Deletions of the miR-424(322)/503 locus were more frequent in molecular subtypes with aggressive behavior (Luminal B, HER2+ and Basal) and both deletions and low expression of the cluster were associated with poor prognosis and reduced survival.

Some of the miR-424(322)/503 targets that we have previously validated (BCL2 and IGF1R) are involved in resistance to chemotherapy. Importantly, primary breast tumors presenting deletion of this miR-cluster locus presented significantly worse response to chemotherapy than the rest of tumors. Thus, we investigated in vivo, utilizing our knock-out mouse model, if loss of miR-424(322)/503 induces resistance to chemotherapeutic drugs. For this we crossed our miR-424(322)/503-/- animals with the HER2+ model FVB/N-Tg(MMTVneu)202Mul/J. Tumors emerging in HER2+/miR-424(322)/503-/- animals presented higher levels of BCL2 and hyperactivation of the IGF1R-AKT signaling compared to HER2+/miR-424(322)/503+/+ counterparts. Furthermore, these tumors were resistant to standard chemotherapy. Importantly, inhibition of BCL2 with ABT-199 and IGF1R with BMS-754807, two compounds currently in clinical trial, completely reverted chemotherapy resistance.

Overall, our data present evidence supporting a tumor suppressor role for miR-424(322)/503 cluster and its implication in resistance to chemotherapy.

#2667

Platelet microparticles infiltrating solid tumors transfer miRNAs and modulate tumor angiogenesis and growth.

James V. Michael,1 Jeremy G.T. Wurtzel,1 Guang Fen Mao,1 A. Koneti Rao,1 Nicholas E. Hoffman,1 Sudarsan Rajan,1 Muniswamy Madesh,1 Fabian Jana,2 Marvin Nieman,3 Jesse W. Rowley,4 Andrew S. Weyrich,4 Lawrence E. Goldfinger1. 1 _Temple University School of Medicine, Philadelphia, PA;_ 2 _University of Chile, Santiago, Chile;_ 3 _Case Western Reserve University, Cleveland, OH;_ 4 _University of Utah, Salt Lake City, UT_.

Platelet-derived microparticles are associated with enhancement of metastasis and poor cancer outcomes. Platelet microparticles can transfer platelet microRNAs (miRNAs) to vascular cells upon co-incubation in vitro, but the contributions of platelet microparticles and miRNAs to tumor progression are still poorly understood. Tumor vasculature is highly permeable, allowing the possibility of platelet microparticle-tumor cell interaction in primary solid tumors. Here we show that platelet microparticles infiltrate solid tumors in humans and mice, attach to tumor cells, and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo as well as in vitro. MiR-24 was a major species in this transfer. We identified RNA targets of platelet-derived miR-24 in lung carcinoma cells, which included mt-Nd2, a mitochondrial mRNA which lacks a 3'-UTR, and Snora75, a non-coding small nucleolar RNA. These RNAs were depleted in platelet microparticle-treated tumor cells, resulting in mitochondrial dysfunction and tumor cell growth inhibition, in a miR-24-dependent manner. Blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by platelet microparticle transfusion. Thus, platelet microparticles inhibit lung carcinoma cell proliferation and solid tumor growth via transfer of miR-24. However, global depletion of platelet miRNAs by platelet-specific deletion of Dicer1 inhibited tumor angiogenesis, platelet microparticle infiltration, and delayed tumor growth in mice. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via microparticles, regulate tumor cell gene expression, and modulate tumor progression, whereas platelet miRNAs promote tumor angiogenesis. These findings shed novel insight onto mechanisms of horizontal gene transfer and add multiple layers to the regulatory roles of miRNAs and platelet microparticles in tumor progression.

#2668

The exosomes derived from BM-hMSCs home to glioma and deliver synthetic microRNA mimics after systemic administration.

Shinji Yamashita,1 Tal Shahar,2 Anwar Hossain,3 Brittany Parker Kerrigan,3 Joy Gumin,3 Shoudong Li,3 Feng Gao,3 Kouji Yamasaki,3 Yuzaburo Shimizu,3 Frederick F. Lang3. 1 _Faculty of Medicine, University of Miyazaki, Miyazaki-city, Japan;_ 2 _Tel Aviv Medical Center, Israel;_ 3 _University of TX, MD Anderson Cancer Center, TX_.

Glioblastoma is the most malignant type of glioma (World Health Organization grade 4) and has a median survival time of only 14.6 months even after the standard treatment of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide administration. Although some novel and promising anti-glioma agents have been developed, therapeutic advancement has remained disappointing due to the lack of efficient delivery tools. Here we focused on exosomes derived from mesenchymal stem cells (MSCs) as carriers to deliver biological therapeutic agents for the treatment of gliomas. These exosomes are expected to cross the blood-brain barrier and selectively home to the glioma tissue, similar to the parental cells (MSCs). Additionally, MSCs could produce sufficient amounts of exosomes on a clinically applicable scale. Thus, MSC exosomes present a potentially ideal cell-free delivery vehicle for glioma therapy.

We hypothesized that exosomes derived from human bone marrow-derived MSCs (BM-hMSCs) can target gliomas and can be used as delivery vehicles following systemic administration. In this study, we employed microRNA (miR-128) as the therapeutic cargo, and exosomes were isolated from a cultured media of BM-hMSCs using ultracentrifugation. At first, exosomes labeled with fluorescent dye were incubated with glioma cells, and exosomes taken up by glioma cells were evaluated by confocal fluorescence microscopy and flow cytometry. Labeled exosomes were also systemically injected into a glioma xenograft model and the ability to home to glioma tissue was evaluated by an in vivo imaging system and histological study. The results showed that MSC exosomes can be taken up by glioma cells and can home to glioma tissues. Next, we cultured miR-128-enriched MSC exosomes with glioma cells. To check the function of miR-128 delivered to glioma cells by MSC exosomes, the down-regulation of the BMI-1 target gene of miR128 was evaluated using real-time polymerase chain reaction and western blot analyses. We also systemically injected these exosomes into glioma xenograft models and evaluated the function of miR-128 by analyzing the downregulation of BMI-1 at the tumor site and the animal survival time in vivo. We confirmed that miR-128 delivered by MSC exosomes could function at the target site both in vitro and in vivo.

These results indicate that MSC exosomes may be an ideal delivery vehicle for the treatment of gliomas, especially for microRNA replacement therapy.

### Mechanisms and Vulnerabilities of Metabolic Reprogramming

#2669

Glutaminase alternative splicing controls tumor growth and metabolism.

Zachary Stine,1 Zandra E. Walton,1 Lin Zhang,1 Teresa MW Fan,2 Andrew N. Lane,2 Chi V. Dang1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _University of Kentucky, Lexington, KY_.

Cancer cells reprogram their metabolism to meet their energetic and biosynthetic needs, with many cancer cells showing increased aerobic glycolysis and glutamine consumption. In addition to providing carbon to the TCA cycle to compensate for the diversion of glucose to lactate, glutamine is an important carbon and nitrogen donor for the synthesis of nucleotides, amino sugars and non-essential amino acids. Glutaminase (GLS) catalyzes the first step of glutamine metabolism, the hydrolysis of glutamine to glutamate, which can then be converted to alpha-ketoglutarate, glutathione or non-essential amino acids. GLS is alternatively spliced to make the KGA or more catalytically active GAC isoforms. Analysis of publicly available TCGA RNA-seq data from primary human tumors shows that GAC is the predominant splice form of GLS in many tumor types. However, the roles played by GAC versus KGA in tumor metabolism and progression are poorly understood. To facilitate the study of GLS alternative splicing, CRISPR/Cas9 mediated deletion of the GAC specific exon was used to convert predominantly GAC-expressing parental cell lines to solely KGA-expressing cell lines (ΔGAC). Despite showing decreased glutamine-derived glutamate and α-ketoglutarate, ΔGAC cells do not show impaired growth in nutrient-replete in vitro conditions compared to parental cells. 13C-isotopic labeling and metabolomics showed that ΔGAC cells have decreased levels of proline and alanine, both of which can be derived from glutamine-derived glutamate. This led us to ask if ΔGAC cells are dependent on exogenous non-essential amino acids. We found that depletion of non-essential amino acids slowed the growth of ΔGAC cells compared with parental cells, rescuable by exogenous proline and alanine. Importantly, the growth of lung (A549) and breast cancer (MDA-MB231) ΔGAC xenograft models was diminished compared with parental tumor xenografts in vivo, which is consistent with a role for GAC in nutrient challenged conditions. In summary, the GAC form of glutaminase fuels tumor growth partly through synthesis of non-essential amino acids, particularly under nutrient limiting conditions. As GLS inhibitors enter the clinic, a more thorough understanding of the role of GLS in tumor metabolism will aid in the development of treatment strategies to best make use of these drugs.

#2670

Non-metabolic function of asparagine in regulating global protein translation.

Ji Zhang, Natalya Pavlova, Richard White, Craig Thompson. _Mem. Sloan-Kettering Cancer Ctr., New York, NY_.

Cancer cells rely on extracellular glutamine for growth and survival. Previously, we showed that asparagine, a nonessential amino acid that can be synthesized de novo from glutamine, is both necessary and sufficient for glutamine-dependent cell survival. In addition, asparagine synthetase (ASNS) expression positively correlates with the progression of the disease in human glioma patients, suggesting a critical role of asparagine during tumor progression. Interestingly, mammalian cells cannot catabolize asparagine for use as either a nitrogen source or a bioenergetic substrate, potentially due to the loss of asparaginase activity during evolution. Ectopic expression of yeast or zebrafish asparaginase restores the capacity of mammalian cells to utilize asparagine as both bioenergetic and biosynthetic substrates to support cell growth. Furthermore, asparagine restores translation of fast-turn-over proteins under glutamine starvation. Together, the data suggests that asparagine has been evolved to be a metabolic reserve to regulate cellular adaptation to nonessential amino acid limitation in mammalian cells and is therefore a potential target for cancer therapy.

#2671

BRAF inhibitor resistance reprograms metabolic and survival pathways to sensitize melanoma cells to arginine deprivation.

Ying-Ying Li,1 Chunjing Wu,1 Sumedh Shah,2 Shu-Mei Chen,3 Medhi Wangpaichitr,1 Lynn Feun,4 Macus Kuo,5 Miguel Suarez,1 Niramol Savaraj1. 1 _University of Miami/VA Medical Center, Miami, FL;_ 2 _University of Miami, Miami, FL;_ 3 _Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan;_ 4 _University of Miami/Sylvester Comprehensive Cancer Center, Miami, FL;_ 5 _University of Texas M. D. Anderson Cancer Center, Houston, TX_.

The combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) has been FDA approved to treat melanomas harboring (V600E) mutation. Although most BRAF mutant melanomas are highly responsive to these treatments, they all relapse ultimately. In this study, we found a unique mechanism which can be used to treat BRAFi resistant patients. Five BRAFi resistant (BR) cells were established from known BRAF mutant cell lines A375, MEL-1220, A2058, UACC-62, and SK-MEL28 by long-term exposure to BRAFi. These BR cells are hypersensitive to ADI-PEG20, an enzyme which degrades arginine to citrulline. ADI-PEG20 treatment resulted in a 2-3-fold decrease in BR cell viability (MTT assay) and 10-30% increase in apoptosis (Annexin V/PI) in BR cells compared to their paternal cells. The mechanisms involved are as follows: All BR cells express very low levels or do not express argininosuccinate synthetase (ASS, a key enzyme to generate arginine from citrulline). Thus, these BR cells rely primarily on exogenous arginine. Furthermore, these BR cells also have attenuation of glucose uptake which makes them rely mainly on amino acids. Importantly, their ability to undergo autophagy upon nutritional stress is also impaired. The underlying mechanism for low levels of ASS is due to diminished c-Myc expression which is a positive regulator for ASS. Additionally, AMPK-α1, which possesses phosphorylation site at Thr172 governing autophagy and glucose uptake, was attenuated in BR cells. Overexpression of AMPK-α1 using plasmid transfection in A2058BR and MEL-1220BR cells rescued 8-28% ADI-PEG20-induced apoptosis and enhanced autophagosome formation (detected by LysoTacker). Conversely, knockdown of AMPK-α1 using siRNA significantly enhanced ADI-PEG20-reduced cell viability (compared to non-targeting siRNA) in A2058 and MEL-1220 cells through attenuation of autophagy and glucose uptake. This is evidenced by decreased GLUT1 and LC3-II expression, and low activity of 2-NBDG uptake, which also can be seen in most BR cells. The xenograft model also showed that ADI-PEG20 aborted tumor growth of A2058BR and A375BR cells but retarded growth of A2058 and A375 cells. Immunohistochemical staining further confirmed lower levels of ASS and AMPK-α1 in A2058BR xenograft and in tumor tissues from 10 BR patients relative to their parental counterparts (average H-scores of ASS and AMPK in parental tissues vs. BR tissues are 58.2 vs. 7.8, and 146 vs. 78.3, respectively, p <0.03). Importantly, these findings also apply to cell lines and tumor samples from patients who failed both BRAF and MEK inhibitor. In summary, our data suggest that attenuated AMPK-α1 mediated autophagy and glucose uptake and decreased c-Myc mediated ASS re-expression sensitize BR cells to arginine deprivation. Thus, ADI-PEG20 is a potential salvage therapy for BR patients (Supported by R01CA152197).

#2672

Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures.

Nicholas A. Graham,1 Aspram Minasyan,1 Heather R. Christofk,1 Ingo K. Mellinghoff,2 Thomas G. Graeber1. 1 _UCLA, Los Angeles, CA;_ 2 _Memorial-Sloan Kettering Cancer Center, New York, NY_.

Copy number alteration (CNA) profiling of human tumors has revealed recurrent patterns of DNA amplifications and deletions across diverse cancer types. These patterns are suggestive of conserved selection pressures during tumor evolution but cannot be fully explained by known oncogenes and tumor suppressor genes. Using a pan-cancer analysis of CNA data from patient tumors and experimental systems, here we show that principal component analysis-defined CNA signatures are predictive of glycolytic phenotypes, including FDG-avidity of patient tumors, and increased proliferation. The primary CNA signature is enriched for p53 mutations and is associated with glycolysis through coordinate amplification of glycolytic genes and other cancer-linked metabolic enzymes. A cross-species comparison of CNA identified 21 conserved altered DNA regions containing 13 enzymes in the glycolysis and pentose phosphate pathways in addition to known cancer driving genes. Furthermore, exogenous expression of hexokinase and enolase enzymes in an experimental immortalization system altered the copy number status of the corresponding endogenous loci, supporting the hypothesis that these metabolic genes act as drivers within the conserved CNA amplification regions. Taken together, these results demonstrate that metabolic stress acts as a selective pressure underlying the recurrent CNAs observed in human tumors, and further cast genomic instability as an enabling event in tumorigenesis and metabolic evolution.

#2673

Inhibition of fatty-acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer.

Roman Camarda,1 Alicia Y. Zhou,1 Rebecca A. Kohnz,2 Sanjeev Balakrishnan,1 Celine Mahieu,1 Brittany Anderton,1 Henok Eyob,1 Shingo Kajimura,1 Aaron Tward,1 Gregor Krings,1 Daniel K. Nomura,2 Andrei Goga1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University of California, Berkeley, Berkeley, CA_.

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC) compared to estrogen, progesterone and/or human epidermal growth factor 2 receptor-positive (RP) breast tumors. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains largely unexplored. We hypothesized that pharmacologic inhibition of MYC-driven metabolic pathways may serve as a therapeutic strategy for this clinically challenging subtype of breast cancer. Using a targeted metabolomics approach, we identified fatty-acid oxidation (FAO) intermediates as dramatically upregulated in a MYC-driven model of TNBC. A lipid metabolism gene signature was identified in patients with TNBC in the TCGA and multiple other clinical datasets, implicating FAO as a dysregulated pathway critical for TNBC metabolism. We find that MYC-overexpressing TNBC, including a transgenic model and patient-derived xenograft (PDX), display increased bioenergetic reliance upon FAO. Pharmacologic inhibition of FAO catastrophically decreases energy metabolism of MYC-overexpressing breast cancer, blocks growth of a MYC-driven transgenic TNBC model and MYC-overexpressing PDX. Our results demonstrate that inhibition of FAO is a novel therapeutic strategy against TNBCs that overexpress MYC.

#2674

High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring.

David P. Labbe',1 Giorgia Zadra,1 Meng Yang,2 Charles Y. Lin,1 Jaime M. Reyes,1 Stefano Cacciatore,3 Ericka M. Ebot,4 Maura B. Cotter,5 Amanda L. Creech,6 Jacob D. Jaffe,6 Philip W. Kantoff,7 James E. Bradner,1 Lorelei A. Mucci,4 Jorge E. Chavarro,2 Massimo Loda,1 Myles Brown1. 1 _Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 2 _Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA, Boston, MA;_ 3 _Imperial College London, London, United Kingdom;_ 4 _Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA;_ 5 _Department of Pathology and Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA;_ 6 _The Broad Institute of MIT and Harvard University, Cambridge, MA;_ 7 _Memorial Sloan Kettering Cancer Center, New York, MA_.

Introduction: The mechanisms underlying the association between high dietary fat intake and prostate cancer (PCa) are unknown. Using a MYC-driven PCa mouse model, we sought to identify metabolic and epigenomic alterations driven by high fat diet (HFD) that facilitate PCa progression. Additionally, we investigated whether these alterations were relevant to PCa progression and lethality in humans.

Material and Methods: Wild-type (WT) and transgenic Hi-MYC (MYC) mice were assigned either a HFD or control diet and were sacrificed at 12, 24, and 36 weeks of age for histologic and phenotypic characterization. Metabolic and epigenomic analyses were carried on the ventral prostates of 12-week old mice. Human PCa gene expression profiling data were obtained from 319 men with PCa and well-annotated post-diagnostic saturated fat intake (SFI) data from the Physicians' Health Study and Health Professionals Follow-up Study prospective cohorts.

Results: HFD does not affect the incidence of MYC-induced murine prostate intraepithelial neoplasia (mPIN) at 12 weeks, but increases mPIN proliferative index (Ki-67) at 24 weeks and tumor burden at 36 weeks.

MYC overexpression, as expected, induces a significant metabolic reprogramming and HFD further enhances this rewiring to provide additional anabolic metabolites to sustain the increased proliferation of MYC prostate while having little effect on the WT prostate. Moreover, MYC altered key metabolites of the methionine cycle in a direction suggestive of a global hypomethylation, again amplified by HFD. Targeted quantitative histone mass spectrometry revealed a robust MYC-driven signature, including a global demethylation of H3K27 and H4K20 marks, the latter enhanced by HFD. Moreover, ChIP-seq revealed an intricate crosstalk between MYC and the H4K20me1 demethylase PHF8, resulting in enhanced genomic instability in the context of HFD. Finally, RNA-seq and ATAC-seq analyses showed that HFD rewires MYC-driven PCa through the alteration of genes implicated in chromatin function and remodeling. In humans, SFI was associated with enrichment in genes associated with increased MYC transcriptional activity in the prostate. Furthermore, this MYC transcriptional signature was associated with PCa lethality overall (OR = 3.21; 95% CI = 1.47, 7.35 comparing extreme score tertiles), and the association was stronger among men with high post-diagnostic SFI (OR = 1.32; 95%CI=1.11, 1.66) than those with low SFI (OR= 1.05; 95%CI=0.98, 1.12).

Conclusions: HFD supports a coordinated metabolomic and epigenomic rewiring to increase epigenomic plasticity and MYC transcriptional activity prior to the appearance of phenotypic alterations in the prostate. Importantly, HFD requires MYC-mediated transformation to trigger its deleterious effects. In humans, SFI also enhances MYC transcriptional activity, which is associated with increased PCa lethality.

#2675

Metabolic addiction in IDH1 mutant cancers.

Andrew S. Chi,1 Kensuke Tateishi,2 Wakimoto Hiroaki,2 Tracy T. Batchelor,2 Anthony J. Iafrate,2 Daniel P. Cahill2. 1 _NYU School of Medicine, New York, NY;_ 2 _Massachusetts General Hospital, Boston, MA_.

Introduction: Recent efforts have revealed IDH1 mutation not only results in marked accumulation of 2-hydroxyglutarate (2-HG), it generates genomic chromatin alterations, altered HIF activity and reprogrammed metabolic flux. We characterized the metabolic perturbations caused by heterozygous mutant IDH1 by comparing endogenous IDH1 mutant glioma cells treated with direct inhibitors of mutant IDH1 (IDH1i) to control cells without treatment, with the overall aim of identifying novel metabolic therapeutic targets.

Experimental Procedures: We tested the effect of IDH1i on the in vitro and in vivo phenotype of a panel of patient-derived endogenous IDH1 mutant solid cancer cells (including gliomas, melanoma, and sarcomas). We then assessed the effect of IDH1i on the metabolome of endogenous IDH1 mutant glioma cells using liquid chromatography-mass spectrometry. We further investigated specifically perturbed metabolic pathways using lentiviral knockdown and overexpression systems.

Results: We found that IDH1i exposure and the resulting 2-HG depletion had a mixed effect in 12 endogenous mutant IDH1 lines studied both in vitro or in vivo, including an orthotopic glioma xenograft model. For 10 of 12 lines, IDH1i treatment had no demonstrable effect on proliferation or survival, while 2 lines displayed growth inhibition after IDH1i treatment. However, no significant changes were detected in the genomic DNA methylation profiles or the global levels of histone tail marks including H3K4me3, H3K9me2, H3K9me3 and H3K27me3 in IDH1 mutant glioma lines, even after inhibition of mutant IDH1 for 12 months in vitro. Broader metabolite profiling revealed that inhibition of mutant IDH1 significantly altered levels of the canonical metabolite NAD+. We tested the effect of NAD+ depletion using NAD+ biosynthesis inhibitors, and found that endogenously mutant IDH1 cells were highly dependent on NAD+ for survival, whereas proliferation and survival of IDH1/2 wild-type cancer cells were unaffected by NAD+ depletion. Using an inducible mutant IDH1 expression system we discovered that mutant IDH1 reduced intracellular NAD+ levels, rendering mutant IDH1 cells susceptible to further NAD+ depletion. Lack of sufficient NAD+ induced metabolic crisis with reduced ATP levels in IDH1 mutant cells, triggering the intracellular energy sensor AMPK and autophagy. In vivo, NAD+ depletion significantly extended the survival of mice bearing IDH1 mutant xenograft tumors, including intracerebral gliomas.

Conclusions: Although most IDH1 mutant cell lines derived from solid cancers were resistant to direct IDH1 inhibition, a subset of IDH1 mutant cancers were found to be sensitive. Mutant IDH1 reprograms metabolism and renders cancer cells highly dependent on NAD+ for survival. This metabolic addiction presents a potential opportunity for metabolically targeted therapeutic development.

## TUMOR BIOLOGY:

### Mechanism and Dynamics of Cancer Metastasis

#2676

**Epithelial-mesenchymal transition does not require transcriptional repression of the epithelial program** in vivo **.**

Nicole M. Aiello, David Balli, Robert Norgard, Jinyang Li, Amine Sahmoud, Ben Z. Stanger. _University of Pennsylvania, Philadelphia, PA_.

Epithelial-mesenchymal transition (EMT), the process by which an epithelial cell loses its adhesion to neighboring cells and acquires a motile, fibroblast-like phenotype, is critical for many aspects of embryogenesis and has been strongly implicated in tumor cell invasion and metastasis. EMT is thought to be primarily regulated at the transcriptional level through repression of epithelial genes by transcription factors such as Snail, Zeb and Twist. To date, the mechanisms driving EMT have been parsed out almost exclusively in vitro under pre-defined conditions (ie, using TGFβ as an EMT inducer), but it remains to be seen whether the machinery that regulates in vitro EMT is physiologically relevant. Using a lineage labeled mouse model of pancreatic ductal adenocarcinoma (PDAC), we isolated tumor cells that have spontaneously undergone EMT to interrogate the molecular mechanisms driving this process in vivo. By comparing the transcriptomes of epithelial (E-cadherin+) and mesenchymal (E-cadherin-) tumor cells, we found that for a majority of PDAC tumors, EMT does not require transcriptional repression of the epithelial program. These cells were fully capable of upregulating a mesenchymal program, as evidenced by the increased expression of collagens, SPARC, MMP2, PDPN, and PRRX1; however, despite the loss of E-cadherin protein, we detected no change in transcript levels of E-cadherin or other epithelial genes after EMT. Our results suggest that in vivo, cells that undergo EMT retain epithelial gene transcription, which may confer the ability to rapidly oscillate between epithelial and mesenchymal states.

#2677

Unfolded protein response is required for EMT-driven metastasis by inducing CREB3L1.

Yuxiong Feng, Dexter X. Jin, Piyush B. Gupta. _MIT Whitehead Inst. for Biomed. Research, Cambridge, MA_.

Cancer initiates and progresses in the presence of a diversity of stresses ranging from nutrient deficiency, low oxygen supply to pH fluctuations. A set of stress response pathways, termed unfolded protein response (UPR), was evolved to maintain homeostasis of the endoplasmic reticulum (ER) from disruption by the above stressors. While it is broadly acknowledged that the UPR is involved in tumorigenesis, as tumor cells can employ the UPR as a critical survival strategy to grow and resist chemotherapy, the role of UPR in metastasis remains largely elusive. Epithelial-to-mesenchymal transition (EMT) is a cell transdifferentiation program frequently hijacked by cancer cells to migrate and invade. Here we showed cancer cells that have undergone an EMT employ the PERK-ATF4 branch of the UPR to drive metastasis, since loss of ATF4 abrogates the cell invasion upon an EMT. By conducting gene expression profiling, we found that the PERK-ATF4 pathway mediates the expression of a subset of the essential pro-invasion EMT-signature genes. In particular, CREB3L1, an ER-associated transcription factor (TF), is highly induced upon EMT in an ATF4-dependent manner. Knockdown of CREB3L1 effectively inhibits the EMT-driven invasion, while overexpression of CREB3L1 not only promotes invasion but also rescues the decrease of invasion caused by loss of ATF4. Mechanistically, CREB3L1 facilitates invasion through an ECM-FAK cascade. In breast cancer patients, the expression of CREB3L1 is significantly upregulated in breast cancer metastases compared to primary lesions, and predicts a poor prognosis. Most importantly, unlike other EMT-TFs, CREB3L1 could be inhibited by known chemicals, since the activation of CREB3L1 is mediated by the S1P- and S2P- dependent proteolysis. In an orthotopic breast cancer model, we found that chemical inhibition of CREB3L1 drastically suppresses lung metastasis. As a summary, we discovered that an ER stress-associated transcription factor CREB3L1 is required for cancer metastasis, and the susceptibility of CREB3L1 to chemical inhibition makes it a valuable target for drug development.

#2678

Notch1 regulates epithelial-mesenchymal transition and tumor-initiating capability in esophageal squamous-cell carcinoma.

Koji Tanaka,1 Kelly A. Whelan,1 Naryan L. Rustgi,1 Prasanna M. Chandramouleeswaran,1 Seiji Naganuma,2 Shingo Kagawa,3 Mitsuteru Natsuizaka,4 Yoshiaki Kita,5 Shoji Natsugoe,5 Que Jianwen,6 Devraj Basu,7 Andres J. Klein-Szanto,8 Adam Bass,9 J. Alan Diehl,10 Hiroshi Nakagawa1. 1 _Gastroenterology Division, Department of Medicine, University of Pennsylvania, Philadelphia, PA;_ 2 _Department of Pathology, Kochi University Medical School, Japan;_ 3 _Department of General Surgery, Graduate School of Medicine, Chiba University, Japan;_ 4 _Department of Gastroenterology and Hepatology, Hokkaido University, Japan;_ 5 _Department of Digestive Surgery, and Breast and Thyroid Surgery, Kagoshima University School of Medicine, Japan;_ 6 _Department of Biomedical Genetics, University of Rochester Medical Center, NY;_ 7 _Departments of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania, Philadelphia, PA;_ 8 _Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA;_ 9 _Division of Cellular and Molecular Oncology, Dana-Farber Cancer Institute, Boston, MA;_ 10 _Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC_.

Introduction: Notch signaling may act as a tumor suppressor during the development of squamous cell carcinomas (SCCs); yet, Notch activation promotes tumor growth in a subset of SCC cells. The roles of Notch in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remain elusive.

Methods: Notch1 activation and epithelial-mesenchymal transition (EMT) were determined in an esophageal epithelium-targeted cell-lineage traceable (K5CreERT2-Rosa26tdTomatolsl) mouse model of ESCC induced by 4-nitroquinoline 1-oxide (4NQO), which was coupled with flow cytometry and single cell-derived ESCC organoid formation assays. Tumor-initiating capability was assessed in xenograft transplantation experiments with TE11 human ESCC cells carrying either Crispr-Cas9-deleted Notch1 loci or tetracycline-inducible expression of the activated form of Notch1 (ICN1). Surgically resected primary tumors and adjacent normal mucosa from ESCC patients (n=152) were analyzed by immunohistochemistry for Notch1 activation and the EMT marker ZEB1.

Results: 4NQO-treated mice developed tdTomato-positive primary and metastatic ESCC tumors with EpCAM-negative ESCC cells displaying traits compatible with EMT. Notch1 activation and ZEB1 expression were co-localized in ESCC cells at the stromal interface, a finding that was further recapitulated in ESCC tumor organoids. Interestingly, Cre-mediated ex vivo Notch1 deletion in a single cell suspension from Notch1loxP/loxP mouse-derived ESCC tumors decreased organoid formation rate. TE11 xenograft tumors appeared to contain a unique ESCC cell fraction containing EpCAM-negative cells, where ICN1 conferred tumorigenicity upon serial transplantation. This population displayed upregulation of Notch1 target genes and the ESCC-lineage survival factor/oncogene SOX2. Moreover, Notch1 deletion in TE11 not only limited tumor formation, but also decreased EMT in culture. A subset of ESCC patients (49/140, 33%) showed ICN1-positive ESCC cells with concurrent ZEB1 expression at the tumor invasive front. The presence of such ESCC cells was associated with poor 5-year survival (P=0.001), tumor depth (P=0.01), lymphatic and venous invasion (P=0.003) and distant metastasis (P=0.002). Moreover, such ICN1-expressing cells were increased in ESCC patients (7/12, 58%) who received pre-surgical neoadjuvant therapy.

Conclusions: Cell-lineage tracing experiments validate for the first time Notch1 activation and EMT in the 4NQO-induced mouse model of ESCC. Analyses of single cell-derived ESCC tumor organoids, xenograft and primary ESCC tumors reveal that Notch1 activation may be associated with tumor initiating capability, EMT and chemotherapy resistance, implicating Notch1 activation in the pathogenesis of ESCC and potentially other SCCs.

#2679

Induction of β-globin protects circulating tumor cells from oxidative stress during dissemination.

Yu Zheng,1 David T. Miyamoto,1 Ben S. Wittner,1 James P. Sullivan,2 Nicola Aceto,3 Nicole Vincent Jordan,1 Min Yu,4 Nezihi Murat Karabacak,5 Valentine Comaills,1 Robert Morris,1 Rushil Desai,1 Niyati Desai,1 Erin Emmons,1 Richard J. Lee,1 Chin-Lee Wu,1 Lecia V. Sequist,1 Wilhelm Haas,1 David T. Ting,1 Mehmet Toner,5 Sridhar Ramaswamy,1 Shyamala Maheswaran,1 Daniel A. Haber1. 1 _MGH Cancer Center, Charlestown, MA;_ 2 _Google Life Sciences, Mountain View, CA;_ 3 _University of Basel, Basel, Switzerland;_ 4 _University of Southern California, Los Angeles, CA;_ 5 _MGH Center for Bioengineering in Medicine, Charlestown, MA_.

Identification of candidate metastasis genes has traditionally resulted from comparison of primary and metastatic tumor specimens. However, Circulating Tumor Cells (CTCs) contain metastatic precursors that are present transiently in the bloodstream and their analysis may reveal additional pathways that are induced for a limited time, as they invade and survive within the vasculature. By comparing transcriptome profiles of CTCs from breast, prostate and lung cancers with their primary tumor of origin, we observed consistent and significant induction of the β-globin gene (ΗΒΒ) within CTCs. In contrast, expression of α-globin, its binding partner within hematopoietic cells, is not coordinately upregulated. The tumor-specific origin of ΗΒΒ was further confirmed by analysis of human xenografts-derived CTCs in mice, where human-specific HBB polymorphisms are readily distinguishable in the murine background. In cultured cancer cells, we further demonstrate that induction of ΗΒΒ is triggered by exposure to reactive oxygen species (ROS), and we identify KLF-family transcriptional regulators that mediate this effect. To investigate the function of aberrant β-globin expression within CTCs, we performed shRNA-mediated knockdown of HBB in breast CTC-derived cultures. Cells with depleted HBB expression displayed elevated intracellular ROS levels, increased sensitivity to hydrogen peroxide, and impaired metastatic potential in mouse models. Taken together, these observations suggest that β-globin, a component of functional hemoglobin in red blood cells, is deregulated in disseminated tumor cells, where it may function as a ROS scavenger, reducing oxidative stress and facilitating cancer metastasis.

#2680

Development and application of a novel model of human lung-to-brain metastasis: identification of TWIST2 and SPOCK1 as unique regulators of brain metastases.

Sheila K. Singh,1 Mohini Singh,1 Chitra Venugopal,1 Nicole McFarlane,1 David Bakhshinyan,1 Manvir Dhillon,1 Sujeivan Mahendram,1 Kevin Brown,2 Amy Tong,2 Kathrin Durrer,2 Tomas Tokar,3 Robin Hallett,4 John Hassell,5 Igor Jurisica,3 Jason Moffat2. 1 _McMaster Stem Cell and Cancer Research Institute, Hamilton, Ontario, Canada;_ 2 _Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada;_ 3 _BM Life Sciences Discovery Centre, Toronto Medical Discovery Centre, Toronto, Ontario, Canada;_ 4 _Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada;_ 5 _McMaster Department of Biochemistry and Biomedical Sciences, Hamilton, Ontario, Canada_.

Brain Metastases (BM) are the most common type of cerebral tumor in adult, occurring at a rate ten times greater than that of primary brain cancers. The inherent properties of a primary tumor cell capable of initiating a BM resemble that of a cancer stem cell (CSC). Previous work conducted in our lab identified a CSC population within lung-derived BM. We hypothesize that a subgroup of CSC-like cells, termed brain metastasis-initiating cells (BMICs), are responsible for the initiation of BM and are identifiable by an exclusive subset of genes that regulate self-renewal and metastasis. Despite the prevalence and lethality of BM, there is no clinically relevant model that fully reflects metastasis in patients. The selection of an appropriate metastatic in vivo model is crucial for the identification of genes that regulate BM formation, prognostic and/or predictive markers, and evaluation of anti-metastatic therapeutics. We recently generated a novel human-mouse xenotransplantation model of BM that allows for interrogation of each phase of the metastatic process from lung to brain, through injection of human patient-derived GFP-expressing BMICs into immunocompromised mice via three routes: 1) intracranial (IC), 2) intrathoracic (IT) and 3) intravascular/intracardiac (IV). GFP+ BMICs were harvested from the lungs and/or brains from each injection route, and RNA was submitted for microarray analysis to identify a unique metastatic and tissue-specific gene signature from BMICs isolated from IT injections. We performed RNA interference screens in vitro and in vivo on BMIC lines against 150 genes implicated in BM formation in order to identify genes involved in self-renewal, tumor initiation and metastasis. We validated two top hits, TWIST2 and SPOCK1, using our novel BM mouse model, and determined that their knockdown functionally blocked BM formation. Future work will examine the expression of these genes in primary lung FFPE samples to determine if they are predictive biomarkers of lung-to-brain metastasis in prospective cohorts of newly diagnosed lung cancer patients, and to determine their potential as therapeutic targets.

#2681

Intravital microscopy of prostate cancer lesions in bone: kinetics of osteolysis and therapy response.

Eleonora Dondossola,1 Stephanie Alexander,1 Boris Holzapfel,2 Christopher J. Logothetis,1 DIetmar Hutmacher,2 Peter Friedl1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Queensland University, Brisbane, Australia_.

Bone metastases are the initial site of progression and account for many of the complications experienced by men with metastatic prostate cancer (PCa), including therapy resistance. Besides cell intrinsic mechanisms, the interaction between PCa cells and the bone microenvironment critically contributes to lesion expansion and drug sensitivity. We here developed a mouse model amenable to intravital multiphoton microscopy (iMPM) to longitudinally study PCa-stromal cell interactions and therapy response in a partially humanized neobone, established in the dermis of the mouse. Tissue engineered bone constructs, TEBCs, were generated by functionalizing polymeric polycaprolactone scaffolds with human mesenchymal stem cells (hMSCs) differentiated to osteoblasts followed by cell-derived calcification of the inter-scaffold space. 30 days after implantation of functionalized scaffolds under the skin of the mouse, bone and bone marrow maturation resulted in a 30mm3 cavity surrounded by optically transparent cortical bone of 50-60 μm thickness, as monitored by microCT and iMPM. Intra-bone tumor growth and osteolysis caused by human fluorescent PCa cells (PC3) were three-dimensionally reconstructed by multi-parameter detection through a body window, including collagen and bone matrix (SHG), calcified bone (fluorescent bisphosphonates), osteoclasts (cathepsin K), bone surface (THG), blood vessels and stromal phagocytes (fluorescent dextran), and PC3 cells (nuclear H2B/eGFP, cytoplasmatic DsRed2). As a proof of concept, the efficacy to halt bone remodeling during bisphosphonate therapy was detected, revealing the composition and kinetics of the bone-tumor interface, including oscillating bone loss followed by neo-apposition. Thus, combining engineered neobone with an optical window is suited to detail the 3D organization and dynamics of cancer lesions in bone and provides mechanistic insight and efficacy predictions for therapy response.

#2682

Sympathetic activation alters the bone vasculature: implication for osteotropic breast cancer metastasis.

Patrick L. Mulcrone,1 J. Preston Campbell,1 Ana Lia Anbinder,2 Julie A. Sterling,1 Florent Elefteriou3. 1 _Vanderbilt University, Nashville, TN;_ 2 _Universidade Estadual Paulista, Sao Paulo, Brazil;_ 3 _Baylor College of Medicine, Houston, TX_.

Emotional stresses chronically activate the Hypothalamic-Pituitary-Adrenal axis and the sympathetic nervous system (SNS), and have been associated with the progression and recurrence of breast cancer, as well as with reduced survival of breast cancer patients. We observed that isoproterenol (ISO), a β1/2 adrenergic receptor (βAR) agonist used to mimic sympathetic activation, induced the expression of Vegfa in mouse bone in vivo and in osteoblast cultures in vitro. Because VEGF is pro-angiogenic, and tumor cells depend on the vasculature to form distant metastases, we hypothesized that SNS chronic activation may increase bone marrow vascular density via an effect on β2AR-expressing osteoblasts, thus promoting the possibility of metastatic breast cancer establishment in the skeleton. To address this question, we measured the formation of CD31+ vessels in femurs in vivo by histomorphometric analyses, following daily ISO treatment of adult mice for 6 weeks. Vessel number was significantly increased by ISO treatment in WT mouse femurs, and this effect was blunted in mice lacking the β2AR. Further supporting our hypothesis, we found that the conditioned media (CM) from MC3T3 osteoblasts treated with ISO augmented blood vessel formation in an ex vivo metatarsal assay as compared to the CM of PBS-treated osteoblasts. Lastly, the CM of ISO-treated MC3T3 osteoblasts promoted primary bone endothelial cell tube formation, and inhibition of Vegf:Vegfr2 interaction with the antibody mcr84 reduced this formation. These results suggest that the effect of chronic stress on metastatic breast cancer cell establishment in the skeleton may be mediated in part due to changes in the bone microenvironment, specifically an increase in osteoblastic VEGF that changes host bone marrow vascular density.

### Mechanisms of Tumorigenesis in Mouse Models of Human Cancer

#2683

The landscape of chromosomal aberrations in mouse models of breast cancer reveals driver-specific routes to tumor development.

Uri Ben-David,1 Gavin Ha,1 Prasidda Khadka,1 Xin Jin,1 Lude Franke,2 Todd R. Golub1. 1 _The Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _University of Groningen, Groningen, Netherlands_.

Aneuploidy and large copy number alterations (CNAs) are a hallmark of human cancer. Although genetically engineered mouse models (GEMMs) are commonly used to model human cancer, their chromosomal landscape remains largely unexplored because large-scale CNA data have not been generated. Here we used gene expression profiles to infer CNAs in 3,108 samples from 45 mouse models, providing the first comprehensive catalog of chromosomal aberrations in cancer GEMMs. Mining this expansive resource, we found that most chromosomal aberrations accumulated late during breast tumorigenesis, and we observed marked differences in CNA prevalence between mouse mammary tumors initiated with distinct drivers. Some of these aberrations were recurrent and unique to specific GEMMs, suggesting distinct driver-dependent routes to tumor development. Synteny-based comparison of mouse and human tumors narrowed critical regions in CNAs, thereby identifying candidate driver genes that were not obvious from the analysis of either dataset alone. Specifically, we experimentally validated that loss of Stratifin (SFN) promotes HER2-induced tumorigenesis in human cells. These results demonstrate the power of GEMM CNA analysis in the understanding of human cancer pathogenesis.

#2684

Modeling a prototypical type I ovarian carcinoma in the mouse based on oviductal versus ovarian epithelial transformation: which is superior.

Rong Wu,1 Yali Zhai,1 Rork Kuick,2 Paloma Garcia,1 Anum Naseem,1 Eric R. Fearon,1 kathleen R. Cho1. 1 _Univ. of Michigan Medical School, Ann Arbor, MI;_ 2 _Univ. of Michigan School of Public Health, Ann Arbor, MI_.

Type I ovarian carcinomas are relatively indolent tumors that are nonetheless deadly if detected too late. Endometrioid carcinoma (EC) is a prototypical Type I ovarian cancer subtype. Existing mouse models of the disease based on transformation of the ovarian surface epithelium (OSE) take advantage of known EC genetic signatures but fail to capture fully the clinical disease progression and tumor phenotype. We previously described a mouse ovarian EC model in which the Apc and Pten tumor suppressor genes are conditionally deleted in the OSE via bursal injection of adenovirus expressing Cre recombinase (AdCre). We now report a new mouse model of EC in which the Ovgp1 (oviductal glycoprotein) promoter regulates expression of tamoxifen-inducible Cre recombinase specifically in the mouse oviductal epithelium (equivalent to human fallopian tube epithelium [FTE]), and not in the OSE or endometrium. Ovgp1-iCreERT2;Apcfl/fl;Ptenfl/fl mice treated with tamoxifen (TAM) develop oviductal tumors over months rather than weeks. The oviductal EC morphology closely resembles that of human ovarian ECs, with overt gland formation throughout, whereas ovarian bursal injection of AdCre in Ovgp1-iCreERT2;Apcfl/fl;Ptenfl/fl mice results in OSE-derived ECs that are poorly differentiated and include a prominent spindle cell component, consistent with our prior work. OSE-derived tumors progress rapidly, with none of the mice surviving beyond 14 weeks post-AdCre injection. Littermates with FTE-derived tumors survive much longer (>30 weeks post TAM). The slow progression and late (typically >16 weeks post TAM) metastasis to ovary and omentum associated with the FTE-derived tumors are more akin to the relatively indolent behavior characteristic of Type I ovarian carcinoma in humans. To further evaluate which of the two types of mouse tumors more closely resemble human ovarian tumors, we profiled gene expression in 6 FTE- and 5 OSE-derived Apc/Pten-deficient tumors, and compared the groups to each other and to data from 99 human ovarian carcinomas we analyzed previously (including 37 endometrioid, 41 serous, and 13 clear cell, and 8 mucinous carcinomas). Based on gene expression, the FTE-derived tumors are more correlated to every class of human ovarian tumors (p < 2 X10-6 in all 4 comparisons), with highest correlation to human ECs (particularly those with mutant CTNNB1) and mucinous carcinomas. This new model of EC, based on transformation of Mullerian epithelium rather than the OSE, will likely prove more suitable for translational applications, including pre-clinical testing of novel therapeutics for ovarian EC.

#2685

**Ultraviolet light cooperates with** NrasQ61R **mutations to drive malignant melanoma.**

Rebecca C. Hennessey, Andrea M. Holderbaum, James E. Gillahan, Conor Delaney, Xing Tang, Raleigh Kladney, Christin E. Burd. _The Ohio State University, Columbus, OH_.

Of the four molecular subtypes of melanoma (BRAF, NRAS, NF1, and triple WT), NRAS mutant tumors are notoriously more aggressive and challenging to treat. The frequent occurrence of NRAS mutations

in benign, congenital nevi suggests that these genetic alterations are early events in melanoma genesis. Since NRAS mutant nevi can remain indolent for years, or even a lifetime, secondary genetic events must be required to drive melanoma initiation. Ultraviolet (UV) light, a major risk factor for melanoma, is known to directly damage DNA and alter the skin microenvironment. Therefore, we hypothesized that the cell-extrinsic and

intrinsic events triggered by UV exposure would cooperate with NrasQ61R mutations to drive melanoma formation in vivo. To test this hypothesis we employed a genetically engineered mouse model (i.e. TpN61R) that combines CRE-inducible expression of NrasQ61R with the loss of p16INK4a in melanocytes. Following neonatal CRE induction, we exposed TpN61R mice to a single, nonerythemic dose of UVB radiation and monitored them for the development of melanoma. Exposure to UVB radiation dramatically reduced the melanoma-free survival of TpN61R mice by 80% and increased tumor multiplicity (average 1.2 to 3.4 tumors/mouse); however, it had no impact on tumor growth rates, overall skin proliferation, immune infiltration, or vascularity. To test the respective roles of NrasQ61R and p16INK4a loss in UV-induced melanoma genesis, we generated mice with either melanocytic NrasQ61R expression or p16INK4a loss. Mice with p16INK4a loss alone did not develop tumors in the presence or absence of UV. By contrast, in UV treated TN61R mice, NrasQ61R was sufficient to initiate melanoma formation, albeit with a 66% longer latency than UV exposed TpN61R animals (9.14wks vs. 5.5wks). To determine if NrasQ61R mutations had to be present at the time of UV exposure to drive early melanoma formation, TpN61R mice were exposed to UV prior to CRE activation. Even when NrasQ61R expression was induced three days after UV exposure, melanoma formation was rapidly accelerated. Therefore, NRAS mutations do not need to be present at time of UV exposure to promote early melanoma formation. Genomic analyses comparing spontaneous and UV-induced TpN61R melanomas failed to uncover common secondary mutations that explain the exquisite cooperativity between NrasQ61R and UV light in driving melanoma formation. For this reason, we sought to identify UV-induced, cell-extrinsic factors that might facilitate the initiation of NrasQ61R mutant melanomas via cytokine profiling. Through these analyses we identified UV-induced pro-inflammatory cytokines that could be therapeutically targeted to limit the initiation of NRAS-mutant melanomas. Together, this work explains why UV exposure is such a significant risk factor for melanoma and provides original mechanistic insight into how this deadly disease might be prevented.

#2686

The genomic landscape of Kras mutant genetically engineered mouse models of human cancers.

Wei-Jen Chung, Anneleen Daemen, Jason Long, Jason Cheng, Chris Tran, Zora Modrusan, Oded Foreman, Melissa Junttila. _Genentech, Inc., South San Francisco, CA_.

Genetically engineered mouse models (GEMM) of cancer are invaluable tools for oncology research. They recapitulate critical aspects of tumor etiology and allow for unique interrogation of tumor evolution and maintenance. In addition, these models are poised for assessing therapeutic drug efficacy and resistance mechanisms. Although these models are widely used, the mutational landscape and transcriptional profile of GEMMs have yet to be thoroughly characterized.

In this study we sought to investigate the precise genomic landscape of three well-validated GEMMs representing two types of Kras mutant cancers, non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC). Using next generation sequencing techniques, we profiled an adenovirally-induced Kras mutant NSCLC model (KrasLSL.G12D/+;p53frt/frt) and two Kras mutant models of spontaneous PDAC: KPP (KrasLSL.G12D/+;p16/p19fl/fl;Pdx1.Cre) and KPR (KrasLSL.G12D/+;p16/p19fl/+;p53LSL.R270H/+;Pdx1.Cre). All three GEMMs incorporate genomic aberrations that are documented to occur in the human patient population. Additionally these models have been reported to approximate histological and phenotypic aspects of their human correlate.

We carried out whole exome sequencing for each model (NSCLC n=115; PDAC n=73 and n=40, respectively) to comprehensively characterize the genomes. Interestingly, both intra and inter-tumoral genomic heterogeneity was evident both within and amongst Kras mutant tumor models. The models most significantly diverge with respect to copy number alterations. The NSCLC model demonstrated whole chromosome 6 gain and focal deletion on chromosome 9, in addition to other genomic alterations. However, the KPP PDAC model tumors exhibited significant focal amplification of mutant Kras in 75% (55/73) of cases. In combination with RNA-seq data, we can show that these genomic alterations correlate with divergent transcriptional signatures despite the shared Kras mutation, especially in MAPK pathway signatures. This data suggests tumors within these models have unique signaling pathway dependencies.

Finally, we compared the tumors resulting in the murine models to that of Kras altered human tumors from TCGA at the genomic level. The genomic landscapes of Kras mutant GEMMs and corresponding Kras mutant patients share significant similarities. We identified mutations in oncogenes and tumor suppressors, in addition to alterations in gene copy number. Taken together, the results provide a clearer understanding of the genomic landscape of these GEMMs and their relationship with human patients. More importantly, these analyses will allow us to better interpret results from previous and future experiments using these Kras mutant GEMMs of cancer.

#2687

Rapid in vivo testing of tumor suppressors in ILC by CRISPR-Cas9 mediated somatic gene editing of the mammary gland.

Stefano Annunziato, Sjors Kas, Micha Nethe, Hatice Yucel, Jessica del Bravo, Colin Pritchard, Rahmen Bin Ali, Bas van Gerwen, Bjorn Siteur, Anne Paulien Drenth, Eva Schut-Kregel, Sjoerd Klarenbeek, Ivo Huijbers, Martine van Miltenburg, Jos Jonkers. _NKI-AVL, Amsterdam, Netherlands_.

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype, accounting for 5% to 15% of breast tumors.The majority of ILCs are characterized by the complete loss of the cell adhesion protein E-cadherin encoded by the CDH1 gene. However, WAPcre;Cdh1F/F mice with mammary gland-specific E-cadherin loss do not develop ILC, unless coupled with the additional disruption of a tumor suppressor gene, like Pten or Trp53. Compound mutant mice develop lesions that closely resemble the human disease in terms of histology and invasivity. However, genome-wide sequencing projects and forward genetic screens identified a number of additional putative ILC-initiating loci. Hence, there is now an urgent need for validation and characterization of these candidate cancer genes. We sought to determine if it was possible to deploy CRISPR/Cas9 technology to somatically inactivate candidate tumor suppressor genes in mammary gland tissue of adult mice. For this purpose we performed in Cdh1F/F mice intraductal injections of high-titer lentiviral vectors carrying different combinations of Cre, Cas9 and sgRNAs targeting several candidate genes, including Pten, Tgfbr2, Myh9, Nf1 and Fbxw7. We found that Cas9-bearing vectors elicited strong immune responses against transduced cells, which resulted in small ILC lesions surrounded by immune cells. However, when Cas9 was expressed endogenously in the mammary tissue from a knock-in allele, intraductal injection of lentiviral vectors encoding the sgRNA moiety of the CRISPR system was sufficient to induce penetrant and multifocal ILC formation in WAPcre;Cdh1F/F;Cas9 female mice. Sequencing revealed specific mutations clustered in the CRISPR targeted gene, and positive selection for loss-of-function indels. Indeed, immunohistochemistry and immunofluorescence analysis confirmed that tumors were largely deficient for E-cadherin and the targeted gene. In sum, we have successfully established a new platform for in vivo CRISPR-Cas9 mediated somatic gene editing in the mouse mammary gland. This rapid and versatile platform can be used to identify novel tumor drivers (e.g.by employing forward genetic screens) and validate candidate cancer genes in ILC and other breast cancer types.

#2688

A forward genetics screen of murine brain tumors identifies novel candidate genes involved in gliomagenesis.

Matko Cancer,1 Holger Weishaupt,1 Gabriela Rosen,1 Ignas Bunikis,1 Yiwen Jiang,2 Smitha Sreedharan,1 Sara Bolin,1 Ulf Gyllensten,1 Oren J. Becher,3 Lene Uhrbom,1 Adam Ameur,1 Fredrik J. Swartling1. 1 _Uppsala University, Uppsala, Sweden;_ 2 _Karolinska Institutet, Stockholm, Sweden;_ 3 _Duke University, Durham, NC_.

Glioma is the most frequent malignant brain tumor in adults. Platelet-derived growth factor (PDGF) signaling is commonly activated in glioma. We have used a retrovirus-driven PDGFB-induced murine glioma model that causes tumors that closely resemble human gliomas of various grades. Knowing that retroviruses have a capacity to induce insertional mutagenesis, we have employed whole genome sequencing to identify potential genes that, together with PDGFB, drive glioma development.

Gliomas were induced by RCAS virus injection into the brains of mice expressing the RCAS retroviral receptor from specific promoters. Genomic DNA from tumor cell lines was probed for retroviral tags and sequenced to identify genomic targets of the retrovirus. A streamlined analysis pipeline was developed for retrovirus integration detection and mapping to the reference mouse genome. Integration sites were analyzed and a common integration site (CIS) label was assigned to a gene, given that it was either tagged by a retrovirus more than once within a discovery set or found within the Retroviral Tagged Cancer Gene Database (RTCGD).

In a small discovery subset of 15 murine gliomas, we have identified 40 CIS, of which 37 were validated by Sanger sequencing. When compared with previously identified CIS in RTCGD, 5.5% of them were shared with our older screen, where we overexpressed PDGFB from another retrovirus in order to induce glioma. Less CIS genes were shared with other published tumor models induced by viruses driven by other cancer genes/viruses.

The majority of genes identified in our screen were tagged twice. However, Nfic, Cuecd1, Thra, Foxj1 and Nrxn1 were tagged three times, Ppfibp1 and Rhbg four times, and Mir29a/29b-1 seven times. As compared to control tumor lines, two top candidate genes, Mir29a and Ppfibp1, demonstrated significantly increased expression in tumor lines in were they were respectively tagged. Mir29a is often found downregulated in human tumors including gliomas, still high levels of Mir29a are sometimes found in certain aggressive cancers and in metastases.

Interestingly, we found that specific PDGFR inhibition negatively regulates Mir29a, indicating a possible role for PDGF signaling in Mir29a regulation. Ppfibp1 has not been extensively studied in cancer. However, Ppfibp1 seems to have a subgroup-specific expression in human glioblastoma, making it an interesting candidate for further analysis.

Here we present a new screening method that can be employed to identify genes involved in PDGFB-driven gliomagenesis. So far, we have identified 37 candidate genes by whole genome sequencing. Two of the most frequently tagged candidates, Mir29a and Ppfibp1 were upregulated as a consequence of retroviral mutagenesis. Their precise role in driving glioma formation in collaboration with PDGF is currently explored.

#2689

First MACC1 transgenic mice demonstrate tumor progression via the newly discovered MACC1/Nanog/Oct4 axis.

Clara Lemos,1 Markus S. Hardt,1 Manisha Juneja,1 Cynthia Voss,1 Susann Förster,2 Boris Jerchow,2 Wolfram Haider,3 Hendrik Bläker,4 Ulrike S. Stein1. 1 _ECRC, Charité and Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany;_ 2 _Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany;_ 3 _Institute for Animal Pathology, Berlin, Germany;_ 4 _Institute for Pathology, Charité, Berlin, Germany_.

We have previously identified the gene Metastasis-Associated in Colon Cancer 1 (MACC1). MACC1 acts a strong prognostic biomarker for colorectal cancer metastasis and patient survival. Meanwhile, MACC1 has been established as a biomarker for tumor progression, metastasis and patient survival for a broad variety of solid cancer types.

Here, we report for the first time the generation of transgenic mouse models for MACC1. We generated mice with transgenic overexpression of MACC1 in the intestine driven by the villin promoter (vil-MACC1) and crossed them with ApcMin mice (vil-MACC1/ApcMin). vil-MACC1/ApcMin mice significantly increased the total number of tumors (P = 0.0056). This was particularly apparent in large sized tumors (≥ 3 mm diameter; P = 0.0024). A detailed histopathological analysis of these lesions demonstrated that the tumors from the vil-MACC1/ApcMin mice had a more invasive phenotype together with an accelerated adenoma-carcinoma-sequence. Consequently, vil-MACC1/ApcMin mice showed a significantly reduced survival time as compared to ApcMin mice (P = 0.03). Molecular analysis revealed an increased Wnt and pluripotency signaling in the tumors of vil-MACC1/ApcMin mice. Specifically, we observed a prominent upregulation of the pluripotency markers Oct4 and Nanog in these tumors as compared to ApcMin controls. Finally, we could also validate that Oct4 and Nanog are regulated by MACC1 in vitro, and strongly correlate with MACC1 levels in a cohort of 60 tumors of colorectal cancer patients (r = 0.7005 and r = 0.6808, respectively; P > 0.0001 and P > 0.0002, respectively).

Taken together, we provide first evidence that MACC1-induced tumor progression acts, at least in part, via the newly discovered MACC1/Nanog/Oct4 axis. The evidence is accumulated from experiments in cell culture, MACC1 transgenic animals as well as colorectal cancer patients. This is the first time that the function of MACC1 is linked to pathways crucial in cancer stem cells. Based on the hypothesis that cancer stem cells are the main engine behind tumor progression and metastasis, these findings might have important therapeutic implications. Specifically, MACC1's relevance as a biomarker might further be augmented by focusing on its expression in cancer stem cells. Furthermore, the connection of MACC1 with Oct4 and Nanog might offer novel options for therapeutic intervention to restrict tumor progression.

### New Molecular Advances in Pediatric Cancer

#2690

A novel antibody-drug conjugate directed to the ALK receptor tyrosine kinase demonstrates efficacy in models of neuroblastoma.

Renata Sano,1 Kateryna Krytska,1 Colleen Larmour,1 Gwenda F. Ligon,2 Jay S. Lillquist,2 Ulisse Cucchi,3 Paolo Orsini,3 Simona Rizzi,3 Gerald McMahon,2 Diego Alvarado,2 Yael P. Mosse1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _Kolltan Pharmaceuticals, New Haven, CT;_ 3 _Nerviano Medical Sciences, Nerviano, Italy_.

Activated ALK by mutation or amplification is a validated therapeutic target in a subset of patients with high-risk neuroblastoma, and tyrosine kinase inhibitors are being developed to abrogate ALK signaling (Bresler et al, 2014). Native ALK is expressed on the surface of the majority of neuroblastoma tumors, but not on normal tissue, giving it properties of a tumor antigen. We hypothesized that ALK-targeted antibodies may be useful in neuroblastoma as single-agent immunotherapy or in combination with small-molecule ALK inhibitors - especially where mutations reduce kinase inhibitor sensitivity. In this work, an anti-ALK monoclonal antibody (anti-ALK2) was conjugated to a cytotoxic agent (ALK2-ADC) and its anti-tumor activity was investigated in a panel of extensively characterized human neuroblastoma cell lines (n=10) harboring wild type and mutant ALK. Cell surface ALK was quantified using flow cytometry and revealed differential antigen expression. Using in vitro growth inhibition assays, there was evidence for a dose-dependent cytotoxicity to neuroblastoma cells at subnanomolar concentrations that correlated with ALK expression and was independent of underlying mutation status. In order to evaluate the therapeutic potential of ALK2-ADC in vivo, mice bearing Felix-patient-derived xenograft (PDX) tumors, containing the third most common ALK mutation (R1245C), were used to demonstrate that ALK2-ADC led to a significant reduction in tumor growth compared to unconjugated antibody and a control ADC (p<0.0001). Although ALK2-ADC binds potently to mouse ALK as well as human ALK, doses of up to 10 mg/kg in the rodent model appeared to be well tolerated with no overt toxicity noted. Additional in vivo studies are ongoing to assess the antitumor potential of the ADC in a broader range of ALK-expressing neuroblastoma models. Thus, targeting human ALK-expressing with an ALK antibody-drug conjugate demonstrated early signs of favorable efficacy and tolerability supporting future development of this approach as a potential novel therapeutic for patients with neuroblastoma.

#2691

Genome-wide association study identifies two susceptibility loci that modify radiation-related risk for breast cancer after childhood cancer: A report from the Childhood Cancer Survivor Study and St. Jude Lifetime Cohort.

Lindsay M. Morton,1 Joshua N. Sampson,1 Gregory T. Armstrong,2 Ting-Huei Chen,1 Melissa Hudson,3 Eric Karlins,4 Casey L. Dagnall,4 Shenchao Li,4 Carmen L. Wilson,2 Kumar Srivastava,5 Wei Liu,5 Guolian Kang,5 Kevin Oeffinger,6 Tara O. Henderson,7 Chaya S. Moskowitz,6 Todd M. Gibson,2 Diana M. Merino,1 Jeannette R. Wong,1 Sue Hammond,8 Joseph P. Neglia,9 Lucie M. Turcotte,9 Jeremy Miller,10 Laura Bowen,10 William A. Wheeler,10 Wendy M. Leisenring,11 John A. Whitton,11 Laurie Burdette,4 Belynda D. Hicks,4 Mitchell J. Machiela,1 Aurelie Vogt,4 Zhaoming Wang,4 Meredith Yeager,4 Geoffrey Neale,12 Matthew Lear,13 Louise C. Strong,14 Yutaka Yasui,2 Marilyn Stovall,15 Rita E. Weathers,15 Susan A. Smith,15 Rebecca Howell,15 Stella M. Davies,16 Gretchen A. Radloff,16 Amy Berrington de González,1 Peter D. Inskip,1 Preetha Rajaraman,1 Joseph F. Fraumeni,1 Smita Bhatia,17 Stephen J. Chanock,1 Margaret A. Tucker,1 Leslie L. Robison2. 1 _Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, MD;_ 2 _Department of Epidemiology and Cancer Control, St. Jude Children's Research Hospital, Memphis, TN;_ 3 _Division of Cancer Survivorship, Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN;_ 4 _Cancer Genomics Research Laboratory, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research & Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, MD; _5 _Department of Biostatistics, St. Jude Children's Research Hospital, Memphis, TN;_ 6 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 7 _Department of Pediatrics, Section of Hematology, Oncology and Stem Cell Transplantation, University of Chicago, Chicago, IL;_ 8 _Ohio State University, Columbus, OH;_ 9 _Department of Pediatrics, University of Minnesota, Minneapolis, MN;_ 10 _Information Management Services, Inc., Calverton, MD;_ 11 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 12 _Hartwell Center for Bioinformatics and Biotechnology, Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN;_ 13 _Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN;_ 14 _Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 15 _Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 16 _Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH;_ 17 _School of Medicine, University of Alabama at Birmingham, Birmingham, AL_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2016 Official Press Program. It will be posted online following its presentation.

#2692

ETV6-RUNX1 targets a developmentally restricted embryonic human B-myeloid progenitor.

Simon E. Richardson,1 Charlotta Böiers,2 Alya Zriwil,2 Virginia Turati,1 John Brown,1 Dapeng Wang,1 Javier Herrero,1 Stefan Karlsson,2 Andrew J. H. Smith,3 Sten Erik Jacobsen,4 Tariq Enver1. 1 _University College London Cancer Institute, London, United Kingdom;_ 2 _BMC A12, Gene Therapy, Lund University, Lund, Sweden;_ 3 _MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom;_ 4 _Haematopoietic Stem Cell Laboratory and MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom_.

Childhood acute lymphoblastic leukemia (cALL) is distinct from that in adults with higher incidence, better prognosis and a distinct mutational spectrum. One hypothesis for this difference is that cALL arises in transient cells unique to early human development. We explored this in ETV6-RUNX1 cALL where evidence from twins and neonatal heel prick testing has shown that this mutation arises in utero and is an initiating event. We characterized B cell development in first trimester human fetal liver (FL) to identify compartments vulnerable to ETV6-RUNX1. Using CD19 as a marker of B lineage commitment we found the first CD19+ B cells emerge in the human FL at Carnegie Stage (CS) 17 and were distinct from adult in that the majority expressed surface IL7 receptor. We used IL7R to identify a CD19-IL7R+ B progenitor compartment that produced B cells in vitro, possessed DJH recombination, but also had monocytic potential. Single cell analysis of CS20 IL7R+ progenitors revealed co-expression of lymphoid and myeloid programmes, whereas at CS17 they were strongly myeloid primed indicating that IL7R+ progenitors acquire lymphoid potential in this developmental window. Some co-expression of lymphoid and myeloid programmes also persisted in CS20 FL B cells. We tested whether FL B cell development could be modeled using human pluripotent stem cells (hPSCs). In vitro B cell differentiation of hPSCs produced IL7R expressing pro and preB cells as well as an IL7R+ progenitor that switched from myeloid to B-myeloid priming during culture. At the global transcriptional level the hPSC lymphoid hierarchy mapped closely with FL, with both separating from adult suggesting that hPSCs provide a developmentally relevant model of early FL B lymphopoiesis. We next used CRIPSR-directed homologous recombination to engineer the expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B cell differentiation at the level of the IL7R+ progenitor. ETV6-RUNX1 expressing B cells co-expressed an abnormal B-myeloid gene expression signature akin to that seen in the IL7R+ progenitor. Both the transcriptional and differentiation phenotypes were dependent on ETV6-RUNX1 as demonstrated by their reversion upon cre-mediated excision of the knock-in cassette. Our data support a model where expression of ETV6-RUNX1 inhibits lymphoid specification in an early FL IL7R+ lymphomyeloid progenitor, arresting B lineage differentiation and resulting in the production of myeloid-primed B cells. This may explain the relatively high levels of myeloid antigen expression lineage promiscuity seen in cALL. ETV6-RUNX1 hPSCs will afford the systematic evaluation of the contribution of additional mutations seen in cALL and may offer a tractable platform for drug screening. In conclusion we propose that a novel IL7R+ lymphomyeloid progenitor in the human FL is a candidate target cell for in utero pre-leukemic initiation in cALL.

#2693

Relapse in BCP-ALL predicted by activated signaling in pro-B cell subsets.

Zinaida Good,1 Jolanda Sarno,2 Astraea Jager,1 Nikolay Samusik,1 Wendy Fantl,1 Nima Aghaeepour,1 Robert Tibshirani,1 Sean C. Bendall,1 Giuseppe Gaipa,2 Andrea Biondi,2 Garry P. Nolan,1 Kara L. Davis1. 1 _Stanford University, Stanford, CA;_ 2 _Pediatric Clinic University of Milano Bicocca, Monza, Italy_.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common type of childhood cancer and is characterized by the malignant expansion of B-lymphocyte progenitors in the bone marrow (BM). Current therapy improves the relapse-free survival in children to over 80%. However, the ~20% of patients who relapse have a poor prognosis and there are no reliable tests that predict relapse using diagnostic samples. We reasoned that aligning BCP-ALL cells according to a formalized context of normal B-lymphocyte development would reveal hidden cell states associated with relapse, and potentially expose targets to augment therapy for patients at risk. Until recently, our ability to pinpoint the identities of B-cell progenitors had been hindered by the vast cellular diversity within the BM and by the scarcity of the primary BM samples. We applied a single-cell proteomics platform termed mass cytometry by time-of-flight (CyTOF). In CyTOF, elemental mass reporter tagged antibodies probe proteins defining cellular identity and signaling within those cells. CyTOF simultaneously quantifies > 40 proteins per cell in millions of individual cells. We defined a cell-state signature for 15 developmental populations of B lymphocytes within the normal human BM. Using this signature we assigned each leukemia cell from 52 primary diagnostic samples to its closest match in B lymphopoiesis using a classifier based on Mahalanobis distance. When applied to BM samples from 4 healthy donors our classifier correctly assigned cells to the true developmental population (accuracy = 0.92, F-measure = 0.92). Using this classifier it was determined that each BCP-ALL sample contains a mix of developmental populations - with 97% of samples enriched in populations that span the pre-pro-B to pre-B transition. We identified 20 predictors (using a machine learning approach) in diagnostic samples that perfectly separate patients who will relapse from those who will not (lasso; predictive AUC = 0.83). This is superior to the NCI risk that is currently employed at clinical diagnosis. These predictors are informative and suggest that high basal activation of IL-7 signaling nodes (pSTAT5, pAKT) in pre-pro-B to pro-BII cells and poor response following pre-B-cell receptor engagement in pre-BI cells portend relapse. As such, these pathways might eventually be targeted via drug repurposing to improve outcomes and to guide therapy in the high-risk childhood BCP-ALL patients identified with our predictor signature. Such an approach to cancer cell developmental classification could be generally applicable across various investigations on understanding and preventing relapse.

#2694

Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis.

Daria G. Valerio,1 Haiming Xu,1 Chun-Wei Chen,1 Takayuki Hoshii,1 Meghan Eisold,1 Christopher Delaney,1 Monica Cusan,1 Aniruddha J. Deshpande,2 Chun-Hao Huang,1 Amaia Lujambio,3 George Zheng,4 Tej K. Pandita,5 Scott W. Lowe,1 Scott A. Armstrong1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA;_ 3 _Mount Sinai School of Medicine, New York, NY;_ 4 _University of Georgia, Athens, GA;_ 5 _Houston Methodist, Houston, TX_.

Chromosomal rearrangements of the Mixed-Lineage Leukemia (MLL) gene are found in 5-10% of all patients with acute leukemia and associated with a poor prognosis. MLL-rearrangements are more frequently present in pediatric and infant patients where AF9 is one of the most common fusion partners.

In order to identify novel druggable targets in MLL-AF9 rearranged leukemia, we conducted a chromatin regulator focused RNAi screen in murine MLL-AF9 leukemia cells and found hairpins targeting (K)Lysine Acetyltransferase 8 (Kat8, also known as Mof) and the previously identified target Bromodomain Containing 4 (Brd4), to be the most potent suppressors of cell growth. MOF is a histone 4 lysine 16 (H4K16) acetyltransferase and member of the MYST family of histone acetyltransferases (HATs). MOF has been shown to be crucial for murine embryogenesis and is a cell-type dependent regulator of chromatin state and various cellular processes such as T-cell differentiation, DNA damage response and cell cycle progression.

Using a conditional murine Mof knockout system, we studied the role of MOF in MLL-AF9 leukemogenesis in detail. In vitro inactivation of Mof in MLL-AF9 transformed mouse hematopoietic stem and progenitor cells led to impaired colony-forming capacity. The specificity of this phenotype was shown by expression of exogenous full-length Mof, which fully rescued transformed cells from the dramatic phenotype. Inactivation of Mof in vivo, lead to reduced tumor burden and prolonged survival of mice bearing MLL-AF9 leukemia cells. RNA sequencing data comparing MLL-AF9 cells with homozygous Mof loss to a wild type control, showed a significant enrichment of genes within the apoptosis (NES 1.98, FDR-q <0.0001) and p53 (NES 2.23, FDR-q <0.0001) pathway. These gene expression data suggest that the importance of MOF in MLL-AF9 leukemogenesis may be through interaction with p53, inducing proliferation and suppressing apoptosis. In addition, we found a reduction of actively cycling cells and a loss of global H4K16 acetylation (H4K16ac) upon Mof knockout. In line with this finding of H4K16ac loss, rescue experiments with HAT domain mutated MOF illustrated that the HAT activity of MOF is indispensable for MLL-AF9 leukemia maintenance. Finally, experiments with the selective MYST protein HAT inhibitor MG149, showed a strong anti-proliferative effect on murine, as well as human MLL-AF9 leukemia cell lines, and MG149 inhibition induced global H4K16ac loss in these cells.

These results indicate that MOF HAT activity is required for MLL-AF9 leukemia maintenance. Our data further suggest that MOF HAT activity may be a good target for new small molecule inhibitor development for the treatment of patients with MLL-AF9 rearranged leukemia.

#2695

Targeted therapy of leukemia and Ewing's sarcoma by human antibody-targeted nanoparticles.

Hyunggyoo Kang,1 Jon Nagy,2 Timothy Trivhe1. 1 _USC/Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _Nano Valent Phamaceuticals, Inc., Boneman, MT_.

Current cancer therapies have shown limited success due to the poor selectivity. The recent chemotherapy could be far more effective if higher doses could be specifically delivered to the tumor and not to normal tissues. Therefore a reliable tumor specific therapy is urgently needed to treat cancer patients. Targeted nanoparticle can attenuate the off-target toxicity and enhance the efficacy of existing therapies, which would be highly beneficial to the cancer patients. In spite of a lot of effort still the optimal formulation of nanoparticle for the actual translational applications was not established yet. Liposomes, unilamellar vesicles composed of natural and/or synthetic lipids have been an intensively studied system. By refining the early formulations and after extensive testing, recently we could invent a new type of lipid bilayer nanoparticle delivery system, HPLN (Hybrid Polymerized Liposomal Nanoparticles) and demonstrate that this new type of polymeric nanoparticle overcomes many, if not most, of the deficiencies noted and has tremendous potential as an effective delivery vehicle for treating many types of cancer. In our study it was shown that the targeted HPLN with human antibodies (antiCD99 or antiCD19) can inhibit the growth of Ewing's sarcoma and leukemia (ALL) in a murine model. The antitumor effects of of HPLN were diminished pretty much by the removal of antibody or drug (Doxorubicin), which proves efficacy of the targeted HPLNs is specifically dependent upon the targeting antibody and drug. In addition, no abnormalities in liver and kidney function tests, complete blood counts or pathology of major organs are observed from tail-vein administrations. These data provide strong evidence for the safety and efficacy of this targeted HPLN delivery system of anticancer drugs to Ewing's sarcoma and leukemia (ALL). In conclusion, our result showed a potential of targeted HPLN as a selective delivery vehicle of cytotoxic drugs to cancers. Hopefully this technology will be applied to treat many other different types of tumor cells in the future.

#2696

Four new brain tumor entities emerge from molecular classification of CNS-PNETs.

Dominik Sturm,1 Brent Orr,2 Umut H. Toprak,1 Volker Hovestadt,1 David T.W. Jones,1 David Capper,1 Peter Lichter,1 Andrey Korshunov,1 Stefan M. Pfister,1 David W. Ellison,2 Marcel Kool1. 1 _German Cancer Research Center DKFZ, Heidelberg, Germany;_ 2 _St Jude Children's Research Hospital, Memphis, TN_.

CNS-primitive neuroectodermal tumors (CNS PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Histological diagnosis is complicated by morphological heterogeneity and divergent differentiation. Recent studies suggest the existence of molecular subgroups of CNS-PNETs sharing biological characteristics with other CNS tumors. To investigate this we have analyzed 323 fresh-frozen or paraffin-embedded institutionally diagnosed CNS-PNETs using DNA methylation and expression arrays. Data were compared to 211 reference cases of other pediatric and adult brain tumors representing more than 20 well-known entities.

DNA methylation and gene expression patterns showed that a significant proportion of CNS PNETs display molecular profiles indistinguishable from those of various other well defined CNS tumor entities. Hallmark genetic alterations for each of these well-defined entities, such as amplification of 19q13.42, mutations in IDH1 or H3F3A, mutations/deletions of the SMARCB1 locus, or RELA fusions, were frequently detected among the reclassified cases. From the remaining fraction of CNS PNETs we have identified four new CNS tumor entities, each associated with a unique recurrent genetic alteration and distinct histopathological and clinical features. Based on these findings we designated these four new entities as "CNS neuroblastoma with FOXR2 activation (CNS NB FOXR2)", "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT CIC)", "CNS high grade neuroepithelial tumor with MN1 alteration (CNS HGNET MN1)", and "CNS high grade neuroepithelial tumor with BCOR alteration (CNS HGNET BCOR)".

Our findings reinforce the importance of incorporating molecular information into the next revision of the WHO classification of CNS tumors, and warrant a replacement of the term "CNS PNET" with biologically specific designations. Our study provides an innovative framework for improving diagnostic accuracy and prognostication in malignant CNS tumors. The approach is amenable to retrospective analyses of patients treated with current regimens and will facilitate the design of more meaningful clinical trials for patients with malignant brain tumors.

### Advocates Poster Session 1

#ADV01

Check your neck- The importance of education for thyroid cancer.

Brittany Avin. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Thyca: Thyroid Cancer Survivors' Association, Inc. (ThyCa) is a non-profit 501(c)(3) organization of thyroid cancer survivors, family members, and health care professionals. ThyCa is dedicated to supporting research for a future free of thyroid cancer, education for patients and loved ones to better understand the disease, and communication between patients and health care professionals to better comprehend each other's needs. ThyCa emphasizes education for early detection, as well as raising thyroid cancer research funding and awarding over 47 scientists research grants worldwide. With current information about thyroid cancer available in seven languages to people at any stage of testing, treatment, or lifelong monitoring for thyroid cancer, we reach annually over 65,000. Thyroid cancer awareness is of high importance as it is the most rapidly increasing cancer in the country. Three-fourths of those diagnosed are women, and the disease is fifth in leading sites of new cancer cases estimated in 2015. As we see an increase in diagnoses, including 9/11 first responders with an incidence of thyroid cancer 239% higher than a comparable population, education and awareness is critical. The organization holds hundreds of events for this goal. Offering support through online support groups for over 30,000, over 100 local support groups in 5 countries, and 1-on-1 support toll-free in multiple languages. Free handbooks and educational brochures are downloaded online, mailed to individuals, and given in bulk to physicians and hospitals. We spread awareness through free online webinars with experts, thyroid cancer awareness month collaborations, and the annual International Thyroid Cancer Survivors' Conference to reach patients. Through all these avenues and more ThyCa is highly committed to support and education in all realms concerning thyroid cancer. With a simple "Check Your Neck" we bring awareness to an important cause, and strive towards a better future for all patients with thyroid cancer.

#ADV02

Working to double pancreatic cancer survival by 2020.

Maurice Bason. _Pancreatic Cancer Action Network, Manhattan Beach, CA_.

The Pancreatic Cancer Action Network is the national organization creating hope in a comprehensive way through research, patient support, community outreach and advocacy for a cure. The organization is leading the way to increase survival for people diagnosed with this devastating disease through a bold initiative — The Vision of Progress: Double Pancreatic Cancer Survival by 2020. Together, we can Wage Hope in the fight against pancreatic cancer by intensifying our efforts to heighten awareness, raise funds for comprehensive private research, and advocate for dedicated federal research to advance early diagnostics and better treatments and increase chances of survival.

#ADV03

Empowering breast cancer patients/survivors through education and support.

Marina C. George. _North New Jersey Young Breast Cancer Support Group, Hackensack, NJ_.

The North NJ Young Breast Cancer Support Group is designed for breast cancer patients and survivors diagnosed in their 40's. Our mission is to empower and educate breast cancer patients and survivors through information and support. Our group is comprised of women who are newly diagnosed, in active treatment and post-treatment. This is a place where they can come and provide mutual support to one another. The group is facilitated by an oncology social worker and a breast patient nurse navigator. We learn about available resources, ask questions, make new friends and be assured that we are not alone. Over the years we have developed our own educational resource for our members on various topics such as informations and tips on mastectomy & recover tips, tattoo artistry, chemotherapy care kits, guidance on talking to daughters about breast health, information on tattoo artistry, and applying for life and long-term disability insurance after cancer diagnosis. We also have a closed Facebook group that is comprised of breast cancer patients/survivors only. We have about 160 constituencies in the group. Our group respects the fact that our members approach our diagnoses, surgery options, treatment choices, fears, feelings, heath, nutrition and wellness, and communication styles differently. Our group is a safe place to ask questions and share feeling and concerns that we may be not comfortable sharing elsewhere. We use caution when sharing our experiences or advice, both generally and specifically regarding doctors, nurses, hospitals, treatment centers medications, treatments, surgery choices (lumpectomy vs mastectomy), health, nutritions, etc. We are mindful of the powerful impact our words can have on the newly diagnosed and those who have had different experiences. In short, the NNJ YBCSG strives to maintain a warm and welcoming environment where all thoughts and feelings are valued and respected.

#ADV04

Precision medicine for all: educating promotores to educate the Latino community.

Ysabel Duron. _Latinas Contra Cancer, San Jose, CA_.

Latinas Contra Cancer (LCC), a non profit based in San Jose, CA, serves an underserved, uninsured Spanish speaking and immigrant population by addressing the cancer continuum from prevention to detection and screening, patient support, survivorship to end of life. We are currently working on a 3yr research project with Georgetown University on a PCORI grant focused on Breast Cancer Patients and their Caregivers. When President Obama announced in early 2015 his Precision Medicine Initiative, LCC was concerned about Latino access to this cutting edge medicine. So LCC will spotlight the debate on Precision Medince at its 5th Biennial National Latino Cancer Summit, focusing on among other things, access, cost, and how PM might exacerbate cancer disparities. In order to prepare Latino communities about the potential impact of precision medicine on the delivery of health care to all, we will bring together all stakeholders to Summit in July 2016 to discuss comparative effectiveness of different information techniques (audio, visual, written). LCC will be testing one approach. We've developed a genetic training module for promotores(community health workers) who are considered some of the most trusted sources of support and information in the Latino community. LCC's poster titled 'Precision Medicine for All: Educating Promotores to educate the Latino Community' will demonstrate 1.results of a recent survey of promotores in an effort to understand their attitudes toward and understanding of genetics, testing and clinical trials, and shows their overwhelming interest in educating the community 2. Describe development of the genetic training module for promotores and share some of the content 3. and the methods of implementation including an introductive training session at the Summit.

#ADV05

Online collaboration and support in the lung cancer social media community.

Janet Freeman-Daily. _Bonnie J. Addario Lung Cancer Foundation, Federal Way, CA_.

The Lung Cancer Social Media hashtag #LCSM fosters social media collaboration across all stakeholders in the lung cancer community. It focuses on using social media to educate, end the stigma, connect the community, enable advocacy, and facilitate successful treatments for lung cancer, the leading cause of cancer deaths worldwide. Since the #LCSM hashtag was registered with Symplur.com in June 2013, it has been used by over 20,000 participants, tweeted over 185,000 times, and generated over 535 million impressions. Participating Twitter accounts include lung cancer patients, caregivers, family members, advocates, healthcare providers, hospitals, researchers, nonprofits, government agencies, journalists, pharma and the biotech industry. They use the hashtag to track new treatments, find patient resources, broadcast new research findings, develop advocacy activities, and connect with other lung cancer community members they might not find otherwise. Biweekly chats on a wide variety of topics enable #LCSM stakeholders to share ideas, support research initiatives, coordinate advocacy activities, and address issues within the lung cancer community. Participants span the globe, yet are able to connect and collaborate across patient-clinician-researcher silos to share their common goal of defeating lung cancer. The Founders of #LCSM Chat have spearheaded several initiatives, such as curating lung cancer social media resources, a petition to gather signatures to support CMS coverage of low-dose CT screening, and participating in a PCORI grant titled "Empowering Patients and Their Families to Improve Outcomes That Are Most Important to Them after Surgery and Other Therapies for Lung Cancer."

#ADV06

Navigating prostate cancer by three time cancer survivor Gerald Green.

Gerald Green. _Men's Health Committee / UCSF Helen Diller Family Comphensive Cancer Center, Oakland, CA_.

The Prostate Health support Group for African American Men's mission is to provide a safe and supportive place where men can come together for dialogue and education. We conduct outreach through faith based communities and the Alameda County Health Department, where we host educational symposiums coupled with Prostate-specific Antigen (PSA) testing. These activities are coordinated through the Community Advisory Board of the Men's Health Committee/ UCSF Helen Diller Family Comprehensive Cancer Center.Navigating Prostate Cancer by Three Time Cancer Survivor Gerald Green, will inform and help men make decisions. However, before deciding cancer therapy options, one must understand some basic arithmetic about prostate cancer, starting with the PSA test. A blood test that produces a single data point. It takes two data points to make a line. The collection of those points produces a historical graph of prostate activity. Note: Prostate activity not prostate cancer activity.

On the poster, I share my personal PSA and Gleason scores to illustrate how that data guided my path to a biopsy and then treatment. A personal journey I share with men in our support group, where hopefully our stories help families navigate choices.

Choices complicated by dueling medical options as expressed by Oleksandr Kryvenko, MD, an assistant professor of pathology and urology at Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine in his article Time to Rethink Active Surveillance in African American Men With Prostate Cancer, as compared to the article Watchful Waiting Becoming More Common For Prostate Cancer Patients, by Matthew R. Cooperberg, MD, MPH, Associate Professor of Urology; Epidemiology & Biostatistics Helen Diller Family Chair in Urology at the University of California, San Francisco. A perplexing dichotomy, unfortunately Black men's mortality rates are twice those of White men.

#ADV07

Van Andel Research Institute / Stand Up To Cancer Epigenetics dream team.

Patrick Gavin. _Grand Rapids Clinical Onclogy Program, Marne, MI_.

The Cancer Research Consortium of West Michigan is a National Cancer Institute Community Oncology Research Program (NCORP). CRCWM is headquartered in Grand Rapids Michigan and serves patients throughout West Michigan. CRCWM is dedicated to assuring every person in the West Michigan region served by CRCWM has the opportunity for education about cancer and participation in nationwide research studies in cancer prevention and treatment. I serve as the Chair of The Patient Advisory Committee and a member of the Executive Operations Board. The Van Andel Institute (VAI) is an independent biomedical research and science education organization in Grand Rapids, Michigan, The Van Andel Research Institute (VARI) is the research division of VAI. VARI is dedicated to determining the epigenetic, genetic, molecular and cellular origins of cancer, Parkinson's and other diseases and translating those findings into effective therapies. The VARI is one of the core member institutions of CRCWM. The VARI is located in Grand Rapids. Through my activities at CRCWM, I was introduced to scientific leaders at the Van Andel Research Institute and invited to become a member of their Stand Up 2 Cancer Epigenetics Research Dream Team. My role of this project is to provide the perspective of the patient to the research team and to provide my input on their work in epigenetics research

#ADV08

Energizing and educating to bridge the scientists/patient advocate survivors gap.

Naomi Greenwood. _Georgetown Breast Cancer Advocacy, Bethesda, MD_.

Input from informed cancer advocates can improve and clarify clinical relevance of researcher's project. However, anxiety about scientific and experiential competence can affect both advocates a with scientific backgrounds and even those who have backgrounds, but little interaction with cancer patients or survivors. To recruit advocates and encourage enthusiastic participation in reviewing grants and contributing to the community, several factors must be recognized and addresses. Opportunities for advocates to be well informed and develop their knowledge must be provided and encourages. Approached to increase knowledge include advocate attendance at educational programs, seminars and conferences. Furthermore, creating a safe atmosphere for open discussion of concerns and anxieties keeps energy positive and flowing. Many cancer survivors may wonder "what do I have to contribute as an advocate?" Therefore, it is vital that each member realized she has much to offer from her particular perspective and experience. These points were carefully considered when forming our research advocacy group called Georgetown Breast cancer Advocates (GBCA). It was formed in April 2011 as part of the Georgetown University Medical Center. GBCA works with researchers to help link cancer research and the community it is intended to impact.

#ADV09

Sharing the lived experience of cancer.

Judy K. Houlette. _Friend for Life Cancer Support Network, Louisville, KY_.

The Mission of Friend for Life Cancer Support Network is to help persons recently diagnosed with cancer and their loved ones navigate the path through diagnosis, treatment and recovery by pairing them with a trained survivor of a similar experience so they can face cancer with someone who's been there. Variously described as mentors, coaches or supporters, Friend for Life Cancer Support Network's Peer Navigators are persons who have experienced cancer firsthand and have participated in training to prepare them to mentor others. The poster will illustrate our Peer Navigation Model and highlight ways Peer Navigation benefits patients, families, oncology professionals, healthcare systems, and communities. It will provide an overview of the training and screening process, as well as lessons learned about what goes into making an optimal peer match. In 2007, Friend for Life Cancer Support Network and the University of Louisville School of Medicine initiated Patient-Practitioner Sessions that connect cancer survivors (Peer Navigators) with first-year students in order to enhance understanding, compassion, and improve overall communication and care. The program has since been expanded to three local schools of nursing. The poster will report impact on participating survivors, students and faculty.The constituents of Friend for Life Cancer Support Network are adults diagnosed with any form of cancer and/or their loved ones. We provide free Peer Navigation nationwide (and sometimes internationally). We also actively and continually recruit cancer survivors and caregivers to serve as Peer Navigators, in order to meet increasing demand for this form of psychosocial support. At the same time, the organization is also deeply dedicated to enhancing awareness and use of our services throughout our home state of Kentucky, where cancer incidence and mortality rates are among the worst.

#ADV10

Colon cancer awareness.

Julia M. Santiago. _Juliastrong Colon Cancer Awareness Foundation, San Juan, PR_.

Our vision: We want a world that's positive, healthy and free of colon cancer.Our mission: Educate on how to prevent colon cancer and any other malignancy using a holistic mind-body-spirit approach, which encourages an active, positive lifestyle filled with love and gratitude.Our pledge: Educate the 40+ adult population on the risk factors and prevention of colon cancer via timely screenings, such as a colonoscopy; educate on the preventive and self-healing power of adequately managing our feelings and emotions, practicing compassion, loving ourselves and loving selflessly our fellow men/women; build awareness on the benefits of holistic health and its power to prevent malignancies via educational, cultural and sport-oriented campaigns/activities; raise funds to support our Foundation's educational efforts and to contribute financially to other organizations supporting cancer patients/survivors during their treatment and recovery, physically and emotionally.

#ADV11

Clinical trial education and training: Priorities un a new era of trials.

Patricia Spears. _Cancer Information and Support Network, Raleigh, NC_.

The mission of Cancer Information & Support Network (CISN) is to assist in the building of bridges between all organizations involved with cancer research. To use the internet to provide education, information and support to cancer patients, their caregivers, advocates, and those working in the field of oncology.New and improved treatments for cancer depend on the completion of successful clinical trials. A major CISN priority is to educate and train patients, advocates and communities about clinical trials, increasing knowledge and engagement, to ultimately enhance accrual and outcomes. Key initiatives include providing patient focused feedback at all phases of clinical trial development, developing strategies to increase health literacy about clinical trials at both the national and community levels, to foster collaborations to provide decision aids for individual trials and to provide patient focused trainings.CISN recently completed a series of 14 webinars to train National Clinical Trials Network (NCTN) advocates in clinical trials, the science of cancer and practical based trainings on how to be an effective advocate. This program was initiated to ensure each advocate is well trained and able to address patient-centered issues during clinical trial development, which is essential to rapid accrual and completion of trials. It is not merely the inclusion of patients and advocates on committees, but the ability of each advocate to participate in the conversation by being educated on important issues and trained on the practical aspects of engagement. Another current project involves the education of all stakeholders on the informed consent process by addressing the lack of psychosocial training of professionals who administer consent. An additional current and future direction involves the incorporation of patient reported outcome (PRO) measurements in clinical trials. Assessing how to best incorporate, utilize and report PRO results in a meaningful and patient-centered manner.

#ADV12

Reducing Liver Cancer Risk by Screening for Hepatitis B Virusin African-born People in the U.S.: Pilot.

Noah Cohen. _The Mount Sinai Hospital, Brooklyn, NY_.

Currently, I conduct research within the lab of Lina Jandorf, MA, as a Project Coordinator of Oncological Sciences at the Icahn School of Medicine at Mount Sinai in East Harlem, NY. The lab's mission is to increase awareness and education for different cancers and diseases that affect the underserved populations of New York City (NYC). The lab initiatives include unique programs (e.g., The Witness Project of Harlem) that aim to effectively interact with the surrounding community through cancer outreach, educational programs and health fairs. Chronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma and is the third most common cause of cancer death in the world (Bosch, et. al., 2004 & Parkin, 2000). My poster will present data from one of our current projects, in collaboration with the Liver Division and Dr. Ponni Perumalswami, that aims to reduce liver cancer incidence amongst African-born immigrants in NYC by increasing screening for and education of HBV. We are in the process of conducting a qualitative study with focus groups to refine our educational program through discussions with African-born immigrants. Previous literature suggests a significant gap in patient-centered, culturally sensitive educational materials for this population in addition to disparities within HBV screening practices and knowledge.Thus far, we have conducted three focus groups (n=17) with NYC-dwelling African-born immigrants. The group demographic thus far is primarily 50 years of age or less (76.4%) and male (52.9%). Once our educational material is refined we will pilot test the program by holding 15 educational programs at distinct sites throughout NYC. Additionally, we will administer pre- and post-assessment questionnaires that will provide data to reflect the efficacy of the project. This poster will present findings from the focus groups, as well as the preliminary data from the initial educational programs.

#ADV13

Community Health Advisors (CHA) Affecting Change.

Sandra Hamilton. _Patient Advocate, Memphis, TN_.

Overall Community Health Advisor (CHA) Project Goals: to impact breast and cervical cancer in African- American women in Shelby and Fayette County Tennessee through screening, treatment, and care to reduce breast and cervical cancer incidences and mortality rates. CHAs provide peer education, support and referrals. Women with abnormal screening results are provided information on community resources as they navigate through the health system. The inception of the Memphis CHA program was 2010. The University of Alabama at Birmingham (UAB) was granted funds by Centers for Disease Control and Prevention (CDC) under the national REACH US (Racial and Ethnic Approaches to Community Health in the United States) program to establish the Mid-South Center of Excellence in the Elimination of Disparities (CEED) for breast and cervical cancer. To achieve the goals of the Mid-South CEED, UAB worked with well-established coalitions of national, state and community partners; among them the American Cancer Society (ACS). In 2013, UAB was not re-funded and the ACS became the sole sponsor for the CHA Mid-South program. Through community collaborations, engagement and mobilization, a community infrastructure was developed that was conducive for the implementation of the CHA program. Working with Community Network Partners (CNP) whose objectives was similar to those of the ACS, including Tennessee Cancer Coalition and the National Black Leadership Initiative on Cancer, a more effective and efficient program was designed. CNP recruited CHA volunteers. After being trained and becoming knowledgeable breast and cervical cancer, screening and treatment, they recognized the significance and health benefits it would bring to African American women in Shelby and Fayette Counties. These volunteers recruited other lay volunteers to act as catalysts for change in the community.

#ADV14

LRF advocacy.

Robert Mesloh. _Lymphoma Research Foundation, The Villages, FL_.

The Lymphoma Research Foundation (LRF's) mission is to eradicate lymphoma and serve those by this disease. LRF is the nation's largest non-profit organization devoted exclusively to funding innovative lymphoma research. It provides to those people with a lymphoma diagnosis/caregivers and healthcare professionals up-to-date information about this type of cancer. To date, the foundation has awarded more than $57 million of lymphoma specific research grants. Many people do not know much about lymphoma until being diagnosed. Lymphoma is type of blood cancer that may develop in many parts of body, including the lymph nodes, spleen, bone marrow, blood or other organs.

Establishing an LRF presence in The Villages, Florida, and surrounding Central Florida is a needed advocacy activity about lymphoma diseases. LRF is dedicated to funding biomedical research focused on the origins, treatment, and identification of a cure for lymphoma. LRF assists members of the lymphoma community by providing comprehensive, disease-specific programs and services to more than 70,000 people each year, so that people can better understand and navigate their lymphoma diagnosis.

The Foundation's board of directors is comprised of business leaders, medical professionals, lymphoma survivors. LRF has a scientific advisory board, comprised of 45 world-renowned lymphoma experts who guide LRF research activities, seeking out the most innovative and promising lymphoma research activities, seeking out the most innovative and promising lymphoma research projects for support.

#ADV15

A prescription to learn.

Sarah Krug. _Cancer 101, New York, NY_.

Mission: To empower, inform and engage patients and their caregivers to take control over their diagnoses, navigate the cancer journey, and partner with their healthcare team to make informed decisions.

To fulfill its mission, CANCER101, founded in 2002: Meets the cancer patient and caregiver on the front line and turns a chaotic experience into a calm and organized plan of attack. Provides innovative tools and resources patients and caregivers need to manage chronic condition in partnership with the healthcare team, allowing them to document their day to day life experiences.

Personalization of the patient experience -one patient at a time Creates a comprehensive roadmap for patients by filtering "garbage from gold", aggregating best practice content through partnerships with other organizations. Connections Matter. Imagine if patients and their caregivers could access a YELP for healthcare to empower them to navigate the health journey and make smarter decisions through a platform that cut through the clutter and aggregated the most credible resources available? Imagine if the search could then be further personalized based on the patient's preferences, their phase in the journey, their medium of choice/learning style, and the ratings of other patients and caregivers who have been in their shoes. The Prescription to Learn Platform™ is designed to help navigate patients and caregivers through the information overload they are often confronted with throughout the cancer journey. We have established criteria and have already assessed thousands of resources available to patients and selected the highest quality education, which will be portrayed by disease state. Whether it's an advocacy organization's website or hotline, a key book or brochure in navigating the journey, a mobile application to track symptoms or medications or a hotline to learn more about clinical trial options, the Prescription to Learn (P2L) platform will provide the patient and their caregiver the option to search within a safe zone that reduces the tsunami of information available today. A Prescription to Learn ignites the shared decision making model and partnership approach to care in that it allows the patient to become more empowered to navigate the journey, learn about their condition and get informed, assess the risk and benefits associated with treatments, partner with the clinician to make informed medical decisions, and take a more active role in symptom and medicine identification/management. 
